WO2014190035A2 - Compositions and methods for identification, assessment, prevention, and treatment of cancer using histone h3k27me2 biomarkers and modulators - Google Patents

Compositions and methods for identification, assessment, prevention, and treatment of cancer using histone h3k27me2 biomarkers and modulators Download PDF

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WO2014190035A2
WO2014190035A2 PCT/US2014/038938 US2014038938W WO2014190035A2 WO 2014190035 A2 WO2014190035 A2 WO 2014190035A2 US 2014038938 W US2014038938 W US 2014038938W WO 2014190035 A2 WO2014190035 A2 WO 2014190035A2
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biomarkers
level
expression
activity
cancer
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WO2014190035A3 (en
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Andrew Lane
David Weinstock
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Dana-Farber Cancer Institute, Inc.
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Publication of WO2014190035A3 publication Critical patent/WO2014190035A3/en

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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
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    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
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    • C12Q2600/00—Oligonucleotides characterized by their use
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Definitions

  • B- ALL B cell acute lymphoblastic leukemia
  • polysomy 21 ⁇ i.e., extra copies of chromosome 21 ⁇ is the most frequent somatic aneuploidy in B-ALL (Heerema et al. (2007) Genes Chrom. Cancer 46:684-693; Pui et al N. Engl J. Med. 350:1535-1548).
  • Additional B-ALLs harbor an iiitrachromosoinal amplification of chr.21q22 (iAmp21) (Moorman et al Lancet Oncol 11 :429-438; Rand et al. Blood 1 1 :6848-6855) that overlaps with the putative "Down Syndrome Critical Region (DSCR)" on chromosome 2 iq22.
  • DSCR Down Syndrome Critical Region
  • B-ALL B cell acute lymphoblastic leukemia
  • B cells with amplified DSCR e.g., copy number gains, enhanced expression, and the like
  • amplified DSCR e.g., copy number gains, enhanced expression, and the like
  • PRC2 porycomb repressor complex 2
  • H3K27 memyltransferases pharmacological inhibition of H3K27 memyltransferases is sufficient to promote self- renewal in wild-type B cells while enhancement of H3 27mc3 levels (*.&, by inhibiting demethylases mat remove H3K27me3) completely block self-renewal induced by DSCR triplication. It has further been discovered that self-renewal in B cells with DSCR triplication requires overexprcssion of the DSCR locus encoding HMON I , a nucleosome remodeling protein encoded on chr.21a22 (Catez et al. (2002) FMBORep. 3:760-766; Lim et al. (2005) UMBO J. 24:3038-3048; Rattner et al.
  • H3f 27me3 suppresses H3f 27me3 levels.
  • Overexprcssion of HMGN1 suppresses H3 27mc3 and promotes both B cell proliferation in vitro and B-ALL in vivo.
  • HMON 1 overexprcssion and loss of H3K27me3 are implicated in progenitor B cell transformation and provide strategics to therapeutically target leukemias with potysomy 21.
  • a method of dctainining whether a subject afflicted with a cancer or at risk for developing a cancer would benefit from modulating bJstone H3K27mc3 levels comprises: a) obtaining a biological sample from die subject b) determining the copy number, levd of expression, or level of activity ofoneormore biomarkcrs listed in Tables 1-5 or a fragment thereof in a subject sample c) determining die copy number, level of expression, or level of activity of die one or more biomarkers in a control; and d) comparing the copy number, levd of expression, or level of activity of said one or more biomarkers detected in steps b) and c); wherein a significant modulation in die copy number, lcvd of expression, or level of activity of die one or more biomarkers in the subject sample relative to the control copy number, level of expression, or level of activity of the one or more biomarkers indicates that the subject afflicted with the
  • the one or more biomarkcrs are selected from the group consisting of the set of a) "top 150 UP” biomarkers shown in Table 1, b) "the 50 UP core” biomarkers shown in Table l,c) u top 150 DOWN” biomarkers shown in Table 1, d), "the 50 DOWN core” biomarkers shown in Table 1, c) the "triplicated gene” biomarkers shown m Table 1 , f) the *chr2lq22 overlap” biomarkcrs shown in Table 2, g) the "PRC2 cluster” biomarkers shown in Table 3, h) the "overlap” biomarkers shown in Table 4, i) the "SUZ 12 target,"
  • Mikkebcn MEF and/or “Mikkdsen NPC biomarkers shown in Table 5, j) DM6A. k) DM6 , 1) EZH2, m) HMGNl , and subsets and/or conAinations thereof.
  • a method for monitoring the progression of a cancer in a subject comprises: a) detecting in a subject sample at a first point in time die copy number, level of expression, or level of activity of one or more biomarkcrs listed in Tables I -5 OT a fragrnent thereof, b) repeat ⁇
  • the one or more biomarkcrs are selected from the group consisting of the set of a) "top 150 UP” bkmwrkers shown in Table I, b) "the 50 UP core” biomarkers shown in Table 1, c) "top 150 DOWN” biomarkers shown in Table I, d), "the 50 DOWN core” biomarkers shown in Table 1, c) the "triplicated gene” biomarkers shown in Table 1 , die “chr21 q22 overlap” biomarkers shown in Table 2, g) die “PRC2 cluster” biomarkers shown in Table 3, h) the "overlap” biomarkers shown in Table 4, i) the "SUZ12 target.”
  • Mikkebcn MEF and or “Mikkdsen NPC” biomarkers shown in Table 5, j) DM6A. k) D 6B, 1) EZH2, m) HMGNl , and subsets and/or combinations thereof.
  • an at least twenty percent increase or an at least twenty percent decrease between the copy number, level of expression, or level of activity of the one or more biomarkers in tbe subject sample at a first point in time relative to tbe copy iitimber, level of expression, or level of acti ity of the one or more biomarkers m the subject sample at a subsequent point in time indicates progression of the cancer
  • wbcrein less than a twenty percent increase or less than a twenty percent decrease between the copy number, level of expression, or level of activity of tbe one or more biomarkers in the subject sample at a first point in time relative to tbe copy number, level of expression, or level of activity of the one or more biomarkers in the subject sample at a subsequent point in time indicates a lack of significant progression of the cancer.
  • the subject has undergone treatment to modulate histone H3K27rae3 levels between die first point in time and tbe subsequent point in time.
  • a method for stratifying subjects afflicted with a cancer according to predicted clinical outcome of treatment with one or more modulators of histone H3K27me3 levels comprises: a) aetenriming the copy number, level of expression, or level of activity of one or more biomarkers listed in Tables 1-5 or a fragment thereof in a subject sample; b) determining the copy number, level of expression, or level of activity of the one or more biomarkers in a control sample; and c) comparing the copy number, level of expression, or level of activity of said one or more biomarkers detected in steps a) and b); wherein a significant modulation in the copy number, level of expression, or level of activity of u ⁇ e one or more biomarkers in tbe subject sample relative to the normal copy number, level of expression, or level of activity of the one or more biomarkers in the control sample predicts the clinical outcome of the patient to treatment with one or more niodulatore of histone H3
  • the predicted clinical outcome is (a) cellular growth, (b) cellular proliferation. or (c) survival time resulting from treatment with one or more modulators of histone H3K27mc3 levels.
  • the one or more biomarkers are selected from die group consisting of die set of a) "top 150 UP” biornarkers shown in Table l, b) "ihe 50 UP core” biomarkers shown in Table I, c) "top 150 DOWN” biomarkers shown in Table 1. d).
  • the method further comprises treating the subject with a therapeutic agent that specifically modulates the copy number, level of expression, or level of activity of the one or more biomarkcrs.
  • the method further comprises treating the subject with one or more modulators of his tone H3K27mc3 levels.
  • a method of dctennining the efficacy of a test compound for inhibiting a cancer in a subject comprises: a) determining the copy number, level of expression, or level of activity of one or more biomarkcrs listed in Tables l-5 cf a fragrnem thereof ma first sample obtained from the subject and exposed to the test compound; b) ddcrmining the copy number, level of expression, or level of activity of the one or more biomarkcrs in a second sample obtained from the subject, wherein the second sample is not exposed to me test compound, and c) comparing the copy number, level of expression, or level of activity of the one or more biomarkers in the first and second samples, wherein a significantly modulated copy number, level of expression, or level of activity of the biomarker, relative to die second sample, is an indication that the test compound is efficacious for inhibiting the cancer in the subject, b one embodiment, the one or more biomarkers are selected rom
  • the first and second samples are portions of a single sample obtained from the subject or portions of pooled samples obtained from the subject in another aspect, a method of determining the efficacy of a therapy for inhibiting a cancer in a subject is provided, wherein the method conphses: a) determining the copy number, level of expression, or level of acti ity of one or more biomarkcrs listed in Tables 1 -5 or a fragment thereof in a first sample obtained f om the subject prior to providing at least a portion of the therapy to the subject; b) determining the copy number, level of expression, or level of activity of the one or more biomarkcrs in a second sample obtained from the subject following provision of the portion of the therapy; and c) comparing the copy number, level of expression, or level of activity of the one or more biomark
  • the one or more biomarkcrs are selected from the group consisting of the set of a) "top 150 UP” biomarkcrs shown in Tabic I , b) "the 50 UP core” braraarkers shown in Table 1.
  • a method for identifying a compound which inhibits a cancer comprises: a) contacting one or more biomarkcrs listed in Tables 1-5 or a fragment thereof with a test compound; and b) determining the effect of the test compound on the copy number, level of expression, or level of activity of the one or more biornarkers to thereby identify a compound which inhibits the cancer.
  • the method comprises: a) contacting one or more biomarkcrs listed in Tables 1-5 or a fragment thereof with a test compound; and b) determining the effect of the test compound on the copy number, level of expression, or level of activity of the one or more biornarkers to thereby identify a compound which inhibits the cancer.
  • the one or more biomarkers is expressed on or in a cell (eg., cdts isolated from an animal modd of a cancer or cells from a subject afflicted with a cancer).
  • a method for inhibiting a cancer comprises contacting a cell with an agent that modulates the copy number, level of expression, or level of activity of one or more biomarkers listed in Tables 1-5 or a fragment thereof to thereby inhibit the cancer.
  • the one or more biomarkers are selected from the group consisting of the set of a) "top 150 UP" biomarkers shown in Table
  • the copy number, level of expression, or level of activity of the one or more biomarkers is downmodulated or unmodulated.
  • the step of contacting occurs in vivo, ex vivo, or in vitro.
  • the method further comprises contacting the cell with an additional agent mat inhibits (he cancer.
  • a method for treating a subject afflicted with a cancer comprises administering an agent that modulates the copy number, level of expression, or level of activity of one or more biomarkers listed in Tables 1-5 or a fragment thereof such that the cancer is treated
  • the one or more biomarkers are selected from the group consisting of the set of a) "top 150 UP” biomarkers shown in Table I, b) "the 50 UP core” biomarkers shown in Table 1, c) "top 150 DOWN” biomarkers shown in Table I , d), "the 50 DOWN core” biomarkers shown in Table 1, e) the ''triplicated gene” biomarkers shown in Table l, f) tbc "chr21q22 overlap” biomarkers shown in Table 2, g) the "PRO cluster” biomarkers shown in Table 3, h) the "overlap” biomarkers shown in Table 4.
  • the agent downmodulatcs or upmodulates the copy number, level of expression, or level of activity of the one or more biomarkers.
  • the method further comprises administering one or more additional agents that treats the cancer, in yet another embodiment, the agent is one or more modulators of iristone H3K27mc3 levels.
  • a pharmaceutical composition comprising a polynucleotide encoding one or more biomarkers listed in Tables 1 -5 or a fragment thereof useful for treating cancer in a pharmaceutically acceptable carrier, hi one embodiment, the polynucleotide encoding the one or more biomarkers listed in Tables 1-5 or a fragment thereof further comprises an expression vector.
  • the pharmaceutical composition is used in a method for treating a cancer.
  • kits comprising an agent which selectively binds to one or more biomarkers listed in Tables 1-5 or a fragment thereof and instructions for use.
  • kits cotnprising an agent which selectively hybridizes to a polynucleotide encoding one or more biomarkers listed in Tables 1-5 or f agment thereof and instructions for use.
  • a biochip apprising a solid substrate, said substrate comprising a plurality of probes capable of detecting one or more biomarkers listed in Tables 1-5 or a fragment thereof wherein each probe is attached to the substrate at a spatially defined address.
  • die probes are complementary to a genomic or transcribed polynucleotide associated with the one or more biomarkers.
  • a method of increasing the number of lymphoid progenitor cells from an initial population of lymphoid progeni tor cells comprises contactin the lymphoid progenitor cells with an agent that inhibits poJycomb repressor complex 2 (PRO) activity or reduces H3K27me3 levels to thereby increase the number of lymphoid progenitor cells.
  • the agent inhibits die activity of the EZH2 histone H3K27 rnethyltransfcrasc subunit of PRC2.
  • the agent is an inhibitor selected from the group consisting of a small molecule, antisense nucleic acid, interfering RNA, shRNA, siRNA, miRNA, aptamer, r bozyme, and dominant-negative protein binding partner.
  • the lymphoid progenitor cells are comprised within bone marrow with marker selection or without marker selection.
  • the lymphoid progenitor cells ccinprise pre-pro B cells, pro B cells, large pre-B cells, small prc-B cells, immature B cells, or any combination thereof.
  • contacting the lymphoid progenitor cells with the agent is performed in vivo, ex vivo, or in vitro.
  • compositions, kits, or biochips described above can use one or more biomarkers selected from the group consisting of the set of a) "top 150 UP” bkrattrkers shown in Table 1, b) "the 50 UP core” biomarkers shown in Table I, c) "top 150 DOWN” biomarkers shown in Table 1. d), "the 50 DOWN core” biomarkers shown in Tabic 1.
  • ikkelscn MEF and or "Mikkelsen NPC biomarkers shown in Table 5, j) K.DM6A, k) KDM6B, 1) EZH2, m) HMG 1 , and subsets and or cc ⁇ nbinatioro thereof.
  • the control is determined from a non-cancerous sample from the subject or member of the same species to which the subject belongs.
  • die sample comprises cells, cell lines, histological slides, paraffin embedded tissue, fresh frozen tissue, fresh tissue- biopsies, blood, plasma, serum, buccal scrape, saliva, cerebrospinal fluid, urine, stool, mucus, or bone marrow, obtained from the subject
  • the copy number is assessed by microarray, quantitative PCR (qPCR), high-throughput sequencing, conipaiatrve enomk liybrid ⁇
  • the expression level of the one or more biomarkers is assessed by detecting the presence in the samples of a polynucleotide molecule encoding the biomarkcr or a portion of said polynucleotide molecule.
  • the polynucleotide molecule is a mRNA, cDNA, or functional variants or fragments thereof.
  • die step of detecting further comprises amplifying the polynucleotide molecule.
  • the expression level of the one or more biomarkers is assessed by annealing a nucleic acid probe with the sample of the polynucleotide encoding the one or more biomarkers or a portion of said polynucleotide molecule under stringent hybridization conditions.
  • the expression level of the biomarker is assessed by detecting the presence in the samples of a protein of the biomarker, a polypeptide, or protein fragment thereof comprising said protein.
  • the presence of said protein, polypeptide or protein fragment thereof is detected using a reagent which specifically binds with said protein, polypeptide or protein fragment thereof (eg. , a reagent selected from the group consisting of an antibody, an antibody derivative, and an antibody fragment).
  • a reagent which specifically binds with said protein, polypeptide or protein fragment thereof eg. , a reagent selected from the group consisting of an antibody, an antibody derivative, and an antibody fragment.
  • the activity level of the biomarkcr is assessed by determining the magnitude of modulation of the activity or expression level of downstream targets of the one or more biomarkers.
  • the agent or test compound modulates historic H3 27me3 levels.
  • the agent or test compound inhibits the expression and/or activity of Jumonji D3 family of histooe HeK27 demethylases.
  • the agent or test compound is a small molecule inhibitor of KMD6A (UTX) and/or KDM6B (J JD3).
  • the agent or test compound inhibits the expression and/or activity of HMGN1.
  • the agent or test compound is an inhibitor selected from die group consisting of a small molecule, anbsense nucleic acid, interfering RNA, shRNA, siRNA, aptamcr, ribozyme, and dominant-negative protein binding partner.
  • the cancer is a leukemia (eg., B-cell acute lymphoblastic leukemia).
  • the subject has an increased copy number of a) human chromosome 2 i or the human DSCR region thereof, b) mouse chromosome 16 or the mouse iAmp, Ts65Dn, TslRhr, Dp(16)l Yu, or Runxl locus thereof, ore) orthologs of a) or b relative to a wild type control.
  • the subject is a human.
  • Figures 1A-1G show mat segmental trisomy orthologous to human chr.21q22 promotes progenitor B cell transformation.
  • Figure 1 A shows regions orthologous to human chromosome 21 mat are triplicated in TslRhr and Ts65Dn mice or amplified in ⁇ 21 B- ALL.
  • Figure 1C shows subtractions from mixed populations in recipient BM 16 weeks after competitive transplantation (n m 5/group).
  • Figure 1G shows leukemia-free survival of recipient mice after
  • FIGHrcs 2A-2F show the results of abnormal differentiation in vivo and colony growth in vitro of B cells with triplication of chr.21 orthologs.
  • Figure 2A shows B220 and CD 3 staining of bone marrow from Ts I Rhr and wild-type mice, highlighting the more immature B220+CD43+ and more mature B220+CD43- B cell populations (top panel) and CD24 and BPi staining of the B220+CD43+ subpopulation demonstrates the car y Hardy fractions: A (CD24- BP1-), B (CD24+BP1-), and C (CD24+BPI+).
  • Figure 2C shows a schematic for the competitive bone marrow
  • FIG. 2D shows representative Hardy f action staining in bone marrow gated on CD45.2 negative (left) competitor ceils or CD45.2 positive (right) test cells.
  • the top rows are wild-type test cells, and the bottom rows arc Tsl Rhr test cells.
  • Figure 2E shows a schematic of the mcthylccihilose replating assay.
  • Figure 3 shows that cell surface phenotype of passage I B cell colonies from wild- type and Tsl Rhr animals are similar. A representative flow cytometry plots of Hardy fraction cell surface phenotype of passage 1 wild-type and Tsl Rhr B cell colonies is shown. All cells are also B2204CD43+.
  • FIG. 4 shows that passage 6 Tsl Rhr B cell colonies can form serially
  • Figures 5A-5G show characterization of the B-ALL that arises in Tsl Rhr bone marrow.
  • Figure SA snows a representative phenotype of C2/J2/P5 Tsl + D 6 B-ALL demonstrating expression of human CRLF2 in Ac leukemic B cells that also co-express dominant negative Ikaros (Ik6).
  • FIG. 5C shows transduced Ts I Rhr and wild-type bone marrow using flow cytometry for B220 and GFP (BCR-ABL) demonstrating approximately equal proportions of GFP+ cells at the time of transplantation.
  • Figure SD shows that Tsl Rhr and wild-type BCR-ABL B-ALLs demonstrate similar splenomegaly at the time of death with leukemia. Red dotted line represents upper limit of normal spleen weight.
  • Figure 5E shows bone marrow and spleen histology by hematoxylin and eosin staining demonstrating similar infiltration with B-ALL cells in Tsl Rhr and wild-type B-ALLs (scale bar ⁇ 50 ⁇ ).
  • Figure 5F snows survival curves for recipients of TslRhr or wild-type bone marrow cells (on a C57BL 6 background) transduced with BCR-ABL (tr*9 mice per group, curves compared by log-rank test).
  • Figure SG shows an increase in B-ALL from Ts Rhr bone marrow is progenitor B ceil autonomous. Hardy B cells were sorted from TslRhr or wild-type bone marrow, transduced with BCR-ABL, and equal numbers of cells were transplanted into wild-type recipients ( ⁇ »5 mice per group, curves compared by log-rank test).
  • Figares 6A-6D show that triplication of die DSCR cooperates with BCR-ABL to promote B-ALL tn vtvo.
  • Figure 6B shows limiting dilution analysis of recipient survival at 80 days after transplantation using a Poisson distribution calculation (Wang era/.
  • FIG. 6C shows cell surface phenorype of leukernias arising in wild-type or TslRhr bone marrow cells.
  • Figure 6D shows the probability of B-ALl free survival in wild-type recipients of 10* wild-type or Tsl Rhr sorted Hardy fraction A, B, or C bone marrow cells transduced with a BCR-ABL- expressing retrovirus (n»15 per genotype, n»5 per Hardy fraction, compared by log-rank tests).
  • FIG. 7 shows that recipients of Tsl Rhr bone marrow transduced with BCR-ABL have more significant hematologic abnormalities after 3 weeks compared to recipients of wild-type bone-marrow.
  • WBC Whits blood cell counts
  • HB hemoglobin
  • PHT platelet
  • Figure 8 shows a schematic of Hardy fraction sorting followed by BCR-ABL transduction and transplantation experiment
  • Hardy fraction A, B, and C cells from wild- type or TslRhr B220+CD43+ bone marrow cells were sorted, transduced with MSCV- BCR-ABL-ircs-GFP, and 10 5 cells were transplanted into lethalty irradiated wild-type recipients (see Figure 2A for the Hardy fraction flow sorting strategy).
  • Figures 9A-9J show that trisomy and tctrasomy 21 retinal pigment epithelium (RPE) cells generated by microcell-mediated chromosome transfer (MMCT) do not have differences m DNA repair after I-Scel or RAG-induced cleavage.
  • Figure 9A shows single nucleotide polymorphism (SNP) array data for a tetrasomy 21 RPE clone (tetra 21-1), two trisomy 21 (tri21-2 and tri21-3) clones, and a diploid clone are shown across the entire genome (top) or chranosome 21 (bottom).
  • SNP single nucleotide polymorphism
  • Figure 9C shows representative G-banding karyotype for a tetrasomy 21 RPE cell line.
  • Figure 9D snows that the DR-GFP construct was targeted to the p84 locus in RPE cells containing 2 or more copies of cm ⁇ 21. A single double-strand DNA break induced by 1-SceI can be repaired by multiple pathways.
  • Figure 9E shows that repair after I-Scel cleavage in cells lacking classical nonhomologous end-joining (NHEJ) factors (e.g. KU7O/80, XRCC4 L1G4) is characterized by higher rates of homologous NHEJ) factors (e.g. KU7O/80, XRCC4 L1G4) is characterized by higher rates of homologous
  • FIG. 9G shows that the DR-GFP-CE construct targeted to the p84 iocus can be used to assess repair after RAO cleavage. Cleavage at the paired RAG recognition signal sequences (white and black triangles) results in removal of the intervening sequence (in yellow) and nonhomoiogous end joining (NHEJ) between the double-strand break ends.
  • Figure 9H shows that PCR shows no difference in the frequencies of the RAG- induced deletion between diploid and tetrasomy 21 cells. Two biologic replicates are shown for each genotype.
  • Figure 91 shows tfiat repair junctions after RAG cleavage in cells lacking classical NHEJ factors (e.g., KU70/80.
  • XRCC4/LIG4 typically have longer deletions and more extensive use of short stretches of homology than in wild-type cells (Wetnstock etal. (2006) A /. Cell. Biol. 26:131-139). However, the number of base pairs deleted after cleavage by RAG and NHEJ did not signifkandy differ between disomic and tetrasomy 21 cells (n Z clones per genotype).
  • Figure 9J shows junction sequences for disomic (n-27) and tetrasomy 21 (n-70) RPE clones. A single nucleotide insertion is shown in Tctra-I B-3-7 (yellow).
  • Figure 10 shows that RNA-seq expression of the triplicated genes in TslRhr compared to wild-type B cells.
  • RNA sequencing of TslRhr and wild-type B cells yielded relative expression levels among die 25 expressed triplicated genes (absolute fragments per kilobase per million reads
  • Figures 12A-12G show that polysemy 21 B-ALL is associated with the overcxprcssion of PRC2 targets.
  • Figure 12A shows a beat map of human genes orthologous to the 150 roost upregulated genes from TslRhr B cells in primary human pediatric B-ALLs. Unsupervised hierarchical clustering by gene revealed the "core TslRhr" gene set (boxed).
  • Figure 12B shows GSEA plots for the full and core TslRhr gene sets in die AIEOP data set ES, enrichment score.
  • Figure 12C shows a GSEA plot of the core TslRhr gene set in an independent ICH validation cohort.
  • Figure 12D shows a network enrichment map of MSigDB gene sets enriched (FDRO.05) in die TslRhr expression signature.
  • Figure 12E shows unsupervised hierarchical clustering of
  • H3 27me3-marked genes from the MIKKELSEN_MEF_H3K27me3 gene set in the AIEOP pediatric B-ALL cohort (karyotype shown).
  • Figure 12F shows GSEA plots of the top 100 genes from three PRC2/H3 27me3 gene sets as defmed in the AIEOP patient cohort in the ICH validation cohort.
  • Figures 13A-I3E shows mat DS-ALL is associated with ovcrexpression of PRC2 targets and genes marked by H3K27mc3, TslRhr and PRC2 H3K27mc3 gene signatures distinguish non-DS-ALL with somatic gain of chromosome 21 or ⁇ 21 , and Ts I Rhr B- ALLs arc associated with H3K27 hypomethylation.
  • Figure 13A shows heat maps of all genes comprising three of the top five scoring target gene sets enriched in die core Ts 1 Rhr signature in DS-ALL* and non-DS-ALLs.
  • Figure 13B shows unsupervised clustering results of a validation cohort of 30 non-DS pediatric B-ALL gene expression signatures (the AIEOP-2 cohort) using a lOO-gcne SUZ12 target gene set Four patients with somatic gain of chr.21 and two with iAMP21 cluster within a distinct group with 5 additional cases (P-0.001 by Fisher's exact test).
  • Figure 1 C shows GSEA plots of the TslRhr gene set and the top 100 discriminating genes in the Mikkeben NPC and MEF H3 27mc3 gene sets from the AIEOP cohort, queried in the primary human B-ALLs in die AIEOP-2 cohort containing cases with somatic +21 and iAMP2l. ES indicates enrichment score.
  • Figure 13E shows
  • FIG. 14A-14H show that TslRhr B cells have reduced H3 27mc3 that results in overexpression of bivalendy marked genes.
  • Figure 14A shows gene tracks showing occupancy of historic marks at the Plod2 promoter (one of the SO core Tsl Rhr genes) in reads per million per base pair (rpen bp).
  • Figure 14B shows levels of H3K27me3 in TslRhr and wild-type B cells at regions enriched for H3K27me3 in wild-type celts (***P ⁇ le-l 6).
  • Figure I4C shows histone marks at the promoters of genes mat are unregulated or ckywiircgulatcd in TslRhr vs. wild-type cells (**? ⁇ le-S).
  • Figure 14D shows chromatin marks in wild-type B cells present at promoters of all genes (left) or genes that arc upregulated in Tsl Rhr B cells (right, ***PO.0001 compared to all genes by Chi-square with Yates' correction).
  • Figure I4E shows colony counts in the presence of DMSO or GSK-J4 (n»3 biological replicates per genotype, *P ⁇ 0.05 compared to DMSO for same genotype).
  • Figure 14F shows colony counts in the presence of GSK-126 or after wrthdrawal at passage 5 (*P 0.05 compared to OSK-126 for same genotype, WPO.05 compared to odier genotype or no withdrawal). Arrow indicates GSK-126 withdrawal.
  • Figure 14G shows Western blotting results of passage 2 colonies after 14 total days in culture with DMSO. 1 ⁇ GSK-J4, or 1 uM GSK-126.
  • Figure 14H shows Western blotting results of colonics one passage (7 days) after continuation (+) or removal (-) of GSK-126.
  • FIGS 15A-15H show that ChlP-seq and CHIP-qPCR exhibit decreased
  • H3K27mc3 at promoters in Tsl Rhr B cells the TslRhr gene set is enriched for E2A TCF3 and LEF1 targets, and DS-ALLs are sensitive to OSK-J4.
  • Figure 15B shows H3K27mc3 enriched regions in wild-type B cells.
  • the promoter region is defined as the Skb flanking annotated transcription start sites. Overlap of H3K27me3 regions with die promoter region was significant in comparison to a random background model of the genome (P ⁇ 10 *10 ).
  • Figure ISC shows a Venn diagram showing die number and overlap between H3K27me3 enriched regions in wild-type (WT) or TslRhr B cells.
  • Figure 1SD shows the logj fold difference in density of H3K27mc3 at promoters between TslRhr and wild-type B cells is shown.
  • Figure 15E shows the top three ranked transcription factors with predicted binding sites among promoters of genes in the listed sets as queried in MSigDB "c3.ttT defined in the TRANS FAC database (version 7.4, available on the World Wide Web at gene-fegulation.com).
  • Figure 15F shows the relative fraction of genes that have proximal E2A TCF3 occupancy among all genes (7129 of 20671 ), genes with only H3K27me3 (557 of 1994) or H3K4me3 (4032 of 360) at the promoter in wild-type B cells, or genes in the TslRhr gene set (85 of 150) (**P ⁇ 0.01, ***P ⁇ 0.0001 versus the TslRhr gene set by Chi-square with Yates * ccHrrcction).
  • Figure 15G shows that expression of genes in the TslRhr and Core TslRhr sets are increased compared to all probesets in wild-type B cell progenitors as compared to E2A" ⁇ (expression data from 2 *; ***P ⁇ 0.0001 by Student t- test, center bars - median, box - 25-75% confidence interval, whiskers - 10-90% confidence interval).
  • Figure 15H shows the 1C» for five DS-ALLs treated in vitro with GSK-J4 (error bars represent 95% confidence intervals).
  • Figures 16 ⁇ -16 ⁇ show the sensitivity of murine and human B-ccll ALL to OSK-J4.
  • Figure I6A shows that a subset of murine B-cell acute rvmphoblastic leukemias that harbor triplication of the Down Syndrome Critical Region (lower panel) arc 100-foW more sensitive to GSK-J4 compared to teukemias that lack triplication (upper panel)-
  • Figure 16B shows that a human primary B-cell ALL xenograft from a patient with Down Syndrome is 1(M 00-fold more sensitive to GSK-J4 compared to a similar xenograft that lacks an extra copy of chromosome 21.
  • Figures 17A-17E show that HMG 1 overcx ressRm decreases H3K27me3 and promotes transformed B cell phenotypes.
  • Figure 17 A shows Western blotting results of Ba F3 cells transchtccd with empty virus or murine HMGN1 (n»3 irtdependent biological replicates).
  • Figure 17C shows GSEA plots for the full and core TslRhr gene sets in
  • H GN1_0E transgenic B cells H GN1_0E transgenic B cells.
  • Figures 18A-18G show that HMGN1 overexpression alone results in multiple B ceU phenotypes observed with triplication of the entire 21q22 orthologous region.
  • Figure 1 KA shows relative quantitation of H3K27me3 and H GN i in BaF3 lymphoblasts transduced with empty vector of mouse HMGN1.
  • Figure 18B shows a beat map showing RNA expressionof the 31 triplicated genes in passages 1, 3, and 6 in triplicate TslRhr cultures (bhie-rcd « low to high log 2 FPK values, genes listed in genomic order).
  • Figure 18D show normalized quantitation of negative (non- largeting) and positive (known to be toxic) control shRNAs in passage 6 Tsl Rhr colonies relative to input (left) demonstrates preferential loss of positive control shRNAs. Neither positive nor negative control shRNAs were preferentially lost from Tsl Rhr passage 3 cells compared to wild-type (right, Tukey box and whiskers plots, horizontal bar is the median and plus is the mean; *P 0.05; NS, not significant).
  • Figure 18E show Western blotting results of BaF3 rymphoblasts confirming knockdown of HMGN1. Antibodies are: A (Abeam), B (A viva), mHMGNl (affinity purified murine HMGN1 antibody).
  • Figure 18F show Western blotting results of HMGN 1 in B cell colonies from wild-type and
  • FIG. 180 shows Hardy B cell subtractions as percentages of bone marrow cells from wild-type (black) and HMGNljOE (orange) littermates (n*4 per group, *P ⁇ 0.05).
  • Figure 19 shows a schematic of B-cell developmental lineages and associated molecular markers according to murine genetics nomenclature.
  • Table 1 shows genes differentially expressed in TslRhr as compared to wild-type B cells.
  • the top 150 higher (UP) and lower (DOWN) expressed genes in TslRhr relative to wild-type passage 1 B cells by RNAseq and EdgcR analysis (p ⁇ 0.05, false discovery rate ⁇ 0.25) is shown (n 3 independent biologic replicates per genotype).
  • Differential expression is annotated as log] fold change in TslRhr relative to wild-type.
  • the SO UP genes that constitute the Core TslRhr gene set ( Figure 12A) are annotated.
  • Table 2 shows the results of a query of the top 150 TslRhr UP gene set against the Molecular Signatures Database (MSigDB) * c I ' positional dataset
  • Table 3 shows the results of gene set enrichment and network enrichment mapping for TslRhr B cells.
  • MSigDB Molecular Signatures Database 4 c2 cgp' chemical and genetic perturbations dataset
  • Table 5 shows die top 100 differentially expressed genes in the SUZ12 target gene, Mikkelsen MEF and NPC H3 27me3 signatures between DS-ALU and non-DS-ALLs.
  • Table 6 shows shR As used in the cornpetitrve growth assay targeting DSCR genes. Gene symbols fix DSCR genes (tab 1 TEST) and controls (tab 2 "CONTROLS") are shown, with clone names in The RNAi Consortium (TRQ database, target sequence, and location of the target sequence within the gene. Data arc the normalized ratio of the quantitation of each shRNA in TslRhr to wild-type B cells during passaging relative to input within each genotype.
  • the present invention is based, at least in part, on the novel discovery of gene profiles useful for distinguishing among cancer subtypes (e.g., lymphoid cancers, such as leukemia) and for predicting the clinical outcome of such cancer subtypes to therapeutic regimens, particularly to modulators of histone mcthylation (e.g., H3K27me3).
  • cancer subtypes e.g., lymphoid cancers, such as leukemia
  • histone mcthylation e.g., H3K27me3
  • agents such as miRNAs, miRNA analogues, small molecules, RNA imerferencc, aptamer, peptides, peptklornirnctics, antibodies that specifically bind to one or more biomarkers of the invention (eg., bmmarkers listed in Tables 1-5 and or described in the Examples, such as H3K27 demethylases, PRC2 complexes, EZH2, and HMON1) and fragments thereof can be used to identify, diagnose, prognose, assess, prevent, and treat cancers lymphoid cancers, such as leukemia).
  • biomarkers of the invention eg., bmmarkers listed in Tables 1-5 and or described in the Examples, such as H3K27 demethylases, PRC2 complexes, EZH2, and HMON1
  • lymphoid progenitor cells e.g., wild type and/or genorracalry altered cells
  • an agent mat inhibits polycomb repressor complex 2 (PRC2) activity or reduces H3K27me3 levels
  • PRC2 polycomb repressor complex 2
  • H3K27me3 levels can increase the rrumbcr of lymphoid progenitor cells (e.g., increase self-renewal and cell proliferation) from the initial population of such lymphoid progenitor cells.
  • PRC2 polycomb repressor complex 2
  • an element means one clement or more than one element.
  • allogeneic refers to deriving from, originating in, or being members of the same species, where the members are genetically related or genetically unrelated bat genetically similar.
  • An “allogeneic transplant” refers to transfer of cells or organs from a donor to a recipient, where the recipient is the same species as the donor.
  • mismatched alfogencic refers to deriving from, originating in, or being members of the same species having non-identical major histocorapatability complex (MHQ antigens (ie., proteins) as typically determined by standard assays used in the art, such as serological or molecular analysis of a defined number of MHC antigens.
  • MHQ antigens ie., proteins
  • a “partial mismatch” refers to partial match of die MHC antigens tested between members, typically between a donor and recipient
  • a "rialf mismatch' * refers to 50% of the MHC antigens tested as showing different MHC antigen type between two members.
  • a roU" or “complete” mismatch refers to all MHC antigens tested as being different between two members.
  • a "xenogeneic transplant” refers to transfer of cells or organs from a donor to a recipient where the recipient is a species different from that of the donor.
  • the term “syngeneic'” refers to deriving from, originating in, or being members of the same species that are genetically identical, particularly with respect to antigens or inmwnoiogicai reactions. These include identical twins having matching MHC types.
  • a “syngeneic transplant” refers to transfer of cells or organs from a donor to a recipient who is genetically identical to the donor.
  • altered amount of a marker or altered level of a marker refers to increased or decreased copy mtmber of the marker andor increased or decreased expression level of a particular marker gene or genes in a cancer sample, as compared to die expression level or copy number of the marker in a control sample.
  • altered amount of a marker also includes an increased or decreased protein level of a marker in a sample, e.g., a cancer sample, as compared to the protein kvd of the marker in a normal, control sample.
  • the "amount ** of a marker, e.g.. expression or copy number of a marker or minimal common region (MCR), or protein level of a marker, in a subject is "significantly'' higher or lower than the normal amount of a marker, if the amount of the marker is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, and preferably at least twice, and more preferably three, four, five, ten or more times that amount
  • the amount of the marker in the subject can be considered "sigrufrcant-y * '' higher or lower than the normal amount if the amount is at least about two, and preferably at least about three, four, or five times, higher or lower, respectively, than the normal amount of die marker.
  • the amount of the marker in the subject can be considered "significantly * ' higher or lower than the normal amount if the amount is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, higher or lower, respectively, than the normal amount of the marker.
  • altered level of expression of a marker refers to an expression level or copy number of a marker in a test sample e.g., a sample derived from a subject suffering from cancer, that is greater or less than the standard error of the assay employed to assess expression or copy mrrnber, and is preferably at least twice, and more rnefcrably three, four, five or ten or more times the expression level or copy number of the marker or
  • chrornosornal region in a control sample e.g., sample from a heafthy subject not having the associated disease
  • a control sample e.g., sample from a heafthy subject not having the associated disease
  • the altered level of expression is greater or less than the standard error of the assay employed to assess expression or copy number, and is preferably at least twice, and more preferably three, four, five or ten or more times the expression level or copy number of the marker in a control sample (e.g., sample f om a healthy subject not having the associated disease) and preferably, the average expression level or copy number of the marker in several control samples.
  • altered activity of a marker refers to an activity of a marker which is increased or decreased in a disease state, e.g.. in a cancer sample, as compared to the activity of the marker in a normal, control sample.
  • Altered activity of a marker may be the result of, for example, altered expression of the marker, altered protein level of the marker, altered structure of the marker, or, e.g., an altered interaction with other proteins involved in the same or different pathway as the marker, or altered interaction with franscriptional activators or inhibitors.
  • altered structure of a marker refers to the presence of mutations or allelic variants within the marker gene or maker protein, e.g., mutations which affect expression or activity of the marker, as compared to the normal or wild-type gene or protein.
  • mutations include, but are not limited to substitutions, deletions, or addition mutations. Mutations may be present in the coding or non-coding region of the marker.
  • altered subcellular localization of a marker refers to the misiocalization of the marker within a cell relative to the normal localization within the cell eg., within a healthy and/or wild-type cell.
  • An indication of normal localization of the marker can be determined through an analysis of subcellular localization motifs known in me field that are harbored by marker polypeptides.
  • antibody ** and “antibodies'' broadly encompass naturally-occurring forms of antibodies (*.#., IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site.
  • Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.
  • antibody as used herein also includes an "antigen-binding portion" of an antibody (or simply “antibody portion”).
  • u antigcn- binding portion refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the anUgen-bindtng function of an antibody can be perfbrmed by fragments of a full-length antibody.
  • binding f agments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab*)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH 1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et at., (1989) Nature 3 1:544-546), which consists of a VH domain; and (vi) an isolated romplcrncntarity (letennining region (CDR).
  • CDR an isolated romplcrncntarity
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker mat enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); sec e.g., Bird et al. ( 1988) Science 242:423-426; and Huston et al (1988) Prvc. Natl. Acad. Sci. USA 85:5879-5883; and Osboum et al. 1998. Nature Biotechnology 16: 778).
  • scFv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "ar ge -binding portion ** of an antibody.
  • Any VH and VL sequences of specific scFv can be linked to human immunoglobulin constant region cO A or genomic sequences, in order to generate expression vectors encoding complete IgG polypeptides or other isotypes.
  • VH and VL can also be used in the generation of Fab , Fv or other fragments of immunoglobulins using eitber protein chemistry or recombinant DNA technology.
  • Other forms of single chain antibodies, such as dtabodies arc also
  • Dtabodies are bivalent, bispeciflc antibodies in which VH and VL domains are expressed on a single poiypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding si es (see e.g., Holligcr, P., et al. (1993) Proc Nad. Acad. Set. l/SA 90:6444-6448; Poljalc, R. J., el al (1994) Structure 2:1121-1123).
  • an antibody or antigen nnding portion thereof may be part of larger immunoadhesion polypeptides, formed by covaknt or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides.
  • immunoadhesion polypeptides include use of the streptavidin core region to make a tetrameric scFv polypeptide (Kipriyanov, S.M., et al.
  • Antibody portions such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies.
  • antibodies, antibody portions and itnnsunc ⁇ dhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein.
  • Antibodies may be polyclonal or monoclonal; xenogeneic allogeneic or syngeneic; or modified forms thereof ( «.£., humanized, chimeric, etc.). Antibodies may also be fully human.
  • a monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.
  • the term "'amiscnee” nucleic acid polypeptide comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein. eg. , complementary to the coding strand of a double-stranded cDNA polypeptide, complementary to an mR A sequence or complementary to the coding strand of a gene. Accordingly, an antisensc nucleic acid polypeptide can hydrogen bond to a sense nucleic acid polypeptide.
  • autologous refers to deriving from or originating in the same subject or patient.
  • An “autologous transplant” refers to the harvesting and reinfusion or transplant of a subject's own cells or organs. Exclusive or supplemental use of autologous cells can diminate or reduce many adverse effects of administration of the cells back to die host, particular graft versus host reaction.
  • the term * 1riochip n refers to a solid substrate comprising an attached probe or plurality of probes of the invention, wherein the probers) comprise at least 1, 2, 3, , 5, 6, 7, 8, , 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 5, 50, 55, 0, 65, 70, 5, 80, 85. 0, 95, 100, 150.200 or more probes.
  • the probes may be capable of hybridizing to a target sequence ondcr stringent hybridization conditions.
  • the probes may be attached at spatially defined address on the substrate. More than one probe per target sequence may be used, with either overlapping probes or probes to different sections of a particular target sequence.
  • the probes may be capable of hybridizing to target sequences associated with a single disorder.
  • the probes may be attached to the biocnip in a wide variety of ways, as will be appreciated by those in the art
  • the probes may either be synthesized first, with subsequent attachment to the biocnip, or may be directly synthesized on thebiochip.
  • the solid substrate may be a material that may be modified to contain discrete individual sites appropriate for the attachment or association of the probes and is amenable to at least one detection method.
  • substrates include glass and modified or iunctionalized glass, plastics (including acrylics, polystyrene and copolymers of styrcne and other materials, polypropylene, polyethylene, porybutylene, poiyurethanes, TeflonJ, etc), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses and plastics.
  • the substrates may allow optical detection without appreciably fluorescing.
  • the substrate may be planar, although other configurations of substrates may be used as well. For example, probes may be placed on the inside surface of a tube, for ffow-through sample analysis to minimize sample volume.
  • me substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.
  • the biocnip and the probe may be derivatized with chemical functional groups for subsequent attachment of the two.
  • the biochtp may be derivatized with a chemical functional group including, bat not limited to, amino groups, carboxyi groups, oxo groups or thiol groups.
  • the probes may be attached using functional groups on the probes either directly or indirectly using a linker.
  • the probes may be attached to the solid support by either the S' terminus, 3' terminus, or via an internal nucleotide.
  • the probe may also be attached to the solid support non-covalently.
  • bio tiny la ted oligonucleotides can be made, which may bind to surfaces covalently coated with streptavidin, resulting in attachment.
  • probes may be synthesized on the surface using techniques such as photoporyincrizarion and pbotoKthography.
  • body fluid refers to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g. amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowpcr's fluid or pre-cjaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, peritoneal fluid, pus, saliva, sebum, semen, scrum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit).
  • fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g. amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowpcr's fluid or pre
  • cancer or "tamof or “hyperproli feranve disorder” refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled prouferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell.
  • Cancers include, but arc not limited to, B cell cancer, eg., multiple myeloma, Waldenstrom's macroglobdinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowd or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, and the like.
  • the heavy chain diseases such as, for example
  • cancers include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotbeliosarcotna, lymphangiosarcoma, lyn ⁇ hangiociidou ⁇ iosarcoma, synovioma, mesothelioma, E wing's tumor,
  • human sarcomas and carcinomas e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotbeliosarcotna, lymphangiosarcoma, lyn ⁇ hangiociidou ⁇ iosarcoma, synovioma, mesothelioma, E
  • leiomyosarcoma rhabck>rnyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adetiocarciiiomas, cystadciiocarciiMma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, hmg carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, mcdulloblastoma,
  • craniopharyngioma ependymoma, pineal oma, hemangioblastoma, acoustic neuroma, oUgodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, &£., acute lymphocytic leukemia and acute myelocytic leukemia (mycloblastic
  • the cancer whose phenotype is determined by the method of the invention is an epithelial cancer such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, bead and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.
  • the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer.
  • die epithelial cancer is non-small-cdi lung cancer, nonpapillary renal ceU carcinoma, cervical carcinoma, ovarian carcinoma (e.g. , serous ovarian carunmtu), c breast carcmoma.
  • the epithelial cancers may be
  • the present invention is used in the treatment, diagnosis, and or prognosis of lymphoma or its subtypes, including, but not limited to, iympbocytc-rich classical Hodgkin lymphoma, mixed ce!hilarity classical Hodgkin lymphoma, lymphocyte-depleted classical Hodgkin lymphoma, nodular sclerosis classical Hodgkin lymphoma, anaplastic large cell lymphoma. diffuse large B-cell lymphomas, MLL' pre B-cdl ALL) based upon analysis of markers described herein.
  • classifying includes “to associate” or “to categorize” a sample with a disease state. In certain instances, “classifying” is based on statistical evidence, empirical evidence, or both. In certain embodiments, the methods and systems of classifying use of a so-called training set of samples having known disease states. Once established, the training data set serves as a basis, model, or template against whkh the features of an unknown sample are compared, in order to classify the unknown disease state of the sample. In certain instances, classifying the sample is akin to diagnosing the disease state of the sample. In certain other instances, classifying the sample is akin to diiTerenriating the disease state of the sample from another disease state.
  • coding region refers to regions of a nucleotide sequence comprising codons which arc translated into amino acid residues
  • noncoding region' refers to regions of a nucleotide sequence that are not translated into amino acids (&g., 5 * and 3' untranslated regions).
  • an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil.
  • base pairing specific hydrogen bonds
  • a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to die first strand if the residue is guanine.
  • a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
  • control refers to any reference standard suitable to provide a cornparison to the expression products in the test sample.
  • control comprises obtaining a "control sample” from which expression product levels arc detected and compared to the expression product levels from the test sample.
  • Such a control sample may comprise any suitable sample, including but not limited to a sample from a control cancer patient (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a normal patient or the cancer patient, cultured primary cells/ tissues isolated from a subject such as a normal subject or the cancer patient, adjacent normal cells/tissues obtained from the same organ or body location of the cancer patient, a tissue or cell sample isolated from a normal subject, or a primary cells tissues obtained from a depository.
  • a sample from a control cancer patient can be stored sample or previous sample measurement
  • normal tissue or cells isolated from a subject such as a normal patient or the cancer patient
  • cultured primary cells/ tissues isolated from a subject such as a normal subject or the cancer patient
  • adjacent normal cells/tissues obtained from the same organ or body location of the cancer patient
  • tissue or cell sample isolated from a normal subject or a primary cells tissues obtained from a depository.
  • control may comprise a reference standard expression product level from any suitable source, including but not limited to housekeeping genes, an expression product level range from normal tissue (or other previously analyzed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, survival for one, two, three, four years, etc.) or receiving a certain treatment It will be understood by those of skill in the art that such control samples and reference standard expression product levels can be used in combination as controls in the methods of the present mvention.
  • control may comprise normal or r >n-cancerous cell tissue sample.
  • control may comprise an expression level for a set of patients, such as a set of cancer patients, or for a set of cancer patients receiving a certain treatment, or for a set of patients with one outcome versus another outcome.
  • the specific expression product level of each patient can be assigned to a percentile level of expression, or expressed as either higher or lower than the mean or average of the reference standard expression level
  • the control may comprise normal cells, cells from patients treated with combination chernotherapy and cells from patients having benign cancer.
  • the control may also comprise a measured value for example, average level of expression of a particular gene in a population compared to the level of expression of a housekeeping gene in the same population.
  • control comprises a ratio transformation of expression product levels. including bat not limited to determining a ratio of expression product levels of two genes in tbe test sample and comparing i to any suitable ratio of the same two genes in a reference standard determining expression product levels of the two or more genes in the test sample and determining a difference in expression product levels in any suitable control; and detenrjining expression product levels of the two or more genes in the test sample, normalizing their expression to expression of housekeeping genes in the test sample, and comparing to any suitable control.
  • control comprises a control sample which is of the same lineage and/or type as the test sample.
  • control may comprise expression product levels grouped as percentiles within or based on a set of patient samples, such as all patients with cancer.
  • a control expression product level is established wherein higher or lower levels of expression product relative to, for instance, a particular percentile, are used as the basis for predicting outcome.
  • a control expression product level is established using expression product levels from cancer control patients with a known outcome, and the expression product levels from the test sample are compared to the control expression product level as the basis for predicting outcome.
  • the methods of die invention are not limited to use of a specific cut-point in comparing die level of expression product in the test sample to the control.
  • diagnosing cancer includes the use of the methods, systems, and code of the present invention to determine the presence or absence of a cancer or subtype thereof in an individual.
  • the term also includes methods, systems, and code for assessing the level of disease activity in an individual.
  • the term "diagnostic marker” includes markers described herein which are useful in die diagnosis of cancer, eg., over- or under- activity, emergence, expression, growth, remission, recurrence or resistance of tumors before, during or after therapy.
  • the predictive functions of the marker may be conftrmed by, eg., (1) increased or decreased copy number (eg., by FISH, FISH plus SKY, single-molecule sequencing, e.g., as described in die art at least at J. BiotechnoL, 86:289-301 , or qPCR).
  • ovcrexpression or underexpression e.g, by SH, Northern Blot, or qPCRX increased or decreased protein level (e.g., by 1HC), or increased or decreased activity (determined by, for example, modulation of a pathway in which the marker is involved), eg., in more than about 5%, 6%, 7%, 8%, 9%, io I ]%, 12%, 13%, 14%, 15%, 20%, 25%, or more of human cancers types or cancer samples; (2) its presence or absence in a biological sample, eg., a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, or bone marrow, from a subject, e.g. a human, afflicted with cancer, (3) its presence or absence in clinical subset of subjects with cancer (e.g., those responding to a particular therapy or those developing resistance). Diagnostic markers also include
  • diagnostic markers e.g., markers which are indirect markers of cancer progression. Such diagnostic markers may be useful to identify populations of subjects amenable to treatment with modulators of H3K27me3 levels (e.g., subjects having Down syndrome-type ALL as described herein) and to thereby treat such stratified patient populations.
  • DS Down syndrome'' or "DS" refers to a condition caused by trisomy for human chromosome 21 (Hsa21 ) and is the most common genetic cause of mental retardation in humans. DS occurs in 1 in 800- 1 (XX) live births arid results in over 80 different clinical phenotypes, including craniofacial abnormalities, a small hypocellular brain with a disproportionately small cerebellum, Alzheimer-like histopathoiogy, and an elevated risk for congenital bcart defects, Hirschsprung's disease, and leukemia. DS is associated with two contrary cancer-related phenotypes.
  • Acute megakaryoblastic leukemia occurs approximately 500-fbld more frequently in individuals with DS than in the general population. AMKL almost always occurs in concert with a somatic mutation in the GATAJ transcription factor.
  • Ts65Dn mouse is trisomic for orthologs of approximately half of the 261 protein coding genes on Hsa21 (Patterson and Costa (2005) ⁇ 3 ⁇ 4 ⁇ . Rev. Genet. 6:137- 147; Davisson (2005) Drug Disc.
  • TslRlr mouse has segmental trisomy for a subset of die genes represented in Ts65Dn which correspond to a "critical region" on Hsa21 which harbors genes sufficient to cause a number of DS phenotypes.
  • the Dp( 16)1 Yu mouse harbors an extra copy of all of the segments on mouse chromosome 16 that are syntenic to human chiomosome 21 and such mice display learning, memory, and heart defects coinpatable to those observed in human DS (Li et al. (2007) Hum. Mol. Genet. 16:1359-66).
  • studies of partial trisomy 21 (“Down Syndrome Critical Region” (DSCR) indicate that only parts of the chromosome are necessary to recapitulate the Down syndrome phenotype (Patterson and Costa (2005) Nat. Rev. Genet. 6:137-147; Olson el al. (2004) Science 306:687-690).
  • the TslRhr mouse is trisomic only fix the region of mouse chromosome 16 that is comparable to the DSCR.
  • expansion in die context of ceils refers to increase in the number of a characteristic cell type, or cell ty es, from an initial population of cells, which may or may not be identical.
  • the initial cells used for expansion need not be the same as the ceils generated from expansion.
  • the expanded cells may be produced by growth and differentiation of the initial population of cells. Excluded from the term expansion are limiting dilution assays used to characterize the differentiation potential of cells.
  • a molecule is "fixed” or "affixed” to a substrate if it is covalently or non-covalently associated with the substrate such the substrate can be rinsed with a fluid (e.g. standard saline citrate, pH 7.4) without a substantial fraction of the molecule dissociating from the substrate.
  • a fluid e.g. standard saline citrate, pH 7.4
  • gene expression data or “gene expression level” as used herein refers to uformation regarding the relative or absolute level of expression of a gene or set of genes in a cell or group of cells.
  • the level of expression of a gene may be determined based on die level of RNA, such as mRNA, encoded by the gene. Alternatively, the level of expression may be determined based on the level of a polypeptide or fragment thereof encoded by the gene.
  • Gene expression data may be acquired for an individual cell, or for a group of cells such as a tumor or biopsy sample.
  • Gene expression data and gene expression levels can be stored on computer readable media, e.g., the computer readable medium used in conjunction with a mkroarray or chip reading device. Such gene expression data can be manipulated to generate gene expression signatures.
  • gene expression signature refers to a group of coordinatery expressed genes.
  • the genes making up this signature may be expressed in a specific cell lineage, stage of differentiation, or during a particular biological response.
  • the genes can reflect biological aspects of the tumors in which they are expressed, such as die cdl of origin of the cancer, the nature of the non-malignant cells in the biopsy, and the oncogenic mechanisms responsible for the cancer.
  • the gene expression signatures described herein stratify Down Syndrome- ALL (DS-ALL from general ALL conditions that are especially amenable to treatment with modulators of H3K27me3 levels.
  • hematological cancer refers to cancers of cells derived from the blood.
  • the hematological cancer is selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML). chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), multiple myeloma (MM), non- Hodgkin's lymphoma (NHLX Hodgkin's lymphoma, mantle cell lymphoma (MCL), follicular lymphoma, Waldenstrom's macroglobulmemia (WM), B-cell lymphoma and diffuse large B-ccll lymphoma (DLfiCL).
  • NHL may include indolent Non-Hodgkin's Lymphoma (iNHL) or aggressive Non-Hodgkin's Lymphoma (aNHL).
  • hematopoietic stem cell refers to a clonogcnic, self-renewing phnipotent ceil capable of ultimately differentiating into all ceil types of the hematopoietic system, including B cells T cells, N cells, lymphoid dendritic cells, myeloid dendritic cells, granulocytes, macrophages, megakaryocytes, and erythroid cells.
  • HSCs are typically defined by the presence of a characteristic set of cell markers.
  • homologous * refers to miclcotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions arc homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that arc occupied by the same nucleotide residue.
  • a region having die nucleotide sequence 5- ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of die portions are occupied by the same nucleotide residue.
  • ** host vi is intended to refer to a cell into which a nucleic acid of the invention, such as a rccornbiriant expression vector of the invention, has been introduced.
  • the terms "host ceil” and 'Recombinant host DC arc used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifkations may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the patent cell, but are still included within the scope of the terra as used herein.
  • humanized antibody is intended to include antibod es made by a ixm-hnman cell having variable and constant regions which have been altered to more closely resemble antibodies mat would be made by a human cell, for example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences.
  • Humanized antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (eg., nutations introduced by random or site-spedrk mutagenesis vftro or by som
  • humanized antibody also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • Immune cell refers to cells mat play a role in the immune response.
  • Immune cells arc of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • immune response includes T cell mediated and/or B cell mediated immune responses.
  • exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity.
  • immune response includes immune responses that are indirectly effected by T cell activation, &£., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages.
  • the term 'inhibit' includes the decrease, limitation, or blockage, of, for example a particular action, function, or interaction.
  • cancer is "inhibited” if at least one symptom of the cancer, such as hyperprob fcrau ve growth, is alleviated, terminated, slowed, or prevented.
  • cancer is also “inhibited” if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.
  • interaction when referring to an interaction between two molecules, refers to the physical contact (e.g., binding) of the molecules with one another. Generally, such an interaction results in an activity (which produces a biological effect) of one or both of said molecules.
  • the activity may be a direct activity of one or both of the molecules.
  • one or both molecules in the interaction may be prevented from binding their Iigand, and thus be held inactive with respect to iigand binding activity [e.g., binding its iigand and triggering or inhibiting an immune response).
  • To inhibit such an interaction results in the disruption of the activity of one or more molecules involved in the interaction.
  • To enhance such an interaction is to prolong or increase the likelihood of said physical contact, and prolong or increase the likelihood of said activity.
  • an "isolated antibody,' * as used herein, is intended to refer to an antibody mat is substantially free of other antibodies having different antigenic specificities. Moreover, an isolated antibody may be sqbstantially free of other cellular material and/or chemicals.
  • an "isolated protein” refers to a protein that is substantially free of other proteins, cellular material, separation medium, and culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • An ''isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide or fusion protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations, in which compositions of the invention are separated from cellular components of the cells from which they arc isolated or recombnantly produced In one embodiment, the language “substantially free of cellular material” includes preparations of having less than about 30%, 20%, 10%, or 5% (by dry weight) of cellular material.
  • an antibody, polypeptide, peptide or fusion protein or fragment thereof, e.g., a biologically active fragment thereof is recoTnbmantry produced, it is also preferably substantially free of culture medium, te., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • kits are any nianufacturc (e.g. a package or container) cotnprising at least one reagent, eg. a probe, for specifically detecting or modulating the expression of a marker of the invention.
  • the kit may be promoted, distributed, or sold as a unit for pcrfbrrning the methods of the present invention.
  • leukemia refers to a group of diseases that arc cancers of the marrow and Mood, where the malignant cells are white blood cells (leukocytes).
  • the two major groups are lyrnphatic. and myeloid leukemia. Both groups are considered as either acute or chronic depending on various factors.
  • lymphoid leukemias can thus be divided into four main types: acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia and chronic myelogenous leukemia. Acute and chronic leukemias are usually studied as groups separated by the cells which arc affected.
  • heterogeneous groups are usually considered together and are considered as a group of diseases characterized by infiltration of the bone marrow and other tissues by the cells of the hematopoietic system.
  • the infiltration is called neoplastic, meaning new growth of cells, but all of the cells seen in the marrow, and peripheral circulation in leukemia are normal in a normal bone marrow, except for one structure, seen in myelocytic leukemia called Auer rods.
  • Auer rods myelocytic leukemia
  • Acute lymphoblastic leukemia is also referred to as acute lymphocytic leukemia and acute lymphoid leukemia and is a form of leukemia characterized by excess lymphoblasts. Malignant, immature white blood cells continuously multiply and arc overproduced in the bone marrow. ALL causes damage and death by crowding out normal cells in the bone marrow, and by spreading (infiltrating) to other organs. ALL is most common in childhood with a peak incidence at 2-5 years of age, and another peak in old age.
  • Standard of care for treating ALL focuses on treatment of different phases in order to control bone marrow and systemic (whole-body) disease as well as to prevent leukemic cells from spreading to other sites, particularly the central nervous system (CNS), eg., monthly lumbar punctures: a) induction chemotherapy is used to bring about bone marrow remission.
  • standard induction plans include prednisone, vincristine, and an anthracyclinc drug; other drug plans may include L- asparaginase or cyctopho ahamide.
  • Standard prophylaxis may include radiation of the head andor drugs delivered directly into the spine; and/or d) maintenance treatments with cfcrnothcrapcutic drugs prevent disease recurrence oocc nraission has been achieved. Maintenance therapy usually involves lower drug doses. and may continue for up to three yean. Alternatively, allogeneic bone marrow
  • Chronic rympbocyrJc leukemia also known as “chronic lymphoid leukemia” or "CLL"
  • CLL chronic rympbocyrJc leukemia
  • Oympbocytcs blood cells
  • CLL chronic lymphoid leukemia
  • the DNA of aB cell is damaged * so mat it cannot fight infection, but grows out of control and crowds out the healthy blood cells that can fight infection.
  • CLL isan abiiorrnal neoplastic proliferation of B cells. The cells accumulate mainly in die bone marrow and blood.
  • CLL is now thought to be identical to a disease called small lymphocytic lymphoma (SLL), a type of non-Hodgkin's lymphoma which presents primarily in the lymph nodes.
  • SLL small lymphocytic lymphoma
  • Most people arc diagnosed without symptoms as the result of a routine blood test that returns a high white blood cell count, but as it advances, CLL results in swollen lymph nodes, spleen, and liver, and eventually anemia and infections.
  • Early CLL is not usually treated, and late CLL is treated with chemotherapy and monoclonal antibodies. Survival varies from 5 years to more than 25 years.
  • Chronk myelogenous leukemia also known as chronic granulocytic leukemia (CGL)
  • CML Chronk myelogenous leukemia
  • CGL chronic granulocytic leukemia
  • this disease is characterized by leukocytosis, the presence of increased numbers of immature granulocytes in the peripheral blood, splenomegaly and anemia.
  • immature granulocytes include basophils, eosinophils, and neutrophils.
  • the immature granulocytes also accumulate in the bone marrow, spleen, liver, and occasionally in other tissues.
  • CytoiogicaUy CML is characterized by a translocation between chromosome 22 and chromosome 9. This translocation juxtaposes a purported proto-oncogenc with tyrosine kinase activity, a drcumstance that apparently leads to uncontrolled cell growth. The resulting translocated chromosome is sometimes referred to as die Philadelphia chromosome.
  • lymphoid progenitor celP refers to an oligopotent or unipotent progenitor cell capable of ultimately developing into any of die terminally differentiated cells of the lymphoid lineage, such as T cell, B cell, N cell or lymphoid dendritic cells, but which do not typically differentiate into cells of die myeloid lineage.
  • lymphoid progenitor cell As with cells of die myeloid lineage, different cell populations of lymphoid progenitors are distinguishable from other cells by their differentiation potential, and die presence of a characteristic set of cell markers.
  • CLP common lymphoid progenitor cell
  • BCP B-cdl progenitors
  • TCP T-ccU progenitors
  • NK cells NK cells
  • dendritic cells These progenitor cells have little or no sdf-renewing capacity, but are capable of giving rise to T lymphocytes, B lymphocytes. NK cells, and lymphoid dendritic cells.
  • myeloid progenitor cell refers to a multipotent or unipotent progenitor cell capable of ultimately developing into any of die terminally differentiated cells of die myeloid lineage, but which do not typically differentiate into cells of die lymphoid lineage.
  • myeloid progenitor cell refers to any progenitor cell in the myeloid lineage.
  • Committed progenitor cells of the myeloid lineage include oligopotent common myeloid progenitor cells, gratmlocyic.monocytc progenitor cells, and megakaryocyte/erythroid cells, but also encompass unipotent erythroid progenitor, megakaryocyte progenitor, granulocyte progenitor, and macrophage progenitor cells.
  • Different cell populations of myeloid progenitor ceils are distinguishable from other cells by their differentiation potential, and the presence of a characteristic set of cell markers.
  • CMP common myeloid progenitor cdl
  • GFP granulocyte/monocyte
  • EP rnegakaryocyie/eryihroid
  • progenitor cells have limited or no self-renewing capacity, but are capable of giving rise to myeloid dendritic, myeloid erythroid, erythroid, megakaryocytes, granulocytc/macnjphage, granulocyte, and macrophage cells.
  • Lymphoma refers to cancers that originate in the lymphatic system. Lymphoma is characterized by malignant neoplasms of lymphocytes— B lymphocytes and T lymphocytes (/.*., B-cclls and T-cclb). Lymphoma generally starts in lymph nodes or collccrions of lymphatic tissue in organs including, but not limited to, the stomach ⁇ intestines. Lymphoma may involve die marrow and the blood in some cases. Lymphoma may spread from one site to other parts of the body.
  • Lymphomas include, but arc not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous B-cell lymphoma, activated B- ell lyraptorna, diffuse large B-cdi lymphoma (DLBCLX mantle cell lymphoma (MCL), follicular center fymptoma, traiisformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate ryrnphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma and mantle zone lymphoma and low grade follicular lymphoma.
  • DLBCLX mantle cell lymphoma MCL
  • follicular center fymptoma
  • a “marker” or “biomarkeT includes a nucleic acid or polypeptide whose altered level of expression in a tissue or cell from its expression level in a control (eg., normal or healthy tissue or cell) is associated with a disease state, such as a cancer or subtype thereof ⁇ e.g., lymphoid cancers, such as leukemia).
  • a “marker nucleic acid” is a nucleic acid ⁇ eg., mRNA, cDNA, mature miRNA, prc-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding site, or a variant thereof and other classes of small RNAs known to a skilled artisan) encoded by or corresponding to a marker of the invention.
  • marker nucleic acids include DNA (eg., cDNA) comprising die entire or a partial sequence of any of the nucleic acid sequences set forth in Tables 1-5 and Examples or the complement of such a sequence.
  • the marker nucleic acids also include R A cornprising the entire or a partial sequence of any of the nucleic acid sequences set forth in the Sequence Listing or the complement of such a sequence, wherein all thymidine residues arc replaced with uridine residues.
  • a "marker protein” includes a protein encoded by or corresportding to a marker of the invention.
  • a marker protein comprises the entire or a partial sequence of any of the sequences set forth in Tables 1-5 and Examples or the Examples.
  • biomarkcrg proteins and polypeptide ate used interchangeably.
  • specific corrtbinan'om; of biomarkcrg are preferred.
  • marker ptenotyping in the context of cell identification refers to identification of markers or antigens on cells for determining their phenotypc (e.g.. differentiation state and/or cell type). This may be done by irmnunophenotyping, which uses antibodies that recognize antigens present on a cell.
  • the antibodies may be monoclonal or polyclonal, but are generally chosen to have minimal crossr cact vity with other cell markers. It is to be understood mat certain cell differentiation or cell surface markers are unique to the animal species from which the cells are derived, while other cell markers will be common between species. These markers d Fating equivalent cell types between species are given the same marker identification even though mere are species differences in structure (eg., amino acid sequence).
  • Cell markers include cell surfaces molecules, also referred to in certain situations as cell differentiation (CD) markers, and gene expression markers.
  • the gene expression markers are those sets of expressed genes indicative of the cell type or differentiation state. In part, the gene expression profile will reflect the cell surface markers, although they may include non-cell surface molecules.
  • modulate includes up-regulation and down-regulation, eg. , enhancing or inhibiting a response.
  • the "normal” or “control” level of expression of a marker is the level of expression of the marker in cells of a subject, e.g., a human patient, not afflicted with a cancer.
  • An "ovcr- €xpressk» or "significantly higher level of expression” of a marker refers to an expression level in a test sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least twice, and more preferably 2.1, 2.2, 2.3.2.4.2.5.2.6.2.7.2.8.2.9.3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8J5, , 9.5, 10, 10.5, 11. 12, 13, 14, 15, 16.
  • a "significantly lower kvd of expression" of a marker refers to an expression level in a test sample that is at least twice, and more preferably 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, .5, 10, 10J, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more lower than the expression level of the marker in a control sample (e.g., sample from a healthy subject not having the marker associated disease) and preferably, the average expression level of the marker in several control samples.
  • peripheral blood cell subtypes refers to cell types normally found in the peripheral blood including, but is not limited to, eosinophils, neutrophils, T cells, monocytes, NK cells, granulocytes, and B cells.
  • probe refers to any molecule which is capable of selectively binding to a specifically intended target molecule, fix example, a nucleotide transcript or protein encoded by or corresponding to a marker. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of die target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.
  • prognosis' * includes a prediction of the probable course and outcome of cancer or the likelihood of recovery from the disease.
  • the use of statistica] algorithms provides a prognosis of cancer in an individual
  • the prognosis can be surgery, development of a clinical subtype of cancer (e.g., lymphoid cancers, such as leukemia), development of one or more clinical factors, development of intestinal cancer, or recovery from the disease.
  • response to cancer therapy or "outcome of cancer therapy * ' relates to any response of fe hypcrprohterativedisor ⁇
  • Hyperprdiferative disorder response may be assessed, for example for efficacy or in a neoadjuvant or adjuvant situation, where the size of a tumor after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, rriarnmogram, ultrasound or palpation. Response may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection for solid cancers.
  • Responses may be recorded in a quantitative fashion like percentage change in tumor volume or in a qualitative fashion like "pathological complete response * '' (pCR), “clinical complete remission'' (cCR), “clinical partial remission' * (cPR), “clinical stable disease” (cSDX, “clinical progressive disease** (cPD) or other qualitative criteria.
  • Assessment of hypeiproHterative disorder response may be done early after the onset of neoadjuvant or adjuvant therapy, eg., after a few hours, days, weeks or preferably after a few months.
  • a typical endpoint for response assessment is upon termination of neoadjuvant d>cinotherapy or upon surgical removal of residual tumor cells and or the tumor bed. This is typically three months after initiation of neoadjuvant therapy.
  • clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR .
  • the clinical benefit rate is measured by determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out rom the end of therapy.
  • the CBR for a particular cancer therapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%.80%, 85%, or more.
  • Additional criteria for evaluating die response to cancer therapies are related to "survival,'' which includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be cither irrespective of cause or tumor related); "recurrence- free survivaT (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein die term disease shall include cancer and diseases associated therewith).
  • the length of said survival may be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence or metastasis).
  • criteria for efficacy of treatment can be expanded to include response to chernotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.
  • a particular cancer therapeutic regimen can be administered to a population of subjects and the outcome can be correlated to copy number, level of expression, level of activity, etc. of one or more biornarkers listed in Tables 1-5 and Examples or the Examples that were determined prior to administration of any cancer therapy.
  • the outcome rneasurernent may be pathologic response to therapy given in die neoadjuvant setting.
  • outcome measures such as overall survival and disease-free oirvival can be monitored over a period of time for subjects following cancer therapy for whom the measurement values are known.
  • the same doses of cancer therapeutic agents are administered to each subject.
  • the doses administered are standard doses known in the art for cancer therapeutic agents.
  • the period of time for which subjects are monitored can vary. For example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 0, 45, 50, 55, or 60 months.
  • Biomarkcr threshold values that correlate to outcome of a cancer therapy can be determined using methods such as those described in the Examples section.
  • Outcomes can also be measured in terms of a "hazard ratio” (the ratio of death rates for one patient group to another, provides likelihood of death at a certain time point), "overall survival " (OS), and/or "progression free survivaL”
  • the prognosis comprises likelihood of overall survival rate at 1 year, 2 years, 3 years, 4 years, or any other suitable time point.
  • significance associated with the prognosis of poor outcome in all aspects of the present invention is measured by techniques known in the art For example, significance may be measured with calculation of odds ratio. In a further embodiment, the significance is measured by a percentage.
  • a significant risk of poor outcome is measured as odds ratio of 0.8 or less or at least about 12, including by not limited to: 0.1, 0.2, 03, 0.4, 05, 0.6, 0.7, 0.8, 12, 13, 1.4, 15, 1.6, 1.7, 1.8, 1.9, 2.0, 25, 3.0, 4.0, 5.0, 10.0, 15.0, 20.0.25.0.30.0 and 40.0.
  • a significant increase or reduction in risk is at least about 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 98%.
  • a significant increase in risk is at least about 50%.
  • the present invention further provides methods for making a treatment decision for a cancer patient, comprising carrying out the methods for prognosing a cancer patient according to the different aspects and embodiments of the present invention, and then weighing the results in light of other known clinical and pathological risk factors, in determining a course of treatment for the cancer patient.
  • a cancer patient that is shown by the methods of the invention to have an increased risk of poor outcome by combination chemotherapy treatment can be treated with more aggressive therapies, including but not limited to radiation therapy, peripheral blood stem cell transplant, bone marrow transplant, or novel or experimental therapies under clinical investigation.
  • resistance * '' refers to an acquired or natural resistance of a cancer sample or a mammal to a cancer therapy ( Le., being nonresponsivc to or having reduced or limited response to the therapeutic treatment), such as having a reduced response to a rberapeutic treatment by 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2- fold, 3-fokt 4-fold, 5-fbld, 10-fold, 15-fold, 20-f ld or more.
  • the reduction in response can be measured by compering with the same cancer sample or mammal before the resistance is acquired, or by comparing with a different cancer sample or a mammal who is known to have no resistance to the therapeutic treatment
  • a typical acquired resistance to chemotherapy is called "multidrug resistance.''
  • the multidrug resistance can be mediated by P-grycoprotein or can be mediated by other mechanisms, or it can occur when a mammal is infected with a multi-drug-resistam microorganism or a combination of nncroorganisms.
  • the term "reverses resistance” means that the use of a second agent in combination with a primary cancer therapy (eg., chemotherapeutic or radiation therapy) is able to produce a significant decrease in tumor volume at a level of statistical significance (e g.
  • tbc primary cancer therapy eg., chemotherapeutic or radiation therapy
  • tbc primary cancer therapy eg., chemotherapeutic or radiation therapy
  • sample used for detecting or dctenxuning the presence or level of at least one biomarker is typically whole Mood, plasma, scrum, saliva, urine, stool (eg., feces), tears, and any other bodily fluid (eg., as described above under the definition of "body fluids"), or a tissue sample (eg., biopsy) such as a small intestine, colon sample, or surgical resection tissue.
  • the method of the present invention further comprises obtaining the sample from the individual prior to detecting or determining die presence or level of at least one marker in the sample.
  • cancer means to alter cancer cells or tumor cells in a way mat allows for more effective treatment of the associated cancer with a cancer therapy (e.g., chemotberapeutic or radiation therapy.
  • a cancer therapy e.g., chemotberapeutic or radiation therapy.
  • normal cells are not affected to an extent that causes the normal cells to be unduly injured by the cancer therapy (eg., chemotherapy or radiation therapy).
  • An increased sensitivity or a reduced sensitivity to a therapeutic treatment is measured according to a known method in the art for the particular treatment and methods described herein below, including, but not limited to, cell proliferative assays (Tanigawa N, Kern D H, Kikasa Y, Morton D L, Cancer Res 1982; 42: 2159-2164), cell death assays (Weisenthal L M, Shoemaker R H, Marsden J A, Dill P L, Baker J A, Moran E M, Cancer Res 1984; 94: 161-173; Weisenthal L M. Lippman M E.
  • the sensitivity or resistance may also be measured in animal by measuring die tumor size reduction over a period of time, for cxample, 6 month for human and 4-6 weeks for mouse.
  • a composition or a method sensitizes response to a therapeutic treatment if die increase in treatment sensitivity or the reduction in resistance is 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2-fold, 3-fold, 4-fbld, 5-fbld, 10-fold, 15-fbld, 20-fold or more, compared to treatment sensitivity or resistance in the absence of such composition or method.
  • the determination of sensitivity or resistance to a therapeutic treatment is routine in the art and withm the sluli ofanoi lmariiy skilled clinician. It is to be understood that any method described herein for enhancing the efficacy of a cancer therapy can be equally applied to methods for sensitizing hyperproliferative or otherwise cancerous cells (e.g., resistant cells) to the cancer therapy.
  • the term “synergistic effect” refers to the combined effect of two ⁇ more anticancer agents or chcrnotherapy drugs can be greater than the sum of the separate effects ofthe anticancer agents CT chemotherapy drugs alone.
  • subject refers to any beaithy animal, mammal or human, or any animal, mammal or human afflicted with a condition of interest (e.g., cancer).
  • condition of interest e.g., cancer
  • subject*' is interchangeable with "patient.”
  • the language "substantially free of chemical precursors or other chemicals'' includes preparations of antibody, polypeptide, peptide or fusion protein in which the protein is separated from chemical precursors or other chemicals which arc involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals'' includes preparations of antibody, polypeptide, peptide or fusion protein having less than about 30% (by dry weight) of chemical precursors or non- antibody, polypeptide, peptide or fusion protein chemicals, more preferably less than about 20% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, still more preferably less than about 10% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, and most preferably less than about 5% chemical precursors or non- antibody, polypeptide, peptide or fusion protein chemicals.
  • substantially pure cell population refers to a population of cells having a specified cell marker characteristic and differentiation potential that is at least about 50%, preferably at least about 75-80%, more preferably at least about 85-90%, and most preferably at least about 95% ofthe cells making up the total cell population.
  • a “substantially pure cell population” refers to a population of cells mat contain fewer than about 50%, preferably fewer than about 20-25%, more preferably fewer than about 1 -15%, and most preferably fewer than about 5% of cells that do not display a specified marker characteristic and differentiation potential under designated assay conditions.
  • the term "survival” includes alt of the following: survival until mortality, also known as overall survival (wherein said mortality may be citber irrespective of cause or tumor related); "recurrcncc-frec survival" (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith).
  • the length of said survival may be calculated by reference to a defined start point (e.g. time of diagnosis or start of treatment) and end point (eg. death, recurrence ⁇ metastasis).
  • criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.
  • a "transcribed por/nuc!ccttde n or ' iucleoddc transcript” is a polynucleotide (e.g. an mRNA, hnRNA, cD A, mature rniRNA, pre-miRNA, pri-rniRNA, nriRNA*, anti- imRNA, or a rniRNA binding site, or a variant thereof or an analog of such RNA or cDNA) which is cornplementary to or homologous with all or a portion of a mature mRNA made by transcription of a marker of the invention and normal post-transcriptional processing (e.g. splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript
  • normal post-transcriptional processing e.g. splicing
  • vector refers to a nucleic acid capable of ttaitsporting another nucleic acid to which it has been linked.
  • plasmi T refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors eg., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are opcrativery linked.
  • Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors.”
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmidT and "vector” may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors ⁇ e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • An * tadeicxprasion" or "significantly tower level of expression or copy number" of a marker refers to an expression level or copy number in a test sample that is greater than the standard error of the assay employed to assess expression or copy number, but is preferably at least twice, and more preferably three, four, five or ten or more times less than the expression level or copy number of the marker in a control sample (e.g., sample from a healthy subject not afflicted with cancer) and preferably, the average expression level or copy number of the marker in several control samples.
  • Arginine AGA, ACG, CGA, CGC,
  • Glycine Gly, G
  • GGC GGG, GGT
  • Isoleucine (lie, I) ATA, ATC, ATT
  • Trp Tryptophan
  • Tyrosine TAC, TAT
  • nucleotide triplet An important and well known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefbrc, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences arc considered functionally equivalent since they result in die production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrirmdine may be found in a given nucleotide sequence. Such methylatioro do not affect the coding relationship between the trinucleotide codon and the conxsponding amino acid.
  • nucleotide sequence of a DNA or RNA coding for a fusion protein or polypeptide of die invention can be used to derive the fusion protein or polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence.
  • coiTesponding nucleotide sequences that can encode the fusion protein or polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given arnino acid sequence).
  • description and or disclosure herein ofa nucleotide sequence which encodes a fusion protein or polypeptide should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence.
  • description and/or disclosure of a fusion protein or polypeptide amino acid sequence herein should be considered to also include description and or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.
  • nucleic acid and amino acid sequence information for the loci and biomarkcrs of the present invention are well known in die art and readily available on publicly available databases, such as the National Center for Biotechnology Infbnnation (NCBQ-
  • NCBQ- National Center for Biotechnology Infbnnation
  • exemplary nucleic acid and amino acid sequences derived from publicly available sequence databases arc provided below.
  • the nucleic acid and amino acid sequences of a representative human DM6A biomarker also known as UTX or MGC141941 or bA386N14.2 or DKFZp686A03225
  • GenBank database under NMJD21140.2 and NP_0066963.2.
  • Nucleic acid and polypeptide sequences of DM6A ortnologs in organisms other man bumans are well known and include, for example, mouse KD 6A (NM_009483.1 and NP_033509. IX rat KDM6A (XM.002730I 85.2 and XP 002730231.1). chimpanzee KDM6A (XM_002806207.1 and XP_002806253.1), chicken KDM6A (X .416762J and XP_416762.3 fruit fly DM6A (NM 001201844.1 and NP_001188773.1 ), and worm KDM6A ( MJ077049J and NP.509450.1).
  • KDM6B biomarker also known as JMJD3 or 1AA0346
  • JMJD3 or 1AA0346 The nucleic acid and amino acid sequences of a representative human KDM6B biomarker (also known as JMJD3 or 1AA0346) is available to die public at the GenBank database under NM_001080424.1 and NP 001073893.1.
  • Nucleic acid and polypeptide sequences of KDM6B ortnologs in organisms other than humans are well known and include, for example, dog KDM6B (XM.546599.3 and XP.546599.2X mouse DM6B (NMJXHOl 7426.1 and NP_001017426.1 ), rat D 6B (NM_001108829.1 and
  • NP.OOl 102299.1 NP.OOl 102299.1
  • zebrafish DM6B XM 00 198938.1 and XP_003198986.1 and NM_001030178.1 and NP_001025349.1.
  • human EZH2 transcript variant 1 is the canonical sequence, all positional information described with respect to the remaining isoforms are determined from this sequence, and the sequences are available to the public at the GenBank database under NM_004456.4 and PJ0O4447.2.
  • the sequences of human EZH2 transcript variant 2 can be found under NM_ 152998.2 and NP_694543.1 and the encoded protein replaces the residues HP of positions 297-298 of the canonical sequences with HR CNYS.
  • the sequences of human EZH2 transcript variant 3 can be found under NM_001203247.1 and NPjOOl 190176.1 and the encoded protein deletes residues 83-121 of die canonical sequence.
  • the sequences of human EZH2 transcript variant 4 can be found under
  • NM_001203248.1 and NP.OOl 190177.1 and the encoded protein deletes residues 74-82 of the canonical sequence.
  • the sequences of human EZH2 transcript variant 5 can be found under NM_001203249.1 and NP_001190178.1 and fee eiKoded rjrotem o ⁇ lctcs n»idues 74- 82 of the canonical sequence, as well as replaces the residues
  • the catalytic site of EZH2 is believed to reside in a conserved domain of the protein known as the SET domain.
  • the amino acid sequence of the SET domain of EZH2 is provided by the following partial sequence spanning amino acid residues 613-726 of human EZH2 isoform 1 described above and as follows:
  • Nucleic acid and polypeptide sequences of EZH2 ortbologs in organisms other than humans are wdl known and include, for example, mouse EZH2 (NM_0O707l .2 and NP_031997.2 and NMJXH 146689.1 and NP.OOI 140161), chimpanzee EZH2
  • nucleic acid and amino acid sequences of a representative human HMGN1 biornarker is available to the public at the GenBank database under NM_004%5.6 and NP_0049565.
  • Nucleic acid and polypeptide sequences of H GN1 orthologs in organisms other than humans are well known and include, for example, monkey H GN1
  • eukaryotes have chromatin arranged around proteins in the form of nucleosomes, which arc the smallest sublimits of chromatin and includes approximately 146-147 base pain ofDNA wrapped around an octamerofec ⁇ histoner ⁇ teins (two each of H2A, H2B, H3, and H4).
  • Triinethyiation of histone H3 on Lys 27 (H3K27me3) is key for cell fate regulation.
  • Mammalian cells have three known sequence variants of histone H3 proteins, denoted H3.1, H3.2 and H3.3, that are highly conserved differing in sequence by only a few amino acids.
  • the terra "histone H3" can refer to H3.1, H3.2, or H3.3 individually or collectively. The sequences are as follows:
  • amino acid sequences include a methionine as residue No. I that is cleaved off when the protein is processed, hence what is lysine 28 in the amino acid sequences above corresponds to lysine ( ) 27.
  • lysine ( ) 27 These three protein variants arc encoded by at least fifteen different genes/transcripts.
  • HIST1H3A (NM.003S2 .2; NP_003520.1)
  • HIST1H3B (NM_003537.3; NP_003528.1X HIST1 H3C (NM_0035312; PJ)03522.1)
  • HISTIH3D (NM_003530.3; NP_003521.2)
  • H1ST1H3E (NM 003532.2; NP_003523.1 )
  • HISTIH3F (NM 021018.2; NPJ0662 8.I)
  • H1ST1H3G (NM_003534.2; NP_003525.I), HIST1H3H (NM_003536.2; NP_003527.1), H1ST1H3I (NM 003533.2; NP_003524.1), and HIST1H3J (NM_003535.2; NP_003526.1). Sequences encoding the histone H3.2 variant are publicly available as HIST2H3A
  • Agents and compositions of the present invention are provided for us in the diagnosis, prognosis, prevention, and treatment of cancer lymphoid cancers, such as leukemia) and cancer subtypes thereof.
  • Such agents and compositions can detect and/or modulate, e.g., up- or down-rcguiaie, expression andor activity of gene products or fragments thereof encoded by biomarkers of the invention, including the biornarkcrs listed in Tables 1-5 and Examples.
  • Exemplary agents include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural Kenya, that can either bind and/or activate or inhibit protein biomarkers of the invention, including the biomarkers listed in Tables 1-5 and Examples, or fragments thereof; RNA interference, amisense, nucleic acid aptamcrs, etc. that can downregulate the expression and/or activity of the biomarkers of the invention, including the biomarkers listed in Tables 1-5 and Examples, or fragments thereof.
  • nucleic acid molecules that specifically hybridize with or encode one or more biomarkers listed in Tables 1-5 and Examples or biologically active portions thereof.
  • nucleic acid molecule is intended to include DNA molecules (i.e., cDNA or genomic DNA) and RNA molecules (/.&, mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded- but preferably is double-stranded DNA.
  • An "isolated' * nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (Le., sequences located at die 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which tbc nucleic acid is derived.
  • die isolated nucleic acid molecules corresponding to the one or more biomarkers listed in Tables I -5 and Examples can contain less than about 5 kb, 4kb, 3kb, 2kb. 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (Le., a leukemic cell).
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially f ee of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of one or more biomarkers listed in Tables 1-5 and Examples or a nncleotidc sequence which is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more (eg., about 98%) homologous to the nucleotide sequence of one or more biomarkers listed in Tables I- 5 and Examples or a portion thereof ( .e.
  • a human cDNA can be isolated from a human cell line (from Stratagcne, La Jolla, CA, or Ckmtech, Palo Alto, CA) using all or portion of the nucleic acid molecule, or fragment thereof, as a hybridization probe and standard hybridization techniques (/. «., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor laboratory. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • mRNA can be isolated from muscle cells (Le., by the guamdiniura-thiocyanate extraction procedure of Chirgwin el al. (1979) Biochemistry 18: 5294-5299) and cDNA can be prepared using reverse transcriptase (i.e., Moloney MLV reverse transcriptase, available from GibccVBRL. Bcthesda, MD; or AMV reverse transcriptase, available from Setkagaku America, Inc., St. Russia, FL).
  • reverse transcriptase i.e., Moloney MLV reverse transcriptase, available from GibccVBRL. Bcthesda, MD; or AMV reverse transcriptase, available from Setkagaku America, Inc., St. Russia, FL.
  • Synthetic oligonucleotide primers for PCR amplification can be designed according to well-known methods in the art
  • a nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers accenting to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to the nucleotide sequence of one or more biomarkers listed in Tables 1-5 and Examples can be prepared by standard synthetic techniques, te., using an automated DNA synthesizer.
  • Probes based on the nucleotide sequences of one or more biomarkers listed in Tables 1-5 and Examples can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, i.e., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cc-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which express one or more biomarkers listed in Tables 1 -5 and Examples, such as by measuring a level of nucleic acid in a sample of cells from a subject, Le. , detecting mRNA levels of one or more biomarkers listed in Tables I -5 and Examples.
  • Nucleic acid molecules encoding protons comspomung to one or more biomarkers listed in Tables 1-5 and Examples from different species are also contemplated.
  • rat or monkey cDNA can be identified based on the nucleotide sequence of a human and/or mouse sequence and such sequences are well known in the art.
  • the nucleic acid molecule(s) of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of one or more biomarkers listed in Tables 1-5 and Examples, such that the protein or portion thereof modulates (eg-, enhance), one or more of the following biological activities: a) binding to the biomarker, b) modulating the copy number of the biomarkcr, c) tnodulaong the expression level of the biomarker, and d) modulating the activity level of the biomarkcr.
  • the language "sufficiently homologous' * refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent (eg., an amino acid residue which has a similar side chain as an amino acid residue in one or more biomarkers listed in Tables 1-5 and Examples, or fragment thereof) amino acid residues to an amino acid sequence of me biornarkcr, or fragment thereof, such that the protein or portion thereof modulates ( g., enhance) one or more of die following biological activities: a) binding to the biornarkcr, b) modulating die copy number of die biornarkcr. c) modulating the expression level of the biornarkcr; and d) modulating the activity level of tbe biornarkcr.
  • a minimum number of identical or equivalent eg., an amino acid residue which has a similar side chain as an amino acid residue in one or more biomarkers listed in Tables 1-5 and Examples, or fragment thereof
  • the protein is at least about 50%, preferably at least about
  • Portions of proteins encoded by nucleic acid molecules of the one or more biomarkers listed in Tables 1 -5 and Examples are preferably biologically active portions of the protein.
  • biologically active portion of one or more biomarkers listed in Tables 1-5 and Examples is intended to include a portion, e.g., a domain motif, that has one or more of the biological activities of die full-length protein.
  • Standard binding assays eg., immunoprecim ' tations and yeast two-hybrid assays, as described herein, or functional assays, .g., RNAi or overexpression experiments, can be performed to determine the ability of the protein or a biologically active fragment thereof to maimain a biological activity of die full-length protein.
  • Tbe invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of the one or more biomarkers listed in Tables 1 -5 and Examples, or fragment thereof due to degeneracy of the genetic code and thus encode tbe same protein as that encoded by tbe nucleotide sequence, or fragment thereof.
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence of one or more biomarkers listed in Tables 1-5 and Examples, or fragment thereof, or a protein having an amino acid sequence which is at least about 70%.75%, 80%, 85%.90%, 91%, 92%.93%, 94%, 95%.96%, 97%, 98%.99% or more homologous to the amino acid sequence of the one or more biomarkers listed in Tables 1-5 and Examples, or fragment thereof.
  • a nucleic acid encoding a polypeptide consists of nucleic acid sequence encoding a portion of a full-length fragment of interest that is tees than 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, or 70 amino acids in length.
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of the one or more biomarkcrs listed in Tables 1 -5 and Examples may exist within a population (eg., a mammalian and/or human population). Such genetic polymorphisms may exist among individuals within a population doe to natural allelic variation.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding one or more biomarkcrs listed in Tables 1-5 and Examples, preferably a mammalian, e.g., human, protein.
  • Such natural allelic variations can typically result in 1 -5% variance in the nucleotide sequence of the one or more biomarkcrs listed in Tables 1-5 and Examples. Any and all such nucleotide variations and resulting amino acid polymorphisms in the one or more biomarkcrs listed in Tables 1-5 and Examples mat are the result of natural allelic variation and mat do not alter the functional activity of the one or more btornarkers listed in Tables 1-5 and Examples are intended to be within the scope of the invention. Moreover, nucleic acid molecules encoding one or more biomarkcrs listed in Tables 1-5 and Examples from other species.
  • allelic variants of the one or more biomarkers listed in Tables 1-5 and Examples sequence that may exist in the population
  • changes can be introduced by mutation into the nucleotide sequence, or fragment thereof, thereby leading to changes in the amino acid sequence of the encoded one or more biomarkcrs listed in Tables 1-5 and Examples, without altering the functional ability of the one or more biomarkcrs listed in Tables 1-5 and Examples.
  • nucleotide substitutions leading to amino acid substitutions at non-cssentiar amino acid residues can be made in the sequence, or fragment thereof.
  • a '*rtc£-csscntiar amino acid residue is a residue that can be altered from the wild-type sequence of the one or more biomarkers listed in Tables 1-5 and Examples without altering the activity of the one or more biomarkcrs listed in Tables 1-5 and Examples, whereas an "essenriar amino acid residue is required for the activity of the one or more biomarkcrs listed in Tables 1-5 and Examples.
  • Other amino acid residues may not be essential for activity and thus are likely to be amenable to alteration without altering the activity of die one or more biomarkcrs listed in Tables 1-5 and Examples.
  • sequence identity or homology refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or sequence identical at that position.
  • the percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shared by die two sequences divided by die number of positions compared x 100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences arc 60% homologous or have 60% sequence identity.
  • the DNA sequences ATTOCC and TATOGC share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology. Unless otherwise specified "loop out regions", e.g., those arising from, from deletions or insertions in one of the sequences are counted as mismatches.
  • the alignment can be performed using the Ciustal Method.
  • Multiple alignment parameters include GAP Penalty "10, Gap Length Penalty » 10.
  • the pairwise alignment parameters can be Htuplc ot 2, Gap
  • the pairwise alignment parameters can be Ktuple ⁇ l, Gap pcnalty-3, Window-5, and Diagonals Savcd*5.
  • the percent identity between two amino acid sequences is deterrnined using the Needleman and Wunsch (/. Mol. Biol. ⁇ 48):444- 53 ( 1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available online), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available online), using a
  • percent identity between two amino acid or nucleotide sequences is determined using die algorithm of E. Meyers and W. Miller (CABIOS, 4: 11 - 17 ( 1989)) which has been incorporated into the ALIGN program (version 2.0) (available online), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • An isolated nucleic acid molecule encoding a protein homologous to ooe or more biomarlccrs listed in Tables 1-5 and Examples, or fragment thereof, can be created by crizroducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence, or fragment thereof, or a homologous nucleotide sequence such that one or more amino acid substitutions, additions or deletions arc introduced into die encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in die art. These families include amino acids with basic side chains (e.£, lysine, argininc, histidine).
  • acidic side chains &g., aspartic acid, grutarnic acid
  • uncharged polar side chains eg., glycine, asparaginc, ghitamine, serine, threonine, tyrosine, cysteine
  • nonpotar side chains e.g., alanine, valine, leucine, isokucine, proline, phenylalanine, mettuonine, tryptophan
  • branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in one or more biornarkers listed in Tables 1 -5 and Examples is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along ail or part of the coding sequence of the one or more biornarkers listed in Tables 1-5 and Examples, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity described herein to identify mutants that retain desired activity.
  • the encoded protein can be expressed recombinant ⁇ according to well-known methods in the art and the activity of the protein can be determined using, for example, assays described herein.
  • the levels of one or more biornarkers listed in Tables 1 -5 and Examples levels may be assessed by any of a wide variety of well-known methods or detecting expression of a transcribed molecule or protein. Non-limiting examples of such methods include immunological methods for detection of proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods. in preferred embodiments, the levels of one or more biomarkcrs listed in Tables 1-5 and Examples levels are ascertained fay measuring gene transcript (e.g., mR A), by a measure of the quantity of translated protein, or by a measure of gene product activity.
  • mR A measure of the quantity of translated protein
  • Expression levels can be monitored in a variety of ways, including by detecting mR A lev s, protein levels, or protein activity, any of which can be measured using standard techniques. Detection can involve quantification of the level of gene expression (eg.. genomic DNA, cDNA, mRNA, protein, or enzyme activity), or. alternatively, can be a qualitative assessment of die level of gene expression, in particular in comparison with a control level. The type of level being detected will be clear from tbe context
  • the mRNA expression level can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art
  • biological sample'' is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject
  • Many expression detection methods use isolated RNA.
  • any RNA isolation technique mat does not select against the isolation of mRNA can be utilized for the purification of RNA from cells (see, e.g., Ausubel et at., ed.. Current Protocols in Molecular Biology, John Wiley ft Sons, New York 1987-1999).
  • large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of
  • the isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays.
  • One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by die gene being detected.
  • the nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding one or more biomarkcrs listed in Tables 1 -5 and Examples.
  • Other suitable probes fix use in the diagnostic assays of the invention ate described herein. Hybridization of an mRNA with the probe indicates that one or more biomarkcrs listed in Tables 1-3 and Examples is being expressed.
  • die mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and tntrisferring the mRNA from die gd to a membrane, such as nitrocellulose.
  • the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in a gene chip array, e.g., an Afiymetrix TM gene chip array.
  • a skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of the One or more biomarkers listed in Tables 1-5 and Examples mRNA expression levels.
  • An alternative method for determining mRNA expression level in a sample involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, US. Patent No.4,683,202), ligase chain reaction (Barany, 19 1 , Proc. Nari. Acad. Sci. USA. 88: 189-1 3), sdf-sustained sequence replication (Guateili ei a/., 1990. Proc. Natl. Acad. Scf. USA 87: 1874-1878), transcriptional amplification system ( woh ei ai, 1989, Proc. Natl Acad.
  • amplification primers are defined as being a pair of nucleic acid molecules mat can anneal to 5 ' or 3 * regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between.
  • amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising die nucleotide sequence flanked by the primers.
  • mRNA does not need to be isolated from the cells prior to detection.
  • a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted wi ih a probe that can h bridize to the One or more biomarkers listed in Tables 1 -5 and Examples mRNA.
  • determinations may be based on the normalized expression level of one or more biomarkers listed in Tables 1-5 and Examples.
  • Expression levels are normalized by collecting the absolute expression level by comparing its expression to the expression of a non-biomarker gene, e.g., a housekeeping gene that is constitutivefy expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, eg., a subject sample, to another sample, .g., a normal sample, or between samples from different sources.
  • the level or activity of a protein corresponding to one or more biornarkers listed in Tables 1 -5 and Examples can also be detected and/or quantified by detecting or quantifying the expressed polypeptide.
  • the polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art.
  • analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLQ, hyperdifraskm chromatography, and the like, or various immunological methods such as fluid or gd precipitin reactions, immunodiffusion (single or double), Immunoelectrophoresis, radioimmunoassay ( IA), enzyme-linked immunosorbent assays (EULSAsX
  • immunofluorescent assays Western blotting, and the like.
  • a skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether cells express the biomarker of interest.
  • the present invention further provides soluble, purified and or isolated polypeptide forms of one or more biornarkers listed in Tables 1-5 and Examples, or fragments thereof.
  • any and all attributes of the polypeptides described herein, such as percentage identities, polypeptide lengths, polypeptide fragments, biological activities, antibodies, etc. can be combined in any order or combination with respect to any biomarker listed in Tables 1-5 and Examples and combinations thereof.
  • a polypeptide may comprise a full-length amino acid sequence corresponding to one or more biornarkers listed in Tables 1-5 and Examples or a full-length amino acid sequence with 1 to about 20 conservative amino acid substitutions.
  • An amino acid sequence of any described herein can also be at least SO, SS, 60, 65, 70, 75, 80, 85, 90, 91 , 2, 93, 94, 95, 96, 97, 98, 9, or 99.5% identical to the full-length sequence of one or more biomarkers listed in Tables 1 -5 and Examples, which is either described herein, well known in die art, or a fragment thereof.
  • the present invention contemplates a composition comprising an isolated polypeptide corresponding to one or more biomarkers listed in Tables 1-5 and Examples polypeptide and less than about 25%, or alternatively 15%, or alternatively 5%, contaminating biological macromolecules or polypeptides.
  • the present invention further provides compositions related to producing, detecting, characterizing, or modulating the level or activity of such polypeptides, or fragment thereof, such as nucleic acids, vectors, host ceils, and the like. Such compositions may serve as compounds mat modulate the expression and/or activity of one or more biomarkcrs listed in Tables 1-5 and Examples.
  • HMGN1 polypeptides can be used to reduce H3K27me3 and thereby allow lymphoid cells, such as lymphoid progenitors, to proliferate or, alternatively, agents that reduce HMGN1 polypeptide levels or activity can be used to stop proliferation of lymphoid cell (e.g., DS-ALL cells).
  • lymphoid cells such as lymphoid progenitors
  • An isolated polypeptide or a fragment thereof (or a nucleic acid encoding such a polypeptide) corresponding to one or more biomarkcrs of the invention, including the biomarkcrs listed in Tables 1-5 and Examples or fragments thereof, can be used as an immunogen to generate antibodies that bind to said immunogen, using standard techniques for polyclonal and monoclonal antibody preparation according to well-known methods in the art.
  • An antigenic peptide comprises at least 8 amino acid residues and encompasses an epitope present in the respective full length molecule such that an antibody raised against the peptide forms a specific immune complex with the respective full length molecule.
  • the antigenic peptide comprises at least 10 amino acid residues.
  • such epitopes can be specific for a given polypeptide molecule from one species, such as mouse or human (I.e., an antigenic peptide that spans a region of the polypeptide molecule that is not conserved across species is used as immunogen; such non conserved residues can be determined using an alignment such as that provided herein).
  • a polypeptide immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • a suitable subject e.g., rabbit, goat, mouse or other mammal
  • An appropriate immunogenic preparation can contain, for example, a recombinandy expressed or chemically synthesized molecule or fragment thereof to which the immune response is to be generated.
  • the preparation can further include an adjuvant, such as Freunrfs complete or incomplete adjuvant, or similar inummostmiulatory agent. Immunization of a suitable subject with an immunogenic preparation induces a polyclonal antibody response to the antigenic peptide contained therein.
  • Polyclonal antibodies can be prepared as described above by imrminizing a suitable subject with a polypeptide immunogen.
  • the polypeptide antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • the antibody directed against the antigen can be isolated from the mammal ⁇ e.g., from die blood) and further purified by well-known techniques, such as protein A chromatography, to obtain the IgG fraction.
  • amfoody-rxoducing cells can be obtained from tbc subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique (originally described by ohier and Milstein (1975) Nature 256:495-497) (see also Brown etal. (1981) J. Immunol. 127:539-46; Brown el al. (1980) J. Biol. Ckem. 255:4980-83; Yen el al. (1976) Proc. Natl. Acad. Set. 76:2927-31; Yen et al. (1982) Int. J.
  • an immortal cell line typically a myeloma
  • lymphocytes typically spienocytes
  • the culture supernatants of the resulting hybridoma cells arc screened to identify a hybridoma producing a monoclonal antibody that binds to the polypeptide antigen, preferably specifically.
  • any of the many well-known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating a monoclonal antibody against one or more biornarkers of the invention, including the biomarkers listed in Tables 1-5 and Examples, or a fragment thereof (see, e.g., Galfre, G. et al (1977) Nature 266:550- 52; Gefter el al. ( 1977) supra; Lerner (1981) supra, Kenneth (1980) supra).
  • the immortal cell line e.g., a myeloma cell line
  • the immortal cell line is derived from the same mammalian species as the lymphocytes.
  • rnurine bybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthinc, aminopterin and myrnidinc ("HAT medium * ').
  • HAT medium * ' any of a number of myeloma celt lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/0-Agl4 myeloma lines.
  • HAT-sensitive mouse myeloma cells are rased to mouse splenocytes using polyeth lene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supematants for antibodies that bind a given polypeptide, eg., using a standard EUSA assay.
  • a monoclonal specific for one of the above described polypeptides can be identified and isolated by screening a recombinant combinatorial inmuinogiobulin library (e.g., an antibody phage display library) with the appropriate polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide.
  • Kits for generating and screening phage display libraries are «jmmercialry available (e.g., the Pharmacia
  • recombinant polypeptide antibodies such as chimeric and humanized monoclonal antibodies, comprising bom human and non-human portions, whkh can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Patent Publication PCT/US86/02269; Akira et al. European Patent
  • humanized antibodies can be made according to standard protocols such as those disclosed in U.S. Patent 5,565,332.
  • antibody chains or specifk binding pair members can be produced by recombination between vectors comprising nucleic acid molecules encoding a fusion of a polypeptide chain of a specific binding pair member and a component of a replicable generic display package and vectors containing nucleic acid molecules encoding a second polypeptide chain of a single binding pair member using techniques known in die art, e.g. , as described in U.S. Patents 5,565,332, 5,871,907, or 5.733.743.
  • Fully human antibodies can be made in mice that are transgenic for human immunoglobulin genes, eg. according to Hogan, et «/., "Manipulating the Mouse Embryo: A Laboratory Manuel," Cold Spring Harbor Laboratory. Briefly, transgenic mice are immunized with purified immunogen. Spleen cells are harvested and fused to myeloma cells to produce hybridomas. Hybridomas are selected based on their ability to produce antibodies which bind to the immunogen. Fully human antibodies would reduce the immunogenki y of such antibodies in a human.
  • an antibody for use in the instant invention is a bispecifk antibody.
  • a bispecifk antibody has binding sites for two different antigens within a single antibody polypeptide. Antigen binding may be sirmiltaneous or sequential.
  • Triomasand hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Examples of bispecific antibodies produced by a hybrid rrybridoma or a trioma are disclosed in U.S. Patent 4,474,893. Bispecific antibodies have been constructed by chemical means (Stacrz ei oL ( 1985) Nattire 314.-628, and Perez et at. (198S) Nature 316:354) and hybridoma technology (Stacrz and Bcvan (1986) Proc.
  • Bispecific antibodies are also described in U.S. Patent 5,959,084. Fragments of bispecific antibodies are described in U.S. Patent 5.798,229.
  • Bispecific agents can also be generated by making heterohybridomas by fusing hybridomas or other cells making different antibodies, followed by identification of clones producing and co-assembling both antibodies. Tbey can also be generated by chemical or genetic conjugation of complete immunoglobulin chains or portions thereof such as Fab and Fv sequences.
  • the antibody componcni can bind to a polypeptide or a fragment thereof of one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples, or a fragment thereof.
  • the bispecifk antibody could specifically bind to both a polypeptide or a fragment thereof and its natural binding partnc s) ⁇ * * fragments) thereof.
  • peptides ⁇ peptide nrimetics can be used to antagonize or promote the activity of one or more biomarkers of the invention, inducting one or more biomarkers listed in Tables 1 -5 and Examples, or a fragments) thereof.
  • variants of one or more biomarkers listed in Tables 1-5 and Examples which fiinctkm as a modulating agent for Ac respective full length protein can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, for antagonist activity.
  • a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of variants can be produced, for instance, by enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such mat a degenerate set of potential polypeptide sequences is expressible as individual polypeptides containing the set of polypeptide sequences therein.
  • methods which can be used to produce libraries of polypeptide variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector.
  • degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential polypeptide sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see. e&, Narang. S. A. (1983)
  • libraries of fragments of a polypeptide coding sequence can be used to generate a variegated population of polypeptide fragments for screening and subsequent selection of variants of a given polypeptide.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a polypeptide coding sequence with a nuclease under conditions wherein nicking occurs only about once per polypeptide, denaturing the double stranded DNA, tenanting the DNA to form double stranded DNA which can include sense antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-tcrminal, C-terminal and internal fragments of various sizes of the polypeptide.
  • combinatorial libraries made by point mutations or truncation and for screening cDNA libraries for gene products having a selected property.
  • Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of polypeptides.
  • the most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected.
  • REM Recursive ensemble mutagenesis
  • a library of expression vectors can be transfected into a ceil line which ordinarily synthesizes one or more biomarkers of the invention, including one or more biomarlccrs listed in Tables 1-5 and Examples, or a fragment thereof.
  • the transfected cells are then cultured such that the full length polypeptide and a particular mutant polypeptide are produced and the effect of expression of the mutant on the full length polypeptide activity in cell supernaranrs can be detected, &g., by any of a number of functional assays.
  • Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of full length polypeptide activity, and the individual clones further characterized.
  • Systematic substitution of one or more amino acids of a polypeptide amino acid sequence with a D-amino acid of the same type can be used to generate more stable peptides.
  • D-amino acid of the same type e.g., D-iysinc in place of L-lysine
  • constrained peptides comprising a polypeptide amino acid sequence of interest or a substantially identical sequence variation can be generated by methods known in die an (Rizo and Gierasch (1992) Amu. Rev.
  • polypeptides corresponding peptide sequences and sequence variants thereof.
  • Such polypeptides can be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding the peptide sequence, f equently as part of a larger polypeptide.
  • polypeptides can be synthesized by chemical methods.
  • Peptides can be produced, typically by direct chemical synthesis. Peptides can be produced as modified peptides, with nonpeptide moieties attached by covalent linkage to the N-lcrminus and/or C-tcnninus. In certain preferred embodiments, either the carboxy- terminus or the amino-tcrminus. or both, are chemically modified. The most common modifications of the terminal amino and carboxyl groups are acctylation and amidation, respectively.
  • Araino-4erminal modifications such as acylation (e.g., acctylation) or alkyiation (e.g., methylation) and carboxy ⁇ tcrnunal-n ⁇ hTcations such as amidation, as well as other terminal nrodifkations, including cyciization, can be incorporated into various embodiments of the invention.
  • Certain amino-terminal and or carboxy-tcrminal modifications and/or peptide extensions to the core sequence can provide advantageous physical, chemical, biochemical, and pharmacological properties, such as: enhanced stability, increased potency and/or efficacy, resistance to scrum proteases, desirable pharmacokinetic properties, and others.
  • Peptides disclosed herein can be used
  • pcptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or piuumacological activity), but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting o - CH2NH-, -CH2S-, -CH2-CH2-, -CH-CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and - CH2SO-, by methods known m the art and further described in the following references: Spatola, A.
  • peptide mimettcs may nave significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
  • Labeling of pcptidciramctics usually involves covalcnt attachment of one or more labels, directly or through a spacer ⁇ e.g., an amide group), to non-interfering positions) on the pe tidomimctje that are predicted by quantitative structure-activity data and/or molecular modeling.
  • Such non-interfering positions generally are positions mat do not form direct contacts with the rnaaopolypeptidcs(s) to which the peptidomimetic binds to produce the therapeutic effect
  • Derivitizaiion (eg., labeling) of peptidonrimetics should not substantially interfere with the desired biological or pharmacological activity of the peptktomimctic.
  • small molecules which can modulate (either enhance or inhibit) inteiactkms, eg., between biornarkers listed in Tables 1-5 and Examples and their natural binding partners, or inhibit activity.
  • the small molecules of the present invention can be obtained using any of die numerous approaches in combinatorial library methods known in the art, including spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. (Lam, K. S. (1997) Anticancer DrugDes. 12:145).
  • chemical inhibitors of one or more historic H3K27 demethylascs are useful.
  • Such inhibitors are well known in the art and include GSK-J4 (ethyl 3- ( ⁇ htydrc ⁇ lH-beii ⁇ dk ⁇
  • Compounds can be screened in cell based or non-cell based assays.
  • the invention also relates to chimeric or fusion proteins of the biomarkers of the invention, including the biornarkcrs listed in Tables 1-5 and Examples, or fragments thereof.
  • a "ch rteric protein” or “fusion protein” comprises one or more bomarkers of the mvention, including one or more biornarkcrs listed in Tables 1-5 and Examples, or a fragment thereof, opcranvc!y linked to another polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the respective biomarker.
  • the fusion protein comprises at least one biologically active portion of one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples, or fragments thereof.
  • the term "operatively linked" is intended to indicate that the bionnrkcr sequences and the rjon-biomarker sequences are fused in-frame to each other in such a way as to preserve functions exhibited when expressed independently of the fusion.
  • the "another" sequences can be fused to the N-terminus or C-terrainus of the biomarkcr sequences, respectively.
  • Such a fusion protein can be produced by recombinant expression of a nucleotide sequence encoding the first peptide and a nucleotide sequence encoding the second peptide.
  • the second peptide may optionally correspond to a moiety mat alters the solubility, affinity, stability or valency of the first peptide, for example, an immunoglobulin constant region.
  • the first peptide consists of a portion of a biologically active molecule (e.g. the extracellular portion of the polypeptide or the ligand binding portion).
  • the second peptide can include an irnmunoglobulin constant region, for example, ahuman ⁇ domain or Cy domain (e.g., the hinge, CH2 and CH3 regions of human IgCy 1. or human IgCy , see eg.. Capon etat. U.S. Patents 5.116,964; 5,580,756; 5,844,095 and the like, incorporated herein by reference).
  • Such constant regions may retain regions which mediate effector function (e.g. Fc receptor binding) or may be altered to reduce effector function.
  • a resulting fusion protein may have altered solubility, binding affinity, stability and/or valency (i.e., the number of binding sites available per polypeptide) as compared to the independently expressed first peptide, and may increase the efficiency of protein purification.
  • Fusion proteins and peptides produced by recombinant techniques can be secreted and isolated from a mixture of cells and medium containing the protein or peptide. Alternatively, the protein or peptide can be retained cytoplasm icalry and the cells harvested, h/sed and the protein isolated.
  • a cell culture typically includes host cells, media and other byproducts. Suitable media for cdl culture arc wdl known in the art. Protein and peptides can be isolated from cell culture media, host cells, or both using techniques known in the art for purifying proteins and peptides. Techniques for inmsfccting host cells and purifying proteins and peptides are known in the art.
  • a fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-fhtme in accordance with conventional techniques, for example employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, fiUing-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols to Molecular Biology, eds. Ausubcl et al. John Wiley & Sons: 1992).
  • the fusion protein contains a heterologous signal sequence at its N-terrninus.
  • host cells e.g., mammalian host ccllsX expression and/or secretion of a polypeptide can be increased through use of a heterologous signal sequence.
  • the fusion proteins of the invention can be used as immunogens to produce antibodies in a subject Such antibodies may be used to purify the respective natural polypeptides from which the fusion proteins were generated, or in screening assays to identify polypeptides which inhibit the interactions between one or more biomarkers polypeptide or a fragment thereof and its natural binding partncits) or a fragments) thereof.
  • compositions comprising one or more nucleic acids comprising or capable of expressing at least 1.2, 3,4, 3, 10, 20 cr more small nucleic acids or antisense oligonucleotides or derivatives thereof, wherein said small nucleic acids or antisense oligonucleotides or derivatives thereof in a ceil specifically hybridize (e.g., bind) under cellular conditions, with cellular nucleic acids (e.g., small non-coding RNAS such as miRNAs, pre-miRNAs, pri-miRNAs, miRNA*, anti-miRNA, a miR A binding site, a variant and/or functional variant thereof cellular mR As or a fragments thereof).
  • small non-coding RNAS such as miRNAs, pre-miRNAs, pri-miRNAs, miRNA*, anti-miRNA, a miR A binding site, a variant and/or functional variant thereof cellular mR As or a fragments thereof.
  • expression of the small nucleic acids or antisense oligonucleotides or derivatives thereof in a cell can enhance or upregulate one or more biological acti vities associated with the corresponding wild-type, naturally occurring, or synthetic small nucleic acids.
  • expression of the small nucleic acids or antisense oligonucleotides or derivatives thereof in a cell can inhibit expression or biological activity of cellular nucleic acids andor proteins, e.g. , by inhibiting transcription, translation andor small nucleic acid processing of, for example, one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples, or fragments) thereof.
  • die small nucleic acids or antisense oligonucleotides or derivatives thereof arc small RNAs (e.g., inicroRNAs) or complements of small RNAs.
  • the small nucleic acids or antisense oligonucleotides or derivatives thereof can be single or double stranded and are at least six nucleotides in length and are less than about 1000, 900, 800, 700, 00, 500, 400, 300, 200, 100, 50, 40, 0, 25, 24, 23, 22, 21,20, 19, 18, 17, 16, IS, or 10 nucleotides in length.
  • a composition may comprise a library of nucleic acids comprising or capable of expressing small nucleic acids or antiscnse oligonucleotides or derivatives thereof, or pools of said small nucleic acids or antiscnse oligonucleotides or derivatives thereof.
  • a pool of nucleic acids may comprise about 2-5, 5-10, 10-20, 10-30 or more nucleic acids comprising or capable of expressing small nucleic acids or antiscnse oligonucleotides or derivatives thereof.
  • binding may be by conventional base pair conmlemcntarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
  • antisense refers to the range of techniques generally employed in the art, and includes any process that relics on specific binding to oligonucleotide sequences.
  • the term "functional variant" of a miRNA sequence refers to an oligonucleotide sequence that varies from the natural miRNA sequence, but retains one or more functional characteristics of the miRNA (e.g. cancer cell proliferation inhibition, induction of cancer cell apoptosis, enhancement of cancer cell susceptibility to chemo therapeutic agents, specific miRNA target inhibition).
  • a functional variant of a miRNA sequence retains all of the functional characteristics of the miRNA.
  • a functional variant of a miRNA has a nucleobasc sequence that is a least about 60%, 65%, 70%.75%, 80%, 85%.90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the miRNA or precursor thereof over a region of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 0, 5, 70, 75, 80, 85, 90, 5, 100 or more nuclcobases, or that the functional variant hybridizes to the complement of the miRNA or precursor thereof under stringent hybridization conditions.
  • the nucleobase sequence of a functional variant is capable of hybridizing to one or more target sequences of the miRNA.
  • imRNAs and their corresponding stem-loop sequences described herein may be found in miRBase, an online searchable database of miRNA sequences and annotation, found on the worid wide web at rniarorna.sanger.ac.uk.
  • Entries in the miRBase Sequence database represent a predicted hairpin portion of a miRNA transcript (the stem-loop), with information an the location and sequence of the mature miRNA sequence.
  • the miRNA stem-loop sequences in the database are not strictly precursor miRNAs (pre-miR As), and may in some instances include the pre-miRNA and some flanking sequence from the presumed primary transcript
  • the miRNA nucleobase sequences described herein encompass any version of the miRNA, including the sequences described in Release 10.0 of the miRBase sequence database and sequences described in any earlier Release of the miRBase sequence database.
  • a sequence database release may result in the re-naming of certain miRNAs.
  • a sequence database release may result in a variation of a mature miRNA sequence.
  • miRNA sequences of the invention may be associated with a second RNA sequence that may be located on the same RNA molecule or on a separate RNA molecule as the miRNA sequence, fn such cases, the miRNA sequence may be referred to as the active strand, while the second RNA sequence, which is at least partially complementary to die miRNA sequence, may be referred to as the complementary strand.
  • the active and complementary strands are hybridized to create a double-stranded RNA that is similar to a naturally occurring miRNA precursor.
  • the activity of a miRNA may be optimized by maximizing uptake of the active strand and minimizing uptake of the complementary strand by the miRNA protein complex that regulates gene translation. This can be done through rnodificarion and/or design of the complementary strand.
  • the complementary strand is modified so that a chemical group other than a phosphate or bydroxyi at its 5' terminus.
  • the presence of the 5' modification apparently eliminates uptake of the complementary strand and subsequently favors uptake of die active strand by die miRNA protein complex.
  • the 5' modification can be any of a variety of molecules known n the an, including Nrfe, NHCOCHb, and biotin.
  • the uptake of the complementary strand by the miRNA pathway is reduced by m ⁇ xrporating nucleotides with sugar modifications in the first 2-6 nucleotides of the complementary strand. It should be noted that such sugar modifications can be combined with the 5 * terminal modifications described above to further enhance miRNA activities.
  • die complementary strand is designed so that nucleotides in the 3' end of die complementary strand are not complementary to the active strand. This results in double-strand hybrid RNAs that are stable at the 3' end of the active strand but relatively unstable at die 5* end of the active strand. This difference in stability enhances the uptake of the active strand by the nriRNA pathway, while reducing uptake of the complementary strand, thereby enhancing miRNA activity.
  • Small nucleic acid and/or antisensc constructs of the methods and compositions presented herein can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of cellular nucleic acids (e.g., small RNAs, mRNA, and/or genomic DNA).
  • the small nucleic acid molecules can produce RNA which encodes mRNA, miRNA, pre- miRNA, pri-miRNA, miRNA*, ami-miRNA, or a miRNA binding site, or a variant thereof.
  • small nucleic acids and/or antisense constructs are oligonucleotide probes that arc generated ex vivo and which, when introduced into the cell, results in hybridization with cellular nucleic acids.
  • Such oligonucleotide probes are preferably modified oligonucleotides that are resistant to endogenous nucleases, eg, exonucleascs and/or endonuc!eascs, and are therefore stable in vivo.
  • Exemplary nucleic acid molecules for use as small nucleic acids and/or antisense oligonucleotides are phosphoramidate, phospbothioate and memylphospbonate analogs of DNA (see also U.S.
  • Patents 5,176,996; 5,264,564; and 5,256,775) are reviewed, for example, by Van der rol el at. ( 1988) BioTechniqucs 6:958-976; and Stein etal (1988) Cancer Res 48:2659-2668.
  • Antisense approaches may involve the design of ohgonucleotides (either DNA or RNA) mat are complementary to cellular nucleic acids (&g., complementary to biomarkers listed in Tables 1 -5 and Examples). Absolute complementarity is not required. In die case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. Tbc ability to hybridize will depend on both the degree of ⁇ rnplementariry and die length of the antisensc nucleic acid.
  • nucleic acid e.g., UNA
  • a nucleic acid e.g., UNA
  • One skilled in the an can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • Oligonucleotides that are complementary to the 5' end of the mRNA should work most efficiently at inhibiting translation.
  • sequences complementary to the 3' untranslated sequences of mRNAs have recently been shown to be effective at inhibiting translation of mRNAs as well (Wagner, R. (1994) Nature 372:333). Therefore, oligonucleotides complementary to either die 5 * or 3 * untranslated, non-coding regions of genes could be used in an antisense approach to inhibit translation of endogenous mRNAs.
  • Oligonucleotides complementary to the 5 * untranslated region of the mRN A may include the complement of the AUG start codon.
  • Antisense ougonucleoudcs complementary to mRNA coding regions are less efficient inhibitors of translation but could also be used in accordance with the methods and compositions presented herein. Whether designed to hybridize to thc5', 3' or coding region of cellular mRNAs, small nucleic acids andor antisense nucleic acids should be at least six nucleotides in length, and can be less than about 1000, 00, 800.700, 00, 500, 00, 300, 200, 100, SO, 40, 30, 25, 24.23, 22, 21,20, 19, 18, 17, 16, 15, or 10 nucleotides in length.
  • in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression.
  • these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides.
  • these studies compare levels of the target nucleic acid or protein with that of an internal control nucleic acid or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide arc compared with those obtained using a control oligonucleotide.
  • control oligonucleotide is of approximately die same length as the test oligonucleotide and that die nucleotide sequence of the oligonucleotide differs from die antisense sequence no more man is necessary to prevent specific hybridization to the target sequence.
  • Small nucleic acids and/or antisense oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded.
  • Small nucleic acids and or antisense oligonucleotides can be modified at die base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc., and may include other appended groups such as peptides (e.g., for targeting host cell receptors), or agents facilitating transport across the cell membrane (see, eg., Lctsinger el al. (1989) Proc. Natl. Acad. Sci. U.S.A.86:6553-6556; Lemaitrc ei al (1987) Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No.
  • small nucleic acids and/or antisense otigonucleotidce may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, Irybridization-triggered cleavage agent, etc.
  • Small nucleic acids and/or antisense oiigonuclcotides may comprise at least one modified base moiety which is selected from the group including but not limited to 5- flucfouracii, 5-bromouracil, 5 hlorouraci1, 5-iodouracil, hypoxanthine, xanttne, 4- acetylcytosine, McarboxyhyoYoxytiethyl) uracil 5 ⁇ arboxyme laminomethyl"2- thiouridine, 5 ⁇ aiboxymethylaminomemyluracil, dihydrouracil, beta-D-galactosylqueostne, inosine, N6-iscpent ⁇ ytadeninc, 1-methylguanine, 1-nwthylinosine, 2,2-dimethylguaninc, 2-methyladcnine, 2-mem i guanine, 3-metfaylcyt06ine, 5-memylcytostne, N
  • Small nucleic acids and/or antisense oligonucleotides may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fhwroarabinose, xylulose, and bexose.
  • a compound comprises an oligonucleotide (e.g.. a miRNA or miRNA encoding oligonucleotide) conjugated to one or more moieties which enhance the activity, cellular distribution or cellular uptake of the resulting oligonucleotide.
  • the moiety is a cholesterol moiety (e.g. , antagomirs) or a lipid moiety or liposome conjugate. Additional moieties for conjugation include carbohydrates, phospholipids, biotin, pbenazme, folate, phenanthridine, amtiraquinone, acridine.
  • a conjugate grou is attached directly to die oligonucleotide.
  • a conjugate group is attached to the oligonucleotide by a linking moiety selected from amino, hydroxy!, carboxylic acid, thiol, unsaturations (eg., double or triple bonds), fr-anuno-3,6- dioxaoctanoic acid (ADO), succinimidyl 4-( -inaleimidemcthyi) cyclohcxanc-1- carboxylate (SMCO.6-amioobexanoic acid (AH EX or AHA), substitutedCI-ClO alkyl, substituted or unsubstitutcd C2-C10 aikenyl.
  • a linking moiety selected from amino, hydroxy!, carboxylic acid, thiol, unsaturations (eg., double or triple bonds), fr-anuno-3,6- dioxaoctanoic acid (ADO), succinimidy
  • a substituent group is selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyi, aryi, aikenyl and alkynyl.
  • the compound comprises the oUgonucieotide having one or more stabilizing groups that are attached (o one or both termini of the
  • oligonucleotide to enhance properties such as, for example, nuclease stability.
  • stabilizing groups include cap structures. These terminal rnodifications protect the oligonucleotide from cxonuclcase degradation, and can help in delivery and/or localization within a cell.
  • the cap can be present at the ⁇ -terminus (5 * -cap), or at the 3'4enninus (3- cap), or can be present on both termini.
  • Cap structures include, for example, inverted deoxy abasic caps.
  • Suitable cap structures include a 4 ⁇ 5 * -methylene nucleotide, a 1 - ⁇ bcta-D- erythrofuranosyl) nucleotide a 4'-thio nucleotide, a carbocyclic nucleotide, a 1,5- anhydrohexitol nucleotide, an L-nuclcotidc, an alpha-nuclcotide, a modified base nucleotide, a phosptorodithioate linkage, a thro-pettofuranosyl nucleotide, an acyclic S' ⁇ '-seco nucleotide, an acyclic 3,4-dilrydroxybutyi nucleotide, an acyclic 3,5- dihydroxypentyl nucleotide, a 3-3 -inverted nucleotide moiety, a 3-3 -inverted abasic moiety, a 3'-2'
  • Small nucleic acids and/or antiscnsc oligonucleotides can also contain a neutral peptidc-likc backbone.
  • Such molecules are termed peptide nucleic acid (PNA)-oiigomcrs and are described, e.g., in Pcrry-O'Kcefc etal (1996) Proc. Nad. Acad. Sci. U.SA
  • PNA oligomers are their capability to bind to complementary DNA essentially independently from the ionic strength of the medium due to the neutral backbone of me DN A.
  • small nucleic acids and/or antiscnsc oligonucleotides comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioafc, a phc ⁇ )horodithioate, a phosphc arriklothioatc, a phosptoiamidate, a ptasphordunrtidatc, a rrKthylphospbonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • modified phosphate backbone selected from the group consisting of a phosphorothioafc, a phc ⁇ )horodithioate, a phosphc arriklothioatc, a phosptoiamidate, a ptasphordunrtidatc, a rrKthylphospbonate, an alky
  • small nucleic acids and or a ti sense oligonucleotides are a-arttmeric oligonucleotides.
  • An a-anomeric oligonocleotide forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al. (1987) Nucl. Acids Res. 15:6625-6641).
  • the oligonucleotide is a 2'-0HT thylr3 ⁇ 4onucleotidc (Inouc tt al (1987) Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al ( 1987) FEBS Lett 215:327-330).
  • Small nucleic acids and/or antisense oligonucleotides of the methods and compositions presented herein may be synthesized by standard methods known in the art. e. ., by use of an automated DNA synthesizer (such as are commercially available from Bioscarch, Applied Biosysfcrm, etc.).
  • an automated DNA synthesizer such as are commercially available from Bioscarch, Applied Biosysfcrm, etc.
  • phosphoromtoate oligonucleotides may be synthesized by the method of Stein tt al (1988) Nucl. Acids Res. 16:3209
  • metnylphospnonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al (1988) Proc. Natl. Acad. Sci.
  • an isolated miRNA can be chemically synthesized or recombinant ⁇ pt duccd using methods known in the art
  • miRNA are chemically synthesized using appropriately protected ribonucleotide phospborarmdites and a conventional DNA RNA synthesizer.
  • Corrtmcrcial suppliers of synthetic RNA molecules or synthesis reagents include, e.g., Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part ofPerbio Science, Rockf rd.111., USA), Glen Research (Sterling, Va., USA), ChemGencs (Ashland, Mass., USA), Cruachcm (Glasgow, UK), and Exiqon (Vedbaek, Denmark). Small nucleic acids and/or antiscnsc oligonucleotides can be delivered ID cells in vtvo.
  • antiscnsc molecules can be injected directly into the tissue she, or modified antisense molecules, designed to target the desired cells (*?.£., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on die target cell surface) can be administered systematically.
  • small nucleic acids and/or antisense oligonucleotides may comprise or be generated from double stranded small interfering R As (siRNAs), in which sequences fully complementary to cellular nucleic acids (eg. mRNAs) sequences mediate degradation or in which sequences incompletely complementary to cellular nuckic acids (e.g., mRNAs) mediate transiational repression when expressed within cells.
  • siRNAs double stranded small interfering R As
  • double stranded siRNAs can be processed into single stranded antisense RNAs mat bind single stranded cellular RNAs (e.g.. microRNAs) and inhibit their expression.
  • RNA mterfcrence is the process of sequence-specific, post- transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) mat is homologous in sequence to the silenced gene, in vivo, long dsRNA is cleaved by ribonuclcase HI to generate 21 ⁇ and 22-miclcotide siRNAs. It has been shown that 21 -nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells (Elbashir et at. (2001) Nature 411 -.494-498).
  • RNAs having a length of about IS to 30 nucleotides or of about 18 to 21 nucleotides or of about 1 to 21 nucleotides.
  • a vector encoding for such siRNAs or short !iaiipin RNAs (shRNAs) that are metabolized into siRNAs can be introduced into a target cell (see, t g.. McManus et af. (2002) RNA 8:842; Xia el ah (2002) Nature Biotechnology 20: 1006; and Brummelkamp el al. (2002) Science 296:550).
  • Vectors that can be used are commercially available, e.g., from OligoEngine under the name pSuper RNAi System 1 ".
  • Ribozyme molecules designed to catalyticalr cleave cellular mRNA transcripts can also be used to prevent translation of cellular mRNAs and expression of cellular polypeptides, or both (Sec, e.g. , PCT International Publication WO90 11364, published October 4, 1990 Sarver etal. (1990) Science 247:1222-1225 and U.S. Patent No.
  • ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy cellular mRNAs, the use of iiammerhead ribozymes is preferred.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3 ⁇
  • the construction and production of hammerhead ribozymes is well known in the an and is described more fully in HaselofTand Gerlach (1988) Nature 334:585-591.
  • the ribozyme may be engineered so that the cleavage recognition site is located near the 5' end of cellular mRNAs; i.e., to increase efficiency and minimize the intracellular accumulation of non-ftincrional mRNA transcripts.
  • RNA endoribomicleases such as the one which occurs naturally in Teirah mena thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, er al. (1984) Science 224:574-578; Zaug, etal. (1986) Science 231:470-475; Zaug. et al. (1986) ature 324:429-433; published International patent application No. WO88/04300 by University Patents Inc.; Been, etal. (1986) Cell 47:207-216).
  • Cech-type ribozymes such as the one which occurs naturally in Teirah mena thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, er al. (1984) Science 224:574-578; Zaug, etal. (1986) Science 231:470-475; Zaug. et al. (19
  • the Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place.
  • the rncthods and compositions presented herein encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in cellular genes.
  • the ribozymes can be composed of modified oligonucleotides (&£., for improved stability, targeting, etc.).
  • a preferred method of delivery involves using a DN A construct "encoding'' the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transacted cells will produce sufficient quantities of the ribozyme to destroy endogenous cellular messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
  • Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription of cellular genes are preferably single stranded and composed of
  • deoxyribonucleotides The base composition of these oiigonucleoidcs should promote triple helix formation via Hoogsteen base pairing rules, which generally require sizable stretches of either purines or pyrimidincs to be present on one strand of a duplex.
  • Nucleotide sequences may be pyrimtdine-based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix.
  • the pvrimidme-rich molecules provide base coinplcmentarrty to a purineHrich region of a single strand of die dupkx in a parallel orientation to that strand
  • nucleic acid molecules may be chosen that are purine-rich, for example, containing a stretch of G residues.
  • the potential sequences that can be targeted for triple helix formation may be increased by creating a so called "switchback' * nuclck acid molecule.
  • Switchback molecules are synthesized in an alternating 5'-3 ⁇ 3 * -5 * manner, such that they base pair with first one strand of a duplex and then the other, diminating the necessity for a sizable stretch of either purines or pyrimidincs to be present on one strand of a duplex.
  • Small nucleic acids e.g., miRNAs, pre-miRNAs, pri-miRNAs, mi RNA*, and- nuR A, or a rraRNA binding site, or a variant thereof
  • antisense oligonucleotides, ribozymes, and triple helix molecules of die methods and compositions presented herein may be prepared by any method known in the art for the synthesis of ONA and R A molecules. These include techniques for chemically synthesizing
  • RNA molecules may be generated by In vitro and in vtvo transcription of DN A sequences erodin the antisense RNA molecule.
  • DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as die T7 or SP6 polymerase promoters.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • nucleic acid molecules may be irutoduccd as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotKlcs or dcoxyribonucleoudes to the 5' andor 3' ends of the molecule or the use of phosphoroduoatc or 2 * O-methyl rather than phosphodiesterase linkages within the oligodcoxyribonucleotide backbone.
  • polypeptides, small nucleic acids, and antisense oUgonuclcotides can be urther linked to another peptide or polypeptide (e.g.
  • label peptide or polypeptide moieties useful for detection in the invention include, without limi cation, suitable enzymes such as horseradish peroxidase, alkaline phosphatase, bcta-galactosidase, or acetylcholinesterase; epitope tags, such as FLAG, MYC, HA, or HIS tags; fluorophorcs such as green fluorescent protein; dyes; radioisotopes; digoxygenin; biotin; antibodies; polymers as well as others known in the art, for example, in Principles of Fluorescence Spectroscopy, Joseph R.
  • the modulatory agents described herein can be incorporated into pharmaceutical compositions and administered to a subject in vivo.
  • the compositions may contain a single such molecule or agent or any combination of agents described herein. Based on the genetic pathway analyses described herein, it is believed that such combinations of agents is especially effective in diagnosing, prognosing, preventing, and treating cancer.
  • single active agents' described herein can be combined with other pharmacologically active compounds (“second active agents") known m the art according to the methods and compositions provided herein. It is believed that certain conibinations work syncrgistically in the treatment of particular types of cancer.
  • Second active agents can be large molecules (e.g., proteins) or small molecules (e.g., synthetic inorganic, organomctallic, or organic molecules).
  • large molecule active agents include, but are not limited to, hematopoietic growth factors, cytokines, and monoclonal and polyclonal antibodies.
  • Typical large molecule active agents arc biological molecules, such as naturally occurring or artificially made proteins.
  • Proteins that arc particularly useful in this mvention include proteins that stimulate the survival and/or proliferation of hematopoietic precursor cells and immunologically active poietk cells in vitro or in vivo. Others stimulate die division and differentiation of committed crythroid progenitors in ceils in vitro or in vivo.
  • interleukins such as IL-2 (including recombinant IL-II ( H riL2 w and canarypox IL-2), IL-10, lL-12, and IL-18; interferons, such as interferon alia- 2a, interferon alfa*2b, interferon alpha-nl , interferon alpha-n3, interferon beta-la, and interferon gamma-lb; GM-CF and OM-CSF; and EPO.
  • interleukins such as IL-2 (including recombinant IL-II ( H riL2 w and canarypox IL-2), IL-10, lL-12, and IL-18
  • interferons such as interferon alia- 2a, interferon alfa*2b, interferon alpha-nl , interferon alpha-n3, interferon beta-la, and interferon gamma-lb
  • Recombinant and mutated forms of GM-CSF can be prepared as described in U.S. Pat Nos. 5.391.485; 5,393,870; and 5.229.496; all of which are incorporated herein by reference.
  • Recombinant and mutated forms of G-CSF can be prepared as described in U.S. Pat Nos.4,810,643; 4,999,29] ; 5,528,823; and 5,580,755; all of which are incorporated herein by reference.
  • Antibodies that can be used in conrinnation form include monoclonal and polyclonal antibodies.
  • antibodies include, but are not limited to, trastuzumab (Herceotin ⁇ ), rituximab (Ritttxantt), bevaciznmab (Avastinft), pertuzumab (Omnitarg ⁇ ), tositumomab (Bexxar$), edrecolomab (Panorex$>). and O250.
  • Compounds of the invention can also be combined with, or used in combination with, anti-TNF-a antibodies. Large molecule active agents may be administered in the form of anti-cancer vaccines.
  • vaccines that secrete, or cause the secretion of. cytokines such as lL-2, G-CSF, and GM-CSF can be used in the methods, pharmaccuticai compositions, and kits provided herein. See, e.g., Emens, L. A.. etai, Curr. Opinion Mol. Ther. 3 ⁇ 1): 77-84 (2001).
  • Second active agents that arc small molecules can also be used to in combination as provided herein.
  • small molecule second active agents include, but are not limited to, anti-cancer agents, antibiotics, imnumosuppressive agents, and steroids.
  • the combination chemotherapy comprises a combination of two or more of cyclophosphamide, hydroxydaunorubtcin (also known as doxorubicin or adriamycin), oncovorin (vincristine), and prednisone.
  • the combination chemotherapy comprises a combination of cyclcfhsophamide, oncovorin, prednisone, and one or more chernotbcrapcufjcs selected from the group consisting of anmracycUne, hydroxydaunorubicin, epirubicin, and motbantrone.
  • anti-cancer agents include, but are not limited to: acivicin;
  • aclarubicin acodazole hydrochloride; acronine; adozeiesin; aldesleukin; altretamine;
  • ambomyciri ametantrone acetate; amsacrine; anastrozole; anthramycin; asparaginase; aspcrlin; azacitidinc; azetepa; azotomycin; batimastat; benzodepa bicalutanude; bisantrene hydrochloride; bisnafidc diroesylate; bizelcsin; bleomycin sulfate; brequirurr sodium;
  • dexormaplatin dezaguanine; dezaguanine mesylate; diaziquooe; docctaxel; doxorubicin; doxorubicin hydrochloride droloxifenc; droloxifenc citrate; dromostanolone propionate; duazomycin; cdatrexale; eflcnuthine hydrochloride; clsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozolc; esorubicin hydrochloride cstramustine; esiramustine phosphate sodium etanidazole; etoposide; etopotide phosphate; etoprine; fadrozole hydrochloride; camrabine; fenretinide; floxaridinc; fludarabinc phosphate;
  • fluorooracil fluorocitabioc; fosqoidone; f striccin sodium; gemcitabinc; gemcitabinc hydrochloride; hydroxyurea; idarabicin hydrochloride; iiosfanride; effetofbsine; iproplatin; irinotecaiu irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate;
  • masoprocol mavtansine; inechloreihaminc bydrocluoride; megestrol acetate; mekngestroi acetate; meb halan; meaogaril; nicrcaptopurine; methotrexate; methotrexate sodium;
  • metoprine meturedepa; rratindomidc; mitocarcin; mitocromin; mitogilhn; iratomalcin; mitomycin; mitospcr, mitotanc; mitoxantrone bydrochioride; mycopbenolic acid;
  • nocodazole nogalamycin; ormaplarin; oxisuran; paclitaxd; pegaspargase; peliomycin; pentamustine; pcploniycin sulfate; pcrfbsfamide; pipobroman; piposutfan; piroxantrone hydrochloride pUcarnycin; plomestanc; porfimer sodium; porfiromycin; predmraustine; procarbazine bydrochioride; puromyctn; puromycin hydrochloride; pyiazofurin; riboprine; safingoi; safmgol hydrochloride; semustine; suppressrazene; sparfbsate sodium; sparsomycin; spirogcrmanium hydrochloride; spiromustine; spiroplatin strcptonigrin streptozocin; suiofenur.
  • talisomyctn tecogalan sodium; taxotere; tcgafur, teioxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; miamiprine thiognanine; thiotcpa;
  • trimetrexafc trimetrexate giucuronatc; triptorclin; rubulozoic hydrochloride; uracil mustard; uredepa; vapreotide; vertcporfin; vinblastine sulfate; vincristine sulfate; vindesinr, vindesine sulfate; vinepidine sulfate; vraglycinate sulfate; vmleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zenipiatin; zinostatin; and zorubkin hydrochloride.
  • arm-cancer drugs include, but are not limited to: 20-cpi-l,25
  • amifbstine aminolevulinic acid; amrobicin; amsacrine; anagrdidc; anastrozoic; andrographolide; angiogencsis inhibiton; antagonist D; antagonist G; antareiix; anti- dorsalizing moiphogenetic protein-1 ; antiandrogen, prostatic carcinoma; anuestrogen; antineoplaston; antiscnse oKgonuclcotidcs; aphidkoltn glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestanc; atrimustine; axinastatin 1 ; axinastatin 2; axinastatin 3; azasetron; aza toxin; azarvrosine; baccatin III derivatives; balanol; batim
  • B betulinic acid; bFGF inhibitor; bkahitamide; bisantrene; bisaziridiiryisperimne;
  • bisnafidc bistratene A; bizelesin; breflate; biopirimine; budotitane; buthiomne sutfoximinc; caknpotriol calpbostin C; camptothecin derivatives; capecitabme; carboxamide-amino- triazole; carboxyarriootriazole CaRcst M3; CARN 700; cartilage derived inhibitor, carzelesin; casein kinase inhibitors (ICOS); casuuMspcrminc; cecropin B; cetrordix;
  • chiorins chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clormrene analogues; clotrimazole; coUismycin A; collismycin B; cc ⁇ nbretastatin A4; combrctastatin analogue; conagenm; crambcsckHn 816; crisnatol; cryptophycin 8; crvptophycin A derivatives; curacin A; cyctopentanAraquinones; cycloplatam; cyclosporin A; cypemycin; cytarabinc oefbsfate; cytolytic factor; cytostatin; daclixtmab; decitabine; dehydrodidemnin
  • doxorubicin doxorubicin
  • droloxifcnc dronabinol
  • duocarmycin SA ebsekn
  • ccomus ine eddfbsinc edrecolomab
  • cfl ornithine elemene
  • emitemr epirubicin
  • epristcridc estramustine analogue
  • estrogen antagonists etanidazole
  • droloxifcnc dronabinol
  • duocarmycin SA ebsekn
  • ccomus ine eddfbsinc edrecolomab
  • cfl ornithine elemene
  • emitemr epirubicin
  • epristcridc estramustine analogue
  • estrogen agonists e
  • flezelasrine fluasterone fhidarabine
  • rhjorodaunorunicin hydrochloride forfemmcx
  • fb mestane fbstriecin
  • fbtemustine gadolinium texaphyrin
  • gallium nitrate gatocitabine
  • ganirelix getatinase inhibitors
  • gemcitabine glutathione inhibitors hepsulfam; heregulin; hexamemylene bisacetamide; hypericin; ibandronic acid; idarubtcin; idoxifene;
  • imrnunosu ' mulant peptides insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interieukins; iobenguanc; iododoxorobicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohonwhalicondrin B; hasctron; jasplakinotide; kahalaiide F; lamellarin-N triacetate lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; lctrozolc; leukemia inhibiting factor; leukocyte alpha interferon;
  • miitefosinc mirimostim; mhoguazone; nritolactol; mitomycin analogues; mitonafidc; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; Erbitux, human chorionic gonadotropin; monophosphoryl Kpid A- myobacterium cell wall sk; mopidamol; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acerykKnaline; N-substituted benzamides; ludarelin; nagrestip;
  • naloxoac+pentaxocine napavin; naphtcrpin; nartograstim; nedaplatin; nemorubicin;
  • neridronic acid nihitamidc; nisarnycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; oblimersen (Gcnascnae$); 06-benzylguaninc; octreotide; okkenone;
  • oligonucleotides onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer ormaplatin; osatcronc; oxaiipiatin; oxaunomycin; paclitaxei; paclitaxel analogues;
  • paclitaxel derivatives palauamine; palmitoylrhizoxtn; pamklronic acid; panaxytriol;
  • panomifene parabactin; pazelliptine pegaspargase; peldesinc pentosan porysulfate sodium; peniostatin; pentrozole perflubron; perfosfamide; pcrillyl alcohol;
  • hydrochloride pimrabtcin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor, platinum complex; platinum compounds; platinum-triamine complex; Dorfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prosuujlandin J2; proteasome inhibitors; protein A-bascd immune modulator, protein kinase C inhibitor, protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pymzobacridine; pyridoxylated hemoglobin polyoxycthylenc conjugate; raf antagonists; raltitrexcd; ramosetron; ras rarnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retcliiptinc dcmethylated
  • spiromustine spknopentin spongistatin 1; squal amine; stipiamide; stromelysin inhibitors sulfinosinc; supcractive vasoactive intestinal peptide antagonist; suradista; suramin;
  • thrombopoictin mimetic wymalfesin; thymopoietin icceplor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tiiapazamine; titanocene bicbloride; topsendn; toremifene; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimctrexatc;
  • triptorelin triptorelin; tropisetron; turosteride tyrosine kinase inhibitors; tyrphostms; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor, urokinase receptor antagonists; vaprcotide; variolin B; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxtn; vorozolc; zanoterone; zcniplaun; zilascorb; and zinostatin stimaUuncr.
  • Specific second active agents include, but are not limited to, chlorambucil, fludarabinc, dcxamethasonc (Decadron$), hydrocortisone, mcthylprcdnisolone, cilostamide, doxorubicin (Doxilft), forskolin, rituximab, cyclosporin A, cisplatin, vu ristine, PDE7 inhibitors such as BRL-50481 and IR-202, dual PDE47 inhibitors such as IR-284, cilostazol, meribendan, milrinone, vesnarionone, enoximone and pimobendan, Syk inhibitors such as fbstamatinib disodium (R406/R788), R343, R-l 12 and Exccliair$ (ZaBeCor Phannaceuticals, Bala Cynwyd, Pa.).
  • Another aspect of the invention relates to methods of selecting agents (e.g., antibodies, fusion proteins, peptides, small molecules, or small nucleic acids) which bind to, uprcgulate, downregulatc, or modulate one or more biomarkcrc of the invention listed in
  • Tables 1-5 and Examples and/or a cancer eg., a lymphoid cancer, such as leukemia.
  • a cancer eg., a lymphoid cancer, such as leukemia.
  • Such methods utilize can use screening assays, including cell based and nan -cell based assays.
  • the invention relates to assays for screening candidate or test compounds which bind to or modulate die expression or activity level of, one or more bramarkers of the invention, including one or more biomarkers listed in Tables 1-5 and
  • an assay is a cell-based assay, comprising contacting a cell expressing one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples, or a fragment thereof, with a test compound and determining the ability of the (est compound to modulate (eg. stimulate or inhibit) the level of interaction between the biomarker and its natural binding partners as measured by direct binding or by measuring a parameter of cancer.
  • the biomarker polypeptide, a binding partner polypeptide of die biomarker, or a fragments) thereof can be coupled with a radioisotope or enzymatic label such that binding of the biomarker polypeptide or a fragment thereof to its natural binding partner(s) or a fragments) thereof can be determined by detecting the labeled molecule in a complex.
  • die biomarker polypeptide, a binding partner polypeptide of the biomarker, or a fragments) thereof can be labeled with l25 l, "S, M C, or 3 H, either directly or indirectly, and die radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • polypeptides of interest a can be enzyrnaticalry labeled with, for example, horseradish peroxidase, alkaline phosphatase, or loaferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product
  • a M niicrophysiometer n e.g., Cytosensor
  • a M niicrophysiometer n is an analytical instrument that measures the rate at which a cell acidifies its environment using a light- addressable potentiomctric sensor (LAPS). Changes in this ackhfication rate can be used as an indicator of the interaction between compound and receptor.
  • LAPS light- addressable potentiomctric sensor
  • determining the ability of the blocking agents eg. antibodies, fusion proteins, peptides, nucleic acid molecules, or small molecules
  • determining the ability of the blocking agents can be accomplished by dbacnwning the activity of one or more members of the set of interacting molecules.
  • the activity of one or more biomarkers of the invention can be determined by detecting induction of cytokine or chemokine response, detecting cataiytic/enzyrnatic activity of an appropriate substrate, detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., chloramphenicol acetyl transferase), or detecting a cellular response regulated by the biomarker or a fragment thereof (e.g., modulations of biological pathways identified herein, such as modulated proliferation, apoptosis, cell cycle, and/or E2F transcription facto binding activity).
  • a reporter gene comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., chloramphenicol acetyl transferase
  • detecting a cellular response regulated by the biomarker or a fragment thereof
  • Determining the ability of the blocking agent to bind to or interact with said polypeptide can be accomplished by measuring the ability of an agent to modulate iiranunc responses, for example, by detecting changes in type and amount of cytokine secretion, changes in apoptosis or proliferation, changes in gene expression or activity associated with cellular identity, or by irtterfering with the ability of said polypeptide to bind to antibodies that recognize a portion thereof.
  • an assay of the present invention is a cell-free assay in which one or more biomarker* of the invention, including one or more biomarkcrs listed in Tables 1 -5 and Examples or a fragment thereof, e.g. a biologically active fragment thereof, is contacted with a test compound, and the ability of the test compound to bind to the polypeptide, or biologically active portion thereof, is determined. Binding of the test compound to the biomarker or a fragment thereof, can be a1 ⁇ 2tcrrmned either directly or indirectly as described above.
  • Determining die ability of die biomarker or a fragment thereof to bind to its natural binding partners) or a fragment(s) thereof can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA) (Sjolander, S. and Uibaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. ()pin. Struct. Biol. 5:699-705).
  • BIOA Biomolecular Interaction Analysis
  • Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological polypeptides.
  • One or more biomarkcrs polypeptide or a fragment thereof can be immobilized on a BlAcore chip and multiple agents, e.g., blocking antibodies, fusion proteins, peptides, or small molecules, can be tested for binding to the immobilized biomarker polypeptide or fragment thereof.
  • agents e.g., blocking antibodies, fusion proteins, peptides, or small molecules.
  • the cell-free assays of the present invention arc amenable to use of bom soluble and/or rriembrane-bound forms of proteins.
  • a sohibilizing agent such that the mraibrane-bound form of the protein is maintained in solution.
  • solu ilizing agents include non-ionic detergents such as n-octylglucosidc, n- dodecyiglucoside, n-dodccytma1toside, c «tanoyl-N-inethylglucamide, decanoyl-N- metylghjcamide, Triton* X-100, Triton* X-l 14, Thesii* lsotridecypoly(eihylcr_e glycol ether),,, 3-((3-cholamidopiopy ) ⁇ 3 ⁇ 4imeihy 1 -propane sulfonate (CHAPS), 3- ⁇ (3- cholamio ⁇ propyl)dimemytammm ⁇ sulfonate (CHAPSO), or dodccyl «N,N- ⁇ innethyl-3-arnnK>do-1 -propane sulfonate
  • binding of a test compound in the assay can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microliter plates, test tubes, and irjicrc-cenmruge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transfcrasc-base fusion proteins can be adsorbed onto glutathione Scpharoec$ beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microliter plates, which are then combined with the test cotnpound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microliter plate welts are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity determined using standard techniques.
  • detennining the ability of the test compound to modulate the activity of one or more biomarkcrs of the invention can be accomplished by determining the ability of the lest compound to modulate the expression or activity of a gene, e&, nucleic acid, or gene product, eg., polypeptide, that functions downstream of the interaction. For example, mflammation cytokine and chemokine) responses can be determined, the activity of the interactor polypeptide on an appropriate target can be determined, or the binding of the intcractor to an appropriate target can be determined as previously described.
  • modulators of one or more biomarkers of the invention including one or more biomarkers listed in Tables 1-S and Examples, or a fragment thereof, are identified in a method wherein a cell is contacted with a candidate compound and the expression or activity level of the biomarker is determined. The level of expression of biomarker mRNA or polypeptide or fragments thereof in the presence of the candidate compound is compared to the level of expression of biomarker mRNA or polypeptide or fragments thereof in the absence of the candidate compound. The candidate compound can then be identified as a modulator of biomarker expression based on this comparison.
  • biomarker mRNA or polypeptide or fragments thereof when expression of biomarker mRNA or polypeptide or fragments thereof is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of biomarker expression.
  • the candidate compound when expression of biomarker mRNA or polypeptide or fragments thereof is reduced (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of biomarker expression.
  • the expression level of biomarker mRNA or polypeptide or fragments thereof in the cells can be determined by methods described herein for detecting biomarker mRNA or polypeptide or fragments thereof,
  • activity of histonc methyl modifying proteins are evaluated.
  • the effect of a test compound can be evaluated, for example, by measuring methylation of a substrate in the presence of a stimulating agent at the beginning of a time course, and then comparing such levels after a prcdctcnruncd time (&£., 0.1 , 0.25, 0.5, 1 , 15, 2, 2.5, 3, or more hours) in a reaction that includes the test compound and in a parallel control reaction that does not include the test compound.
  • a prcdctcnruncd time &£., 0.1 , 0.25, 0.5, 1 , 15, 2, 2.5, 3, or more hours
  • an assay involves preparing a reaction mixture of a histone methyl modifying enzyme, a substrate, a stimulating agent, and one or more test compounds under coiklitions and for a time sufficient to allow components to interact Methylation can be evaluated directly or indirccdy. For example, H3 27 mono-, ⁇ h-, and/or tri-methylation or the relative proportions or relative changes from one species to another over time, can be assessed.
  • a component of an assay reaction mixture e.g., a substrate
  • a component anchored on the solid phase can be detected at the end of a reaction, e.g., a mcthylase reaction.
  • Any vessel suitable reactants can be used. Examples of suitable vessels include mkrodter plates, test tubes, and micro-centrifuge tubes.
  • Activity of methyl modifying enzymes can be evaluated by any available means.
  • a mediylation state of a substrate is evaluated by mass spectrometry analysis of a substrate.
  • methylation of a substrate is evaluated with an antibody specific for a methylated or demethylated substrate.
  • antibodies are conunercially available (e.g., from Upstate Group, NY, or Abeam Ltd., UK).
  • Suitable imimtnoassay techniques for detecting methylation state of a substrate include
  • Methylation reactions can be carried out in the presence of a labeled methyl donor (e.g., a S-edenosyH methyl-' *C ⁇ -L- methionine, or S ⁇ enosyHmemyl- j H] ⁇ methionine), allowing detection of label into a mcthylase substrate, or release of label from a demethylase substrate.
  • a labeled methyl donor e.g., a S-edenosyH methyl-' *C ⁇ -L- methionine, or S ⁇ enosyHmemyl- j H] ⁇ methionine
  • activity of a methyl modifying enzyme is evaluated using fluorescence energy transfer (FET or FRET for fluorescence resonance energy transfer) (see, for example, Lakowicz et a!., U.S. Pat. No. 5.631.169; Stavrianopoulos, et al. U.S. Pat No. 4,868,103).
  • FET fluorescence energy transfer
  • a fluorophorc label on a 'donor * e.g., a DNA molecule of a nucleosome
  • an 'acceptor' e.g., an antibody specific for a histone methyl modification of interest
  • a reaction can be carried out using an unlabeled substrate, and histone modification is determined by detecting antibody binding using a fluorimeter (see, U.S. Pat. Pub.2008/0070257).
  • demethylation is evaluated by direct or indirect detection of release of a reaction product such as formaldehyde and/or succinate.
  • a reaction product such as formaldehyde and/or succinate.
  • release of formaldehyde is detected.
  • Release of formaldehyde can be detected using a fbrmaidehyde dehydrogenase assay in which fbrmaidchyde dehydrogenase converts released fbrmaidchyde to formic acid using NAD+ as electron acceptor.
  • Reduction ofNAD+ can be detected specuiophotonietrically (Lizcano et aL, Anal. Biochem.286:75- 79, 2000).
  • release of formaldehyde is detected by converting formaldehyde to 3,5- ⁇ iiaccmyM,4- ⁇ i%dr >Ju ⁇ (DDL) and detecting the DDL, for example, by detecting radiolabeled DDL (e.g. , 3 H-DDL).
  • a substrate can be labeled so that a labeled reaction product is released (e.g., fbrmaidchyde and or succinate) by a done thy tation reaction.
  • a substrate is methylated win J H-SAM (S- adenosytmethioiiine), demethyiation of which releases ⁇ -formaldehyde, which can detected directly, or which can be converted to J H-DDL, which is detected.
  • Methods of detecting reaction products such as formaldehyde and/or succinate include mass spectrometry, gas chromatography, liquid chromatography, immunoassay, electrophoresis, and the like, and combinations thereof Dcmctfaylase assays are also described in Shi eta)., Cell 119:9 1-953, 2004.
  • An alternative means for detecting dcmethylase activity employs analysis of release of radioactive carbon dioxide (see, e.g., Pappalardi et al (2008) Btochem. 47:11165-11167 and Supporting Information, which describes use of a radioactive assay in which capture of 14 C(1 ⁇ 4 is captured and detected following release from ct
  • Such assays can involve use of an immobilized scintillant (e.g., immobilized on a bead or microp!atc) and a radioactive methyl donor.
  • a scintillation proximity assay employs scintillant-coated microplatcs such as FlashPlatesK> (Perkin Elmer).
  • components of an assay reaction mixture are conjugated to biotin and strcptavidin.
  • Biotinylated components eg., biotinytated substrate or biotinylatcd stimulating agent
  • biotin-NHS N-hydioxy-euccmmiide
  • Biotinylated components can be captured using strcptavidm-coated beads or immobilized in the wells of streptavidin-coated plates (Pierce Qtcmical).
  • assays can also employ any of a nutnoer of standard techniques for preparation and/or analysis of enzymatic activity, including but not limited to: differential
  • centrifugation see, for example, RJrvas, G., and Minton, A. P.. (1993) Trends Btochem Sci 18:284-7
  • chromatography gd filtration chromatography, ion-exchange chnrniatography
  • dectrophorcsts see, eg., Ausubcl, F. et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.
  • iimmmoprec ⁇ itation see, for example, Ausubcl, F. et al., eds. (1 99) Current Protocols in Molecular Biology, J. Wiley: New York).
  • a biomatker of the invention including one or more biomarkers listed in Tables 1-5 and Examples, or a fragment thereof, can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., VS. Pat No.
  • biomarker-binding proteins are also likely to be involved in die propagation of signals by the biomarkcr polypeptides or biomarkcr natural binding partners) as, for example, downstream elements of one or more biomarkers -mediated signaling pathway.
  • die assay utilizes two different DNA constructs.
  • the gene mat codes for one or more biomarkers polypeptide is fused to a gene encoding tbc DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence from a library of DNA sequences, mat encodes an unidentified polypeptide ("prey" or "sample") is fused to a gene mat codes for tbc activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transchptioa factor are brought into close proximity. This proximity allows transchptioa of a reporter gene ( g., LacZ) which is opcrably linked to a trartscriptiortal regulatory site responsive to the transcription factor.
  • a reporter gene g., LacZ
  • reporter gene Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain tbc cloned gene which encodes me polypeptide which interacts with one or more biomarkers polypeptide of the invention, including one or more biomarkers listed in Tables 1-5 and Examples or a fragment thereof.
  • the invention pertains to a combination of two or more of the assays described herein.
  • a modulating agent can be identified using a cell- based or a cell-free assay, and the ability of the agent to modulate the activity of one or more biomarkers polypeptide or a fragment thereof can be confirmed in vivo, e.g., in an animal such as an animal model for cellular tram formation and/or tumorigencsis.
  • This invention further pertains to novel agents identified by the above-described screening assays.
  • an agent identified as described herein in an appropriate animal model
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein. in. ⁇ 3 ⁇ 43 ⁇ 4 and Methods of the Inventio
  • biomarkers of the invention described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine diagnostic assays, prognostic assays, and monitoring of clinical trials); and c) methods of treatment (&g., therapeutic and prophylactic, e.g.. by up- or down-modulating the copy number, level of expression, and/or level of activity of the one or more biomarkcrs).
  • the isolated nucleic acid molecules of the invention can be used, for example, to (a) express one or more biomarkers of the invention, including one or more biomarkcrs listed in Tables 1-5 and Examples or a fragment thereof (e.g., via a recombinant expression vector in a host cell in gene therapy applications or synthetic nucleic acid molecule), (b) detect bio marker mRNA or a fragment thereof (#.£., in a biological sample) or a genetic alteration in one or more biomarkers gene, and or (c) modulate biomarker activity, as described further below.
  • biomarker polypeptides or fragments thereof can be used to treat conditions or disorders characterized by insufficient or excessive production of one or more biomarkcrs polypeptide or fragment thereof or production of biomarker polypeptide inhibitors.
  • biomarker polypeptides or fragrr nts thereof can be used to screen for naturally occurring biomarker binding partner(s), to screen for drugs or compounds which modulate biomarker activity, as well as to treat conditions or disorders characterized by insufficient or excessive production of biomarker polypeptide or a fragment thereof or production of biomarker polypeptide forms which have decreased, aberrant or unwanted activity compared to biomarker wild-type polypeptides or fragments thereof (&&, cancers, including lymphoid cancers, such as leukemia).
  • && cancers, including lymphoid cancers, such as leukemia.
  • the present invention relates to a method for preventing in a subject, a disease or condition associated with an unwanted, more than desintbk, or less than desirable, expression and/or activity of one or more btornarkers described herein.
  • Subjects at risk for a disease that would benefit from treatment with the claimed agents or methods can be identified, for example, by any one or combination of diagnostic or prognostic assays known in the art and described herein (see, for example, agents and assays described in III. tibotb of ScfoBog Agents and Cnmp wtions) B. Pre ⁇ QveMedjqag
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring of clinical trials arc used for prognostic (predictive) purposes to thereby treat an individual prophylactically.
  • one aspect of the present invention relates to diagnostic assays for detennining the expression anoVor activity level of biomarkers of the invention, including biomarkcrs listed in Tables 1-5 and Examples or fragments thereof, in the context of a biological sample (e.g., blood, scram, cells, or tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant or unwanted biomarkcr expression or activity.
  • the present invention also provides for prognostic (or predictive) assays for dctermming whether an individual is at risk of developing a disorder associated with biomarkcr polypeptide, nucleic acid expression or activity. For example, mutations in one or more biomarkcrs gene can be assayed in a biological sample.
  • Such assays can be used for prognostic or predictive purpose to thereby prophylacticaliy treat an individual prior to the onset of a disorder characterized by or associated with biomarkcr polypeptide, nucleic acid expression or activity.
  • Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds, and small nucleic acid-based molecules) on the expression or activity ofbiotnarkcrs of the mvention, including biomarkcrs listed in Tables 1-5 and Examples, or fragments thereof, in clinical trials.
  • agents e.g., drugs, compounds, and small nucleic acid-based molecules
  • biomarkcrs listed in Tables 1-5 and Examples, or fragments thereof in clinical trials.
  • the present invention provides, in part, methods, systems, and code for accurately classifying whether a biological sample is associated with a cancer or a clinical subtype thereof (eg., lymphoid cancers, such as leukemia), fn some embodiments, the present invention is useful for classifying a sample (e.g., from a subject) as a cancer sample using a statistical algorithm and or empirical data (*.#., the presence or level of one or biomarkers described herein).
  • a sample e.g., from a subject
  • empirical data *.#., the presence or level of one or biomarkers described herein.
  • An exemplary method for detecting die level of expression or activity of one or more biomarkers of die invention including one or more biomarkers listed in Tables 1-5 and Examples or fragments thereof, and thus useful for classifying whether a sample is associated with cancer or a clinical subtype thereof (e.g.
  • lymphoid cancers such as leukemia
  • a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting the biomarker (e.g., polypeptide or nucleic acid mat encodes the biomarker or fragments thereof) such that the level of expression or activity of the biomarker is detected in the biological sample
  • the presence or level of at least one, two, three, four, five, six, seven, eight, nine, ten, fifty, hundree, or more biomarkers of the invention are determined in the individual's sample.
  • the statistical algorithm is a single learning statistical classifier system. Exemplary statistical analyses are presented in the Examples and can be used in certain embodiments.
  • a single learning statistical classifier system can be used to classify a sample as a cancer sample, a cancer subtype sample, or a non-cancer sample based upon a prediction or probability value and the presence or level of one or more biomarkers described herein.
  • a single learning statistical classifier system typically classifies the sample as a cancer sample with a sensitivity, specificity, positive predictive value, negative predictive value, and/or overall accuracy of at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%.89%, 90%, 91%.92%, 93%, 94%.95%, 96%, 97%.98%, or 99%.
  • learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex data sets (e.g., panel of markers of interest) and making decisions based upon such data sets.
  • a single teaming statistical classifier system such as a classification tree (e.g., random forest) is used.
  • a combination of 2, , 4, 5, , 7, , , 10, or more learning statistical classifier systems are used, preferably in tandem.
  • Examples of learning statistical classifier systems include, but are not limited to, those using inductive learning (eg.,
  • dedskm/classirkation trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.).
  • PAC Probably Approximately Correct
  • coniKcdonist learning e.g., neural networks ( N), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptions such as multi-layer perceptrong, multi-layer feed-forward networks, applications of neural networks, Baycsian learning in belief networks, etc.
  • reinforcement learning eg., passive learning in a known environment such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action- value functions, applications of reinforcement learning, ctc.X and genetic algorithms and evolutionary programming.
  • the method of the present invention further comprises sending the cancer classification results to a clinician, e.g., an oncologist or hematotogist
  • the method of the present invention further provides a diagnosis in the form of a probability that the individual has a cancer or a clinical subtype thereof.
  • the individual can have about a 0% 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater probability of having cancer or a clinical subtype thereof.
  • the method of the present invention further provides a prognosis of cancer in the iixfcvidual.
  • the prognosis can be surgery, development of a clinical subtype of the cancer (e.g., subtype of leukemia), development of one or more symptoms, development of malignant cancer, or recovery from the disease.
  • the method of classifying a sample as a cancer sample is further baaed on the symptoms (e.g., clinical factors) of the individual from which the sample is obtained.
  • the symptoms or group of symptoms can be, for example, those associated with the 1PI.
  • the diagnosis of an individual as having cancer or a clinical subtype thereof is followed by adnunistering to the individual a therapeutically effective amount of a drug useful for treating one or more symptoms associated with cancer or me cancer.
  • an agent for detecting biomarker raRNA, genomic DNA, or fragments thereof is a labeled nuclek acid probe capable of hybridizing to biomarker raRNA, genomic DNA., or fragments thereof.
  • the nucleic acid probe can be, for example, full-length biomarker nucleic acid, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, l(X) t 250 or 500 nucleotides in lengm anl su cient to specifically hybridize under stringent conditions well known to a skilled artisan to biomarker mKNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays of die invention arc described herein.
  • a preferred agent for detecting one or more biomarkers listed in Tables 1-5 and Examples or a fragment thereof is an antibody capable of binding to the biomarker.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (eg., Fab or F(ab')2) can be used.
  • labeled with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling ⁇ i.e., physically linking) a detectable substance to tbc probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluoresccntry labeled secondary antibody and end-labeling of a DN A probe with bkXin such that it can be detected with ftuofcscently labeled streptavidin.
  • biological sample is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject That is, the detection method of the invention can be used to detect biomarker mRNA, polypeptide, genomic DNA, or fragments thereof, in a biological sample to vitro as well as in vivo.
  • in vitro techniques for detection of biomarker mRNA or a fragment thereof include Northern hybridizations and in situ hybridizations.
  • biomarker polypeptide examples include enzyme linked immunosorbent assays (ELISAs), Western blots, immunopredpitations and immunofluorescence.
  • In vitro techniques for detection of biomarker genomic DNA or a fragment thereof include Southern hybridizations.
  • in vivo techniques for detection of one or more biomarkers polypeptide or a fragment thereof include introducing into a subject a labeled anti- biomarker antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains polypeptide molecules from me test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a preferred biological sample is a hematological tissue (e.g. , a sample comprising blood, plasma, B cell, bone marrow, etc.) sample isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting polypeptide, mRNA, cD A, small RNAs, mature rrriRNA, prc- rruRNA, pri-miRNA, miRNA*, arrti-rniRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof of one or more bioraarkcrs listed in Tables I -5 and Examples such mat the presence ofbiomarkcr polypeptide, mRNA, genomic DNA, or fragments thereof, is detected in the biological sample, and comparing the presence of biomarker polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri- miRNA, miRNA*, anti-mi R A, or a miR A binding site, or a variant thereof, genomic DNA, or fragments thereof in tbc control sample with the presence of biomarl cr
  • the mvention also encompasses kits for detecting the presence of a polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti- nuRNA, or a miRNA binding she, or a variant thereof, genomic DNA, or fragments thereof, of one or more biomarkers listed in Tables 1-5 and Examples in a biological sample.
  • the kit can comprise a labeled compound or agent capable of detecting one or more biomarkers polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRN A, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof, in a biological sample means for detenroining the amount of the biomarker polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thcreof,f in the sample; and means for comparing the amount of the biomarker polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding site, or a variant thereof, genomic
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect the biomarker polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miR A, miRNA*, anti-miRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof.
  • therapies tailored to treat stratified patient populations based on the described diagnostic assays are further administered.
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant expression or activity of one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples, or a fragment thereof.
  • the term "aberrant" includes biomarker expression or activity levels which deviates from the normal expression or activity in a control.
  • the assays described herein can be utilized to identify a subject having or at risk of developing a disorder associated with a nrisrcgulation of biomarker activity or expression, such as in a cancer (e.g., lymphoid cancers, such as leukemia).
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a disorder associated with a misregulation of biomarker activity or expression.
  • the present invention provides a method for identifying and/or classifying a disease associated with aberrant expression or activity of one or more biomarkers of the invention, including one or more biomarkers listed in Tables I -5 and Examples, or a fragment thereof.
  • die prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimctic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant biomarker expression or activity.
  • agent e.g., an agonist, antagonist, peptidomimctic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate
  • agent e.g., an agonist, antagonist, peptidomimctic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate
  • agent e.g., an agonist, antagonist, peptidomimctic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate
  • such methods can be used to determine whether a subject can be effectively treated with an agent fix a cancer (e.g., lymphoid cancers,
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disease associated with aberrant biomarker expression or activity in which a test sample is obtained and biomarker polypeptide or nucleic acid expression or activity is detected (e.g. , wherein a significant increase or decrease in biomarker polypeptide or nucleic acid expression or activity relative to a control is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant biomarker expression or activity).
  • significant increase or decrease in biomarker expression or activity comprises at least 22.1, 22, 2J, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4.4.5.5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, JO, 10.5. 11. 12, 13, 14, 15, 16. 17. 18, 19, 20 times or more higher or kw ⁇
  • the methods of the invention can also be used to detect genetic alterations in one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples or a fragment thereof, thereby detenruning if a subject with the altered biomarker is at risk for cancer (eg., lymphoid cancers, such as leukemia) characterized by aberrant biomarker activity or expression levels.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one alteration affecting the integrity of a gene encoding one or more biomarkers polypeptide, or the mis-expression of the biomarker.
  • such genetic alterations can be detected by ascertaining die existence of at least one of 1) a deletion of one or more nucleotides from one or more biomarkers gene, 2) an addition of one or more nucleotides to one or more biomarkers gene, 3) a substitution of one or more nucleotides of one or more biomarkers gene, 4) a chromosomal rearrangement of one or more biomarkers gene, 5) an alteration in the level of a messenger RNA transcript of one or more biomarkers gene.6) aberrant rnodification of one or more biomarkers gene, such as of the metfaylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of one or more biomarkers gene, 8) a non-wild type level of one or more biomarkers polypeptide, 9) allelic loss of one or more biomarkers gene, and 10) inappropriate poet-translational modification of one or more biomarkers polypeptide.
  • detection of the alteration involves the use of a probe primer in a polymerase chain reaction (PGR) (see, e.g., U.S. Patents 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR. or, alternatively, in a ligation chain reaction (LCR) (see, eg., Landegran etal. (1988) Science 241:1077-1080; and Nakazawa etal. (1994) Proc. Natl. Acad. Sci. USA 91:360-364), the latter of which can be particularly useful for detecting point mutations in one or more biomarkers gene (sec Abravaya et al.
  • PGR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic DNA, mRNA, cDNA, small RNA, mature miR A, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding site, or a variant thereof) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to one or more biomarkers gene of the invention, including the biomarker genes listed in Tables 1 -5 and Examples, or fragments thereof, under conditions such that hybridization and amplification of the biomarker gene (if present) occurs, and detecting the presence or absence of an
  • PCR and/or LCR may be desirable to use as a preliminary amplification step m conjunction with any of the techniques used for detecting mutations described herein.
  • Alternative amplification methods include: self-sustained sequence replication (Ouatdli, J. C. et al. (1990) Proc. Nad. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. * al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q-Beta Replicase (Lizardi, P. M. el al (1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for me detection of nucleic acid molecules rf such molecules are present in very low numbers.
  • mutations in one or more biomarkers gene of the invention can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified
  • fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, for example, U.S. Patent 5,498,531 ) can be used to score for die presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in one or more biomarkers gene of the invention can be identified by hybridizing a sample and control nucleic acids, &g., DNA, RNA, mRNA, small RNA, cDN A, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miR A binding site, or a variant thereof to high density arrays containing hundreds or thousands of oligonucleotide probes (Cronin, M. T. et al. (1996) Hum Mutat 7:244-255; Kozal, . J. et al. (1996) Nat Med. 2:753-739).
  • a first hybridization array of probes can be used to scan through k>ng stretches of DN A in a sample and control to identify base changes between the sequences by making linear arrays of sequential, overlapping probes. This step allows the idcruifkation of point miitations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected.
  • Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence one or more biomarkers gene of the mvention, including a gene listed in Tables 1 -5 and Examples, or a fragment thereof, and detect mutations by comparing the sequence of the sample biomarkcr gene with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert (1977) Proc. Natl. Acad. Set. USA 74:360 or Sanger ( 977) Proc. Nad. Acad Sci. USA 74:3463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Nacve, C. W.
  • RNA/RNA or RNA/DNA hctcroduplcxcs examples include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA hctcroduplcxcs (Myer etal. (1985) Science 230:1242).
  • the art technique of "mismatch cleavage" starts by providing heteroduplcxcs formed by hybridizing (labeled) RNA or DNA containing die wild-type sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to eriz rnaticalry digest the mismatched regions.
  • either DNA DNA or RNA/DNA duplexes can be treated with hydroxy laminc or osmium tetroxidc and with piperidinc in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacryiarnide gels to determine the site of mutation.
  • control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called ' ⁇ mismatch repair" enzymes) in defined systems for delecting and mapping point mutations in biomarker genes of the invention, including genes listed in Tables 1-5 and Examples, or fragments thereof, obtained from samples of cells.
  • the mutY enzyme of E. coli cleaves A at G A mismatches and the thymidine DNA glycosylasc f om HcLa cells cleaves TatG/T mismatches (Hsu etai. (1 94) Carcinogenesis 15: 1657- 1662).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in biomarker genes of the mvention, including genes listed in Tables 1-5 and Examples, or fragments thereof.
  • SSCP single strand conformation polymorphism
  • Single-stranded DNA f agments of sample and control nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of die assay may be enhanced by using RNA (rather than DNA), in which die secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes betcroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen el al (1991) Trends Genet 7:5).
  • the movement of mutant or wild-type fragments in polyacr lamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. ( 1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to ensure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high- melting OC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to kientify differences in the mobility of control and sample DNA (Roscnbaum and Reissner (1987) Biophys. Chem. 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163; Saiki el al. (1989) Proc. Nad. Acad. Sci. USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides arc attached to the hybridizing membrane and hybridized with labeled target DNA.
  • the hybridization reactions can occur using btochips, raicroarrays, etc, or other array technology that arc well known in the art
  • PCR amplification may be used in conjunction with the instant invention.
  • Oligonucleotides used as primers for specific amplification may cany the mutation of interest in the center of the molecule (so mat amplification depends on differential hybridization) (Gibbs et al. ( 1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one prima where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtcch 11:238).
  • amplification may also be performed using Taq hgase for amplification (Barany (1991) Proc. Nad. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3* end of the 5 * sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving ooc or more biomarkent of me mvention, including ooc or more biomarkers listed in Tables 1-5 and Examples, or fragments thereof.
  • Monitoring the influence of agents (eg., drags) on the expression or activity of one or more biomarkers of the invention, including one or more biomarkers listed in Tables l-S and Examples, or a fragment thereof (e.g., the modulation of a cancer state) can be applied not only in basic drug screening, but also in clinical trials.
  • the effectiveness of an agent determined by a screening assay as described herein to increase expression and/or activity of one or more biomarkers of the invention, mcluding one or more biomarkers listed in Tables 1 -5 and Examples or a fragment thereof, can be mortitorcd in clinical trials of subjects exhibiting decreased expression andor activity of one or more biomarkers of the invention, inducting one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1 -5 and Examples, or a fragment thereof, relative to a control reference.
  • the effectiveness of an agent determined by a screening assay to decrease expression and/or activity of one or more biomarkers of the invention can be monitored in clinical trials of subjects exhibiting decreased expression and/or activity of the biomarker of the invention, including one or more biomarkers listed in Tables l-S and Examples or a fragment thereof relative to a control reference.
  • the expression and/or activity of the biomarker can be used as a "read out" or marker of the phenotype of a particular cell.
  • the present invention provides a method for momtoring the effectiveness of treatment of a subject with an agent (e.g.. an agonist, antagonist, peptidomimctic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) including the steps of (i) obtaining a pit-administration sample from a subject prior to administration of the agent (ii) detecting die level of expression andor activity of one or more biomarkers of the invention, including one or more biomarkers listed in Tables l-S and Examples or fragments thereof in the prcad ministration sample; (iii) obtaining one or more pc -adnunistra ion samples from the subject (iv) detecting the level of expression or activity of the biomarker in die post- administration samples: (v) coinparing the level of expression or activity of the biomarker or fragments thereof in the prc-administration sample with the that of the biomarker in the poet administration sample or samples;
  • an agent
  • increased admmisCration of die agent may be desirable to increase the expression or activity of one or more biornarkcrs to higher levels than detected (e.g., to increase the effectiveness of the agent)
  • decreased administration of the agent may be desirable to decrease expression or activity of the biomarker to lower levels than detected (&#., to decrease the effectiveness of the agent).
  • biomarker expression or activity may be used as an indicator of the effectiveness of an agent, even in die absence of an observable pbenotypic response.
  • the present invention provides for bom prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder characterized by insufficient or excessive production of biornarkcrs of the invention, including biornarkcrs listed in Tables 1-5 and Examples or fragments thereof, which have aberrant expression or activity compared to a control.
  • agents of the invention described herein can be used to detect and isolate the biornarkcrs or fragments thereof, regulate the bioavailability of the biornarkcrs or fragments thereof, and modulate biomarker expression levels or activity.
  • the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant expression or activity of one or more biornarkcrs of the invention, including one or more biomarkers Ksted in Tables 1-5 and Examples or a fragment thereof, by administering to the subject an agent which modulates biomarker expression or at least one activity of the biomarker.
  • Subjects at risk for a disease or disorder which is caused or contributed to by aberrant biomarker expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. A ⁇ rniinistration of a prophylactic agent can occur prior to the
  • Another aspect of the invention pertains to methods of modulating the expression or activity or interaction with natural binding partners) of one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1 -5 and Examples or fragments thereof for therapeuiic purposes.
  • the biomarkers of the invention have been demonstrated to correlate with cancer (e.g., lymphoid cancers, such as leukemia).
  • the activity and/or expression of the biomarker as well as the interaction between one or more biomarkers or a fragment thereof and its natural binding partner(s) or a fragments) thereof can be modulated in order to modulate the immune response.
  • Modulatory methods of the invention involve contacting a ceil with one or more biomarkers of the invention, including one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples or a fragment thereof or agent that modulates one or more of the activities of biomarker activity associated with the cell.
  • An agent that modulates biomarker activity can be an agent as described herein, such as a nucleic acid or a polypeptide, a naturally-c curring binding partner of the biomarker, an antibody against die biomarker, a combination of antibodies against the biomarker and antibodies against other immune related targets, one or more biomarkers agonist or antagonist, a peptidomiractk of one or more biomarkers agonist or antagonist, one or more biomarkers peptidomimetic, other small molecule, or small RNA directed against or a mimic of one or more biomarkers nucleic acid gene expression product
  • An agent that modulates the expression of one or more biomarkers of the invention including one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples or a fragment thereof is, eg., an antisense nucleic acid molecule, RNAi molecule, shRNA, mature raiRNA, pre-miRNA, pri-miRNA, mi RNA*, anti-miRNA, or a miRN binding site, or a variant thereof, or other small RNA molecule, triplex oligonucleotide, ribozyroe, or recombinant vector for expression of one or more biomarkers polypeptide.
  • an oligonucleotide complementary to the area around one or more biomarkers polypeptide translation initiation site can be synthesized.
  • One or more antisense oligonucleotides can be added to cell media, typically at 200 ug/nu, or administered to a patient to prevent the synthesis of one or more biomarkers polypeptide.
  • the antisense oligonucleotide is taken up by cells and hybridizes to one or more biomarkers mRNA to prevent translation.
  • an oligonucleotide which binds double- stranded DNA to form a triplex construct to prevent DNA unwinding and transcription can be used. As a result of either, synthesis of biomatker polypeptide is blocked.
  • biomarker expression is modulated, preferably, such modulation occurs by a means other than by knocking out the biomarker gene.
  • Agents which modulate expression by virtue of the fact that they control tbc amount of biomarker in a cell, also modulate the total amount of biomarker activity in a cell.
  • the agent stimulates one or more activities of one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples or a fragment thereof.
  • stimulatory agents include active biomarker polypeptide or a fragment thereof and a nucleic acid molecule encoding the biomarker or a fragment thereof that has been introduced into die cell (e.g. , cDNA, mRNA, shRNAs, siRNAs, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti- nuRNA, or a miRNA binding site, or a variant thereof, or other functionally equivalent molecule known to a skilled artisan).
  • the agent inhibits one or more biomarker activities.
  • the agent inhibits or enhances the interaction of die biomarker with its natural binding partners).
  • inhibitory agents include antisense nucleic acid molecules, anti-biomarker antibodies, biomarker inhibitors, and compounds identified in die screening assays described herein.
  • ⁇ present invention provides methods of treating an individual afflicted with a condition or disorder that would benefft fromup- or downHtnodulation of one or more biomarkers of the invention listed in Tables 1-5 and Examples or a fragment thereof, eg., a disorder characterized by unwanted, insufficient, or aberrant expression or activity of the biomarker or fragments thereof, in one embodiment, die method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (eg., upregulates or downrcgulatcs) biomarker expression or activity. In another embodiment, the method involves administering one or more biomarkers polypeptide or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted bio
  • Stimulation of biomarker activity is desirable in sitiiztiom in which the biomarker is abnormally downrcgulatcd and or in which increased biomarker activity is likely to have a beneficial effect likewise, inhibition of biomarker activity is desirable in situations in which toomarker is abnormally unregulated and/or in which decreased biomarker activity is likely to have a beneficial effect
  • modulatory agents can also be administered in combination therapy with, eg., cbcnxxherapcutic agents, hormones, antiangiogens, radiolabciled, compounds, or with surgery, cryotherapy, and/or radiodierapy.
  • cbcnxxherapcutic agents e.g., cbcnxxherapcutic agents, hormones, antiangiogens, radiolabciled, compounds, or with surgery, cryotherapy, and/or radiodierapy.
  • the preceding treatment methods can be administered in conjunction with other forms of conventional therapy (*.g., standard-o care treatments for cancer well known to the skilled artisan), either
  • these modulatory agents can be administered with a therapeutically effective dose of chcmothcrapeutic agent
  • these modulatory agents are administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemothcrapeutic agent.
  • the Physicians' Desk Reference discloses dosages of chernotherarjcutic agents that have been used in the treatment of various cancers.
  • c ernc ⁇ ierapcutic drugs that are therapeutically effective will depend on the particular cancer (eg., lymphoid cancers, such as leukemia), being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician.
  • cancer eg., lymphoid cancers, such as leukemia
  • the present invention provides methods of increasing the number of lymphoid progenitor cells from an initial population of lymphoid progenitor cells comprising contacting the lymphoid progenitor cells with an agent that inhibits polycomb repressor complex 2 (PRC2) activity to thereby increase the number of lymphoid progenitor cells.
  • PRC2 polycomb repressor complex 2
  • lymphoid progenitor celts and cellular sources comprising same can be used.
  • Descriptions of cells herein are well known to the skilled artisan and are further described with the understanding that these (inscriptions reflect the current state of knowledge in the art and the invention is not limited thereby to only those rAenotypk markers described herein.
  • lymphoid progenitor cell refers to a cell capable differentiating into any of the terminally cbffercnoated cells of the lymphoid lineage. Encompassed within the lymphoid progenitor cells arc the common lymphoid progenitor cells (CLP), a cell population characterized by limited or non-self-renewai capacity but which is capable of cell division to form T lymphocyte and B lymphocyte progenitor cells, K cells, and lymphoid dendritic cells.
  • CLP common lymphoid progenitor cells
  • the marker pbenotypes useful for identifying CLPs will be those commonly known in the art
  • CLP cells of mouse the cell population is characterized by the presence of markets as described m Kc ⁇ ⁇ a/. (1997) Ce 7 1: ⁇ 1 ⁇ 72, wmk for hurnan CLPs, a marker phenotype of CD34+ CD38+ CD10+ IL7R+ may be used (Gary et at. (1995) Immunity t 3:459-473; Akashi et al. (1 99) Int. J. Hematol. 69:217-226). Additional iUustrations of B cell lineage deveiopmem and associated molecular markers defining each cell stage in mouse models are provided in Figure 19 (Iritani etal. (1997) EMBOJ.
  • cc mittcd myeloid progenitor cells refer to cell populations capable of differentiating into any of the terminally differentiated cells of die myeloid lineage.
  • CMP common myeloid progenitor cells
  • GMP granulocyfc/rnacrophage progenitor cells
  • MEP megakaryocyte erythroid progenitor cells
  • the marker phenotypes useful for identifying CMPs include those commonly known in the an For CMP cdls of murine origin, the cell population is characterized by the marker phenotype c-Kit(high) (CD117) CD16(low) CD34(tow) Sca-l(ncg) Lin(neg) and further characterized by the marker phenotypes FcyR(k>) IL-7Rct(neg) (GDI 27).
  • the murine CMP cell population is also characterized by the absence of expression of markers that include B220, CD4, CDS, CD3, Ter 119, Gr- 1 and Mac- 1.
  • the cell population is chanuftrrized by CD34+CD38+ and further characterized by die marker phenotypes CD123+ (IL-3Ra) CD45R(ncg).
  • the human CMP cell population is also characterized by the absence of cell markers CD3, CD4, CD7, CDS, CD 10, GDI lb, CD14. CD 19, CD20. CD56, and CD234a.
  • Descriptions of marker phenotypes for various myeloid progenitor cells are described in, for example, U.S. Pat. Nos. 6,465,247 and 6,761,883; Akas (2000) Nomre 404:193-197.
  • GMP granulocyte/macrophage progenitor cell
  • the cells of mis progenitor cell population arc characterized by their capacity to give rise to granulocytes (e.g., basophils, eosinophils, and neutrophils) and macrophages.
  • GMPs lack seif-renewal capacity.
  • Marine GMPs are characterized by the marker phenotype c- itfhi) (CD! 17) Sea- i (neg) Fc (CD16) ]L-7R7(neg) CD34(pos).
  • Murine GMPs also lack expression of markers B220.
  • Human GMPs are characterized by me marker phenotype CD34+ CD38+ CD 123+ CD 5RA+. Human GMP cell populations are also characterized by the absence of markers CD3, CD4, CD7, CDS, CD 10, CDi lb, CD 14, CD 19, CD20, CD56, and CD235a.
  • inegakaryocyuVciythroid progenitor cells which are derived from the CMPs, are characterized by their capability of differentiating into committed
  • megakaryocyte progenitor and crythroid progenitor cells Mature megakaryocytes are polyploid cells that are precursors for formation of platelets, a developmental process regulated by thrornbopoietin. Erythroid cells arc formed from the committed crythroid progenitor cells through a process regulated by erythropoietin, and ultimately differentiate into mature red blood cells.
  • Murine MEPs arc diaracterizcd by cell marker phenotype c- Kit(hi) and IL-7R and further characterized by marker phenotypes Fc and CD34(low).
  • Murine MEP ceil populations arc also characterized by the absence of markers B220. CD4, CD8, CD3, Gr-1 , and CD 0. Another exemplary marker phenotype for mouse MEPs is c- kit(high) Sca-l(ncg) Lin (ncg/low) CD 16 (low) CD34dow).
  • Human MEPs are
  • ncg characterized by marker phenotypes CD3 +- CD38+ CDI 23(ncg) CD45RA(ncg).
  • Human MEP cell populations are also characterized by the absence of markers CD3, CD4, CD7, CD8, CD10,CDllb, CD14,CD19, CD20,CD56, aDdCD235a.
  • Further restricted progenitor cells in the myeloid lineage are the granulocyte progenitor, macrophage progenitor, rdegakaryocytc progenitor, and erythroid progenitor.
  • Granulocyte progenitor cells are characterized by their capability to differentiate into terminally differentiated granulocytes, inducting eosinophils, basophils, neutrophils.
  • the GPs typically do not differentiate into other cells of the myeloid lineage.
  • M P megakaryocyte progenitor cell
  • the cells to be expanded are comprised within tissues or other cellular sources, such as bone marrow, peripheral blood, cord blood, and the like.
  • Peripheral and cord blood is a rich source of HSCs and progenitor cells. Cells are obtained using methods known and commonly practiced in die art. For example, methods for preparing bone marrow cells are described in Sutherland et aL, Bone Marrow Processing and Purging: A Practical Guide (Gee, A. P. ed.), CRC Press Inc. (1 91)).
  • Umbilical cord blood or placental cord blood is typically obtained by puncture of the umbilical vein, in both term or preterm, before or after placental detachment (sec, eg. Turner, C. W. et a!., Bone Marrow Transplant 10:89 (1992); Bcrtolini, F. et al., J. Hematoma.4:29 (1995)).
  • the starting cells to be expanded are isolated cells. Such cells can farther be selected and purified, which can include both positive and negative selection methods, to obtain a substantially pure population of cells.
  • fluorescence activated cell sorting also referred to as flow cytometry, is used to sort and analyze the different cell populations.
  • Cells having the cellular nurkers specific for a lymphoid progenitor cell population are tagged with an antibody, or typically a mixture of antibodies, that bind the cellular markers.
  • Each antibody directed to a different marker is conjugated to a detectable molecule, particularly a fluorescent dye that can be distinguished from other fluorescent dyes coupled to other antibodies.
  • a stream of tagged or "stained" cells is passed through a light source that excites the fluorochromc and the emission spectrum from the cells detected to detenrune the presence of a particular labeled antibody.
  • fluorochrorocs also referred to in the art as multicolor fluorescence cell sorting
  • cells displaying different sets of ceil markers may be identified and isolated from other cells in the population.
  • Other FACS parameters including, by way of example and not limitation, side scatter (SSC), forward scatter (FSQ, and vital dye staining ⁇ e.g., with propidium iodide) allow selection of cells based on size and viability.
  • FACS sorting and analysis of HSC and progenitor cells is described in, among others, U.S.
  • Another method of isolating the initial cell populations uses a solid or insoluble substrate to which is bound antibodies or ligands that interact with specific cell surface markers.
  • cells are contacted with the substrate (e.g., column of beads, flasks, magnetic particles) containing the antibodies and any unbound cells removed.
  • substrates include, by way of example and not limitation, plastic, cellulose, dextran, polyacrylamide, agarose, and others known in the ait (e.g., Pharmacia Scpharose 6 MB macrobeads).
  • Affinity chromatographic cell separations typically involve passing a suspension of cells over a support bearing a selective ligand immobilized to its surface. The ligand interacts with its specific target molecule on the cell and is captured on the matrix. The bound cell is released by tbc addition of an elation agent to me running buffer of the column and die free cell is washed through the column and harvested as a homogeneous population.
  • adsorption techniques are not limited to those employing specific antibodies, and may use nonspecific adsorption. For example, adsorption to silica is a simple procedure for removing phagocytes from cell preparations.
  • FACS and most batch wise tmmunoadsorption techniques can be adapted to both positive and negative selection procedures (see, e.g., U.S. Pat. No. 5,877,299).
  • positive selection tbc desired cells are labeled with antibodies and removed away from the remaining un labeled/unwanted cells.
  • negative selection the unwanted cells arc labeled and removed.
  • Another type of negative selection that can be employed is use of antibody/complement treatment or immunotoxins to remove unwanted cells.
  • a typical combination may cotnprise an initial procedure that is effective in removing the bulk of unwanted cells and cellular material, for example leukapharcsis.
  • a second step may include isolation of cells expressing a marker common to one or more of the progenitor cell populations by immunoadscrption on antibodies bound to a substrate.
  • a marker common to one or more of the progenitor cell populations by immunoadscrption on antibodies bound to a substrate.
  • magnetic beads containing anti-B220+ antibodies are able to bind and capture lymphoid progenitors that commonly express the B220 antigen.
  • An additional step providing higher resolution of different cell types, such as FACS sorting with antibodies to a set of specific cellular markers, can be used to obtain substantially pure populations of the desired cells.
  • Another combination may involve an initial separation using magnetic beads bound with anti-B220 antibodies followed by an additional round of purification with FACS.
  • stem cells and lymphoid progenitor cells can be mobilized from the bone marrow into the peripheral blood by prior administration of cytokines or drugs to de subject (see, «#, Lapidot, T. et al., Exp. Hematol.30:973-981 (2002)).
  • Cytokines and cberookines capable of hxiucing mobilization include, by way of example and not limitation, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSFX erythropoietin (Kicssinger, A. et at., Exp. Hcmatol.
  • SCF stem cell factor
  • AMD3100 AnorMed, Vancouver, Canada
  • IL-8 inter leukh 8
  • variants of these factors e.g. , pegfilgastrim, darbopoictin.
  • Combinations of cytokines and or chemokmes can act synergistically to promote mobilization and may be used to increase die number of lymphoid progenitor cells in the peripheral blood, particularly for subjects who do not show efficient mobilization with a single cytokine or chemokine (Morris, C. et al., J. Haematol. 120:413-423 (2003)).
  • Cytoablative agents can also be used at inducing doses (I.e., cytoreductive doses) to mobilize lymphoid progenitor cells, and are useful cither alone or in combination with cytokines. This mode of mobilization is applicable when the subject is to undergo mycloablativc treatment and is carried out prior (o the higher dose cheniotherapy.
  • Cytarcductivc drugs for mobilization include, among others,
  • Determining the differentiation potential of cells, and thus the type of stem cells or progenitor cells isolated is typically conducted by exposing the cells to conditions that permit development into various tenninally differentiated cells. These conditions generally comprise a mixture of cytokines and growth factors in a culture medium permissive for development of the lymphoid lineage. Colony forming culture assays rely on culturing die cells in vitro via limiting dilution and assessing the types of cells that arise from their continued development A common assay of this type is based on methylceilulose medium supplemented with cytokines (*.g. , MethoCult, Stem Cell Technologies, Vancouver, Canada; Kennedy, M.
  • Cytokines include SCF, FLT-3 ligand, GM-CSF, 1L-3, TPO, and EPO.
  • Another in vitro assay is long-term culture initiating cell (LTC-1C) assay, which typically uses stromal cells to support honatopoicsis (see. e.g., Ploemacher. R. E. ct al.. Blood.74:2755-2763 (1989); and Sutherland, H. J. et al., Proc. Natl. Acad. Sci. USA 87:3745 (1995)).
  • LTC-1C long-term culture initiating cell
  • Another type of assay suitable for determining the differentiation potential of isolated cells relies upon in vtvo administration of cells into a host animal and assessment of the repopulation of the hematopoietic system.
  • the recipient is immunocoiruHomised or immunodcficicnl to limit rejection and permit acceptance of allogeneic or xenogeneic cell transplants.
  • a useful animal system of this kind is the NOD/SCID (Pfiumio, F. et at, Blood 88:3731 (1996); Szih/assym S. J. et al., "Hematopoietic Stem Cell Protocol," in Methods in Molecular Medicine, Humana Press (2002); Gremer, D. L. et al..
  • pbenotyping typically by FACS analysis. Detection of markers specific to the transplanted cells permits distinguishing between endogenous and transplanted cells. For example, antibodies specific to human forms of the cell markers (e.g., HLA antigens) identify human cells when they are transplanted into suitable immunodeficient mouse (see, eg., Piacibello. W. ctal., supra).
  • HLA antigens e.g., HLA antigens
  • the initial populations of cells obtained by the methods above are used directly for expansion or frozen for use at a later date.
  • a variety of mediums and protocols for freezing cells are known in the an.
  • the freezing medium will comprise DMSO from about 5-10%, 10-90% scrum albumin, and 50-90% culture medium.
  • Other additives useful for preserving cells include, by way of example and not limitation, disaccharides such as trehalose (Scheinkonig, C. et al., Bone Marrow Transplant.34(6):531 -6 (2004)), or a plasma volume expander, such as hetaslarch (/.*., faydroxyethyi starch).
  • isotonic buffer solutions such as phosphate-buffered saline
  • An exemplary cryopreservative composition has cell-culture medium with 4% HSA, 7.5% dimethyl sulfoxide (DMSO), and 2% hetastarch.
  • Other compositions and methods for cryopreservation are well known and described in the art (see, e.g., Broxmeyer et al. (2003 ) Proc. Nad. Acad. Set. USA 100:645-650). Cells are preserved at a final temperature of less than about -135*C.
  • Expansion of lymphoid progenitor cells is carried out in a basal medium, which can be supplemented with the mixture of cytokines and growth factors described herein, sufficient to support expansion of lymphoid progenitor ceils.
  • the basal medium will comprise amino acids, carbon sources (e.g., pyruvate, glucose, etc.), vitamins, serum proteins fa£., albumin), inorganic salts, divalent cations, antibiotics, buffers, and other preferably defined components that support expansion of myeloid progenitor cells.
  • Suitable basal mediums include, by way of example and not limitation, RPM1 medium, Iscovc's medium, minimum essential medium, Dulbeccos Modified Eagles Medium, and others known in the art (see, .g., U.S. Pat. No. 6,733,746).
  • Commercially available basal mediums include, by way of example and not limitation, Stemline.TM. (Sigma Aldrich), StcmSpan.TM. (StemCdl Technologies, Vancouver, Canada), Stempro.TM. (Life
  • HPGM.TM ((Otmbrcx, Walkersvillc, Md., USA), QBSF.TM. (Quality Biological, Gaitfacrsburg, Md., USA), X-VTVO (Cambrex Corp., Walkersville, Md.. USA) and Mesencult.TM. (StemCdl Technologies, Vancouver, Canada).
  • HPGM.TM ((Otmbrcx, Walkersvillc, Md., USA), QBSF.TM. (Quality Biological, Gaitfacrsburg, Md., USA), X-VTVO (Cambrex Corp., Walkersville, Md.. USA) and Mesencult.TM. (StemCdl Technologies, Vancouver, Canada).
  • HPGM.TM ((Otmbrcx, Walkersvillc, Md., USA)
  • QBSF.TM Quality Biological, Gaitfacrsburg, Md., USA
  • X-VTVO Cellular Corp., Walkersville, Md
  • the initial population of cells arc contacted with die mixture of cytokines and growth factors in the basal rncd rm, and cultured to expand the population of myeloid progenitor cells. Expansion is done for from about 2 days to about 14 days, preferably from about 4 days to 10 days, more preferably about 4 days to 8 days and or until the indicated fold expansion and the characteristic cell populations are obtained.
  • the final cell culture preparation is characterized by a lymphoid progenitor cell population that is expanded at least about 0.5 fold, about I fold, about 5 fold, about 10 fold, about 20 fold, or more.
  • the lyinphoid progenitor cell population can comprise at least about 60%, 65%.70%, 75%, 80%.85%, 90%, 95%. 99%, or more of the total cells in the culture.
  • feeder cell cultures can be used to aher the growth media environment (Feugier, P. et a!., J Hematother Stem Cell Res 11(1): 127-38 (2002)).
  • co-cultures of various cell populations can be created.
  • Cells expanded by the methods described herein can be used without further purification, or can be isolated into different cell populations by various techniques known in the art, such as by immunoaffinity chromatography,
  • a FACS gating strategy has an initial selection for live cells based on characteristic forward scatter (cell size) and side scatter (ceil density) parameters, and a second selection for expression of cell markers for lymphoid progenitor cells or non-lymphoid cells.
  • the PRC2 complex directs historic methyltransfcrase activity.
  • the compositions of the complexes isolated by different groups are slightly different, they generally contain EED, EZH2, SUZ12, and bAp48 or Drotophih horaoiogs thereof.
  • a reconstituted complex comprising only EED, EZH2, and SUZ12 retains historic nKthyltransferase activity ( «.£., mono- through tri-mcthylation) for lysine 27 of histonc H3 (eg., H3K27mc3; see U.S. Pat No.
  • the PRC2 complex may also interact with DN TI, DNMT3A, DNMT3B and PHFl via the EZH2 subunit and with SIRT1 via the SUZ12 subunit Of the various proteins making up PRC2 complexes, EZH2 (Enhancer of Zeete Homolog 2) is the catalytic subunit (Vire et al. (2006) Nature 439:871-874).
  • the catalytic site of EZH2 in turn is present within a SET domain, a highly conserved sequence motif (named after Su(var)3- , Enhancer of Zeste, Trithorax) that is found in several chronmtm-associated proteins, including members of bom the Trithorax group and Polycomb group.
  • the SET domain is characteristic of all known histonc lysine niethyitransfcrascs except the H3- 79 memyhransferasc DOT1.
  • any agent that disrupts the catalytic rnemy I transferase activity of PRC2 can be used according to the methods described herein.
  • agents include small molecules, antisense nucleic acids, interfering RNA, shRNA, siRNA, aptamcrs, ribozymes, and dominant- negative protein binding partners.
  • knockout or knockdown of EZH2 or other PRC2 complex components such as through reduction of mRNA or protein, will reduce H3K27me3 methylarion.
  • H3K27mc3 activity can be achieved by disrupting the protein-protein interactions necessary for the PRC2 to form and/or maintain catalytic activity.
  • dominant negative proteins such as EZH2 lacking a functional catalytic domain and/or having reduced hi stone methyltransferase activity, but maintaining the ability to bind to PRC2 complex binding partne s) wilt reduce PRC2 H3 27me3 activity.
  • chemical ( «.#., small molecule) inhibitors of PRC2 activity such as small molecule inhibitors of EZH2 are particularly useful because expansion of cell populations can be easily reversed by withdrawal of die compound.
  • Such chemical inhibitors are well known in die ait and are described, for example, in US Pat Pubis.
  • a chemical inhibitor of EZH2 is used, such as GS -126 (S>- 1 - ⁇ $cc-butyl N-((4,6-dimcihyl-2-oxo- 1 ,2-dihydfopyridiii-3-yl)i ⁇
  • Treatment can refer to tbcrarxutic or prophylactic treatment, or a suppressive measure for a disease, disorder or undesirable condition.
  • Treatment encompasses adnunistmtton of the subject cells in an appropriate form prior to the onset of disease symptoms and/or after clinical manifestations, or other manifestations of the disease or condition to reduce disease severity, halt disease progression, or eliminate the disease.
  • Prevention of the disease includes prolonging or delaying the onset of sy nptorns of the disorder or disease, preferably in a subject with increased susceptibility to the disorder.
  • the amount of the cells needed for achieving a therapeutic effect will be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for adrrurUstering the cells for therapeutic purposes, the cells are given at a pharmacologically effective dose.
  • pharmacologicalfy effective amount or “pharmacologically effective dose” is an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease condition, including reducing or eliminating one or more symptoms or rnanifestations of the disorder or disease.
  • Cell populations expanded in vivo will already be comprised within a subject's body for use therein.
  • Cells for infusion such as those prepared in vitro or ex vivo, include expanded ceil populations without additional purification, or isolated ceil populations having defined cell marker pbenotype and characteristic differentiation potential as described herein.
  • Expanded cells may be derived from a single subject where the ceils arc autologous or allogeneic to the recipient ft is to be understood that cells isolated directly from a donor subject without expansion in culture may be used for the same therapeutic purposes as die expanded cells.
  • the isolated cells are a substantially pure population of cells. These unexpended cells may be autologous, where the cells to be infused are obtained from the recipient, such as before treatment with cytoablative agents.
  • the unexpended ceils are allogeneic to the recipient, where die cells have a complete match, or partial or full mismatch with the MHC of the recipient.
  • the isolated unexpended cells are preferably obtained from different donors to provide a mixture of allogeneic lymphoid cells.
  • Transplantation of cells into an appropriate host can be accomplished by methods generally used in die art
  • the preferred method of admmistration is imravenous infusion.
  • the number of cells transfused will take into consideration factors such as sex. age, weight, the types of disease or disorder, stage of the disorder, the percentage of the desired cells in the cell population ⁇ e.g., purity of cell population), and the cell number needed to produce a therapeutic benefit.
  • the numbers of expanded cells infused may be from about 1x10* to about 1x10 s cells kg, from about Ixl ⁇ 5 to about Hbil ⁇ cells/kg, preferably about lxl( ceUs to about 5xl( ⁇ c ls/kg of body wdgh ⁇
  • the cells are in a pharmaceutically acceptable carrier at about IxlO 9 to about 1x10* cells.
  • Cells can be administered in one infusion, or through successive infusions over a defined time period sufficient to generate a therapeutic effect Different populations of cells may be infused when treatment involves successive infusions.
  • a pharmaceutically acceptable carrier may be used for infusion of die cells into the patient
  • These will typically cornprise, for example, buffered saline (eg., phosphate buffered saline) or unsuppkmented basal cell culture medium, or medium as known in the art
  • Conditions suitable for treatment include genetic and or acquired irnrminodcficicncy or autoimmune diseases where, for example, patients have decreased numbers of lymphocytes leading to susceptibility to infection and shortened lifespan.
  • exemplary, non-limiting genetic unmunodeficiencies include combined immunodeficiencies (SCIDX such as ADA-defkiency (adenosine deaminase), X-SC1D (X linked SOD), ZAP-70 deficiency, Rag 1/2 deficiency, Jak3 deficiency, IL7RA deficiency or CD3 deficiencies; primary imimmodeficiencics, such as the acqaired imtmmodefkiency syndrome (AIDS), DtGeorge's (vclocardiofacial) syndrome, adenosine deaminase (ADA) deficiency, reticular dysgenesis, Wiskott Aidrich syndrome, ataxia-telangiectasia, severe combined immunodeficiency; and secondary immunodeficiencies
  • Expanded lymphoid cell populations are also useful for various transplantation conditions, such as transplantation of stem cells, bone marrow, and/or umbilical cord blood. Lymphoid progenitors expanded in vitro, ex vivo, or in vivo can shorten the time to immune reconsutution, thereby decreasing the likelihood of infectious complications.
  • lymphoid ceil populations have numerous additional applications to biotechnologicai and biomedical research in addition to or outside the context of treating subjects.
  • lymphocytes that produce antibodies can be expanded in order to improved immune responses in vivo or to improve the yields of diagnostic or therapeutic antibodies produced in vitro or ex vivo.
  • B cells or other tymphoid cells such as those useful for research purposes that have been genetically modified, could be indefinitely cultured to perpetuate clonal cell populations.
  • the present invention provides pharmaceutically acceptable compositions which comprise a therapeuticalh/ flective amount of an agent that modulates ⁇ e.g., increases or decreases) PRC2 activity and/or H3K27mc3 levels, formulated together with one or more pharrnaceutically acceptable carriers (additives) and or diluents.
  • die pharmaceutical compositions of die present invention may be specially formulated for administration in solid or liquid form, including those adapted for die following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes (2) parenteral administration, for example, by subcutaneous, mtramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravagfnalry or intrarectal r , for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes
  • parenteral administration for example, by subcutaneous
  • therapeutically-cfTectivc amount means that amount of an agent that modulates (e.g., inhibits) PRC2 activity and/or H3K27me3 levels, or expression and/or activity of the complex, or composition comprising an agent that modulates (e.g., inhibits) PRC2 activity and/or H3K27me3 levels, or expression and/or activity of the complex, which is effective for producing some desired therapeutic effect, e.g. , cancer treatment, at a reasonable benefit/risk ratio.
  • phrases "pharmaceutically acceptable * ' is employed herein to refer to those agents, rnatehals, compositions, and or dosage forms which arc, within tbc scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, corntnensurate with a reasonable benefit/risk ratio.
  • phanroacculicalry-acceptablc carrier means a pharniaccurically-accc tabic material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting tbc subject chemical from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • materials which can serve as pharmaceutical ly-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cdhilose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (°) oils, such as peanut oil, cottonseed oil, safFlower oil, sesame oil, olive oil, corn oil and soybean oil; (1 ) glycols, such as propylene glycol; ( 11) potyols, such as glycerin, sorbitol, marmitol and
  • polyethylene glycol polyethylene glycol
  • esters such as ethyl oteatc and ethyl laurate
  • agar (14) buffering agents, such as magnesium hydroxide and aiurninum hydroxide;
  • IS alginic acid
  • pyrogen-free water 17.
  • isotonic saline 17.
  • Ringer's solution 19.
  • ethyl alcohol (20) phosphate buffer solutions; and (21 ) other non-toxic compatible substances employed in pharmaceutical formulations.
  • phannaccuficaUy-acccptablc salts refers to the relatively non-toxic, inorganic and organic acid addition salts of the agents that modulates (e.g., inhibits) PRC2 activity and/or H3K27me3 levels, or expression and/or activity of the complex
  • These salts can be prepared in situ during tbc final isolation and purification of the respiration uncoupling agents, or by separately reacting a purified respiration uncoupling agent in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed.
  • Representative salts include the hydrobroraidc, Hydrochloride, sulfate, bisulfatc, phosphate, nitrate, acetate, valerate, oleate, palnritatc, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, rumarate, succinate, tartrate, nap thy late, mesylate, glucoheptonate, lactobionate, and lauryunuphonate salts and the like (See. for example, Bcrge elal. (1977) "Pharrnaccutical Salts", J. Pham. Sci.66:1- 19).
  • the agents useful in the methods of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutical hy- acccptable salts with phannaceutically-acccptable bases.
  • pharmaceutically- acceptable salts refers to the relatively non-toxic, inorganic and organic base addition salts of agents mat modulates (e.g.. inhibits) PRC2 activity andor H3K27mc3 levels, or expression andor activity of the complex.
  • These salts can likewise be prepared in situ during the final isolation and purification of the respiration uncoupling agents, or by separately reacting the purified respiration uncoupling agent in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically- acceptable metal cation, with ammonia, or with a ptannaceutically-acceptable organic primary, secondary or tertiary amine.
  • a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically- acceptable metal cation, with ammonia, or with a ptannaceutically-acceptable organic primary, secondary or tertiary amine.
  • Representative alkali or alkaline earth salts include die lithium, sodium, potassium, calcium, magnesium, and amminum salts and the like.
  • Organic amines useful for the formation of base addition salts include emylarnine, diethylamine, emylcnediamine, efhanolamine, diethanc4amine, piperazine and the like (see, for example, Bcrge el al., supra).
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and aritioxidants can also be present in the compositions.
  • antioxidants examples include: ( 1 ) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfatc, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylalcd hydroxyanisote (BHA), butyiatcd hydroxytotucne (BUT), lecithin, propyl gailate, alpna-tocopherol. and the like; and (3) metal chelating agents, such as citric acid, cthylenediainine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfatc, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylalcd hydroxyanis
  • Formulations useful in the methods of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administratioa.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, die particular mode of administration.
  • the amount of active ingredient, which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 % to about 99% of active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%.
  • Methods of preparing these fbrrmilations or compositions include the step of bringing into association an agent that modulates (e.g., increases or decreases) PRC2 activity and or H3 27mc3 levels, with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by um ' formly and intimately bringing into association a respiration uncoupling agent with liquid carriers, or finery divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragaca th), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a respiration uncoupling agent as an active ingredient.
  • a compound may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more prtanriaccurically-acceptable carriers, such as sodium citrate or dkalchim phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, andor silicic acid; (2) binders, such as, for example, carboxymethylceltulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodhim carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds: (7) we
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excrpients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydraxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegram (for example, sodium starch grycolatc or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent
  • Tablets, and other solid dosage forms may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharaurauttcal-fccraulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, bydroxypiopylmcthyi cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes andor microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredients) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding
  • compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of die above-described excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the ait, such as, for example, water or other solvents, soiubilizing agents and crau ifiers, such as ethyl alcohol, isopropyl alcohoL ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butykne glycol, oils (in particular, cottonseed, groundnut, com, genu, olive, castor and sesame oils), glycerol, tctrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the ait such as, for example, water or other solvents, soiubilizing agents and crau ifiers, such as ethyl alcohol, isopropyl alcohoL ethyl carbon
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active agent may contain suspending agents as, for example, cthoxylated isostearyl alcohols, poiyoxyemylene sorbitol and sorbitan esters, macrocrystalline cellulose, aluminum metahydroxide. bentonite. agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, cthoxylated isostearyl alcohols, poiyoxyemylene sorbitol and sorbitan esters, macrocrystalline cellulose, aluminum metahydroxide. bentonite. agar-agar and tragacanth, and mixtures thereof.
  • Forrnulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more respiration uncoupling agents with one or more suitable ⁇ taring excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.
  • suitable ⁇ taring excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.
  • Formulations which are suitable for vaginal adnrinistration also include pessaries, tampons, creams, gels, pastes, foams or spray fbnnulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration of an agent that modulates (e.g., increases or decreases) PRC2 activity and/or H3 27me3 levels include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active component may be mixed under sterile conditions win a pharrnaccutically- acccptablc carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to a respiration uncoupling agent, excipients, such as animal and vegetable fats, oils, waxes, paraffins. starch, tragacartth, vahiiose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof
  • Powders and sprays can contain, in addition to an agent that modulates (eg., increases or decreases) PRC2 activity and/or H3K27mc3 levels, exctpients such as lactose, lak, silicic acid, aluminum hydroxide, calcium silicates and poh amide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellents, such as chlorofluorohydrcxarbons and volatile unsubstitutcd rjydrocarbom, such as butane and propane.
  • the agent that modulates (e.g., increases or decreases) PRC2 activity and/or H3K27me3 levels can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles comaining die compound. A nonaqueous (e.g., fluorocarbon propeliant) suspension could be used. Sonic nebulizers arc preferred because they minimize exposing the agent to shear, which can result in degradation of die compound.
  • an aqueous aerosol is made by fbnnulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers.
  • the carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Twecns, Piuronics. or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols.
  • Aerosols generally are prepared from isotonic solutions.
  • Transdermal patches have the added advantage of providing controlled delivery of a respiration uncoupling agent to the body.
  • dosage forms can be made by dissolving or dispersing the agent in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the peptidomimetic across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the
  • Ophthalmic formulations are also contemplated as being within die scope of this invention.
  • compositions of this invention suitable for parenteral administration ccinprise one or more respiration uncoupling agents in combination with one or more pharmaceutical iy-acccptablc sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bactcriostats, sohites which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the likeX and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl o!catc.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the likeX and suitable mixtures thereof
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl o!catc.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsif ing agents and dispersing agents.
  • microorganisms may be ensured by the inclusion of various antibacterial and anti fungal agents, for example, paraben, chlorobutanol, phenol sorbtc acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aruminum monosteatate and gelatin.
  • various antibacterial and anti fungal agents for example, paraben, chlorobutanol, phenol sorbtc acid, and the like.
  • isotonic agents such as sugars, sodium chloride, and the like into the compositions.
  • prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aruminum monosteatate and gelatin.
  • the absorption of the drug in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drag then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenterally-adrmnislered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are made by teeming mjcroencapsuk matrices of an agent that modulates ( «?.#., increases or decreases) PRC2 activity andor H3K27me3 levels, in biodegradable polymers such as polylacodc-polygfycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include po1y(orthocsters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or nucroemulsions. which are compatible with body tissue.
  • respiration uncoupling agents of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example.0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • Actual dosage levels of the active ingredients in the pharmaceutical competitions of this invention may be determined by the methods of the present invention so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc Natl. Acad. Sci. USA 91 :3054 3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • the cancer diagnostic, prognostic, prevention, and or treatment modulating agents of the invention are administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo, to either enhance or suppress immune cell mediated immune responses.
  • biologically compatible form suitable for administration In vivo is meant a form of the protein to be administered in which any toxic effects are outweighed by die therapeutic effects of the protein.
  • subject is intended to include living organisms in which an immune response can be elicited, eg., mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • Administration of an agent as described herein can be in any pharmacological form including a therapeutically active amount of an agent alone or in combination with a pharmaceutically acceptable carrier.
  • a therapeutically active amount of the therapeutic composition of de present invention is defined as an amount effective, at dosages and for periods of time necessary, to achieve the desired result.
  • a therapeutically active amount of a blocking antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of peptide to chert a desired response in the individual. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • agents of the invention described herein can be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal adrmnistration.
  • the active compound can be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound.
  • An agent can be administered to an individual in an appropriate carrier, diluent or adjuvant, co-administer cd with enzyme inhibitors or in an appropriate carrier such as liposomes.
  • Piutrmaceuticalry acceptable diluents include saline and aqueous buffer solutions.
  • Adjuvant is used in its broadest sense and includes any immune stimulating compound such as mterfcron.
  • Adjuvants contemplated herein include rcsorcinols, non- ionic surfactants such as polyoxyethyiene olcyl ether and n-bexadecyl polyethylene ether.
  • Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphatc (DEEP) and trasylol.
  • Liposomes include water-m-o ⁇ tn-water entuisions as well as conventional liposomes (Sterna ei al (1984) J. N uroimmunoL 7:27).
  • the agent may also be administered parenteralry or intraperitonealry.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • compositions of agents suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the composition will preferably be sterile and must be fluid to the extent mat easy
  • Tb carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyoi (for example, glycerol, propyiene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of rmcroorgamsms can be achieved by various antibacterial and antifungal agents, for example, parabens, cbJorobutanol, phenol, ascorbic acid, thimcrosal, and the like.
  • isotonic agents for example, sugars, polyalcohob such as manhol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, ahnninum monostcarate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating an agent of the invention (e.g., an antibody, peptide, fusion protein or small molecule) in die required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • agent of the invention e.g., an antibody, peptide, fusion protein or small molecule
  • dispersions arc prepared by incorporating the active compound into a sterile vehicle which contains a bask dispersion medium and the required other ingredients from those enumerated above.
  • die preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the agent phis any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the protein can be orally adiiunistcred, for example, with an inert diluent or an assimilable edible carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the requited pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by, and directly dependent on, (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of corr ⁇ ounding such an active compound for the treatment of sensitivity in individuals.
  • an agent of the invention is an antibody.
  • Asdefuicdhcrein a therapeutically effective amount of antibody ie., m effective dosage
  • m effective dosage ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mgkg body weight, more preferably about 0. ] to 20 mgkg body weight, and even more preferably about 1 to 10 mg kg, 2 to 9 mgkg, 3 to 8 mgkg, 4 to 7 mgkg, or 5 to 6 mgkg body weight.
  • certain factors may influence the dosage required to effectively treat a subject, including but not limited to die severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
  • treatment of a subject with a therapeutically effective amount of an antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with antibody in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of antibody used for treatment may increase or decrease over the course of a particular treatment Changes in dosage may result from the results of diagnostic assays.
  • an antibody of the invention can also be administered in combination therapy with, .g., chemotherapeutic agents, hormones, arttiangiogens, radio labelled, compounds, or with surgery, cryotherapy, and/or radiotherapy.
  • An antibody of the invention can also be administered in conjunction with other forms of conventional therapy, either consecutively with, pre- or post- conventional therapy.
  • the antibody can be administered with a therapeutically effective dose of chemotherapeutic agent
  • the antibody can be administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent
  • the Physicians' Desk Reference discloses dosages of temotheiapeutic agents that have been used in the treatment of various cancers.
  • chcrnotherapcutic drugs that are thetapcutically effective will depend on the particular immune disorder, e.g., Hodgkin lymphoma, being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician.
  • die agents of the invention described herein can be administered using nanc?artklc-based composition and delivery methods well known to the skilled artisan.
  • nattcpartfcle-based delivery for improved nucleic acid (eg., small RNAs) therapeutics are well known in the art (Expert Opinion on Biological Therapy 7:1811- 1822).
  • TslRhr B6.129S6-Dp(16Cbrl-ORF )l Rhr/J; stock #005838
  • Ts65Dn B6EiC3Sn.BLiA- Ts(17 ,6 >65Dn DnJ; stock #005252 mice were obtained from Jackson Laboratories.
  • HMG l_OE mice were described in Bustin etal. (1995) DNA Cell Biol. 14:997-1005.
  • Pax5 * mice (Urbanek ef al. (1994) Cell 79:901-912) backcrossed to C57BL/6 were obtained from M. BussUnger.
  • Eu-CRLF2 and Ep-JAK2 R683G were generated by subcloning cD As expressing human CRLF2 or mouse JAK2 R683G (MulKghan et al. (2009) Nat. Genet. 41:1243-1246; Yodae/o/. (XHQ) Pmc. Natl. Acad. Sci. U.S .
  • TslRhr were wild-type Uttermates from crosses with either C57BI/6 (Jackson; #000664) or FVB (Jackson; #001800) mice as indicated.
  • Controls for Ts65Dn were littermates from the colony (B6EiC3Sn.BLiAF 1 /J; Jackson #003647).
  • HMGNl.OE mice (Bustin etal (1995) DNA Cell Biol 14.997-1005) had been backcrossed >10 generations to C57BL6 (Abunatrira et al. (2011) J. Biol. Cheat
  • HMGNljOE human immunodeficiency virus
  • Donors for competitive transplantation were eongenic CD45.1 + B6.SJLr Ptpnf Pep /Boyi (Jackson; stock #002014) crossed with FVB (CD45.1), C57BL 6 x FVB Fl (CD45.1/2X or Tsl Rhr (C57BL 6) crossed with FVB Fl (CD45.1/2).
  • Recipients for competitive transplant, and BCR/ABL and lk6 bone marrow transplants were
  • mice C57BL 6 FVB Fl female mice. No randomization was performed for experiments involving mice or samples collected from animals.
  • CWP-seq antibodies were H3K27me3 (Cell Signaling Technologies, #9733X H3 4me3 (Abeam, #ab8580X and H3 27ac (Abeam, #ab4729). c. F ow cytometry for tone marrow B crfis
  • mice were transplanted. Mice were followed daily for clinical signs of leukemia and were sacrificed when moribund. Investigators were not blinded to the experimental groups. Ten mice were used per arm for 80% power to detect a 60% difference in survival at a specific time point with alpha of 0.05. No animals were excluded from analysis.
  • MMCT was performed as described in Yang and Shen (2011 ) Methods Mol Biol 325:59-66 with modifications.
  • A9 cells were cultured to approximately 70% confluence, and treated with 75 ng/ml colcemid for 48 hours. Cells were collected and rcsuspended in 1 : 1 DMEM:PercoU (GE Healthcare Biosciences) with 10 ugml Cytochalasin B (Sigma- Aldrich), and spun at 17,000 rpm for 75 minutes in a Beckman JA17 rotor. Supernatant was collected and filtered through 10 and 5 urn filters.
  • FACS using FACSCalibur for iiomology-directed repair.
  • the remaining cells were used to extract genomic DNA.
  • One ug DNA was digested with 20U I-Scel (Roche) overnight, purified, and amplified with a two-step PCR protocol.
  • Accuprime GO- rich polymerase was used for the first step PCR (20 cycles), and Taq polymerase (Qiagcn) was used for the second step PCR (20 cycles).
  • PCR products were cloned with the TOPO TA cloning kit for sequencing (Life Technologies).
  • pCAGGS-RAGl and pCAGGS-RAG2 vectors were co-transfeeted.
  • One genomic DNA was digested with 10U Mfel and I0U Ndel (NEB) overnight to exclude templates that had not been cleaved by RAG- 1 and RAG-2 before PCR amplification.
  • shRNAs targeting triplicated Ts 1 Rhr genes and controls were obtained from The RNAi Consortium (available on the World Wide Web at bfoadmstitute.oig rnai trc) as pL O lctti viral supernatant* (Ashton et al. (2012) Cell Stem Cell 1 1 :359-372) (n» 185 total shRNAs; see Table 5 for clone IDH and target sequences). Wild-type or TslRhr passage 1 B cell colonies were collected and plated at 5x10* cells per well of a 96 well plate in 100 ⁇ of RPM1 with 20% FBS, and 10 ng ml each of murine IL-7.
  • the shRNA encoded in the genomic DNA was amplified using two rounds of PCR.
  • Primary PCR reactions were performed using up to lO pg ofgcnomic PNA in 100 ⁇ reactions consisting of 10 ⁇ buffer, 8 ul dNTPs (2 J mM each), 10 ⁇ of 5 uM primary PCR primer mix (see below) and 1.5 ⁇ Talcara exTaq.
  • Primary PCR reactions were performed using up to lO pg ofgcnomic PNA in 100 ⁇ reactions consisting of 10 ⁇ buffer, 8 ul dNTPs (2 J mM each), 10 ⁇ of 5 uM primary PCR primer mix (see below) and 1.5 ⁇ Talcara exTaq.
  • RNA was harvested from B cell colonics (n 3 independent biologic replicates per genotype per passage). RNA sequencing was performed at The Center for Cancer Computational Biology at the Dana-Farbcr Cancer Institute (DFCI). Quality control of total RNA was performed using the RNA Qubtt Assay (Invhrogen) and me Bioanalyzer RNA Nano 6000 Chip Kit (Agilent). At least 100 ng of total R A and a Bioanalyzer RNA Integrity Number of >7.0 were required Library construction was performed using a TruSeq RNA Library Prep Kit (Ilrununa).
  • GTF gene transfer format
  • EdgeR uses empirical Bayes estimation and exact tests based on the negative binomial distribution model of die genome-scale count data.
  • EdgeR estimates the gene-wisc dispersions by conditional maximum likelihood, conditioning on the total count for that gene.
  • the gene-wise dispersion is "normalized” by shrinking towards a consensus value based on an empirical Bayes procedure (Robinson and Smyth (2007) Bioinformatlcs 23:2881-2887).
  • the differential expression is estimated separately for each gene based on an exact test analogous to Fisher's exact test adopted for over-dispersed data (Robinson and Smyth (2008) Biostattsiics 9:321-332).
  • RNA from HMGNl transgenic (HMGN 1 _OE) or wild-type littermate B cell colonies was processed and hybridized to Affymctrix Mouse Gene 2.0 ST array at the DFCI Microarray Core per the manufacturer's instructiom.
  • Raw probe-level data from the AIEOP-2 non-DS-ALL cohort and the mouse HMGNl JOE GEP were summarized using the Robust Multiarray Average (RMA) (Irizarry et al. (2003) Nucl Acids Res. 31 :e15) and Brainarray custom chip identification files based on Entrez IDs (Version 17) (Dai et al. (2005) Nud. Acids Res. 33:el 75) using die ExpressictfikCreator module in Gene Pattern (Reich et al (2005) Nat. Genet. 38:500-501 ).
  • RMA Robust Multiarray Average
  • GSEA the expression file was converted to human gene ortboiogs using Bio art ( inseila et al.
  • MU KELSEN_MEF_NPC_WITH_H3K27ME3 were obtained from MSigDB v3.1.
  • the 100 most differentially expressed genes between the DS-ALLs and die non-DS-ALLs were determined using the MarkerSelecuonModule in GencPattern.
  • RAG1- - proB cells were compared to E2A-/- preproB cells to generate probescts with >1.5-fold change and P ⁇ 0.05 between conditions, exacdy as had been done by tbc authors (Lin et al. (2010) Nat. Immunol. 1 :635-643).
  • the Tsl Rhr and core gene sets were compared to ail probescts for their relative expression in E2A wild-type (RAG! -/- proB) vs E2AV- cells.
  • the gene sets wim significant enrichment in genes up-regulated in TslRhr by GSEA were selected based on the maximum cut-off value 0.05 for P-valuc and FDR, and visualized with Enrichment Map software (Merico et al. (2010) PLoS One 5:cl3 84).
  • This software organizes the significant gene sets into a network, where nodes correspond to gene sets and the edges reflect significant overlap between the nodes according to a Fisher's test The size of the nodes is proportional to the number of genes in the gene set
  • the hubs correspond to collections of genes sets with significant pair-wise overlap which have a unifying functional description according to GO biological processes. Tbc node color is associated to the functional description of the hub.
  • Fcrcalculad ⁇ >n oflcukemia-mitiaririg cell frequency L-Calc software from Stem Cell Technologies (available on the World Wide Web at stet»ccll.con ⁇ en PToducts/All-P ⁇ was used and transplanted BCR/ABL+ cells were calculated by multiplying the number of cells transplanted by the GFP+ cells at the time of transplant (limiting dilution curves compared by ch -squared test) (Wang el at. Blood 89:3 19-3924).
  • cryoprcserved B-ALL splcnocytcs were enriched using ami -CD 19 antibody conjugated to magnetic rnicrobeads (#]30- ⁇ 52 ⁇ 201) and an MS MACS column (#130-042-201), both (torn Miltenyi Biotec u. Hiaoiig mag sasaimtn
  • GSK-J4 KDM6A/UTX and DM6B JMJD3 inhibitor, catalog #M60063-2) (KriridcnicT ⁇ o/. (2012) Nature 488:404-408) and GSK-126 (EZH2 inhibitor, catalog #M60071-2) (McCabe /e/. (2012) Nature 492:108-112) were purchased from Xccssbio.
  • DMSO methylcellulose
  • GSK-J4 KDM6A/UTX and DM6B JMJD3 inhibitor, catalog #M60063-2)
  • GSK-126 EZH2 inhibitor, catalog #M60071-2
  • DS-ALLs (dddentiftcd specimens obtained wirh inibrmed consent under DFCI IRB protocol 05-001) were treated in vitro in quadruplicate with GSK-J4 at two-fold dilutions from 40 nM to 10 uM in RPMI with 20% calf serum supplemented with 10 ngmL IL3, IL7, SCF, FLT3 ligand, and 50 uM beta- mercaptoethanol. After 3 days, viability was measured using CellTitcr-Glo reagent and normalized to DMSO control (Promega). w. fo rtt ( -J4 aiways
  • Leukemia cells were murine BCR ABL-positive B-ALLs as described above, or human Down syndrome or non-Down syndrome primary xenografed B-ALLe.
  • Viable cells were plated in white opaque 384-well plates (50 ⁇ well; Corning) using EL406 Qmibination Washer Dispenser (BioTek) at a density of 0.25 * 10* cells/ml.
  • GSK-J4 or vehicle (DMSO) were added using a JANUS Automated Workstation (PcrkinElmer) at d e indicated concentrations.
  • CcllTitcr-Gk I ⁇ rninesccnt Cdl Viability Assay reagent (Promega) was added (25 ul each well) and read by the 2104 EnVision Multi label Reader (PcrkinElmer) per the numufacturcrs' instructions. Each data point was quantified in quadruplicate. Dosc-responae curves and plots were generated with GraphPad Prism software.
  • B cell colonics ( 5,000 colonies per genotype) from 3 wild-type and 3 Tsl Rhr animals were pooled after 7 days in methylccllulose culture. ChIP was performed as described in Vera etal. (2010) Dev. Cell 19:713-726. Libraries for sequencing were prepared following die Illurmna TruSeq DNA Sample Preparation v2 kh protocol. After end-repair and A-taOing, imtnuiMpcecipitated DNA (10-50 ng) or whole cell extract DNA (50 ng) was ligated to a 1:50 dihitkm of Iliunrina Adaptor Oligo Mix assigning one of 24 unique indexes in the kit to each sample.
  • RNA-scq, ChEP-seq, and microarray expression data are deposited with GEO under GEO accession number GSE4 555.
  • This differentiation defect essentially phenocopies human fetal livers with trisomy 21, which have reduced pre- pro-B (CD34+CD 19+CD 10-) and pro-B ceils (CD34+CD 1 +CD 10+X as well as other hematopoictk defects (Roy el al. (2012) Proc. Nail. Acad. Sci. USA. 109:17579-17584).
  • CD45.1/45-2 bone marrow had equal representations of both populations in Hardy A, B and C fractions, as well as whole bone marrow ( Figures 1C and 2D).
  • mice that received wild-type CD45.1 mixed with Ts I Rhr CD45.1/452 recapitulated the Ts 1 Rhr defect, with significant reductions in CD45.1/45.2 Hardy B and C fractions ( Figures 1C and 2D).
  • the differentiation effect is independent of non-liematopoietic cells.
  • progenitor B cell colonies were generated from unselected TslRhr and wild-type bone marrow in three-dimensional cultures with 1L7 ( Figures 2E-2F). Wild-type bone marrow forms colonics (termed 'passage 1 ') under these conditions that can be replated to form new colonies for 1-2 additional passages.
  • Tsl Rhr bone marrow generated more colonics in early passages and serially replated indefinitely ( Figure 1 D), which indicates self-renewal capacity.
  • Tsl Rhr and wild-type colonies from early passages were universally Hardy C (CD24+BP-1+) by flow cytometry (Figure 3). After passage 2, wild-type cells formed few if any colonies while Tsl Rhr cells obtained from all mice (np9) expanded exponentially after passages 3 or 4 ( Figure 1 D) and continued to repassage fbr more than 10 platings. In contrast, there were no significant differences between Tsl Rhr and wild-type bone marrow in the number or repassaging potential of myeloid colonies ( Figure IE).
  • mice were transduced with a lower titer of either empty virus or Ik6 virus. Mice transplanted with
  • B- ALL was induced by transplanting nnsdected bone marrow transduced with p210 BCR- ABL (KraiBetf e/. (200 ⁇ Mtf. A/ «* 12:1175-1180). Although BCR-ABL ALL is uncommon in children with DS, pol somy 21 is the most common somatic aneuploidy among BCR-ABL ALU ( Wetzier el al (2004) Br. J. Haematol. 124:275-288). Limiting dilution analysis was performed by transplanting 10*, 10* or 10 transduced bone marrow cells from TslRhr mice or wild-type littermatcs into wild-type recipients (Figure 6A).
  • mice C57BL/6 and FVBxC57BlJ6 Fi backgrounds
  • mice that received transduced Tst Rhr bone marrow succumbed to B-ALL with shorter latency and increased penetrance
  • mice mat received transduced Tsl Rhr bone marrow had higher white blood cell counts and lower hemogiobin concentrations in peripheral blood compared with mice that received transduced wild-type bone marrow ( Figure 7).
  • mice transplanted with either wild-type or TslRhr bone marrow succumbed to progenitor (B220+CD43+) B-ALLs with similar histology mat infiltrated the bone marrow and spleen ( Figure 5D-5E).
  • B-ALLs in mice transplanted with TslRhr marrow developed with shorter latency and, in cohorts transplanted with 10 s or 10* cells, increased penetrance ( Figures 6A and SF).
  • the frequency of B-ALl ⁇ ininating cells was over 4-fbld higher in Tsl Rhr bone marrow ( Figure 6B; 1 :244 versus 1 :60 transduced cells, p ⁇ O.01).
  • B-ALLs (based on GFP+ B220+ phenotype) derived from wild-type bone marrow were homogenous populations of CD24+BP-1+ (equivalent to Hardy Q cells.
  • nearly one-half of B-ALLs derived from Tsl Rhr bone marrow were primarily CD24+BP-1- (Hardy B; Figure 6C, p O.003 compared to wild-type by Wilcoxon rank sum test), with some cases harboring CD24-BP- 1 - (Hardy A) cells.
  • B-ALL differentiation phenotype raised the possibility that DSCR triplication affects the B cell stage that is transformed by BCR-ABL.
  • Hardy A, B and C fractions were sorted from Tsl Rhr and wild-type bone marrow, individually transduced with BCR-ABL. and then transplanted 10 3 cells into wild-type recipients (Figure 8).
  • polysemy 21 could also contribute to B cell transformation by promoting aberrant DNA double-strand break repair (DSBR), which mediates kukemogenic alterations at CRLF2, IKZFJ, PAX5 and other loci (Multighan et al. (2009) Nat. Genet. 41 : 1243-1246; Russell et al (2009) Blood 114:2688-2698; Yoda et al (2010) Proc. Natl Acad. Sci. U! . 107:232-257).
  • DSBR DNA double-strand break repair
  • Zinc finger tnic lease-mediated recombination was used to target DSBR reporters (Weinstock and Jasin (2006) Mol. Cell Biol 26 131-139) ⁇ otbcp 4 locus of cells with different numbers of chr.21 , which avoids confounding locus-specific differences (Smith et al (2008) Stem Cells 26:496-504).
  • Polysemy 21 had no effect on cither homology ⁇ urected repair frequency or junction characteristics formed by nonhomoiogous end-joining, whether DSBs were induced by the I-Scel endoouc lease ( Figures 9D-9F) or by the V(D)J recombinase ( Figures 9G-9J). Although a subtle defect or one specific to progenitor B cells remains possible, these results indicate for the first time in an isogenic system that poh/somy 21 does not drastically affect DSBR pbenotype.
  • RNA-seq Whole transcriptomc sequencing (RNA-seq) of passage 1 B cells was also performed; triplicated loci in Tsl Rhr cells were expressed at approximately 1.5-fold higher levels compared to wild-type cells ( Figure 10) while absolute expression among die 25 genes differed markedly ( Figure 11).
  • GSEA gene set enrichment analysis
  • the TsIRhr B cell signature was enriched among DS-ALLs by GSEA ( Figures 12A-12B; FDR-0.019), indicating that transcriptional differences defined in Tsl Rhr B cells are biologically relevant to human DS-ALL.
  • GSEA GSEA
  • a "core TsIRhr set" of only 50 genes Table 1 was observed that distinguished DS-ALLs ( Figure 12 A).
  • the most highly enriched cluster consisted of polycomb repressor complex 2 (PRC2) targets and sites of tri-methylated histone H3 27 (H3K27me3), the repressive mark added by PRC2, that were defined across multiple lineages (Table 3).
  • PRC2 polycomb repressor complex 2
  • H3K27me3 tri-methylated histone H3 27
  • the additional clusters consisted of gene sets that distinguish either stem cells from lineage-matched differentiated cells, cancer cells from nonmalignant cells, or less dtffeentiated f om more differentiated lymphoid cells (Table 3).
  • BCR-ABL B-ALLs from TsIRhr bone marrow also had reduced H3K27me3 by bom mass spectrometry and immunoblotting ( Figures 13D-13E).
  • triplication of only 31 genes directly suppresses H3 27mc3.
  • Bivalent marks may indicate genes that are modulated during lincagc-*pecinc differentiation (Bernstein el al. (2006) Cell 125: 15-326).
  • the enrichment of bivalentry- marked genes within the TsIRhr gene set therefore suggests that the global loss of H3K27me3 from chr.2 Iq22 triplication selectively drives the overexpression of genes defined by a progenitor B cell-specific developmental program.
  • the TsIRhr and PRC2/H3K27m 3 gene sets were highly enriched for rxedicted binding sites of the master B cell transcription factors E2A TCF3 and LEF1 ( Figure 15E) (Kruidcnicr et al.
  • Hmgn encodes a nucleosome binding protein that modulates transcription and promotes chromatin decompaction
  • mice with transgenic overexpression of human HMGN1 (HMGN l_OE) at levels comparable to mouse HMGN1 were analyzed ( Figure 18F) (Bustin et al. (1 95) DM4 Cell Biol. 14:997-1005).
  • Figure 18F A gene expression signature of HMGNl_OE passage 1 B cells (compared to littcrmatc controls) was highly enriched for the Tsl Rhr and core Tsl Rhr gene sets ( Figure 17Q.
  • HMGNl_OE bone marrow had reduced Hardy C cells in vivo (Figure 18G), generated more B cell colonics in passages 1 -4 tn vitro ( Figure 17D), and resulted in greater penetrance and shorter latency of BCR-ABL- induced B-ALL ( Figure 17E).
  • overexpression of HMGN 1 alone recapitulates transcriptional and phenotypic alterations observed from triplication of all 31 Tsl Rhr genes.

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Abstract

The present invention relates to methods for identifying, assessing, preventing, and treating cancer (e.g., lymphoid and/or myeloid malignancies such as B-ALL in humans). A variety of histone H3K27rne3 biomarkets are provided, wherein alterations in the copy number of one or more of the bioniarkers and/or alterations in the amount, structure, and/or activity of one or more of the biomarkers is associated with cancer status and indicates amenability to treatment or prevention by modulating H3K27me3 levels. The present invention further relates to methods of increasing the number of lymphoid progenitor cells (e.g., increase self-renewal and cell proliferation) by contacting the lymphoid progenitor ceils (e.g., wild type and/or genomicaily altered cells) with an agent that inhibits polycomb repressor complex 2 (PRC2) activity or reduces H3K27roe3 levels.

Description

COMPOSITIONS AND METHODS FOR IDENTIFICATION,
ASSESSMENT, PREVENTION, A D TREATMENT OF CANCER
USING HISTONE H3K27ME3 BIOMARKERS AND MODULATORS
This application claims the benefit of U.S. Provisional Application No. 61/825,710, filed on 21 May 2013 and U.S. Provisional Application No. 61/981 ,317, filed on 18 April 2014; the entire cootcois of said applications arc incorporated herein io their entirety by this reference.
This invention was made with government support under Grant ΝΪΗ RO 1
CA15 i.98-01 and Grant NIH ROi CA.172387- A0.1 awarded by the National institutes of Health. The U.S. government lias certain rights in the invention. This statement is included solely to comply with 37 C.F.R. § 401 , 14(a)(i)(4) and should not be taken as an assertion or admission that the application discloses and/or claims only one invention.
Background of the Invention
Up to 3% of children with Down syndrome (.OS) will develop B cell acute lymphoblastic leukemia (B- ALL) (Rabin and Whitlock, Oncologist 14: 164-173) and polysomy 21 {i.e., extra copies of chromosome 21} is the most frequent somatic aneuploidy in B-ALL (Heerema et al. (2007) Genes Chrom. Cancer 46:684-693; Pui et al N. Engl J. Med. 350:1535-1548). Additional B-ALLs harbor an iiitrachromosoinal amplification of chr.21q22 (iAmp21) (Moorman et al Lancet Oncol 11 :429-438; Rand et al. Blood 1 1 :6848-6855) that overlaps with the putative "Down Syndrome Critical Region (DSCR)" on chromosome 2 iq22.
The mechanistic links between loci in these regions {e.g., polysomy, gone copy modulation, gene expression modulation, and the like) and precursor B ceil transformation remain undefined. A series of studies across four decades have attempted to define phenotypes within cells from patients wit DS that could underlie the association with B- ALL and other lymphoid and/or myeloid malignancies. However, comparisons between patients with DS and controls may he confounded by genetic or environmental differences distinct from trisomy 21 itself. Accordingly, there is a great need io identify the genetic, molecular, and biochemical underpinnings of such lymphoid and/or myeloid malignancies in such subjects, including the generation of diagnostic, prognostic, and therapeutic agents to effectively control such disorders in subjects.
Children with Down syndrome (DS) have a 20-fbld increased risk of developing B cell acute lymphoblastic leukemia (B-ALL) (Rabin and Whitlock (2009) Oncologist 14: 164-173), yet die mechanisms underlying mis association are undefined. The present invention is based in part on the discovery that polysemy (eg., triplication) of only 31 gene orthologous to the putative DS Critical Region (DSCR) on human chromosome 2lq22 is sufficient to confer and promote B cell autonomous self-renewal in vitro. B cell maturation defects in vivo, and B-ALL in concert with either BCR-ABL or CRLP2 with activated JAK. Chr.21q22 triplication suppresses H3 27me3 in murine progenitor B cells and B-ALLs, and "bivalent" genes with bom H3K27mc3 and H3K4mc3 at their promoters in wild-type progenitor B cells are preferentially overexpressed in triplicated cells. Human B-ALLs with polysorny 21 are distinguished by tbeir overexprcssion of genes known to be marked with H3K27me3 in multiple cell types. B cells with amplified DSCR (e.g., copy number gains, enhanced expression, and the like) relative to wild type harbor a transcriptional signature characterized by de-repression of porycomb repressor complex 2 (PRC2) components and/or targets mat is highly enriched among B-ALLs in children with DS. Inhibition of PRC2 function and/or modulation of H3K27me3 levels (e.g., by
pharmacological inhibition of H3K27 memyltransferases) is sufficient to promote self- renewal in wild-type B cells while enhancement of H3 27mc3 levels (*.&, by inhibiting demethylases mat remove H3K27me3) completely block self-renewal induced by DSCR triplication. It has further been discovered that self-renewal in B cells with DSCR triplication requires overexprcssion of the DSCR locus encoding HMON I , a nucleosome remodeling protein encoded on chr.21a22 (Catez et al. (2002) FMBORep. 3:760-766; Lim et al. (2005) UMBO J. 24:3038-3048; Rattner et al. (2009) Mol Cell 34:620-626), suppresses H3f 27me3 levels. Overexprcssion of HMGN1 suppresses H3 27mc3 and promotes both B cell proliferation in vitro and B-ALL in vivo. HMON 1 overexprcssion and loss of H3K27me3 are implicated in progenitor B cell transformation and provide strategics to therapeutically target leukemias with potysomy 21.
In one aspect, a method of dctainining whether a subject afflicted with a cancer or at risk for developing a cancer would benefit from modulating bJstone H3K27mc3 levels is provided, wherein the method comprises: a) obtaining a biological sample from die subject b) determining the copy number, levd of expression, or level of activity ofoneormore biomarkcrs listed in Tables 1-5 or a fragment thereof in a subject sample c) determining die copy number, level of expression, or level of activity of die one or more biomarkers in a control; and d) comparing the copy number, levd of expression, or level of activity of said one or more biomarkers detected in steps b) and c); wherein a significant modulation in die copy number, lcvd of expression, or level of activity of die one or more biomarkers in the subject sample relative to the control copy number, level of expression, or level of activity of the one or more biomarkers indicates that the subject afflicted with the cancer or at risk for developing the cancer would benefit from modulating histone H3K27me3 levels. In one embodiment, the one or more biomarkcrs are selected from the group consisting of the set of a) "top 150 UP" biomarkers shown in Table 1, b) "the 50 UP core" biomarkers shown in Table l,c) utop 150 DOWN" biomarkers shown in Table 1, d), "the 50 DOWN core" biomarkers shown in Table 1, c) the "triplicated gene" biomarkers shown m Table 1 , f) the *chr2lq22 overlap" biomarkcrs shown in Table 2, g) the "PRC2 cluster" biomarkers shown in Table 3, h) the "overlap" biomarkers shown in Table 4, i) the "SUZ 12 target,"
"Mikkebcn MEF," and/or "Mikkdsen NPC biomarkers shown in Table 5, j) DM6A. k) DM6 , 1) EZH2, m) HMGNl , and subsets and/or conAinations thereof.
In another aspect, a method for monitoring the progression of a cancer in a subject is provided, wherein die method comprises: a) detecting in a subject sample at a first point in time die copy number, level of expression, or level of activity of one or more biomarkcrs listed in Tables I -5 OT a fragrnent thereof, b) repeat^
and c) comparing the copy number, level of expression, or level of activity of said one or more biomarkers detected in steps a) and b) to monitor the progression of the cancer. In one method, the one or more biomarkcrs are selected from the group consisting of the set of a) "top 150 UP" bkmwrkers shown in Table I, b) "the 50 UP core" biomarkers shown in Table 1, c) "top 150 DOWN" biomarkers shown in Table I, d), "the 50 DOWN core" biomarkers shown in Table 1, c) the "triplicated gene" biomarkers shown in Table 1 , die "chr21 q22 overlap" biomarkers shown in Table 2, g) die "PRC2 cluster" biomarkers shown in Table 3, h) the "overlap" biomarkers shown in Table 4, i) the "SUZ12 target."
"Mikkebcn MEF," and or "Mikkdsen NPC" biomarkers shown in Table 5, j) DM6A. k) D 6B, 1) EZH2, m) HMGNl , and subsets and/or combinations thereof. In another embodiment, an at least twenty percent increase or an at least twenty percent decrease between the copy number, level of expression, or level of activity of the one or more biomarkers in tbe subject sample at a first point in time relative to tbe copy iitimber, level of expression, or level of acti ity of the one or more biomarkers m the subject sample at a subsequent point in time indicates progression of the cancer, or wbcrein less than a twenty percent increase or less than a twenty percent decrease between the copy number, level of expression, or level of activity of tbe one or more biomarkers in the subject sample at a first point in time relative to tbe copy number, level of expression, or level of activity of the one or more biomarkers in the subject sample at a subsequent point in time indicates a lack of significant progression of the cancer. In still another embodiment, the subject has undergone treatment to modulate histone H3K27rae3 levels between die first point in time and tbe subsequent point in time.
In still another aspect, a method for stratifying subjects afflicted with a cancer according to predicted clinical outcome of treatment with one or more modulators of histone H3K27me3 levels is provided, wherein the method comprises: a) aetenriming the copy number, level of expression, or level of activity of one or more biomarkers listed in Tables 1-5 or a fragment thereof in a subject sample; b) determining the copy number, level of expression, or level of activity of the one or more biomarkers in a control sample; and c) comparing the copy number, level of expression, or level of activity of said one or more biomarkers detected in steps a) and b); wherein a significant modulation in the copy number, level of expression, or level of activity of u\e one or more biomarkers in tbe subject sample relative to the normal copy number, level of expression, or level of activity of the one or more biomarkers in the control sample predicts the clinical outcome of the patient to treatment with one or more niodulatore of histone H3 27me3 levels. In one embodiment, the predicted clinical outcome is (a) cellular growth, (b) cellular proliferation. or (c) survival time resulting from treatment with one or more modulators of histone H3K27mc3 levels. In another embodiment, the one or more biomarkers are selected from die group consisting of die set of a) "top 150 UP" biornarkers shown in Table l, b) "ihe 50 UP core" biomarkers shown in Table I, c) "top 150 DOWN" biomarkers shown in Table 1. d). "the SO DOWN core" biomarkers shown in Table l, e) thc ''triplicated gene" biomarkers shown in Table 1, f) the *chr2lq22 overlap" biomarkers shown in Table 2, g) the **PRC2 cluster" biomarkers shown in Table 3, h) the "overlap** biomarkers shown in Table 4, i) the "SUZ12 target," "Mikkelsen MEF," and/or "Mikkelsen NPC biomarkers shown in Table 5 J) KDM6A, k) KDM6B, I) EZH2, m) HMGN1 , and subsets and/or combinations thereof In still another embodiment, an at least twenty percent increase or an at least twenty percent decrease between the copy number, level of expression, or level of activity of the one or more biomarkcrs in the subject sample compared to the control sample predicts that the subject has a poor clinical outcome; or wherein less than a twenty percent increase or less than a twenty percent decrease between the copy number, level of expression, or level of activity of the one or more biomarkcrs in the subject sample compared to the control sample predicts that the subject has a favorable clinical outcome. In yet another embodimcnt, the method further comprises treating the subject with a therapeutic agent that specifically modulates the copy number, level of expression, or level of activity of the one or more biomarkcrs. In another embodiment, the method further comprises treating the subject with one or more modulators of his tone H3K27mc3 levels.
In yet another aspect, a method of dctennining the efficacy of a test compound for inhibiting a cancer in a subject is provided, wherein die method comprises: a) determining the copy number, level of expression, or level of activity of one or more biomarkcrs listed in Tables l-5 cf a fragrnem thereof ma first sample obtained from the subject and exposed to the test compound; b) ddcrmining the copy number, level of expression, or level of activity of the one or more biomarkcrs in a second sample obtained from the subject, wherein the second sample is not exposed to me test compound, and c) comparing the copy number, level of expression, or level of activity of the one or more biomarkers in the first and second samples, wherein a significantly modulated copy number, level of expression, or level of activity of the biomarker, relative to die second sample, is an indication that the test compound is efficacious for inhibiting the cancer in the subject, b one embodiment, the one or more biomarkers are selected rom the group consisting of the set of a) "top 150 UP" biomarkers shown in Table 1, b) "the 50 UP core" biomarkers shown in Table I, c) "top 150 DOWN" biomarkcrs shown in Tabk 1 , d), "the 50 DOWN core" biomarkers shown in Table 1 , e) the "triplicated gene" biomarkers shown in Table 1 , f) the "chr21q22 overlap" biomarkers shown in Table 2, g) the MPRC2 cluster" biomarkers shown in Table 3, h) the "overlap" biomarkers shown in Table 4, i) the "SUZ12 target," "Mikkelscn MEF," and/or "Mikk scn NPC" biomarkcrs shown in Table 5. j) DM6A, k) KD 6B, 1) EZH2, m) HMGN I , and subsets and/οτ rombiiurtions thereof In another embodiment, the first and second samples are portions of a single sample obtained from the subject or portions of pooled samples obtained from the subject in another aspect, a method of determining the efficacy of a therapy for inhibiting a cancer in a subject is provided, wherein the method conphses: a) determining the copy number, level of expression, or level of acti ity of one or more biomarkcrs listed in Tables 1 -5 or a fragment thereof in a first sample obtained f om the subject prior to providing at least a portion of the therapy to the subject; b) determining the copy number, level of expression, or level of activity of the one or more biomarkcrs in a second sample obtained from the subject following provision of the portion of the therapy; and c) comparing the copy number, level of expression, or level of activity of the one or more biomarkcrs in the first and second samples, wherein a significantly modulated copy number, level of expression, or level of activity of the one or more biornarkers in the second sample, relative to the first sample, is an indication that the therapy is efficacious for inhibiting the cancer in the subject. In one embodiment, the one or more biomarkcrs are selected from the group consisting of the set of a) "top 150 UP" biomarkcrs shown in Tabic I , b) "the 50 UP core" braraarkers shown in Table 1. c) "top ISO DOWN" biomarkcrs shown in Table I, d), "the 50 DOWN core" biomarkcrs shown in Table 1, c) the "triplicated gene" biornarkers shown in Table 1 , i) the Mcnr2 lq22 overlap" biomarkcrs shown in Table 2, g) the "PRC2 cluster" biomarkcrs shown in Table 3, h) the "overlap" biomarkcrs shown in Table 4, i) the "SUZ12 target," "Mikkdsen MEF," and/or "Mikkelscn NPC biornarkers shown in Table 5, j) KDM6A, k) DM6B, I) EZH2, m) HMGN 1, and subsets and/or combinations thereof, or wherein said therapy farther cornprises standard of care therapy for treating the cancer.
In still another aspect, a method for identifying a compound which inhibits a cancer is provided, wherein the method comprises: a) contacting one or more biomarkcrs listed in Tables 1-5 or a fragment thereof with a test compound; and b) determining the effect of the test compound on the copy number, level of expression, or level of activity of the one or more biornarkers to thereby identify a compound which inhibits the cancer. In one ernbodin^t, thcone ormorebiomaiicm
of a) "top 150 UP" biornarkers shown in Table 1, b) "the 50 UP core" biomarkcrs shown in Table I , c) "top 150 DOWN" biomarkers shown in Table 1, d), "(he 50 DOWN core" biornarkers shown in Table 1, c) the "triplicated gene" biomarkcrs shown in Table 1 , f) the "chr21q22 overlap" biornarkers shown in Table 2, g) the "PRC2 cluster" biomarkers shown in Table 3, h) the "overlap" biomarkers shown in Table 4, i) the "SUZ 12 target,"
"Mikkclscn MEF," and or "Mikkcf sen NPC" biomarkers shown in Table 5, j) KDM6A, k) KDM6B, I) EZH2, m) HMGN1 , and subsets and/or combinations thereof In another embodiment, the one or more biomarkers is expressed on or in a cell (eg., cdts isolated from an animal modd of a cancer or cells from a subject afflicted with a cancer).
In yet another aspect, a method for inhibiting a cancer is provided, wherein the method comprises contacting a cell with an agent that modulates the copy number, level of expression, or level of activity of one or more biomarkers listed in Tables 1-5 or a fragment thereof to thereby inhibit the cancer. In one embodiment, the one or more biomarkers are selected from the group consisting of the set of a) "top 150 UP" biomarkers shown in Table
1, b) "the 50 UP core" biomarkers shown in Table l, c) "top 150 DOWN" biomarkers shown in Table 1, d), "the 50 DOWN core" biomarkers shown in Table 1 , e) the "triplicated gene" biomarkers shown in Table I, f) the "chr21q22 overlap" biomarkers shown in Table
2, g) the "PRC2 cluster" biomarkers shown in Table 3, h) the "overlap*' biomarkers shown in Table 4, i) the "SUZ12 target," "Mikkelsen MEF," and/or " ikkelsen NPC biomarkers shown in Table 5, j) KDM6A. k) KDM6B, 1) EZH2, m) HMGN1 , and subsets and or combinations thereof. In another embodiment, the copy number, level of expression, or level of activity of the one or more biomarkers is downmodulated or unmodulated. Instill another embodiment, the step of contacting occurs in vivo, ex vivo, or in vitro. In yet another embodiment, the method further comprises contacting the cell with an additional agent mat inhibits (he cancer.
In another aspect, a method for treating a subject afflicted with a cancer is provided, wherein the method comprises administering an agent that modulates the copy number, level of expression, or level of activity of one or more biomarkers listed in Tables 1-5 or a fragment thereof such that the cancer is treated In one embodiment, the one or more biomarkers are selected from the group consisting of the set of a) "top 150 UP" biomarkers shown in Table I, b) "the 50 UP core" biomarkers shown in Table 1, c) "top 150 DOWN" biomarkers shown in Table I , d), "the 50 DOWN core" biomarkers shown in Table 1, e) the ''triplicated gene" biomarkers shown in Table l, f) tbc "chr21q22 overlap" biomarkers shown in Table 2, g) the "PRO cluster" biomarkers shown in Table 3, h) the "overlap" biomarkers shown in Table 4. i) the "SUZ12 target," "Mikkelsen MEF," and or "Mikkelsen NPC" biomarkers shown in Table 5. j) DM6A, k) KDM6B, 1) EZH2, m) HMGN1, and subsets and/or combustions thereof. In another embodiment, the agent downmodulatcs or upmodulates the copy number, level of expression, or level of activity of the one or more biomarkers. In still another embodiment, the method further comprises administering one or more additional agents that treats the cancer, in yet another embodiment, the agent is one or more modulators of iristone H3K27mc3 levels.
In still another aspect, a pharmaceutical composition comprising a polynucleotide encoding one or more biomarkers listed in Tables 1 -5 or a fragment thereof useful for treating cancer in a pharmaceutically acceptable carrier, hi one embodiment, the polynucleotide encoding the one or more biomarkers listed in Tables 1-5 or a fragment thereof further comprises an expression vector. In another ernbodirnent, the pharmaceutical composition is used in a method for treating a cancer.
In yet another aspect, a kit is provided comprising an agent which selectively binds to one or more biomarkers listed in Tables 1-5 or a fragment thereof and instructions for use.
In another aspect, a kit is provided cotnprising an agent which selectively hybridizes to a polynucleotide encoding one or more biomarkers listed in Tables 1-5 or f agment thereof and instructions for use.
In still another aspect, a biochip is provided apprising a solid substrate, said substrate comprising a plurality of probes capable of detecting one or more biomarkers listed in Tables 1-5 or a fragment thereof wherein each probe is attached to the substrate at a spatially defined address. In one embodiment, die probes are complementary to a genomic or transcribed polynucleotide associated with the one or more biomarkers.
In yet another aspect, a method of increasing the number of lymphoid progenitor cells from an initial population of lymphoid progeni tor cells is provided, wherein die method comprises contactin the lymphoid progenitor cells with an agent that inhibits poJycomb repressor complex 2 (PRO) activity or reduces H3K27me3 levels to thereby increase the number of lymphoid progenitor cells. In one embodiment, the agent inhibits die activity of the EZH2 histone H3K27 rnethyltransfcrasc subunit of PRC2. In another embodiment, the agent is an inhibitor selected from the group consisting of a small molecule, antisense nucleic acid, interfering RNA, shRNA, siRNA, miRNA, aptamer, r bozyme, and dominant-negative protein binding partner. In still another embodiment, the lymphoid progenitor cells are comprised within bone marrow with marker selection or without marker selection. In yet another embodiment, the lymphoid progenitor cells ccinprise pre-pro B cells, pro B cells, large pre-B cells, small prc-B cells, immature B cells, or any combination thereof. In another embodiment, contacting the lymphoid progenitor cells with the agent is performed in vivo, ex vivo, or in vitro. it is to be understood that any embodiments of the present invention can be combined and/or adapted for use in any of the compositions, methods, kits, biochips, and the like described herein. For example, pharmaceutical compositions, kits, or biochips described above can use one or more biomarkers selected from the group consisting of the set of a) "top 150 UP" bkrattrkers shown in Table 1, b) "the 50 UP core" biomarkers shown in Table I, c) "top 150 DOWN" biomarkers shown in Table 1. d), "the 50 DOWN core" biomarkers shown in Tabic 1. c) the "triplicated gene" biomarkers shown in Tabic 1 , f) the chr2lq22 overlap" biomarkers shown in Tabic 2, g) the "PRC2 cluster" biomarkers shown in Table 3, h) the "overlap" biomarkers shown in Table 4, i) the "SUZ12 target,"
" ikkelscn MEF," and or "Mikkelsen NPC biomarkers shown in Table 5, j) K.DM6A, k) KDM6B, 1) EZH2, m) HMG 1 , and subsets and or cc^nbinatioro thereof.
Regarding methods of the present invention, in one embodiment, the control is determined from a non-cancerous sample from the subject or member of the same species to which the subject belongs. In another embodiment die sample comprises cells, cell lines, histological slides, paraffin embedded tissue, fresh frozen tissue, fresh tissue- biopsies, blood, plasma, serum, buccal scrape, saliva, cerebrospinal fluid, urine, stool, mucus, or bone marrow, obtained from the subject In still another embodiment the copy number is assessed by microarray, quantitative PCR (qPCR), high-throughput sequencing, conipaiatrve enomk liybrid^ In yet another embodiment the expression level of the one or more biomarkers is assessed by detecting the presence in the samples of a polynucleotide molecule encoding the biomarkcr or a portion of said polynucleotide molecule. In another embodiment the polynucleotide molecule is a mRNA, cDNA, or functional variants or fragments thereof. In still another embodiment die step of detecting further comprises amplifying the polynucleotide molecule. In yet another embodiment, the expression level of the one or more biomarkers is assessed by annealing a nucleic acid probe with the sample of the polynucleotide encoding the one or more biomarkers or a portion of said polynucleotide molecule under stringent hybridization conditions. In another embodiment the expression level of the biomarker is assessed by detecting the presence in the samples of a protein of the biomarker, a polypeptide, or protein fragment thereof comprising said protein. In still another embodiment the presence of said protein, polypeptide or protein fragment thereof is detected using a reagent which specifically binds with said protein, polypeptide or protein fragment thereof (eg. , a reagent selected from the group consisting of an antibody, an antibody derivative, and an antibody fragment). In yet another embodiment, the activity level of the biomarkcr is assessed by determining the magnitude of modulation of the activity or expression level of downstream targets of the one or more biomarkers. In another embodiment, the agent or test compound modulates historic H3 27me3 levels. In still another embodiment, the agent or test compound inhibits the expression and/or activity of Jumonji D3 family of histooe HeK27 demethylases. In yet another embodiment, the agent or test compound is a small molecule inhibitor of KMD6A (UTX) and/or KDM6B (J JD3). In another embodiment, the agent or test compound inhibits the expression and/or activity of HMGN1. In still another embodiment, the agent or test compound is an inhibitor selected from die group consisting of a small molecule, anbsense nucleic acid, interfering RNA, shRNA, siRNA, aptamcr, ribozyme, and dominant-negative protein binding partner. In yet another embodiment, the cancer is a leukemia (eg., B-cell acute lymphoblastic leukemia). In another embodiment, the subject has an increased copy number of a) human chromosome 2 i or the human DSCR region thereof, b) mouse chromosome 16 or the mouse iAmp, Ts65Dn, TslRhr, Dp(16)l Yu, or Runxl locus thereof, ore) orthologs of a) or b relative to a wild type control. In still another embodiment, the subject is a human.
Figures 1A-1G show mat segmental trisomy orthologous to human chr.21q22 promotes progenitor B cell transformation. Figure 1 A shows regions orthologous to human chromosome 21 mat are triplicated in TslRhr and Ts65Dn mice or amplified in ΪΑΜΡ21 B- ALL. Figure IB shows progenitor B cells (B220+CD43+) and Hardy subtractions as percentages of bone marrow (BM) cells (n=*vgroup in 2 independent experiments). Figure 1C shows subtractions from mixed populations in recipient BM 16 weeks after competitive transplantation (nm5/group). Figure I D shows B cell colonies across 6 passages (n=3 biological replicates genotype representative of 3 independent experiments, mean values shown, *P<0.05, **P<0.01), and bright field microscopy of 3 TslRhr and 3 WT passage 2 cultures. Figure 1 E shows myeloid colonies across 4 passages (n=3 mice per genotype; NS, not significant). Figure 1 F shows leukemia-free survival of recipient mice after tnmspiantation ofEu-CRLF2 (C2) Eu-JAK2 R683G (J2yPax5 (P5), with or without TslRhr (Tsl ) BM transduced with vector or dominant negative Ikaros (Ik6) (n=10 mice/group). Figure 1G shows leukemia-free survival of recipient mice after
transplantation of BM transduced with BCR-ABL (n=10 mice group).
FigHrcs 2A-2F show the results of abnormal differentiation in vivo and colony growth in vitro of B cells with triplication of chr.21 orthologs. Figure 2A shows B220 and CD 3 staining of bone marrow from Ts I Rhr and wild-type mice, highlighting the more immature B220+CD43+ and more mature B220+CD43- B cell populations (top panel) and CD24 and BPi staining of the B220+CD43+ subpopulation demonstrates the car y Hardy fractions: A (CD24- BP1-), B (CD24+BP1-), and C (CD24+BPI+). Figure 2B shows Hardy subtactions of the B220+CD43+ population as absolute percentages of bone marrow mononuclear ceils by flow cytometry from Ts65Dn (blue) or C57BL 6 Ts I Rhr (orange) animals compared to wild-type littermate (black) mice (n=» mice per genotype) (bottom panel). Figure 2C shows a schematic for the competitive bone marrow
transplantation assay. Figure 2D shows representative Hardy f action staining in bone marrow gated on CD45.2 negative (left) competitor ceils or CD45.2 positive (right) test cells. The top rows are wild-type test cells, and the bottom rows arc Tsl Rhr test cells. There are fewer Ts 1 Rhr Hardy B C cells and greater numbers of Ts 1 Rhr Hardy A cells in recipients of wild-typc:Tsl Rhr competitive transplants (bottom right). Figure 2E shows a schematic of the mcthylccihilose replating assay. Whole BM from Tsl Rhr or wild-type mice was plated in semi -solid medium containing cytokines favoring B cell or myeloid colony growth.50,000 cells were collected from pooled colonies every seven days and rcplated in fresh media. Figure 2F shows that the cell surface phototype of passage 1 B cell colonies from Ts IRhr and wild-type animals is similar. Representative flow cytometry plots of Hardy fraction cell surface phenotype of passage 1 Ts IRhr and wild-type B cell colonies are shown. All cells are also B220+CD43+.
Figure 3 shows that cell surface phenotype of passage I B cell colonies from wild- type and Tsl Rhr animals are similar. A representative flow cytometry plots of Hardy fraction cell surface phenotype of passage 1 wild-type and Tsl Rhr B cell colonies is shown. All cells are also B2204CD43+.
Figure 4 shows that passage 6 Tsl Rhr B cell colonies can form serially
transplantable B-ALL in vivo. Passage 6 TslRhr B cells were transplanted into
immunodeftcknt Nod.Scid.IL2R "'" (NSG) primary recipients (left). Primary recipient mice (n»3) died within 150 days with progenitor B cell proliferations similar in disease phenotype to those seen with BCR-ABL tr*ns<hiction and transplantation. When splcnocytes from a moribund mouse were transplanted info secondary sublethaily-irradiaied syngeneic (FVB x C57BL 6 Fl ) imniunocompetent animals (n=5), all mice succumbed to rapidly progressive fatal B-ALL within two weeks (right).
Figures 5A-5G show characterization of the B-ALL that arises in Tsl Rhr bone marrow. Figure SA snows a representative phenotype of C2/J2/P5 Tsl + D 6 B-ALL demonstrating expression of human CRLF2 in Ac leukemic B cells that also co-express dominant negative Ikaros (Ik6). Figure 5B shows leukemia-free survival for wild-type mice after transplantation with bone marrow of the genotypes listed transduced with dominant negative Ikaros (Ik6) (n=6-8 mice group, **P<0.01 for C2 J2 P5 + Dc6 versus any other genotype by log-rank test). Figure 5C shows transduced Ts I Rhr and wild-type bone marrow using flow cytometry for B220 and GFP (BCR-ABL) demonstrating approximately equal proportions of GFP+ cells at the time of transplantation. Figure SD shows that Tsl Rhr and wild-type BCR-ABL B-ALLs demonstrate similar splenomegaly at the time of death with leukemia. Red dotted line represents upper limit of normal spleen weight. Figure 5E shows bone marrow and spleen histology by hematoxylin and eosin staining demonstrating similar infiltration with B-ALL cells in Tsl Rhr and wild-type B-ALLs (scale bar■ 50 μηι). Figure 5F snows survival curves for recipients of TslRhr or wild-type bone marrow cells (on a C57BL 6 background) transduced with BCR-ABL (tr*9 mice per group, curves compared by log-rank test). Figure SG shows an increase in B-ALL from Ts Rhr bone marrow is progenitor B ceil autonomous. Hardy B cells were sorted from TslRhr or wild-type bone marrow, transduced with BCR-ABL, and equal numbers of cells were transplanted into wild-type recipients (η»5 mice per group, curves compared by log-rank test).
Figares 6A-6D show that triplication of die DSCR cooperates with BCR-ABL to promote B-ALL tn vtvo. Figure 6A shows Kaplan-Meier survival curves showing the probability of B-ALL-free survival among wild-type recipients of 10*. 10s, or 104 wild-type or Tsl Rhr bone marrow cells transduced with BCR-ABL (n=20 per genotype at 10*, n«IO per genotype at 10s and I0 ; curves compared by the log-rank test). Figure 6B shows limiting dilution analysis of recipient survival at 80 days after transplantation using a Poisson distribution calculation (Wang era/. (1997) Blood 89:3919-3924) to estimate B- AlX-initiating cell f equency in wild-type (1:244 cells) and TslRhr bone marrow ( 1 :60 cells). Figure 6C shows cell surface phenorype of leukernias arising in wild-type or TslRhr bone marrow cells. All teukemias were B220+CD43+, consistent with a precursor-B cell acute lymphoblastic leukemia (Morse et al (2002) Blood I 00: 246-258), and shown are the percentages of cells with surface immunophenotypes equivalent to normal Hardy A, B, and C fractions from individual leukemia* (p=0.U03 tor the difference in Hardy C B ratio between wild-type and TslRhr by a two-sided exact Wilcoxon rank sum test). Figure 6D shows the probability of B-ALl free survival in wild-type recipients of 10* wild-type or Tsl Rhr sorted Hardy fraction A, B, or C bone marrow cells transduced with a BCR-ABL- expressing retrovirus (n»15 per genotype, n»5 per Hardy fraction, compared by log-rank tests).
Figure 7 shows that recipients of Tsl Rhr bone marrow transduced with BCR-ABL have more significant hematologic abnormalities after 3 weeks compared to recipients of wild-type bone-marrow. Peripheral blood analysis 3 weeks after transplantation of 10* or 104 wild-type + BCR-ABL or TslRhr + BCR-ABL bone marrow cells is shown (n=5 mice per dose per genotype). Whits blood cell counts (WBC), hemoglobin (HB). and platelet (PLT) counts are shown. BCR-ABL positivity is expressed as the percentage of peripheral blood mononuclear cells (%) or the absolute number (Absolute * GFP+ percentage x total WBC) per μί. Groups were compared by Student t test
Figure 8 shows a schematic of Hardy fraction sorting followed by BCR-ABL transduction and transplantation experiment Hardy fraction A, B, and C cells from wild- type or TslRhr B220+CD43+ bone marrow cells were sorted, transduced with MSCV- BCR-ABL-ircs-GFP, and 105 cells were transplanted into lethalty irradiated wild-type recipients (see Figure 2A for the Hardy fraction flow sorting strategy).
Figures 9A-9J show that trisomy and tctrasomy 21 retinal pigment epithelium (RPE) cells generated by microcell-mediated chromosome transfer (MMCT) do not have differences m DNA repair after I-Scel or RAG-induced cleavage. Figure 9A shows single nucleotide polymorphism (SNP) array data for a tetrasomy 21 RPE clone (tetra 21-1), two trisomy 21 (tri21-2 and tri21-3) clones, and a diploid clone are shown across the entire genome (top) or chranosome 21 (bottom). Figure 9B shows representative fluorescence in situ hybridization for human chr.21 in trisomy 21 and tetrasomy 21 RPE cells (red » chr.21 probe, blue = DAP1). Figure 9C shows representative G-banding karyotype for a tetrasomy 21 RPE cell line. Figure 9D snows that the DR-GFP construct was targeted to the p84 locus in RPE cells containing 2 or more copies of cm\21. A single double-strand DNA break induced by 1-SceI can be repaired by multiple pathways. Figure 9E shows that repair after I-Scel cleavage in cells lacking classical nonhomologous end-joining (NHEJ) factors (e.g. KU7O/80, XRCC4 L1G4) is characterized by higher rates of homologous
recombination and more extensive deletions at NHEJ junctions (Pierce et al. (2001) Genes Dev. 15:3237-42). However, the frequencies of homologous recombination (shown as percent GFP-positivc) induced by I-Sccl do not significantly differ between disomic (Di) and trisomy 21 (Tri) RPE clones. Two clones rom each genotype were assayed on two occasions in triplicate. Figure 9F show mat the phenotype of nonhomologous end-joining induced by I-Sccl did not significantly differ between disomic and trisomy 21 RPE clones. The number of base pairs deleted at junction formed by NHEJ rm
genotype is shown. Figure 9G shows that the DR-GFP-CE construct targeted to the p84 iocus can be used to assess repair after RAO cleavage. Cleavage at the paired RAG recognition signal sequences (white and black triangles) results in removal of the intervening sequence (in yellow) and nonhomoiogous end joining (NHEJ) between the double-strand break ends. Figure 9H shows that PCR shows no difference in the frequencies of the RAG- induced deletion between diploid and tetrasomy 21 cells. Two biologic replicates are shown for each genotype. Figure 91 shows tfiat repair junctions after RAG cleavage in cells lacking classical NHEJ factors (e.g., KU70/80. XRCC4/LIG4) typically have longer deletions and more extensive use of short stretches of homology than in wild-type cells (Wetnstock etal. (2006) A /. Cell. Biol. 26:131-139). However, the number of base pairs deleted after cleavage by RAG and NHEJ did not signifkandy differ between disomic and tetrasomy 21 cells (n Z clones per genotype). Figure 9J shows junction sequences for disomic (n-27) and tetrasomy 21 (n-70) RPE clones. A single nucleotide insertion is shown in Tctra-I B-3-7 (yellow).
Figure 10 shows that RNA-seq expression of the triplicated genes in TslRhr compared to wild-type B cells. RNA sequencing of TslRhr and wild-type B cells (n-3 mice per genotype) yielded relative expression levels among die 25 expressed triplicated genes (absolute fragments per kilobase per million reads |FP ] > 0. I and the flanking centromeric and tdomeric regions.
Figure II shows the absolute expression of DSCR genes in wild-type and TslRhr B cells by RNAscq. Fragments per kilobase per million reads (FPKM) values for wild-type and Tsl Rhr passage 1 B cells are plotted (n=3 independent biologic replicates per genotype).
Figures 12A-12G show that polysemy 21 B-ALL is associated with the overcxprcssion of PRC2 targets. Figure 12A shows a beat map of human genes orthologous to the 150 roost upregulated genes from TslRhr B cells in primary human pediatric B-ALLs. Unsupervised hierarchical clustering by gene revealed the "core TslRhr" gene set (boxed). Figure 12B shows GSEA plots for the full and core TslRhr gene sets in die AIEOP data set ES, enrichment score. Figure 12C shows a GSEA plot of the core TslRhr gene set in an independent ICH validation cohort. Figure 12D shows a network enrichment map of MSigDB gene sets enriched (FDRO.05) in die TslRhr expression signature. Figure 12E shows unsupervised hierarchical clustering of
H3 27me3-marked genes from the MIKKELSEN_MEF_H3K27me3 gene set in the AIEOP pediatric B-ALL cohort (karyotype shown). Figure 12F shows GSEA plots of the top 100 genes from three PRC2/H3 27me3 gene sets as defmed in the AIEOP patient cohort in the ICH validation cohort. Figure I2G shows quantitative histone MS for H3 27-K36 peptides (*P<0.05, n=3 samples per group per genotype).
Figures 13A-I3E shows mat DS-ALL is associated with ovcrexpression of PRC2 targets and genes marked by H3K27mc3, TslRhr and PRC2 H3K27mc3 gene signatures distinguish non-DS-ALL with somatic gain of chromosome 21 or ΪΑΜΡ21 , and Ts I Rhr B- ALLs arc associated with H3K27 hypomethylation. Figure 13A shows heat maps of all genes comprising three of the top five scoring target gene sets enriched in die core Ts 1 Rhr signature in DS-ALL* and non-DS-ALLs. Figure 13B shows unsupervised clustering results of a validation cohort of 30 non-DS pediatric B-ALL gene expression signatures (the AIEOP-2 cohort) using a lOO-gcne SUZ12 target gene set Four patients with somatic gain of chr.21 and two with iAMP21 cluster within a distinct group with 5 additional cases (P-0.001 by Fisher's exact test). Figure 1 C shows GSEA plots of the TslRhr gene set and the top 100 discriminating genes in the Mikkeben NPC and MEF H3 27mc3 gene sets from the AIEOP cohort, queried in the primary human B-ALLs in die AIEOP-2 cohort containing cases with somatic +21 and iAMP2l. ES indicates enrichment score. Figure 13D shows unstipervised hierarchical clustering results of histone H3 post-tianslational modifkations in spienocytes from mice with TslRhr and wild-type BCR-ABL B-ALLs quantitated by mass spectrometry (blue-red * low-to-high relative amount of each listed peptide, n=3 independent leukemias for each genotype). Peptides containing H3 27mc3 with lower abundance in TslRhr B-ALLs are indicated by arrows. Figure 13E shows
Western blotting results in sorted CDI9+ Tsl Rhr and wild-type B-ALLs (n=5 independent leukemias for each genotype, distinct from those in panel D). Figures 14A-14H show that TslRhr B cells have reduced H3 27mc3 that results in overexpression of bivalendy marked genes. Figure 14A shows gene tracks showing occupancy of historic marks at the Plod2 promoter (one of the SO core Tsl Rhr genes) in reads per million per base pair (rpen bp). Figure 14B shows levels of H3K27me3 in TslRhr and wild-type B cells at regions enriched for H3K27me3 in wild-type celts (***P<le-l 6). Figure I4C shows histone marks at the promoters of genes mat are unregulated or ckywiircgulatcd in TslRhr vs. wild-type cells (**?< le-S). Figure 14D shows chromatin marks in wild-type B cells present at promoters of all genes (left) or genes that arc upregulated in Tsl Rhr B cells (right, ***PO.0001 compared to all genes by Chi-square with Yates' correction). Figure I4E shows colony counts in the presence of DMSO or GSK-J4 (n»3 biological replicates per genotype, *P<0.05 compared to DMSO for same genotype). Figure 14F shows colony counts in the presence of GSK-126 or after wrthdrawal at passage 5 (*P 0.05 compared to OSK-126 for same genotype, WPO.05 compared to odier genotype or no withdrawal). Arrow indicates GSK-126 withdrawal. Figure 14G shows Western blotting results of passage 2 colonies after 14 total days in culture with DMSO. 1 μΜ GSK-J4, or 1 uM GSK-126. Figure 14H shows Western blotting results of colonics one passage (7 days) after continuation (+) or removal (-) of GSK-126.
Figures 15A-15H show that ChlP-seq and CHIP-qPCR exhibit decreased
H3K27mc3 at promoters in Tsl Rhr B cells, the TslRhr gene set is enriched for E2A TCF3 and LEF1 targets, and DS-ALLs are sensitive to OSK-J4. Figure I SA shows ChfP for H3K27me3 (IcftX H3K27me3 (right), or control rabbit IgG followed by quantitative PCR on a representative set of genes from the TslRhr signature in an independent validation set of wild-type and Ts 1 Rhr mice (n=3 mice per genotype, one representative of two independent experiments). Data represented as fold enrichment over input re
negative control inicrgenic region on chr.5 (Chr 5 IN) (**PO 01 , *P<0.0S). Figure 15B shows H3K27mc3 enriched regions in wild-type B cells. The promoter region is defined as the Skb flanking annotated transcription start sites. Overlap of H3K27me3 regions with die promoter region was significant in comparison to a random background model of the genome (P<10*10). Figure ISC shows a Venn diagram showing die number and overlap between H3K27me3 enriched regions in wild-type (WT) or TslRhr B cells. Figure 1SD shows the logj fold difference in density of H3K27mc3 at promoters between TslRhr and wild-type B cells is shown. Figure 15E shows the top three ranked transcription factors with predicted binding sites among promoters of genes in the listed sets as queried in MSigDB "c3.ttT defined in the TRANS FAC database (version 7.4, available on the World Wide Web at gene-fegulation.com). Figure 15F shows the relative fraction of genes that have proximal E2A TCF3 occupancy among all genes (7129 of 20671 ), genes with only H3K27me3 (557 of 1994) or H3K4me3 (4032 of 360) at the promoter in wild-type B cells, or genes in the TslRhr gene set (85 of 150) (**P<0.01, ***P<0.0001 versus the TslRhr gene set by Chi-square with Yates* ccHrrcction). Figure 15G shows that expression of genes in the TslRhr and Core TslRhr sets are increased compared to all probesets in wild-type B cell progenitors as compared to E2A"~ (expression data from2*; ***P<0.0001 by Student t- test, center bars - median, box - 25-75% confidence interval, whiskers - 10-90% confidence interval). Figure 15H shows the 1C» for five DS-ALLs treated in vitro with GSK-J4 (error bars represent 95% confidence intervals).
Figures 16Λ-16Β show the sensitivity of murine and human B-ccll ALL to OSK-J4. Figure I6A shows that a subset of murine B-cell acute rvmphoblastic leukemias that harbor triplication of the Down Syndrome Critical Region (lower panel) arc 100-foW more sensitive to GSK-J4 compared to teukemias that lack triplication (upper panel)- Figure 16B shows that a human primary B-cell ALL xenograft from a patient with Down Syndrome is 1(M 00-fold more sensitive to GSK-J4 compared to a similar xenograft that lacks an extra copy of chromosome 21.
Figures 17A-17E show that HMG 1 overcx ressRm decreases H3K27me3 and promotes transformed B cell phenotypes. Figure 17 A shows Western blotting results of Ba F3 cells transchtccd with empty virus or murine HMGN1 (n»3 irtdependent biological replicates). Figure 17B shows relative shRNA representation over passages 1 -3. Each line represents an individual shRNA (n=155 total). The five shR As targeting H gnl are indicated. Figure 17C shows GSEA plots for the full and core TslRhr gene sets in
H GN1_0E transgenic B cells. Figure 17D show B cell colonies during repassaging of WT and H GN l_OE BM (n=6 biological replicates per genotype in two independent experiments, *P<0.05). Figure 17E shows lcukemia-frce survival of recipient mice after transplantation of wild-type or HMGNIjOE bone marrow transduced with BCR-ABL (aggregate of three independent experiments, n=20 [ WT] or n=28 (HMGN l_OEJ per group, curves compared by log-rank test).
Figures 18A-18G show that HMGN1 overexpression alone results in multiple B ceU phenotypes observed with triplication of the entire 21q22 orthologous region. Figure 1 KA shows relative quantitation of H3K27me3 and H GN i in BaF3 lymphoblasts transduced with empty vector of mouse HMGN1. Figure 18B shows a beat map showing RNA expressionof the 31 triplicated genes in passages 1, 3, and 6 in triplicate TslRhr cultures (bhie-rcd « low to high log2 FPK values, genes listed in genomic order). Figure 18C shows a schematic of the primary B cell shRNA experiment Passage 1 B cells from Tsl Rhr or wild-type bone marrow were pooled after infection with individual lentiviral shRNAs targeting cither a triplicated gene (5 shRNA gcne) or a control (n»30). DNA was collected post-infection (baseline) and after each passage (indicated by arrows), and the relative representation of each shRNA was quantitatcd by next generation sequencing. Data represent the average of independent biological replicates from wild-type (n=3) and Tsl Rhr (n=4) animals. Figure 18D show normalized quantitation of negative (non- largeting) and positive (known to be toxic) control shRNAs in passage 6 Tsl Rhr colonies relative to input (left) demonstrates preferential loss of positive control shRNAs. Neither positive nor negative control shRNAs were preferentially lost from Tsl Rhr passage 3 cells compared to wild-type (right, Tukey box and whiskers plots, horizontal bar is the median and plus is the mean; *P 0.05; NS, not significant). Figure 18E show Western blotting results of BaF3 rymphoblasts confirming knockdown of HMGN1. Antibodies are: A (Abeam), B (A viva), mHMGNl (affinity purified murine HMGN1 antibody). Figure 18F show Western blotting results of HMGN 1 in B cell colonies from wild-type and
HMGNjOE mice using the Abeam HMGN1 antibody. **Endo~ represents endogenous mouse HMGN1 and "Tg" represents transgenic human HMGN I . Figure 180 shows Hardy B cell subtractions as percentages of bone marrow cells from wild-type (black) and HMGNljOE (orange) littermates (n*4 per group, *P<0.05).
Figure 19 shows a schematic of B-cell developmental lineages and associated molecular markers according to murine genetics nomenclature.
Brief DeacriBtios of Table*
Table 1 shows genes differentially expressed in TslRhr as compared to wild-type B cells. The top 150 higher (UP) and lower (DOWN) expressed genes in TslRhr relative to wild-type passage 1 B cells by RNAseq and EdgcR analysis (p<0.05, false discovery rate < 0.25) is shown (n=3 independent biologic replicates per genotype). Differential expression is annotated as log] fold change in TslRhr relative to wild-type. The SO UP genes that constitute the Core TslRhr gene set (Figure 12A) are annotated. Table 2 shows the results of a query of the top 150 TslRhr UP gene set against the Molecular Signatures Database (MSigDB) *c I ' positional dataset
Table 3 shows the results of gene set enrichment and network enrichment mapping for TslRhr B cells.
Tabfe 4 snows tte results tf a query of u^e50C^
Molecular Signatures Database (MSigDB) 4c2 cgp' chemical and genetic perturbations dataset
Table 5 shows die top 100 differentially expressed genes in the SUZ12 target gene, Mikkelsen MEF and NPC H3 27me3 signatures between DS-ALU and non-DS-ALLs.
Table 6 shows shR As used in the cornpetitrve growth assay targeting DSCR genes. Gene symbols fix DSCR genes (tab 1 TEST) and controls (tab 2 "CONTROLS") are shown, with clone names in The RNAi Consortium (TRQ database, target sequence, and location of the target sequence within the gene. Data arc the normalized ratio of the quantitation of each shRNA in TslRhr to wild-type B cells during passaging relative to input within each genotype.
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The present invention is based, at least in part, on the novel discovery of gene profiles useful for distinguishing among cancer subtypes (e.g., lymphoid cancers, such as leukemia) and for predicting the clinical outcome of such cancer subtypes to therapeutic regimens, particularly to modulators of histone mcthylation (e.g., H3K27me3). Thus, agents such as miRNAs, miRNA analogues, small molecules, RNA imerferencc, aptamer, peptides, peptklornirnctics, antibodies that specifically bind to one or more biomarkers of the invention (eg., bmmarkers listed in Tables 1-5 and or described in the Examples, such as H3K27 demethylases, PRC2 complexes, EZH2, and HMON1) and fragments thereof can be used to identify, diagnose, prognose, assess, prevent, and treat cancers lymphoid cancers, such as leukemia). In addition, the present invention is based, at least in part, on the novel discovery that contacting lymphoid progenitor cells (e.g., wild type and/or genorracalry altered cells) with an agent mat inhibits polycomb repressor complex 2 (PRC2) activity or reduces H3K27me3 levels can increase the rrumbcr of lymphoid progenitor cells (e.g., increase self-renewal and cell proliferation) from the initial population of such lymphoid progenitor cells. 1, fcfftfltotti
The articles "a" and "an" are used herein to refer to one or to more than one (/.*. to at least one) of the grammatical object of the article. By way of example, "an element" means one clement or more than one element.
The term "allogeneic" refers to deriving from, originating in, or being members of the same species, where the members are genetically related or genetically unrelated bat genetically similar. An "allogeneic transplant" refers to transfer of cells or organs from a donor to a recipient, where the recipient is the same species as the donor. The term "mismatched alfogencic" refers to deriving from, originating in, or being members of the same species having non-identical major histocorapatability complex (MHQ antigens (ie., proteins) as typically determined by standard assays used in the art, such as serological or molecular analysis of a defined number of MHC antigens. A "partial mismatch" refers to partial match of die MHC antigens tested between members, typically between a donor and recipient For instance, a "rialf mismatch'* refers to 50% of the MHC antigens tested as showing different MHC antigen type between two members. A roU" or "complete" mismatch refers to all MHC antigens tested as being different between two members. These terms contrast with the term "xenogeneic," which refers to deriving from, originating in, or being members of different species, eg., human and rodent, human and swine, human and chimpanzee, etc. A "xenogeneic transplant" refers to transfer of cells or organs from a donor to a recipient where the recipient is a species different from that of the donor. The term "syngeneic'" refers to deriving from, originating in, or being members of the same species that are genetically identical, particularly with respect to antigens or inmwnoiogicai reactions. These include identical twins having matching MHC types. Thus, a "syngeneic transplant" refers to transfer of cells or organs from a donor to a recipient who is genetically identical to the donor.
The term "altered amount" of a marker or "altered level" of a marker refers to increased or decreased copy mtmber of the marker andor increased or decreased expression level of a particular marker gene or genes in a cancer sample, as compared to die expression level or copy number of the marker in a control sample. The term "altered amount" of a marker also includes an increased or decreased protein level of a marker in a sample, e.g., a cancer sample, as compared to the protein kvd of the marker in a normal, control sample.
The "amount** of a marker, e.g.. expression or copy number of a marker or minimal common region (MCR), or protein level of a marker, in a subject is "significantly'' higher or lower than the normal amount of a marker, if the amount of the marker is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, and preferably at least twice, and more preferably three, four, five, ten or more times that amount Alternately, the amount of the marker in the subject can be considered "sigrufrcant-y*'' higher or lower than the normal amount if the amount is at least about two, and preferably at least about three, four, or five times, higher or lower, respectively, than the normal amount of die marker. In some embodiments, the amount of the marker in the subject can be considered "significantly*' higher or lower than the normal amount if the amount is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, higher or lower, respectively, than the normal amount of the marker.
The term "altered level of expression" of a marker refers to an expression level or copy number of a marker in a test sample e.g., a sample derived from a subject suffering from cancer, that is greater or less than the standard error of the assay employed to assess expression or copy mrrnber, and is preferably at least twice, and more rnefcrably three, four, five or ten or more times the expression level or copy number of the marker or
chrornosornal region in a control sample (e.g., sample from a heafthy subject not having the associated disease) and preferably, the average expression level or copy number of the marker or chromosomal region in several control samples. The altered level of expression is greater or less than the standard error of the assay employed to assess expression or copy number, and is preferably at least twice, and more preferably three, four, five or ten or more times the expression level or copy number of the marker in a control sample (e.g., sample f om a healthy subject not having the associated disease) and preferably, the average expression level or copy number of the marker in several control samples.
The term "altered activity" of a marker refers to an activity of a marker which is increased or decreased in a disease state, e.g.. in a cancer sample, as compared to the activity of the marker in a normal, control sample. Altered activity of a marker may be the result of, for example, altered expression of the marker, altered protein level of the marker, altered structure of the marker, or, e.g., an altered interaction with other proteins involved in the same or different pathway as the marker, or altered interaction with franscriptional activators or inhibitors.
The term "altered structure" of a marker refers to the presence of mutations or allelic variants within the marker gene or maker protein, e.g., mutations which affect expression or activity of the marker, as compared to the normal or wild-type gene or protein. For example, mutations include, but are not limited to substitutions, deletions, or addition mutations. Mutations may be present in the coding or non-coding region of the marker.
The term "altered subcellular localization" of a marker refers to the misiocalization of the marker within a cell relative to the normal localization within the cell eg., within a healthy and/or wild-type cell. An indication of normal localization of the marker can be determined through an analysis of subcellular localization motifs known in me field that are harbored by marker polypeptides.
Unless otherwise specified herein, the terms "antibody** and "antibodies'' broadly encompass naturally-occurring forms of antibodies (*.#., IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.
The term "antibody as used herein also includes an "antigen-binding portion" of an antibody (or simply "antibody portion"). The term uantigcn- binding portion", as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the anUgen-bindtng function of an antibody can be perfbrmed by fragments of a full-length antibody. Examples of binding f agments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab*)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH 1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et at., (1989) Nature 3 1:544-546), which consists of a VH domain; and (vi) an isolated romplcrncntarity (letennining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker mat enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); sec e.g., Bird et al. ( 1988) Science 242:423-426; and Huston et al (1988) Prvc. Natl. Acad. Sci. USA 85:5879-5883; and Osboum et al. 1998. Nature Biotechnology 16: 778). Such single chain antibodies are also intended to be encompassed within the term "ar ge -binding portion** of an antibody. Any VH and VL sequences of specific scFv can be linked to human immunoglobulin constant region cO A or genomic sequences, in order to generate expression vectors encoding complete IgG polypeptides or other isotypes. VH and VL can also be used in the generation of Fab , Fv or other fragments of immunoglobulins using eitber protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as dtabodies arc also
encompassed. Dtabodies are bivalent, bispeciflc antibodies in which VH and VL domains are expressed on a single poiypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding si es (see e.g., Holligcr, P., et al. (1993) Proc Nad. Acad. Set. l/SA 90:6444-6448; Poljalc, R. J., el al (1994) Structure 2:1121-1123).
Still further, an antibody or antigen nnding portion thereof may be part of larger immunoadhesion polypeptides, formed by covaknt or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include use of the streptavidin core region to make a tetrameric scFv polypeptide (Kipriyanov, S.M., et al. ( 199S) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a maifccT peptide and a C-temiinal polyhistidine tag to make bivalent and biotiny!ated scFv polypeptides (Kipriyanov, S.M., et al. (1994) Mol Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and itnnsunc^dhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein.
Antibodies may be polyclonal or monoclonal; xenogeneic allogeneic or syngeneic; or modified forms thereof («.£., humanized, chimeric, etc.). Antibodies may also be fully human. The terms "monoclonal antibodies" and "monoclonal antibody composition", as used heroin, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of irnrmmorea ing with a particular epitope of an antigen, whereas die term "polyclonal antibodies" and "polyclonal antibody composition" refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts. The term "'amiscnee" nucleic acid polypeptide comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein. eg. , complementary to the coding strand of a double-stranded cDNA polypeptide, complementary to an mR A sequence or complementary to the coding strand of a gene. Accordingly, an antisensc nucleic acid polypeptide can hydrogen bond to a sense nucleic acid polypeptide.
The term "autologous" refers to deriving from or originating in the same subject or patient. An "autologous transplant" refers to the harvesting and reinfusion or transplant of a subject's own cells or organs. Exclusive or supplemental use of autologous cells can diminate or reduce many adverse effects of administration of the cells back to die host, particular graft versus host reaction.
The term *1riochipn refers to a solid substrate comprising an attached probe or plurality of probes of the invention, wherein the probers) comprise at least 1, 2, 3, , 5, 6, 7, 8, , 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 5, 50, 55, 0, 65, 70, 5, 80, 85. 0, 95, 100, 150.200 or more probes. The probes may be capable of hybridizing to a target sequence ondcr stringent hybridization conditions. The probes may be attached at spatially defined address on the substrate. More than one probe per target sequence may be used, with either overlapping probes or probes to different sections of a particular target sequence. The probes may be capable of hybridizing to target sequences associated with a single disorder. The probes may be attached to the biocnip in a wide variety of ways, as will be appreciated by those in the art The probes may either be synthesized first, with subsequent attachment to the biocnip, or may be directly synthesized on thebiochip. The solid substrate may be a material that may be modified to contain discrete individual sites appropriate for the attachment or association of the probes and is amenable to at least one detection method. Representative examples of substrates include glass and modified or iunctionalized glass, plastics (including acrylics, polystyrene and copolymers of styrcne and other materials, polypropylene, polyethylene, porybutylene, poiyurethanes, TeflonJ, etc), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses and plastics. The substrates may allow optical detection without appreciably fluorescing. The substrate may be planar, although other configurations of substrates may be used as well. For example, probes may be placed on the inside surface of a tube, for ffow-through sample analysis to minimize sample volume. Similarly, me substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics. The biocnip and the probe may be derivatized with chemical functional groups for subsequent attachment of the two. For example, the biochtp may be derivatized with a chemical functional group including, bat not limited to, amino groups, carboxyi groups, oxo groups or thiol groups. Using these functional groups, the probes may be attached using functional groups on the probes either directly or indirectly using a linker. The probes may be attached to the solid support by either the S' terminus, 3' terminus, or via an internal nucleotide. The probe may also be attached to the solid support non-covalently. For example, bio tiny la ted oligonucleotides can be made, which may bind to surfaces covalently coated with streptavidin, resulting in attachment. Alternatively, probes may be synthesized on the surface using techniques such as photoporyincrizarion and pbotoKthography.
The term "body fluid" refers to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g. amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowpcr's fluid or pre-cjaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, peritoneal fluid, pus, saliva, sebum, semen, scrum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit).
The terms "cancer" or "tamof or "hyperproli feranve disorder" refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled prouferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. Cancers include, but arc not limited to, B cell cancer, eg., multiple myeloma, Waldenstrom's macroglobdinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowd or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, and the like. Other non- limiting examples of types of cancers applicable to the methods encompassed by the present invention include human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotbeliosarcotna, lymphangiosarcoma, lyn^hangiociidou^iosarcoma, synovioma, mesothelioma, E wing's tumor,
leiomyosarcoma, rhabck>rnyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adetiocarciiiomas, cystadciiocarciiMma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, hmg carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, mcdulloblastoma,
craniopharyngioma, ependymoma, pineal oma, hemangioblastoma, acoustic neuroma, oUgodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, &£., acute lymphocytic leukemia and acute myelocytic leukemia (mycloblastic
promydocytic, myelomonocytic, monocytic and crythrolcukcmia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic foikemia); and polycythemia vera, rymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobditKrma, and heavy chain disease. In some embodiments, the cancer whose phenotype is determined by the method of the invention is an epithelial cancer such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, bead and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, die epithelial cancer is non-small-cdi lung cancer, nonpapillary renal ceU carcinoma, cervical carcinoma, ovarian carcinoma (e.g. , serous ovarian carunmtu), c breast carcmoma. The epithelial cancers may be
characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, brenner, or umiiffcrcntiated. In some embodiments, the present invention is used in the treatment, diagnosis, and or prognosis of lymphoma or its subtypes, including, but not limited to, iympbocytc-rich classical Hodgkin lymphoma, mixed ce!hilarity classical Hodgkin lymphoma, lymphocyte-depleted classical Hodgkin lymphoma, nodular sclerosis classical Hodgkin lymphoma, anaplastic large cell lymphoma. diffuse large B-cell lymphomas, MLL' pre B-cdl ALL) based upon analysis of markers described herein.
The term "classifying" includes "to associate" or "to categorize" a sample with a disease state. In certain instances, "classifying" is based on statistical evidence, empirical evidence, or both. In certain embodiments, the methods and systems of classifying use of a so-called training set of samples having known disease states. Once established, the training data set serves as a basis, model, or template against whkh the features of an unknown sample are compared, in order to classify the unknown disease state of the sample. In certain instances, classifying the sample is akin to diagnosing the disease state of the sample. In certain other instances, classifying the sample is akin to diiTerenriating the disease state of the sample from another disease state.
The term "coding region" refers to regions of a nucleotide sequence comprising codons which arc translated into amino acid residues, whereas the term "noncoding region'" refers to regions of a nucleotide sequence that are not translated into amino acids (&g., 5* and 3' untranslated regions).
The term "comrrfemcntary" refers to the broad concept of sequence
complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to die first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion arc capable of base pairing with nucleotide residues in the second portion. Tbe term "control" refers to any reference standard suitable to provide a cornparison to the expression products in the test sample. In one embodiment, the control comprises obtaining a "control sample" from which expression product levels arc detected and compared to the expression product levels from the test sample. Such a control sample may comprise any suitable sample, including but not limited to a sample from a control cancer patient (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a normal patient or the cancer patient, cultured primary cells/ tissues isolated from a subject such as a normal subject or the cancer patient, adjacent normal cells/tissues obtained from the same organ or body location of the cancer patient, a tissue or cell sample isolated from a normal subject, or a primary cells tissues obtained from a depository. In another preferred embodiment, the control may comprise a reference standard expression product level from any suitable source, including but not limited to housekeeping genes, an expression product level range from normal tissue (or other previously analyzed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, survival for one, two, three, four years, etc.) or receiving a certain treatment It will be understood by those of skill in the art that such control samples and reference standard expression product levels can be used in combination as controls in the methods of the present mvention. In one embodiment, the control may comprise normal or r >n-cancerous cell tissue sample. In another preferred embodiment, the control may comprise an expression level for a set of patients, such as a set of cancer patients, or for a set of cancer patients receiving a certain treatment, or for a set of patients with one outcome versus another outcome. In the former case, the specific expression product level of each patient can be assigned to a percentile level of expression, or expressed as either higher or lower than the mean or average of the reference standard expression level, in another prefcrred embodiment, the control may comprise normal cells, cells from patients treated with combination chernotherapy and cells from patients having benign cancer. In another embodiment, the control may also comprise a measured value for example, average level of expression of a particular gene in a population compared to the level of expression of a housekeeping gene in the same population. Such a population may comprise normal subjects, cancer patients who have not undergone any treatment (£e., treatment naive), cancer patients undergoing therapy, or patients having benign cancer. In another preferred embodiment, the control comprises a ratio transformation of expression product levels. including bat not limited to determining a ratio of expression product levels of two genes in tbe test sample and comparing i to any suitable ratio of the same two genes in a reference standard determining expression product levels of the two or more genes in the test sample and determining a difference in expression product levels in any suitable control; and detenrjining expression product levels of the two or more genes in the test sample, normalizing their expression to expression of housekeeping genes in the test sample, and comparing to any suitable control. In particularly preferred embodiments, the control comprises a control sample which is of the same lineage and/or type as the test sample. In another embodiment, the control may comprise expression product levels grouped as percentiles within or based on a set of patient samples, such as all patients with cancer. In one embodiment a control expression product level is established wherein higher or lower levels of expression product relative to, for instance, a particular percentile, are used as the basis for predicting outcome. In another preferred embodiment, a control expression product level is established using expression product levels from cancer control patients with a known outcome, and the expression product levels from the test sample are compared to the control expression product level as the basis for predicting outcome. As demonstrated by the data below, the methods of die invention are not limited to use of a specific cut-point in comparing die level of expression product in the test sample to the control.
The term "diagnosing cancer" includes the use of the methods, systems, and code of the present invention to determine the presence or absence of a cancer or subtype thereof in an individual. The term also includes methods, systems, and code for assessing the level of disease activity in an individual.
As used herein, the term "diagnostic marker" includes markers described herein which are useful in die diagnosis of cancer, eg., over- or under- activity, emergence, expression, growth, remission, recurrence or resistance of tumors before, during or after therapy. The predictive functions of the marker may be conftrmed by, eg., (1) increased or decreased copy number (eg., by FISH, FISH plus SKY, single-molecule sequencing, e.g., as described in die art at least at J. BiotechnoL, 86:289-301 , or qPCR). ovcrexpression or underexpression (e.g, by SH, Northern Blot, or qPCRX increased or decreased protein level (e.g., by 1HC), or increased or decreased activity (determined by, for example, modulation of a pathway in which the marker is involved), eg., in more than about 5%, 6%, 7%, 8%, 9%, io I ]%, 12%, 13%, 14%, 15%, 20%, 25%, or more of human cancers types or cancer samples; (2) its presence or absence in a biological sample, eg., a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, or bone marrow, from a subject, e.g. a human, afflicted with cancer, (3) its presence or absence in clinical subset of subjects with cancer (e.g., those responding to a particular therapy or those developing resistance). Diagnostic markers also include
"surrogate markers," e.g., markers which are indirect markers of cancer progression. Such diagnostic markers may be useful to identify populations of subjects amenable to treatment with modulators of H3K27me3 levels (e.g., subjects having Down syndrome-type ALL as described herein) and to thereby treat such stratified patient populations.
The term "Down syndrome'' or "DS" refers to a condition caused by trisomy for human chromosome 21 (Hsa21 ) and is the most common genetic cause of mental retardation in humans. DS occurs in 1 in 800- 1 (XX) live births arid results in over 80 different clinical phenotypes, including craniofacial abnormalities, a small hypocellular brain with a disproportionately small cerebellum, Alzheimer-like histopathoiogy, and an elevated risk for congenital bcart defects, Hirschsprung's disease, and leukemia. DS is associated with two contrary cancer-related phenotypes. The first observation of a patient with leukemia and DS was made in 1930, and an increased risk of leukemia among individuals with DS was established by 1 SS. Acute megakaryoblastic leukemia (AMKL) occurs approximately 500-fbld more frequently in individuals with DS than in the general population. AMKL almost always occurs in concert with a somatic mutation in the GATAJ transcription factor. Several genetic mouse models of DS exist The most widely-used of these models is the Ts65Dn mouse, which is trisomic for orthologs of approximately half of the 261 protein coding genes on Hsa21 (Patterson and Costa (2005) Λ¾ν. Rev. Genet. 6:137- 147; Davisson (2005) Drug Disc. Today: Disease Models 2: 103- 109). This mouse recapitulates in detail several phenotypes of DS, including impainncnts in learning and memory degeneration of basal fbrebrain cholinergic neurons with aging, small cerebellum, fewer granule cell neurons and reduced cdl proliferation in the dentate gyrus, and dysmorphology of the craniofacial skeleton, mandible and cranial vault The TslRlr mouse has segmental trisomy for a subset of die genes represented in Ts65Dn which correspond to a "critical region" on Hsa21 which harbors genes sufficient to cause a number of DS phenotypes. In addition, the Dp( 16)1 Yu mouse harbors an extra copy of all of the segments on mouse chromosome 16 that are syntenic to human chiomosome 21 and such mice display learning, memory, and heart defects coinpatable to those observed in human DS (Li et al. (2007) Hum. Mol. Genet. 16:1359-66). In humans, studies of partial trisomy 21 ("Down Syndrome Critical Region" (DSCR) indicate that only parts of the chromosome are necessary to recapitulate the Down syndrome phenotype (Patterson and Costa (2005) Nat. Rev. Genet. 6:137-147; Olson el al. (2004) Science 306:687-690). The TslRhr mouse is trisomic only fix the region of mouse chromosome 16 that is comparable to the DSCR.
The term "expansion" in die context of ceils refers to increase in the number of a characteristic cell type, or cell ty es, from an initial population of cells, which may or may not be identical. The initial cells used for expansion need not be the same as the ceils generated from expansion. For instance, the expanded cells may be produced by growth and differentiation of the initial population of cells. Excluded from the term expansion are limiting dilution assays used to characterize the differentiation potential of cells.
A molecule is "fixed" or "affixed" to a substrate if it is covalently or non-covalently associated with the substrate such the substrate can be rinsed with a fluid (e.g. standard saline citrate, pH 7.4) without a substantial fraction of the molecule dissociating from the substrate.
The term "gene expression data" or "gene expression level" as used herein refers to uformation regarding the relative or absolute level of expression of a gene or set of genes in a cell or group of cells. The level of expression of a gene may be determined based on die level of RNA, such as mRNA, encoded by the gene. Alternatively, the level of expression may be determined based on the level of a polypeptide or fragment thereof encoded by the gene. Gene expression data may be acquired for an individual cell, or for a group of cells such as a tumor or biopsy sample. Gene expression data and gene expression levels can be stored on computer readable media, e.g., the computer readable medium used in conjunction with a mkroarray or chip reading device. Such gene expression data can be manipulated to generate gene expression signatures.
The term "gene expression signature" or "signature" as used herein refers to a group of coordinatery expressed genes. The genes making up this signature may be expressed in a specific cell lineage, stage of differentiation, or during a particular biological response. The genes can reflect biological aspects of the tumors in which they are expressed, such as die cdl of origin of the cancer, the nature of the non-malignant cells in the biopsy, and the oncogenic mechanisms responsible for the cancer. For example, the gene expression signatures described herein stratify Down Syndrome- ALL (DS-ALL from general ALL conditions that are especially amenable to treatment with modulators of H3K27me3 levels. The term "hematological cancer" refers to cancers of cells derived from the blood. In some embodiments, the hematological cancer is selected from the group consisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML). chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), multiple myeloma (MM), non- Hodgkin's lymphoma (NHLX Hodgkin's lymphoma, mantle cell lymphoma (MCL), follicular lymphoma, Waldenstrom's macroglobulmemia (WM), B-cell lymphoma and diffuse large B-ccll lymphoma (DLfiCL). NHL may include indolent Non-Hodgkin's Lymphoma (iNHL) or aggressive Non-Hodgkin's Lymphoma (aNHL).
The term "hematopoietic stem cell" or "HSC refers to a clonogcnic, self-renewing phnipotent ceil capable of ultimately differentiating into all ceil types of the hematopoietic system, including B cells T cells, N cells, lymphoid dendritic cells, myeloid dendritic cells, granulocytes, macrophages, megakaryocytes, and erythroid cells. As with other cells of the hematopoietic system, HSCs are typically defined by the presence of a characteristic set of cell markers.
The term "homologous*' as used herein, refers to miclcotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions arc homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that arc occupied by the same nucleotide residue. By way of example, a region having die nucleotide sequence 5- ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of die portions are occupied by the same nucleotide residue.
The term **host cei is intended to refer to a cell into which a nucleic acid of the invention, such as a rccornbiriant expression vector of the invention, has been introduced. The terms "host ceil" and 'Recombinant host cei arc used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifkations may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the patent cell, but are still included within the scope of the terra as used herein.
The term humanized antibody," as used herein, is intended to include antibod es made by a ixm-hnman cell having variable and constant regions which have been altered to more closely resemble antibodies mat would be made by a human cell, for example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. Humanized antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (eg., nutations introduced by random or site-spedrk mutagenesis vftro or by som
for example in the CDRs. The term humanized antibody", as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
As used herein, the term "immune cell" refers to cells mat play a role in the immune response. Immune cells arc of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
As used herein, the term "immune response" includes T cell mediated and/or B cell mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity. In addition, the term immune response includes immune responses that are indirectly effected by T cell activation, &£., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages.
As used herein, the term 'inhibit'" includes the decrease, limitation, or blockage, of, for example a particular action, function, or interaction. For example, cancer is "inhibited" if at least one symptom of the cancer, such as hyperprob fcrau ve growth, is alleviated, terminated, slowed, or prevented. As used herein, cancer is also "inhibited" if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.
As used herein, the term "interaction,'' when referring to an interaction between two molecules, refers to the physical contact (e.g., binding) of the molecules with one another. Generally, such an interaction results in an activity (which produces a biological effect) of one or both of said molecules. The activity may be a direct activity of one or both of the molecules. Alternatively, one or both molecules in the interaction may be prevented from binding their Iigand, and thus be held inactive with respect to iigand binding activity [e.g., binding its iigand and triggering or inhibiting an immune response). To inhibit such an interaction results in the disruption of the activity of one or more molecules involved in the interaction. To enhance such an interaction is to prolong or increase the likelihood of said physical contact, and prolong or increase the likelihood of said activity.
An "isolated antibody,'* as used herein, is intended to refer to an antibody mat is substantially free of other antibodies having different antigenic specificities. Moreover, an isolated antibody may be sqbstantially free of other cellular material and/or chemicals.
As used herein, an "isolated protein" refers to a protein that is substantially free of other proteins, cellular material, separation medium, and culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. An ''isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide or fusion protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations, in which compositions of the invention are separated from cellular components of the cells from which they arc isolated or recombnantly produced In one embodiment, the language "substantially free of cellular material" includes preparations of having less than about 30%, 20%, 10%, or 5% (by dry weight) of cellular material. When an antibody, polypeptide, peptide or fusion protein or fragment thereof, e.g., a biologically active fragment thereof is recoTnbmantry produced, it is also preferably substantially free of culture medium, te., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
A "kit" is any nianufacturc (e.g. a package or container) cotnprising at least one reagent, eg. a probe, for specifically detecting or modulating the expression of a marker of the invention. The kit may be promoted, distributed, or sold as a unit for pcrfbrrning the methods of the present invention.
The term "leukemia" refers to a group of diseases that arc cancers of the marrow and Mood, where the malignant cells are white blood cells (leukocytes). The two major groups are lyrnphatic. and myeloid leukemia. Both groups are considered as either acute or chronic depending on various factors. Also included are lymphoid leukemias. Leukemias can thus be divided into four main types: acute lymphocytic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia and chronic myelogenous leukemia. Acute and chronic leukemias are usually studied as groups separated by the cells which arc affected. These heterogeneous groups are usually considered together and are considered as a group of diseases characterized by infiltration of the bone marrow and other tissues by the cells of the hematopoietic system. The infiltration is called neoplastic, meaning new growth of cells, but all of the cells seen in the marrow, and peripheral circulation in leukemia are normal in a normal bone marrow, except for one structure, seen in myelocytic leukemia called Auer rods. These structures arc repeated in this kind of leukemia, and arc unknown as to structure, and relationship to any other material. Acute lymphoblastic leukemia (ALL) is also referred to as acute lymphocytic leukemia and acute lymphoid leukemia and is a form of leukemia characterized by excess lymphoblasts. Malignant, immature white blood cells continuously multiply and arc overproduced in the bone marrow. ALL causes damage and death by crowding out normal cells in the bone marrow, and by spreading (infiltrating) to other organs. ALL is most common in childhood with a peak incidence at 2-5 years of age, and another peak in old age. Standard of care for treating ALL focuses on treatment of different phases in order to control bone marrow and systemic (whole-body) disease as well as to prevent leukemic cells from spreading to other sites, particularly the central nervous system (CNS), eg., monthly lumbar punctures: a) induction chemotherapy is used to bring about bone marrow remission. For adults, standard induction plans include prednisone, vincristine, and an anthracyclinc drug; other drug plans may include L- asparaginase or cyctopho ahamide. For children with low-risk ALL, standard therapy usually consists of three drugs (prednisone, ^asparaginase, and vincristine) for the first month of treatment; b) consolidation therapy or intensification therapy eliminates any remaining leukemia cells. There are many different approaches to consolidation, but it is typically a high-dose, multi-drug treatment that is unclertakcn for a few months. Patients with low- to average-risk ALL receive therapy with antimetabolite drugs such as methotrexate and 6-racrcaptopuhne (6-MP). High-risk patients receive higher drug doses of these drugs, plus additional drugs; c) CNS prophylaxis (preventive therapy) stops die cancer from spreading to die brain and nervous system in high-risk patients. Standard prophylaxis may include radiation of the head andor drugs delivered directly into the spine; and/or d) maintenance treatments with cfcrnothcrapcutic drugs prevent disease recurrence oocc nraission has been achieved. Maintenance therapy usually involves lower drug doses. and may continue for up to three yean. Alternatively, allogeneic bone marrow
transplantation may be appropriate for higb-risk or relapsed patients. Chronic rympbocyrJc leukemia (also known as "chronic lymphoid leukemia" or "CLL"), is a leukemia of the while blood cells Oympbocytcs) that affects a particular lymphocyte, the B cell, which originates in the bone marrow, develops in the lymph nodes, and normally fights infection. In CLL, the DNA of aB cell is damaged* so mat it cannot fight infection, but grows out of control and crowds out the healthy blood cells that can fight infection. CLL isan abiiorrnal neoplastic proliferation of B cells. The cells accumulate mainly in die bone marrow and blood. Although not originally appreciated, CLL is now thought to be identical to a disease called small lymphocytic lymphoma (SLL), a type of non-Hodgkin's lymphoma which presents primarily in the lymph nodes. Most people arc diagnosed without symptoms as the result of a routine blood test that returns a high white blood cell count, but as it advances, CLL results in swollen lymph nodes, spleen, and liver, and eventually anemia and infections. Early CLL is not usually treated, and late CLL is treated with chemotherapy and monoclonal antibodies. Survival varies from 5 years to more than 25 years. It is now possible to diagnose patients with short and long survival more precisely by examining the DNA mutations, and patients with slowfy -progressing disease can be reassured and may not need any treatment in their lifetimes fChiorazzi et al., (2005) N. Engl. J. Med. 352(8):804- 815). Chronk myelogenous leukemia (CML), also known as chronic granulocytic leukemia (CGL), is a neoplastic disorder of the hematopoietic stem cell. In its early phases, this disease is characterized by leukocytosis, the presence of increased numbers of immature granulocytes in the peripheral blood, splenomegaly and anemia. These immature granulocytes include basophils, eosinophils, and neutrophils. The immature granulocytes also accumulate in the bone marrow, spleen, liver, and occasionally in other tissues.
Patients presenting with this disease characteristically have more than 75,000 white blood cells per microliter, and the count may exceed 500,000 ul. CytoiogicaUy, CML is characterized by a translocation between chromosome 22 and chromosome 9. This translocation juxtaposes a purported proto-oncogenc with tyrosine kinase activity, a drcumstance that apparently leads to uncontrolled cell growth. The resulting translocated chromosome is sometimes referred to as die Philadelphia chromosome.
The term "lymphocytes" refers to cells of the immune system which arc a type of white blood cell. Lymphocytes include, but arc not limited to, T-cells (cytotoxic and helper T-cells), B-cells and natural killer cells ( K cells). The term lymphoid progenitor celP refers to an oligopotent or unipotent progenitor cell capable of ultimately developing into any of die terminally differentiated cells of the lymphoid lineage, such as T cell, B cell, N cell or lymphoid dendritic cells, but which do not typically differentiate into cells of die myeloid lineage. As with cells of die myeloid lineage, different cell populations of lymphoid progenitors are distinguishable from other cells by their differentiation potential, and die presence of a characteristic set of cell markers. Similarly, the term "common lymphoid progenitor cell" or "CLP" refers to an oligopotent cell characterized by its capacity to give rise to B-cdl progenitors (BCP), T-ccU progenitors (TCP), NK cells, and dendritic cells. These progenitor cells have little or no sdf-renewing capacity, but are capable of giving rise to T lymphocytes, B lymphocytes. NK cells, and lymphoid dendritic cells. By contrast, the term "myeloid progenitor cell" refers to a multipotent or unipotent progenitor cell capable of ultimately developing into any of die terminally differentiated cells of die myeloid lineage, but which do not typically differentiate into cells of die lymphoid lineage. Hence, "myeloid progenitor cell" refers to any progenitor cell in the myeloid lineage. Committed progenitor cells of the myeloid lineage include oligopotent common myeloid progenitor cells, gratmlocyic.monocytc progenitor cells, and megakaryocyte/erythroid cells, but also encompass unipotent erythroid progenitor, megakaryocyte progenitor, granulocyte progenitor, and macrophage progenitor cells. Different cell populations of myeloid progenitor ceils are distinguishable from other cells by their differentiation potential, and the presence of a characteristic set of cell markers. Similarly, the term "common myeloid progenitor cdl" or "CMP" refers to a cell characterized by its capacity to give rise to granulocyte/monocyte (GMP) progenitor cells and rnegakaryocyie/eryihroid ( EP) progenitor cells. These progenitor cells have limited or no self-renewing capacity, but are capable of giving rise to myeloid dendritic, myeloid erythroid, erythroid, megakaryocytes, granulocytc/macnjphage, granulocyte, and macrophage cells.
The term "lymphoma" refers to cancers that originate in the lymphatic system. Lymphoma is characterized by malignant neoplasms of lymphocytes— B lymphocytes and T lymphocytes (/.*., B-cclls and T-cclb). Lymphoma generally starts in lymph nodes or collccrions of lymphatic tissue in organs including, but not limited to, the stomach οτ intestines. Lymphoma may involve die marrow and the blood in some cases. Lymphoma may spread from one site to other parts of the body. Lymphomas include, but arc not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous B-cell lymphoma, activated B- ell lyraptorna, diffuse large B-cdi lymphoma (DLBCLX mantle cell lymphoma (MCL), follicular center fymptoma, traiisformed lymphoma, lymphocytic lymphoma of intermediate differentiation, intermediate ryrnphocytic lymphoma (ILL), diffuse poorly differentiated lymphocytic lymphoma (PDL), centrocytic lymphoma, diffuse small-cleaved cell lymphoma (DSCCL), peripheral T-cell lymphomas (PTCL), cutaneous T-Cell lymphoma and mantle zone lymphoma and low grade follicular lymphoma.
A "marker" or "biomarkeT includes a nucleic acid or polypeptide whose altered level of expression in a tissue or cell from its expression level in a control (eg., normal or healthy tissue or cell) is associated with a disease state, such as a cancer or subtype thereof {e.g., lymphoid cancers, such as leukemia). A "marker nucleic acid" is a nucleic acid {eg., mRNA, cDNA, mature miRNA, prc-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding site, or a variant thereof and other classes of small RNAs known to a skilled artisan) encoded by or corresponding to a marker of the invention. Such marker nucleic acids include DNA (eg., cDNA) comprising die entire or a partial sequence of any of the nucleic acid sequences set forth in Tables 1-5 and Examples or the complement of such a sequence. The marker nucleic acids also include R A cornprising the entire or a partial sequence of any of the nucleic acid sequences set forth in the Sequence Listing or the complement of such a sequence, wherein all thymidine residues arc replaced with uridine residues. A "marker protein" includes a protein encoded by or corresportding to a marker of the invention. A marker protein comprises the entire or a partial sequence of any of the sequences set forth in Tables 1-5 and Examples or the Examples. The terms "protein" and "polypeptide" ate used interchangeably. In some embodiments, specific corrtbinan'om; of biomarkcrg are preferred. For example, a combiruuion or subgroup of one or more of the biomarkers selected from the group consisting of a) "top 150 UP" biomarkers shown in Table I , b) "the 50 UP core" biomarkers shown in Table 1, c) "top 150 DOWN" biomarkers shown in Table 1 , d), "the 50 DOWN core" biomarkers shown in Table 1, e) the "triplicated gene" biomarkers shown in Table 1, f) the "chr2lq22 overlap" biomarkers shown in Table 2, g) the "PRC2 cluster" biomarkers shown in Table 3. h) the "overlap" biomarkers shown in Table 4, i) the "SUZ12 target," "Mikkelscn MEF," and/or "Mikkdsen NPC biomarkers shown in Table 5, j) KDM6A, k) KDM6B, I) EZH2, m) HMGN1, and subsets and/or combinations thereof.
The term "marker ptenotyping" in the context of cell identification refers to identification of markers or antigens on cells for determining their phenotypc (e.g.. differentiation state and/or cell type). This may be done by irmnunophenotyping, which uses antibodies that recognize antigens present on a cell. The antibodies may be monoclonal or polyclonal, but are generally chosen to have minimal crossr cact vity with other cell markers. It is to be understood mat certain cell differentiation or cell surface markers are unique to the animal species from which the cells are derived, while other cell markers will be common between species. These markers d Fating equivalent cell types between species are given the same marker identification even though mere are species differences in structure (eg., amino acid sequence). Cell markers include cell surfaces molecules, also referred to in certain situations as cell differentiation (CD) markers, and gene expression markers. The gene expression markers are those sets of expressed genes indicative of the cell type or differentiation state. In part, the gene expression profile will reflect the cell surface markers, although they may include non-cell surface molecules.
As used herein, the term "modulate" includes up-regulation and down-regulation, eg. , enhancing or inhibiting a response.
The "normal" or "control" level of expression of a marker is the level of expression of the marker in cells of a subject, e.g., a human patient, not afflicted with a cancer. An "ovcr-€xpressk» or "significantly higher level of expression" of a marker refers to an expression level in a test sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least twice, and more preferably 2.1, 2.2, 2.3.2.4.2.5.2.6.2.7.2.8.2.9.3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8J5, , 9.5, 10, 10.5, 11. 12, 13, 14, 15, 16. 17, 18, 19, 20 times or more higher than the expression activity or level of the marker in a control sample (e.g., sample from a healthy subject not having die marker associated disease) and preferably, the average expression level of the marker in several control samples. A "significantly lower kvd of expression" of a marker refers to an expression level in a test sample that is at least twice, and more preferably 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, .5, 10, 10J, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more lower than the expression level of the marker in a control sample (e.g., sample from a healthy subject not having the marker associated disease) and preferably, the average expression level of the marker in several control samples.
The term "peripheral blood cell subtypes" refers to cell types normally found in the peripheral blood including, but is not limited to, eosinophils, neutrophils, T cells, monocytes, NK cells, granulocytes, and B cells. The term "probe" refers to any molecule which is capable of selectively binding to a specifically intended target molecule, fix example, a nucleotide transcript or protein encoded by or corresponding to a marker. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of die target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.
The term "prognosis'* includes a prediction of the probable course and outcome of cancer or the likelihood of recovery from the disease. In some embodiments, the use of statistica] algorithms provides a prognosis of cancer in an individual For example, the prognosis can be surgery, development of a clinical subtype of cancer (e.g., lymphoid cancers, such as leukemia), development of one or more clinical factors, development of intestinal cancer, or recovery from the disease.
The term "response to cancer therapy" or "outcome of cancer therapy*' relates to any response of fe hypcrprohterativedisor^
change in tumor mass and or volume after initiation of neoadjuvant or adjuvant chemotherapy. Hyperprdiferative disorder response may be assessed, for example for efficacy or in a neoadjuvant or adjuvant situation, where the size of a tumor after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, rriarnmogram, ultrasound or palpation. Response may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection for solid cancers. Responses may be recorded in a quantitative fashion like percentage change in tumor volume or in a qualitative fashion like "pathological complete response*'' (pCR), "clinical complete remission'' (cCR), "clinical partial remission'* (cPR), "clinical stable disease" (cSDX, "clinical progressive disease** (cPD) or other qualitative criteria.
Assessment of hypeiproHterative disorder response may be done early after the onset of neoadjuvant or adjuvant therapy, eg., after a few hours, days, weeks or preferably after a few months. A typical endpoint for response assessment is upon termination of neoadjuvant d>cinotherapy or upon surgical removal of residual tumor cells and or the tumor bed. This is typically three months after initiation of neoadjuvant therapy. In some embodiments, clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR . The clinical benefit rate is measured by determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out rom the end of therapy. The shorthand for this formula is CBR=CR PR+SD over 6 months. In some embodiments, the CBR for a particular cancer therapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%.80%, 85%, or more. Additional criteria for evaluating die response to cancer therapies are related to "survival,'' which includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be cither irrespective of cause or tumor related); "recurrence- free survivaT (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein die term disease shall include cancer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence or metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to chernotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence. For example in order to determine appropriate threshold values, a particular cancer therapeutic regimen can be administered to a population of subjects and the outcome can be correlated to copy number, level of expression, level of activity, etc. of one or more biornarkers listed in Tables 1-5 and Examples or the Examples that were determined prior to administration of any cancer therapy. The outcome rneasurernent may be pathologic response to therapy given in die neoadjuvant setting. Alternatively, outcome measures, such as overall survival and disease-free oirvival can be monitored over a period of time for subjects following cancer therapy for whom the measurement values are known. In certain embodiments, the same doses of cancer therapeutic agents are administered to each subject. In related embodiments, the doses administered are standard doses known in the art for cancer therapeutic agents. The period of time for which subjects are monitored can vary. For example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 0, 45, 50, 55, or 60 months. Biomarkcr threshold values that correlate to outcome of a cancer therapy can be determined using methods such as those described in the Examples section. Outcomes can also be measured in terms of a "hazard ratio" (the ratio of death rates for one patient group to another, provides likelihood of death at a certain time point), "overall survival " (OS), and/or "progression free survivaL" In certain embodiments, the prognosis comprises likelihood of overall survival rate at 1 year, 2 years, 3 years, 4 years, or any other suitable time point. The significance associated with the prognosis of poor outcome in all aspects of the present invention is measured by techniques known in the art For example, significance may be measured with calculation of odds ratio. In a further embodiment, the significance is measured by a percentage. In one embodiment, a significant risk of poor outcome is measured as odds ratio of 0.8 or less or at least about 12, including by not limited to: 0.1, 0.2, 03, 0.4, 05, 0.6, 0.7, 0.8, 12, 13, 1.4, 15, 1.6, 1.7, 1.8, 1.9, 2.0, 25, 3.0, 4.0, 5.0, 10.0, 15.0, 20.0.25.0.30.0 and 40.0. In a further embodiment, a significant increase or reduction in risk is at least about 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and 98%. In a further embodiment, a significant increase in risk is at least about 50%. Thus, the present invention further provides methods for making a treatment decision for a cancer patient, comprising carrying out the methods for prognosing a cancer patient according to the different aspects and embodiments of the present invention, and then weighing the results in light of other known clinical and pathological risk factors, in determining a course of treatment for the cancer patient. For example, a cancer patient that is shown by the methods of the invention to have an increased risk of poor outcome by combination chemotherapy treatment can be treated with more aggressive therapies, including but not limited to radiation therapy, peripheral blood stem cell transplant, bone marrow transplant, or novel or experimental therapies under clinical investigation.
The term "resistance*'' refers to an acquired or natural resistance of a cancer sample or a mammal to a cancer therapy ( Le., being nonresponsivc to or having reduced or limited response to the therapeutic treatment), such as having a reduced response to a rberapeutic treatment by 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2- fold, 3-fokt 4-fold, 5-fbld, 10-fold, 15-fold, 20-f ld or more. The reduction in response can be measured by compering with the same cancer sample or mammal before the resistance is acquired, or by comparing with a different cancer sample or a mammal who is known to have no resistance to the therapeutic treatment A typical acquired resistance to chemotherapy is called "multidrug resistance.'' The multidrug resistance can be mediated by P-grycoprotein or can be mediated by other mechanisms, or it can occur when a mammal is infected with a multi-drug-resistam microorganism or a combination of nncroorganisms. The determination of resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician, for example, can be measured by cell proliferative assays and cell death assays as described herein as "sensitizing." In some embodiments, the term "reverses resistance" means that the use of a second agent in combination with a primary cancer therapy (eg., chemotherapeutic or radiation therapy) is able to produce a significant decrease in tumor volume at a level of statistical significance (e g. , p<0.05) wben compared to tumor volume of untreated tumor in the circumstance where tbc primary cancer therapy (eg., chemotherapeutic or radiation therapy) alone is unable to produce a statistically significant decrease in tumor volume compared to tumor volume of untreated tumor. This generally applies to tumor volume measurements made at a time when the untreated tumor is growing log rhythmically.
The term "sample" used for detecting or dctenxuning the presence or level of at least one biomarker is typically whole Mood, plasma, scrum, saliva, urine, stool (eg., feces), tears, and any other bodily fluid (eg., as described above under the definition of "body fluids"), or a tissue sample (eg., biopsy) such as a small intestine, colon sample, or surgical resection tissue. In certain instances, the method of the present invention further comprises obtaining the sample from the individual prior to detecting or determining die presence or level of at least one marker in the sample.
The term "sensitize" means to alter cancer cells or tumor cells in a way mat allows for more effective treatment of the associated cancer with a cancer therapy (e.g., chemotberapeutic or radiation therapy. In some embodiments, normal cells are not affected to an extent that causes the normal cells to be unduly injured by the cancer therapy (eg., chemotherapy or radiation therapy). An increased sensitivity or a reduced sensitivity to a therapeutic treatment is measured according to a known method in the art for the particular treatment and methods described herein below, including, but not limited to, cell proliferative assays (Tanigawa N, Kern D H, Kikasa Y, Morton D L, Cancer Res 1982; 42: 2159-2164), cell death assays (Weisenthal L M, Shoemaker R H, Marsden J A, Dill P L, Baker J A, Moran E M, Cancer Res 1984; 94: 161-173; Weisenthal L M. Lippman M E. Cancer Treat Rep 1985; 69: 15-632; Weisenthal L M, In: aspers G J L, Pictcrs R, Twentyman PR, Weisenthal L M, Veerman A J P, eds. Drug Resistance in Leukemia and Lymphoma. Langhornc, P A: Harwood Academic Publishers, 1993: 415-432; Weisenthal L M, Contrib Gynecol Obstet 1994; 19: 82-90). The sensitivity or resistance may also be measured in animal by measuring die tumor size reduction over a period of time, for cxample, 6 month for human and 4-6 weeks for mouse. A composition or a method sensitizes response to a therapeutic treatment if die increase in treatment sensitivity or the reduction in resistance is 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2-fold, 3-fold, 4-fbld, 5-fbld, 10-fold, 15-fbld, 20-fold or more, compared to treatment sensitivity or resistance in the absence of such composition or method. The determination of sensitivity or resistance to a therapeutic treatment is routine in the art and withm the sluli ofanoi lmariiy skilled clinician. It is to be understood that any method described herein for enhancing the efficacy of a cancer therapy can be equally applied to methods for sensitizing hyperproliferative or otherwise cancerous cells (e.g., resistant cells) to the cancer therapy.
The term "synergistic effect" refers to the combined effect of two οτ more anticancer agents or chcrnotherapy drugs can be greater than the sum of the separate effects ofthe anticancer agents CT chemotherapy drugs alone.
The term "subject" refers to any beaithy animal, mammal or human, or any animal, mammal or human afflicted with a condition of interest (e.g., cancer). The term "subject*' is interchangeable with "patient."
The language "substantially free of chemical precursors or other chemicals'' includes preparations of antibody, polypeptide, peptide or fusion protein in which the protein is separated from chemical precursors or other chemicals which arc involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals'' includes preparations of antibody, polypeptide, peptide or fusion protein having less than about 30% (by dry weight) of chemical precursors or non- antibody, polypeptide, peptide or fusion protein chemicals, more preferably less than about 20% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, still more preferably less than about 10% chemical precursors or non-antibody, polypeptide, peptide or fusion protein chemicals, and most preferably less than about 5% chemical precursors or non- antibody, polypeptide, peptide or fusion protein chemicals.
The term "substantially pure cell population" refers to a population of cells having a specified cell marker characteristic and differentiation potential that is at least about 50%, preferably at least about 75-80%, more preferably at least about 85-90%, and most preferably at least about 95% ofthe cells making up the total cell population. Thus, a "substantially pure cell population" refers to a population of cells mat contain fewer than about 50%, preferably fewer than about 20-25%, more preferably fewer than about 1 -15%, and most preferably fewer than about 5% of cells that do not display a specified marker characteristic and differentiation potential under designated assay conditions. As used herein, the term "survival" includes alt of the following: survival until mortality, also known as overall survival (wherein said mortality may be citber irrespective of cause or tumor related); "recurrcncc-frec survival" (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point (e.g. time of diagnosis or start of treatment) and end point (eg. death, recurrence οτ metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.
A "transcribed por/nuc!ccttden or ' iucleoddc transcript" is a polynucleotide (e.g. an mRNA, hnRNA, cD A, mature rniRNA, pre-miRNA, pri-rniRNA, nriRNA*, anti- imRNA, or a rniRNA binding site, or a variant thereof or an analog of such RNA or cDNA) which is cornplementary to or homologous with all or a portion of a mature mRNA made by transcription of a marker of the invention and normal post-transcriptional processing (e.g. splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript
As used herein, the term "vector" refers to a nucleic acid capable of ttaitsporting another nucleic acid to which it has been linked. One type of vector is a "plasmi T, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are opcrativery linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmidT and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors {e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
An *tadeicxprasion" or "significantly tower level of expression or copy number" of a marker refers to an expression level or copy number in a test sample that is greater than the standard error of the assay employed to assess expression or copy number, but is preferably at least twice, and more preferably three, four, five or ten or more times less than the expression level or copy number of the marker in a control sample (e.g., sample from a healthy subject not afflicted with cancer) and preferably, the average expression level or copy number of the marker in several control samples.
There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can code for the protein, as defined by die genetic code (shown below). Likewise, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.
GENETIC CODE
Alanine (Ala, A) GCA, GCC, GCG, GCT
Arginine (Arg, R) AGA, ACG, CGA, CGC,
Asparagine (Asn, N) AAC, AAT
Aspartlc acid (Asp, D) GAC, GAT
Cysteine (Cys, C) TGC, TGT
Glutamic acid (Glu, E) GAA, GAG
Glutamine (Gin, Q) CAA, CAG
Glycine (Gly, G) GGA, GGC, GGG, GGT
Histidine (His, H) CAC, CAT
Isoleucine (lie, I) ATA, ATC, ATT
Leucine (Leu, L) CTA, CTC, CTG, CTT,
Lysine (Lys, K) AAA, AAG
Methionine (Met, M) ATG
Phenylalanine (Phe, F) TTC, TTT
Pzoline (Pro, P) CCA, CCC, CCG, CCT
Serine (Ser, S) AGC, AGT, TCA, TCC,
Threonine (Thr, T) ACA, ACC, ACG, ACT
Tryptophan (Trp, W) TGG Tyrosine (Tyr, Y) TAC, TAT
Valine (Val, V) GTA, GTC, GTG, GTT
Termination signal (end) TAA, TAG, TGA
An important and well known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefbrc, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences arc considered functionally equivalent since they result in die production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrirmdine may be found in a given nucleotide sequence. Such methylatioro do not affect the coding relationship between the trinucleotide codon and the conxsponding amino acid.
In view of the foregoing, the nucleotide sequence of a DNA or RNA coding for a fusion protein or polypeptide of die invention (or any portion thereof) can be used to derive the fusion protein or polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence. Likewise, for a fusion protein or polypeptide amino acid sequence, coiTesponding nucleotide sequences that can encode the fusion protein or polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given arnino acid sequence). Thus, description and or disclosure herein ofa nucleotide sequence which encodes a fusion protein or polypeptide should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence.
Similarly, description and/or disclosure of a fusion protein or polypeptide amino acid sequence herein should be considered to also include description and or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.
Finally, nucleic acid and amino acid sequence information for the loci and biomarkcrs of the present invention (eg., biomarkcrs listed in Tables 1-5 and Examples) are well known in die art and readily available on publicly available databases, such as the National Center for Biotechnology Infbnnation (NCBQ- For example, exemplary nucleic acid and amino acid sequences derived from publicly available sequence databases arc provided below. The nucleic acid and amino acid sequences of a representative human DM6A biomarker (also known as UTX or MGC141941 or bA386N14.2 or DKFZp686A03225) is available to the public at the GenBank database under NMJD21140.2 and NP_0066963.2. Nucleic acid and polypeptide sequences of DM6A ortnologs in organisms other man bumans are well known and include, for example, mouse KD 6A (NM_009483.1 and NP_033509. IX rat KDM6A (XM.002730I 85.2 and XP 002730231.1). chimpanzee KDM6A (XM_002806207.1 and XP_002806253.1), chicken KDM6A (X .416762J and XP_416762.3 fruit fly DM6A (NM 001201844.1 and NP_001188773.1 ), and worm KDM6A ( MJ077049J and NP.509450.1).
The nucleic acid and amino acid sequences of a representative human KDM6B biomarker (also known as JMJD3 or 1AA0346) is available to die public at the GenBank database under NM_001080424.1 and NP 001073893.1. Nucleic acid and polypeptide sequences of KDM6B ortnologs in organisms other than humans are well known and include, for example, dog KDM6B (XM.546599.3 and XP.546599.2X mouse DM6B (NMJXHOl 7426.1 and NP_001017426.1 ), rat D 6B (NM_001108829.1 and
NP.OOl 102299.1), and zebrafish DM6B (XM 00 198938.1 and XP_003198986.1 and NM_001030178.1 and NP_001025349.1).
At least five splice variants encoding five human EZH2 isoforms exist The sequence of human EZH2 transcript variant 1 is the canonical sequence, all positional information described with respect to the remaining isoforms are determined from this sequence, and the sequences are available to the public at the GenBank database under NM_004456.4 and PJ0O4447.2. The sequences of human EZH2 transcript variant 2 can be found under NM_ 152998.2 and NP_694543.1 and the encoded protein replaces the residues HP of positions 297-298 of the canonical sequences with HR CNYS. The sequences of human EZH2 transcript variant 3 can be found under NM_001203247.1 and NPjOOl 190176.1 and the encoded protein deletes residues 83-121 of die canonical sequence. The sequences of human EZH2 transcript variant 4 can be found under
NM_001203248.1 and NP.OOl 190177.1 and the encoded protein deletes residues 74-82 of the canonical sequence. The sequences of human EZH2 transcript variant 5 can be found under NM_001203249.1 and NP_001190178.1 and fee eiKoded rjrotem o^lctcs n»idues 74- 82 of the canonical sequence, as well as replaces the residues
O!^1WNYQK3)tu^QKI^ of positions 511-553 with G.
The catalytic site of EZH2 is believed to reside in a conserved domain of the protein known as the SET domain. The amino acid sequence of the SET domain of EZH2 is provided by the following partial sequence spanning amino acid residues 613-726 of human EZH2 isoform 1 described above and as follows:
HMJAPSDVAGWGrH IX^^
ATR GT^KIRFAMISVNFNCYA VM V ^ Additional sequences and structural information is publicly available in the art (e.g. , U.S. Pat PubL 2013- 0040906). Nucleic acid and polypeptide sequences of EZH2 ortbologs in organisms other than humans are wdl known and include, for example, mouse EZH2 (NM_0O707l .2 and NP_031997.2 and NMJXH 146689.1 and NP.OOI 140161), chimpanzee EZH2
(NM_001266503.1 and NPjOOl 253432.1), cow EZH2 (NM_O0l 193024.1 and
NP_001179953.1 ), and rat EZH2 (N .001134979.1 and NP.OOl 128451.1).
The nucleic acid and amino acid sequences of a representative human HMGN1 biornarker is available to the public at the GenBank database under NM_004%5.6 and NP_0049565. Nucleic acid and polypeptide sequences of H GN1 orthologs in organisms other than humans are well known and include, for example, monkey H GN1
(XM OOl 113912.2 and XPJJ01113912.1), chimpanzee HMGN1 (XM.514899.4 and XP.514899.2X and cow HMGN1 (XM.002697394.1 and ZP.002697440).
In addition, eukaryotes have chromatin arranged around proteins in the form of nucleosomes, which arc the smallest sublimits of chromatin and includes approximately 146-147 base pain ofDNA wrapped around an octamerofec^histoner^teins (two each of H2A, H2B, H3, and H4). Triinethyiation of histone H3 on Lys 27 (H3K27me3) is key for cell fate regulation. Mammalian cells have three known sequence variants of histone H3 proteins, denoted H3.1, H3.2 and H3.3, that are highly conserved differing in sequence by only a few amino acids. As used herein, the terra "histone H3" can refer to H3.1, H3.2, or H3.3 individually or collectively. The sequences are as follows:
Histone H3.1:
MAR1K<^AR STCX)KAPRKQI 1KA^
Histone H3.2:
MARTK^ARKSTGGKAPRKQIAIXA^
Histone H3..V.
MARTX(^ARKi»K}GKAP QLAri AAR^
These amino acid sequences include a methionine as residue No. I that is cleaved off when the protein is processed, hence what is lysine 28 in the amino acid sequences above corresponds to lysine ( ) 27. These three protein variants arc encoded by at least fifteen different genes/transcripts. Sequences encoding the histone H3.1 variant arc publicly available as HIST1H3A (NM.003S2 .2; NP_003520.1), HIST1H3B (NM_003537.3; NP_003528.1X HIST1 H3C (NM_0035312; PJ)03522.1), HISTIH3D (NM_003530.3; NP_003521.2), H1ST1H3E (NM 003532.2; NP_003523.1 ), HISTIH3F (NM 021018.2; NPJ0662 8.I),
H1ST1H3G (NM_003534.2; NP_003525.I), HIST1H3H (NM_003536.2; NP_003527.1), H1ST1H3I (NM 003533.2; NP_003524.1), and HIST1H3J (NM_003535.2; NP_003526.1). Sequences encoding the histone H3.2 variant are publicly available as HIST2H3A
(NM_0i)1005 64.2; NP_001005464.1 ), HIST2H3C (NM_021059J2; NP_066403.2), and H1ST2H3D ( M 001123375.1; NP 001116847.1). Sequences encoding the histone H3.3 variant are publicly available as H3F3A (NM_002107.3; NP_002098.I ) and H3F3B ( .00S324.3; NP_005315.1). See U.S. Pat. Publ. 2012/0202843 for additional details. Antibodies for die detection of H3K27mc3 and methods for making diem are known in die art
SEQ IP NO; 1 Human K NftA cPNA StwwMfC
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
SEQI W;2 Hwron KP ATOWP Arid Swsaa.
Figure imgf000053_0002
SEQI W; ? MWHC PMftA sPNA fiwunra
Figure imgf000053_0003
Figure imgf000054_0001
Figure imgf000055_0001
SEP ID NO: 4 Mouse DM6A Amino Acid Sequence
Figure imgf000055_0002
01PNQ;? Human ΚΡΜ0Β cPNA S ware
Figure imgf000056_0001
Figure imgf000057_0001
SEQ1PNQ;$ Human KPMfl? Amino Aci^l fawncc
Figure imgf000058_0001
SEP ID NO: 7 Mouse KDM6B cDNA Sequence
Figure imgf000058_0002
Figure imgf000059_0001
Figure imgf000060_0001
SEO!P O.? MpBtc KP ffl Amia Arid SWBCTK
Figure imgf000060_0002
Figure imgf000061_0001
SEQt Nfr? Human BZH2 (iwfonn 1 ) ¾PWA jtoware
Figure imgf000061_0002
Figure imgf000062_0001
SEQ1 W;.Q HwTKW E¾TO (i»fam I) Amino A«d SWWCTW
Figure imgf000062_0002
SEQIPNQ; 11 Human EZH2 (iwform 2) sPNA SWUCTSC
Figure imgf000062_0003
Figure imgf000063_0001
SEQ PNQ; 12 Huron EZH2 (iwfofm 3) cPNAJkaMtaa,
Figure imgf000063_0002
Figure imgf000064_0001
SEO ID NO: 13 Human EZH2 (inform 41 cDNA Sequence
Figure imgf000064_0002
Figure imgf000065_0001
SEP IP NO: 14 Human EZH2 eform SI cDNA Square
Figure imgf000065_0002
Figure imgf000066_0001
SK P O. IS Mouse EZH2 (iwfam 1 ) cPNA SWKTKC
Figure imgf000066_0002
Figure imgf000067_0001
Figure imgf000067_0002
SEQI NO.P Ntotfc EZtiB (reform 2) cPNA SwifflK?
Figure imgf000067_0003
Figure imgf000068_0001
SEP ID NO: 18 Momte EZH2 fanforin ¾ϊ Amino Acid Sequence
Figure imgf000068_0002
SEQI NO;.? Hwnm H Q 1 tPNA Swum*
Figure imgf000068_0003
SEQ I NQ-, ? Hunan HMGN1 Amino Ackt SWWBW
Figure imgf000069_0001
SEP ID NO 21 Rhesus Monkey HMGN1 cDNA Samace
Figure imgf000069_0002
SEP ID NO: 22 Rhesus Monkey HMGN1 Amino Acid Sequence
Figure imgf000069_0003
II, Agents and ona^QOBs
Agents and compositions of the present invention are provided for us in the diagnosis, prognosis, prevention, and treatment of cancer lymphoid cancers, such as leukemia) and cancer subtypes thereof. Such agents and compositions can detect and/or modulate, e.g., up- or down-rcguiaie, expression andor activity of gene products or fragments thereof encoded by biomarkers of the invention, including the biornarkcrs listed in Tables 1-5 and Examples. Exemplary agents include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural Uganda, that can either bind and/or activate or inhibit protein biomarkers of the invention, including the biomarkers listed in Tables 1-5 and Examples, or fragments thereof; RNA interference, amisense, nucleic acid aptamcrs, etc. that can downregulate the expression and/or activity of the biomarkers of the invention, including the biomarkers listed in Tables 1-5 and Examples, or fragments thereof.
In one embodiment, isolated nucleic acid molecules that specifically hybridize with or encode one or more biomarkers listed in Tables 1-5 and Examples or biologically active portions thereof. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (i.e., cDNA or genomic DNA) and RNA molecules (/.&, mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded- but preferably is double-stranded DNA. An "isolated'* nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (Le., sequences located at die 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which tbc nucleic acid is derived. For example, in various embodiments, die isolated nucleic acid molecules corresponding to the one or more biomarkers listed in Tables I -5 and Examples can contain less than about 5 kb, 4kb, 3kb, 2kb. 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived (Le., a leukemic cell). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially f ee of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of one or more biomarkers listed in Tables 1-5 and Examples or a nncleotidc sequence which is at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more (eg., about 98%) homologous to the nucleotide sequence of one or more biomarkers listed in Tables I- 5 and Examples or a portion thereof ( .e. , 100, 200, 300, 00, 50, 500, or more nucleotides), can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a human cDNA can be isolated from a human cell line (from Stratagcne, La Jolla, CA, or Ckmtech, Palo Alto, CA) using all or portion of the nucleic acid molecule, or fragment thereof, as a hybridization probe and standard hybridization techniques (/.«., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor laboratory. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Moreover, a nucleic acid molecule er Xxnpassing all or a portion of the nucleotide sequence of one or more biomarkers listed in Tables 1-5 and Examples or a nucleotide sequence which is at least about 50%, preferably at least about 60% more preferably at least about 70%, yet more preferably at least about 80%, still more preferably at least about 90%, and most preferably at least about 95% or more homologous to the nucleotide sequence, or fragment thereof, can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon the sequence of the one or more biomarkers listed in Tables 1-5 and Examples, or fragment thereof, or the homologous nucleotide sequence. For example, mRNA can be isolated from muscle cells (Le., by the guamdiniura-thiocyanate extraction procedure of Chirgwin el al. (1979) Biochemistry 18: 5294-5299) and cDNA can be prepared using reverse transcriptase (i.e., Moloney MLV reverse transcriptase, available from GibccVBRL. Bcthesda, MD; or AMV reverse transcriptase, available from Setkagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for PCR amplification can be designed according to well-known methods in the art A nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers accenting to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to the nucleotide sequence of one or more biomarkers listed in Tables 1-5 and Examples can be prepared by standard synthetic techniques, te., using an automated DNA synthesizer.
Probes based on the nucleotide sequences of one or more biomarkers listed in Tables 1-5 and Examples can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, i.e., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cc-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which express one or more biomarkers listed in Tables 1 -5 and Examples, such as by measuring a level of nucleic acid in a sample of cells from a subject, Le. , detecting mRNA levels of one or more biomarkers listed in Tables I -5 and Examples.
Nucleic acid molecules encoding protons comspomung to one or more biomarkers listed in Tables 1-5 and Examples from different species are also contemplated. For example, rat or monkey cDNA can be identified based on the nucleotide sequence of a human and/or mouse sequence and such sequences are well known in the art. in one embodiment, the nucleic acid molecule(s) of the invention encodes a protein or portion thereof which includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of one or more biomarkers listed in Tables 1-5 and Examples, such that the protein or portion thereof modulates (eg-, enhance), one or more of the following biological activities: a) binding to the biomarker, b) modulating the copy number of the biomarkcr, c) tnodulaong the expression level of the biomarker, and d) modulating the activity level of the biomarkcr. As used herein, the language "sufficiently homologous'* refers to proteins or portions thereof which have amino acid sequences which include a minimum number of identical or equivalent (eg., an amino acid residue which has a similar side chain as an amino acid residue in one or more biomarkers listed in Tables 1-5 and Examples, or fragment thereof) amino acid residues to an amino acid sequence of me biornarkcr, or fragment thereof, such that the protein or portion thereof modulates ( g., enhance) one or more of die following biological activities: a) binding to the biornarkcr, b) modulating die copy number of die biornarkcr. c) modulating the expression level of the biornarkcr; and d) modulating the activity level of tbe biornarkcr.
In another embodiment, the protein is at least about 50%, preferably at least about
60%, more preferably at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the entire amino acid sequence of the bfomarker, or a fragment thereof.
Portions of proteins encoded by nucleic acid molecules of the one or more biomarkers listed in Tables 1 -5 and Examples are preferably biologically active portions of the protein. As used herein, the term biologically active portion" of one or more biomarkers listed in Tables 1-5 and Examples is intended to include a portion, e.g., a domain motif, that has one or more of the biological activities of die full-length protein.
Standard binding assays, eg., immunoprecim'tations and yeast two-hybrid assays, as described herein, or functional assays, .g., RNAi or overexpression experiments, can be performed to determine the ability of the protein or a biologically active fragment thereof to maimain a biological activity of die full-length protein.
Tbe invention further encompasses nucleic acid molecules that differ from the nucleotide sequence of the one or more biomarkers listed in Tables 1 -5 and Examples, or fragment thereof due to degeneracy of the genetic code and thus encode tbe same protein as that encoded by tbe nucleotide sequence, or fragment thereof. In another erribodimcnt, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence of one or more biomarkers listed in Tables 1-5 and Examples, or fragment thereof, or a protein having an amino acid sequence which is at least about 70%.75%, 80%, 85%.90%, 91%, 92%.93%, 94%, 95%.96%, 97%, 98%.99% or more homologous to the amino acid sequence of the one or more biomarkers listed in Tables 1-5 and Examples, or fragment thereof. In another embodiment, a nucleic acid encoding a polypeptide consists of nucleic acid sequence encoding a portion of a full-length fragment of interest that is tees than 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, or 70 amino acids in length.
It will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the one or more biomarkcrs listed in Tables 1 -5 and Examples may exist within a population (eg., a mammalian and/or human population). Such genetic polymorphisms may exist among individuals within a population doe to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding one or more biomarkcrs listed in Tables 1-5 and Examples, preferably a mammalian, e.g., human, protein. Such natural allelic variations can typically result in 1 -5% variance in the nucleotide sequence of the one or more biomarkcrs listed in Tables 1-5 and Examples. Any and all such nucleotide variations and resulting amino acid polymorphisms in the one or more biomarkcrs listed in Tables 1-5 and Examples mat are the result of natural allelic variation and mat do not alter the functional activity of the one or more btornarkers listed in Tables 1-5 and Examples are intended to be within the scope of the invention. Moreover, nucleic acid molecules encoding one or more biomarkcrs listed in Tables 1-5 and Examples from other species.
In addition to naturally-occurring allelic variants of the one or more biomarkers listed in Tables 1-5 and Examples sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequence, or fragment thereof, thereby leading to changes in the amino acid sequence of the encoded one or more biomarkcrs listed in Tables 1-5 and Examples, without altering the functional ability of the one or more biomarkcrs listed in Tables 1-5 and Examples. For example, nucleotide substitutions leading to amino acid substitutions at non-cssentiar amino acid residues can be made in the sequence, or fragment thereof. A '*rtc£-csscntiar amino acid residue is a residue that can be altered from the wild-type sequence of the one or more biomarkers listed in Tables 1-5 and Examples without altering the activity of the one or more biomarkcrs listed in Tables 1-5 and Examples, whereas an "essenriar amino acid residue is required for the activity of the one or more biomarkcrs listed in Tables 1-5 and Examples. Other amino acid residues, however, (eg., those that are not conserved or only sertu-conscrved between mouse and human) may not be essential for activity and thus are likely to be amenable to alteration without altering the activity of die one or more biomarkcrs listed in Tables 1-5 and Examples. The term "sequence identity or homology" refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or sequence identical at that position. The percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shared by die two sequences divided by die number of positions compared x 100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences arc 60% homologous or have 60% sequence identity. By way of example, the DNA sequences ATTOCC and TATOGC share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology. Unless otherwise specified "loop out regions", e.g., those arising from, from deletions or insertions in one of the sequences are counted as mismatches.
Tbc comparison of sequences and a½terniraation of percent homology
between two sequences can be accomplished using a mathematical algorithm.
Preferably, the alignment can be performed using the Ciustal Method. Multiple alignment parameters include GAP Penalty "10, Gap Length Penalty » 10. For
DNA alignments, the pairwise alignment parameters can be Htuplcot2, Gap
penalty3^, Window*" , and Diagonal savcoV . For protein alignments, the pairwise alignment parameters can be Ktuple^l, Gap pcnalty-3, Window-5, and Diagonals Savcd*5.
In a preferred embodiment, the percent identity between two amino acid sequences is deterrnined using the Needleman and Wunsch (/. Mol. Biol. <48):444- 53 ( 1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available online), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. to yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available online), using a
N Sgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using die algorithm of E. Meyers and W. Miller (CABIOS, 4: 11 - 17 ( 1989)) which has been incorporated into the ALIGN program (version 2.0) (available online), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
An isolated nucleic acid molecule encoding a protein homologous to ooe or more biomarlccrs listed in Tables 1-5 and Examples, or fragment thereof, can be created by iritroducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence, or fragment thereof, or a homologous nucleotide sequence such that one or more amino acid substitutions, additions or deletions arc introduced into die encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in die art. These families include amino acids with basic side chains (e.£, lysine, argininc, histidine). acidic side chains (&g., aspartic acid, grutarnic acid), uncharged polar side chains (eg., glycine, asparaginc, ghitamine, serine, threonine, tyrosine, cysteine), nonpotar side chains (e.g., alanine, valine, leucine, isokucine, proline, phenylalanine, mettuonine, tryptophan), branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in one or more biornarkers listed in Tables 1 -5 and Examples is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along ail or part of the coding sequence of the one or more biornarkers listed in Tables 1-5 and Examples, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity described herein to identify mutants that retain desired activity. Following mutagenesis, the encoded protein can be expressed recombinant^ according to well-known methods in the art and the activity of the protein can be determined using, for example, assays described herein.
The levels of one or more biornarkers listed in Tables 1 -5 and Examples levels may be assessed by any of a wide variety of well-known methods or detecting expression of a transcribed molecule or protein. Non-limiting examples of such methods include immunological methods for detection of proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods. in preferred embodiments, the levels of one or more biomarkcrs listed in Tables 1-5 and Examples levels are ascertained fay measuring gene transcript (e.g., mR A), by a measure of the quantity of translated protein, or by a measure of gene product activity. Expression levels can be monitored in a variety of ways, including by detecting mR A lev s, protein levels, or protein activity, any of which can be measured using standard techniques. Detection can involve quantification of the level of gene expression (eg.. genomic DNA, cDNA, mRNA, protein, or enzyme activity), or. alternatively, can be a qualitative assessment of die level of gene expression, in particular in comparison with a control level. The type of level being detected will be clear from tbe context
In a particular embodiment, the mRNA expression level can be determined both by in situ and by in vitro formats in a biological sample using methods known in the art The term biological sample'' is intended to include tissues, cells, biological fluids and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject Many expression detection methods use isolated RNA. For in vitro methods, any RNA isolation technique mat does not select against the isolation of mRNA can be utilized for the purification of RNA from cells (see, e.g., Ausubel et at., ed.. Current Protocols in Molecular Biology, John Wiley ft Sons, New York 1987-1999). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of
Chomczymki (1989, U.S. Patent No.4,843,155).
The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One preferred diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by die gene being detected. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to a mRNA or genomic DNA encoding one or more biomarkcrs listed in Tables 1 -5 and Examples. Other suitable probes fix use in the diagnostic assays of the invention ate described herein. Hybridization of an mRNA with the probe indicates that one or more biomarkcrs listed in Tables 1-3 and Examples is being expressed.
In one format, die mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and tntrisferring the mRNA from die gd to a membrane, such as nitrocellulose. In an alternative format, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in a gene chip array, e.g., an AfiymetrixTM gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of the One or more biomarkers listed in Tables 1-5 and Examples mRNA expression levels.
An alternative method for determining mRNA expression level in a sample involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, US. Patent No.4,683,202), ligase chain reaction (Barany, 19 1 , Proc. Nari. Acad. Sci. USA. 88: 189-1 3), sdf-sustained sequence replication (Guateili ei a/., 1990. Proc. Natl. Acad. Scf. USA 87: 1874-1878), transcriptional amplification system ( woh ei ai, 1989, Proc. Natl Acad. Set USA 86:1173-1177), Q-Beta Replicase (Lizardi ei ai, 1988, Bio/Technology 6: 1197), rolling circle replication (Lizardi ei /., U.S. Patent No. 3,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well-known to those of skill in die art These detection schemes arc especially useful for the detection of nucleic acid molecules if such molecules arc present in very low numbers. As used hereto, amplification primers are defined as being a pair of nucleic acid molecules mat can anneal to 5 ' or 3 * regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising die nucleotide sequence flanked by the primers.
For in situ methods, mRNA does not need to be isolated from the cells prior to detection. In such methods, a cell or tissue sample is prepared/processed using known histological methods. The sample is then immobilized on a support, typically a glass slide, and then contacted wi ih a probe that can h bridize to the One or more biomarkers listed in Tables 1 -5 and Examples mRNA.
As an alternative to making determinations based on the absolute expression level, determinations may be based on the normalized expression level of one or more biomarkers listed in Tables 1-5 and Examples. Expression levels are normalized by collecting the absolute expression level by comparing its expression to the expression of a non-biomarker gene, e.g., a housekeeping gene that is constitutivefy expressed. Suitable genes for normalization include housekeeping genes such as the actin gene, or epithelial cell-specific genes. This normalization allows the comparison of the expression level in one sample, eg., a subject sample, to another sample, .g., a normal sample, or between samples from different sources.
The level or activity of a protein corresponding to one or more biornarkers listed in Tables 1 -5 and Examples can also be detected and/or quantified by detecting or quantifying the expressed polypeptide. The polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art. These may include analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLQ, hyperdifraskm chromatography, and the like, or various immunological methods such as fluid or gd precipitin reactions, immunodiffusion (single or double), Immunoelectrophoresis, radioimmunoassay ( IA), enzyme-linked immunosorbent assays (EULSAsX
immunofluorescent assays. Western blotting, and the like. A skilled artisan can readily adapt known protein/antibody detection methods for use in determining whether cells express the biomarker of interest.
The present invention further provides soluble, purified and or isolated polypeptide forms of one or more biornarkers listed in Tables 1-5 and Examples, or fragments thereof. n addition, it is to be understood that any and all attributes of the polypeptides described herein, such as percentage identities, polypeptide lengths, polypeptide fragments, biological activities, antibodies, etc. can be combined in any order or combination with respect to any biomarker listed in Tables 1-5 and Examples and combinations thereof.
In one aspect, a polypeptide may comprise a full-length amino acid sequence corresponding to one or more biornarkers listed in Tables 1-5 and Examples or a full-length amino acid sequence with 1 to about 20 conservative amino acid substitutions. An amino acid sequence of any described herein can also be at least SO, SS, 60, 65, 70, 75, 80, 85, 90, 91 , 2, 93, 94, 95, 96, 97, 98, 9, or 99.5% identical to the full-length sequence of one or more biomarkers listed in Tables 1 -5 and Examples, which is either described herein, well known in die art, or a fragment thereof. In another aspect, the present invention contemplates a composition comprising an isolated polypeptide corresponding to one or more biomarkers listed in Tables 1-5 and Examples polypeptide and less than about 25%, or alternatively 15%, or alternatively 5%, contaminating biological macromolecules or polypeptides. The present invention further provides compositions related to producing, detecting, characterizing, or modulating the level or activity of such polypeptides, or fragment thereof, such as nucleic acids, vectors, host ceils, and the like. Such compositions may serve as compounds mat modulate the expression and/or activity of one or more biomarkcrs listed in Tables 1-5 and Examples. For example, HMGN1 polypeptides can be used to reduce H3K27me3 and thereby allow lymphoid cells, such as lymphoid progenitors, to proliferate or, alternatively, agents that reduce HMGN1 polypeptide levels or activity can be used to stop proliferation of lymphoid cell (e.g., DS-ALL cells).
An isolated polypeptide or a fragment thereof (or a nucleic acid encoding such a polypeptide) corresponding to one or more biomarkcrs of the invention, including the biomarkcrs listed in Tables 1-5 and Examples or fragments thereof, can be used as an immunogen to generate antibodies that bind to said immunogen, using standard techniques for polyclonal and monoclonal antibody preparation according to well-known methods in the art. An antigenic peptide comprises at least 8 amino acid residues and encompasses an epitope present in the respective full length molecule such that an antibody raised against the peptide forms a specific immune complex with the respective full length molecule. Preferably, the antigenic peptide comprises at least 10 amino acid residues. In one embodiment such epitopes can be specific for a given polypeptide molecule from one species, such as mouse or human (I.e., an antigenic peptide that spans a region of the polypeptide molecule that is not conserved across species is used as immunogen; such non conserved residues can be determined using an alignment such as that provided herein).
For example, a polypeptide immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for example, a recombinandy expressed or chemically synthesized molecule or fragment thereof to which the immune response is to be generated. The preparation can further include an adjuvant, such as Freunrfs complete or incomplete adjuvant, or similar inummostmiulatory agent. Immunization of a suitable subject with an immunogenic preparation induces a polyclonal antibody response to the antigenic peptide contained therein.
Polyclonal antibodies can be prepared as described above by imrminizing a suitable subject with a polypeptide immunogen. The polypeptide antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the antibody directed against the antigen can be isolated from the mammal {e.g., from die blood) and further purified by well-known techniques, such as protein A chromatography, to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the antibody titers arc highest, amfoody-rxoducing cells can be obtained from tbc subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique (originally described by ohier and Milstein (1975) Nature 256:495-497) (see also Brown etal. (1981) J. Immunol. 127:539-46; Brown el al. (1980) J. Biol. Ckem. 255:4980-83; Yen el al. (1976) Proc. Natl. Acad. Set. 76:2927-31; Yen et al. (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor el al. ( 1983) Immunol. Today 4:72), the EBV-hybrido na technique (Cole e al. (1985) Monoclonal Antibodies and Cancer Therapy* Alan R. Use, Inc., pp. 77-96) or triorna techniques. The technology for producing monoclonal antibody bybridomas is well known (see generally Kenneth, R. H. in
Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp.. New York. New York (1980); Umer, E. A. (1981) Yale J. Biol. Med. 54:387-402; Gefter, M. L. etal. (\ 977) Somatic Cell Genet 3:23 -36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically spienocytes) from a mammal immunized with an immunogen as described above, and the culture supernatants of the resulting hybridoma cells arc screened to identify a hybridoma producing a monoclonal antibody that binds to the polypeptide antigen, preferably specifically.
Any of the many well-known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating a monoclonal antibody against one or more biornarkers of the invention, including the biomarkers listed in Tables 1-5 and Examples, or a fragment thereof (see, e.g., Galfre, G. et al (1977) Nature 266:550- 52; Gefter el al. ( 1977) supra; Lerner (1981) supra, Kenneth (1980) supra). Moreover, the ordinary skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, rnurine bybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthinc, aminopterin and myrnidinc ("HAT medium*'). Any of a number of myeloma celt lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/0-Agl4 myeloma lines. These myeloma tines are available from the American Type Cubure Collection (ATCC), Rockville, MD. Typically, HAT-sensitive mouse myeloma cells are rased to mouse splenocytes using polyeth lene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not
transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supematants for antibodies that bind a given polypeptide, eg., using a standard EUSA assay.
As an alternative to preparing monoclonal ami body-secreting hybridomas, a monoclonal specific for one of the above described polypeptides can be identified and isolated by screening a recombinant combinatorial inmuinogiobulin library (e.g., an antibody phage display library) with the appropriate polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide. Kits for generating and screening phage display libraries are «jmmercialry available (e.g., the Pharmacia
Recombinant Phage Antibody System, Catalog No.27-9400-01 ; and the Stratagene
S rfZAP™ Phage Display Kit, Catalog No.240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening an antibody display library can be found in, for example, Ladner el al U.S. Patent No. 5,223,409; ang et al Intcrnatkmal Publication No. WO 92/18619; Dower et al mternarjonal Publication No. WO 91/17271; Winter et at. International Publication WO 92/207 1; arkland et al.
mernatJonal Publication No. WO 92 15679; Breitling elal nteriiationai Publication WO 93 01288; McCafferty etai International Publication No. WO 92/01(>47; Garrard */ a . Imcnutfjonal Publication No. WO 92/09690; Ladner et al. International Publication No. WO 9002809; Fuchs et al. (\99\) Biotechnology (NY) 9A 69-\372, Hay et al. (1992) Num. Anttbod. Hybrtdt>mas 3:Hl-85-, H\iic etai (1989)5c/em»246:l27S-128l; Griffimser al. (1993) EMBOJ. 12:725-734; Hawkins (1992)7. Mol. Biol. 226:889-896; Clarkson et al. (1991 ) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Set. USA 89:3576- 3580; Garrard etai. (1991 ) Biotechnology (NY) 9: 1373-1377; Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137; Barbas et al. (1991 ) Proc. Natl. Acad. ScL USA 88:7978- 7982; and McCaficrty et al. ( 1990) Nature 348:552-554.
Additionally, recombinant polypeptide antibodies, such as chimeric and humanized monoclonal antibodies, comprising bom human and non-human portions, whkh can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Patent Publication PCT/US86/02269; Akira et al. European Patent
Application 184,187; Ta iguchi, M. European Patent Application 171,496; Morrison era/. European Patent Application 173,494; Neuberger et al PCT Application WO 86/01533; Cabiliy et al. U.S. Patent No.4,816,567; Cabilly et al. European Patent Application 125.023; Better el al (1988) Science 240:1041-1043; Lhi elal. (1987) Proc. Natl. Acad. Scl. USA 84:3439-3443; Liu etal. (1987) Immunol 139:3521-3526; Sun etaL (1987) Proc. Natl. Acad. Set. 84:214-218; Nishimura etal. (1987) Cancer Res. 47:999-1005;
Wood* a/. (1985) Atom*? 314:446-449; Shaw elal (1 88) J Nail. Q cer/nst. 80:1553- 1559); orrison, S. L. (1985) Science 229:1202-1207; Qi etal. (1986) Btotechniques 4:214; Winter U.S. Patent 5,225,539; Jones et al. (1986) Nature 321:552-525; Vcrboeyan et al (1988) Science 239:1534; and Beidlcr etal (1988) J. Immunol. 141:4053-4060.
In addition, humanized antibodies can be made according to standard protocols such as those disclosed in U.S. Patent 5,565,332. fn another embodiment, antibody chains or specifk binding pair members can be produced by recombination between vectors comprising nucleic acid molecules encoding a fusion of a polypeptide chain of a specific binding pair member and a component of a replicable generic display package and vectors containing nucleic acid molecules encoding a second polypeptide chain of a single binding pair member using techniques known in die art, e.g. , as described in U.S. Patents 5,565,332, 5,871,907, or 5.733.743. The use of intracellular antibodies to inhibit protein function in a ceil is also known in the art (see e.g., Carlson, J. R. (1988) Mol. Cell Biol. 8:2638-2646; Biocca, S. etal (1990) EMBOJ. 9:101-108; Werge, T. ML etal (1990) FEBSLett.
274:193-198; Carlson, J. R. (1993) Proc. Natl. Acad. Scl USA 90:7427-7428; Marasco. W. A. etal. (1993) Proc. Natl Acad. Set. USA 90:7889-7893; Biocca, S. el al (1994)
Biotechnology (NY) 12:396-399; Chen, S-Y. etal (1994) Hum. Gene Ther. 5:595-601; Duan, Letal (1994) Proc. Natl. Acad. Scl USA 91:5075-5079; Chen, S-Y. etal. (1994) Proc. Natl. Acad. Sci. USA 91:5932-5936; Beerli, R. R. etal. (1994)/ Biol. Che .
269:23931-23936; Beerli. R. R. elal. (1994) Biochem. Btophys. Res. Commun. 204:666- 672; Mhashilkar. A. M. etal. (1995) EMBOJ. 14 1542-1551; Richardson, J. H. etal (1995) Proc. Natl Acad. Sci. USA 92:3137-3141; PCT Publication No. WO 94/02610 by Marasco et al ; and PCT Publication No. WO 95 03832 by Duan et al). Additionally, fully human antibodies could be made against biomarkers of the invention, including die biomarkers listed in Tables 1-5 and Examples, or fragments thereof. Fully human antibodies can be made in mice that are transgenic for human immunoglobulin genes, eg. according to Hogan, et «/., "Manipulating the Mouse Embryo: A Laboratory Manuel," Cold Spring Harbor Laboratory. Briefly, transgenic mice are immunized with purified immunogen. Spleen cells are harvested and fused to myeloma cells to produce hybridomas. Hybridomas are selected based on their ability to produce antibodies which bind to the immunogen. Fully human antibodies would reduce the immunogenki y of such antibodies in a human.
In one embodiment, an antibody for use in the instant invention is a bispecifk antibody. A bispecifk antibody has binding sites for two different antigens within a single antibody polypeptide. Antigen binding may be sirmiltaneous or sequential. Triomasand hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Examples of bispecific antibodies produced by a hybrid rrybridoma or a trioma are disclosed in U.S. Patent 4,474,893. Bispecific antibodies have been constructed by chemical means (Stacrz ei oL ( 1985) Nattire 314.-628, and Perez et at. (198S) Nature 316:354) and hybridoma technology (Stacrz and Bcvan (1986) Proc. Natl. Acad Set. USA, 83: 1453, and Staerz and Bevan ( 1 86) Immunol. Today 7:241). Bispecific antibodies are also described in U.S. Patent 5,959,084. Fragments of bispecific antibodies are described in U.S. Patent 5.798,229.
Bispecific agents can also be generated by making heterohybridomas by fusing hybridomas or other cells making different antibodies, followed by identification of clones producing and co-assembling both antibodies. Tbey can also be generated by chemical or genetic conjugation of complete immunoglobulin chains or portions thereof such as Fab and Fv sequences. The antibody componcni can bind to a polypeptide or a fragment thereof of one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples, or a fragment thereof. In one embodiment, the bispecifk antibody could specifically bind to both a polypeptide or a fragment thereof and its natural binding partnc s) <* * fragments) thereof.
In another aspect of this invention, peptides οτ peptide nrimetics can be used to antagonize or promote the activity of one or more biomarkers of the invention, inducting one or more biomarkers listed in Tables 1 -5 and Examples, or a fragments) thereof. In one embodiment, variants of one or more biomarkers listed in Tables 1-5 and Examples which fiinctkm as a modulating agent for Ac respective full length protein, can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, for antagonist activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of variants can be produced, for instance, by enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such mat a degenerate set of potential polypeptide sequences is expressible as individual polypeptides containing the set of polypeptide sequences therein. There are a variety of methods which can be used to produce libraries of polypeptide variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential polypeptide sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see. e&, Narang. S. A. (1983)
Tetrahedron 393; Itakura elaL (\ 84) Amu. Rev. Biochem. 33:323; Itakura et al ( 1984) Science 198: 1056; Ike et al. (1983) Nucleic Acid Res. 11 :477.
h addition, libraries of fragments of a polypeptide coding sequence can be used to generate a variegated population of polypeptide fragments for screening and subsequent selection of variants of a given polypeptide. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a polypeptide coding sequence with a nuclease under conditions wherein nicking occurs only about once per polypeptide, denaturing the double stranded DNA, tenanting the DNA to form double stranded DNA which can include sense antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-tcrminal, C-terminal and internal fragments of various sizes of the polypeptide.
Several techniques are known m the art for screening gene products of
combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of polypeptides. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of interest (Arktn and Youvan (1992) Proc. Natl. Acad Scl. (JSA 89:7811-7815; Ddagravc etoL (1993) Protein Eng. 6(3): 327-331). In one embodiment, ceil based assays can be exploited to analyze a variegated polypeptide library. For example, a library of expression vectors can be transfected into a ceil line which ordinarily synthesizes one or more biomarkers of the invention, including one or more biomarlccrs listed in Tables 1-5 and Examples, or a fragment thereof. The transfected cells are then cultured such that the full length polypeptide and a particular mutant polypeptide are produced and the effect of expression of the mutant on the full length polypeptide activity in cell supernaranrs can be detected, &g., by any of a number of functional assays. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of full length polypeptide activity, and the individual clones further characterized.
Systematic substitution of one or more amino acids of a polypeptide amino acid sequence with a D-amino acid of the same type (e.g., D-iysinc in place of L-lysine) can be used to generate more stable peptides. In addition, constrained peptides comprising a polypeptide amino acid sequence of interest or a substantially identical sequence variation can be generated by methods known in die an (Rizo and Gierasch (1992) Amu. Rev.
Btochem. 61:387, incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
The amino acid sequences disclosed herein will enable those of skill in the art to produce polypeptides corresponding peptide sequences and sequence variants thereof. Such polypeptides can be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding the peptide sequence, f equently as part of a larger polypeptide. Alternatively, such peptides can be synthesized by chemical methods. Methods for expression of heterologous proteins in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in die art and are described further in Maniatis el al Molecular Cloning: A laboratory Manual ( 1989), 2nd Ed., Cold Spring Harbor, N.Y.; Berger and imroel, Methods in Enzyrnology, Volume 152, Guide to Molecular Ooning Techniques (1 87), Academic Press, Inc., San Diego, Cali£; Mem field, J. (1969)7. Am. Chan. Soc. 91:501; Chaiken I. M. (1981) CRCCrit. Rev. Bioc em. 11: 255; Kaiser etal. (1989) Science 243:187; Mcrrifteld, B. (\9U) Science 232:342; Kent, S. B. H. (1988) Annu. Rev. Biochem. 57:957; and Oflord, R_ E. (1980) Semisynthetic Proteins, Wiley Publishing, which are incorporated herein by reference).
Peptides can be produced, typically by direct chemical synthesis. Peptides can be produced as modified peptides, with nonpeptide moieties attached by covalent linkage to the N-lcrminus and/or C-tcnninus. In certain preferred embodiments, either the carboxy- terminus or the amino-tcrminus. or both, are chemically modified. The most common modifications of the terminal amino and carboxyl groups are acctylation and amidation, respectively. Araino-4erminal modifications such as acylation (e.g., acctylation) or alkyiation (e.g., methylation) and carboxy^tcrnunal-n^hTcations such as amidation, as well as other terminal nrodifkations, including cyciization, can be incorporated into various embodiments of the invention. Certain amino-terminal and or carboxy-tcrminal modifications and/or peptide extensions to the core sequence can provide advantageous physical, chemical, biochemical, and pharmacological properties, such as: enhanced stability, increased potency and/or efficacy, resistance to scrum proteases, desirable pharmacokinetic properties, and others. Peptides disclosed herein can be used
therapeutically to treat disease, by ateringcosuflc ilatkm in a patient
Pcptidomimetics (Fauchere, J. (1986) Adv. Drug Res. 15:29; Veber and Freidinger (1985) TINS p.392; and Evans eloL (1987)7. Med. Chem. 30:1229, which are
incorporated herein by reference) are usually developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce an equivalent therapeutic or prophylactic effect. Generally, pcptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or piuumacological activity), but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting o - CH2NH-, -CH2S-, -CH2-CH2-, -CH-CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and - CH2SO-, by methods known m the art and further described in the following references: Spatola, A. F. in "Chemistry and Biochemistry of Amino Acids, Peptides, and ProteimT Wemstcin, B.. cd., Marcel Dekker. New York. p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1 , Issue 3, "Peptide Backbone Modif-cations" (general review); Morlcy, J. S. ( 1980) Trends Pharm. Scl. pp.463-468 (general review); Hudson, D. et al. (1979) Int. J. Pcpl. Prof. Res. 14: 177-185 (-CH2NH-, CH2CH2-); Spatola. A. F. et al (\9*6)lJ/eSci. 38:1243-1249 (-CH2-S); Hano, M. M. (1982)7. Chem. Soc. PeHin Trans. I. 307-314 (-CH-CH-, cis and trans); Almquist, R. G. el al (1 0) J. Med. Chem. 23: 1392- 1398 (-COCH2-); Jennings-White, C. et al. (1982) Tetrahedron Lett. 23:2533 (-COCH2-); Swike, M. et al European Appln. EP 4566S (1982) CA: 97:39405 (1982X-CH(OH)CH2- ); Hollada . M. W. et al. (1983) Tetrahedron Lett. (1983) 24:4401-4404 (-C(OH)CH2-); and Hruby, V. J. (\9H2) IJfe Sci. (1982) 31:189-199 (-CH2-S-); each of which is incoiporated herein by reference. A particularly preferred non-peptidc linkage is
-CH2NH-. Such peptide mimettcs may nave significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others. Labeling of pcptidciramctics usually involves covalcnt attachment of one or more labels, directly or through a spacer {e.g., an amide group), to non-interfering positions) on the pe tidomimctje that are predicted by quantitative structure-activity data and/or molecular modeling. Such non-interfering positions generally are positions mat do not form direct contacts with the rnaaopolypeptidcs(s) to which the peptidomimetic binds to produce the therapeutic effect Derivitizaiion (eg., labeling) of peptidonrimetics should not substantially interfere with the desired biological or pharmacological activity of the peptktomimctic.
Also encompassed by the present invention are small molecules which can modulate (either enhance or inhibit) inteiactkms, eg., between biornarkers listed in Tables 1-5 and Examples and their natural binding partners, or inhibit activity. The small molecules of the present invention can be obtained using any of die numerous approaches in combinatorial library methods known in the art, including spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. (Lam, K. S. (1997) Anticancer DrugDes. 12:145). In some embodiments, chemical inhibitors of one or more historic H3K27 demethylascs (e.g, MD6A and/or MD6B) are useful. Such inhibitors are well known in the art and include GSK-J4 (ethyl 3- ( <htydrc~lH-beii^dk^
4-y1)ammo)propanoate), which has the chemical formula:
Figure imgf000088_0001
(see, tbc World Wide Web at xccssbio.eom/h«fcx.php new-piow
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et at. (1993) Proc. Nail. Acad. Sci. USA 90:6909; Erb et at. (1994)
Proc. Nad. Acad. Sci. USA 91:11422; Zockeraiatm era/. (1994) J. Med. ( hem. 37:2678;
Cboetal. (1993) Science 261:1303; Carrcll el at. (1994) Angew. Chem. Int. Ed. Engl.
33:2059; Garell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061 ; and in Gallop et al.
(1994) J. Med. Chem. 37:1233.
Libraries of compounds can be presented in solution (e.g., Houghton ( 1992)
Btotechniques 13:412-421 ), or on beads (Lam ( 1991 ) Nature 354:82-84), chips (Fodor
(1993) Nature 364:535-556), bacteria (Ladner USP 5,223,409), spores (Ladncr USP '409), piasmids (Cull et at. (1992) Proc. Natl Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith (1 90) Science 249:386-390) (Devlin (1990) Science 249:404-406); (Cwuia et al. (1990) Proc. Nad. Acad. Sci. USA 87:6378-6382); (Felici (1991) J. Mot. Biol. 222:301-
310); (Ladner supra. ). Compounds can be screened in cell based or non-cell based assays.
(Propounds can be screened in pools (e.g. multiple compounds in each testing sample) or as individual compounds.
The invention also relates to chimeric or fusion proteins of the biomarkers of the invention, including the biornarkcrs listed in Tables 1-5 and Examples, or fragments thereof. As used herein, a "ch rteric protein" or "fusion protein" comprises one or more bomarkers of the mvention, including one or more biornarkcrs listed in Tables 1-5 and Examples, or a fragment thereof, opcranvc!y linked to another polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the respective biomarker. In a preferred embodiment, the fusion protein comprises at least one biologically active portion of one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples, or fragments thereof. Within die fusion protein, the term "operatively linked" is intended to indicate that the bionnrkcr sequences and the rjon-biomarker sequences are fused in-frame to each other in such a way as to preserve functions exhibited when expressed independently of the fusion. The "another" sequences can be fused to the N-terminus or C-terrainus of the biomarkcr sequences, respectively.
Such a fusion protein can be produced by recombinant expression of a nucleotide sequence encoding the first peptide and a nucleotide sequence encoding the second peptide. The second peptide may optionally correspond to a moiety mat alters the solubility, affinity, stability or valency of the first peptide, for example, an immunoglobulin constant region. In another preferred embodiment, the first peptide consists of a portion of a biologically active molecule (e.g. the extracellular portion of the polypeptide or the ligand binding portion). The second peptide can include an irnmunoglobulin constant region, for example, ahuman Ογΐ domain or Cy domain (e.g., the hinge, CH2 and CH3 regions of human IgCy 1. or human IgCy , see eg.. Capon etat. U.S. Patents 5.116,964; 5,580,756; 5,844,095 and the like, incorporated herein by reference). Such constant regions may retain regions which mediate effector function (e.g. Fc receptor binding) or may be altered to reduce effector function. A resulting fusion protein may have altered solubility, binding affinity, stability and/or valency (i.e., the number of binding sites available per polypeptide) as compared to the independently expressed first peptide, and may increase the efficiency of protein purification. Fusion proteins and peptides produced by recombinant techniques can be secreted and isolated from a mixture of cells and medium containing the protein or peptide. Alternatively, the protein or peptide can be retained cytoplasm icalry and the cells harvested, h/sed and the protein isolated. A cell culture typically includes host cells, media and other byproducts. Suitable media for cdl culture arc wdl known in the art. Protein and peptides can be isolated from cell culture media, host cells, or both using techniques known in the art for purifying proteins and peptides. Techniques for inmsfccting host cells and purifying proteins and peptides are known in the art.
Preferably- a fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-fhtme in accordance with conventional techniques, for example employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, fiUing-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols to Molecular Biology, eds. Ausubcl et al. John Wiley & Sons: 1992).
In another embodiment, the fusion protein contains a heterologous signal sequence at its N-terrninus. In certain host cells (e.g., mammalian host ccllsX expression and/or secretion of a polypeptide can be increased through use of a heterologous signal sequence.
The fusion proteins of the invention can be used as immunogens to produce antibodies in a subject Such antibodies may be used to purify the respective natural polypeptides from which the fusion proteins were generated, or in screening assays to identify polypeptides which inhibit the interactions between one or more biomarkers polypeptide or a fragment thereof and its natural binding partncits) or a fragments) thereof.
Also provided herein are compositions comprising one or more nucleic acids comprising or capable of expressing at least 1.2, 3,4, 3, 10, 20 cr more small nucleic acids or antisense oligonucleotides or derivatives thereof, wherein said small nucleic acids or antisense oligonucleotides or derivatives thereof in a ceil specifically hybridize (e.g., bind) under cellular conditions, with cellular nucleic acids (e.g., small non-coding RNAS such as miRNAs, pre-miRNAs, pri-miRNAs, miRNA*, anti-miRNA, a miR A binding site, a variant and/or functional variant thereof cellular mR As or a fragments thereof). In one embodiment, expression of the small nucleic acids or antisense oligonucleotides or derivatives thereof in a cell can enhance or upregulate one or more biological acti vities associated with the corresponding wild-type, naturally occurring, or synthetic small nucleic acids. In another embodiment, expression of the small nucleic acids or antisense oligonucleotides or derivatives thereof in a cell can inhibit expression or biological activity of cellular nucleic acids andor proteins, e.g. , by inhibiting transcription, translation andor small nucleic acid processing of, for example, one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples, or fragments) thereof. In one embodiment, die small nucleic acids or antisense oligonucleotides or derivatives thereof arc small RNAs (e.g., inicroRNAs) or complements of small RNAs. In another embodiment, the small nucleic acids or antisense oligonucleotides or derivatives thereof can be single or double stranded and are at least six nucleotides in length and are less than about 1000, 900, 800, 700, 00, 500, 400, 300, 200, 100, 50, 40, 0, 25, 24, 23, 22, 21,20, 19, 18, 17, 16, IS, or 10 nucleotides in length. In another embodiment, a composition may comprise a library of nucleic acids comprising or capable of expressing small nucleic acids or antiscnse oligonucleotides or derivatives thereof, or pools of said small nucleic acids or antiscnse oligonucleotides or derivatives thereof. A pool of nucleic acids may comprise about 2-5, 5-10, 10-20, 10-30 or more nucleic acids comprising or capable of expressing small nucleic acids or antiscnse oligonucleotides or derivatives thereof.
In one embodiment, binding may be by conventional base pair conmlemcntarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. In general, "antisense" refers to the range of techniques generally employed in the art, and includes any process that relics on specific binding to oligonucleotide sequences.
It is well known in the art that modifications can be made to the sequence of a miRNA or a prc-miRNA without disrupting miRNA activity. As used herein, the term "functional variant" of a miRNA sequence refers to an oligonucleotide sequence that varies from the natural miRNA sequence, but retains one or more functional characteristics of the miRNA (e.g. cancer cell proliferation inhibition, induction of cancer cell apoptosis, enhancement of cancer cell susceptibility to chemo therapeutic agents, specific miRNA target inhibition). In some emrx)diments, a functional variant of a miRNA sequence retains all of the functional characteristics of the miRNA. In certain ernbodiments, a functional variant of a miRNA has a nucleobasc sequence that is a least about 60%, 65%, 70%.75%, 80%, 85%.90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the miRNA or precursor thereof over a region of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 0, 5, 70, 75, 80, 85, 90, 5, 100 or more nuclcobases, or that the functional variant hybridizes to the complement of the miRNA or precursor thereof under stringent hybridization conditions. Accordingly, in certain embodiments the nucleobase sequence of a functional variant is capable of hybridizing to one or more target sequences of the miRNA.
imRNAs and their corresponding stem-loop sequences described herein may be found in miRBase, an online searchable database of miRNA sequences and annotation, found on the worid wide web at rniarorna.sanger.ac.uk. Entries in the miRBase Sequence database represent a predicted hairpin portion of a miRNA transcript (the stem-loop), with information an the location and sequence of the mature miRNA sequence. The miRNA stem-loop sequences in the database are not strictly precursor miRNAs (pre-miR As), and may in some instances include the pre-miRNA and some flanking sequence from the presumed primary transcript The miRNA nucleobase sequences described herein encompass any version of the miRNA, including the sequences described in Release 10.0 of the miRBase sequence database and sequences described in any earlier Release of the miRBase sequence database. A sequence database release may result in the re-naming of certain miRNAs. A sequence database release may result in a variation of a mature miRNA sequence.
bi some embodiments, miRNA sequences of the invention may be associated with a second RNA sequence that may be located on the same RNA molecule or on a separate RNA molecule as the miRNA sequence, fn such cases, the miRNA sequence may be referred to as the active strand, while the second RNA sequence, which is at least partially complementary to die miRNA sequence, may be referred to as the complementary strand. The active and complementary strands are hybridized to create a double-stranded RNA that is similar to a naturally occurring miRNA precursor. The activity of a miRNA may be optimized by maximizing uptake of the active strand and minimizing uptake of the complementary strand by the miRNA protein complex that regulates gene translation. This can be done through rnodificarion and/or design of the complementary strand.
In some embodiments, the complementary strand is modified so that a chemical group other than a phosphate or bydroxyi at its 5' terminus. The presence of the 5' modification apparently eliminates uptake of the complementary strand and subsequently favors uptake of die active strand by die miRNA protein complex. The 5' modification can be any of a variety of molecules known n the an, including Nrfe, NHCOCHb, and biotin. In another embodiment, the uptake of the complementary strand by the miRNA pathway is reduced by m<xrporating nucleotides with sugar modifications in the first 2-6 nucleotides of the complementary strand. It should be noted that such sugar modifications can be combined with the 5* terminal modifications described above to further enhance miRNA activities.
In some embodiments, die complementary strand is designed so that nucleotides in the 3' end of die complementary strand are not complementary to the active strand. This results in double-strand hybrid RNAs that are stable at the 3' end of the active strand but relatively unstable at die 5* end of the active strand. This difference in stability enhances the uptake of the active strand by the nriRNA pathway, while reducing uptake of the complementary strand, thereby enhancing miRNA activity.
Small nucleic acid and/or antisensc constructs of the methods and compositions presented herein can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of cellular nucleic acids (e.g., small RNAs, mRNA, and/or genomic DNA). Alternatively, the small nucleic acid molecules can produce RNA which encodes mRNA, miRNA, pre- miRNA, pri-miRNA, miRNA*, ami-miRNA, or a miRNA binding site, or a variant thereof. For example, selection of plasnrids suitable for expressing die mi RNAs, methods tor inserting nucleic acid sequences into the plasmid, and methods of delivering the reco nbinam plasmid to the ceils of interest are within tbc skill in the art See, for example, Zeng et al (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat BiotechnoL 20:446- 448; Brurranelkamp et al. (2002), Science 296:550-553; Miyagishi et al. (2002), Nat BiotechnoL 20:497-500; Paddison etai. (2002). Genes Dev. 16:948-958; Lee et al (2002), Nat. BiotechnoL 20300-505; and Paul et al (2002), Nat. Biotechnol.20:505-508, the entire disclosures of which are herein incorporated by reference.
Alternatively, small nucleic acids and/or antisense constructs are oligonucleotide probes that arc generated ex vivo and which, when introduced into the cell, results in hybridization with cellular nucleic acids. Such oligonucleotide probes are preferably modified oligonucleotides that are resistant to endogenous nucleases, eg, exonucleascs and/or endonuc!eascs, and are therefore stable in vivo. Exemplary nucleic acid molecules for use as small nucleic acids and/or antisense oligonucleotides are phosphoramidate, phospbothioate and memylphospbonate analogs of DNA (see also U.S. Patents 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van der rol el at. ( 1988) BioTechniqucs 6:958-976; and Stein etal (1988) Cancer Res 48:2659-2668.
Antisense approaches may involve the design of ohgonucleotides (either DNA or RNA) mat are complementary to cellular nucleic acids (&g., complementary to biomarkers listed in Tables 1 -5 and Examples). Absolute complementarity is not required. In die case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. Tbc ability to hybridize will depend on both the degree of∞rnplementariry and die length of the antisensc nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with a nucleic acid (e.g., UNA) it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the an can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
Oligonucleotides that are complementary to the 5' end of the mRNA, eg., the 5' untranslated sequence up to and including me AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3' untranslated sequences of mRNAs have recently been shown to be effective at inhibiting translation of mRNAs as well (Wagner, R. (1994) Nature 372:333). Therefore, oligonucleotides complementary to either die 5* or 3* untranslated, non-coding regions of genes could be used in an antisense approach to inhibit translation of endogenous mRNAs. Oligonucleotides complementary to the 5* untranslated region of the mRN A may include the complement of the AUG start codon. Antisense ougonucleoudcs complementary to mRNA coding regions are less efficient inhibitors of translation but could also be used in accordance with the methods and compositions presented herein. Whether designed to hybridize to thc5', 3' or coding region of cellular mRNAs, small nucleic acids andor antisense nucleic acids should be at least six nucleotides in length, and can be less than about 1000, 00, 800.700, 00, 500, 00, 300, 200, 100, SO, 40, 30, 25, 24.23, 22, 21,20, 19, 18, 17, 16, 15, or 10 nucleotides in length.
Regardless of the choice of target sequence, it is preferred that in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. In one embodiment these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. In another embodiment these studies compare levels of the target nucleic acid or protein with that of an internal control nucleic acid or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide arc compared with those obtained using a control oligonucleotide. It is preferred that the control oligonucleotide is of approximately die same length as the test oligonucleotide and that die nucleotide sequence of the oligonucleotide differs from die antisense sequence no more man is necessary to prevent specific hybridization to the target sequence.
Small nucleic acids and/or antisense oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded. Small nucleic acids and or antisense oligonucleotides can be modified at die base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc., and may include other appended groups such as peptides (e.g., for targeting host cell receptors), or agents facilitating transport across the cell membrane (see, eg., Lctsinger el al. (1989) Proc. Natl. Acad. Sci. U.S.A.86:6553-6556; Lemaitrc ei al (1987) Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No.
W088/09810, published December 15, 1988) or the blood-brain barrier (see, *&, PCT Publication No. W089/10134, published April 25, 1988), hybridization-triggered cleavage agents. (Sec, e.g., rol el al. (1 88) BwTcchniqucs 6:958-976) or intercalating agents. (See, eg., Zon (1988), Pharm. Res. 5:539-549). To this end, small nucleic acids and/or antisense otigonucleotidce may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, Irybridization-triggered cleavage agent, etc.
Small nucleic acids and/or antisense oiigonuclcotides may comprise at least one modified base moiety which is selected from the group including but not limited to 5- flucfouracii, 5-bromouracil, 5 hlorouraci1, 5-iodouracil, hypoxanthine, xanttne, 4- acetylcytosine, McarboxyhyoYoxytiethyl) uracil 5^arboxyme laminomethyl"2- thiouridine, 5^aiboxymethylaminomemyluracil, dihydrouracil, beta-D-galactosylqueostne, inosine, N6-iscpent∞ytadeninc, 1-methylguanine, 1-nwthylinosine, 2,2-dimethylguaninc, 2-methyladcnine, 2-mem i guanine, 3-metfaylcyt06ine, 5-memylcytostne, N6-adenine, 7-methylguanine, 5-methylammonieayiuracil, 5-metlioxyaminometnyl-2-u^ beta- D-niannosylqueosine, S'-metlioxycartwxynieayiuraci 5-methoxyuracil, 2-mcthylthio-N6- isopcntcnyladcninc, uracil-5-oxyacctic acid (vX wybutoxosine, pscudouractl, queosine,
2- thiocytostne, 5-inclhyl-2-thiouracil 2-thiouraci 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-duouracil, 3-{3-amino-
3- N-2-carboxypropyl) uracil (acp3)w, and 2.6-diaminopurme. Small nucleic acids and/or antisense oligonucleotides may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fhwroarabinose, xylulose, and bexose.
In certain embodiments, a compound comprises an oligonucleotide (e.g.. a miRNA or miRNA encoding oligonucleotide) conjugated to one or more moieties which enhance the activity, cellular distribution or cellular uptake of the resulting oligonucleotide. In certain such embodiments, the moiety is a cholesterol moiety (e.g. , antagomirs) or a lipid moiety or liposome conjugate. Additional moieties for conjugation include carbohydrates, phospholipids, biotin, pbenazme, folate, phenanthridine, amtiraquinone, acridine. fluoresceins, rfoodamines, coumarins, and dyes, in certain embodiments, a conjugate grou is attached directly to die oligonucleotide. In certain embodiments, a conjugate group is attached to the oligonucleotide by a linking moiety selected from amino, hydroxy!, carboxylic acid, thiol, unsaturations (eg., double or triple bonds), fr-anuno-3,6- dioxaoctanoic acid (ADO), succinimidyl 4-( -inaleimidemcthyi) cyclohcxanc-1- carboxylate (SMCO.6-amioobexanoic acid (AH EX or AHA), substitutedCI-ClO alkyl, substituted or unsubstitutcd C2-C10 aikenyl. and substituted or unsubstitu ed C2-C10 alkynyl. In certain such embodiments, a substituent group is selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyi, aryi, aikenyl and alkynyl.
In certain such embodiments, the compound comprises the oUgonucieotide having one or more stabilizing groups that are attached (o one or both termini of the
oligonucleotide to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal rnodifications protect the oligonucleotide from cxonuclcase degradation, and can help in delivery and/or localization within a cell. The cap can be present at the ^-terminus (5*-cap), or at the 3'4enninus (3- cap), or can be present on both termini. Cap structures include, for example, inverted deoxy abasic caps.
Suitable cap structures include a 4\5*-methylene nucleotide, a 1 -{bcta-D- erythrofuranosyl) nucleotide a 4'-thio nucleotide, a carbocyclic nucleotide, a 1,5- anhydrohexitol nucleotide, an L-nuclcotidc, an alpha-nuclcotide, a modified base nucleotide, a phosptorodithioate linkage, a thro-pettofuranosyl nucleotide, an acyclic S'^'-seco nucleotide, an acyclic 3,4-dilrydroxybutyi nucleotide, an acyclic 3,5- dihydroxypentyl nucleotide, a 3-3 -inverted nucleotide moiety, a 3-3 -inverted abasic moiety, a 3'-2'-in verted nucleotide moiety, a 3 -2 -inverted abasic moiety, a 1 ,4-butancdiol phosphate, a 3'-phc*pr«rarriidaie, a hcxylphosphate, an annnobexyi phosphate, a 3- phosphate, a 3'-phc«phoro.hioate, a phospborodithioate, a bridging methylpbosphonate moiety, and a non-bridging methylphosphonate moiety S'-amuxKdkyi phosphate, a 1 ,3- diamino-2-propyl phosphate, 3-aminopropy1 phosphate, a 6-aminohexy 1 phosphate, a 1 -2- aminododecyl phosphate, a hydroxypropyl phosphate, a 5 -S nverted nucleotide moiety, a S'-S'-invcrtcd abasic moiety, a 5'-phosDhoramidate, a 5'-pl»sphoroduoatc, a 5'-amino, a bridging and or non-bridging 5-pliosphoramidate, a phoephorothioatc, and a 5'-mercapto moiety. Small nucleic acids and/or antiscnsc oligonucleotides can also contain a neutral peptidc-likc backbone. Such molecules are termed peptide nucleic acid (PNA)-oiigomcrs and are described, e.g., in Pcrry-O'Kcefc etal (1996) Proc. Nad. Acad. Sci. U.SA
93: 14670 and in Eglora e( al (1993) Nature 365:566. One advantage ofPNA oligomers is their capability to bind to complementary DNA essentially independently from the ionic strength of the medium due to the neutral backbone of me DN A. In yet another embodiment, small nucleic acids and/or antiscnsc oligonucleotides comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioafc, a phc^)horodithioate, a phosphc arriklothioatc, a phosptoiamidate, a ptasphordunrtidatc, a rrKthylphospbonate, an alkyl phosphotriester, and a formacetal or analog thereof.
In a further embodiment, small nucleic acids and or a ti sense oligonucleotides are a-arttmeric oligonucleotides. An a-anomeric oligonocleotide forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al. (1987) Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a 2'-0HT thylr¾onucleotidc (Inouc tt al (1987) Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al ( 1987) FEBS Lett 215:327-330).
Small nucleic acids and/or antisense oligonucleotides of the methods and compositions presented herein may be synthesized by standard methods known in the art. e. ., by use of an automated DNA synthesizer (such as are commercially available from Bioscarch, Applied Biosysfcrm, etc.). As examples, phosphoromtoate oligonucleotides may be synthesized by the method of Stein tt al (1988) Nucl. Acids Res. 16:3209, metnylphospnonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al (1988) Proc. Natl. Acad. Sci. U.S.A.85:7448-7451), etc. For example, an isolated miRNA can be chemically synthesized or recombinant^ pt duccd using methods known in the art In some instances, miRNA are chemically synthesized using appropriately protected ribonucleotide phospborarmdites and a conventional DNA RNA synthesizer. Corrtmcrcial suppliers of synthetic RNA molecules or synthesis reagents include, e.g., Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part ofPerbio Science, Rockf rd.111., USA), Glen Research (Sterling, Va., USA), ChemGencs (Ashland, Mass., USA), Cruachcm (Glasgow, UK), and Exiqon (Vedbaek, Denmark). Small nucleic acids and/or antiscnsc oligonucleotides can be delivered ID cells in vtvo. A number of methods have been developed for delivering small nucleic acids and or antiscnsc oligonucleotides DNA or RNA to cells; eg, antiscnsc molecules can be injected directly into the tissue she, or modified antisense molecules, designed to target the desired cells (*?.£., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on die target cell surface) can be administered systematically.
In one embodiment, small nucleic acids and/or antisense oligonucleotides may comprise or be generated from double stranded small interfering R As (siRNAs), in which sequences fully complementary to cellular nucleic acids (eg. mRNAs) sequences mediate degradation or in which sequences incompletely complementary to cellular nuckic acids (e.g., mRNAs) mediate transiational repression when expressed within cells. In another embodiment, double stranded siRNAs can be processed into single stranded antisense RNAs mat bind single stranded cellular RNAs (e.g.. microRNAs) and inhibit their expression. RNA mterfcrence (RNAi) is the process of sequence-specific, post- transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) mat is homologous in sequence to the silenced gene, in vivo, long dsRNA is cleaved by ribonuclcase HI to generate 21· and 22-miclcotide siRNAs. It has been shown that 21 -nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells (Elbashir et at. (2001) Nature 411 -.494-498). Accordingly, translation of a gene in a cell can be inhibited by contacting the cell with short double stranded RNAs having a length of about IS to 30 nucleotides or of about 18 to 21 nucleotides or of about 1 to 21 nucleotides. Alternatively, a vector encoding for such siRNAs or short !iaiipin RNAs (shRNAs) that are metabolized into siRNAs can be introduced into a target cell (see, t g.. McManus et af. (2002) RNA 8:842; Xia el ah (2002) Nature Biotechnology 20: 1006; and Brummelkamp el al. (2002) Science 296:550). Vectors that can be used are commercially available, e.g., from OligoEngine under the name pSuper RNAi System1".
Ribozyme molecules designed to catalyticalr cleave cellular mRNA transcripts can also be used to prevent translation of cellular mRNAs and expression of cellular polypeptides, or both (Sec, e.g. , PCT International Publication WO90 11364, published October 4, 1990 Sarver etal. (1990) Science 247:1222-1225 and U.S. Patent No.
5,093,246). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy cellular mRNAs, the use of iiammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3\ The construction and production of hammerhead ribozymes is well known in the an and is described more fully in HaselofTand Gerlach (1988) Nature 334:585-591. The ribozyme may be engineered so that the cleavage recognition site is located near the 5' end of cellular mRNAs; i.e., to increase efficiency and minimize the intracellular accumulation of non-ftincrional mRNA transcripts.
The ribozymes of the methods and compositions presented herein also include RNA endoribomicleases (hereinafter "Cech-type ribozymes") such as the one which occurs naturally in Teirah mena thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, er al. (1984) Science 224:574-578; Zaug, etal. (1986) Science 231:470-475; Zaug. et al. (1986) ature 324:429-433; published International patent application No. WO88/04300 by University Patents Inc.; Been, etal. (1986) Cell 47:207-216). The Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place. The rncthods and compositions presented herein encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in cellular genes.
As in the antisense approach, the ribozymes can be composed of modified oligonucleotides (&£., for improved stability, targeting, etc.). A preferred method of delivery involves using a DN A construct "encoding'' the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transacted cells will produce sufficient quantities of the ribozyme to destroy endogenous cellular messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription of cellular genes are preferably single stranded and composed of
deoxyribonucleotides. The base composition of these oiigonucleoidcs should promote triple helix formation via Hoogsteen base pairing rules, which generally require sizable stretches of either purines or pyrimidincs to be present on one strand of a duplex.
Nucleotide sequences may be pyrimtdine-based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix. The pvrimidme-rich molecules provide base coinplcmentarrty to a purineHrich region of a single strand of die dupkx in a parallel orientation to that strand In addition, nucleic acid molecules may be chosen that are purine-rich, for example, containing a stretch of G residues. These molecules will form a triple helix with a ONA duplex that is rich in GC pairs, in which die majority of the purine residues are located on a single strand of the targeted duplex, resulting inCGC triplets across the three strands in the triplex.
Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so called "switchback'* nuclck acid molecule. Switchback molecules are synthesized in an alternating 5'-3\ 3*-5* manner, such that they base pair with first one strand of a duplex and then the other, diminating the necessity for a sizable stretch of either purines or pyrimidincs to be present on one strand of a duplex.
Small nucleic acids (e.g., miRNAs, pre-miRNAs, pri-miRNAs, mi RNA*, and- nuR A, or a rraRNA binding site, or a variant thereof), antisense oligonucleotides, ribozymes, and triple helix molecules of die methods and compositions presented herein may be prepared by any method known in the art for the synthesis of ONA and R A molecules. These include techniques for chemically synthesizing
oligodeoxyribonucleotidcs and oligoribonuclcotides well known in the art such as for example solid phase ptosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by In vitro and in vtvo transcription of DN A sequences erodin the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as die T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
Moreover, various well-known modifications to nucleic acid molecules may be irutoduccd as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotKlcs or dcoxyribonucleoudes to the 5' andor 3' ends of the molecule or the use of phosphoroduoatc or 2* O-methyl rather than phosphodiesterase linkages within the oligodcoxyribonucleotide backbone. One of skill in the art will readily understand that polypeptides, small nucleic acids, and antisense oUgonuclcotides can be urther linked to another peptide or polypeptide (e.g. , a heterologous peptideX eg., that serves as a means of protein detection. Non-limiting examples of label peptide or polypeptide moieties useful for detection in the invention include, without limi cation, suitable enzymes such as horseradish peroxidase, alkaline phosphatase, bcta-galactosidase, or acetylcholinesterase; epitope tags, such as FLAG, MYC, HA, or HIS tags; fluorophorcs such as green fluorescent protein; dyes; radioisotopes; digoxygenin; biotin; antibodies; polymers as well as others known in the art, for example, in Principles of Fluorescence Spectroscopy, Joseph R.
kowkz (Editor), Plenum Pub Corp, 2nd edition (Jury 1999).
The modulatory agents described herein (&g. antibodies, small molecules, peptides, fusion proteins, or small nucleic acids) can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The compositions may contain a single such molecule or agent or any combination of agents described herein. Based on the genetic pathway analyses described herein, it is believed that such combinations of agents is especially effective in diagnosing, prognosing, preventing, and treating cancer. Thus, "single active agents'" described herein can be combined with other pharmacologically active compounds ("second active agents") known m the art according to the methods and compositions provided herein. It is believed that certain conibinations work syncrgistically in the treatment of particular types of cancer. Second active agents can be large molecules (e.g., proteins) or small molecules (e.g., synthetic inorganic, organomctallic, or organic molecules).
Examples of large molecule active agents include, but are not limited to, hematopoietic growth factors, cytokines, and monoclonal and polyclonal antibodies.
Typical large molecule active agents arc biological molecules, such as naturally occurring or artificially made proteins. Proteins that arc particularly useful in this mvention include proteins that stimulate the survival and/or proliferation of hematopoietic precursor cells and immunologically active poietk cells in vitro or in vivo. Others stimulate die division and differentiation of committed crythroid progenitors in ceils in vitro or in vivo. Particular proteins include, but are not limited to: interleukins, such as IL-2 (including recombinant IL-II (HriL2w and canarypox IL-2), IL-10, lL-12, and IL-18; interferons, such as interferon alia- 2a, interferon alfa*2b, interferon alpha-nl , interferon alpha-n3, interferon beta-la, and interferon gamma-lb; GM-CF and OM-CSF; and EPO.
Particular proteins that can be used in the methods and compositions provided herein include but are not limited to: filgrastim, which is sold in the United States under die trade name Ncupogcn 5> (Amgen, Thousand Oaks, Calif.); sargramoetim, which is sold in the United States under the trade name Leukincft (Immune x, Seattle, Wash.); and recombinant EJPO, which is sold in the United Slates under the trade name Epogenft (Amgen, Thousand Oaks, Calif). Recombinant and mutated forms of GM-CSF can be prepared as described in U.S. Pat Nos. 5.391.485; 5,393,870; and 5.229.496; all of which are incorporated herein by reference. Recombinant and mutated forms of G-CSF can be prepared as described in U.S. Pat Nos.4,810,643; 4,999,29] ; 5,528,823; and 5,580,755; all of which are incorporated herein by reference.
Antibodies that can be used in conrinnation form include monoclonal and polyclonal antibodies. Examples of antibodies include, but are not limited to, trastuzumab (Herceotin^), rituximab (Ritttxantt), bevaciznmab (Avastinft), pertuzumab (Omnitarg^), tositumomab (Bexxar$), edrecolomab (Panorex$>). and O250. Compounds of the invention can also be combined with, or used in combination with, anti-TNF-a antibodies. Large molecule active agents may be administered in the form of anti-cancer vaccines. For example, vaccines that secrete, or cause the secretion of. cytokines such as lL-2, G-CSF, and GM-CSF can be used in the methods, pharmaccuticai compositions, and kits provided herein. See, e.g., Emens, L. A.. etai, Curr. Opinion Mol. Ther. 3<1): 77-84 (2001).
Second active agents that arc small molecules can also be used to in combination as provided herein. Examples of small molecule second active agents include, but are not limited to, anti-cancer agents, antibiotics, imnumosuppressive agents, and steroids.
In some embodiments, well known "combination chemotherapyn regimens can be used. In one embodiment, the combination chemotherapy comprises a combination of two or more of cyclophosphamide, hydroxydaunorubtcin (also known as doxorubicin or adriamycin), oncovorin (vincristine), and prednisone. In another preferred cn-bodimcnt, the combination chemotherapy comprises a combination of cyclcfhsophamide, oncovorin, prednisone, and one or more chernotbcrapcufjcs selected from the group consisting of anmracycUne, hydroxydaunorubicin, epirubicin, and motbantrone.
Examples of other anti-cancer agents include, but are not limited to: acivicin;
aclarubicin; acodazole hydrochloride; acronine; adozeiesin; aldesleukin; altretamine;
ambomyciri; ametantrone acetate; amsacrine; anastrozole; anthramycin; asparaginase; aspcrlin; azacitidinc; azetepa; azotomycin; batimastat; benzodepa bicalutanude; bisantrene hydrochloride; bisnafidc diroesylate; bizelcsin; bleomycin sulfate; brequirurr sodium;
bropirimine; busulfan; cactinomycin; calustcronc; caracemidc; carbetimer, carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; celecoxib (COX-2 inhibitor); chleranibuctl; cirolemycin cisplatin; cladribine; crisnatol mesylate; cydophosphamide; cytarabine; dacarbazinc; dactuwrnycm; daunorubictn hydrochloride; decitabinc;
dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquooe; docctaxel; doxorubicin; doxorubicin hydrochloride droloxifenc; droloxifenc citrate; dromostanolone propionate; duazomycin; cdatrexale; eflcnuthine hydrochloride; clsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozolc; esorubicin hydrochloride cstramustine; esiramustine phosphate sodium etanidazole; etoposide; etopotide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxaridinc; fludarabinc phosphate;
fluorooracil; fluorocitabioc; fosqoidone; f striccin sodium; gemcitabinc; gemcitabinc hydrochloride; hydroxyurea; idarabicin hydrochloride; iiosfanride; ihnofbsine; iproplatin; irinotecaiu irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate;
liarozole hydrochloride; lometrexol sodium; lomustinc; losoxamronc hydrochloride;
masoprocol; mavtansine; inechloreihaminc bydrocluoride; megestrol acetate; mekngestroi acetate; meb halan; meaogaril; nicrcaptopurine; methotrexate; methotrexate sodium;
metoprine; meturedepa; rratindomidc; mitocarcin; mitocromin; mitogilhn; iratomalcin; mitomycin; mitospcr, mitotanc; mitoxantrone bydrochioride; mycopbenolic acid;
nocodazole; nogalamycin; ormaplarin; oxisuran; paclitaxd; pegaspargase; peliomycin; pentamustine; pcploniycin sulfate; pcrfbsfamide; pipobroman; piposutfan; piroxantrone hydrochloride pUcarnycin; plomestanc; porfimer sodium; porfiromycin; predmraustine; procarbazine bydrochioride; puromyctn; puromycin hydrochloride; pyiazofurin; riboprine; safingoi; safmgol hydrochloride; semustine; simtrazene; sparfbsate sodium; sparsomycin; spirogcrmanium hydrochloride; spiromustine; spiroplatin strcptonigrin streptozocin; suiofenur. talisomyctn; tecogalan sodium; taxotere; tcgafur, teioxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; miamiprine thiognanine; thiotcpa;
tiazofurin; t rapazainirje; toremifenc citrate; trestolone acetate triciribine phosphate;
trimetrexafc; trimetrexate giucuronatc; triptorclin; rubulozoic hydrochloride; uracil mustard; uredepa; vapreotide; vertcporfin; vinblastine sulfate; vincristine sulfate; vindesinr, vindesine sulfate; vinepidine sulfate; vraglycinate sulfate; vmleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zenipiatin; zinostatin; and zorubkin hydrochloride.
Other arm-cancer drugs include, but are not limited to: 20-cpi-l,25
dihydroxyvitainin D3; 5-cthynyhiracil; abiraterone; aclarubicin; acyMurvene; adecypenol; adozeksin; aldesleukin; ALL-T antagonists altittanune; ambamustine; amidox;
amifbstine; aminolevulinic acid; amrobicin; amsacrine; anagrdidc; anastrozoic; andrographolide; angiogencsis inhibiton; antagonist D; antagonist G; antareiix; anti- dorsalizing moiphogenetic protein-1 ; antiandrogen, prostatic carcinoma; anuestrogen; antineoplaston; antiscnse oKgonuclcotidcs; aphidkoltn glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestanc; atrimustine; axinastatin 1 ; axinastatin 2; axinastatin 3; azasetron; aza toxin; azarvrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzoch!orins; benzoylstauiosporine; beta lactam derivatives; beta-alethine; betaciamycin
B; betulinic acid; bFGF inhibitor; bkahitamide; bisantrene; bisaziridiiryisperimne;
bisnafidc; bistratene A; bizelesin; breflate; biopirimine; budotitane; buthiomne sutfoximinc; caknpotriol calpbostin C; camptothecin derivatives; capecitabme; carboxamide-amino- triazole; carboxyarriootriazole CaRcst M3; CARN 700; cartilage derived inhibitor, carzelesin; casein kinase inhibitors (ICOS); casuuMspcrminc; cecropin B; cetrordix;
chiorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clormrene analogues; clotrimazole; coUismycin A; collismycin B; cc^nbretastatin A4; combrctastatin analogue; conagenm; crambcsckHn 816; crisnatol; cryptophycin 8; crvptophycin A derivatives; curacin A; cyctopentanAraquinones; cycloplatam; cyclosporin A; cypemycin; cytarabinc oefbsfate; cytolytic factor; cytostatin; daclixtmab; decitabine; dehydrodidemnin
B; deslorelin; dcxamethasonc; dexirbsraitude; dexrazoxane; (texverapamii; diaaqnonc; dkiemnin B; didox; <neftyfoorspennme; dihydro-5-azacytidine; dihydrotaxol, 9-;
dioxamycin; diphenyl spiromustine; docctaxel; docosanol; doiasetron; doxifluridine;
doxorubicin; droloxifcnc; dronabinol; duocarmycin SA; ebsekn; ccomus ine eddfbsinc; edrecolomab; cfl ornithine; elemene; emitemr, epirubicin; epristcridc; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate;
exemestane; radrazole; fazarabine; femetinide; filgrastim; finasteride; flavopiridol;
flezelasrine fluasterone; fhidarabine; rhjorodaunorunicin hydrochloride; forfemmcx;
fb mestane; fbstriecin; fbtemustine; gadolinium texaphyrin; gallium nitrate; gatocitabine; ganirelix; getatinase inhibitors; gemcitabine glutathione inhibitors; hepsulfam; heregulin; hexamemylene bisacetamide; hypericin; ibandronic acid; idarubtcin; idoxifene;
idramantone; ihnofbsine; ilomastat; imatinib (e.g., Glecvec$X imtquhnod;
imrnunosu'mulant peptides; insulin-like growth factor- 1 receptor inhibitor; interferon agonists; interferons; interieukins; iobenguanc; iododoxorobicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohonwhalicondrin B; hasctron; jasplakinotide; kahalaiide F; lamellarin-N triacetate lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; lctrozolc; leukemia inhibiting factor; leukocyte alpha interferon;
Icuprolio t-cstrogerHprogcstc^ leuprorelin; levamisole; liarozole; linear poly amine analogue; lipophilic disaccharidc peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricinc; lometrexol; kxridamine; loaoxantrone; loxoribinc; htrtoeccan;
hitetium texaphyrin; lysofylline; lytic peptides; rnaitanainc; matmostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metal proteinase inhibitors; menogaril; merbarone; meterdin; rncthioninase; tnetrxlopiaiiiide; MIF inhibitor, rnifepristonc;
miitefosinc; mirimostim; mhoguazone; nritolactol; mitomycin analogues; mitonafidc; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; Erbitux, human chorionic gonadotropin; monophosphoryl Kpid A- myobacterium cell wall sk; mopidamol; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acerykKnaline; N-substituted benzamides; ludarelin; nagrestip;
naloxoac+pentaxocine; napavin; naphtcrpin; nartograstim; nedaplatin; nemorubicin;
neridronic acid; nihitamidc; nisarnycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; oblimersen (Gcnascnae$); 06-benzylguaninc; octreotide; okkenone;
oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer ormaplatin; osatcronc; oxaiipiatin; oxaunomycin; paclitaxei; paclitaxel analogues;
paclitaxel derivatives; palauamine; palmitoylrhizoxtn; pamklronic acid; panaxytriol;
panomifene; parabactin; pazelliptine pegaspargase; peldesinc pentosan porysulfate sodium; peniostatin; pentrozole perflubron; perfosfamide; pcrillyl alcohol;
phenazinomycin; pbenylacetato; phosphatase inhibitors; picibanil; pilocarpine
hydrochloride; pimrabtcin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor, platinum complex; platinum compounds; platinum-triamine complex; Dorfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prosuujlandin J2; proteasome inhibitors; protein A-bascd immune modulator, protein kinase C inhibitor, protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pymzobacridine; pyridoxylated hemoglobin polyoxycthylenc conjugate; raf antagonists; raltitrexcd; ramosetron; ras rarnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retcliiptinc dcmethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII rctinamidc; rohituldne; romurtidc; roquinimex; rubiginone Bl; niboxyl; safmgol; saintopin; SaiCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1 ; sense oligonucleotides; signal transduction inhibitors; sizofuran; sobuzoxane; sodium borocaptate; sodium pbenylacetate solvcrol; somatomedin binding protein; sonennin sparfbsic acid; spicamycin D;
spiromustine; spknopentin spongistatin 1; squal amine; stipiamide; stromelysin inhibitors sulfinosinc; supcractive vasoactive intestinal peptide antagonist; suradista; suramin;
swainsonine; talliroustine; tamoxif n methiodidc; tauromustinc; tazarotcne; tecogalan sodium; tegaf r; idlurapyrylium; teiomerase inhibitors; temoporfin; tenipoeide;
tetnchlorodecaoxtde; tetn omine; thaliblastine; miocoralme; mrombopoietm;
thrombopoictin mimetic; wymalfesin; thymopoietin icceplor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tiiapazamine; titanocene bicbloride; topsendn; toremifene; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimctrexatc;
triptorelin; tropisetron; turosteride tyrosine kinase inhibitors; tyrphostms; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor, urokinase receptor antagonists; vaprcotide; variolin B; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxtn; vorozolc; zanoterone; zcniplaun; zilascorb; and zinostatin stimaUuncr.
Specific second active agents include, but are not limited to, chlorambucil, fludarabinc, dcxamethasonc (Decadron$), hydrocortisone, mcthylprcdnisolone, cilostamide, doxorubicin (Doxilft), forskolin, rituximab, cyclosporin A, cisplatin, vu ristine, PDE7 inhibitors such as BRL-50481 and IR-202, dual PDE47 inhibitors such as IR-284, cilostazol, meribendan, milrinone, vesnarionone, enoximone and pimobendan, Syk inhibitors such as fbstamatinib disodium (R406/R788), R343, R-l 12 and Exccliair$ (ZaBeCor Phannaceuticals, Bala Cynwyd, Pa.).
TO. Methods of Sctotmg Kcng ir«j Cy^ppsitigrtf
Another aspect of the invention relates to methods of selecting agents (e.g., antibodies, fusion proteins, peptides, small molecules, or small nucleic acids) which bind to, uprcgulate, downregulatc, or modulate one or more biomarkcrc of the invention listed in
Tables 1-5 and Examples and/or a cancer (eg., a lymphoid cancer, such as leukemia). Such methods utilize can use screening assays, including cell based and nan -cell based assays.
In one embodiment, the invention relates to assays for screening candidate or test compounds which bind to or modulate die expression or activity level of, one or more bramarkers of the invention, including one or more biomarkers listed in Tables 1-5 and
Examples, or a fragment thereof. Such compounds include, without limitation, antibodies, proteins, fusion proteins, nucleic acid molecules, and small molecules. in one embodiment, an assay is a cell-based assay, comprising contacting a cell expressing one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples, or a fragment thereof, with a test compound and determining the ability of the (est compound to modulate (eg. stimulate or inhibit) the level of interaction between the biomarker and its natural binding partners as measured by direct binding or by measuring a parameter of cancer.
For example, in a direct binding assay, the biomarker polypeptide, a binding partner polypeptide of die biomarker, or a fragments) thereof, can be coupled with a radioisotope or enzymatic label such that binding of the biomarker polypeptide or a fragment thereof to its natural binding partner(s) or a fragments) thereof can be determined by detecting the labeled molecule in a complex. For example, die biomarker polypeptide, a binding partner polypeptide of the biomarker, or a fragments) thereof, can be labeled with l25l, "S, MC, or 3H, either directly or indirectly, and die radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, the polypeptides of interest a can be enzyrnaticalry labeled with, for example, horseradish peroxidase, alkaline phosphatase, or loaferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product
It is also within the scope of mis invention to determine the ability of a compound to modulate the interactions between one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples, or a fragment thereof, and its natural binding partneris) or a fragmcnt(s) thereof, without die labeling of any of the intcractants (eg., using a nnaophysiometer as described in MeConnell, H. Mrtai (l 992) Science 257:1906-1912). As used herein, a Mniicrophysiometern (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light- addressable potentiomctric sensor (LAPS). Changes in this ackhfication rate can be used as an indicator of the interaction between compound and receptor.
In a preferred embodiment, determining the ability of the blocking agents (eg. antibodies, fusion proteins, peptides, nucleic acid molecules, or small molecules) to antagonize the interaction between a given set of polypeptides can be accomplished by dbacnwning the activity of one or more members of the set of interacting molecules. For example, the activity of one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples, or a fragment thereof, can be determined by detecting induction of cytokine or chemokine response, detecting cataiytic/enzyrnatic activity of an appropriate substrate, detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., chloramphenicol acetyl transferase), or detecting a cellular response regulated by the biomarker or a fragment thereof (e.g., modulations of biological pathways identified herein, such as modulated proliferation, apoptosis, cell cycle, and/or E2F transcription facto binding activity). Determining the ability of the blocking agent to bind to or interact with said polypeptide can be accomplished by measuring the ability of an agent to modulate iiranunc responses, for example, by detecting changes in type and amount of cytokine secretion, changes in apoptosis or proliferation, changes in gene expression or activity associated with cellular identity, or by irtterfering with the ability of said polypeptide to bind to antibodies that recognize a portion thereof.
In yet another embodiment, an assay of the present invention is a cell-free assay in which one or more biomarker* of the invention, including one or more biomarkcrs listed in Tables 1 -5 and Examples or a fragment thereof, e.g. a biologically active fragment thereof, is contacted with a test compound, and the ability of the test compound to bind to the polypeptide, or biologically active portion thereof, is determined. Binding of the test compound to the biomarker or a fragment thereof, can be a½tcrrmned either directly or indirectly as described above. Determining die ability of die biomarker or a fragment thereof to bind to its natural binding partners) or a fragment(s) thereof can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA) (Sjolander, S. and Uibaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. ()pin. Struct. Biol. 5:699-705). As used herein, "BIA" is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BlAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological polypeptides. One or more biomarkcrs polypeptide or a fragment thereof can be immobilized on a BlAcore chip and multiple agents, e.g., blocking antibodies, fusion proteins, peptides, or small molecules, can be tested for binding to the immobilized biomarker polypeptide or fragment thereof. An example of using the BIA technology is described by Fitz et al. (1997) Oncogene 15:613.
The cell-free assays of the present invention arc amenable to use of bom soluble and/or rriembrane-bound forms of proteins. In the case of cell-free assays in which a membrane-bound form protein is used it may be desirable to utilize a sohibilizing agent such that the mraibrane-bound form of the protein is maintained in solution. Examples of such solu ilizing agents include non-ionic detergents such as n-octylglucosidc, n- dodecyiglucoside, n-dodccytma1toside, c«tanoyl-N-inethylglucamide, decanoyl-N- metylghjcamide, Triton* X-100, Triton* X-l 14, Thesii* lsotridecypoly(eihylcr_e glycol ether),,, 3-((3-cholamidopiopy )<¾imeihy 1 -propane sulfonate (CHAPS), 3-{(3- cholamio^propyl)dimemytammm^ sulfonate (CHAPSO), or dodccyl«N,N-<innethyl-3-arnnK>do-1 -propane sulfonate.
In one or more embodiments of the above described assay methods, it may be desirable to immobilize either the biomarker polypeptide, the natural binding partners) polypeptide of the biomarker, or fragments thereof, to facilitate separation of complexcd from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound in the assay can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microliter plates, test tubes, and irjicrc-cenmruge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transfcrasc-base fusion proteins, can be adsorbed onto glutathione Scpharoec$ beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microliter plates, which are then combined with the test cotnpound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microliter plate welts are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity determined using standard techniques.
In an alternative embodiment, detennining the ability of the test compound to modulate the activity of one or more biomarkcrs of the invention, including one or more biomarkers listed in Tables 1-5 and Examples, or a fragment thereof, or of natural binding partners) thereof can be accomplished by determining the ability of the lest compound to modulate the expression or activity of a gene, e&, nucleic acid, or gene product, eg., polypeptide, that functions downstream of the interaction. For example, mflammation cytokine and chemokine) responses can be determined, the activity of the interactor polypeptide on an appropriate target can be determined, or the binding of the intcractor to an appropriate target can be determined as previously described. in another embodiment, modulators of one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-S and Examples, or a fragment thereof, are identified in a method wherein a cell is contacted with a candidate compound and the expression or activity level of the biomarker is determined. The level of expression of biomarker mRNA or polypeptide or fragments thereof in the presence of the candidate compound is compared to the level of expression of biomarker mRNA or polypeptide or fragments thereof in the absence of the candidate compound. The candidate compound can then be identified as a modulator of biomarker expression based on this comparison. For example, when expression of biomarker mRNA or polypeptide or fragments thereof is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of biomarker expression. Alternatively, when expression of biomarker mRNA or polypeptide or fragments thereof is reduced (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of biomarker expression. The expression level of biomarker mRNA or polypeptide or fragments thereof in the cells can be determined by methods described herein for detecting biomarker mRNA or polypeptide or fragments thereof,
In other embodiments, activity of histonc methyl modifying proteins (e.g., enzymes) are evaluated. The effect of a test compound can be evaluated, for example, by measuring methylation of a substrate in the presence of a stimulating agent at the beginning of a time course, and then comparing such levels after a prcdctcnruncd time (&£., 0.1 , 0.25, 0.5, 1 , 15, 2, 2.5, 3, or more hours) in a reaction that includes the test compound and in a parallel control reaction that does not include the test compound. This is one example of a method for determining the effect of a test compound on enzyme activity in vitro using a stimulating agent as provided by the present disclosure. In general, an assay involves preparing a reaction mixture of a histone methyl modifying enzyme, a substrate, a stimulating agent, and one or more test compounds under coiklitions and for a time sufficient to allow components to interact Methylation can be evaluated directly or indirccdy. For example, H3 27 mono-, <h-, and/or tri-methylation or the relative proportions or relative changes from one species to another over time, can be assessed. In some embodiments, a component of an assay reaction mixture (e.g., a substrate) is anchored onto a solid phase. A component anchored on the solid phase can be detected at the end of a reaction, e.g., a mcthylase reaction. Any vessel suitable reactants can be used. Examples of suitable vessels include mkrodter plates, test tubes, and micro-centrifuge tubes.
Activity of methyl modifying enzymes can be evaluated by any available means. In some embodiments, a mediylation state of a substrate is evaluated by mass spectrometry analysis of a substrate. In some embodiments, methylation of a substrate is evaluated with an antibody specific for a methylated or demethylated substrate. Such antibodies are conunercially available (e.g., from Upstate Group, NY, or Abeam Ltd., UK). Suitable imimtnoassay techniques for detecting methylation state of a substrate include
imrnunobloUing, ELISA, and ύτηιτΗΐηορϊτχφί^κ).!. Methylation reactions can be carried out in the presence of a labeled methyl donor (e.g., a S-edenosyH methyl-' *C}-L- methionine, or S ^enosyHmemyl-jH]^methionine), allowing detection of label into a mcthylase substrate, or release of label from a demethylase substrate. In some
embodiments, activity of a methyl modifying enzyme is evaluated using fluorescence energy transfer (FET or FRET for fluorescence resonance energy transfer) (see, for example, Lakowicz et a!., U.S. Pat. No. 5.631.169; Stavrianopoulos, et al. U.S. Pat No. 4,868,103). A fluorophorc label on a 'donor* (e.g., a DNA molecule of a nucleosome) is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on an 'acceptor' (e.g., an antibody specific for a histone methyl modification of interest), which in turn is able to fluoresce doe to the absorbed energy. A reaction can be carried out using an unlabeled substrate, and histone modification is determined by detecting antibody binding using a fluorimeter (see, U.S. Pat. Pub.2008/0070257).
In some embodiments, demethylation is evaluated by direct or indirect detection of release of a reaction product such as formaldehyde and/or succinate. In some
embodiments, release of formaldehyde is detected. Release of formaldehyde can be detected using a fbrmaidehyde dehydrogenase assay in which fbrmaidchyde dehydrogenase converts released fbrmaidchyde to formic acid using NAD+ as electron acceptor. Reduction ofNAD+ can be detected specuiophotonietrically (Lizcano et aL, Anal. Biochem.286:75- 79, 2000). In some embodiments, release of formaldehyde is detected by converting formaldehyde to 3,5-<iiaccmyM,4-<i%dr >Ju^ (DDL) and detecting the DDL, for example, by detecting radiolabeled DDL (e.g. , 3H-DDL). A substrate can be labeled so that a labeled reaction product is released (e.g., fbrmaidchyde and or succinate) by a done thy tation reaction. In some embodiments, a substrate is methylated win JH-SAM (S- adenosytmethioiiine), demethyiation of which releases ^-formaldehyde, which can detected directly, or which can be converted to JH-DDL, which is detected. Methods of detecting reaction products such as formaldehyde and/or succinate include mass spectrometry, gas chromatography, liquid chromatography, immunoassay, electrophoresis, and the like, and combinations thereof Dcmctfaylase assays are also described in Shi eta)., Cell 119:9 1-953, 2004. An alternative means for detecting dcmethylase activity employs analysis of release of radioactive carbon dioxide (see, e.g., Pappalardi et al (2008) Btochem. 47:11165-11167 and Supporting Information, which describes use of a radioactive assay in which capture of 14C(¼ is captured and detected following release from ct| l-'^J-ketoghitaric acid coupled to hydroxylation reactions). Such methods can also be employed for detection of demethylarion. Detection of enzyme activity can include use of fluorescent, radioactive, scintillant, or other type of reagents. In some embodiments, a scintillation proximity assay is used for evaluating enzyme activity. Such assays can involve use of an immobilized scintillant (e.g., immobilized on a bead or microp!atc) and a radioactive methyl donor. In some embodiments, a scintillation proximity assay employs scintillant-coated microplatcs such as FlashPlatesK> (Perkin Elmer). In some embodiments, components of an assay reaction mixture are conjugated to biotin and strcptavidin.
Biotinylated components (eg., biotinytated substrate or biotinylatcd stimulating agent) can be prepared, eg., using biotin-NHS (N-hydioxy-euccmmiide) according to known techniques (e.g., biotinylation kit, Pierce Chemicals, Rockfbrd, flL). Biotinylated components can be captured using strcptavidm-coated beads or immobilized in the wells of streptavidin-coated plates (Pierce Qtcmical). As would be appreciated by those of skill in the art, assays can also employ any of a nutnoer of standard techniques for preparation and/or analysis of enzymatic activity, including but not limited to: differential
centrifugation (see, for example, RJrvas, G., and Minton, A. P.. (1993) Trends Btochem Sci 18:284-7); chromatography (gd filtration chromatography, ion-exchange chnrniatography); dectrophorcsts (see, eg., Ausubcl, F. et al., eds. Current Protocols in Molecular Biology 1999, J. Wiley: New York.); and iimmmoprec^itation (see, for example, Ausubcl, F. et al., eds. (1 99) Current Protocols in Molecular Biology, J. Wiley: New York). Such resins and chromatographic techniques are known to one skilled in the art (see. e.g., Heegaard, N. H., (1998) J Mo! Recognit 11:141-8; Hagc, D. S., and Tweed. S. A. (1997) J Chromatogr B Biomed Sci Appl.699:499-525). Further, fluorescence energy transfer may also be conveniently utilized, as described herein, to detect activity of Mstone methyl modifying enzymes.
• In yet another aspect of the invention, a biomatker of the invention, including one or more biomarkers listed in Tables 1-5 and Examples, or a fragment thereof, can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., VS. Pat No.
5,283,317; Zervos eial (1993) Cell 72:223-232; Madura eial (1993) J. Biol. Chem.
268:12046-12054; Battel ei al (1993) Biotechniques 14:920-924; Iwabucbi et at. (1993) Oncogene 8:1693-1696; and Brent W094/I0300), to identify other polypeptides which bind to or interact with the biomarkcr or fragments thereof and are involved in activity of the biomarkers. Such biomarker-binding proteins are also likely to be involved in die propagation of signals by the biomarkcr polypeptides or biomarkcr natural binding partners) as, for example, downstream elements of one or more biomarkers -mediated signaling pathway.
The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, die assay utilizes two different DNA constructs. In one construct, the gene mat codes for one or more biomarkers polypeptide is fused to a gene encoding tbc DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, mat encodes an unidentified polypeptide ("prey" or "sample") is fused to a gene mat codes for tbc activation domain of the known transcription factor. If the "bait" and the "prey" polypeptides are able to interact, In vtvo, fcrming one or more biomarkers -dependent complex, the DNA-binding and activation domains of the transchptioa factor are brought into close proximity. This proximity allows transchptioa of a reporter gene ( g., LacZ) which is opcrably linked to a trartscriptiortal regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain tbc cloned gene which encodes me polypeptide which interacts with one or more biomarkers polypeptide of the invention, including one or more biomarkers listed in Tables 1-5 and Examples or a fragment thereof.
In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell- based or a cell-free assay, and the ability of the agent to modulate the activity of one or more biomarkers polypeptide or a fragment thereof can be confirmed in vivo, e.g., in an animal such as an animal model for cellular tram formation and/or tumorigencsis. This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model For example, an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein. in. υ¾¾ and Methods of the Inventio
The biomarkers of the invention described herein, including the biomarkcrs listed in Tables 1 -5 and Examples or fragments thereof, can be used in one or more of the following methods: a) screening assays; b) predictive medicine diagnostic assays, prognostic assays, and monitoring of clinical trials); and c) methods of treatment (&g., therapeutic and prophylactic, e.g.. by up- or down-modulating the copy number, level of expression, and/or level of activity of the one or more biomarkcrs).
The isolated nucleic acid molecules of the invention can be used, for example, to (a) express one or more biomarkers of the invention, including one or more biomarkcrs listed in Tables 1-5 and Examples or a fragment thereof (e.g., via a recombinant expression vector in a host cell in gene therapy applications or synthetic nucleic acid molecule), (b) detect bio marker mRNA or a fragment thereof (#.£., in a biological sample) or a genetic alteration in one or more biomarkers gene, and or (c) modulate biomarker activity, as described further below. The biomarker polypeptides or fragments thereof can be used to treat conditions or disorders characterized by insufficient or excessive production of one or more biomarkcrs polypeptide or fragment thereof or production of biomarker polypeptide inhibitors. In addition, the biomarker polypeptides or fragrr nts thereof can be used to screen for naturally occurring biomarker binding partner(s), to screen for drugs or compounds which modulate biomarker activity, as well as to treat conditions or disorders characterized by insufficient or excessive production of biomarker polypeptide or a fragment thereof or production of biomarker polypeptide forms which have decreased, aberrant or unwanted activity compared to biomarker wild-type polypeptides or fragments thereof (&&, cancers, including lymphoid cancers, such as leukemia). A. Swearing AasBPtt
In one aspect, the present invention relates to a method for preventing in a subject, a disease or condition associated with an unwanted, more than desintbk, or less than desirable, expression and/or activity of one or more btornarkers described herein. Subjects at risk for a disease that would benefit from treatment with the claimed agents or methods can be identified, for example, by any one or combination of diagnostic or prognostic assays known in the art and described herein (see, for example, agents and assays described in III. tibotb of ScfoBog Agents and Cnmp wtions) B. Pre^QveMedjqag
The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring of clinical trials arc used for prognostic (predictive) purposes to thereby treat an individual prophylactically.
Accordingly, one aspect of the present invention relates to diagnostic assays for detennining the expression anoVor activity level of biomarkers of the invention, including biomarkcrs listed in Tables 1-5 and Examples or fragments thereof, in the context of a biological sample (e.g., blood, scram, cells, or tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant or unwanted biomarkcr expression or activity. The present invention also provides for prognostic (or predictive) assays for dctermming whether an individual is at risk of developing a disorder associated with biomarkcr polypeptide, nucleic acid expression or activity. For example, mutations in one or more biomarkcrs gene can be assayed in a biological sample.
Such assays can be used for prognostic or predictive purpose to thereby prophylacticaliy treat an individual prior to the onset of a disorder characterized by or associated with biomarkcr polypeptide, nucleic acid expression or activity.
Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds, and small nucleic acid-based molecules) on the expression or activity ofbiotnarkcrs of the mvention, including biomarkcrs listed in Tables 1-5 and Examples, or fragments thereof, in clinical trials. These and other agents are described in further detail in the following sections. The present invention provides, in part, methods, systems, and code for accurately classifying whether a biological sample is associated with a cancer or a clinical subtype thereof (eg., lymphoid cancers, such as leukemia), fn some embodiments, the present invention is useful for classifying a sample (e.g., from a subject) as a cancer sample using a statistical algorithm and or empirical data (*.#., the presence or level of one or biomarkers described herein).
An exemplary method for detecting die level of expression or activity of one or more biomarkers of die invention, including one or more biomarkers listed in Tables 1-5 and Examples or fragments thereof, and thus useful for classifying whether a sample is associated with cancer or a clinical subtype thereof (e.g. , lymphoid cancers, such as leukemia), involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting the biomarker (e.g., polypeptide or nucleic acid mat encodes the biomarker or fragments thereof) such that the level of expression or activity of the biomarker is detected in the biological sample, fn some embodiments, the presence or level of at least one, two, three, four, five, six, seven, eight, nine, ten, fifty, hundree, or more biomarkers of the invention are determined in the individual's sample. In certain instances, the statistical algorithm is a single learning statistical classifier system. Exemplary statistical analyses are presented in the Examples and can be used in certain embodiments. In other cinbodiments, a single learning statistical classifier system can be used to classify a sample as a cancer sample, a cancer subtype sample, or a non-cancer sample based upon a prediction or probability value and the presence or level of one or more biomarkers described herein. The use of a single learning statistical classifier system typically classifies the sample as a cancer sample with a sensitivity, specificity, positive predictive value, negative predictive value, and/or overall accuracy of at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%.89%, 90%, 91%.92%, 93%, 94%.95%, 96%, 97%.98%, or 99%.
Other suitable statistical algorithms are well known to those of skill in the art For example, learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex data sets (e.g., panel of markers of interest) and making decisions based upon such data sets. In some embodiments, a single teaming statistical classifier system such as a classification tree (e.g., random forest) is used. In other embodiments, a combination of 2, , 4, 5, , 7, , , 10, or more learning statistical classifier systems are used, preferably in tandem. Examples of learning statistical classifier systems include, but are not limited to, those using inductive learning (eg.,
dedskm/classirkation trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.). Probably Approximately Correct (PAC) learning, coniKcdonist learning (e.g., neural networks ( N), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptions such as multi-layer perceptrong, multi-layer feed-forward networks, applications of neural networks, Baycsian learning in belief networks, etc.), reinforcement learning (eg., passive learning in a known environment such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action- value functions, applications of reinforcement learning, ctc.X and genetic algorithms and evolutionary programming. Other learning statistical classifier systems include support vector machines Kernel methods), multivariate adaptive regression splines (MARS), Lcvenberg-Marquardt algorithms, Gauss-Newton algorithms, mixtures of Gaussians, gradient descent algorithms, and learning vector quamizaboa (LVQ). In certain embodiments, the method of the present invention further comprises sending the cancer classification results to a clinician, e.g., an oncologist or hematotogist
In another embodiment, the method of the present invention further provides a diagnosis in the form of a probability that the individual has a cancer or a clinical subtype thereof. For example, the individual can have about a 0% 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater probability of having cancer or a clinical subtype thereof. In yet another embodiment, the method of the present invention further provides a prognosis of cancer in the iixfcvidual. For example, the prognosis can be surgery, development of a clinical subtype of the cancer (e.g., subtype of leukemia), development of one or more symptoms, development of malignant cancer, or recovery from the disease. In some instances, the method of classifying a sample as a cancer sample is further baaed on the symptoms (e.g., clinical factors) of the individual from which the sample is obtained. The symptoms or group of symptoms can be, for example, those associated with the 1PI. In some embodiments, the diagnosis of an individual as having cancer or a clinical subtype thereof is followed by adnunistering to the individual a therapeutically effective amount of a drug useful for treating one or more symptoms associated with cancer or me cancer. in some embodiments, an agent for detecting biomarker raRNA, genomic DNA, or fragments thereof is a labeled nuclek acid probe capable of hybridizing to biomarker raRNA, genomic DNA., or fragments thereof. The nucleic acid probe can be, for example, full-length biomarker nucleic acid, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, l(X)t 250 or 500 nucleotides in lengm anl su cient to specifically hybridize under stringent conditions well known to a skilled artisan to biomarker mKNA or genomic DNA. Other suitable probes for use in the diagnostic assays of die invention arc described herein.
A preferred agent for detecting one or more biomarkers listed in Tables 1-5 and Examples or a fragment thereof is an antibody capable of binding to the biomarker.
preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (eg., Fab or F(ab')2) can be used. The term labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling {i.e., physically linking) a detectable substance to tbc probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluoresccntry labeled secondary antibody and end-labeling of a DN A probe with bkXin such that it can be detected with ftuofcscently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject That is, the detection method of the invention can be used to detect biomarker mRNA, polypeptide, genomic DNA, or fragments thereof, in a biological sample to vitro as well as in vivo. For example, in vitro techniques for detection of biomarker mRNA or a fragment thereof include Northern hybridizations and in situ hybridizations. In viiro techniques for detection of biomarker polypeptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunopredpitations and immunofluorescence. In vitro techniques for detection of biomarker genomic DNA or a fragment thereof include Southern hybridizations. Furthermore, in vivo techniques for detection of one or more biomarkers polypeptide or a fragment thereof include introducing into a subject a labeled anti- biomarker antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. in one embodiment, the biological sample contains polypeptide molecules from me test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a hematological tissue (e.g. , a sample comprising blood, plasma, B cell, bone marrow, etc.) sample isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting polypeptide, mRNA, cD A, small RNAs, mature rrriRNA, prc- rruRNA, pri-miRNA, miRNA*, arrti-rniRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof of one or more bioraarkcrs listed in Tables I -5 and Examples such mat the presence ofbiomarkcr polypeptide, mRNA, genomic DNA, or fragments thereof, is detected in the biological sample, and comparing the presence of biomarker polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri- miRNA, miRNA*, anti-mi R A, or a miR A binding site, or a variant thereof, genomic DNA, or fragments thereof in tbc control sample with the presence of biomarl cr polypeptide, mRNA, cD A, small RNAs, mature miRNA, prc-nriR A, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof in the test sample.
The mvention also encompasses kits for detecting the presence of a polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti- nuRNA, or a miRNA binding she, or a variant thereof, genomic DNA, or fragments thereof, of one or more biomarkers listed in Tables 1-5 and Examples in a biological sample. For example, the kit can comprise a labeled compound or agent capable of detecting one or more biomarkers polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRN A, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof, in a biological sample means for detenroining the amount of the biomarker polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thcreof,f in the sample; and means for comparing the amount of the biomarker polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof, in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect the biomarker polypeptide, mRNA, cDNA, small RNAs, mature miRNA, pre-miRNA, pri-miR A, miRNA*, anti-miRNA, or a miRNA binding site, or a variant thereof, genomic DNA, or fragments thereof.
In some embodiments, therapies tailored to treat stratified patient populations based on the described diagnostic assays are further administered.
2. Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant expression or activity of one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples, or a fragment thereof. As used herein, the term "aberrant" includes biomarker expression or activity levels which deviates from the normal expression or activity in a control.
The assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with a nrisrcgulation of biomarker activity or expression, such as in a cancer (e.g., lymphoid cancers, such as leukemia). Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disorder associated with a misregulation of biomarker activity or expression. Thus, the present invention provides a method for identifying and/or classifying a disease associated with aberrant expression or activity of one or more biomarkers of the invention, including one or more biomarkers listed in Tables I -5 and Examples, or a fragment thereof. Furthermore, die prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimctic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant biomarker expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent fix a cancer (e.g., lymphoid cancers, such as leukemia). Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disease associated with aberrant biomarker expression or activity in which a test sample is obtained and biomarker polypeptide or nucleic acid expression or activity is detected (e.g. , wherein a significant increase or decrease in biomarker polypeptide or nucleic acid expression or activity relative to a control is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant biomarker expression or activity). In some enibod ncnts, significant increase or decrease in biomarker expression or activity comprises at least 22.1, 22, 2J, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4.4.5.5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, JO, 10.5. 11. 12, 13, 14, 15, 16. 17. 18, 19, 20 times or more higher or kw^
expression activity or level of the marker in a control sample,
The methods of the invention can also be used to detect genetic alterations in one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples or a fragment thereof, thereby detenruning if a subject with the altered biomarker is at risk for cancer (eg., lymphoid cancers, such as leukemia) characterized by aberrant biomarker activity or expression levels. In preferred embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one alteration affecting the integrity of a gene encoding one or more biomarkers polypeptide, or the mis-expression of the biomarker. For example, such genetic alterations can be detected by ascertaining die existence of at least one of 1) a deletion of one or more nucleotides from one or more biomarkers gene, 2) an addition of one or more nucleotides to one or more biomarkers gene, 3) a substitution of one or more nucleotides of one or more biomarkers gene, 4) a chromosomal rearrangement of one or more biomarkers gene, 5) an alteration in the level of a messenger RNA transcript of one or more biomarkers gene.6) aberrant rnodification of one or more biomarkers gene, such as of the metfaylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of one or more biomarkers gene, 8) a non-wild type level of one or more biomarkers polypeptide, 9) allelic loss of one or more biomarkers gene, and 10) inappropriate poet-translational modification of one or more biomarkers polypeptide. As described herein, there are a large number of assays known in the art which can be used for detecting alterations in one or more biomarkers gene. A preferred biological sample is a tissue or scrum sample isolated by conventional means from a subject.
In certain ernbodiments, detection of the alteration involves the use of a probe primer in a polymerase chain reaction (PGR) (see, e.g., U.S. Patents 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR. or, alternatively, in a ligation chain reaction (LCR) (see, eg., Landegran etal. (1988) Science 241:1077-1080; and Nakazawa etal. (1994) Proc. Natl. Acad. Sci. USA 91:360-364), the latter of which can be particularly useful for detecting point mutations in one or more biomarkers gene (sec Abravaya et al. ( 1995) Nucleic Acids Res.23:675-682). This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic DNA, mRNA, cDNA, small RNA, mature miR A, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding site, or a variant thereof) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to one or more biomarkers gene of the invention, including the biomarker genes listed in Tables 1 -5 and Examples, or fragments thereof, under conditions such that hybridization and amplification of the biomarker gene (if present) occurs, and detecting the presence or absence of an
amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step m conjunction with any of the techniques used for detecting mutations described herein.
Alternative amplification methods include: self-sustained sequence replication (Ouatdli, J. C. et al. (1990) Proc. Nad. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. * al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q-Beta Replicase (Lizardi, P. M. el al (1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for me detection of nucleic acid molecules rf such molecules are present in very low numbers.
In an alternative embodiment, mutations in one or more biomarkers gene of the invention, including one or more biomarkers listed in Tables 1 -5 and Examples, or a fragment thereof, from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified
(optionally), digested with one or more restriction endonucleascs, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Patent 5,498,531 ) can be used to score for die presence of specific mutations by development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in one or more biomarkers gene of the invention, including a gene listed in Tables 1-5 and Examples, or a fragment thereof, can be identified by hybridizing a sample and control nucleic acids, &g., DNA, RNA, mRNA, small RNA, cDN A, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miR A binding site, or a variant thereof to high density arrays containing hundreds or thousands of oligonucleotide probes (Cronin, M. T. et al. (1996) Hum Mutat 7:244-255; Kozal, . J. et al. (1996) Nat Med. 2:753-739). For example, genetic mutations m one or more biomarkers can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin et aL (19%) supra. Briefly, a first hybridization array of probes can be used to scan through k>ng stretches of DN A in a sample and control to identify base changes between the sequences by making linear arrays of sequential, overlapping probes. This step allows the idcruifkation of point miitations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence one or more biomarkers gene of the mvention, including a gene listed in Tables 1 -5 and Examples, or a fragment thereof, and detect mutations by comparing the sequence of the sample biomarkcr gene with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert (1977) Proc. Natl. Acad. Set. USA 74:360 or Sanger ( 977) Proc. Nad. Acad Sci. USA 74:3463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Nacve, C. W. (1993) Btotechniques 19:448-53X including sequencing by mass spectrometry (see, e.g., PCT mternationa) Publication No. WO 94/16101 ; Cohen et aL (1996) Adv. Chromatogr. 36:127-162; and Griffin etal. (1993) AppL Biochcm. Bio techno!. 38:147-159).
Other methods for detecting mutations in one or more biomarkers gene of me invention, including a gene listed in Tables 1 -5 and Examples, or fragments thereof, include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA hctcroduplcxcs (Myer etal. (1985) Science 230:1242). In general, the art technique of "mismatch cleavage" starts by providing heteroduplcxcs formed by hybridizing (labeled) RNA or DNA containing die wild-type sequence with potentially mutant RNA or DNA obtained from a tissue sample. The (louble-strandcd duplexes arc treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to base pair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to eriz rnaticalry digest the mismatched regions. In other embodiments, either DNA DNA or RNA/DNA duplexes can be treated with hydroxy laminc or osmium tetroxidc and with piperidinc in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacryiarnide gels to determine the site of mutation. See, for example. Cotton et al (1988) Proc. Nad. Acad. Sci. USA 85:4397 and Salccoa et al (1992) Methods Enzymol.217:286-295. In a preferred embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called 'ΌΝΑ mismatch repair" enzymes) in defined systems for delecting and mapping point mutations in biomarker genes of the invention, including genes listed in Tables 1-5 and Examples, or fragments thereof, obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G A mismatches and the thymidine DNA glycosylasc f om HcLa cells cleaves TatG/T mismatches (Hsu etai. (1 94) Carcinogenesis 15: 1657- 1662). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in biomarker genes of the mvention, including genes listed in Tables 1-5 and Examples, or fragments thereof. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al (1989) Proc Natl. Acad. Sci USA 86:2766; see also Cotton (1993) MutaL Res.285:125-144 and Hayashi (1992) Genet Anal. Tech. Appl.9:73- 79). Single-stranded DNA f agments of sample and control nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of die assay may be enhanced by using RNA (rather than DNA), in which die secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes betcroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen el al (1991) Trends Genet 7:5). in yet another embodiment the movement of mutant or wild-type fragments in polyacr lamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. ( 1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to ensure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high- melting OC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to kientify differences in the mobility of control and sample DNA (Roscnbaum and Reissner (1987) Biophys. Chem. 265:12753).
Examples of other techniques for detecting point mutations include, but are not limited to. selective oligonucleotide hybridization, selective amphfoation, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163; Saiki el al. (1989) Proc. Nad. Acad. Sci. USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides arc attached to the hybridizing membrane and hybridized with labeled target DNA. In some embodiments, the hybridization reactions can occur using btochips, raicroarrays, etc, or other array technology that arc well known in the art
Alternatively, allele specific amplification technology which depends on selective
PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may cany the mutation of interest in the center of the molecule (so mat amplification depends on differential hybridization) (Gibbs et al. ( 1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one prima where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtcch 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et aL (1992) Mol. Cell Probes 6: 1). It is anticipated that in certain embodiments amplification may also be performed using Taq hgase for amplification (Barany (1991) Proc. Nad. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3* end of the 5* sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification. The methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving ooc or more biomarkent of me mvention, including ooc or more biomarkers listed in Tables 1-5 and Examples, or fragments thereof.
3. MqnitvTiog of Effatt During Clinical Trialt
Monitoring the influence of agents (eg., drags) on the expression or activity of one or more biomarkers of the invention, including one or more biomarkers listed in Tables l-S and Examples, or a fragment thereof (e.g., the modulation of a cancer state) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase expression and/or activity of one or more biomarkers of the invention, mcluding one or more biomarkers listed in Tables 1 -5 and Examples or a fragment thereof, can be mortitorcd in clinical trials of subjects exhibiting decreased expression andor activity of one or more biomarkers of the invention, inducting one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1 -5 and Examples, or a fragment thereof, relative to a control reference. Alternatively, the effectiveness of an agent determined by a screening assay to decrease expression and/or activity of one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1 -5 and Examples, or a fragment thereof, can be monitored in clinical trials of subjects exhibiting decreased expression and/or activity of the biomarker of the invention, including one or more biomarkers listed in Tables l-S and Examples or a fragment thereof relative to a control reference. In such clinical trials, the expression and/or activity of the biomarker can be used as a "read out" or marker of the phenotype of a particular cell.
In some embodiments, the present invention provides a method for momtoring the effectiveness of treatment of a subject with an agent (e.g.. an agonist, antagonist, peptidomimctic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) including the steps of (i) obtaining a pit-administration sample from a subject prior to administration of the agent (ii) detecting die level of expression andor activity of one or more biomarkers of the invention, including one or more biomarkers listed in Tables l-S and Examples or fragments thereof in the prcad ministration sample; (iii) obtaining one or more pc -adnunistra ion samples from the subject (iv) detecting the level of expression or activity of the biomarker in die post- administration samples: (v) coinparing the level of expression or activity of the biomarker or fragments thereof in the prc-administration sample with the that of the biomarker in the poet administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased admmisCration of die agent may be desirable to increase the expression or activity of one or more biornarkcrs to higher levels than detected (e.g., to increase the effectiveness of the agent) Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of the biomarker to lower levels than detected (&#., to decrease the effectiveness of the agent). According to such an embodiment, biomarker expression or activity may be used as an indicator of the effectiveness of an agent, even in die absence of an observable pbenotypic response. D. Methods of Trwtti Pi
The present invention provides for bom prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder characterized by insufficient or excessive production of biornarkcrs of the invention, including biornarkcrs listed in Tables 1-5 and Examples or fragments thereof, which have aberrant expression or activity compared to a control. Moreover, agents of the invention described herein can be used to detect and isolate the biornarkcrs or fragments thereof, regulate the bioavailability of the biornarkcrs or fragments thereof, and modulate biomarker expression levels or activity.
1. Prophylactic Methods
In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant expression or activity of one or more biornarkcrs of the invention, including one or more biomarkers Ksted in Tables 1-5 and Examples or a fragment thereof, by administering to the subject an agent which modulates biomarker expression or at least one activity of the biomarker. Subjects at risk for a disease or disorder which is caused or contributed to by aberrant biomarker expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. A<rniinistration of a prophylactic agent can occur prior to the
manifestation of symptoms characteristic of the biomarker expression or activity aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. 2. TbCTMKUtifi Methods,
Another aspect of the invention pertains to methods of modulating the expression or activity or interaction with natural binding partners) of one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1 -5 and Examples or fragments thereof for therapeuiic purposes. The biomarkers of the invention have been demonstrated to correlate with cancer (e.g., lymphoid cancers, such as leukemia).
Accordingly, the activity and/or expression of the biomarker, as well as the interaction between one or more biomarkers or a fragment thereof and its natural binding partner(s) or a fragments) thereof can be modulated in order to modulate the immune response.
Modulatory methods of the invention involve contacting a ceil with one or more biomarkers of the invention, including one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples or a fragment thereof or agent that modulates one or more of the activities of biomarker activity associated with the cell. An agent that modulates biomarker activity can be an agent as described herein, such as a nucleic acid or a polypeptide, a naturally-c curring binding partner of the biomarker, an antibody against die biomarker, a combination of antibodies against the biomarker and antibodies against other immune related targets, one or more biomarkers agonist or antagonist, a peptidomiractk of one or more biomarkers agonist or antagonist, one or more biomarkers peptidomimetic, other small molecule, or small RNA directed against or a mimic of one or more biomarkers nucleic acid gene expression product
An agent that modulates the expression of one or more biomarkers of the invention, including one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples or a fragment thereof is, eg., an antisense nucleic acid molecule, RNAi molecule, shRNA, mature raiRNA, pre-miRNA, pri-miRNA, mi RNA*, anti-miRNA, or a miRN binding site, or a variant thereof, or other small RNA molecule, triplex oligonucleotide, ribozyroe, or recombinant vector for expression of one or more biomarkers polypeptide. For example, an oligonucleotide complementary to the area around one or more biomarkers polypeptide translation initiation site can be synthesized. One or more antisense oligonucleotides can be added to cell media, typically at 200 ug/nu, or administered to a patient to prevent the synthesis of one or more biomarkers polypeptide. The antisense oligonucleotide is taken up by cells and hybridizes to one or more biomarkers mRNA to prevent translation. Alternatively, an oligonucleotide which binds double- stranded DNA to form a triplex construct to prevent DNA unwinding and transcription can be used. As a result of either, synthesis of biomatker polypeptide is blocked. When biomarker expression is modulated, preferably, such modulation occurs by a means other than by knocking out the biomarker gene.
Agents which modulate expression, by virtue of the fact that they control tbc amount of biomarker in a cell, also modulate the total amount of biomarker activity in a cell.
In one embodiment, the agent stimulates one or more activities of one or more biomarkers of the invention, including one or more biomarkers listed in Tables 1-5 and Examples or a fragment thereof. Examples of such stimulatory agents include active biomarker polypeptide or a fragment thereof and a nucleic acid molecule encoding the biomarker or a fragment thereof that has been introduced into die cell (e.g. , cDNA, mRNA, shRNAs, siRNAs, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti- nuRNA, or a miRNA binding site, or a variant thereof, or other functionally equivalent molecule known to a skilled artisan). In another embodiment the agent inhibits one or more biomarker activities. In one embodiment, the agent inhibits or enhances the interaction of die biomarker with its natural binding partners). Examples of such inhibitory agents include antisense nucleic acid molecules, anti-biomarker antibodies, biomarker inhibitors, and compounds identified in die screening assays described herein.
These modulatory methods can be performed in vitro (eg., by contacting the cell with the agent) or, alternatively, by contacting an agent with cells in vivo (e.g., by administering aw agent to a subject). As such, η present invention provides methods of treating an individual afflicted with a condition or disorder that would benefft fromup- or downHtnodulation of one or more biomarkers of the invention listed in Tables 1-5 and Examples or a fragment thereof, eg., a disorder characterized by unwanted, insufficient, or aberrant expression or activity of the biomarker or fragments thereof, in one embodiment, die method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (eg., upregulates or downrcgulatcs) biomarker expression or activity. In another embodiment, the method involves administering one or more biomarkers polypeptide or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted biomarker expression or activity.
Stimulation of biomarker activity is desirable in sitiiztiom in which the biomarker is abnormally downrcgulatcd and or in which increased biomarker activity is likely to have a beneficial effect likewise, inhibition of biomarker activity is desirable in situations in which toomarker is abnormally unregulated and/or in which decreased biomarker activity is likely to have a beneficial effect
hi addition, these modulatory agents can also be administered in combination therapy with, eg., cbcnxxherapcutic agents, hormones, antiangiogens, radiolabciled, compounds, or with surgery, cryotherapy, and/or radiodierapy. The preceding treatment methods can be administered in conjunction with other forms of conventional therapy (*.g., standard-o care treatments for cancer well known to the skilled artisan), either
consecutively with, pre- or r>ost-conYeritional therapy. For example, these modulatory agents can be administered with a therapeutically effective dose of chcmothcrapeutic agent In another embodiment, these modulatory agents are administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemothcrapeutic agent. The Physicians' Desk Reference (PDR) discloses dosages of chernotherarjcutic agents that have been used in the treatment of various cancers. The dosing regimen and dosages of these aforementioned c ernc^ierapcutic drugs that are therapeutically effective will depend on the particular cancer (eg., lymphoid cancers, such as leukemia), being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician.
E. Methods, of Expanding ymphoid Pr enitorC?u Population.
In another aspect, the present invention provides methods of increasing the number of lymphoid progenitor cells from an initial population of lymphoid progenitor cells comprising contacting the lymphoid progenitor cells with an agent that inhibits polycomb repressor complex 2 (PRC2) activity to thereby increase the number of lymphoid progenitor cells.
l. Ceil typa for expansion
As described herein, lymphoid progenitor celts and cellular sources comprising same can be used Descriptions of cells herein are well known to the skilled artisan and are further described with the understanding that these (inscriptions reflect the current state of knowledge in the art and the invention is not limited thereby to only those rAenotypk markers described herein.
Hematopoietic stem cells give rise to lymphoid or myeloid progenitor cells. A "rymphoid progenitor cell" refers to a cell capable differentiating into any of the terminally cbffercnoated cells of the lymphoid lineage. Encompassed within the lymphoid progenitor cells arc the common lymphoid progenitor cells (CLP), a cell population characterized by limited or non-self-renewai capacity but which is capable of cell division to form T lymphocyte and B lymphocyte progenitor cells, K cells, and lymphoid dendritic cells. The marker pbenotypes useful for identifying CLPs will be those commonly known in the art For example, for CLP cells of mouse, the cell population is characterized by the presence of markets as described m Kc^ ^a/. (1997) Ce 7 1: ^1^72, wmk for hurnan CLPs, a marker phenotype of CD34+ CD38+ CD10+ IL7R+ may be used (Gary et at. (1995) Immunity t 3:459-473; Akashi et al. (1 99) Int. J. Hematol. 69:217-226). Additional iUustrations of B cell lineage deveiopmem and associated molecular markers defining each cell stage in mouse models are provided in Figure 19 (Iritani etal. (1997) EMBOJ.
16:7019-7031; Hardy and Hayakawa (2001) ΛΛ. Rev. Immunol 19:595-621).
By contrast, cc mittcd myeloid progenitor cells refer to cell populations capable of differentiating into any of the terminally differentiated cells of die myeloid lineage.
Encompassed within die myeloid progenitor cells are the common myeloid progenitor cells (CMP), a cell population characterized by limited or non-self-rcnewal capacity but which is capable of cell division to form granulocyfc/rnacrophage progenitor cells (GMP) and megakaryocyte erythroid progenitor cells (MEP). Non-sclf-rencwing cells refers to cells that undergo cell division to produce daughter cells, neither of which have the
differentiation potential of the parent cell type, but instead generates differentiated daughter cells. The marker phenotypes useful for identifying CMPs include those commonly known in the an For CMP cdls of murine origin, the cell population is characterized by the marker phenotype c-Kit(high) (CD117) CD16(low) CD34(tow) Sca-l(ncg) Lin(neg) and further characterized by the marker phenotypes FcyR(k>) IL-7Rct(neg) (GDI 27). The murine CMP cell population is also characterized by the absence of expression of markers that include B220, CD4, CDS, CD3, Ter 119, Gr- 1 and Mac- 1. For CMP cells of human origin, the cell population is chanuftrrized by CD34+CD38+ and further characterized by die marker phenotypes CD123+ (IL-3Ra) CD45R(ncg). The human CMP cell population is also characterized by the absence of cell markers CD3, CD4, CD7, CDS, CD 10, GDI lb, CD14. CD 19, CD20. CD56, and CD234a. Descriptions of marker phenotypes for various myeloid progenitor cells are described in, for example, U.S. Pat. Nos. 6,465,247 and 6,761,883; Akas (2000) Nomre 404:193-197. Another committed progenitor cell of the myeloid lineage is the granulocyte/macrophage progenitor cell (GMP). The cells of mis progenitor cell population arc characterized by their capacity to give rise to granulocytes (e.g., basophils, eosinophils, and neutrophils) and macrophages. Similar to other committed progenitor ceils, GMPs lack seif-renewal capacity. Marine GMPs are characterized by the marker phenotype c- itfhi) (CD! 17) Sea- i (neg) Fc (CD16) ]L-7R7(neg) CD34(pos). Murine GMPs also lack expression of markers B220. CD4, CDS, CD3, Gr-l,Mac-l, and CD 0. Human GMPs are characterized by me marker phenotype CD34+ CD38+ CD 123+ CD 5RA+. Human GMP cell populations are also characterized by the absence of markers CD3, CD4, CD7, CDS, CD 10, CDi lb, CD 14, CD 19, CD20, CD56, and CD235a. In addition, inegakaryocyuVciythroid progenitor cells (MEP), which are derived from the CMPs, are characterized by their capability of differentiating into committed
megakaryocyte progenitor and crythroid progenitor cells. Mature megakaryocytes are polyploid cells that are precursors for formation of platelets, a developmental process regulated by thrornbopoietin. Erythroid cells arc formed from the committed crythroid progenitor cells through a process regulated by erythropoietin, and ultimately differentiate into mature red blood cells. Murine MEPs arc diaracterizcd by cell marker phenotype c- Kit(hi) and IL-7R and further characterized by marker phenotypes Fc and CD34(low). Murine MEP ceil populations arc also characterized by the absence of markers B220. CD4, CD8, CD3, Gr-1 , and CD 0. Another exemplary marker phenotype for mouse MEPs is c- kit(high) Sca-l(ncg) Lin (ncg/low) CD 16 (low) CD34dow). Human MEPs are
characterized by marker phenotypes CD3 +- CD38+ CDI 23(ncg) CD45RA(ncg). Human MEP cell populations are also characterized by the absence of markers CD3, CD4, CD7, CD8, CD10,CDllb, CD14,CD19, CD20,CD56, aDdCD235a. Further restricted progenitor cells in the myeloid lineage are the granulocyte progenitor, macrophage progenitor, rdegakaryocytc progenitor, and erythroid progenitor. Granulocyte progenitor cells are characterized by their capability to differentiate into terminally differentiated granulocytes, inducting eosinophils, basophils, neutrophils. The GPs typically do not differentiate into other cells of the myeloid lineage. With regards to the megakaryocyte progenitor cell (M P), these cells are characterized by their capability to diflerentiaie into tenninally differentiated megakaryocytes but generally not other cells of the myeloid lineage (see, e.g. WO 2004/024875).
In some embodiments, the cells to be expanded are comprised within tissues or other cellular sources, such as bone marrow, peripheral blood, cord blood, and the like. Peripheral and cord blood is a rich source of HSCs and progenitor cells. Cells are obtained using methods known and commonly practiced in die art. For example, methods for preparing bone marrow cells are described in Sutherland et aL, Bone Marrow Processing and Purging: A Practical Guide (Gee, A. P. ed.), CRC Press Inc. (1 91)). Umbilical cord blood or placental cord blood is typically obtained by puncture of the umbilical vein, in both term or preterm, before or after placental detachment (sec, eg. Turner, C. W. et a!., Bone Marrow Transplant 10:89 (1992); Bcrtolini, F. et al., J. Hematoma.4:29 (1995)).
In other ernoodiincnts, the starting cells to be expanded are isolated cells. Such cells can farther be selected and purified, which can include both positive and negative selection methods, to obtain a substantially pure population of cells. In one aspect fluorescence activated cell sorting (FACS), also referred to as flow cytometry, is used to sort and analyze the different cell populations. Cells having the cellular nurkers specific for a lymphoid progenitor cell population are tagged with an antibody, or typically a mixture of antibodies, that bind the cellular markers. Each antibody directed to a different marker is conjugated to a detectable molecule, particularly a fluorescent dye that can be distinguished from other fluorescent dyes coupled to other antibodies. A stream of tagged or "stained" cells is passed through a light source that excites the fluorochromc and the emission spectrum from the cells detected to detenrune the presence of a particular labeled antibody. By concurrent detection of different fluorochrorocs, also referred to in the art as multicolor fluorescence cell sorting, cells displaying different sets of ceil markers may be identified and isolated from other cells in the population. Other FACS parameters, including, by way of example and not limitation, side scatter (SSC), forward scatter (FSQ, and vital dye staining {e.g., with propidium iodide) allow selection of cells based on size and viability. FACS sorting and analysis of HSC and progenitor cells is described in, among others, U.S. Pat Nos. 5,137,809, 5,750,397, 5,840,580; 6,465,249; Manx, M. G. et al., Proc NatL Acad. Sci. USA 99: 11872-11877 (2002); and Alcashi, . etal., Nature 404(6774): 193-197 (2000)). General guidance on fluorescence activated cell sorting is described in, for example, Shapiro, H. M, Practical Flow Cytometry, 4th Ed., Witey-Liss (2003) and Ormerod, M. G., Flow
Cytometry: A Practical Approach, 3rd Ed, Oxford University Press (2000).
Another method of isolating the initial cell populations uses a solid or insoluble substrate to which is bound antibodies or ligands that interact with specific cell surface markers. In immunoadsorption techniques, cells are contacted with the substrate (e.g., column of beads, flasks, magnetic particles) containing the antibodies and any unbound cells removed. Immunoadsorption techniques can be scaled up to deal directly with die large numbers of cells in a clinical harvest Suitable substrates include, by way of example and not limitation, plastic, cellulose, dextran, polyacrylamide, agarose, and others known in the ait (e.g., Pharmacia Scpharose 6 MB macrobeads). When a solid substrate comprising magnetic or paramagnetic beads is used, ceils bound to the beads can be readily isolated by a magnetic separator (see, Kato, K. and Radbruch, A., Cytometry 14<4):384- 2 ( 1993); CD34+ direct isolation kit, Miltenyi Biotec, Bergisch, Gladbach, Germany). Affinity chromatographic cell separations typically involve passing a suspension of cells over a support bearing a selective ligand immobilized to its surface. The ligand interacts with its specific target molecule on the cell and is captured on the matrix. The bound cell is released by tbc addition of an elation agent to me running buffer of the column and die free cell is washed through the column and harvested as a homogeneous population. As apparent to tbe skilled artisan, adsorption techniques are not limited to those employing specific antibodies, and may use nonspecific adsorption. For example, adsorption to silica is a simple procedure for removing phagocytes from cell preparations.
FACS and most batch wise tmmunoadsorption techniques can be adapted to both positive and negative selection procedures (see, e.g., U.S. Pat. No. 5,877,299). In positive selection, tbc desired cells are labeled with antibodies and removed away from the remaining un labeled/unwanted cells. In negative selection, the unwanted cells arc labeled and removed. Another type of negative selection that can be employed is use of antibody/complement treatment or immunotoxins to remove unwanted cells.
It is to be understood that the purification of cells also includes combinations of the methods described above. A typical combination may cotnprise an initial procedure that is effective in removing the bulk of unwanted cells and cellular material, for example leukapharcsis. A second step may include isolation of cells expressing a marker common to one or more of the progenitor cell populations by immunoadscrption on antibodies bound to a substrate. For example, magnetic beads containing anti-B220+ antibodies are able to bind and capture lymphoid progenitors that commonly express the B220 antigen. An additional step providing higher resolution of different cell types, such as FACS sorting with antibodies to a set of specific cellular markers, can be used to obtain substantially pure populations of the desired cells. Another combination may involve an initial separation using magnetic beads bound with anti-B220 antibodies followed by an additional round of purification with FACS.
Where applicable, stem cells and lymphoid progenitor cells can be mobilized from the bone marrow into the peripheral blood by prior administration of cytokines or drugs to de subject (see, «#, Lapidot, T. et al., Exp. Hematol.30:973-981 (2002)). Cytokines and cberookines capable of hxiucing mobilization include, by way of example and not limitation, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSFX erythropoietin (Kicssinger, A. et at., Exp. Hcmatol. 23:609- 612 (1995)), stem cell factor (SCF), AMD3100 ( AnorMed, Vancouver, Canada), inter leukh 8 (IL-8), and variants of these factors (e.g. , pegfilgastrim, darbopoictin).
Combinations of cytokines and or chemokmes, such as G-CSF and SCF or GM-CSF and O- CSF, can act synergistically to promote mobilization and may be used to increase die number of lymphoid progenitor cells in the peripheral blood, particularly for subjects who do not show efficient mobilization with a single cytokine or chemokine (Morris, C. et al., J. Haematol. 120:413-423 (2003)). Cytoablative agents can also be used at inducing doses (I.e., cytoreductive doses) to mobilize lymphoid progenitor cells, and are useful cither alone or in combination with cytokines. This mode of mobilization is applicable when the subject is to undergo mycloablativc treatment and is carried out prior (o the higher dose cheniotherapy. Cytarcductivc drugs for mobilization, include, among others,
cyclophosphamide, rfosfamidc, etoposide, cytosine arabinoside, and carbopiatin (Monti Uo, M et al., Leukemia 18:57-62 (2004); Dasgupta, A. et al., J. mfusional G»cmother.6:12 (1996); Wright. D. E. et al., Blood 97:(8):2278-2285 (2001)).
Determining the differentiation potential of cells, and thus the type of stem cells or progenitor cells isolated, is typically conducted by exposing the cells to conditions that permit development into various tenninally differentiated cells. These conditions generally comprise a mixture of cytokines and growth factors in a culture medium permissive for development of the lymphoid lineage. Colony forming culture assays rely on culturing die cells in vitro via limiting dilution and assessing the types of cells that arise from their continued development A common assay of this type is based on methylceilulose medium supplemented with cytokines (*.g. , MethoCult, Stem Cell Technologies, Vancouver, Canada; Kennedy, M. et al., Nature 386:488-493 (1997)). Cytokine and growth factor formulations permissive for differentiation in the hematopoietic pathway are described in Manzet aL, Proc. Natl. Acad. So. USA 99(18): 11872-11877 (2002); U.S. Pat. No.
6,465.249; and Akashi. K. et al.. Nature 404(6774): 193-197 (2000)). Cytokines include SCF, FLT-3 ligand, GM-CSF, 1L-3, TPO, and EPO. Another in vitro assay is long-term culture initiating cell (LTC-1C) assay, which typically uses stromal cells to support honatopoicsis (see. e.g., Ploemacher. R. E. ct al.. Blood.74:2755-2763 (1989); and Sutherland, H. J. et al., Proc. Natl. Acad. Sci. USA 87:3745 (1995)). Another type of assay suitable for determining the differentiation potential of isolated cells relies upon in vtvo administration of cells into a host animal and assessment of the repopulation of the hematopoietic system. The recipient is immunocoiruHomised or immunodcficicnl to limit rejection and permit acceptance of allogeneic or xenogeneic cell transplants. A useful animal system of this kind is the NOD/SCID (Pfiumio, F. et at, Blood 88:3731 (1996); Szih/assym S. J. et al., "Hematopoietic Stem Cell Protocol," in Methods in Molecular Medicine, Humana Press (2002); Gremer, D. L. et al.. Stem Cells 16(3):166-177 (1998); Piacibdlo, W. et al., Blood 93:( 11): 3736-3749 (1999)) or Rag2 deficient mouse (Shinkai, Y. et at, Cell 68:855-867 (1992)). Cells originating from the infused ceils arc assessed by recovering ceils from the bone marrow, spleen, or blood of the host animal and determining presence of cells displaying specific cellular markers, (/.&, marker
pbenotyping) typically by FACS analysis. Detection of markers specific to the transplanted cells permits distinguishing between endogenous and transplanted cells. For example, antibodies specific to human forms of the cell markers (e.g., HLA antigens) identify human cells when they are transplanted into suitable immunodeficient mouse (see, eg., Piacibello. W. ctal., supra).
The initial populations of cells obtained by the methods above are used directly for expansion or frozen for use at a later date. A variety of mediums and protocols for freezing cells are known in the an. Generally, the freezing medium will comprise DMSO from about 5-10%, 10-90% scrum albumin, and 50-90% culture medium. Other additives useful for preserving cells include, by way of example and not limitation, disaccharides such as trehalose (Scheinkonig, C. et al., Bone Marrow Transplant.34(6):531 -6 (2004)), or a plasma volume expander, such as hetaslarch (/.*., faydroxyethyi starch). In some embodiments, isotonic buffer solutions, such as phosphate-buffered saline, may be used. An exemplary cryopreservative composition has cell-culture medium with 4% HSA, 7.5% dimethyl sulfoxide (DMSO), and 2% hetastarch. Other compositions and methods for cryopreservation are well known and described in the art (see, e.g., Broxmeyer et al. (2003 ) Proc. Nad. Acad. Set. USA 100:645-650). Cells are preserved at a final temperature of less than about -135*C.
Expansion of lymphoid progenitor cells is carried out in a basal medium, which can be supplemented with the mixture of cytokines and growth factors described herein, sufficient to support expansion of lymphoid progenitor ceils. The basal medium will comprise amino acids, carbon sources (e.g., pyruvate, glucose, etc.), vitamins, serum proteins fa£., albumin), inorganic salts, divalent cations, antibiotics, buffers, and other preferably defined components that support expansion of myeloid progenitor cells. Suitable basal mediums include, by way of example and not limitation, RPM1 medium, Iscovc's medium, minimum essential medium, Dulbeccos Modified Eagles Medium, and others known in the art (see, .g., U.S. Pat. No. 6,733,746). Commercially available basal mediums include, by way of example and not limitation, Stemline.TM. (Sigma Aldrich), StcmSpan.TM. (StemCdl Technologies, Vancouver, Canada), Stempro.TM. (Life
Technologies, Gibeo BRL, Gahhcrsburg, MA, USA) HPGM.TM. ((Otmbrcx, Walkersvillc, Md., USA), QBSF.TM. (Quality Biological, Gaitfacrsburg, Md., USA), X-VTVO (Cambrex Corp., Walkersville, Md.. USA) and Mesencult.TM. (StemCdl Technologies, Vancouver, Canada). The formulations of these and other mediums will be apparent to tbc skilled artisan.
The initial population of cells arc contacted with die mixture of cytokines and growth factors in the basal rncd rm, and cultured to expand the population of myeloid progenitor cells. Expansion is done for from about 2 days to about 14 days, preferably from about 4 days to 10 days, more preferably about 4 days to 8 days and or until the indicated fold expansion and the characteristic cell populations are obtained.
In one embodiment, the final cell culture preparation is characterized by a lymphoid progenitor cell population that is expanded at least about 0.5 fold, about I fold, about 5 fold, about 10 fold, about 20 fold, or more. In the final culture, the lyinphoid progenitor cell population can comprise at least about 60%, 65%.70%, 75%, 80%.85%, 90%, 95%. 99%, or more of the total cells in the culture.
Variations on the basic culture techniques described herein readily understood by the skilled artisan are included within the scope of die present invention. For example, feeder cell cultures can be used to aher the growth media environment (Feugier, P. et a!., J Hematother Stem Cell Res 11(1): 127-38 (2002)). Similarly, co-cultures of various cell populations can be created. Cells expanded by the methods described herein can be used without further purification, or can be isolated into different cell populations by various techniques known in the art, such as by immunoaffinity chromatography,
irnrnunoadsorptioii, FACS sorting, or other procedures as described above. Preferably, FACS sorting or mirmuKMdsorption is used. For example, a FACS gating strategy has an initial selection for live cells based on characteristic forward scatter (cell size) and side scatter (ceil density) parameters, and a second selection for expression of cell markers for lymphoid progenitor cells or non-lymphoid cells.
2. Arena to inhibit Myggmfr Repressor Complex, 2 (FR 2) catalytic activity The PRC2 complex directs historic methyltransfcrase activity. Although the compositions of the complexes isolated by different groups are slightly different, they generally contain EED, EZH2, SUZ12, and bAp48 or Drotophih horaoiogs thereof. However, a reconstituted complex comprising only EED, EZH2, and SUZ12 retains historic nKthyltransferase activity («.£., mono- through tri-mcthylation) for lysine 27 of histonc H3 (eg., H3K27mc3; see U.S. Pat No. 7,563,589; Cardoso et at. (2000) Eur. J. Hum. Genet. 8:174-180). The PRC2 complex may also interact with DN TI, DNMT3A, DNMT3B and PHFl via the EZH2 subunit and with SIRT1 via the SUZ12 subunit Of the various proteins making up PRC2 complexes, EZH2 (Enhancer of Zeete Homolog 2) is the catalytic subunit (Vire et al. (2006) Nature 439:871-874). The catalytic site of EZH2 in turn is present within a SET domain, a highly conserved sequence motif (named after Su(var)3- , Enhancer of Zeste, Trithorax) that is found in several chronmtm-associated proteins, including members of bom the Trithorax group and Polycomb group. The SET domain is characteristic of all known histonc lysine niethyitransfcrascs except the H3- 79 memyhransferasc DOT1.
Any agent that disrupts the catalytic rnemy I transferase activity of PRC2 can be used according to the methods described herein. Such agents include small molecules, antisense nucleic acids, interfering RNA, shRNA, siRNA, aptamcrs, ribozymes, and dominant- negative protein binding partners. For example, knockout or knockdown of EZH2 or other PRC2 complex components, such as through reduction of mRNA or protein, will reduce H3K27me3 methylarion. Similarly, functional knockout or knockdown of PRC2
H3K27mc3 activity can be achieved by disrupting the protein-protein interactions necessary for the PRC2 to form and/or maintain catalytic activity. For example, dominant negative proteins, such as EZH2 lacking a functional catalytic domain and/or having reduced hi stone methyltransferase activity, but maintaining the ability to bind to PRC2 complex binding partne s) wilt reduce PRC2 H3 27me3 activity. In some embodiments, chemical («.#., small molecule) inhibitors of PRC2 activity, such as small molecule inhibitors of EZH2, are particularly useful because expansion of cell populations can be easily reversed by withdrawal of die compound. Such chemical inhibitors are well known in die ait and are described, for example, in US Pat Pubis. 2013-0059849, 2013-0053397, 2013-0053383, 2013-0040906, 2012-0264734, 2012-0071418, as well as McCabe etat. (2012) Nat n 492: 1 8-112. In one embodiment, a chemical inhibitor of EZH2 is used, such as GS -126 (S>- 1 -<$cc-butyl N-((4,6-dimcihyl-2-oxo- 1 ,2-dihydfopyridiii-3-yl)i^
(piperazin- 1 -y l)pyridin-3- I )- 1 H-ina\>lc-4-carboxarradc) having (he structure:
Figure imgf000139_0001
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3. Uses of expanded rvnmhoid Proaenitor cells
Expanded cell populations prepared by the methods described herein arc useful for the treatment of various disorders and applicable for many biomedical and biotechnological situations. As used herein, "treatment" can refer to tbcrarxutic or prophylactic treatment, or a suppressive measure for a disease, disorder or undesirable condition. Treatment encompasses adnunistmtton of the subject cells in an appropriate form prior to the onset of disease symptoms and/or after clinical manifestations, or other manifestations of the disease or condition to reduce disease severity, halt disease progression, or eliminate the disease. Prevention of the disease includes prolonging or delaying the onset of sy nptorns of the disorder or disease, preferably in a subject with increased susceptibility to the disorder. The amount of the cells needed for achieving a therapeutic effect will be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for adrrurUstering the cells for therapeutic purposes, the cells are given at a pharmacologically effective dose. By "fiharmacologicalfy effective amount" or "pharmacologically effective dose" is an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease condition, including reducing or eliminating one or more symptoms or rnanifestations of the disorder or disease.
Cell populations expanded in vivo will already be comprised within a subject's body for use therein. Cells for infusion, such as those prepared in vitro or ex vivo, include expanded ceil populations without additional purification, or isolated ceil populations having defined cell marker pbenotype and characteristic differentiation potential as described herein. Expanded cells may be derived from a single subject where the ceils arc autologous or allogeneic to the recipient ft is to be understood that cells isolated directly from a donor subject without expansion in culture may be used for the same therapeutic purposes as die expanded cells. Preferably, the isolated cells are a substantially pure population of cells. These unexpended cells may be autologous, where the cells to be infused are obtained from the recipient, such as before treatment with cytoablative agents. In another embodiment, the unexpended ceils are allogeneic to the recipient, where die cells have a complete match, or partial or full mismatch with the MHC of the recipient. As described above, the isolated unexpended cells are preferably obtained from different donors to provide a mixture of allogeneic lymphoid cells.
Transplantation of cells into an appropriate host can be accomplished by methods generally used in die art The preferred method of admmistration is imravenous infusion. The number of cells transfused will take into consideration factors such as sex. age, weight, the types of disease or disorder, stage of the disorder, the percentage of the desired cells in the cell population {e.g., purity of cell population), and the cell number needed to produce a therapeutic benefit. Generally, the numbers of expanded cells infused may be from about 1x10* to about 1x10s cells kg, from about Ixl©5 to about Hbil^ cells/kg, preferably about lxl( ceUs to about 5xl(^c ls/kg of body wdgh^ In some embodiments, the cells are in a pharmaceutically acceptable carrier at about IxlO9 to about 1x10* cells. Cells can be administered in one infusion, or through successive infusions over a defined time period sufficient to generate a therapeutic effect Different populations of cells may be infused when treatment involves successive infusions. A pharmaceutically acceptable carrier, as further described below, may be used for infusion of die cells into the patient These will typically cornprise, for example, buffered saline (eg., phosphate buffered saline) or unsuppkmented basal cell culture medium, or medium as known in the art
Conditions suitable for treatment include genetic and or acquired irnrminodcficicncy or autoimmune diseases where, for example, patients have decreased numbers of lymphocytes leading to susceptibility to infection and shortened lifespan. Exemplary, non- limiting genetic unmunodeficiencies include combined immunodeficiencies (SCIDX such as ADA-defkiency (adenosine deaminase), X-SC1D (X linked SOD), ZAP-70 deficiency, Rag 1/2 deficiency, Jak3 deficiency, IL7RA deficiency or CD3 deficiencies; primary imimmodeficiencics, such as the acqaired imtmmodefkiency syndrome (AIDS), DtGeorge's (vclocardiofacial) syndrome, adenosine deaminase (ADA) deficiency, reticular dysgenesis, Wiskott Aidrich syndrome, ataxia-telangiectasia, severe combined immunodeficiency; and secondary immunodeficiencies, such as anergy from tuberculosis, drug-induced leukopenia, non-HIV viral illnesses leukopenia, radiation poisoning, toxin exposure, malnutrition, and the like.
Expanded lymphoid cell populations are also useful for various transplantation conditions, such as transplantation of stem cells, bone marrow, and/or umbilical cord blood. Lymphoid progenitors expanded in vitro, ex vivo, or in vivo can shorten the time to immune reconsutution, thereby decreasing the likelihood of infectious complications.
The ability to expand lymphoid ceil populations has numerous additional applications to biotechnologicai and biomedical research in addition to or outside the context of treating subjects. For example, lymphocytes that produce antibodies can be expanded in order to improved immune responses in vivo or to improve the yields of diagnostic or therapeutic antibodies produced in vitro or ex vivo. Similarly, B cells or other tymphoid cells, such as those useful for research purposes that have been genetically modified, could be indefinitely cultured to perpetuate clonal cell populations. IV. Hiajn¾¾¾uti^aj CvlBPfflHtiflM
In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeuticalh/ flective amount of an agent that modulates {e.g., increases or decreases) PRC2 activity and/or H3K27mc3 levels, formulated together with one or more pharrnaceutically acceptable carriers (additives) and or diluents. As described in detail bdow, die pharmaceutical compositions of die present invention may be specially formulated for administration in solid or liquid form, including those adapted for die following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes (2) parenteral administration, for example, by subcutaneous, mtramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravagfnalry or intrarectal r , for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound. The phrase "therapeutically-cfTectivc amount" as used herein means that amount of an agent that modulates (e.g., inhibits) PRC2 activity and/or H3K27me3 levels, or expression and/or activity of the complex, or composition comprising an agent that modulates (e.g., inhibits) PRC2 activity and/or H3K27me3 levels, or expression and/or activity of the complex, which is effective for producing some desired therapeutic effect, e.g. , cancer treatment, at a reasonable benefit/risk ratio.
The phrase "pharmaceutically acceptable*' is employed herein to refer to those agents, rnatehals, compositions, and or dosage forms which arc, within tbc scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, corntnensurate with a reasonable benefit/risk ratio.
The phrase "phanroacculicalry-acceptablc carrier" as used herein means a pharniaccurically-accc tabic material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting tbc subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutical ly-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cdhilose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (°) oils, such as peanut oil, cottonseed oil, safFlower oil, sesame oil, olive oil, corn oil and soybean oil; (1 ) glycols, such as propylene glycol; ( 11) potyols, such as glycerin, sorbitol, marmitol and
polyethylene glycol; (12) esters, such as ethyl oteatc and ethyl laurate; (13) agar (14) buffering agents, such as magnesium hydroxide and aiurninum hydroxide; (IS) alginic acid; (16) pyrogen-free water, (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21 ) other non-toxic compatible substances employed in pharmaceutical formulations.
The term "phannaccuficaUy-acccptablc salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of the agents that modulates (e.g., inhibits) PRC2 activity and/or H3K27me3 levels, or expression and/or activity of the complex
encompassed by die invention. These salts can be prepared in situ during tbc final isolation and purification of the respiration uncoupling agents, or by separately reacting a purified respiration uncoupling agent in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobroraidc, Hydrochloride, sulfate, bisulfatc, phosphate, nitrate, acetate, valerate, oleate, palnritatc, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, rumarate, succinate, tartrate, nap thy late, mesylate, glucoheptonate, lactobionate, and lauryunuphonate salts and the like (See. for example, Bcrge elal. (1977) "Pharrnaccutical Salts", J. Pham. Sci.66:1- 19).
In other cases, the agents useful in the methods of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutical hy- acccptable salts with phannaceutically-acccptable bases. The term "pharmaceutically- acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of agents mat modulates (e.g.. inhibits) PRC2 activity andor H3K27mc3 levels, or expression andor activity of the complex. These salts can likewise be prepared in situ during the final isolation and purification of the respiration uncoupling agents, or by separately reacting the purified respiration uncoupling agent in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically- acceptable metal cation, with ammonia, or with a ptannaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include die lithium, sodium, potassium, calcium, magnesium, and amminum salts and the like. Representative organic amines useful for the formation of base addition salts include emylarnine, diethylamine, emylcnediamine, efhanolamine, diethanc4amine, piperazine and the like (see, for example, Bcrge el al., supra).
Wetting agents, emulsifters and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and aritioxidants can also be present in the compositions.
Examples of pharmaceutically-acceptable antioxidants include: ( 1 ) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfatc, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylalcd hydroxyanisote (BHA), butyiatcd hydroxytotucne (BUT), lecithin, propyl gailate, alpna-tocopherol. and the like; and (3) metal chelating agents, such as citric acid, cthylenediainine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Formulations useful in the methods of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administratioa. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, die particular mode of administration. The amount of active ingredient, which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 % to about 99% of active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%.
Methods of preparing these fbrrmilations or compositions include the step of bringing into association an agent that modulates (e.g., increases or decreases) PRC2 activity and or H3 27mc3 levels, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by um'formly and intimately bringing into association a respiration uncoupling agent with liquid carriers, or finery divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragaca th), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a respiration uncoupling agent as an active ingredient. A compound may also be administered as a bolus, electuary or paste.
In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the KkeX the active ingredient is mixed with one or more prtanriaccurically-acceptable carriers, such as sodium citrate or dkalchim phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, andor silicic acid; (2) binders, such as, for example, carboxymethylceltulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodhim carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds: (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostcarate (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof, and (10) coloring agents. In the case of capsules, tablets and pills, the
pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excrpients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydraxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegram (for example, sodium starch grycolatc or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent
Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharaurauttcal-fccraulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, bydroxypiopylmcthyi cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes andor microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredients) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding
compositions, which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of die above-described excipients. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the ait, such as, for example, water or other solvents, soiubilizing agents and crau ifiers, such as ethyl alcohol, isopropyl alcohoL ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butykne glycol, oils (in particular, cottonseed, groundnut, com, genu, olive, castor and sesame oils), glycerol, tctrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions, in addition to the active agent may contain suspending agents as, for example, cthoxylated isostearyl alcohols, poiyoxyemylene sorbitol and sorbitan esters, macrocrystalline cellulose, aluminum metahydroxide. bentonite. agar-agar and tragacanth, and mixtures thereof.
Forrnulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more respiration uncoupling agents with one or more suitable ΜΜΪΠΪ taring excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.
Formulations which are suitable for vaginal adnrinistration also include pessaries, tampons, creams, gels, pastes, foams or spray fbnnulations containing such carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration of an agent that modulates (e.g., increases or decreases) PRC2 activity and/or H3 27me3 levels include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions win a pharrnaccutically- acccptablc carrier, and with any preservatives, buffers, or propellants which may be required.
The ointments, pastes, creams and gels may contain, in addition to a respiration uncoupling agent, excipients, such as animal and vegetable fats, oils, waxes, paraffins. starch, tragacartth, ceihiiose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof
Powders and sprays can contain, in addition to an agent that modulates (eg., increases or decreases) PRC2 activity and/or H3K27mc3 levels, exctpients such as lactose, lak, silicic acid, aluminum hydroxide, calcium silicates and poh amide powder, or mixtures of these substances. Sprays can additionally contain customary propellents, such as chlorofluorohydrcxarbons and volatile unsubstitutcd rjydrocarbom, such as butane and propane.
The agent that modulates (e.g., increases or decreases) PRC2 activity and/or H3K27me3 levels, can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles comaining die compound. A nonaqueous (e.g., fluorocarbon propeliant) suspension could be used. Sonic nebulizers arc preferred because they minimize exposing the agent to shear, which can result in degradation of die compound.
Ordinarily, an aqueous aerosol is made by fbnnulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Twecns, Piuronics. or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.
Transdermal patches have the added advantage of providing controlled delivery of a respiration uncoupling agent to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the peptidomimetic across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the
peptidomimetic in a polymer matrix or gel.
Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within die scope of this invention.
Pharmaceutical compositions of this invention suitable for parenteral administration ccinprise one or more respiration uncoupling agents in combination with one or more pharmaceutical iy-acccptablc sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bactcriostats, sohites which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
Examples of suitable aqueous and nonaqueous carriers which may be employed in die pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the likeX and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl o!catc. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preservatives, wetting agents, emulsif ing agents and dispersing agents. Prevention of the action of
microorganisms may be ensured by the inclusion of various antibacterial and anti fungal agents, for example, paraben, chlorobutanol, phenol sorbtc acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aruminum monosteatate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drag then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenterally-adrmnislered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by teeming mjcroencapsuk matrices of an agent that modulates («?.#., increases or decreases) PRC2 activity andor H3K27me3 levels, in biodegradable polymers such as polylacodc-polygfycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include po1y(orthocsters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or nucroemulsions. which are compatible with body tissue. When the respiration uncoupling agents of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example.0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
Actual dosage levels of the active ingredients in the pharmaceutical competitions of this invention may be determined by the methods of the present invention so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject
The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc Natl. Acad. Sci. USA 91 :3054 3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system. v. Ajdjm^strariftfi of Agtnw
The cancer diagnostic, prognostic, prevention, and or treatment modulating agents of the invention are administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo, to either enhance or suppress immune cell mediated immune responses. By "biologically compatible form suitable for administration In vivo" is meant a form of the protein to be administered in which any toxic effects are outweighed by die therapeutic effects of the protein. The term "subject" is intended to include living organisms in which an immune response can be elicited, eg., mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
Administration of an agent as described herein can be in any pharmacological form including a therapeutically active amount of an agent alone or in combination with a pharmaceutically acceptable carrier.
Administration of a therapeutically active amount of the therapeutic composition of de present invention is defined as an amount effective, at dosages and for periods of time necessary, to achieve the desired result. For example, a therapeutically active amount of a blocking antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of peptide to chert a desired response in the individual. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.
The agents of the invention described herein can be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal adrmnistration. Depending on the route of *_rrtimstratior the active compound can be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. For example, for administration of agents, by other than parenteral administration, it may be desirable to coat the agent with, or co-administer the agent with, a material to prevent its hiactrvation.
An agent can be administered to an individual in an appropriate carrier, diluent or adjuvant, co-administer cd with enzyme inhibitors or in an appropriate carrier such as liposomes. Piutrmaceuticalry acceptable diluents include saline and aqueous buffer solutions. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as mterfcron. Adjuvants contemplated herein include rcsorcinols, non- ionic surfactants such as polyoxyethyiene olcyl ether and n-bexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphatc (DEEP) and trasylol. Liposomes include water-m-o^tn-water entuisions as well as conventional liposomes (Sterna ei al (1984) J. N uroimmunoL 7:27).
The agent may also be administered parenteralry or intraperitonealry. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
Pharmaceutical compositions of agents suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the composition will preferably be sterile and must be fluid to the extent mat easy
syringcability exists. It will preferably be stable under die conditions of manufacture and storage and preserved against the contsminating action of rmcroorganisms such as bacteria and fungi. Tb carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyoi (for example, glycerol, propyiene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of rmcroorgamsms can be achieved by various antibacterial and antifungal agents, for example, parabens, cbJorobutanol, phenol, ascorbic acid, thimcrosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohob such as manhol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, ahnninum monostcarate and gelatin.
Sterile injectable solutions can be prepared by incorporating an agent of the invention (e.g., an antibody, peptide, fusion protein or small molecule) in die required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions arc prepared by incorporating the active compound into a sterile vehicle which contains a bask dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, die preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the agent phis any additional desired ingredient from a previously sterile-filtered solution thereof.
When the agent is suitably protected, as described above, the protein can be orally adiiunistcred, for example, with an inert diluent or an assimilable edible carrier. As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the therapeutic compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and um'formity of dosage. "Dosage unit form", as used herein, refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the requited pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by, and directly dependent on, (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of corr^ounding such an active compound for the treatment of sensitivity in individuals.
In one embodiment, an agent of the invention is an antibody. Asdefuicdhcrein, a therapeutically effective amount of antibody ie., m effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mgkg body weight, more preferably about 0. ] to 20 mgkg body weight, and even more preferably about 1 to 10 mg kg, 2 to 9 mgkg, 3 to 8 mgkg, 4 to 7 mgkg, or 5 to 6 mgkg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to die severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
Moreover, treatment of a subject with a therapeutically effective amount of an antibody can include a single treatment or, preferably, can include a series of treatments. In a profaned example, a subject is treated with antibody in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of antibody used for treatment may increase or decrease over the course of a particular treatment Changes in dosage may result from the results of diagnostic assays. In addition, an antibody of the invention can also be administered in combination therapy with, .g., chemotherapeutic agents, hormones, arttiangiogens, radio labelled, compounds, or with surgery, cryotherapy, and/or radiotherapy. An antibody of the invention can also be administered in conjunction with other forms of conventional therapy, either consecutively with, pre- or post- conventional therapy. For example, the antibody can be administered with a therapeutically effective dose of chemotherapeutic agent In another embodiment, the antibody can be administered in conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent The Physicians' Desk Reference (PDR) discloses dosages of temotheiapeutic agents that have been used in the treatment of various cancers. The dosing regimen and dosages of these aforementioned chcrnotherapcutic drugs that are thetapcutically effective will depend on the particular immune disorder, e.g., Hodgkin lymphoma, being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician.
In addition, die agents of the invention described herein can be administered using nanc?artklc-based composition and delivery methods well known to the skilled artisan. For example, nattcpartfcle-based delivery for improved nucleic acid (eg., small RNAs) therapeutics are well known in the art (Expert Opinion on Biological Therapy 7:1811- 1822).
This invention is further illustrated by the following examples, which should not be construed as limiting.
Exaasrple 1: Materials aad Methods for Example 2
A. Mice
All animal experiments were performed with approval of the Dana-Farbcr Cancer
Institute (DFCl) Institutional Animal Care And Use Committee (IACUC). All experiments were performed in an FVB x C57BU6 Fl background, unless otherwise specified. TslRhr (B6.129S6-Dp(16Cbrl-ORF )l Rhr/J; stock #005838) and Ts65Dn (B6EiC3Sn.BLiA- Ts(17,6>65Dn DnJ; stock #005252) mice were obtained from Jackson Laboratories.
HMG l_OE mice were described in Bustin etal. (1995) DNA Cell Biol. 14:997-1005. Pax5* mice (Urbanek ef al. (1994) Cell 79:901-912) backcrossed to C57BL/6 were obtained from M. BussUnger. Eu-CRLF2 and Ep-JAK2 R683G were generated by subcloning cD As expressing human CRLF2 or mouse JAK2 R683G (MulKghan et al. (2009) Nat. Genet. 41:1243-1246; Yodae/o/. (XHQ) Pmc. Natl. Acad. Sci. U.S . 107:252- 257) downstream of the immunoglobulin heavy chain enhancer (ΐμ) and generating transgenic founders in FVB fertilized eggs as described in Dildrop et al. (1989) EMBOJ. 8:1121-1128. Controls for TslRhr were wild-type Uttermates from crosses with either C57BI/6 (Jackson; #000664) or FVB (Jackson; #001800) mice as indicated. Controls for Ts65Dn were littermates from the colony (B6EiC3Sn.BLiAF 1 /J; Jackson #003647).
HMGNl.OE mice (Bustin etal (1995) DNA Cell Biol 14.997-1005) had been backcrossed >10 generations to C57BL6 (Abunatrira et al. (2011) J. Biol. Cheat
286:42051-42062). Comrob for HMGNljOE were wild-type uttermates after crossing with FVB mice. Donors for competitive transplantation were eongenic CD45.1 + B6.SJLr Ptpnf Pep /Boyi (Jackson; stock #002014) crossed with FVB (CD45.1), C57BL 6 x FVB Fl (CD45.1/2X or Tsl Rhr (C57BL 6) crossed with FVB Fl (CD45.1/2). Recipients for competitive transplant, and BCR/ABL and lk6 bone marrow transplants were
C57BL 6 FVB Fl female mice. No randomization was performed for experiments involving mice or samples collected from animals.
B. Aflftodia
Western blotting antibodies were against HMGN 1 (Aviva Systems Biology, #ARP38532_P050, rabbit polyclonal), HMGN1 (Abeam, #ab5212, rabbit polyclonal), mouse HMGN I (affinity purified rabbit polyclonal) (Birger ex ai (2003) EMBOJ. 22:1665- 167S; Bustin etal. (1990) J. Biol. Chan. 265:20077-20080), H3K27mc3 (Cell Signaling Technologies. 9733, rabbit polyclonal), total Histone H3 (Cell Signaling Technologies. #9715, rabbit polyclonal), and a- tubulin (Sigma, #T9026, mouse monoclonal)- Flow cytometry antibodies were B220-Pacific Blue (BD Pharmingen. #558108, clone RA3-6B2), CD43-APC (BD, #560663, clone S7) or CD43-F1TC (BD, #561856, clone S7), CD24-PE- Cy7 (BD, #560536, clone Ml/69), BPl-PE ^Biosciences, 12-5891, clone 6C3) or BP1- FITC (eBiosciences, 11-5891 , done 6C3), CD 5.1-PE-Cy7 (eBiosciences, 25-0453, clone A20), and CD45.2-APC (eBiosciences, 17-0454, clone 104). CWP-seq antibodies were H3K27me3 (Cell Signaling Technologies, #9733X H3 4me3 (Abeam, #ab8580X and H3 27ac (Abeam, #ab4729). c. F ow cytometry for tone marrow B crfis
Whole bone marrow was harvested from femurs and tibias of 6-8-week-okl mice. After red blood cell lysis (Qiagen, #158904), B cell progenitors were stained using antibodies and flow cytometry was performed as described in Hardy t at. (1991)7. Exp. Med. 173:1213-1225. Analysis was performed on a BD FACSCanto 11.
D. Competitive bgnc nwrrw transplantation
Whole bone marrow was pooled from femurs and tibias of two 8-week-old donor mice. Donor cells were wild-type or Tsl Rhr CD45.1+/CD45.2+ C57BL/6xFVB Fl (test) and CD45.1+ B6.SJLxFVB Fl (competitor), and were mixed in a 1 :1 ratio. Recipients were lethaliy irradiated (550 cGy x2, spaced 4 hours apart). B6.SJLxFVB Fl mice received 10* total cells (5x10s cells each of test and competitor) via lateral tail vein injection. Bone marrow was harvested 16 weeks after transplantation and analyzed by flow cytometry. ε. Mttfrytolfatoc w mr ftnnimt vssm
Whole bone marrow was harvested from 6-8-week-old mice, and red blood cells were lyscd. Cells were plated in B cell (Methocult M3630, Stem Cell Technologies) or myeloid (Methocult M3434) mcthylcclluose media in gridded 35 mm dishes. Myeloid colonies were plated at 2x10* cellsml per passage. B ceil colonies were plated at 2x10* ceils ml in passage 1, and at 5x10 cclls/rd pcT subsequent passage. Colonics were counted at 7 days, and colonics were then pooled and replated in the same manner. F. BMT models
For BCR-ABL transplantations (Krause et at. (2006) Nat. Med 12: 1175- 1180), 10s transduced cells were transplanted with 10* wild-type untransduced bone marrow cells for rad^oprciection. For generation of BCR-ABL B-ALLs derived from Hardy B cells, 5x10* Hardy B cells from 6 week-old mice were sorted on a BD FACSAria II SORP, spinocubtion was performed as described above, and 10* cells were transplanted into ledially irradiated wild-type recipients with 10* bone marrow cells for radioprotection. Dominant negative Ikaros experiments were performed similarly, except 10* cells spinfectcd with an MSCV retrovirus expressing GFP alone, or coexpressing GFP and H 6 (lacobucci et ai (2008) Blood 1123847-3855; Trageser etal. (1991) Exp. Med.
206: 1739-1753), were transplanted. Mice were followed daily for clinical signs of leukemia and were sacrificed when moribund. Investigators were not blinded to the experimental groups. Ten mice were used per arm for 80% power to detect a 60% difference in survival at a specific time point with alpha of 0.05. No animals were excluded from analysis.
G. Cell culture
Ba F3 experiments were performed as described in Yoda et ai (2010) Proc. Natl. Acad. ScL U.S.A. 107252-257. shRNAs targeting Hmgnl are described below
(competitive shRNA assay), and cDNA expressing HMGN1 was described in Rochman et ai (2011 ) Nucl. Acids Res. 39: 076-4087). One week after selection in puromycin, retroviral cDNA or lentiviral shRNA-transduced cells were harvested for Western blotting. hTERT-RPEl cells were cultured in DMEM/F-12. Mouse A9 cells containing a single human chromosome 21 tagged with neonryem-resistam gene (a gift from Dr. M. Oshimura, Tottori Lhriversity, Japan) were cultured in DMEM All medium was soppiemented with 10% FBS, 100 lU ml penicillin and 100 jig/ml streptomycin.
H. lmmunobiottina and Quantitation
Western blotting was performed as described in Yoda et al. (2010) Proc. Natl.
Acad. Set U.Sui. 107:252-257. Image J (available on the World Wide Web at
iinagej.nib.gov/ij) was used for quantitation of immunoMots, with band intensity normalized to total H3. 1. MW W^roedja ed cfrrompson transfer (M C0
MMCT was performed as described in Yang and Shen (2011 ) Methods Mol Biol 325:59-66 with modifications. A9 cells were cultured to approximately 70% confluence, and treated with 75 ng/ml colcemid for 48 hours. Cells were collected and rcsuspended in 1 : 1 DMEM:PercoU (GE Healthcare Biosciences) with 10 ugml Cytochalasin B (Sigma- Aldrich), and spun at 17,000 rpm for 75 minutes in a Beckman JA17 rotor. Supernatant was collected and filtered through 10 and 5 urn filters. Approximately 2* 10* RPE1 cells were collected and mixed with filtered microcclls, treated with 100 ug/ml PHA-P (Sigma- Aldrich) for 30 minutes, and fused by PEG 1500 (Sigma-Aldrich) in solution. Hybrid cells were plated and cultured for 48 hours, and selected with 500 Mgml Gcncttcin (Life Technologies) for 12-14 days. Standard G-band analysis was performed at Karyotogic, Inc. SNP array was performed at the DFC1 microarray core, using die Human Mapping 250k- Nsp platform. Ruorescent in situ hybridization was performed with the Vysis LSI 21 SpectrumOrange probe (Abbott Molecular) according to the manufacturer's instructions. J. DR-GFP and DR-GFP-CE reporter tantering
Gcnerating and screening of targeted clones were performed as described in Fung and Weinstock (2011 ) PLoS One 6:e20514. with the following modifications. 10* RPE1 cells with 2, 3, or 4 copies of chromosome 21 were nuclcofected with 2 ug pAA VS 1 - DRGFP or pAAVSl-DRGFPCE plasmid together with 2 ug pZFN-AAVSl , using program X-001 of the Amaxa nucleofector II (Lonza). Targeting of mdidtvidual clones was confirmed by PCR using the Accuprime GC-rich DNA polymerase (Life Technologies). The presence of a single integrant was determined by qPCR. . A mm «ΜΥΕ lifting PR-QFP mtff ccJUag
Assays for homologous recombination and imprecise non-homologous end-joining were performed as described in Wcinstock el al. (2006) Methods Enz oi 409:524-340 with the following niodiikations. Trarofections were performed with the Neon transaction system (Life Technologies) using 1600V, 20 ms, and 1 pulse. 4* 103 DR-GFP cells were transfected with 10 ug \ScA expression vector (pCBA&e) or empty vector (pCAGGS), and plated in 6-well plates. proCherry-Cl vector (Gontcch) was trarofected in parallel to confirm equal transaction efficiency. Cells were cultured for 7 days and analyzed by
FACS using FACSCalibur (BD Biosciences) for iiomology-directed repair. The remaining cells were used to extract genomic DNA. One ug DNA was digested with 20U I-Scel (Roche) overnight, purified, and amplified with a two-step PCR protocol. Accuprime GO- rich polymerase was used for the first step PCR (20 cycles), and Taq polymerase (Qiagcn) was used for the second step PCR (20 cycles). PCR products were cloned with the TOPO TA cloning kit for sequencing (Life Technologies). For DR-OFP-CE, pCAGGS-RAGl and pCAGGS-RAG2 vectors were co-transfeeted. One genomic DNA was digested with 10U Mfel and I0U Ndel (NEB) overnight to exclude templates that had not been cleaved by RAG- 1 and RAG-2 before PCR amplification.
L. PCR primcrt vxd in PNA nw sstaa
The following primers sequences were designed and synthesized to amplified the indicated amplkon for the indicated use:
Figure imgf000157_0001
M Competitive shRNA aiwav in primary B gdla
shRNAs targeting triplicated Ts 1 Rhr genes and controls were obtained from The RNAi Consortium (available on the World Wide Web at bfoadmstitute.oig rnai trc) as pL O lctti viral supernatant* (Ashton et al. (2012) Cell Stem Cell 1 1 :359-372) (n» 185 total shRNAs; see Table 5 for clone IDH and target sequences). Wild-type or TslRhr passage 1 B cell colonies were collected and plated at 5x10* cells per well of a 96 well plate in 100 μΐ of RPM1 with 20% FBS, and 10 ng ml each of murine IL-7. stem cell factor, and FLT3 ligand (all from R&D Systems), with 8 pgml porybrene. Ten μΙ of lentiviral supernatant was added and the plate was centrifuged at lOOOxg for 30 minutes, and men placed in a 37"C incubator for 24 hours. Wells were pooled, 10* cells were saved for input shRNA analysis, and 2x10* cells were plated in 6 ml M3630 mediykeUulose with 0.05 pg/rnl puromyctn in a 10 cm non-tissue culture treated dish. At this density of plating, after 7 days of growth there were at least 4xl04 colonies per plate which would represent >200 colonies per individual shRNA on average. After each passage, genomic DNA was harvested from 106 cells (Qiagen QIAmp kit), and 2x10* cells were replated in the same manner. Rcpassaging continued until cultures stopped forming new colonies (3-4 passages for wild-type) or until 6 passages were completed. The entire assay was repeated in n=3 (wild-type) and n«4 (TslRhr) independent biological replicates.
The shRNA encoded in the genomic DNA was amplified using two rounds of PCR. Primary PCR reactions were performed using up to lO pg ofgcnomic PNA in 100 μΐ reactions consisting of 10 μΐ buffer, 8 ul dNTPs (2 J mM each), 10 μΙ of 5 uM primary PCR primer mix (see below) and 1.5 μΐ Talcara exTaq. For the secondary PCR
amplification the reaction was performed as described in Ashton etal. (2012) Cell Stem Cell 11.359-372 using modified forward primers, which incorporated Ilrumina adapters and 6-nucleotidc barcodes. Secondary PCR reactions were pooled and run on a 2% agarose gel. The bands were normalized and pooled based on relative intensity. Equal amount of sample was run on a 2% agarose gel and gd purified. Samples were sequenced using a custom sequencing primer on an Ilhimina Hi-Scq and quantitated as described in Ashton et al. (2012) Cell Stem Cell 11359-372. The following PCR primer sequences were used:
Primary Ρτίπ π
Figure imgf000158_0001
?taflitrt«y PCR Primcn
Figure imgf000159_0001
Curtpm -Jh-ΤίΐίτΜ iWABCTcing ρππκΓ
Figure imgf000159_0002
N. RNA acflUCTc iM. and dm iMWtain
Total RNA was harvested from B cell colonics (n=3 independent biologic replicates per genotype per passage). RNA sequencing was performed at The Center for Cancer Computational Biology at the Dana-Farbcr Cancer Institute (DFCI). Quality control of total RNA was performed using the RNA Qubtt Assay (Invhrogen) and me Bioanalyzer RNA Nano 6000 Chip Kit (Agilent). At least 100 ng of total R A and a Bioanalyzer RNA Integrity Number of >7.0 were required Library construction was performed using a TruSeq RNA Library Prep Kit (Ilrununa). Final library quality control was performed using the DNA High Sensitivity Qubit Kit (InvitrogenX the Bioanalyzer High Sensitivity Chip Kit (Agilent) and the 7 00HT Fast qPCR machine (Applied Biosystems). qPCR was performed using the lUununa Universal Library Quarinfication Kit from KAPA
Biosystems. RNASeq libraries were then normalized to 2 nM, pooled for multiplexing in equal volumes, and sequenced at 10 pM on the lllumina HiScq 2000. Sequencing was performed as 2x50 paired-end reads using the 100 cycles per lane Sanger Iliumina 1.9 deep sequencing protocol. The raw sequence data were subjected to data quality control checks based on per base sequence quality scores, per sequence quality scores, per sequence GC content, sequence length distribution, and overrepresented sequences, which are implemented in the FastQC tool (available on the World Wide Web at
bioinfbrrnmics.babraham.^^ Reads mat passed quality control filters were aligned against the mouse reference genome by using the ultahhigh-throughput long read aligner Bowtie2 (Langmead and Salzbcrg (2012) Nature Methods 9:357-359) available through TopHat 2.0.7 (Trapnell et al. (2012) Nat. Protocols 7:562-578) (available on the World Wide Web at tophat.cbcb.umd.edu). Mapping results were further analyzed with TopHat to identify splice junctions between cxons. Genomic annotations in gene transfer format (GTF) were obtained from Ensembl mouse genome GRCm38 (available on the World Wide Web at uaeastcnseinblorg Musjmuscri^ Gene-level expression measurements for 23,021 Ensembl mouse genes were reported in fragments per kilobase per million reads (FPKM) by Cufflinks 2.0.0 (Trapnell etal. (2010) Nat. Biotech. 28:511- 515) (available on the World Wide Web at cufT1inks.cbcb.umd.cdu ). An FPKM filtering cutoff of 1.0 in at least one of the sample was used to determine expressed transcripts. o. Mfl rentiaj analysis for RNA-Sw transcript CTW¾»wn
Differential analysis was performed by applying the EdgeR method (Robinson et al. (2010) Bfolnfbrmatfcs 26:139-140) implemented in the EdgeR library in Biocondnctor v2.11 (available on ή World Wide Web at bioccflductor.org ). EdgeR uses empirical Bayes estimation and exact tests based on the negative binomial distribution model of die genome-scale count data. EdgeR estimates the gene-wisc dispersions by conditional maximum likelihood, conditioning on the total count for that gene. The gene-wise dispersion is "normalized" by shrinking towards a consensus value based on an empirical Bayes procedure (Robinson and Smyth (2007) Bioinformatlcs 23:2881-2887). The differential expression is estimated separately for each gene based on an exact test analogous to Fisher's exact test adopted for over-dispersed data (Robinson and Smyth (2008) Biostattsiics 9:321-332).
P. Gene expression Profiling (GEF> and Gene Set Enrichment Analvm (OSEA1
The series matrix file for two DS-ALL datasets (AIEOP and ICH) were downloaded from GEO (GEO accession number GSE17459) (Hertzberg et al. (2010) B od 115: 1006- 1017), as were the Ragl" and E2A Tcfl^ B cell progenitors (GSE21 78) (Lin et al. (2010) Nat. Immunol. 11:635-643). RNA from HMGNl transgenic (HMGN 1 _OE) or wild-type littermate B cell colonies was processed and hybridized to Affymctrix Mouse Gene 2.0 ST array at the DFCI Microarray Core per the manufacturer's instructiom. Raw probe-level data from the AIEOP-2 non-DS-ALL cohort and the mouse HMGNl JOE GEP were summarized using the Robust Multiarray Average (RMA) (Irizarry et al. (2003) Nucl Acids Res. 31 :e15) and Brainarray custom chip identification files based on Entrez IDs (Version 17) (Dai et al. (2005) Nud. Acids Res. 33:el 75) using die ExpressictfikCreator module in Gene Pattern (Reich et al (2005) Nat. Genet. 38:500-501 ). For GSEA the expression file was converted to human gene ortboiogs using Bio art ( inseila et al. (201 i) Database 2011 :bat030). GSEA of (he TslRhr, the core Tsl Rhr, and the PRC2 gene sets was performed as described in Subramanian etal. (2005) Proc. Natl Acad. Scl. V.&A
102:15545-15550 using GSEA v2.0.10 (available on the World Wide Web at
broadinstitute.org/gsea/). The TslRhr gene set was tested lor its cnrichmem in the cl
(positional), c2.cgp (chemical and genetic perturbation), c3.tft (transcription factor targets), and c6 (oncogenic signatures) gene sets deposited in die Molecular Signature Database MSigDB v3.1 (Broad Institute available on the World Wide Web at
broadinstitute. orggsca/rnsigdb). The analysis was performed by applying the 2-tailed Fisher test method, as implemented in die Investigate GeneSets module at MSigDB. To define tbc Tsl Rhr B ceil gene set, the top 150 most differentially expressed protein coding genes with an adjusted p-valuc below 0.25 were selected. Hierarchical clustering of this signature in DS-ALL vs. non-DS-ALL revealed a subset of genes most contributing to the distinguishing phenotype and this branch defined the "Core" TslRhr gene set Full gene sets for BENP0RATH_SUZ12_TARGETS,
MIKKELSEN_MEF_HCnP_WITH_H3K27ME3, and
MU KELSEN_MEF_NPC_WITH_H3K27ME3 were obtained from MSigDB v3.1. The 100 most differentially expressed genes between the DS-ALLs and die non-DS-ALLs were determined using the MarkerSelecuonModule in GencPattern. For E2A target gene expression, RAG1- - proB cells were compared to E2A-/- preproB cells to generate probescts with >1.5-fold change and P<0.05 between conditions, exacdy as had been done by tbc authors (Lin et al. (2010) Nat. Immunol. 1 :635-643). The Tsl Rhr and core gene sets were compared to ail probescts for their relative expression in E2A wild-type (RAG! -/- proB) vs E2AV- cells.
Q. Network cuTicbjnCTt mapping
The gene sets wim significant enrichment in genes up-regulated in TslRhr by GSEA were selected based on the maximum cut-off value 0.05 for P-valuc and FDR, and visualized with Enrichment Map software (Merico et al. (2010) PLoS One 5:cl3 84). This software organizes the significant gene sets into a network, where nodes correspond to gene sets and the edges reflect significant overlap between the nodes according to a Fisher's test The size of the nodes is proportional to the number of genes in the gene set The hubs correspond to collections of genes sets with significant pair-wise overlap which have a unifying functional description according to GO biological processes. Tbc node color is associated to the functional description of the hub. The clusters provided by the
Enrichment Map are described in Table 3.
R. Visualization of acne cx∞g¾aion and mass spectrometry data
R AScq-dcrivcd expression data from TslRhr and wild-type B cells, B-ALL gene expression data, and historic mass spectrometry data were visualized as heat maps using GENE-E (available on the World Wide Web at
S. BCR-AB p-Aqrooq^
Generation of B-ALLe by tzansduction of wild-type or TslRhr bone marrow with p210 BCR-ABL in an MSCV-ires-GFP retrovirus was performed as previously described (Krause et al. Sat. Med. 12:1175-1180). with momftamons. For limiting dilution transplantations, 10 or 10* spinoculated cells were transplanted with 10* wild-type untransduccd bone marrow cells for radioprotcctkra. 10* spinoculated cells were transplanted without additional radioprotective cells. Mice were followed daily for clinical signs of leukemia and were sacrificed when moribund Complete blood count analysis was performed with a Hemavet 950 (Drew Scientific). Fcrcalculad<>n oflcukemia-mitiaririg cell frequency, L-Calc software from Stem Cell Technologies (available on the World Wide Web at stet»ccll.con^en PToducts/All-P^ was used and transplanted BCR/ABL+ cells were calculated by multiplying the number of cells transplanted by the GFP+ cells at the time of transplant (limiting dilution curves compared by ch -squared test) (Wang el at. Blood 89:3 19-3924). For generation of BCR- ABL B-ALLs derived from Hardy A, Hardy B or Hardy C cells, staining for Hardy fractions in wild-type or Tsl Rhr 6-8-weck-old bone marrow was performed as described above, and SxlO4 cells from each subpopulation were sotted on a BD FACSAria II SORP. Spinoculation with BCR-ABL retrovirus was performed as described above, and 10* cells were transplanted into lethally irradiated wild-type recipients with 10* bone marrow cells for radioprotection. τ. Column rniriffatirw) of mouse B-AL
For Western blotting of mouse B-ALLs, cryoprcserved B-ALL splcnocytcs were enriched using ami -CD 19 antibody conjugated to magnetic rnicrobeads (#]30-Ο52·201) and an MS MACS column (#130-042-201), both (torn Miltenyi Biotec u. Hiaoiig mag sasaimtn
Mass spectrometry for global histone H3 post-translational moeiiications was performed as described in Peach ei al. (2012) Mol Cell Prote m. 11: 128-137 using wild- type or TslRhr passage B cells and BCR-ABL B-ALLs. H3K27 modifications are prcscmed in conjunction with H3 36, as both arc present in the same measured peptides because of their close proximity.
V. Drug treatment
GSK-J4 (KDM6A/UTX and DM6B JMJD3 inhibitor, catalog #M60063-2) (KriridcnicT^o/. (2012) Nature 488:404-408) and GSK-126 (EZH2 inhibitor, catalog #M60071-2) (McCabe /e/. (2012) Nature 492:108-112) were purchased from Xccssbio. For methylcellulose experiments, at each passage DMSO, GSK-J4, or GSK-126 were added to cultures a final concentration of 1 μΜ. DS-ALLs (dddentiftcd specimens obtained wirh inibrmed consent under DFCI IRB protocol 05-001) were treated in vitro in quadruplicate with GSK-J4 at two-fold dilutions from 40 nM to 10 uM in RPMI with 20% calf serum supplemented with 10 ngmL IL3, IL7, SCF, FLT3 ligand, and 50 uM beta- mercaptoethanol. After 3 days, viability was measured using CellTitcr-Glo reagent and normalized to DMSO control (Promega). w. fo rtt ( -J4 aiways
Leukemia cells were murine BCR ABL-positive B-ALLs as described above, or human Down syndrome or non-Down syndrome primary xenografed B-ALLe. Viable cells were plated in white opaque 384-well plates (50 μΙ weil; Corning) using EL406 Qmibination Washer Dispenser (BioTek) at a density of 0.25 * 10* cells/ml. GSK-J4 or vehicle (DMSO) were added using a JANUS Automated Workstation (PcrkinElmer) at d e indicated concentrations. After 72 hours, CcllTitcr-Gk) I^rninesccnt Cdl Viability Assay reagent (Promega) was added (25 ul each well) and read by the 2104 EnVision Multi label Reader (PcrkinElmer) per the numufacturcrs' instructions. Each data point was quantified in quadruplicate. Dosc-responae curves and plots were generated with GraphPad Prism software.
X. ChlP analyses
B cell colonics ( 5,000 colonies per genotype) from 3 wild-type and 3 Tsl Rhr animals were pooled after 7 days in methylccllulose culture. ChIP was performed as described in Vera etal. (2010) Dev. Cell 19:713-726. Libraries for sequencing were prepared following die Illurmna TruSeq DNA Sample Preparation v2 kh protocol. After end-repair and A-taOing, imtnuiMpcecipitated DNA (10-50 ng) or whole cell extract DNA (50 ng) was ligated to a 1:50 dihitkm of Iliunrina Adaptor Oligo Mix assigning one of 24 unique indexes in the kit to each sample. Following ligation, binaries were amplified by 18 cycles ofPCR using the HiFi NGS Library Amplification kit from APA Biosystems. Amplified libraries were then size-selected using a 2% gel cassette in the Pippin Prep system from Sage Science set to capture fragments between 200 and 400 bp. Libraries were quantified by qPCR using the KAPA Biosystems Ithimma Library Quantification kit according to kit protocols. Libraries with distinct TruSeq indexes were multiplexed by mixing at cquimolar ratios and running together in a lane on the Illumina HiScq 2000 for 40 bases in single read mode. Alignment to mouse genome assembly NCBI37/mm9 and norrnalifflhon were performed as described in Lin etal (2012) Cell 151:56-67. Regions of modified histones enriched in wild type and Tsl Rhr cells were identified using MACS peak calling algorithm at a P-vahte of I e-9 (Zhang et al. (2008) Genome Biol. 9:R 137). Location analysis of ChlP-target enriched regions was performed using the CEAS software suite developed by the Liu lab at DFCI (Shin et al. (2009) Bioinfbrmatics 25:2605-2606).
Promoters states were classified by the presence of H3 4mc3, H3K27me3, or both (bivalent) ChlP-scq enriched regions in the +/- lkb region relative to the transcriptional start site (TSS). ChlP-qPCR was performed on two independent sets of pooled B cell colonies from 3 wild-type and 3 Tsl Rhr mice. For analysis of unregulated genes in Tsl Rhr B cells, the 31 triplicated genes in Tsl Rhr mice were excluded. Data arc presented as boxplots designating median (black line). I SD (box), and 2 SD (whiskers). E2A ChlP-Seq data from Rag ^ proB cells were obtained from GEO (GSE21978) (Lin et al. (2010) Nat. Immunol. 11:635-643) and mapped to the genome as above. Regions of enriched E2A genomic occupancy were defined using the MACS algorithm as above. Genes were considered associated with E2A if their gene body overlapped an E2A enriched region, or if their TSS was within 50kb of an E2A enriched region, as was performed in Loven et al. (2013) Cell 153:320-334.
Y. Statistical analyses
Painvise comparisons are represented as means +/- SE by two-tailed Student t test, except where otherwise specified. Categorica] variables were compared using a Fisher's exact test Kaplan-Meier survival curves were compared using the log-rank test. In addition, RNA-scq, ChEP-seq, and microarray expression data are deposited with GEO under GEO accession number GSE4 555.
Exanple 2: Analysis of DSCR triplication effects
In order to directhy interrogate the effects of polysemy 21, B cell development in TslRhr mice (Figure I A), which harbor a triplication of 31 genes and one non-coding RNA on mouse chr.16 orthologous to human chr.21q22 (Olson et al. (2004) Science 306:687- 690), was assayed. Bone marrow from 6-week-old Tsl Rhr mice had fewer total progenitor (B220+CD43+) B and pro-B (Hardy B and O (Hardy et al. (199) ) J. Exp. Med. 173:1213- 1225) cells man wild-type littennates, while the pre-pro-B (Hardy A) fraction was itna ected (Figures IB and 2A). C57BL/6 TslRhr, FVBxC57BL 6 Fl TslRhr and Ts65Dn mice (Reeves er /. (1995) Nat. Genet. 11:177-184), which harbor a larger triplication (Figure I A), all had similar reductions in pro-B cells (Figure 2B). This differentiation defect essentially phenocopies human fetal livers with trisomy 21, which have reduced pre- pro-B (CD34+CD 19+CD 10-) and pro-B ceils (CD34+CD 1 +CD 10+X as well as other hematopoictk defects (Roy el al. (2012) Proc. Nail. Acad. Sci. USA. 109:17579-17584).
Competitive transplantation was performed using equal mixtures of congenic CD45.1 wiki-type bone marrow and
wild-type mice (Figure 2C). After 16 weeks, recipients of wild-type CD45.1 and
CD45.1/45-2 bone marrow had equal representations of both populations in Hardy A, B and C fractions, as well as whole bone marrow (Figures 1C and 2D). In contrast, mice that received wild-type CD45.1 mixed with Ts I Rhr CD45.1/452 recapitulated the Ts 1 Rhr defect, with significant reductions in CD45.1/45.2 Hardy B and C fractions (Figures 1C and 2D). Thus, the differentiation effect is independent of non-liematopoietic cells.
To address whether chr.21q22 directly confers tiansfbrmed phenotypes like proliferation and sd f-rcnewal, progenitor B cell colonies were generated from unselected TslRhr and wild-type bone marrow in three-dimensional cultures with 1L7 (Figures 2E-2F). Wild-type bone marrow forms colonics (termed 'passage 1 ') under these conditions that can be replated to form new colonies for 1-2 additional passages. In contrast, Tsl Rhr bone marrow generated more colonics in early passages and serially replated indefinitely (Figure 1 D), which indicates self-renewal capacity. Both Tsl Rhr and wild-type colonies from early passages were universally Hardy C (CD24+BP-1+) by flow cytometry (Figure 3). After passage 2, wild-type cells formed few if any colonies while Tsl Rhr cells obtained from all mice (np9) expanded exponentially after passages 3 or 4 (Figure 1 D) and continued to repassage fbr more than 10 platings. In contrast, there were no significant differences between Tsl Rhr and wild-type bone marrow in the number or repassaging potential of myeloid colonies (Figure IE). Passage 6 B cells from Tsl Rhr bone marrow were capable of causing fatal lymphoproiiferation in vivo upon injection into Nod.Scid.lL2R "" mice and rapidly lethal B-ALL upon secondary transplantation into imrnunocompctent recipients (Figure 4). Thus, DSCR triplication is sufficient to confer B cell self-renewal in vitro and that results in serially transplantable B-ALL in vivo.
Sixty percent of DS-associatcd B-ALLs harbor rearrangements of CRLF2 that commonly occur in combination with activating JAK2 mutations (Mullighan et al. (2009) Nat. Genet. 41:1243-1246; Russell etal. (2009) Blood 114:2688-2698; Yoda etal. (2010) Proa Natl. Acad. Sci. U.S.A. 107:232-257). To model this, EM-CRLF2 (hereafter 'C2*) and Eu-JAK2 R683G ('J2') transgenic mice, which have B-ceil restricted transgene expression, were generated. C2 J2 mice did not develop B-ALL by 18 months of age, nor did C2 J2 mice crossed to Pax5*'~ mice. Transduction of C2/J2/Paxf' bone marrow with a dormram-ncgative IKZFI allele (Ik6) (Iacobucci et al. (2008) Blood 112:3847-3855) and transplantation into wild-type recipients resulted in CRLF2-positive B-ALL in all mice by 120 days (Figures SA-5B). Control mice lacking C2, J2 or Pax5 heterozygosity did not develop B-ALL with Hc6 (Figure SB), thus establishing this transgenic combination as the first model of CRLF2 JA ^-drtvtn B-ALL. To assess the effect from the addition of chr.21q22 triplication, C2l}UPax5 ^ and Tsl Rhr C2/J2 Aix5* ' mice were transduced with a lower titer of either empty virus or Ik6 virus. Mice transplanted with
Tsl Rhr C2 J2 ipox5* ' bone marrow transduced with lk6 developed B-ALL with greater penetrance and reduced latency compared to C2/J2/Pax5 ' alone (Figure IF). The same genotypes (C2/i2/Pax5* flk6 with or without polysomy 21 ) occur in high-risk cases of human B-ALL (Mullighan et al. (2009) Proc. Natl. Acad. Sci. USA. 106:9414-9 18), supporting the validity of the model. To confirm the contribution of chr.21q22 triplication in a more tractable model. B- ALL was induced by transplanting nnsdected bone marrow transduced with p210 BCR- ABL (KraiBetf e/. (200^ Mtf. A/«* 12:1175-1180). Although BCR-ABL ALL is uncommon in children with DS, pol somy 21 is the most common somatic aneuploidy among BCR-ABL ALU ( Wetzier el al (2004) Br. J. Haematol. 124:275-288). Limiting dilution analysis was performed by transplanting 10*, 10* or 10 transduced bone marrow cells from TslRhr mice or wild-type littermatcs into wild-type recipients (Figure 6A). TsIRhr and wild-type bone marrow had similar transduction efficiencies (Figure 5Q, but mice (C57BL/6 and FVBxC57BlJ6 Fi backgrounds) that received transduced Tst Rhr bone marrow succumbed to B-ALL with shorter latency and increased penetrance (Figures IG and 5D-5F). Specifically, three weeks after transplantation, mice mat received transduced Tsl Rhr bone marrow had higher white blood cell counts and lower hemogiobin concentrations in peripheral blood compared with mice that received transduced wild-type bone marrow (Figure 7).
Mice transplanted with either wild-type or TslRhr bone marrow succumbed to progenitor (B220+CD43+) B-ALLs with similar histology mat infiltrated the bone marrow and spleen (Figure 5D-5E). However, B-ALLs in mice transplanted with TslRhr marrow developed with shorter latency and, in cohorts transplanted with 10s or 10* cells, increased penetrance (Figures 6A and SF). Based on a Poisson distribution analysis, the frequency of B-ALl^ininating cells was over 4-fbld higher in Tsl Rhr bone marrow (Figure 6B; 1 :244 versus 1 :60 transduced cells, p^O.01). B-ALLs (based on GFP+ B220+ phenotype) derived from wild-type bone marrow were homogenous populations of CD24+BP-1+ (equivalent to Hardy Q cells. In contrast, nearly one-half of B-ALLs derived from Tsl Rhr bone marrow were primarily CD24+BP-1- (Hardy B; Figure 6C, p=O.003 compared to wild-type by Wilcoxon rank sum test), with some cases harboring CD24-BP- 1 - (Hardy A) cells.
The difference in B-ALL differentiation phenotype raised the possibility that DSCR triplication affects the B cell stage that is transformed by BCR-ABL. To address this, Hardy A, B and C fractions were sorted from Tsl Rhr and wild-type bone marrow, individually transduced with BCR-ABL. and then transplanted 103 cells into wild-type recipients (Figure 8). As with unsortcd bone marrow (Figure 6A), B-ALLs developed with greater penetrance and shorter latency among mice transplanted with transduced Ts IRhr Hardy B cells (p=0.002 by log-rank test; Figure 6D) compared with transduced wild-type Hardy B cells. B-ALL also developed in mice transplanted with transduced Tsl Rhr Hardy C ceils but not wild-type (p=0.049; Figure 14D), although with longer latency than among mice uwteplanted with transduced Tsl Rhr Hardy B ceils (p«0.002 for Tsl rhr Hardy B versus Hardy C). No mice transplanted with transduced Hardy A ceils from either genotype developed B-ALL (Figures 6D). Thus, DSCR triplication promotes BCR-ABL transi nnation in both Hardy B and Hardy C fractions, despite the In vivo reduction in absolute numbers of these cells in Tsl Rhr bone marrow (Figure IB). These sorting experiments also confirm that the increased Icukemogcnesis induced with BCR-ABL, like the differentiation abnormality, is a B cell autonomous effect of DSCR triplication.
Transplantation of BCR- ABL-transduccd sorted Hardy B cells from Tsl Rhr or wild-type mice recapitulated the same effect (Figure 5GX indicating that the leukernogenic effect from chr.21q22 triplication is progenitor B-cell autonomous.
In addition to these direct effects, polysemy 21 could also contribute to B cell transformation by promoting aberrant DNA double-strand break repair (DSBR), which mediates kukemogenic alterations at CRLF2, IKZFJ, PAX5 and other loci (Multighan et al. (2009) Nat. Genet. 41 : 1243-1246; Russell et al (2009) Blood 114:2688-2698; Yoda et al (2010) Proc. Natl Acad. Sci. U! . 107:232-257). To address this, otherwise isogenic retinal pigment epithelial (RPE) cells that harbor 2, 3 or 4 copies of human chr.21 by microcell-mediated chromosomal transfer were generated (Figures 9A-9C). Zinc finger tnic lease-mediated recombination was used to target DSBR reporters (Weinstock and Jasin (2006) Mol. Cell Biol 26 131-139) \otbcp 4 locus of cells with different numbers of chr.21 , which avoids confounding locus-specific differences (Smith et al (2008) Stem Cells 26:496-504). Polysemy 21 had no effect on cither homology^urected repair frequency or junction characteristics formed by nonhomoiogous end-joining, whether DSBs were induced by the I-Scel endoouc lease (Figures 9D-9F) or by the V(D)J recombinase (Figures 9G-9J). Although a subtle defect or one specific to progenitor B cells remains possible, these results indicate for the first time in an isogenic system that poh/somy 21 does not drastically affect DSBR pbenotype.
Whole transcriptomc sequencing (RNA-seq) of passage 1 B cells was also performed; triplicated loci in Tsl Rhr cells were expressed at approximately 1.5-fold higher levels compared to wild-type cells (Figure 10) while absolute expression among die 25 genes differed markedly (Figure 11). A transcriptional "Tsl Rhr gene set** of the 150 most differentially expressed genes compared to wild-type was defined (Table 1 ). As expected, this signature was highly enriched by gene set enrichment analysis (GSEA) (Subramanian et al. (2005) Five. Natl. Acad. Sci. U.S.A. 102:15545-15550) for human chr.21q22 genes (Table 2), bat not other human chromosomal segments, based on a query of the Broad Institute Molecular Signatures Database (MSigDB) Mc 1 " positional dataset (Suoramanian et al. (2005) Proc. Natl. Acad. ScL V.S.A. 102:15345-15550). The TsIRhr gene set was next applied to a gene expression dataset of pediatric B-ALLs (AIEOP) (Hertzberg et al. (2010) Blood 115:1006-1017). The TsIRhr B cell signature was enriched among DS-ALLs by GSEA (Figures 12A-12B; FDR-0.019), indicating that transcriptional differences defined in Tsl Rhr B cells are biologically relevant to human DS-ALL. By hierarchical clustering, a "core TsIRhr set" of only 50 genes (Table 1) was observed that distinguished DS-ALLs (Figure 12 A). Although none of the 50 genes are triplicated in TsIRhr cells, the core TsIRhr set was highly enriched among DS-ALLs in both the AIEOP dataset (Figure 12B; FDR-0.001) and an independent validation dataset (ICH) (Figure IX; FDR=0.001).
To identify pathways perturbed by chr.2 Iq22 triplication, the TsIRhr gene set was queried against >3000 functionally defined gene sets in the MSigDB **c2" chemical and genetic perturbations and wc6" oncogenic signatures repositories (Suoramanian et al. (2005) Proc. Nad. Acad. Set. U.S . 102:15545-15550). Arranging the significant gene sets in a network enrichment map (Merico et al. (2010) PLoS One 5:c 13984) defined 4 clusters (Figure 12D). The most highly enriched cluster consisted of polycomb repressor complex 2 (PRC2) targets and sites of tri-methylated histone H3 27 (H3K27me3), the repressive mark added by PRC2, that were defined across multiple lineages (Table 3). The additional clusters consisted of gene sets that distinguish either stem cells from lineage-matched differentiated cells, cancer cells from nonmalignant cells, or less dtffeentiated f om more differentiated lymphoid cells (Table 3).
It was next asked whether differential expression of PRC2/H3K27rac3-classified genes would distinguish DS-ALLs from other B-ALLs. A previous effort using genome- wide expression in the AIEOP cohort failed to define a transcriptional signature specific to DS-ALL (Hertzberg etal (2010) lood 115:1006-1017). Strikingly, expression of H3K27me3 targets defined in murine embryonic fibroblasts distinguished DS-ALLs from non-DS-ALLs (Figure 12E). To validate these findings, the 100 most differentially expressed genes between DS-ALLs and non-DS-ALLs in the AIEOP cohort across three different PftC2/H3 27me3 signatures were detennined (Figure 13A and Table 5). All three signatures were significantly enriched (FDR 0.001) among DS-ALLs in die ICH validation cohort (Figure 12F). In a third cohort of non-DS-ALLs (AlEOP-2), cases with either polysomy 21 or iA P(21) clustered based on expression of PRC2 targets (Figure 13B, P=0.00l by Fisher's exact test), and the Tsl Rhr and H3K27me3 gene sets were enriched among cases with polysomy 21 or iAMP(21 ) by GSEA (Figure 13Q.
Genes from PRC2 H3K27mc3 gene sets that distinguish DS-ALLs arc
predominantly overexpressed in DS-ALL (Figures 12E and 13A). This indicates that DS- ALL is associated with de-repression of P C2 targets and reduced H3 27me3. Consistent with the GSEA, histone H3 mass spectrometry demonstrated a global reduction in
H3K27mc3 peptides in passage I TsIRhr B cells compared to wild-type cells, with reciprocal increases in umnethyiated and inonomctfaylated H3K27 peptides (Figure 12G). BCR-ABL B-ALLs from TsIRhr bone marrow also had reduced H3K27me3 by bom mass spectrometry and immunoblotting (Figures 13D-13E). Thus, triplication of only 31 genes directly suppresses H3 27mc3.
To identify mechanisms that directly link gene triplication, H3K27mc3 levels, and gene expression. ChlP-scq of passage 1 TsIRhr and wild-type B cells was performed. TsIRhr B cells bad a genome-wide reduction of H3K27mc3 at regions enriched for this mark in wild-type cells (Figures 14A-14B) that was confirmed at multiple loci by ChIP followed by quantitative PCR (Figure 15A). Within TsIRhr B cells, H3K27me3 was found almost exclusively at regions enriched for H3K27me3 in wild-type cells, suggesting lttrJe or no redistribution but rather a global reduction in the H3 27me3 density (Figures 15B- 1SD). As expected, reciprocal changes in activating (H3K4me3, H3K27ac) and repressive (H3K27me3) marks were observed at promoters of genes differentially expressed in TsIRhr B cells (Figure 14Q. However, genes "bivalentry marked" with both H3 27me3 and H3K4me3 in wild-type cells were highly enriched among those overexpressed in TsIRhr B cells (Figure 14T . PO.0001 ).
Bivalent marks may indicate genes that are modulated during lincagc-*pecinc differentiation (Bernstein el al. (2006) Cell 125: 15-326). The enrichment of bivalentry- marked genes within the TsIRhr gene set therefore suggests that the global loss of H3K27me3 from chr.2 Iq22 triplication selectively drives the overexpression of genes defined by a progenitor B cell-specific developmental program. In support of this, the TsIRhr and PRC2/H3K27m 3 gene sets were highly enriched for rxedicted binding sites of the master B cell transcription factors E2A TCF3 and LEF1 (Figure 15E) (Kruidcnicr et al. (2012) Nature 488:404-408; McCabc e al. (2012) Nature 492:108-112). To test whether de TsIRhr gene set is enriched for functional E2A TCF3 targets, a previously reported dataset of ChlP-eeq and gene expression from wild-type and E2A" marine B ceil progenitors (Krakfenier et ah (2012) Nature 488:404-408) was analyzed. Genes within the TslRhr gene set had increased proximal occupancy by E2A/TCF3 (Figure 15F). In addition, the expression of genes within both the TslRhr gene set and the core TslRhr set was preferentially increased in the presence of E2A TCF3 (Figure 1SG).
It was next asked whether pharmacologic restoration of H3K27me3 with GSK-J4 (Kraidcnicr et al. (2012) Nature 488:404-408), a selective inhibitor of H3K27 demcthylases, would block Tsl Rhr B cell repassaging. GSK-J4 increased H3 27me3 in Ts 1 Rhr B cells, decreased colony-fonning activity, and blocked indefinite repassaging (Figures 14E and 14G). Previous studies demonstrated mat 10 μΜ GS -J4 reduces lipopolysacchande- induced proinflammatory cytokine production by human primary macrophages (Kruidenier et al. (2012) Nature 488:404-408). 1C» values for GS -J4 across a panel of DS-ALLs ranged from 1.4-2.3 μΜ (Figure I SH). Treatment with OSK-12615, a selective inhibitor of the PRC2 catalytic subunit EZH2, decreased H3 27mc3 and was sufficient to confer indefinite repassaging in wild-type B cells (Figures 14F-14G). In addition, murine and human B-ccIl ALLs harboring increased copies of the Down syndrome critical region were more sensitive to GS -J4 than to leukemias lacking such increased cooks in a limited set of teukemias analyzed (Figure 16). Both die loss of H3 27me3 and indefinite repassaging were reversible upon withdrawal of GS -126 from wild-type cells (Figures 14F and 14H).
Among the 31 triplicated genes in Tsl Rhr cells is Hmgn], which encodes a nucleosome binding protein that modulates transcription and promotes chromatin decompaction (Catez et aL (2002) EMBO Rep. 3:760-766; Rattner et al. (2009) Mol. Cell 34:620-626). Modest increases in HMGN1 induce changes in histone H3 nwdifications and gene expression (Lim ere/. (2005) EMBO J. 24:3038-3048; Rochman etal. (2011) Nucl. Acids Res. 39:4076-4087).
Ovcrexpression of HMGN1 in murine Ba/F3 B eclb suppressed H3K27me3 in a dose-dependent fashion (Figures 17A and 18A). By RNA-seq, Hmgn I was one of only seven triplicated genes mat maintained >70% of its passage 1 expression level at passages 3 and 6 in all Tsl Rhr replicates (Figure 18BX indicating that it may be necessary for serial repassaging To address this, 5 shRNA targeting each of the 31 triplicated genes and controls were individually transduced into TslRhr and wild-type passage 1 B cells (Figure 18Q. Transduced ceils were pooled and passaged in adequate numbers to ensure that each shRNA was represented, on avenge, in >200 colonies at each passage. The relative abundance of each shR A at each passage was deconvoluted by next-generation sequencing.
As expected, positive control shRNAs that reduce viability across cell types were equally depicted at later passages from Tsl Rhr and wild-type backgrounds (Figures 18D and Table 6). Among shRNAs against triplicated genes, two of the top four that most selectively depleted Tsl Rhr B cells targeted Hmgn 1 and die remaining three shRNAs against Hmgnl all scored as preferentially toxic in Tsl Rhr B cells (Figure 17B and Table 6). By passage 6, all 5 shRNAs against Hmgnl were depleted by an average of>99% across replicates. All five shRNAs reduced HMGN I protein in Ba F3 cells (Figure 18E). Together, these data indicate that HMGN 1 contributes to the repassaging phenotype of Tsl Rhr B cells.
To directly address the sufficiency of HMGN 1 overexpression for effects observed in Tsl Rhr cells, mice with transgenic overexpression of human HMGN1 (HMGN l_OE) at levels comparable to mouse HMGN1 were analyzed (Figure 18F) (Bustin et al. (1 95) DM4 Cell Biol. 14:997-1005). A gene expression signature ofHMGNl_OE passage 1 B cells (compared to littcrmatc controls) was highly enriched for the Tsl Rhr and core Tsl Rhr gene sets (Figure 17Q. Compared to control bone marrow, HMGNl_OE bone marrow had reduced Hardy C cells in vivo (Figure 18G), generated more B cell colonics in passages 1 -4 tn vitro (Figure 17D), and resulted in greater penetrance and shorter latency of BCR-ABL- induced B-ALL (Figure 17E). Thus, overexpression of HMGN 1 alone recapitulates transcriptional and phenotypic alterations observed from triplication of all 31 Tsl Rhr genes.
In conclusion, it has been described herein that triplication of chr.21q22 genes confers cell autonomous differentiation and transformation phenotypes in progenitor B cells. By first delineating these biologic consequences of chr.21q22 triplication, human B- ALL datascts were more effectively interrogated and it was demonstrated that DS-ALLs are distinguished by the overexpression of H3K27roc3-markcd genes. The data also highlight die therapeutic potential of H3K27 demethyiase inhibitors for B-ALLs with extra copies of chr.21q22. At the same time, inhibitors of EZH2 are believed to be useful for in vitro or In vivo expansion of precursor B cells. Finally, the nucleosome remodeling protein HMGN 1 promotes the in vitro passaging of B cells, suppresses global H3K27mc3 and functions as a cooperating oncogene in vivo. Table 1
Figure imgf000173_0001
Figure imgf000174_0001
Figure imgf000175_0001
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Figure imgf000177_0001
Figure imgf000178_0001
Table 2
Figure imgf000179_0001
Table 3
Figure imgf000180_0001
Table 4
Figure imgf000181_0001
Tables
Figure imgf000182_0001
Figure imgf000183_0001
Table 6
Figure imgf000184_0001
Figure imgf000185_0001
Figure imgf000186_0001
Controls
Figure imgf000187_0001
The contents of all references, patent applications, patents, and published patent applications, as well as the Figures and the Sequence Listing, cited throughout this application arc hereby incorporated by reference.
EaaivnlCTto
Those skilied in the art will recognize, or be able to ascertain using no more than routine cxpeririKntarion, many equivalents to the specific embodiments of the invention described herein. Such equivalents arc intended to be encompassed by the following claims.

Claims

What is claimed:
1. A medKxl of determining whether a subject afflicted with a cancer or at risk for developing a cancer would benefit from modulating histonc H3 27rac3 levels, the method comprising:
5 a) obtaining a biological sample from the subject;
b) detcimining die copy number, level of expression, or level of activity of one or more biofliarkcrs listed in Tables 1-5 or a fragment thereof in a subject sample;
c) determining the copy number, level of expression, or level of activity of the one or more biomarkcrs in a control; and
0 d) comparing the copy number, level of expression, or level of activity of said one or more biomarkcrs detected in steps b) and c);
wherein a significant modulation in the copy number, level of expression, or level of activity of the one or more biomarkcrs in the subject sample relative to the control copy number, level of expression, or level of activity of the one or more biomarkcrs indicates S that the subject afflicted with the cancer or at risk for developing the cancer would benefit from modulating histonc H3K27mc3 levels.
2. The method of claim 1, wherein the one or more biomarkers are selected from the group consisting of the set of a) "top 150 UP" biomarkcrs shown in Table I, b) "the 50 UP
0 core" biomarkers shown in Table I, c) "top ISO DOWN" biomarkers shown in Table 1, d), "the SO DOWN core" biomarkers shown in Table 1, e) die "triplicated gene" biomarkers shown in Table 1, the Mchr21q22 overlap" biomarkers shown in Table 2, g) the "PRC2 cluster'* biomarkers shown in Table 3, h) die "overlap" biomarkers shown in Table 4, i) the "SUZ12 target," "Mikkclscn MEF " and/or "Mflckclsen NPC bk>rnarkers shown in Table
S 5, j) KDM6A, k) DM6B, 1) EZH2, m) HMGN1, and subsets and/or carnations thereof
3. A method for momtoring the progression of a cancer in a subject, the method comprising:
a) detecting in a subject sample at a first point in time the copy number, level of
0 expression, or level of activity of one or more biomarkers listed in Tables 1-5 or a fragment thereof;
b) repeating step a) at a subsequent point in time; and
c) comparing the copy number, level of expression, or level of activity of said one or more biomarkers detected in steps a) and b) to monitor the progression of the cancer.
4. The method of claim 3. wherein the one or more biomarkers arc selected from the group consisting of the set of a) "top ISO UP" biomarkers shown in Table 1, b) "the 50 UP core" biomarkers shown in Tabic 1, c) "top 150 DOWN" biomarkers shown in Table 1, d "the 50 DOWN core" bkm.arkers shown in Tabic i. c) the "triplicated gene'' biomarkers shown in Tabic 1, f) the "chr2Iq22 overlap" biomarkers shown in Tabic 2, g) the **PRC2 cluster" biomarkers shown in Tabic 3, h) the "overlap** biomarkers shown in Table 4, i) the "SUZ12 target," "Mikkelscn MEF,n and/or "Mikkclscn NPC biomarkers shown in Tabic
5, j) KDM6A, k) KDM6B, I) EZH2, m) HMGNl , and subsets and/or combinations thereof.
5. The method of claim 3, wherein an at least twenty percent increase or an at least twenty percent decrease between the copy number, level of expression, or level of activity of the one or more biomarkers in the subject sample at a first point in time relative to the copy number, level of expression, or level of activity of the one or more biomarkers in the subject sample at a subsequent point in time indicates progression of the cancer, or wherein less than a twenty percent increase or less than a twenty percent decrease between the copy number, level of expression, or level of activity of the one or more biomarkers in die subject sample at a first point in time relative to the copy mtmbcr, level of expression, or level of activity of the one or more bioraarkcrs in the subject sample at a subsequent point in time indicates a lack of significant progression of the cancer.
6. The method of claim 3, wherein between the first point in time and the subsequent point in time, the subject has undergone treatment to modulate histone H3K27roc3 levels.
7. A method tor stratifying subjects afflicted with a cancer according to predicted clinical outcome of treatment with one or more modulators of histone H3K27mc3 levels, the method∞mprising:
a) determining the copy number, level of expression, or level of activity of one or more biomarkers listed in Tables 1-5 or a fragment thereof in a subject sample;
b) clctcrrmning the copy number, level of expression, or level of activity of the one or more biomarkers in a control sample; and
c) cornparing the copy number, level of expression, or level of activity of said one or more biomarkers detected in steps a) and b);
wherein a significant modulation in the copy number, level of expression, or level of activity of the one or more biomarkers in t c subject sample relative to the normal copy number, level of expression, or level of activity of me one or more biomarkers in die control sample predicts tbe clinical outcome of tbe patient to treatment with one or more modulators of histonc H3 27me3 levels.
8. The method of claim 7, wherein the predicted clinical outcome is (a) c hilar growth, (b) cellular proliferation, or (c) survival time resulting from treatment with one or more modulators of histone H3 27me3 levels.
9. The method of claim 7, wherein the one or more biomarkers are selected from the group consisting of die set of a) "top 150 UP" biomarkers shown in Table I, b) "the 50 UP core" biomarkers shown in Table 1, c) "top 150 DOWN** biomarkers shown in Table 1 , d), "the 50 DOWN core" bk>markers shown in Table 1, c) the "triplicated gene" bkraiarkers shown in Table 1 , f) the "chr2 lq22 overlap" biomarkers shown in Table 2, g) the "PRC2 cluster" biomarkers shown in Table 3, h) the "overlap" biomarkers shown in Table 4, i) the "SUZ12 target," "Mikkelscn MEF," and/or "Mikkclsen NFC biomarkers shown in Table 5, j) KDM6A. k) DM6B, I) EZH2, m) HMGN1, and subsets and/or «m*inations thereof.
10. The method of claim 7, wherein an at least twenty percent increase or an at least twenty percent decrease between the copy number, level of expression, or level of activity of the one or more biomarkers in the subject sample compared to the control sample predicts that the subject has a poor clinical outcome; or wherein less than a twenty percent increase or less than a twenty percent decrease between the copy number, level of expression, or levd of activity of the one or more biomarkers in the subject sample compared to the control sample predicts that tbe subject has a favorable clinical outcome.
11. The method of claim 7, further comprising treating the subject whh a therapeutic agent that specifically modulates die copy number, level of expression, or level of activity ofthconc or more biomarkers.
12. The method of claim 7, further comprising treating the subject with one or more modulators of histone H3 27me3 levels.
13. A rncthod of a ennining the efficacy of a test compound for inhibiting a cancer in a subject, die method comprising: a) determining the copy number, level of expression, or level of activity of one or more biotnarkers listed in Tables 1-5 or a fragment thereof in a first sample obtained from the subject and exposed to the test compound;
b) determining the copy number, level of expression, or level of activity of the one or more biomarkers in a second sample obtained from the subject, wherein the second sample is not exposed to the test compound, and
c) comparing the copy number, level of expression, or level of activity of the one or moTC biomarkers in the first and second samples,
wherein a significantly modulated copy number, level of expression, or level of activity of the biomarker, relative to the second sample, is an indication that the test compound is efficacious for inhibiting the cancer in the subject
14. The method of claim 13, wherein the one or more biomarkers are selected from the group consisting of the set of a) ^top 150 UP" biornarkcrs shown in Table l, b)"thc50 UP core" biornarkcrs shown in Table 1, c) "top 150 DOWNT biomarkers shown in Tabk 1, d), "the 50 DOWN core" biomarkers shown in Table 1 , e) the "triplicated gene" biomarkers shown in Tabk 1 , f) the "chr21 q22 overlap" biomarkers shown in Table 2, g) the UPRC2 cluster" biomarkers shown in Table 3, h) the "overlap" biomarkers shown in Table 4, i) the "SUZ12 target," "Mikkclsen MEF." and/or wMikkelsen NPC biomarkers shown in Table 5, j) ΟΜ6Λ, k) DM6B, I) EZH2, m) HMGN1, and subsets and/or combinations thereof.
15. The method of claim 13, wherein the first and second samples are portions of a single sample obtained from the subject or portions of pooled samples obtained from the subject.
16. A method o deteniunmg the efficacy of a u^^
subject, the method comprising:
a) determining the copy number, level of expression, or level of activity of one or more biomarkers listed in Tables 1-5 or a fragment thereof in a first sample obtained from the subject prior to providing at least a portion of the therapy to the subject;
b) determining the copy number, level of expression, or level of activity of me one or more biomarkers in a second sample obtained from the subject following provision of the portion of die therapy; and c) comparing the copy number, level of expression, or level of activity of the one or more biomarkers in the first and second samples,
wherein a significantly modulated copy number, lcvd of expression, or level of activity of die one or more biomarkers in the second sample, relative to the first sample, is an indication that the therapy is efficacious for inhibiting the cancer in the subject
17. The method of claim 16, wherein the one or more biomarkers are selected from the group ccmistmg of the set of a) "top 150 Ul^ bwmaAers shown in Table l, b) "thc 50 UP core" biomarkers shown in Table 1 , c) "lop 150 DOWN** biomarkers shown in Table 1 , d), "the 50 DOWN core" biomarkers shown in Table 1, e) the "triplicated gene" biomarkers shown in Table 1, f) the "chr21q22 overlap" biomarkers shown in Table 2, g) the "PRC2 cluster*' biomarkers shown in Table 3, h) the "overlap" biomarkers shown in Table 4, i) the "SUZ12 target," "Mikkclsen MEF," and or "Mikkelsen NPC biomarkers shown in Table S, j) KDM6A, k) DM6B, I) EZH2, m) HMGN1 , and subsets and or combinations thereof; or wherein said therapy further comprises standard of care therapy for treating the cancer.
18. A method for identifying a compound which inhibits a cancer, the method comprising:
a) cciitacting one or more biomarkers listed in Tables 1-5 or a fragment thereof with a test compound; and
b) determining the effect of the test compound on the copy number, level of expression, or level of activity of the one or more biomarkers to thereby identify a compound which inhibits the cancer.
19. The method of claim 18, wherein the one or more biomarkers are selected from the group consisting of the set of a) "top 150 UP** biomarkers shown in Table 1, b) "the 50 UP core" biomarkers shown in Table 1, c) "top 150 DOWN" biomarkers shown in Table 1, d), "the 50 DOWN core" biomarkers shown in Table 1, e) the "triplicated gene" biomarkers shown in Table 1 , f) the "chr21 q22 overlap" biomarkers shown in Table 2, g) the "PRC2 cluster*' biomarkers shown in Table 3, h) the "overlap" biomarkers shown in Table 4, i) the *SUZ12 target," "Mikkelscn MEF," and or "Mikkelscn NPC biomarkers shown in Table 5, j) KDM6A, k) DM6B, I) EZH2, m) HMGN1 , and subsets and or combinations thereof.
20. The method of claim 18, wherein the one or more biomarkers is expressed on or in a cell.
21. The method of claim 20, wherein said cells are isolated from an animal model of a cancer.
22. The method of claim 20, wherein said cells are from a subject afflicted with a cancer.
23. A method for inhibiting a cancer, the method comprising contacting a cdl with an agent that modulates the copy number, level of expression, or level of activity of one or more biomarkcrs listed in Tables 1-5 or a fragment thereof to thereby inhibit the cancer.
24. The method of claim 23, wherein the one or more biomarkcrs are selected from the group consisting of die set of a) "top 150 UP" biomarkcrs shown in Table l. b) "the SOUP core" biornarkers shown in Tabic 1, c) "top 150 DOWN" biomarkcrs shown in Table 1, d), "the 50 DOWN core" biomarkcrs shown in Table 1 , e) the "triplicated gene" biomarkcrs shown in Table I, f) the "chr2la22 overlap" biornarkers shown in Table 2, g) the "PRC2 cluster" biomarkcrs shown in Table 3, h) the "overlap" biomarkcrs shown in Table 4, i) the "SUZ12 target," "Mildelscn MEF," and/or "Mikkclsen NPC biomarkers shown in Table 5, j) DM6A, k) KDM6B, I) EZH2, m) HMGN1 , and subsets and/or combmaiions thereof
25. The method of claim 23, wherein the copy number, level of expression, or level of activity of the one or more biomarkcrs is downmodulatcd or upmodulated.
26. The method of claim 23, wherein the step of contacting occurs tn vtvo, ex vivo, or in vitro.
27. The method of claim 23, further comprising contacting the cell with an additional agent that inhibits the cancer.
28. A method for treating a subject afflicted with a cancer, the method comprising administering an agent that modulates the copy number, level of expression, or level of activity of one or more biomarkers listed in Tables 1-5 or a fragment thereof such that the cancer is treated.
29. The method of claim 28, wherein the one or more biomarkcrs are selected from the group consisting of the set of a) "top 150 UP** biornarkers shown in Table 1, b) "the 50 UP core" biomarkers shown in Table 1, c) "top ISO DOWN" biomaifcers shown in Table 1, d), "the SO DOWN core" biomatkers shown in Table 1, e) die "triplicated gene" biomarkers shown in Table 1, f) thc achr21q22 overlap" biomarkers shown in Table 2, g) the "PRC2 cluster" biomarkers shown in Tabic 3, h) the "overlap" biomarkcrs shown in Table 4, i) (he "SUZ12 target," "Mikkelsen EF," and/or MMflckelsen NPC" biomarkers shown in Table 5,j) KDM6A, k) K.DM6B, I) EZH2, m) HMON1, and subsets and or combinations thereof.
30. The method of claim 28, wherein said agent O wnmodulates or uproodulates the copy number, level of expression, or level of activity of the one or more biomarkers.
31. Tbe method of claim 28, further comprising administering one or more additional agents that treats the cancer.
32. The method of claim 28, wherein the agent is one or more modulators of historic H3K27me3 levels.
33. A pharmaceutical composition comprising a polynucleotide encoding one or more biomarkers listed in Tables 1-5 or a fragment thereof useful for treating cancer in a pharmaceutically acceptable carrier.
34. The pharmaceutical composition of claim 33, wherein the potynucleotide encoding the one or more biomarkers listed in Tables 1-5 or a fragment thereof further comprises an expression vector.
35. A method of using the pharmaceutical composition of claims 33 or 34 for treating a cancer.
36. A kit comprising an agent which selectively binds to one or more biomarkers listed in Tables 1-S or a f-agmcm thereof and imtru(^icms fo^
37. A kit comprising an agent which selectively hybridizes to a polynucleotide encoding one or more biomarkers listed in Tables 1-5 or fragment thereof and .instructions for use.
38. A biochip comprising a solid substrate, said substrate comprising a plurality of probes capable of detecting one or more biomarkers listed in Tables 1-5 or a fragment thereof wherein each probe is attached to the substrate at a spatially defined address.
39. The biochip of claim 51, wherein the probes are complementary to a genomic or transcribed polynucleotide associated with the one or more biomarkers.
40. The pharmaceutical composition of claim 33, the kit of claim 36, or the biochip of claim 38, wherein the one or more biomarkers are selected from the group consisting of the set of a) "top ISO UP" biomarkers shown in Table I, b) "the SO UP core" biotnarkcrs shown in Table 1, c) "top 150 DOWN" biomarkers shown in Table I, d), "the 50 DOWN core" biomarkers shown in Table l, c) tbek ^Hcated geric" biomaifcro shown in Table 1, the Mchr21q22 overlap" biomarkers shown in Table 2, g) die "PRC2 cluster" biomarkers shown in Table 3, h) the "overlap" biotnarkcrs shown in Table 4, i) the "SUZ12 target,"
" ikkelaen MEF," and/or "Mikkelscn NPC biomarkers shown in Table 5 J) DM6A, k) DM6B. I) ΈΖΗ2, m) HMGN1, and subsets and/or combinations thereof.
41. The method of any one of claims 1, 3, 7 , 13, 16, 18, 23, and 28, n^rem the control is determined from a noncancerous sample from the subject or member of the same species to which the subject belongs.
42. The method of any one of claims 1, 3, 7, 13, 16, 18, 23, and 28, wherein the sample comprises cells, cell lines, histological slides, paraffin embedded tissue, fresh frozen tissue, fresh tissue, biopsies, blood, plasma, serum, buccal scrape, saliva, cerebrospinal fluid, urine, stool, mucus, or bone marrow, obtained from the subject.
43. The method of any one of claims 1, 3, 7, 13, 16, 18, 23, and 28, wherein the copy number is assessed by microarray, quantitative PCR (qPCR), high-diroughput sequencing, comparative genomic hybridization (CGH), or fluorescent in situ hybridization (FISH).
44. The method of any one of claims 1, 3, 7, 13, 16, 18, 23, and 28, wherein the expression level of the one or more biomarkers is assessed by detecting the presence in the samples of a polynucleotide molecule encoding the biomarkcr or a portion of said polynucleotide molecule.
45. The method of claim 44, wherein the polynucleotide molecule is a mRN A, cDNA, or functional variants or fragments thereof.
46. Tbe method of claim 44, wherein the step of detecting further comprises amplifying the polynucleotide molecule.
47. The method of any one of claims 1,3, 7, 13, 16, 18, 23. and 28, wherein the expression level of the one or more biomarkers is assessed by annealing a nucleic acid probe with tbe sample of the polynucleotide encoding die one of more biomarkers or a portion of said polynucleotide molecule under stringent hybridization conditions.
48. The method of any one of claims 1, , 7, 13, 16, 18, 23, and 28, wherein the expression level of die biomarker is assessed by delecting die presence in die samples of a protein of the biomarker, a polypeptide, or protein fragment thereof comprising said protein.
49. The method of claim 48, wherein tbe presence of said protein, polypeptide or protein fragment thereof is detected using a reagent which specifically binds with said protein, polypeptide or protein fragment thereof.
50. The method of claim 49, wherein the reagent is selected from the group consisting of an antibody, an antibody derivative, and an antibody fragment.
51. The method of any one of claims 1, 3, 7, 13, 16, 18, 23, and 28, wherein the activity level of the biomarker is assessed by determining die magnitude of modulation of the activity or expression level of downstream targets of the one or more biomarkers.
52. The method of any one of claims 1, 6, 7, 13, 16, 18, 23, and 28, wherein the agent or test compound modulates histone H3K27me3 levels.
53. The method of claim 52, wherein the agent or test compound inhibits the expression and/or activity of Jumonji D3 family of histone Hc 27 dcmethylascs.
54. The method of claim 53, wherein the agent or test compound is a small molecule inhibitor of KMD6A (UTX) and/or KDM6B (JMJD3).
55. The method of claim 52, wherein the agent or test compound inhibits the expression andor activity of HMGN1.
56. Tbe method of claim S2, wherein the agent or test compound is an inhibitor selected from die group consisting of a small molecule, an ti sense nucleic acid, interfering RNA, shRNA, siRNA, aptamer. ribozymc, and dominant-negati ve protein binding partner.
57. Tbe method of any one of claims 1, 6, 7, 13, 16, 18, 23.28, and 35, wherein the cancer is a leukemia.
58. The method of claim 57, wherein the leukemia is B-cdl acnte lymphoblastic leukemia.
59. The method of any one of claims 1, 3, 7, 13, 16, 18.23, and 28, wherein the subject has an increased copy number of a) human chromosome 21 or the human DSCR region thereof, b) mouse chromosome 16 or the mouse iAmp, Ts65Dn, TslRhr, Dp(l6)lYu, or Runxl locus thereof, or c) orthologs of a) or b), relative to a wild type control.
60. The method of any one of claims 1, 3, 7, 13, 16, 18, 23, and 28, wherein the subject is a human.
61. A method of increasing the number of lymphoid progemtc cdls from sn raitial population of lymphoid progenitor cells comprising contacting the lymphoid progenitor cells with an agent that inhibits poly comb repressor complex 2 (PRC2) activity or reduces H3K27mc3 levels to thereby increase the number of lymphoid progenitor cells.
62. Tbe method of claim 61 , wherein the agent inhibits the activity of the EZH2 histonc H3K27 methyltransferase subunit of PRC2.
63. The method of claim 62, wherein the agent is an inhibitor selected from the group consisting of a small molecule, antisense nucleic acid, interfering RNA, shRNA, siRNA, nuRNA, aptamer, ribozymc. and dominant-negative protein binding partner.
64. The method of claim 61, wherein the lymphoid progenitor cells are comprised within bone marrow with marker selection or without marker selection.
65. The method of claim 1 , wherein the lymphoid progenitor cells comprise pre-pro B cells, pro B cells, large pre-B cells, small prc-B cells, immature B cells, or any combination thereof.
66. The method of claim 61, wherein the contacting the lymphoid progenitor ceils wid¬ ths agent is performed In vivo, ex vtvo, or In vitro.
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