WO2018044906A1 - Compositions and methods for treating cancer - Google Patents

Compositions and methods for treating cancer Download PDF

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Publication number
WO2018044906A1
WO2018044906A1 PCT/US2017/049143 US2017049143W WO2018044906A1 WO 2018044906 A1 WO2018044906 A1 WO 2018044906A1 US 2017049143 W US2017049143 W US 2017049143W WO 2018044906 A1 WO2018044906 A1 WO 2018044906A1
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pten
mmset
cell
cells
agent
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PCT/US2017/049143
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French (fr)
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Wenyi WEI
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Beth Israel Deaconess Medical Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell

Definitions

  • Phosphatase and tensin homolog deleted on chromosome ten is a lipid and protein dual phosphatase, and is a tumor suppressor. PTEN is frequently mutated, deleted, or epigenetically silenced in various types of human cancers. In the cytoplasm, PTEN primarily governs key cellular processes including cell survival, proliferation, aging, angiogenesis and metabolism through its lipid phosphatase activity to antagonize the PI3K-Akt oncogenic pathway. However, compared to its well-studied lipid phosphatase activity, the protein phosphatase function of PTEN remains largely undefined. To this end, increasing evidence argues that PTEN also possesses multiple important functions in the nucleus independent of its lipid phosphatase activity, such as controlling the chromosomal integrity, chromatin structure, DNA replication and DNA damage repair.
  • the present invention provides a method of sensitizing a neoplastic cell to chemotherapy, the method comprising contacting the cell with an agent that inhibits multiple myeloma SET domain protein (MMSET) activity or expression and a
  • MMSET multiple myeloma SET domain protein
  • chemotherapeutic agent thereby sensitizing the cell to chemotherapy.
  • the present invention provides a method of sensitizing a neoplastic cell to radiation, the method comprising contacting the cell with an agent that inhibits MMSET activity or expression and exposing the cell to radiation, thereby sensitizing the cell to ⁇ -irradiation.
  • the present invention provides a method of enhancing cell death or reducing proliferation in a neoplastic cell, the method comprising contacting the cell with an agent that inhibits MMSET activity or expression and a chemotherapeutic agent, thereby enhancing cell death or reducing proliferation in the cell.
  • the present invention provides a method of enhancing cell death or reducing proliferation in a neoplastic cell, the method comprising contacting the cell with an agent that inhibits MMSET activity or expression and exposing the cell to radiation, thereby enhancing cell death or reducing proliferation in the cell.
  • the present invention provides a method of enhancing
  • administering to the subject an agent that inhibits MMSET activity or expression or and a chemotherapeutic agent, thereby enhancing chemotherapy sensitivity in the subject.
  • the present invention provides a method of enhancing radiation sensitivity in a subject having a neoplasia, the method comprising administering to the subject radiation and an agent that inhibits MMSET activity or expression, thereby enhancing radiation sensitivity in the subject.
  • the present invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein; and a chemotherapeutic agent, thereby treating cancer in the subject.
  • MMSET multiple myeloma SET domain
  • the agent that inhibits MMSET activity is a polypeptide, polynucleotide, or small molecule.
  • the agent that inhibits MMSET activity is selected from the group consisting of: (l S,2R,5R)-5-(4-Amino-lH-imidazo[4,5- c]pyridin-l-yl)-3-(hydroxymethyl)-3-cyclopentene-l,2-diol hydrochloride, 3- hydrazinylquinoxaline-2-thiol, and LEM-06.
  • the polynucleotide is an inhibitory nucleic acid molecule that inhibits the expression of MMSET.
  • the inhibitory nucleic acid molecule is an antisense molecule, siRNA, or shRNA.
  • the shRNA comprises or consists essentially of one of the following sequences: MMSET shRNA 1 : 5 ' -GC ACGCTAC AAC ACC AAGTTT;
  • the chemotherapeutic agent is selected from the group consisting of:
  • the chemotherapeutic agent is a PI3 kinase inhibitor.
  • the PI3 kinase inhibitor is BKM120, BYL719 or RP6530.
  • the neoplastic cell is a mammalian cell.
  • the mammalian cell is a murine, rat, or human cell.
  • the cell is in vitro or in vivo.
  • the neoplastic cell or cancer comprises a mutation in PTEN or amplification of MMSET.
  • the method reduces neoplastic cell survival or proliferation.
  • the neoplastic cell is derived from prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, or other types of epithelium derived carcinomas where loss of function of PTEN is frequently observed.
  • the subject has prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, or other types of epithelium derived carcinomas where loss of function of PTEN is frequently observed.
  • the method reduces tumor growth, and/or increases subject survival.
  • the agent that inhibits the expression or activity of MMSET reduces the effective amount of the chemotherapeutic agent necessary to treat the cancer.
  • the cancer is prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, or other types of epithelium derived carcinomas where loss of function of PTEN is frequently observed.
  • the agent that inhibits MMSET activity is a pan-inhibitor of S-adenosylmethionine-dependent
  • the invention provides a kit comprising a therapeutic or prophylactic composition containing (i) an effective amount of an agent of any one of preceding embodiments; and (ii) an effective amount of a chemotherapeutic agent.
  • a therapeutic or prophylactic composition containing (i) an effective amount of an agent of any one of preceding embodiments; and (ii) an effective amount of a chemotherapeutic agent.
  • Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.
  • PTEN polypeptide a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: NP 000305.3, or a fragment thereof, and having phosphatase activity.
  • An exemplary PTEN amino acid sequence is provided below (SEQ ID NO: 1):
  • PTEN polynucleotide is meant a nucleic acid molecule encoding a PTEN polypeptide.
  • An exemplary PTEN polynucleotide sequence is provided at NCBI Reference Sequence: NM_000314.6, and reproduced herein below (SEQ ID NO: 2).
  • MMSET multiple myeloma SET domain
  • MMSET multiple myeloma SET domain
  • MMSET contains a SET domain that is found in many histone methyltransferases (HMTs).
  • HMTs histone methyltransferases
  • Other potential functional motifs in MMSET include nuclear localization signals (NLSs), an HMG box (high mobility group) often representing a DNA-binding domain, 2 PWWP domains (proline-tryptophan-tryptophan-proline) found in other nuclear proteins and 4 PHD (plant homeodomain) zinc fingers recently defined as binding modules for methylated lysines.
  • NLSs nuclear localization signals
  • HMG box high mobility group
  • PWWP domains proline-tryptophan-tryptophan-proline
  • PHD plant homeodomain
  • MMSET polynucleotide is meant a nucleic acid molecule encoding a MMSET polypeptide.
  • An exemplary MMSET polynucleotide sequence is provided at NCBI Reference Sequence: M 133330.2, and reproduced herein below (SEQ ID NO: 4):
  • the MMSET gene is also known as Wolf-Hirschhorn Syndrome Candidate 1 (WHSC1) or Nuclear Receptor-binding SET Domain 2 (NSD2).
  • WHSC1 Wolf-Hirschhorn Syndrome Candidate 1
  • NSD2 Nuclear Receptor-binding SET Domain 2
  • MDC1 polypeptide a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: NP_055456.2, or a fragment thereof, and having cell cycle regulatory activity. MDC1 is required to activate the intra-S phase and G2/M phase cell cycle checkpoints in response to DNA damage.
  • This nuclear protein interacts with phosphorylated histone H2AX, near sites of DNA double-strand breaks through its two BRCAl C-terminal (BRCT) motifs, and facilitates recruitment of the protein kinase ataxia-telangiectasia mutated (ATM) and meiotic recombination 11 protein complex to DNA damage foci.
  • BRCT BRCAl C-terminal
  • ATM protein kinase ataxia-telangiectasia mutated
  • meiotic recombination 11 protein complex to DNA damage foci.
  • DNA damaging agent is meant a chemotherapeutic agent that introduce lesions in the DNA of a cell that lead to replication-associated DNA double-strand breaks (DSBs), which are toxic to the cell.
  • DSBs replication-associated DNA double-strand breaks
  • DNA double-stand breaks (DSBs) repair or “DNA double-stand breaks (DSBs) repair” is meant a collection of processes in which a cell identifies and corrects damage to DNA encoding the cell's genome.
  • agent any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
  • ameliorate decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • alteration is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein.
  • an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.
  • an analog is meant a molecule that is not identical, but has analogous functional or structural features.
  • a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding.
  • An analog may include an unnatural amino acid.
  • Detect refers to identifying the presence, absence or amount of the analyte to be detected.
  • disease is meant any condition or disorder that damages, or interferes with the normal function of a cell, tissue, or organ.
  • diseases include any cancer, including but not limited to breast cancer, prostate cancer, or colon cancer.
  • an effective amount is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient.
  • the effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
  • the invention provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein.
  • the methods of the invention provide a facile means to identify therapies that are safe for use in subjects.
  • the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.
  • fragment is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide.
  • a fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
  • Hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
  • adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
  • inhibitory nucleic acid is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene.
  • a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule.
  • an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.
  • isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state.
  • Isolate denotes a degree of separation from original source or surroundings.
  • Purify denotes a degree of separation that is higher than isolation.
  • a “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • the term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
  • modifications for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
  • isolated polynucleotide is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
  • the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
  • an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it.
  • the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention.
  • An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • marker any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder. Cancers of the invention are those characterized by a reduction in, or an alteration in, or the loss of markers Pten and p53.
  • obtaining as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
  • reference is meant a standard or control condition.
  • a "reference sequence” is a defined sequence used as a basis for sequence
  • a reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids.
  • the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
  • siRNA is meant a double stranded RNA.
  • a siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3' end.
  • These dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream.
  • Such siRNAs are used to downregulate mRNA levels or promoter activity.
  • telomere binding By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
  • Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity.
  • Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
  • hybridize is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency.
  • complementary polynucleotide sequences e.g., a gene described herein
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C.
  • Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA).
  • hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C.
  • wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
  • Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
  • a reference amino acid sequence for example, any one of the amino acid sequences described herein
  • nucleic acid sequence for example, any one of the nucleic acid sequences described herein.
  • such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;
  • BLAST program may be used, with a probability score between e "3 and e "100 indicating a closely related sequence.
  • subject is meant a mammal, including, but not limited to, a human or non- human mammal, such as a bovine, equine, canine, ovine, or feline.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
  • the term "about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • FIGs. 1 A-1K show ATM-mediated phosphorylation of PTEN is required for binding the BRCT domain of MDCl upon DNA damage signaling.
  • FIGs. 1 A and IB Immunoblot (IB) analysis of anti-PTEN immunoprecipitations (IPs) and whole cell lysates (WCL) derived from NIH3T3 cells treatment with 30 ⁇ etoposide (FIG. 1A) or after irradiation (IR) (5 Gy; Gy refers the gray symbol, a derived unit of ionizing radiation does in the International System of Units (SI)) (FIG. IB), at indicated time points before harvesting.
  • FIG. IB Immunoblot analysis of anti-PTEN immunoprecipitations (IPs) and whole cell lysates (WCL) derived from NIH3T3 cells treatment with 30 ⁇ etoposide (FIG. 1A) or after irradiation (IR) (5 Gy; Gy refers the gray symbol, a derived
  • FIG. 1C IB analysis of anti-PTEN IPs and WCL derived from NIH3T3 cells firstly treated using 1 ⁇ Ku55933 (ATM inhibitor), ⁇ VE821 (ATR inhibitor) and 1 ⁇ ⁇ 7026 (DNAPK inhibitor) for 90 minutes (min) followed by addition of 30 ⁇ etoposide for 30 min before harvesting.
  • FIG. ID IB analysis of anti-HA IPs and WCL derived from 293T cells that ectopically express HA-tagged wild type (WT) or S398A mutant mouse PTEN (mPTEN). 36 hours (hr) after transfection, 30 ⁇ etoposide was added and cells were harvested at indicated time points for IP analysis.
  • FIG. ID IB analysis of anti-HA IPs and WCL derived from 293T cells that ectopically express HA-tagged wild type (WT) or S398A mutant mouse PTEN (mPTEN). 36 hours (hr) after transfection, 30 ⁇ etoposide was added
  • FIG. IE A schematic representation of the indicated domains of 53BP1, MDCl and PTEN.
  • FIG. IF IB analysis of GST pull-down precipitations and WCL derived from U20S cells treatment with/without 30 ⁇ etoposide for 30 min before harvesting.
  • FIG. 1G IB analysis of anti-PTEN IPs and WCL derived from U20S cells treatment with 30 ⁇ etoposide at indicated time points.
  • FIG. 1H IB analysis of anti- Flag-IPs and WCL derived from U20S cells co-transfected empty vector (EV), HA-tagged wild type (WT) or T398A human PTEN (hPTEN) with Flag-MDCl, respectively.
  • EV empty vector
  • WT HA-tagged wild type
  • hPTEN T398A human PTEN
  • FIGs. II and 1 J IB analysis of anti-PTEN IPs and whole cell lysates (WCL) derived from U20S cells (FIG. II), or derived ⁇ Mmset +/+ and Mmsef ' MEFs (FIG. 1J), treatment with 30 ⁇ etoposide as indicated time points before harvesting.
  • FIG. IK IB analysis of anti-PTEN IPs and WCL derived from Mdcl +/+ and Mdcl ' ' ' MEFs treatment with 30 ⁇ etoposide as indicated time points before harvesting.
  • FIGs. 2A-2K show DNA-damaging agent induced phosphorylation of PTEN is required for its interaction with the BRCT domain of MDCl .
  • FIGs. 2A and 2B Immunoblot (IB) analysis of anti-PTEN immunoprecipitations (IPs) and whole cell lysates (WCL) derived from MEFs cells treatment after irradiation (FIG. 2A) or with/without 30 ⁇ etoposide (IR, 5 Gy) treatment (FIG. 2B) at indicated time points before harvesting.
  • FIG. 1B Immunoblot analysis of anti-PTEN immunoprecipitations
  • WCL whole cell lysates
  • FIG. 2C IB analysis of anti-HA IPs and WCL derived from U20S cell lines stably expressing shScramble (shScr), shATM, or shATF transfected with indicated constructs. 36 hours (h) after transfection, cells were treated with/without 30 ⁇ etoposide for 30 min before harvesting.
  • FIGs. 2D and 2F IB analysis of GST pull-down precipitations and WCL derived from U20S cells treatment with/without 30 ⁇ etoposide for 30 min before harvesting.
  • FIG. 2E Sequence alignment of PTEN C-tails between human and mouse.
  • FIG. 2G A schematic representation of the indicated domains of PTEN, including N-terminus (N-tail and phosphatase domain) and C- terminus (C2 and C-tail domain).
  • FIG. 2H IB analysis of GST pull-down precipitations and WCL derived from U20S cells treatment with 30 ⁇ etoposide for 30 min before harvesting.
  • FIG. 21 IB analysis of anti-Flag IPs and WCL derived from U20S transfected with indicated constructs. 36 hours after transfection, cells were treated with/without 30 ⁇ etoposide for 30 min before harvesting.
  • FIG. 2J A schematic illustrate to demonstrate that DNA damages induce MDCl -BRCT domain binding with PTEN and MMSET, respectively, largely through a phosphorylation-dependent manner.
  • FIG. 2K A schematic representation of the indicated domains of 53BP1, MDCl and BRCAl .
  • FIGs. 3 A-3B show MMSET methyltransferase specifically interacts with, and promotes the methylati on of PTEN.
  • FIG. 3 A Immunoblot (IB) analysis of anti -Flag immunoprecipitations (IPs) and whole cell lysates (WCL) derived from HEK293T cells with indicated antibodies.
  • FIG. 3B IB analysis of anti -Flag IPs and WCL derived from
  • HEK293T cells transfected with indicated constructs.
  • FIGs. 4A-4M show DNA damage promotes MMSET-mediated di-methylation of PTEN at K349, which is subsequently recognized by the tudor domain of 53BP1.
  • FIG. 4A Immunoblot (IB) analysis of anti-PTEN immunoprecipitation (IPs) and whole cell lysates (WCL) derived from U20S cells treatment with 30 ⁇ etoposide as indicated time points before harvesting.
  • FIG. 4B IB analysis of anti-PTEN IPs and WCL derived from MDC1 +/+ and MDCf ' MEFs treatment with 30 ⁇ etoposide as indicated time points before harvesting.
  • FIG. 4A Immunoblot (IB) analysis of anti-PTEN immunoprecipitation (IPs) and whole cell lysates (WCL) derived from U20S cells treatment with 30 ⁇ etoposide as indicated time points before harvesting.
  • FIG. 4B IB analysis of anti-PTEN IPs and WCL derived from M
  • FIG. 4C IB analysis of anti-HA IPs and WCL derived from 293T cells transfected with the indicated constructs.
  • FIG. 4D IB analysis of anti-HA IP and WCL derived from U20S cells transfected with HA-PTEN WT or K349R mutant and treated with/without etoposide at indicated time points.
  • FIG. 4E IB analysis of anti-PTEN IPs and WCL derived from U20S cells stably expressing shScramble or shMMSET that were treated with irradiation (TR) (5 Gy) at indicated time points before harvesting.
  • TR irradiation
  • FIG. 4F IB analysis of anti-PTEN IPs and WCL derived from U20S cells treated with 1 ⁇ or 2 DZNep for 24 hours followed by addition of 30 ⁇ etoposide for 30 min before harvesting.
  • FIG. 4G IB analysis of anti-PTEN IPs and WCL derived from U20S cells treated with 30 ⁇ etoposide at indicated time points before harvesting.
  • FIG. 4H and 4J IB analysis of GST pull-down precipitates and WCL derived from U20S cells transfected with the indicated constructs. 36 hr after transfection, cells were treated with/without 30 ⁇ etoposide for 30 min and harvested for GST pull-down assays.
  • FIG. 4G IB analysis of anti-PTEN IPs and WCL derived from U20S cells treated with 30 ⁇ etoposide at indicated time points before harvesting.
  • FIG. 4H and 4J IB analysis of GST pull-down precipitates and WCL derived from U20S cells transfected with the
  • FIG. 41 IB analysis of GST pull-down precipitates and WCL derived from U20S cells transfected with the indicated constructs. 36 hr after transfection, cells were harvested for GST pull-down assays.
  • FIG. 4K 1 ⁇ g of indicated biotin-labeled synthetic PTEN peptides were incubated with 250 ng purified recombinant GST-tagged 53BP1 tudor domain, respectively. Streptavidin beads were added to perform pull-down assays and the precipitations were analyzed by IB. Dot blot assays were performed to show equal amount of biotinylated peptides were used for the pull-down assay.
  • FIG. 4K 1 ⁇ g of indicated biotin-labeled synthetic PTEN peptides were incubated with 250 ng purified recombinant GST-tagged 53BP1 tudor domain, respectively. Streptavidin beads were added to perform pull-down assays and the precipitations were analyzed by IB. Dot
  • FIG. 4L IB analysis of anti-PTEN IPs and WCL derived from U20S cells stably expressing shScramble or shMMSET that were treated with/without 30 ⁇ etoposide for 30 min before harvesting.
  • FIG. 4M IB analysis of anti-PTEN IPs and WCL derived from WT or MMSET ' MEFs that were treated with/without 30 ⁇ etoposide for 30 min before harvesting.
  • FIGs. 5A-5C show PTEN binding with MM SET is largely dependent on MDC1 and the T398 phosphorylation status of PTEN.
  • FIG. 5A Immunoblot (IB) analysis of anti-PTEN immunoprecipitations (IPs) and whole cell ly sates (WCL) derived from MDC1 +/+ or MDC1 " " MEFs treated with 30 ⁇ etoposide at indicated time points before harvesting.
  • FIG. 5B IB analysis of anti-Flag IP and WCL derived from U20S cells co-transfected with HA-PTEN WT or T398A mutant with Flag-MMSET and treated with/without etoposide for 60 min before harvesting.
  • FIG. 5A Immunoblot (IB) analysis of anti-PTEN immunoprecipitations (IPs) and whole cell ly sates (WCL) derived from MDC1 +/+ or MDC1 " " MEFs treated with 30 ⁇ etoposide at indicated time points
  • FIG. 6A-6G show MMSET largely binds the C-terminal domain of PTEN to promote di -methyl ati on of PTEN at the K349 residue.
  • FIG. 6A A schematic representation of the indicated domains of PTEN, including N-terminus (N-tail and phosphatase domain) and C-terminus (C2 and C-tail domain), which is required for PTEN interaction with
  • FIG. 6B Immunoblot (IB) analysis of immunoprecipitations (IPs) and whole cell lysates (WCL) derived HEK293T cells transfected with the indicated constructs and treated with 30 ⁇ etoposide for 30 min before harvesting.
  • FIG. 6C IB analysis of anti-HA IP and WCL derived from HEK293T cells transfected with the indicated constructs and treated with/without 30 ⁇ etoposide for 30 min before harvesting.
  • FIGs. 6D and 6E IB analysis of anti-HA IP and WCL derived from HEK293T cells transfected with the indicated constructs, which were treated with/without 30 ⁇ etoposide for 30 min before harvesting.
  • FIG. 6B Immunoblot (IB) analysis of immunoprecipitations (IPs) and whole cell lysates (WCL) derived HEK293T cells transfected with the indicated constructs and treated with 30 ⁇ etoposide for 30 min before harvest
  • FIG. 6E Mass spectrometry analysis was performed with immunoprecipitated HA-PTEN derived from HEK293T cells co-transfected with HA-PTEN and Flag-MMSET. Different peptides derived from PTEN were enriched and the K349 di-methylation site was identified as a +28 Dolton increased.
  • FIG. 6F The sequence alignment of PTEN among different species to illustrate that the K349 di-methylation site (K349me2) is evolutionarily conserved.
  • FIG. 6G A surface diagram of the crystal structure of PTEN (PDB code: 1D5R). The N-terminal domain of PTEN is indicated in the left side of the figure, and C-terminal domain is indicated in the right side of the figure. The identified Lys349, is located on the upper right side of the figure, and is at the outer face of PTEN C-terminal domain. The structure model was analyzed using PyMOL software.
  • FIGs. 7A-7C shows the generation and validation of the antibodies that specifically recognize K349 di-methylation (K349me2) of PTEN.
  • FIG. 7A A schematic representation of the various biotinylated synthetic PTEN-derived peptides covering amino acids 340-358 of PTEN.
  • FIG. 7B Each indicated synthetic peptides in (FIG. 7 A) was diluted and used for the dot immunoblot analysis with the anti-PTEN-K349me2, anti-PTEN-K349me3 or anti-Biotin antibody, respectively.
  • FIG. 7C IB analysis of anti-PTEN IPs and WCL derived from U20S cells treated with 1 ⁇ or 2 DZNep for 24 hours followed by addition of 30 ⁇ etoposide for 30 min before harvesting.
  • FIGs. 8A-8D show 53BP1 recognizes the K349 di-methylation species of PTEN largely through its tudor domain.
  • FIG. 8A A schematic illustration of the indicated domains of 53BP1 to show its tudor domain binding with reported dimethylation lysine 20 of H4 (H4K20me2) and dimethylation lysine 810 of Rb (RbK810me2).
  • FIG. 8B IB analysis of anti-His IPs and WCL derived from U20S cells treatment with/without 30 ⁇ etoposide for 60 min before harvesting.
  • FIG. 8A A schematic illustration of the indicated domains of 53BP1 to show its tudor domain binding with reported dimethylation lysine 20 of H4 (H4K20me2) and dimethylation lysine 810 of Rb (RbK810me2).
  • FIG. 8B IB analysis of anti-His IPs and WCL derived from U20S cells treatment with/without 30 ⁇ e
  • FIG. 8C IB analysis of GST pull-down and WCL derived from U20S cells co-transfected with HA-PTEN WT or K349R mutant with Flag-MMSET and treated with 30 ⁇ etoposide for 60 min before harvesting.
  • FIG. 8D A schematic model to illustrate that DNA damage induces the interaction between the MDC1-BRCT domain and PTEN or MMSET, respectively, through a phosphorylation-dependent manner.
  • MMSET promoted the K349 dimethylation of PTEN, which is recognized by the 53BP1 tudor domain.
  • FIGs. 9A-9P show protein phosphatase activity of PTEN is required for efficient complement of DSBs repair largely through dephosphorylating ⁇ 2 ⁇ .
  • FIG. 9B Immunoblot (IB) analysis of whole cell lysates (WCL) derived from PTEN ⁇ /+ and ⁇ ' HCT116 cells after treatment with IR (5 Gy) as indicated time points.
  • FIG. 9B Immunoblot analysis of whole cell lysates (WCL) derived from PTEN ⁇ /+ and ⁇ ' HCT116 cells after treatment with IR (5 Gy) as indicated time points.
  • FIG. 9A Quantification of protein intensity in FIG. 9A was performed using the ImageJ software. ⁇ 2 ⁇ immunoblot bands were normalized to Vinculin, then normalized to the control (no IR treatment).
  • FIGs. 9C and 9D IB analysis of WCL derived from ⁇ ' HCT116 cells introducing PTEN WT, C124S, G129E, Y138L as well as EV, which were treated with IR (5 Gy) at indicated time points before harvesting. Quantification of protein intensity in FIG. 9C was performed using the ImageJ software FIG. 9D. ⁇ 2 ⁇ immunoblot bands were normalized to Vinculin, then normalized to the control (no IR treatment).
  • FIGs. 9A Quantification of protein intensity in FIG. 9A was performed using the ImageJ software. ⁇ 2 ⁇ immunoblot bands were normalized to Vinculin, then normalized to the control (no IR treatment).
  • FIGs. 9E and 9F IB analysis of WCL derived from ⁇ ' HCT 116 cells introducing PTEN WT, K349R as well as EV, which were treated with IR (5 Gy) at indicated time points before harvesting (FIG. 9D). Quantification of protein intensity in (FIG. 9E) was performed using the ImageJ software (FIG. 9F).
  • FIGs. 9G and 9H IB analysis of anti-HA IPs, anti-Flag IPs and WCL derived from U20S cells transfected with the indicated constructs. 36 hours after transfection, cells were harvested for IP assays after treatment with 30 ⁇ etoposide for 30 min.
  • FIG. 9G and 9H IB analysis of anti-HA IPs, anti-Flag IPs and WCL derived from U20S cells transfected with the indicated constructs. 36 hours after transfection, cells were harvested for IP assays after treatment with 30 ⁇ etoposide for 30 min.
  • FIG. 9J Immunoblot (IB) analysis of whole cell lysates (WCL) derived from PTE ⁇ /+ and ⁇ ' HCT116 cells after treatment with IR (5 Gy) as indicated time points.
  • FIG 9K and FIG. 9L IB analysis of WCL derived from Pten +/+ , Pten G129E/+ and Pten cl24S/+ MEFs, which were treated with IR (5 Gy) at indicated time points before harvesting.
  • FIG. 9K Quantification of protein intensity in (FIG. 9K) was performed using the ImageJ software (FIG. 9L). ⁇ 2 ⁇ immunoblot bands were normalized to Vinculin, and then normalized to the control (no IR treatment).
  • FIG 9M and FIG. 9N Immunohistochemistry (IHC) analysis of spleen tissue derived from Pten +/+ , Pten G129E/+ and Pten cl24S/+ mice, which were treated with IR (3 Gy) and sacrificed at 24 h after irradiation (FIG. 9M). Bar, 50 ⁇ . IB analysis of the sample was performed using indicated antibodies (FIG. 9N). Four mice each group.
  • FIG. 9N Immunohistochemistry
  • FIG. 9P HAPTEN accumulates at sites of laser microirradiation in HCTl 16-PTEN ⁇ ⁇ cells. Scale bar: 10 ⁇ .
  • FIGs. 1 OA- 10E shows the K349R mutation in PTEN does not affect its lipid phosphatase activity.
  • FIG. 10A Immunoblot (IB) analysis of whole cell lysates (WCL) derived from HCTl 16 TEA ⁇ cells reconstituted with the indicated PTEN WT and PTEN- mutant proteins.
  • FIG. 10B A table summary of the lipid versus protein phosphatase activity of reported PTEN WT and various well-characterized PTEN mutants.
  • FIG. IOC IB analysis of WCL derived from HCTl 16 PTEN '1' cells reconstituted with the PTEN WT, K349R mutant, as well as empty vector (EV) using indicated antibodies.
  • FIG. 10A Immunoblot (IB) analysis of whole cell lysates (WCL) derived from HCTl 16 TEA ⁇ cells reconstituted with the indicated PTEN WT and PTEN- mutant proteins.
  • FIG. 10B A table summary of the lipid versus protein phosphatas
  • FIG. 10D IB analysis of anti-HA IPs, anti-Flag IPs and WCL derived from U20S cells transfected with the indicated constructs. 36 hr after transfection, cells were harvested for IP assays after 30 uM etoposide treatment for 60 min.
  • FIG. 10E The amino acid sequences of synthetic H2AX and ⁇ 2 ⁇ peptides used in the present invention.
  • FIGs. 11 A-l 1C show the protein phosphatase activity and K349 methylation of PTEN is required for completing DSB repair process.
  • FIG. 11 A Immunoblot (IB) analysis of whole cell lysates (WCL) derived from JEN-deficient HCTl 16 cells reconstituted with the indicated PTEN WT and the indicated PTEN mutant proteins.
  • FIGs. 1 IB and 11C PTEN- deficient U87MG cells reconstituted with indicated constructs were subjected to
  • FIGs. 12A-12W show MMSET-mediated methylation of PTEN at K349 dictates cellular sensitivity to DNA-damaging agents.
  • FIGs. 12A and 12B TEN-deficient U87MG cells reconstituted with the indicated PTEN constructs were treated with IR (5 Gy) and immunostained at the indicated times with anti-53BPl (FIG. 12A) or anti-yH2AX (FIG. 12B). Quantification of 53BP1 or ⁇ 2 ⁇ foci positive cells (foci > 5 per cell) are shown, respectively.
  • FIGs. 12C and 12D U20S cells stably expressing shScr or shMMSET were treated with IR (5 Gy) and immunostained at the indicated times with anti-53BPl (FIG.
  • FIG. 12E TEN-deficient U87MG cells reconstituted with the indicated PTEN constructs were pre-treated with 1 ⁇ BKM120 for 24 hours followed by an additional 10 IR treatments (5 Gy). 48 hours post-irradiation, cells were harvested for cell viability assay (FIG. 12E) or cell apoptosis assays (FIG. 12F). Data are shown as mean ⁇ s.d. from three independent experiments. * p ⁇ 0.05 (t-test). FIG.
  • FIGs. 12G Immunoblot (IB) analysis of whole cell lysates (WCL) derived from the samples of (FIGs. 12E-12F) with indicated antibodies.
  • FIGs. 12H-12I U20S cells stably expressing shScr or shMMSET were pre-treated with 1 ⁇ BKM120 for 24 hours followed by an additional 10 IR treatments (5 Gy). 48 hours post-irradiation, cells were harvested for cell viability assay (FIG. 12H) or cell apoptosis assays (FIG. 121). Data are shown as mean ⁇ s.d. from three independent experiments. * p ⁇ 0.05 (t-test). FIGs.
  • FIG. 12J and 12K U20S cells stably expressing shScr or shMMSET were pre-treated with 1 ⁇ BKM120 and 2 ⁇ DZNep for 24 hours followed by an additional 10 IR treatments (5 Gy). 48 hours post-irradiation, cells were harvested for cell viability assay (FIG. 12J) or cell apoptosis assays (FIG. 12K). Data are shown as mean ⁇ s.d. from three independent experiments. * p ⁇ 0.05 (t-test).
  • FIG. 12L IB analysis of WCL derived from the samples of (FIG. 12J-12K) with indicated antibodies.
  • FIG. 12L IB analysis of WCL derived from the samples of (FIG. 12J-12K) with indicated antibodies.
  • FIG. 12M HCT116 cells were pre-treated with/without 1 ⁇ BKM120 for 24 h followed by additional IR (0.5 Gy) and/or 2 ⁇ DZNep treatment. After one week, cells were stained with crystal violet.
  • FIG. 1M HCT116 cells were pre-treated with/without 1 ⁇ BKM120 for 24 h followed by additional IR (0.5 Gy) and/or 2 ⁇ DZNep treatment. After one week, cells were stained with crystal violet.
  • FIG 12N ⁇
  • Tumor xenograft mouse assays were performed by inplanting PTEN-/- HCT1 16 cells stably expressing PTEN WT, K349R and empty vector (EV). Tumor growth rate in nude mice treated every other day with
  • FIG. 120 Tumors were dissected after euthanizing the mice and tumors were recorded at the time of sacrifice (FIG. 12P). IB analysis of the samples was performed using indicated antibodies (FIG. 12Q). Statistical analysis of tumor volumes showed significant differences in mean tumor volumes between the PTEN-WT and the PTEN-K349R groups. Four mice each group. *p ⁇ 0.05 (Student's t-test).
  • FIG. 12R PTE /+ and ⁇ ' HCT1 16 cells stably depleting MMSET by shRNA (with shScr as a negative control) were pre-treated with 1 ⁇
  • FIG. 12S Tumor xenograft mouse assays were performed by injection of PTE ⁇ /+ and PTEN ⁇ ⁇ ⁇ £ ⁇ ⁇ 6 cells stably expressing shRNA against MMSET or shScr as a negative control. Tumor growth rate in nude mice treated every other day with a combination of etoposide (20 mg/kg) and BKM120 (25 mg/kg) was shown in FIG. 12S.
  • FIG 12V and FIG. 12W Tumor xenograft mouse assays were performed by subcutaneously implanting PTE ⁇ /+ and ⁇ ' HCT1 16 cells. Tumor growth rate in nude mice treated every other day with a combination of etoposide (20 mg/kg) and BKM120 (25 mg/kg) with DZNep (1 mg/kg) (or with vehicle as a negative control) was shown (FIG. 12V). Tumors were dissected after euthanizing the mice and were performed by IB analysis using indicated antibodies (FIG. 12W). Four mice each group. *p ⁇ 0.05, NS indicates no significant difference (Student's t-test).
  • FIGs. 13A-13B shows depletion of MMSET causes DNA DSB repair defects.
  • FIGs. 13A and 13B U20S cells stably expressing shScr or shMMSET were treated with IR (5 Gy) and immunostained at the indicated times with anti-53BPl (FIG. 13 A) or anti-yH2AX (FIG. 13B). Representative immunofluorescence images of 53BP1 or H2AX foci are shown.
  • FIG. 13A and FIG. 13B Quantification of FIG. 13A and FIG. 13B are shown in FIG. 12C and FIG. 12D.
  • FIGs. 14A-14D show loss of PTEN protein phosphatase activity or K349 methylation event sensitizes cells to IR and BKM120 combination treatment.
  • FIGs. 14A-14C PTEN- deficient HCT1 16 cells reconstituted with the PTEN WT (PTEN ⁇ I+ ) or empty vector
  • FIG. 14A JEN-deficient HCTl 16 cells reconstituted with the indicated PTEN constructs were pre-treated with 1 ⁇ BKM120 for 24 hours followed by an additional 10 IR treatments (5 Gy). 48 hours post-irradiation, cells were harvested for cell apoptosis assays. Data are shown as mean ⁇ s.d. from three independent experiments. * p ⁇ 0.05 or ** p ⁇ 0.001 (t-test)
  • FIGs. 15A-15D show loss of PTEN protein phosphatase activity or K349 methylation event sensitizes cells to etoposide and BKM120 combination treatment.
  • FIGs. 15A and 15B TEN-deficient U87MG cells reconstituted with the PTEN WT (PTEN+/+) or empty vector were treated as indicated. Cells were harvested for cell apoptosis assays.
  • FIGs. 15C and 15D PTEN-deficient U87MG cells reconstituted with the indicated PTEN constructs were pre-treated with 1 ⁇ BKM120 for 24 hours followed by additional 20 uM etoposide treatment. 48 hours after etoposide treatment, cells were harvested for cell apoptosis assays. Data are shown as mean ⁇ s.d. from three independent experiments. * p ⁇ 0.05 (t-test).
  • FIGs. 16A-16B show inhibition of MMSET by shRNA or DZNep sensitizes cells to combination treatment DNA damaging agents and BKM120.
  • FIG. 16 A HCTl 16 cells stably expressing shScr or shMMSET were pre-treated with/without 1 ⁇ BKM120 or 2 ⁇ DZNep for 24 hours followed by additional IR (2 Gy) as indicated. After 48 hours post- IR, cells were harvested for cell apoptosis assays.
  • FIG. 16B HCTl 16 cells were pre-treated with/without 1 uM BKM120 or 2 uM DZNep for 24 hours followed by additional IR (2 Gy). After 48 hours post-IR, cells were harvested for cell apoptosis assays.
  • FIG. 18 shows a schematic representation of how PTEN methylation and protein phosphatase activity response to DNA damage signaling.
  • the MMSET methyltransferase promotes the methylation of PTEN, which was recruited into DNA damage sites to help complete DNA damage repair through dephosphorylating ⁇ - ⁇ 2 ⁇ .
  • FIGs. 19A-19K show ATM-mediated phosphorylation of PTEN is required for binding the BRCT domain of MDC1 upon DNA damage signaling.
  • FIGs. 19A and 19B Immunoblot (IB) analysis of anti-PTEN immunoprecipitations (IPs) and whole cell lysates (WCL) derived from NIH3T3 cells treatment after irradiation (IR) (5 Gy) (FIG.
  • IPs anti-PTEN immunoprecipitations
  • WCL whole cell lysates
  • FIG. 19A or with 30 ⁇ etoposide (FIG. 19B) as indicated time points before harvesting.
  • FIG. 19C IB analysis of anti-PTEN IPs and WCL derived from NIH3T3 cells pre-treated with 1 ⁇ Ku55933 (ATM inhibitor), ⁇ VE821 (ATR inhibitor) or 1 ⁇ ⁇ 7026 (DNAPK inhibitor) for 90 minutes (min) followed by addition of 30 ⁇ etoposide for 30 min before harvesting.
  • FIG. 19D IB analysis of anti-HA IPs and WCL derived from U20S cell lines stably expressing shScr or shATM transfected with indicated constructs.
  • FIG. 19E IB analysis of anti-HA IPs and WCL derived from 293T cells that ectopically express HA-tagged wild type (WT) or S398A mutant mouse PTEN (mPTEN). 36 hours (hr) after transfection, 30 ⁇ etoposide was added and cells were harvested at indicated time points for IP analysis.
  • FIG. 19F IB analysis of GST pull-down precipitations and WCL derived from U20S cells treatment with/without 30 ⁇ etoposide for 30 min before harvesting.
  • FIG. 19E IB analysis of anti-HA IPs and WCL derived from 293T cells that ectopically express HA-tagged wild type (WT) or S398A mutant mouse PTEN (mPTEN). 36 hours (hr) after transfection, 30 ⁇ etoposide was added and cells were harvested at indicated time points for IP analysis.
  • FIG. 19F IB analysis of GST pull-down precipitations and WCL derived from U20S cells treatment with/without 30 ⁇ e
  • FIGs. 19H and 191 IB analysis of anti-PTEN IPs and whole cell lysates (WCL) derived from U20S cells treatment with 30 ⁇ etoposide as indicated time points before harvesting.
  • FIG. 19J IB analysis of anti-PTEN IPs and WCL derived from U20S cell lines stably expressing shScr or shATM transfected with indicated constructs. 36 hours after transfection, cells were treated with/without 30 uM etoposide for 30 min before harvesting.
  • FIG. 19K IB analysis of anti- PTEN IPs and WCL derived from C7 +/+ and MDCJ 'A MEFs treatment with 30 ⁇ etoposide as indicated time points before harvesting.
  • FIGs. 20A-20I show protein phosphatase activity of PTEN is required for efficient complement of DSBs repair largely through dephosphorylating ⁇ 2 ⁇ .
  • FIG. 20B Immunoblot (IB) analysis of whole cell lysates (WCL) derived from PTEN ⁇ /+ and ⁇ ' HCT116 cells after treatment with IR (5 Gy) as indicated time points.
  • FIG. 20B Immunoblot (IB) analysis of whole cell lysates (WCL) derived from PTEN ⁇ /+ and ⁇ ' HCT116 cells after treatment with IR (5 Gy) as indicated time points.
  • FIGs. 20A and 20D Quantification of protein intensity in (FIG. 20A) was performed using the ImageJ software. ⁇ 2 ⁇ immunoblot bands were normalized to Vinculin, then normalized to the control (no IR treatment).
  • FIGs. 20C and 20D IB analysis of WCL derived from PTEK HCTl 16 cells introducing PTEN WT, C124S, G129E, Y138L as well as EV, which were treated with IR (5 Gy) at indicated time points before harvesting (FIG. 20C). Quantification of protein intensity in (FIG. 20C) was performed using the Image J software (FIG. 20D). ⁇ 2 ⁇ immunoblot bands were normalized to Vinculin, then normalized to the control (no IR treatment). FIGs.
  • FIG. 20E and 20F IB analysis of WCL derived from ⁇ ' HCTl 16 cells introducing PTEN WT, K349R as well as EV, which were treated with IR (5 Gy) at indicated time points before harvesting (FIG. 20D). Quantification of protein intensity in (FIG. 20E) was performed using the ImageJ software (FIG. 20F). ⁇ 2 ⁇ immunoblot bands were normalized to Vinculin, then normalized to the control (no IR treatment).
  • FIG. 20G IB analysis of anti -Flag IPs and WCL derived from U20S cells transfected with the indicated constructs. 36 hr after transfection, cells were harvested for IP assays after treatment with IR (5 Gy) for 60 min.
  • FIG. 20G IB analysis of anti -Flag IPs and WCL derived from U20S cells transfected with the indicated constructs. 36 hr after transfection, cells were harvested for IP assays after treatment with IR (5 Gy) for 60 min.
  • FIG. 201 In vitro dephosphorylation assays with bacterially purified recombinant GST-tagged PTEN WT and the indicated PTEN mutants including C124S, G129E, and Y138L incubating with indicated H2AX synthetic peptides, then analyzed by dot immunoblot analyses.
  • FIGs. 21 A-21N show MMSET-mediated methylation of PTEN at K349 dictates cellular sensitivity to DNA-damaging agents.
  • FIGs. 2 IE and 2 IF TEN-deficient U87MG cells reconstituted with the indicated PTEN constructs were pre-treated with 1 ⁇ BKM120 for 24 hours followed by an additional 10 IR treatments (5 Gy).
  • FIG. 21G Immunoblot (IB) analysis of whole cell lysates (WCL) derived from the samples of (FIG. 21E-21F) with indicated antibodies.
  • FIGs. 21H and 211 U20S cells stably expressing shScr or shMMSET were pre-treated with/without 1 ⁇ BKM120 for 24 hours followed by additional IR (10 Gy) treatment as indicated.
  • FIGs. 21J and 21K U20S cells stably expressing shScr or shMMSET were pre-treated with/without 1 ⁇ BKM120 for 24 hours followed by additional IR (0.5 Gy) treatment as indicated. After one week, cells were stained with crystal violet (FIG. 21 J) and the colony number was counted (FIG. 21K).
  • FIGs. 21M U20S cells stably were pre- treated with/without 1 ⁇ BKM120 or 2 ⁇ DZNep for 24 hours followed by additional IR (0.5 Gy) treatment. After one week, cells were stained with crystal violet (FIG.
  • FIG. 22 shows a schematic model to illustrate that PTEN has lipid phosphatase and protein phosphatase activity, which involves in PI3K/Akt signaling pathway and DNA damage/yH2AX pathway, respectively.
  • FIG. 23 shows depletion of MMSET by shRNAs sensitizes cells to IR or etoposide with BKM120 combinational treatment.
  • FIG. 23F and FIG. 23G HCT116 cells stably expressing shScr or shMMSET were pre-treated with/without 1 ⁇ BKM120 for 24 h followed by additional etoposide (20 ⁇ ) treatment as indicated.
  • FIG. 23H and FIG. 231 HCT116 cells stably expressing shScr or shMMSET were pre-treated with/without 1 ⁇ BKM120 for 24 h followed by additional etoposide (20 ⁇ ) treatment as indicated. 24 h after etoposide treatment, cells were replaced with fresh medium. After one week, cells were stained with crystal violet (FIG. 23H) and the colony number was counted (FIG. 231).
  • FIG. 23L and FIG. 23M PTE?
  • FIG. 23N Xenografted tumors in FIG. 12S were dissected after euthanizing the mice and tumors were recorded at the time of sacrifice. Four mice each group.
  • FIG. 24 shows inhibition of MMSET by DZNep (a pan-inhibitor of S- adenosylmethionine-dependent methyltransferase including MMSET) sensitizes cells to IR or etoposide with BKM120 combinational treatment.
  • FIG. 24B and FIG. 24C shows inhibition of MMSET by DZNep (a pan-inhibitor of S- adenosylmethionine-dependent methyltransferase including
  • FIG. 24D Xenografted tumors in FIG. 12V were dissected after euthanizing the mice and tumors were recorded at the time of sacrifice. Four mice each group. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention features compositions and methods of treating cancers by inhibiting the activity of MMSET methyltransferase.
  • the inhibition of MMSET methyltransferase sensitizes cancer cells to chemotherapeutic drugs.
  • This invention is based, at least in part on the discovery of a novel molecular mechanism for PTEN regulation of DSBs repair through its methylation modification event and protein phosphatase activity:
  • DNA DSBs promote ATM-dependent phosphorylation of T398-PTEN, which is specifically recognized by the BRCA1 C
  • MDC1 is pivotal for PTEN and MMSET interaction following DNA DSBs, which subsequently leads to MMSET -mediated di-methylation of K349 on PTEN.
  • the tudor domain of 53BP1 (FIG. IE) recognizes the methylated PTEN and recruits PTEN to DSB sites to govern the timely repair of DSBs in part through dephosphorylation of ⁇ 2 ⁇ .
  • methylation-deficient PTEN mutants e.g., the protein-phosphatase dead mutant C124S or Y138L, or methylation-deficient mutant K349R or T398A
  • MMSET methyltansferase sensitizes cancer cells to chemotherapeutic drugs.
  • PTEN sensitizes cancer cells to chemotherapeutic drugs.
  • the present invention provides a method of treating cancer, the method comprising administering (1) an effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein; and (ii) an effective amount of a chemotherapeutic agent to a subject having a cancer.
  • MMSET multiple myeloma SET domain
  • the agent that inhibits the expression or activity of MMSET protein sensitizes the cancer to the chemotherapeutic agent.
  • the method reduces tumor growth, and/or increases subject survival.
  • the method inhibits the growth of cancer cells.
  • the agent that inhibits the expression or activity of MMSET protein reduces the effective amount of the chemotherapeutic agent necessary to treat the cancer.
  • the cancer is prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, or other types of epithelium-derived carcinomas where PTEN inactivation is frequently observed.
  • DNA damaging agents result in the death of cancer cells by reducing the integrity of the cancer cell's DNA.
  • DNA integrity is critical for proper cellular function and proliferation. High levels of DNA damage are detected by cell-cycle checkpoint proteins, whose activation induces cell-cycle arrest to prevent the transmission of damaged DNA during mitosis.
  • DNA lesions that occur during the S phase of the cell cycle block replication fork progression and can lead to replication-associated DNA double-strand breaks (DSBs), which are among the most toxic of all DNA lesions. If the damaged DNA cannot be properly repaired, cell death may result.
  • DSBs replication-associated DNA double-strand breaks
  • both large-scale proteomic analyses and in vitro specific biochemical kinase assays identified PTEN to be phosphorylated at T398 in human PTEN (S398 in mouse PTEN, thereafter referred as T/S398) by ataxia telangiectasia mutated (ATM), in response to DNA damage.
  • ATM ataxia telangiectasia mutated
  • phosphorylation of PTEN at endogenous levels could be readily detected using the phospho-(Ser/Thr) ATM/ATR substrate antibody after etoposide or irradiation (TR) treatment in NIH3T3 (FIGs. 1 A-1B) and mouse embryonic fibroblasts (MEFs) (FIGs. 2A-2B).
  • DNA DSBs promote the interaction of PTEN with MDC1, following ATM-dependent phosphorylation of T/S398- PTEN. Additionally, DNADSBs enhance MMSET-mediated di-methylation of K349-PTEN, which is recognized by the tudor domain of 53BP1, thereby enhancing the recruitment of PTEN to DSB sites, and governing the repair process and cellular sensitivity to DNA damage in part by dephosphorylating ⁇ - ⁇ 2 ⁇ .
  • DNA damaging agents induce MMSET-mediated di- methylation of PTEN at lysine 349.
  • the di-methylation of PTEN at lysine 349 promotes the integration of PTEN with 53BP1, to facilitate the recruitment of PTEN to sites of DNA damage.
  • the MMSET-mediated di-methylation of PTEN at lysine 349 promotes the repair of damaged DNA, thereby reducing the efficacy of chemotherapeutic agents.
  • the agent that inhibits the expression or activity of MMSET protein is a polypeptide, polynucleotide, or a small molecule.
  • the agent that inhibits the expression or activity of MMSET protein is an inhibitory nucleic acid molecule that inhibits the expression of a MMSET protein.
  • the inhibitory nucleic acid molecule is an antisense molecule, siRNA, or shRNA.
  • Inhibitory nucleic acid molecules are those oligonucleotides that inhibit the expression or activity of a MMSET polypeptide.
  • Such oligonucleotides include single and double stranded nucleic acid molecules (e.g., DNA, RNA, and analogs thereof) that bind a nucleic acid molecule that encodes a MMSET polypeptide (e.g., antisense molecules, siRNA, shRNA), as well as nucleic acid molecules that bind directly to the polypeptide to modulate its biological activity (e.g., aptamers).
  • RNAs Short twenty-one to twenty-five nucleotide double-stranded RNAs are effective at down-regulating gene expression (Zamore et al., Cell 101 : 25-33; Elbashir et al., Nature 411 : 494-498, 2001, hereby incorporated by reference).
  • the therapeutic effectiveness of a siRNA approach in mammals was demonstrated in vivo by McCaffrey et al. (Nature 418: 38- 39.2002).
  • siRNAs may be designed to inactivate that gene.
  • siRNAs could be administered directly to an affected tissue, or administered systemically.
  • the nucleic acid sequence of a gene can be used to design small interfering RNAs (siRNAs).
  • siRNAs small interfering RNAs
  • the 21 to 25 nucleotide siRNAs may be used, for example, as therapeutics to treat cancer.
  • RNAi RNA interference
  • expression of MMSET polypeptide is reduced in a subject having cancer.
  • RNAi is a method for decreasing the cellular expression of specific proteins of interest (reviewed in Tuschl, Chembiochem 2:239-245, 2001; Sharp, Genes & Devel. 15:485- 490, 2000; Hutvagner and Zamore, Curr. Opin. Genet. Devel. 12:225-232, 2002; and
  • siRNAs introduction of siRNAs into cells either by transfection of dsRNAs or through expression of siRNAs using a plasmid-based expression system is increasingly being used to create loss-of-function phenotypes in mammalian cells.
  • a double-stranded RNA (dsRNA) molecule is made that includes between eight and nineteen consecutive nucleobases of a nucleobase oligomer of the invention.
  • the dsRNA can be two distinct strands of RNA that have duplexed, or a single RNA strand that has self-duplexed (small hairpin (sh)RNA).
  • small hairpin (sh)RNA small hairpin
  • dsRNAs are about 21 or 22 base pairs, but may be shorter or longer (up to about 29 nucleobases) if desired.
  • dsRNA can be made using standard techniques (e.g., chemical synthesis or in vitro transcription).
  • Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296:550-553, 2002; Paddison et al. Genes & Devel. 16:948-958, 2002. Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99:6047- 6052, 2002; Miyagishi et al. Nature Biotechnol. 20:497-500, 2002; and Lee et al. Nature Biotechnol. 20:500-505 2002, each of which is hereby incorporated by reference.
  • Small hairpin RNAs comprise an RNA sequence having a stem-loop structure.
  • a "stem-loop structure” refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand or duplex (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion).
  • the term “hairpin” is also used herein to refer to stem-loop structures. Such structures are well known in the art and the term is used consistently with its known meaning in the art.
  • the secondary structure does not require exact base-pairing.
  • the stem can include one or more base mismatches or bulges.
  • the base-pairing can be exact, i.e. not include any mismatches.
  • the multiple stem-loop structures can be linked to one another through a linker, such as, for example, a nucleic acid linker, a miRNA flanking sequence, other molecule, or some combination thereof.
  • small hairpin RNA includes a conventional stem-loop shRNA, which forms a precursor miRNA (pre-miRNA). While there may be some variation in range, a conventional stem-loop shRNA can comprise a stem ranging from 19 to 29 bp, and a loop ranging from 4 to 30 bp. "shRNA” also includes micro-RNA embedded shRNAs (miRNA-based shRNAs), wherein the guide strand and the passenger strand of the miRNA duplex are incorporated into an existing (or natural) miRNA or into a modified or synthetic (designed) miRNA. In some instances the precursor miRNA molecule can include more than one stem-loop structure.
  • MicroRNAs are endogenously encoded RNA molecules that are about 22-nucleotides long and generally expressed in a highly tissue- or developmental - stage-specific fashion and that post-transcriptionally regulate target genes. More than 200 distinct miRNAs have been identified in plants and animals. These small regulatory RNAs are believed to serve important biological functions by two prevailing modes of action: (1) by repressing the translation of target mRNAs, and (2) through RNA interference (RNAi), that is, cleavage and degradation of mRNAs. In the latter case, miRNAs function
  • siRNAs small interfering RNAs
  • shRNAs can be expressed from DNA vectors to provide sustained silencing and high yield delivery into almost any cell type.
  • the vector is a viral vector.
  • Exemplary viral vectors include retroviral, including lentiviral, adenoviral, baculoviral and avian viral vectors, and including such vectors allowing for stable, single-copy genomic integrations.
  • Retroviruses from which the retroviral plasmid vectors can be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • a retroviral plasmid vector can be employed to transduce packaging cell lines to form producer cell lines.
  • packaging cells which can be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14x, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy 1 :5-14 (1990), which is incorporated herein by reference in its entirety.
  • the vector can transduce the packaging cells through any means known in the art.
  • a producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a DNA replication protein. Such retroviral vector particles then can be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a DNA replication protein.
  • Catalytic RNA molecules or ribozymes that include an antisense sequence of the present invention can be used to inhibit expression of a nucleic acid molecule in vivo (e.g., a nucleic acid encoding MMSET).
  • a nucleic acid molecule in vivo e.g., a nucleic acid encoding MMSET.
  • the inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs.
  • the design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334:585-591. 1988, and U.S. Patent Application Publication No. 2003/0003469 Al, each of which is incorporated by reference.
  • the invention also features a catalytic RNA molecule that includes, in the binding arm, an antisense RNA having between eight and nineteen consecutive nucleobases.
  • the catalytic nucleic acid molecule is formed in a hammerhead or hairpin motif. Examples of such hammerhead motifs are described by Rossi et al., Aids Research and Human Retroviruses, 8: 183, 1992. Example of hairpin motifs are described by Hampel et al., "RNA Catalyst for Cleaving Specific RNA
  • any method for introducing a nucleic acid construct into cells can be employed.
  • Physical methods of introducing nucleic acids include injection of a solution containing the construct, bombardment by particles covered by the construct, soaking a cell, tissue sample or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the construct.
  • a viral construct packaged into a viral particle can be used to accomplish both efficient introduction of an expression construct into the cell and transcription of the encoded shRNA.
  • Other methods known in the art for introducing nucleic acids to cells can be used, such as lipid-mediated carrier transport, chemical mediated transport, such as calcium phosphate, and the like.
  • shRNA-encoding nucleic acid construct can be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, promote annealing of the duplex strands, stabilize the annealed strands, or otherwise increase inhibition of the target gene.
  • DNA vectors for example plasmid vectors comprising either an RNA polymerase II or RNA polymerase III promoter can be employed.
  • Expression of endogenous miRNAs is controlled by RNA polymerase II (Pol II) promoters and in some cases, shRNAs are most efficiently driven by Pol II promoters, as compared to RNA polymerase III promoters (Dickins et al., 2005, Nat. Genet. 39: 914-921).
  • expression of the shRNA can be controlled by an inducible promoter or a conditional expression system, including, without limitation, RNA polymerase type II promoters.
  • promoters in the context of the invention are tetracycline- inducible promoters (including TRE-tight), IPTG-inducible promoters, tetracycline transactivator systems, and reverse tetracycline transactivator (rtTA) systems.
  • Constitutive promoters can also be used, as can cell- or tissue-specific promoters. Many promoters will be ubiquitous, such that they are expressed in all cell and tissue types.
  • a certain embodiment uses tetracycline-responsive promoters, one of the most effective conditional gene expression systems in in vitro and in vivo studies. See International Patent Application
  • Naked polynucleotides, or analogs thereof, are capable of entering mammalian cells and inhibiting expression of a gene of interest. Nonetheless, it may be desirable to utilize a formulation that aids in the delivery of oligonucleotides or other nucleobase oligomers to cells (see, e.g., U.S. Pat. Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference).
  • an inhibitory nucleic acid molecule described herein is delivered using a nanoparticle.
  • Nanoparticle compositions suitable for use with inhibitory nucleic acid molecules are known in the art and described for example by Kanasty et al., Nature materials 12: 967-977, 2013, which is incorporated herein by reference.
  • Such nanoparticle delivery compositions include cyclodextrin polymer (CDP)-based nanoparticles, lipid nanoparticles, cationic or ionizable lipid, lipid-anchored PEG, PEGylated nanoparticles, oligonucleotide nanoparticles (ONPs), and siRNA-polymer conjugate delivery systems (e.g., Dynamic Poly Conjugate, Triantennary GalNAc-siRNA).
  • CDP cyclodextrin polymer
  • lipid nanoparticles cationic or ionizable lipid
  • lipid-anchored PEG lipid-anchored PEG
  • PEGylated nanoparticles oligonucleotide nanoparticles
  • siRNA-polymer conjugate delivery systems e.g., Dynamic Poly Conjugate, Triantennary GalNAc-siRNA.
  • Examples of compounds suitable as MMSET methyltransferase inhibitors include the pan-histone methyltransferase inhibitor (l S,2R,5R)-5-(4-Amino-lH-imidazo[4,5-c]pyridin-l- yl)-3-(hydroxymethyl)-3-cyclopentene-l,2-diol hydrochloride (3-Deazaneplanocin A hydrochloride).
  • MMSET methyltransferase inhibitor is the inhibitor 3-hydrazinylquinoxaline-2-thiol, disclosed in U.S. Patent No. 8,697,407, incorporated by reference in its entirety.
  • LEM-06 Another example of a compound suitable as a MMSET methyltransferase inhibitor is the inhibitor LEM-06, disclosed in Journal of Cancer Prevention Vol. 20, No. 2, 2015. pp. 113-120, incorporated by reference in its entirety.
  • LEM-06 has the structure shown in the formula below:
  • the agent that inhibits the expression or activity of MMSET protein inhibits the activity of ataxia telangiectasia mutated (ATM).
  • the agent that inhibits the expression or activity of MMSET protein is the specific ATM kinase inhibitor Ku55933.
  • the agent that inhibits the expression or activity of MMSET protein is the specific ATM kinase inhibitor AZD0156, which is currently under clinical trial to treat advanced solid tumors.
  • AZD0156 has the structure shown in the formula below:
  • Chemotherapeutic agents suitable for use in the methods of the present invention include, but are not limited to alkylating agents. Without intending to be limited to any particular theory, alkylating agents directly damage DNA to keep the cell from reproducing. Alkylating agents work in all phases of the cell cycle and are used to treat many different cancers, including leukemia, lymphoma, Hodgkin disease, multiple myeloma, and sarcoma, as well as cancers of the lung, breast, and ovary.
  • Alkylating agents are divided into different classes, including, but not limited to: (i) nitrogen mustards, such as, for example mechlorethamine (nitrogen mustard), chlorambucil, cyclophosphamide (Cytoxan®), ifosfamide, and melphalan; (ii) nitrosoureas, such as, for example, streptozocin, carmustine (BCNU), and lomustine; (iii) alkyl sulfonates, such as, for example, busulfan; (iv) riazines, such as, for example, dacarbazine (DTIC) and temozolomide (Temodar®); (v) ethylenimines, such as, for example, thiotepa and altretamine
  • nitrogen mustards such as, for example mechlorethamine (nitrogen mustard), chlorambucil, cyclophosphamide (Cytoxan®), ifosf
  • compositions provided herein can be used to treat or prevent progression of a cancer.
  • the effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein; and (ii) the effective amount of a chemotherapeutic agent to a subject having a cancer can be
  • the effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein can be administered concurrently with the effective amount of a chemotherapeutic agent.
  • the effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein can be administered before the effective amount of a chemotherapeutic agent.
  • the effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein can be administered after the effective amount of a
  • Treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk of developing such cancer. Determination of those subjects "at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, family history, and the like). Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
  • a diagnostic test or opinion of a subject or health care provider e.g., genetic test, enzyme or protein marker, family history, and the like. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
  • the effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein; and the effective amount of a chemotherapeutic agent may be administered in combination with one or more of any other standard anti-cancer therapies.
  • an MMSET inhibitor as described herein may be administered in combination with standard chemotherapeutics.
  • the present invention features compositions useful for treating cancer.
  • the methods include administering an effective amount of (i) an effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein; and (ii) an effective amount of a chemotherapeutic agent to a subject having a cancer in a physiologically acceptable carrier.
  • MMSET multiple myeloma SET domain
  • the carrier or excipient for the composition provided herein is a
  • a pharmaceutically acceptable carrier or excipient such as sterile water, aqueous saline solution, aqueous buffered saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, ethanol, or combinations thereof.
  • a carrier or excipient is selected to minimize allergic and other undesirable effects, and to suit the particular route of administration, e.g., subcutaneous, intramuscular, intranasal, and the like.
  • the administration may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing the disease symptoms in a subject.
  • the composition may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline.
  • Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, intrathecal, or intradermal injections that provide continuous, sustained levels of the agent in the patient.
  • the amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of the cancer.
  • compositions are administered at a dosage that ameliorates or decreases effects of the cancer as determined by a method known to one skilled in the art.
  • the therapeutic or prophylactic composition may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition.
  • the composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, intrathecally, or intraperitoneally) administration route.
  • parenteral e.g., subcutaneously, intravenously, intramuscularly, intrathecally, or intraperitoneally
  • the pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
  • compositions according to the invention may be formulated to release the active agent substantially immediately upon administration or at any predetermined time or time period after administration.
  • controlled release formulations which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release
  • composition adjacent to or in contact with an organ, such as the heart comprising (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target a disease using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type.
  • controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.
  • controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings.
  • the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.
  • compositions may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants.
  • suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants.
  • Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.
  • Compositions for parenteral use may be provided in unit dosage forms (e.g., in single- dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below).
  • the composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use.
  • the composition may include suitable parenterally acceptable carriers and/or excipients.
  • the active therapeutic agent(s) e.g., an MMSET inhibitor described herein
  • the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.
  • the composition comprising the active therapeutic is formulated for intravenous delivery.
  • the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection.
  • the suitable therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle.
  • acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution.
  • the aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate).
  • preservatives e.g., methyl, ethyl or n-propyl p-hydroxybenzoate.
  • a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.
  • kits for the treatment or prevention of cancer includes a therapeutic or prophylactic composition containing (i) an effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein; and (ii) an effective amount of a chemotherapeutic agent in unit dosage form.
  • the kit includes (i) an effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein; and (ii) an effective amount of a chemotherapeutic agent in unit dosage form in a sterile container.
  • Such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • a pharmaceutical composition of the invention is provided together with instructions for administering the pharmaceutical composition to a subject having or at risk of contracting or developing cancer.
  • the instructions will generally include information about the use of the composition for the treatment or prevention of cancer.
  • the instructions include at least one of the following: description of the
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • Example 1- DNA Damage Induced Endogenous Phosphorylated PTEN in Cell Lines.
  • Phosphorylation of PTEN at endogenous levels could be readily detected using the phospho-(Ser/Thr) ATM/ATR substrate antibody upon etoposide or irradiation (IR) treatment in Nffl3T3 (FIGs. 1A-1B) and mouse embryonic fibroblasts (MEFs) (FIGs. 2A-2B).
  • Example 2- DNA Damage Promoted the Specific Interaction Between PTEN and the BRCT domain of MDC2.
  • the studies in this example were intended to examine whether ATM-mediated phosphorylation of PTEN involves the DNA repair pathway through interaction with key DDR regulators with pS/pTQ-binding domains, such as MDCl, 53BP1, and BRCA1 (FIG. 2K).
  • pS/pTQ-binding domains such as MDCl, 53BP1, and BRCA1
  • the studies in this example suggest that etoposide treatment enhanced both of hPTEN and mPTEN binding with the BRCT domain of MDCl, but not its FHA domain, nor the BRCT domains derived from 53BP1 or BRCA1 (FIGs. 1F-G and FIG. 2D, and FIG. 2F).
  • methyltransferase that primarily promotes K349 di-methylation of PTEN in cells in response to DNA damaging signals.
  • the interaction between 53BP1 and PTEN is largely mediated through the tudor domain of 53BP1 (FIG. 4H).
  • etoposide treatment only enhanced PTEN interaction with the tudor domain derived from 53BP1, but neither SETDB 1 or KDM4 (FIG. 8C).
  • the methyltransferase MMSET could promote the interaction between WT-PTEN and the tudor domain of 53BP1, indicating the involvement of methyl- K349-PTEN and its subsequent recognition by the 53BP1 -tudor domain (FIG. 41).
  • the PTEN K349R mutant but not K254R, K266R, K289R, K332R, and K402R mutants, was deficient in MMSET-induced (FIG. 41), or etoposide-triggered (FIG. 4J), interaction with the 53BP1 -tudor domain.
  • MMSET-induced FIG. 41
  • etoposide-triggered FIG. 4J
  • interaction with the 53BP1 -tudor domain was deficient in MMSET-induced (FIG. 41), or etoposide-triggered (FIG. 4J) interaction with the 53BP1 -tudor domain.
  • methyl-K349-PTEN using an in vitro pull-down assay with synthetic PTEN peptides with unmodified K349 (K349-me0), mono-methylated (K349-mel), di-methylated (K349-me2) and tri-methylated (K349-me3) (FIG.
  • 53BP1 Upon DNA damage, 53BP1 can be recruited into DNA damage sites through different molecular mechanisms, such as tudor domain-mediated recognition of di-methylated K20 in H4, or through the ubiquitination dependent recruitment (UDR) motif of 53BP1 by recognizing ubiquitinated K15 in H2A, or BRCT domain-mediated interaction with ⁇ 2 ⁇ . On the other hand, 53BP1 also recruits the methylated tumor suppressor proteins including p53 and pRb into DNA damage sites through its tudor domain.
  • UDR ubiquitination dependent recruitment
  • Pten mice displayed high levels of ⁇ 2 ⁇ at 24 h post ⁇ -irradiation in relative to Pten +/+ and Pten G129E/+ mice (FIG. 9M, and FIG. 9N). These results suggested that the protein phosphatase activity, but not lipid phosphatase activity, of PTEN plays a crucial role in regulation of ⁇ - ⁇ 2 ⁇ status in response to DNA damage.
  • PTEN-WT and various mutants including C124S, G129E, Y138L, K349R, T398A as well as the empty vector control were retrovirrally introduced at comparable levels into PTEN- deficient U87MG cells. Consistent with results derived from ⁇ ' HCT116 cells (FIGs. 8A-8B), ectopic expression of lipid phosphatase dead mutants C124S and G129E, but not Y138L, K349R, S398A mutants, failed to reduce pSer437-Akt (FIG. 11 A).
  • the DNA damage response profiles of these cell lines were monitored by immuno-staining against DSBs markers 53BP1 and ⁇ - ⁇ 2 ⁇ at indicated time points post ⁇ -irradiation. Notably, 1 hour post-irradiation, the foci of 53BP1 could be observed in all cell lines (FIG. 12A and FIG. 1 IB), a faithful marker indicative of double strand breaks generated by IR.
  • MMSET deficiency also minimally affects DNA damage foci formation at the early stage (4 hour) of post irradiation and mainly inhibits the foci resolve at the late stage (24 hour) of post irradiation to govern DNA damage repair.
  • combination treatment with etoposide and BKM120 displayed a greater inhibition of xenografted tumor growth bearing PTEN-/- HCTl 16 cells stably expressing the methylation-deficient mutant K349R, accompanied with elevated ⁇ 2 ⁇ and cleaved caspase 3 levels in tumors with the PTEN- K349R status (FIGs. 120-12P).
  • MMSET is a potential therapeutic target in cancer
  • developing MMSET specific inhibitor(s) and combining with DNA-damaging agents and PI3K inhibitors might be useful in treating cancers with AiMSEJoverexpressi on (FIG. 17A-FIG. 17D; FIG. 18).
  • Such inhibitors could efficiently sensitize cancer cells with wild-type PTEN genetic status to chemo- or radio-therapeutics.
  • these studies uncover a critical role of PTEN methylation and its protein phosphatase activity in regulating DSBs repair and sensitivity to DNA damaging-agents, including both chemo- and radio-therapeutics, in part by governing DSB repair process via dephosphorylating ⁇ 2 ⁇ .
  • These studies disclosed herein further extend knowledge about the precise molecular mechanism of how PTEN involve in DNA damage pathway.
  • U87MG and HCT116 PTEN" + and PTEN “1' cells were cultured in DMEM medium supplemented with 10% FBS, 100 units of penicillin and 100 mg/ml streptomycin.
  • Mouse embryonic fibroblasts (MEFs) MDC1 +I+ and MDCF 1' cells were maintained in DMEM medium supplemented with 10% FBS.
  • Cell transfection was performed using lipofectamine and plus reagents according to standard protocols. Packaging of lentiviral and retroviral cDNA expressing viruses, as well as subsequent infection of various cell lines were performed according to standard protocols. Following viral infection, cells were selected in the presence of hygromycin (200 ⁇ g/mL) or puromycin (1 ⁇ g/mL) for 3 days. Etoposide was purchased from Sigma (E1383). KU-55933 (ATM Kinase Inhibitor, S1092), VE-821 (S8007), NU7026 (S2893), BKM120 (S2247), 3-deazaneplanocin A
  • Plasmid Construction and MMSET shRNAs HA-hPTEN and HA-mPTEN were generated by inserting the corresponding cDNAs into pcDNA3-HA vector.
  • Flag-MMSET, Flag-EZH2 and Flag-Set8 were constructed by cloning corresponding cDNAs into pFlag- CMV vector.
  • pCMV-GST-MDCl-BRCT, pCMV-GST-53BPl-BRCT, pCMV-GST-BRCAl- BRCT and pCMV-GST-53BPl tudor domain were cloned into mammalian expression GST- fusion vectors.
  • GST-PTEN was constructed by inserting the cDNA into pGEX-4T-l vector.
  • pLenti-HA-PTEN and pBabe-Super-HA-PTEN were constructed by subcloning the PTEN cDNA into pLenti-HA-puro and pBabe-Super-HA-hygro vector, respectively.
  • Various human PTEN mutants (hPTEN-T398A, K254R, K266R, K289R, K332R, K349R, C124S, G129E, Y138L) were generated using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. All mutants were generated using mutagenesis PCR and the sequences were verified by DNA sequencing.
  • MMSET shRNA vectors to deplete endogenous MMSET were generously offered by Dr. Zhenkun Luo in the Department of Oncology, Mayo Clinic and previously described (34).
  • sequences for MMSET shRNA were listed below:
  • MMSET shRNA 1 5 ' -GC ACGCTAC AAC ACC AAGTTT (SEQ ID NO: 7);
  • MMSET shRNA 2 5 ' -GC AC AGTCTTCGGAAGAGAGAC AC AATC A (SEQ ID NO: 1
  • Antibodies All antibodies were used at a 1 : 1000 dilution in TBST buffer with 5% non-fat milk for western blot.
  • Ant-phospho-ATM/ATR Substrate (S*Q) (9607), anti-pS15- p53 (9284), anti-pT68-Chk2 (2661), anti-Chk2 antibody (3440), anti -Mono-Methyl Lysine Motif (14679), anti-Di-Methyl Lysine Motif (14117), anti-Tri -Methyl Lysine Motif (14680), anti-His tag (2366), anti-yH2AX (9718), anti-pS1618-53BPl (6209), ant-pT543-53BPl (3428), anti-pS25/29-53BPl (2647), anti-pS428-ATR (2853), anti-pS1542-BRCAl (9009), anti-pS296-Chkl (23
  • Anti-PTEN (sc-7974), anti-MDCl (sc-27737), anti-p53 (sc-6243/126), anti-ATM (sc-23921), anti-ATR (sc-1887), anti-BRCAl (sc-6954/641), anti-Chkl (sc-8408), anti-HA (sc-805) and anti-GST (sc-459) were obtained from Santa Cruz.
  • Anti-Flag F-2425
  • anti-Flag F-3165, clone M2
  • anti-Tubulin antibody T-5168
  • anti-Vinculin V9131
  • anti-Flag agarose beads A-2220
  • anti-HA agarose beads A-2095
  • peroxidase-conjugated anti -mouse secondary antibody A-4416
  • peroxidase- conjugated anti-rabbit secondary antibody A-4914
  • the polyclonal anti-PTEN-K349-me2 and anti-PTEN-K349-me3 antibodies generated by Cell Signaling Technology were derived from rabbit, with each antibody produced three clones.
  • the antigen peptide sequence comes from 10 amino acids surrounding the
  • the antibodies were affinity purified using the antigen peptide column, but they were not counter selected on unmodified antigen.
  • the reactions were incubated at 37 °C for 30 min with or without the addition of recombinant GST-fusion PTEN WT, C124S, G129E and Y138L, as well as GST protein as negative control, and were stopped by adding 3 x SDS loading buffer for western blot.
  • Peptide Synthesis The PTEN peptides with/without methylation modification were synthesized at Tufts Medical School. Each contained an N-terminal biotin and free C- terminus and was synthesized in 0.1 mM scale. Peptides were diluted into lmg/ml for further biochemical assays. The sequences were listed below:
  • Biotin-NFKVKLYFTK(mel)TVEEPSNPE (SEQ ID NO: 10)
  • Biotin-NFKVKLYFTK(me2)TVEEPSNPE SEQ ID NO: 1
  • Biotin-NFKVKLYFTK(me3)TVEEPSNPE SEQ ID NO: 12
  • Dot Immunoblot Assays Peptides were spotted onto nitrocellulose membrane allowing solution to penetrates (usually 3-4 mm diameter) by applying it slowly as a volume 1 [iL once. The membrane was dried, and blocked in TBST buffer with 5% non-fat milk for immunoblot analysis with indicated antibodies according to standard protocols.
  • Peptide-binding assays Peptides (2 ⁇ g) were incubated with 1 mg of whole cell lysates in a total volume of 500 ⁇ ⁇ EBC buffer. After incubation for 4 hr at 4°C, 10 ⁇ ⁇ Streptavidin agarose (Thermo Scientific 20353) was added in the sample for another 1 hr. The agarose was washed four times with NETN buffer. Bound proteins were added in 2 x SDS loading buffer and resolved by SDS-PAGE for immunoblot analysis.
  • IP anti-HA-PTEN immunoprecipitations
  • LC-MS/MS microcapillary reversed-phase liquid chromatography -tandem mass spectrometry
  • MS/MS data were searched against the human protein database using Mascot (Matrix Science) and data analysis was performed using the Scaffold 4 software (Proteome Software). Peptides and modified peptides were accepted if they passed a 1% FDR threshold.
  • Immunofluorescence assays Cells cultured on glass cover slips were fixed with 4% formaldehyde in PBS for 15 minutes at room temperature. The cells were permeabilized with 0.1% Triton X-100 in PBS for 5 minutes on ice. After washing 3 times in PBS, the samples were blocked for 30 minutes with 5% control goat serum. The samples were incubated with primary antibodies for 2 hours at room temperature. After rinsed 3 times using PBST containing 0.1% Tween-20, the coverslips incubated with Alexa-594-conjugated goat anti- mouse secondary antibody (Invitrogen) for 1 hour and washed 3 times with PBST, the nucleus was stained with 4, 6-diamidino-2-phenylindole (DAPI) for 10 minutes. Coverslips were rinsed 2 times with PBS and mounted onto slides.
  • Alexa-594-conjugated goat anti- mouse secondary antibody Invitrogen
  • HCT1 16- ⁇ cells stably expressing HA-PTEN were cultured for 48 hr prior to irradiation with 10 ⁇ BrdU4 (Sigma- Aldrich cat. #B9285).
  • UVA laser (50 mW) irradiation was conducted using an inverted microscope (Eclipse Ti; Nikon) with a Palm microbeam laser microdissection workstation. Following irradiation, cells were incubated at 37 °C for 5 minutes, washed once with cold PBS, and then fixed with PBS containing 4% paraformaldehyde (PFA) for 10 min at room temperature.
  • PFA paraformaldehyde
  • mice per genotype were randomly chosen and analyzed at the indicated age.
  • Immunohistochemistry (IHC) assays Individual tumors derived from nude mice were dissected and fixed in 4% paraformaldehyde for IHC analysis. For staining, tissues were fixed in 4% paraformaldehyde overnight, paraffin embedded, and then sectioned at 5 ⁇ . After deparaffinization and rehydration, antigen retrieval was performed in a pressure cooker with sodium citrate buffer at 95°C for 25 minutes. Sections were incubated in a 0.3% H2O2 solution in lx PBS, and then a 10% serum solution in lx PBS for 30 minutes each solution was used to block endogenous peroxidase and background from the secondary antibody, respectively.
  • the sections were stained with the ⁇ 2 ⁇ (Cell Signaling #9718, 1 :500) in lx PBS at 4°C overnight, and incubated in a biotinylated anti-rabbit secondary antibody in lx PBS (1 : 1000) at room temperature for 30 minutes.
  • the Vectastain ABC Elite kit was used to enhance specific staining, and the staining was visualized using a 3 ' -diaminobenzidine (DAB) substrate.
  • Stained sections were counterstained using hematoxylin and dehydrated before they were sealed with a coverslip with Richard-Allan Scientific® CytosealTM XYL Mounting Medium. Stained slides were visualized by a bright-field microscope.
  • mice were left to recover at the incubator for the indicated times.
  • mice were carefully monitored during the recovery period.
  • NVP-BKM120 MedChem Express
  • Etoposide Selleckchem
  • Cell viability and apoptosis assays 2000 cells per well were plated in 96-well plates, and incubated with complete DMEM medium containing different treatments as indicated. Assays were performed with the Cell Titer-Glo
  • Colony formation assays Cells were seeded in 6-well plates (3000 cells/well) and pre-treated with/without BKM120 ( ⁇ ) for 24 hr hours followed by additional IR (0.5 Gy) or etoposide treatment as indicated for 24 hours. Cells were incubated for 6-10 days until formation of visible colonies. Colonies were fixed with 10% acetic acid/10% methanol for 20 min and stained with 0.4% crystal violet/20%) ethanol for 20 min. After staining, the plates were washed with distilled water and air-dried. The colony number was counted.

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Abstract

In some embodiments, the present invention provides a method of treating cancer, method comprising administering (i) an effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein; and (ii) an effective amount of a chemotherapeutic agent to a subject having a cancer.

Description

COMPOSITIONS AND METHODS FOR TREATING CANCER
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of and priority to U.S. Provisional Patent
Application serial number 62/381,346 filed August 30, 2016, which is incorporated herein by reference in its entirety.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY
SPONSORED RESEARCH
This invention was made with government support under Grant No. CA177910 and GM094777 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND OF THE INVENTION
Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a lipid and protein dual phosphatase, and is a tumor suppressor. PTEN is frequently mutated, deleted, or epigenetically silenced in various types of human cancers. In the cytoplasm, PTEN primarily governs key cellular processes including cell survival, proliferation, aging, angiogenesis and metabolism through its lipid phosphatase activity to antagonize the PI3K-Akt oncogenic pathway. However, compared to its well-studied lipid phosphatase activity, the protein phosphatase function of PTEN remains largely undefined. To this end, increasing evidence argues that PTEN also possesses multiple important functions in the nucleus independent of its lipid phosphatase activity, such as controlling the chromosomal integrity, chromatin structure, DNA replication and DNA damage repair.
In support of a role for PTEN in DNA double-stand breaks (DSBs) repair, both large- scale proteomic analyses and in vitro specific biochemical kinase assays identified PTEN to be phosphorylated at T398 in human PTEN (S398 in mouse PTEN, hereafter referred as T/S398) by ataxia telangiectasia mutated (ATM), in response to DNA damage. However, the precise molecular mechanism of ATM-dependent phosphorylation T/S398 of PTEN in regulating its role in DNA damage repair remains largely elusive. SUMMARY OF THE INVENTION
In one aspect, the present invention provides a method of sensitizing a neoplastic cell to chemotherapy, the method comprising contacting the cell with an agent that inhibits multiple myeloma SET domain protein (MMSET) activity or expression and a
chemotherapeutic agent, thereby sensitizing the cell to chemotherapy.
In another aspect, the present invention provides a method of sensitizing a neoplastic cell to radiation, the method comprising contacting the cell with an agent that inhibits MMSET activity or expression and exposing the cell to radiation, thereby sensitizing the cell to γ-irradiation.
In another aspect, the present invention provides a method of enhancing cell death or reducing proliferation in a neoplastic cell, the method comprising contacting the cell with an agent that inhibits MMSET activity or expression and a chemotherapeutic agent, thereby enhancing cell death or reducing proliferation in the cell.
In another aspect, the present invention provides a method of enhancing cell death or reducing proliferation in a neoplastic cell, the method comprising contacting the cell with an agent that inhibits MMSET activity or expression and exposing the cell to radiation, thereby enhancing cell death or reducing proliferation in the cell.
In another aspect, the present invention provides a method of enhancing
chemotherapy sensitivity in a subject having a neoplasia, the method comprising
administering to the subject an agent that inhibits MMSET activity or expression or and a chemotherapeutic agent, thereby enhancing chemotherapy sensitivity in the subject.
In another aspect, the present invention provides a method of enhancing radiation sensitivity in a subject having a neoplasia, the method comprising administering to the subject radiation and an agent that inhibits MMSET activity or expression, thereby enhancing radiation sensitivity in the subject.
In another aspect, the present invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein; and a chemotherapeutic agent, thereby treating cancer in the subject.
In various embodiments of any of the above aspects or any other aspect of the invention delineated herein, the agent that inhibits MMSET activity is a polypeptide, polynucleotide, or small molecule. In other embodiments, the agent that inhibits MMSET activity is selected from the group consisting of: (l S,2R,5R)-5-(4-Amino-lH-imidazo[4,5- c]pyridin-l-yl)-3-(hydroxymethyl)-3-cyclopentene-l,2-diol hydrochloride, 3- hydrazinylquinoxaline-2-thiol, and LEM-06. In still other embodiments, the polynucleotide is an inhibitory nucleic acid molecule that inhibits the expression of MMSET. In still other embodiments, the inhibitory nucleic acid molecule is an antisense molecule, siRNA, or shRNA. In still other embodiments, the shRNA comprises or consists essentially of one of the following sequences: MMSET shRNA 1 : 5 ' -GC ACGCTAC AAC ACC AAGTTT;
MMSET shRNA 2: 5 ' -GC AC AGTCTTCGGAAGAGAGAC AC AATC A. In still other embodiments, the chemotherapeutic agent is selected from the group consisting of:
doxorubicin and etoposide. In still other embodiments, the chemotherapeutic agent is a PI3 kinase inhibitor. In still other embodiments, the PI3 kinase inhibitor is BKM120, BYL719 or RP6530. In still other embodiments, the neoplastic cell is a mammalian cell. In still other embodiments, the mammalian cell is a murine, rat, or human cell. In still other embodiments, the cell is in vitro or in vivo. In still other embodiments, the neoplastic cell or cancer comprises a mutation in PTEN or amplification of MMSET. In still other embodiments, the method reduces neoplastic cell survival or proliferation. In still other embodiments, the neoplastic cell is derived from prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, or other types of epithelium derived carcinomas where loss of function of PTEN is frequently observed. In still other embodiments, the subject has prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, or other types of epithelium derived carcinomas where loss of function of PTEN is frequently observed. In still other embodiments, the method reduces tumor growth, and/or increases subject survival. In still other embodiments, the agent that inhibits the expression or activity of MMSET reduces the effective amount of the chemotherapeutic agent necessary to treat the cancer. In still other embodiments, the cancer is prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, or other types of epithelium derived carcinomas where loss of function of PTEN is frequently observed. In still other embodiments, the agent that inhibits MMSET activity is a pan-inhibitor of S-adenosylmethionine-dependent
methyltransf erase.
In various embodiments of any of the above aspects or any other aspect of the invention delineated herein, the invention provides a kit comprising a therapeutic or prophylactic composition containing (i) an effective amount of an agent of any one of preceding embodiments; and (ii) an effective amount of a chemotherapeutic agent. Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
By "Phosphatase And Tensin Homolog Deleted On Chromosome Ten (PTEN) polypeptide" is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: NP 000305.3, or a fragment thereof, and having phosphatase activity. An exemplary PTEN amino acid sequence is provided below (SEQ ID NO: 1):
1 mtaiikeivs rnkrryqedg fdldltyiyp niiamgfpae rlegvyrnni ddvvrfldsk 61 hknhykiynl caerhydtak fncrvaqypf edhnppqlel ikpfcedldq wlseddnhva 121 aihckagkgr tgvmicayll hrgkflkaqe aldfygevrt rdkkgvtips qrryvyyysy 181 llknhldyrp vallfhkmmf etipmfsggt cnpqfvvcql kvkiyssnsg ptrredkfmy 241 fefpqplpvc gdikveffhk qnkmlkkdkm fhfwvntffi pgpeetsekv engslcdqei 301 dsicsierad ndkeylvltl tkndldkank dkanryfspn fkvklyftkt veepsnpeas 361 sstsvtpdvs dnepdhyrys dttdsdpene pfdedqhtqi tkv
By "PTEN polynucleotide" is meant a nucleic acid molecule encoding a PTEN polypeptide. An exemplary PTEN polynucleotide sequence is provided at NCBI Reference Sequence: NM_000314.6, and reproduced herein below (SEQ ID NO: 2).
1 cctcccctcg cccggcgcgg tcccgtccgc ctctcgctcg cctcccgcct cccctcggtc 61 ttccgaggcg cccgggctcc cggcgcggcg gcggaggggg cgggcaggcc ggcgggcggt 121 gatgtggcgg gactctttat gcgctgcggc aggatacgcg ctcggcgctg ggacgcgact 181 gcgctcagtt ctctcctctc ggaagctgca gccatgatgg aagtttgaga gttgagccgc 241 tgtgaggcga ggccgggctc aggcgaggga gatgagagac ggcggcggcc gcggcccgga 301 gcccctctca gcgcctgtga gcagccgcgg gggcagcgcc ctcggggagc cggccggcct 361 gcggcggcgg cagcggcggc gtttctcgcc tcctcttcgt cttttctaac cgtgcagcct 421 cttcctcggc ttctcctgaa agggaaggtg gaagccgtgg gctcgggcgg gagccggctg 481 aggcgcggcg gcggcggcgg cacctcccgc tcctggagcg ggggggagaa gcggcggcgg 541 cggcggccgc ggcggctgca gctccaggga gggggtctga gtcgcctgtc accatttcca 601 gggctgggaa cgccggagag ttggtctctc cccttctact gcctccaaca cggcggcggc 661 ggcggcggca catccaggga cccgggccgg ttttaaacct cccgtccgcc gccgccgcac 721 cccccgtggc ccgggctccg gaggccgccg gcggaggcag ccgttcggag gattattcgt
781 cttctcccca ttccgctgcc gccgctgcca ggcctctggc tgctgaggag aagcaggccc 841 agtcgctgca accatccagc agccgccgca gcagccatta cccggctgcg gtccagagcc 901 aagcggcggc agagcgaggg gcatcagcta ccgccaagtc cagagccatt tccatcctgc 961 agaagaagcc ccgccaccag cagcttctgc catctctctc ctcctttttc ttcagccaca 1021 ggctcccaga catgacagcc atcatcaaag agatcgttag cagaaacaaa aggagatatc
1081 aagaggatgg attcgactta gacttgacct atatttatcc aaacattatt gctatgggat 1141 ttcctgcaga aagacttgaa ggcgtataca ggaacaatat tgatgatgta gtaaggtttt 1201 tggattcaaa gcataaaaac cattacaaga tatacaatct ttgtgctgaa agacattatg 1261 acaccgccaa atttaattgc agagttgcac aatatccttt tgaagaccat aacccaccac 1321 agctagaact tatcaaaccc ttttgtgaag atcttgacca atggctaagt gaagatgaca
1381 atcatgttgc agcaattcac tgtaaagctg gaaagggacg aactggtgta atgatatgtg 1441 catatttatt acatcggggc aaatttttaa aggcacaaga ggccctagat ttctatgggg 1501 aagtaaggac cagagacaaa aagggagtaa ctattcccag tcagaggcgc tatgtgtatt 1561 attatagcta cctgttaaag aatcatctgg attatagacc agtggcactg ttgtttcaca 1621 agatgatgtt tgaaactatt ccaatgttca gtggcggaac ttgcaatcct cagtttgtgg
1681 tctgccagct aaaggtgaag atatattcct ccaattcagg acccacacga cgggaagaca 1741 agttcatgta ctttgagttc cctcagccgt tacctgtgtg tggtgatatc aaagtagagt 1801 tcttccacaa acagaacaag atgctaaaaa aggacaaaat gtttcacttt tgggtaaata 1861 cattcttcat accaggacca gaggaaacct cagaaaaagt agaaaatgga agtctatgtg 1921 atcaagaaat cgatagcatt tgcagtatag agcgtgcaga taatgacaag gaatatctag
1981 tacttacttt aacaaaaaat gatcttgaca aagcaaataa agacaaagcc aaccgatact 2041 tttctccaaa ttttaaggtg aagctgtact tcacaaaaac agtagaggag ccgtcaaatc 2101 cagaggctag cagttcaact tctgtaacac cagatgttag tgacaatgaa cctgatcatt 2161 atagatattc tgacaccact gactctgatc cagagaatga accttttgat gaagatcagc 2221 atacacaaat tacaaaagtc tgaatttttt tttatcaaga gggataaaac accatgaaaa
2281 taaacttgaa taaactgaaa atggaccttt ttttttttaa tggcaatagg acattgtgtc 2341 agattaccag ttataggaac aattctcttt tcctgaccaa tcttgtttta ccctatacat 2401 ccacagggtt ttgacacttg ttgtccagtt gaaaaaaggt tgtgtagctg tgtcatgtat 2461 ataccttttt gtgtcaaaag gacatttaaa attcaattag gattaataaa gatggcactt 2521 tcccgtttta ttccagtttt ataaaaagtg gagacagact gatgtgtata cgtaggaatt
2581 ttttcctttt gtgttctgtc accaactgaa gtggctaaag agctttgtga tatactggtt 2641 cacatcctac ccctttgcac ttgtggcaac agataagttt gcagttggct aagagaggtt 2701 tccgaagggt tttgctacat tctaatgcat gtattcgggt taggggaatg gagggaatgc 2761 tcagaaagga aataatttta tgctggactc tggaccatat accatctcca gctatttaca 2821 cacacctttc tttagcatgc tacagttatt aatctggaca ttcgaggaat tggccgctgt
2881 cactgcttgt tgtttgcgca ttttttttta aagcatattg gtgctagaaa aggcagctaa 2941 aggaagtgaa tctgtattgg ggtacaggaa tgaaccttct gcaacatctt aagatccaca 3001 aatgaaggga tataaaaata atgtcatagg taagaaacac agcaacaatg acttaaccat 3061 ataaatgtgg aggctatcaa caaagaatgg gcttgaaaca ttataaaaat tgacaatgat 3121 ttattaaata tgttttctca attgtaacga cttctccatc tcctgtgtaa tcaaggccag
3181 tgctaaaatt cagatgctgt tagtacctac atcagtcaac aacttacact tattttacta 3241 gttttcaatc ataatacctg ctgtggatgc ttcatgtgct gcctgcaagc ttcttttttc 3301 tcattaaata taaaatattt tgtaatgctg cacagaaatt ttcaatttga gattctacag 3361 taagcgtttt ttttctttga agatttatga tgcacttatt caatagctgt cagccgttcc 3421 acccttttga ccttacacat tctattacaa tgaattttgc agttttgcac attttttaaa
3481 tgtcattaac tgttagggaa ttttacttga atactgaata catataatgt ttatattaaa 3541 aaggacattt gtgttaaaaa ggaaattaga gttgcagtaa actttcaatg ctgcacacaa 3601 aaaaaagaca tttgattttt cagtagaaat tgtcctacat gtgctttatt gatttgctat 3661 tgaaagaata gggttttttt tttttttttt tttttttttt ttaaatgtgc agtgttgaat
3721 catttcttca tagtgctccc ccgagttggg actagggctt caatttcact tcttaaaaaa
3781 aatcatcata tatttgatat gcccagactg catacgattt taagcggagt acaactacta
3841 ttgtaaagct aatgtgaaga tattattaaa aaggtttttt tttccagaaa tttggtgtct
3901 tcaaattata ccttcacctt gacatttgaa tatccagcca ttttgtttct taatggtata
3961 aaattccatt ttcaataact tattggtgct gaaattgttc actagctgtg gtctgaccta
4021 gttaatttac aaatacagat tgaataggac ctactagagc agcatttata gagtttgatg
4081 gcaaatagat taggcagaac ttcatctaaa atattcttag taaataatgt tgacacgttt
4141 tccatacctt gtcagtttca ttcaacaatt tttaaatttt taacaaagct cttaggattt
4201 acacatttat atttaaacat tgatatatag agtattgatt gattgctcat aagttaaatt
4261 ggtaaagtta gagacaacta ttctaacacc tcaccattga aatttatatg ccaccttgtc
4321 tttcataaaa gctgaaaatt gttacctaaa atgaaaatca acttcatgtt ttgaagatag
4381 ttataaatat tgttctttgt tacaatttcg ggcaccgcat attaaaacgt aactttattg
4441 ttccaatatg taacatggag ggccaggtca taaataatga cattataatg ggcttttgca
4501 ctgttattat ttttcctttg gaatgtgaag gtctgaatga gggttttgat tttgaatgtt
4561 tcaatgtttt tgagaagcct tgcttacatt ttatggtgta gtcattggaa atggaaaaat
4621 ggcattatat atattatata tataaatata tattatacat actctcctta ctttatttca
4681 gttaccatcc ccatagaatt tgacaagaat tgctatgact gaaaggtttt cgagtcctaa
4741 ttaaaacttt atttatggca gtattcataa ttagcctgaa atgcattctg taggtaatct
4801 ctgagtttct ggaatatttt cttagacttt ttggatgtgc agcagcttac atgtctgaag
4861 ttacttgaag gcatcacttt taagaaagct tacagttggg ccctgtacca tcccaagtcc
4921 tttgtagctc ctcttgaaca tgtttgccat acttttaaaa gggtagttga ataaatagca
4981 tcaccattct ttgctgtggc acaggttata aacttaagtg gagtttaccg gcagcatcaa
5041 atgtttcagc tttaaaaaat aaaagtaggg tacaagttta atgtttagtt ctagaaattt
5101 tgtgcaatat gttcataacg atggctgtgg ttgccacaaa gtgcctcgtt tacctttaaa
5161 tactgttaat gtgtcatgca tgcagatgga aggggtggaa ctgtgcacta aagtgggggc
5221 tttaactgta gtatttggca gagttgcctt ctacctgcca gttcaaaagt tcaacctgtt
5281 ttcatataga atatatatac taaaaaattt cagtctgtta aacagcctta ctctgattca
5341 gcctcttcag atactcttgt gctgtgcagc agtggctctg tgtgtaaatg ctatgcactg
5401 aggatacaca aaaataccaa tatgatgtgt acaggataat gcctcatccc aatcagatgt
5461 ccatttgtta ttgtgtttgt taacaaccct ttatctctta gtgttataaa ctccacttaa
5521 aactgattaa agtctcattc ttgtcattgt gtgggtgttt tattaaatga gagtttataa
5581 ttcaaattgc ttaagtccat tgaagtttta attaatgggc agccaaatgt gaatacaaag
5641 ttttcagttt ttttttttcc tgctgtcctt caaagcctac tgtttaaaaa aaaaaaaaaa
5701 aaaaaacatg gcctgagagt agagtatctg tctactcatg tttaattaag gaaaaacact
5761 tatttttagg gctttagtca tcacttcata aattgtataa gcacattaaa tagcgttcta
5821 gtcctgaaaa agtccaagat tcttagaaaa ttgtgcatat ttttattatg acagatgttt
5881 gaagataatt ccccagaatg gatttgatac tttagatttc aattttgtgg cttttgtcta
5941 ttattctgta ctctgccatc agcatatgga aagcttcatt tactcatcat gacttgtgcc
6001 atataaaaat tgatatttcg gaatagtcta aaggactttt tgtacttgaa tttaatcatg
6061 ttgtttctaa tattcttaaa agcttgaaga ctaaagcata tcctttcaac aaagcatagt
6121 aaggtaataa gaaagtgtag tttgtacaag tgttaaaaaa ataaagtaga caatgttaca
6181 gtgggactta ttatttcaag tttacatttt ctccatgtaa ttttttaaaa agtaaatgaa
6241 aaaatgtgca ataatgtaaa atatgaagtg tatgtgtaca cacattttat ttttcggtat
6301 cttgggtata cgtatggttg aaaactatac tggagtctaa aagtattcta atttataaga
6361 agacattttg gtgatgtttg aaaaatagaa atgtgctagt tttgttttta tatcatgtcc
6421 tttgtacgtt gtaatatgag ctggcttggt tcagtaaatg ccatcaccat ttccattgag
6481 aatttaaaac tcaccagtgt ttaatatgca ggcttccaaa ggcttatgaa aaaaatcaag
6541 acccttaaat ctagttaatt tgctgctaac atgaaactct ttggttcttt tatttttgcc
6601 agataattag acacacatct aaagcttagt cttaaatggc ttaagtgtag ctattgatta
6661 gtgctgttgc tagttcagaa agaaatgttt gtgaatggaa acaagaatat tcagtccaaa
6721 ctgttgtaag gacagtacct gaaaaccagg aaacaggata atggaaaaag tcttttaaag
6781 atgaaatgtt ggagccaact ttcttataga attaattgta tgtggctata gaaagcctaa 6841 tgattgttgc ttatttttga gagcatatta ttcttttatg accataatct tgctgttttt 6901 ccatcttcca aaagatcttc cttctaatat gtatatcaga atgtgggtag ccagtcagac 6961 aaattcatat tggttggtag ctttaaaaag tttgtaatgt gaagacagga aaggacaaaa 7021 tagtttgctt tggtggtagt actctggttg ttaagctagg tattttgaga ctacttcccc 7081 atcacaacaa caataaaata atcactcata atcctatcac ctggagacat agccatcgtt 7141 aatatgttag tgactataca atcatgtttt cttctgtata tccatgtata ttctttaaaa 7201 atgaaattta tactgtacct gatctcaaag ctttttagct tagtatatct gtcatgaatt 7261 tgtaggatgt tccattgcat cagaaaacgg acagtgattt gattactttc taatgccaca 7321 gatgcagatt acatgtagtt attgagaatc ctttcgaatt cagtggctta atcatgaatg 7381 tctaaatatt gttgacatta ggatgataca tgtaaattaa agttacattt gtttagcata 7441 gacaagctta acattgtaga tgtttctctt caaaaatcat cttaaacatt tgcatttgga 7501 attgtgttaa atagaatgtg tgaaacactg tattagtaaa cttcatcacc tttctacttc 7561 cttatagttt gaacttttca gtttttgtag ttcccaaaca gttgctcaat ttagagcaaa 7621 ttaatttaac acctgccaaa aaaaggctgc tgttggctta tcagttgtct ttaaattcaa 7681 atgctcatgt gacttttatc acatcaaaaa atatttcatt aatgattcac ctttagctct 7741 gaaaattacc gcgtttagta attatagtgg gcttataaaa acatgcaact ctttttgata 7801 gttatttgag aattttggtg aaaaatattt agctgagggc agtatagaac ttataaacca 7861 atatattgat atttttaaaa catttttaca tataagtaaa ctgccatctt tgagcataac 7921 tacatttaaa aataaagctg catattttta aatcaagtgt ttaacaagaa tttatatttt 7981 ttatttttta aaattaaaaa taatttatat ttcctctgtt gcatgaggat tctcatctgt 8041 gcttataatg gttagagatt ttatttgtgt ggaatgaagt gaggcttgta gtcatggttc 8101 tagtgtttca gtttgccaag tctgtttact gcagtgaaat tcatcaaatg tttcagtgtg 8161 gttttctgta gcctatcatt tactggctat ttttttatgt acacctttag gattttctgc 8221 ctactctatc cagttgtcca aatgatatcc tacattttac aaatgccctt tcagtttcta 8281 ttttcttttt ccattaaatt gccctcatgt cctaatgtgc agtttgtaag tgtgtgtgtg 8341 tgtgtctgtg tgtgtgtgaa tttgattttc aagagtgcta gacttccaat ttgagagatt 8401 aaataattta attcaggcaa acatttttca ttggaatttc acagttcatt gtaatgaaaa 8461 tgttaatcct ggatgacctt tgacatacag taatgaatct tggatattaa tgaatttgtt 8521 agtagcatct tgatgtgtgt tttaatgagt tattttcaaa gttgtgcatt aaaccaaagt 8581 tggcatactg gaagtgttta tatcaagttc catttggcta ctgatggaca aaaaatagaa 8641 atgccttcct atggagagta tttttccttt aaaaaattaa aaaggttaat tattttgact 8701 aaaaaaaaaa aaaaaaaa
By "multiple myeloma SET domain (MMSET) polypeptide" is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: 096028.1, or a fragment thereof, and having histone methyl transferase activity. MMSET contains a SET domain that is found in many histone methyltransferases (HMTs). Other potential functional motifs in MMSET include nuclear localization signals (NLSs), an HMG box (high mobility group) often representing a DNA-binding domain, 2 PWWP domains (proline-tryptophan-tryptophan-proline) found in other nuclear proteins and 4 PHD (plant homeodomain) zinc fingers recently defined as binding modules for methylated lysines. An exemplary MMSET amino acid sequence is provided below (SEQ ID NO: 3):
1 mefsikqspl svqsvvkcik mkqapeilgs angktpscev nrecsvflsk aqlssslqeg 61 vmqkfnghda lpfipadklk dltsrvfnge pgahdaklrf esqemkgigt ppnttpikng 121 speiklkitk tymngkplfe ssicgdsaad vsqseengqk penkarrnrk rsikydslle 181 qglveaalvs kisspsdkki pakkescpnt grdkdhllky nvgdlvwskv sgypwwpcmv 241 sadpllhsyt klkgqkksar qyhvqffgda perawifeks lvafegegqf eklcqesakq 301 aptkaekikl lkpisgklra qwemgivqae eaasmsveer kakftflyvg dqlhlnpqva 361 keagiaaesl gemaessgvs eeaaenpksv reecipmkrr rraklcssae tleshpdigk 421 stpqktaead prrgvgsppg rkkttvsmpr srkgdaasqf lvfcqkhrde vvaehpdasg 481 eeieellrsq wsllsekqra ryntkfalva pvqaeedsgn vngkkrnhtk riqdptedae 541 aedtprkrlr tdkhslrkrd titdktarts sykameaass lksqaatknl sdackplkkr 601 nrastaassa lgfsksssps asltenevsd spgdepsesp yesadetqte vsvsskkser 661 gvtakkeyvc qlcekpgsll lcegpccgaf hlaclglsrr pegrftcsec asgihscfvc 721 kesktdvkrc vvtqcgkfyh eacvkkyplt vfesrgfrep lhscvschas npsnprpskg 781 kmmrcvrcpv ayhsgdacla agcsviasns iictahftar kgkrhhahvn vswcfvcskg 841 gsllccescp aafhpdclni empdgswfcn dcragkklhf qdiiwvklgn yrwwpaevch 901 pknvppniqk mkheigefpv fffgskdyyw thqarvfpym egdrgsryqg vrgigrvfkn 961 alqeaearfr eiklqreare tqeserkppp ykhikvnkpy gkvqiytadi seipkenckp 1021 tdenpcgfds eclnrmlmfe chpqvcpage fcqnqcftkr qypetkiikt dgkgwglvak 1081 rdirkgefvn eyvgelidee ecmarikhah endithfyml tidkdriida gpkgnysrfm 1141 nhscqpncet Ikwtvngdtr vglfavcdip agteltfnyn Idclgnektv crcgasncsg 1201 flgdrpktst tlsseekgkk tkkktrrrra kgegkrqsed ecfrcgdggq lvlcdrkfct 1261 kayhlsclgl gkrpfgkwec pwhhcdvcgk psts fchlep ns fckehqdg tafsctpdgr 1321 syccehdlga asvrstktek pppepgkpkg krrrrrgwrr vtegk
By "MMSET polynucleotide" is meant a nucleic acid molecule encoding a MMSET polypeptide. An exemplary MMSET polynucleotide sequence is provided at NCBI Reference Sequence: M 133330.2, and reproduced herein below (SEQ ID NO: 4):
1 tggtcttgaa ctcctgacct tgtgatccgc tcgcctcagc ctcccaaagt gctgggatta 61 caggcatgag ccaccgtgcc tgtcctagaa ccactggtaa ctcttaactg agacaagatt 121 tcgccatgtt gcccaggctg gtctcgaact tctgagctca agegatctae ctgcctcgtc 181 ctcccaaagt gttggaattg caggtgtgag ccactgcacc cggcccttgc ctcatgtttg 241 aatgataatt tacctgggca taaaattctg gacacttgtg gtggcacgag ctgtctgctt 301 ttctgtggag actgegggtt gcagacacca gcagccccct attgetgeae gtcaggtgtc 361 ctccacctca gtactgtgga catctcgggc cgcatagccc tgttgcgcgg gcatcctgtg 421 cgttgcagga gatgeagcag catccatgac cgcaacccat cagagtgttc taagaacgga 481 agcatctggg ctggatggaa tttagcatca agcagagtcc cctttctgtt cagagtgttg 541 taaagtgcat aaagatgaag caggcaccag aaatcctegg cagtgccaac gggaagactc 601 egagctgega ggtgaaccgc gagtgttctg tgttcctcag caaagcccag ctctccagta 661 gectgeagga gggggtcatg cagaagttta acggccacga cgccctgccc tttattccag 721 ccgacaagct gaaagatctt acttcccggg tgtttaatgg agaacccggc gcacacgatg 781 ccaaactgcg ttttgagtcc caggaaatga aagggattgg gacaccccct aacactaccc 841 ctatcaaaaa tggctctcca gaaattaagc tgaaaatcac caaaacatac atgaatggga 901 agcctctctt tgaatcttcc atttgtggtg acagtgctgc tgatgtgtct cagtcagaag 961 aaaatggaca aaaaccagaa aacaaggega gaaggaacag gaagaggagc ataaaatatg 1021 actccttgct ggagcagggc cttgtcgaag cagctcttgt gtctaagatc tcaagtcctt 1081 cagataaaaa gattccagct aagaaagagt cttgtccaaa cactggaaga gacaaagacc 1141 acctgttgaa atacaacgtt ggtgatttgg tgtggtccaa agtgtcgggt tacccttggt 1201 ggecttgeat ggtttctgca gatccactcc ttcacagcta taccaaactt aaaggtcaga 1261 aaaagagtgc aegecagtat caegtacagt tctttggtga cgccccagaa agagcttgga 1321 tatttgagaa gagcctcgta gcttttgaag gagaaggaca gtttgaaaaa ttatgccagg 1381 aaagtgccaa gcaggcaccc acgaaagctg agaaaattaa gctattgaaa ccaatttcag 1441 ggaaattgag ggcccagtgg gaaatgggca ttgttcaagc agaagaagct gcaagcatgt 1501 cagtggagga geggaaagee aagttcacct ttctctatgt gggggaccag cttcatctca 1561 accctcaagt agecaaggag gctggcattg ctgcagagtc tttgggagaa atggcagaat 1621 cctcaggagt cagtgaagaa getgetgaaa accccaagtc tgtgagagaa gagtgeatte 1681 ccatgaagag aaggeggagg gecaaactgt gtagctctgc agagaccctg gagagtcacc 1741 ccgacatagg gaagagtact cctcaaaaga eggcagagge tgaccccaga agaggagtag 1801 ggtctcctcc tgggaggaag aagaccacag tctccatgcc acgaagcagg aagggagatg 1861 cagcatccca gtttttggtc ttctgtcaaa aacacaggga tgaggtggta gctgagcacc 1921 cagatgcttc aggtgaggag attgaagagc tgctcaggtc acagtggagt ctgctgagtg 1981 agaagcagag agcacgctac aacaccaagt ttgccctggt ggcccctgtc caggctgaag 2041 aagactctgg taatgtaaat gggaaaaaaa gaaaccacac aaagaggata caggacccta
2101 cagaagatgc tgaagctgag gacacaccca ggaaaagact caggacggac aagcacagtc 2161 ttcggaagag agacacaatc actgacaaaa cggccagaac aagctcttac aaggccatgg 2221 aggcagcctc ctcgctcaag agccaggcag caacgaaaaa tctgtctgat gcatgtaaac 2281 cactgaagaa gcgaaatcgg gcttccacgg cagcatcttc agctcttggg tttagcaaaa 2341 gttcatctcc ttctgcatcc ttaactgaga atgaggtctc ggacagcccg ggagacgagc
2401 cctcggagtc cccatacgaa agtgcagacg aaacacaaac tgaagtatct gtctcatcca 2461 aaaagtctga gcgaggagtg actgccaaaa aggagtatgt gtgccagctg tgtgagaagc 2521 cgggcagcct cctgctctgt gaaggaccct gctgcggagc tttccacctc gcctgccttg 2581 ggctttcccg gaggccagaa gggaggttca cctgcagcga gtgtgcctca gggattcact 2641 catgtttcgt gtgtaaagag agcaagacag atgttaagcg ctgtgtggta actcagtgtg
2701 gaaaatttta ccatgaggct tgtgtgaaaa aataccctct gactgtattt gagagccgag 2761 gtttccgctg ccccctccac agctgtgtga gctgccatgc ttccaaccct tcaaacccaa 2821 ggccgtcaaa aggtaaaatg atgcggtgtg tccgctgccc cgttgcctat cacagcgggg 2881 atgcttgtct ggcagcagga tgctcagtga tcgcctccaa cagcatcatc tgcactgccc 2941 acttcactgc tcggaagggg aagcgacacc acgcccacgt caacgtgagc tggtgcttcg
3001 tgtgctccaa aggggggagc cttctgtgct gtgagtcctg cccagcggcc ttccaccctg 3061 actgcctgaa catcgagatg cctgacggca gctggttctg caatgactgc agggctggga 3121 agaagctgca cttccaggat atcatttggg tgaaacttgg gaactacaga tggtggccgg 3181 cagaagtttg ccatcccaaa aatgttcccc caaatattca gaaaatgaag cacgagattg 3241 gagaattccc tgtgtttttc tttgggtcta aagattatta ctggacgcat caggcgcgag
3301 tgttcccgta catggagggg gaccggggca gccgctacca gggggtcaga gggatcggaa 3361 gagtcttcaa aaacgcactg caagaagctg aagctcgttt tcgtgaaatt aagcttcaga 3421 gggaagcccg agaaacacag gagagcgagc gcaagccccc accatacaag cacatcaagg 3481 tgaataagcc ttacgggaaa gtccagatct acacagcgga tatttcagaa atccctaagt 3541 gcaactgcaa gcccacagat gagaatcctt gtggctttga ttcggagtgt ctgaacagga
3601 tgctgatgtt tgagtgccac ccgcaggtgt gtcccgcggg cgagttctgc cagaaccagt 3661 gcttcaccaa gcgccagtac ccagagacca agatcatcaa gacagatggc aaagggtggg 3721 gcctggtcgc caagagggac atcagaaagg gagaatttgt taacgagtac gttggggagc 3781 tgatcgacga ggaggagtgc atggcgagaa tcaagcacgc acacgagaac gacatcaccc 3841 acttctacat gctcactata gacaaggacc gtataataga cgctggcccc aaaggaaact
3901 actctcgatt tatgaatcac agctgccagc ccaactgtga gaccctcaag tggacagtga 3961 atggggacac tcgtgtgggc ctgtttgccg tctgtgacat tcctgcaggg acggagctga 4021 cttttaacta caacctcgat tgtctgggca atgaaaaaac ggtctgccgg tgtggagcct 4081 ccaattgcag tggattcctc ggggatagac caaagacctc gacgaccctt tcatcagagg 4141 aaaagggcaa aaagaccaag aagaaaacga ggcggcgcag agcaaaaggg gaagggaaga
4201 ggcagtcaga ggacgagtgc ttccgctgcg gtgatggcgg gcagctggtg ctgtgtgacc 4261 gcaagttctg caccaaggcc taccacctgt cctgcctggg ccttggcaag cggcccttcg 4321 ggaagtggga atgtccttgg catcattgtg acgtgtgtgg caaaccttcg acttcatttt 4381 gccacctctg ccccaattcg ttctgtaagg agcaccagga cgggacagcc ttcagctgca 4441 ccccggacgg gcggtcctac tgctgtgagc atgacttagg ggcggcatcg gtcagaagca
4501 ccaagactga gaagcccccc ccagagccag ggaagccgaa ggggaagagg cggcggcgga 4561 ggggctggcg gagagtcaca gagggcaaat agcgccaggc ggccgcttgg ccggatccag 4621 gggcggtgca gggcggccgg ccctgcctgc gggagagggc gagcatgaac tggcccggag 4681 gacccagctc gagccgccag gacacagacg tacaggcctc ctcgggaggg agcgcctccc 4741 caccactgag ccatcctcag cagcgtccgc tgcgtctgca ctgatgaccg tctgagccca
4801 gctcagcgtt cctggacaaa cagcctcact cctcagcgtt accgccacac ttgaatttct 4861 ccgaatgtca aggttccctc ccactctatt tttttaggtt aaagttaatt ggcatatgga 4921 atgttttaat ctcctctgaa atgtgtagcg taggcttttc ccaagggtcg ctagaaactc 4981 gtcttcgcgt tgcccccttt ctggctctca gcgccgtcgc cactcgggag aggctgggtg
5041 aggcccgtgt gaggactgac cctggattcc tcgaaactgc cattgtgatc attactctgc
5101 tctttggaaa tggctgtatc atttttttgt actaatgtga attgttcctc agaaacgctt
5161 cttttccatc ctagtgagaa gctggccctg caggtggtgg cagcaatggt gttgtaagat
5221 ttcctcccgt agttttttct cctcatggat ttgaatgaaa tgccaataac acgtccactt
5281 tcaacgtgta gtttacgcgg agcactttcg aggcctggcc gggttgggcc tacttctcac
5341 ctgggcctat cttctgaact cgctaggttc ttatcaacat ttgggggata actttgtata
5401 tttttttcat ttggcttttc tttaccagtt tctgattttt attctcaata tatttttgct
5461 aaacctattt cacaaatcac caccgactga agtgtgtgtt tactgatgcg gccctgagct
5521 ccatggcgaa aggagtgact ttgcagggcg tgagaccgca gtctgcttag agcacaggaa
5581 gtgacaactt agggagcccc gtagggcgct gcaggccccg gggaccccag cacgtgggtc
5641 taaagagaga cggagtctag ctctcctgcc acccagagtg gcttccatct cagcactctg
5701 tgggtctggt gatggaagat gcagtctctg ctgatcacat gtgccctctg ccagggcacc
5761 tactgagagg tgcggtcctg ggggtggagg cctgcctggc aggtgtgcgt gcctcgtacg
5821 tgtgttatgg gcactggtct aggccaggta tgacacccac tctcctgtga gatttcactt
5881 tagtttttaa aaggtccagt tctacagagt gagacctatc tatctgagta ctacatatgt
5941 tttaagactt ggttcttttt ttgagggatc cttgaccctg ggaagtctgg agcaccctga
6001 gaagggggca ccatgtgtgc ctttgcccac gtgtcctgag gggctgcttg tctgggaggg
6061 agggagagaa cattcagcag caggtgcttt tttatggcct tttcttaaaa taacctaagg
6121 gggacacatc catcttgcag agaagtttac agaactcccc ttgaaaactg ctgctgaggc
6181 tcctgttaaa ttttctgtgg catcttttat gccttggtaa aaactgcagt gtctttggac
6241 ctgagagtgg ctactccgtg gttttgtgac ctgtaagcgt ggggttcagg ggtgtgtggc
6301 cctgcagggt cccacgcctc cctgagcact gactggaagt ttcactggct ggtggctgtc
6361 ccttctccca tcagggtccc cagcaaagtt aactacacag aggacccagg ggaaacgagc
6421 tgtgtagcca ctgacttgct cgcgcggccg tggcctctga ggggcactcg ccggttaaga
6481 cagggtggga gtagtgcttt ccagttcaga ctctaacttc tcccaaagtg tcctaagaaa
6541 atactggatc ggctcataga tttatgctcc ttatgatgcc ctaacttgga aggttgttct
6601 agggacaggc cgggcagtgt ccccacacac accttagagt cgaaggcccc agggccccgc
6661 tgtcacttgc ccaaaagatc ccttccggca ggtaagggac taccaatgct tacgtcaaaa
6721 cagcagaatc ggctttgcag tgcactttgg ggagcagata ttaacttatt tttgtgttgg
6781 acagtagtga aatcttgtga tttttaatcg ctttgataat acttccaaat tttatgattt
6841 ttctgaagga aataatgcaa acattttaaa tatgtttctc cccctttcca aaaactgtta
6901 aactaatgag caagtaacac taactttgaa tgtctctaca atacccgttg ataactcagt
6961 ggagccaggc tttggggtag cggccctgag cttgcagggt ttctcgccac tggggctgac
7021 cacgccccca gctgtgaccg tgggtgtggc tggctctcgg ccctgcccag ctttgttctg
7081 aggacgtggt gacttcctga acatcagctt caatcctcca tcattaatgt gaagcaaaac
7141 acaaaaaccg ccccaatccc tcaggattcc ttggcatccg aaaccagcat ctgcacctaa
7201 acccataccc acccgtgtgc gcccacaggg ggatgtgtcc gaatgggcag cttaaaatgt
7261 ggtcacctgt gggggaaact cttcaggcac ctgaagtgag aacccagctg tccgtcctca
7321 ggccggcctt tcttccggcg acacccgtcc atggctggct gggtcccctt cgcagtgttt
7381 gtctgtcttg acatctaaac cccggcgtgt gcagtgccca tcttccagga ctaccttatt
7441 ttccagaatt aaacctgttt tataattcaa gttaatgcaa atgactgtca gttgccaaat
7501 atcttgatcc tatgagtgta gttgatgact gtttgttagt cagtagagta aaatgctgtg
7561 tccacggggt gtcacagcct caccataccc tgttgaggtg tgaaatgccc cgtcagaaat
7621 taaatacaaa cttaaatgtg cctattggtg tctaaacttc atacaatgta aggtcagatt
7681 ccttttagga atactgggtg ctgtcaccag gtttgatagt tagacttaaa aacttgaaat
7741 tcactttttg gggggaggga tatactgaaa tagagagttg agacttgcca gttgggggaa
7801 aatagcattt aaaatggaaa gctgtgtttg gaaaattgtg tatgagtatt tttgtattaa
7861 aaacatttta aaggcttttt tcttaa
The MMSET gene, is also known as Wolf-Hirschhorn Syndrome Candidate 1 (WHSC1) or Nuclear Receptor-binding SET Domain 2 (NSD2). By "mediator of DNA damage checkpoint 1 (MDC1) polypeptide" is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: NP_055456.2, or a fragment thereof, and having cell cycle regulatory activity. MDC1 is required to activate the intra-S phase and G2/M phase cell cycle checkpoints in response to DNA damage. This nuclear protein interacts with phosphorylated histone H2AX, near sites of DNA double-strand breaks through its two BRCAl C-terminal (BRCT) motifs, and facilitates recruitment of the protein kinase ataxia-telangiectasia mutated (ATM) and meiotic recombination 11 protein complex to DNA damage foci. An exemplary MDC1 amino acid sequence is provided below (SEQ ID NO: 5):
1 medtqaidwd veeeeeteqs seslrcnvep vgrlhifsga hgpekdfplh lgknvvgrmp 61 dcsvalpfps iskqhaeiei lawdkapilr dcgslngtqi lrppkvlspg vshrlrdqel 121 ilfadllcqy hrldvslpfv srgpltveet prvqgetqpq rlllaedsee evdflserrm 181 vkksrttsss vivpesdeeg hspvlgglgp pfafnlnsdt dveegqqpat eeassaarrg 241 atveakqsea evvteiqlek dqplvkerdn dtkvkrgagn gvvpagvile rsqppgedsd 301 tdvdddsrpp grpaevhler aqpfgfidsd tdaeeeripa tpvvipmkkr kifhgvgtrg 361 pgapglahlq esqagsdtdv eegkapqavp leksqasmvi nsdtddeeev saaltlahlk 421 esqpaiwnrd aeedmpqrvv llqrsqttte rdsdtdveee elpvenreav lkdhtkiral 481 vrahsekdqp pfgdsddsve adksspgihl ersqasttvd intqvekevp pgsaiihikk 541 hqvsvegtnq tdvkavggpa kllvvsleea wplhgdcetd aeegtsltas vvadvrksql 601 paegdagaew aaavlkqera hevgaqggpp vaqveqdlpi srenltdlvv dtdtlgestq 661 pqregaqvpt grereqhvgg tkdsednygd sedldlqatq cflenqglea vqsmedeptq 721 afmltppqel gpshcsfqtt gtldepwevl atqpfclres edsetqpfdt hleaygpcls 781 ppraipgdqh pespvhtepm giqgrgrqtv dkvmgipket aervgpergp leretekllp 841 erqtdvtgee eltkgkqdre qkqllardtq rqesdknges asperdresl kveietseei 901 qekqvqkqtl pskafereve rpvanrecdp aeleekvpkv ilerdtqrge peggsqdqkg 961 qassptpepg vgagdlpgpt sapvpsgsqs ggrgspvspr rhqkgllnck mppaekasri 1021 raaekvsrgd qespdaclpp tvpeapappq kplnsqsqkh lapppllspl lpsikptvrk 1081 trqdgsqeap eaplsselep fhpkpkirtr kssrmtpfpa tsaapephps tstaqpvtpk 1141 ptsqatrsrt nrssvktpep vvptapelqp ststdqpvts eptsqvtrgr ksrssvktpe 1201 tvvptalelq pststdrpvt septsqatrg rknrssvktp epvvptapel qpststdqpv 1261 tseptyqatr grknrssvkt pepvvptape lrpststdrp vtpkptsrtt rsrtnmssvk 1321 tpetvvptap elqiststdq pvtpkptsrt trsrtnmssv knpestvpia pelppstste 1381 qpvtpeptsr atrgrknrss gktpetlvpt apklepstst dqpvtpepts qatrgrtnrs 1441 svktpetvvp tapelqpsts tdqpvtpept sqatrgrtdr ssvktpetvv ptapelqasa 1501 stdqpvtsep tsrttrgrkn rssvktpetv vpaapelqps tstdqpvtpe ptsratrgrt 1561 nrssvktpes ivpiapelqp stsrnqlvtp eptsratrcr tnrssvktpe pvvptapeph 1621 pttstdqpvt pkltsratrr ktnrssvktp kpvepaasdl epftptdqsv tpeaiaqggq 1681 sktlrsstvr ampvpttpef qspvttdqpi spepitqpsc ikrqraagnp gslaapidhk 1741 pcsaplepks qasrnqrwga vraaesltai pepaspqlle tpihasqiqk vepagrsrft 1801 pelqpkasqs rkrslatmds pphqkqpqrg evsqktviik eeeedtaekp gkeedvvtpk 1861 pgkrkrdqae eepnripsrs lrrtklnqes tapkvlftgv vdargeravl alggslagsa 1921 aeashlvtdr irrtvkflca lgrgipilsl dwlhqsrkag fflppdeyvv tdpeqeknfg 1981 fslqdalsra rerrllegye iyvtpgvqpp ppqmgeiisc cggtylpsmp rsykpqrvvi 2041 tcpqdfphcs iplrvglpll speflltgvl kqeakpeafv lsplemsst By "MDC1 polynucleotide" is meant a nucleic acid molecule encoding a MDC1 polypeptide. An exemplary MDC1 polynucleotide sequence is provided at NCBI Reference Sequence: M_014641.2, and reproduced herein below (SEQ ID NO: 6):
1 caccatgtct aggaggaccg aggaaaggcg ctctggcctt accagacacg tcggacgtct 61 atgacacagc ccctctatcc gttgccggca gctggcgcca gactctctgg tcgcggtttg 121 gaactgcgcg ggaagtgggt ggtgggcggg caagcggtag tgggttgtcc cttggagctg 181 cccaatcgac gtgcattatt ctgttggcgc acggcggcct tcaattaccg tctcattaac 241 tgatctcagc agcctgggag acaccaccta tttgaactct aagggggcgg ggctttgggt 301 gtgcctccgc tcgactggct gcggttgtga aagacagcgg cagaagccaa tcagcaaata 361 agctcttttt cggcacacgc agtcgctcca cctgggtcgc gaccgttact ggtggcgcgc 421 gcggggactt aaagtagatc atggaggaca cccaggctat tgactgggat gttgaagaag 481 aggaggagac agagcaatcc agtgaatcct tgaggtgtaa cgtggagcca gtagggcggc 541 tacatatctt tagtggtgcc catggaccag aaaaagattt cccactacac ctcgggaaga 601 atgtggtagg ccgaatgcct gactgctctg tggccctgcc ctttccatct atctccaaac 661 aacatgcaga gattgaaatc ttagcctggg acaaggcacc tatcctccga gactgtggga 721 gccttaatgg tactcaaatc ctgagacctc ctaaggtttt gagccctggg gtgagtcacc 781 gtctgaggga ccaggaattg attctctttg ctgacttgct ctgccagtac catcgcctgg 841 atgtctctct gccctttgtc tcccggggcc ctctgacagt agaagagaca cccagagtac 901 agggagaaac tcaaccccag aggcttctgt tggctgagga ctcggaggag gaagtagatt 961 ttctttctga aaggcgtatg gtaaaaaaat caaggaccac atcttcctct gtgatagttc 1021 cagagagtga tgaagagggg cattccccgg tcctgggcgg ccttgggccg ccttttgcct 1081 tcaatttgaa cagtgacaca gatgtggaag aaggtcagca accagccaca gaggaggcct 1141 cctcagctgc cagaagaggt gccactgtag aggcaaagca gtctgaagct gaagttgtaa 1201 ctgaaatcca gcttgaaaag gatcagcctt tagtgaagga gagggacaat gatacaaaag 1261 tcaagagggg tgcagggaat ggggtggttc cagctggggt gattctggag aggagccaac 1321 ctcctggaga ggacagtgac acagatgtgg atgatgacag caggcctcct ggaaggccag 1381 ctgaggtcca tttggaaagg gctcagcctt ttggcttcat cgacagcgac actgatgcgg 1441 aagaagagag gatcccagca accccagttg tcattcctat gaagaagagg aagatcttcc 1501 atggagtagg tacaaggggt cctggagcac caggcctggc ccatctgcag gagagccagg 1561 ctggtagtga tacagatgtg gaagaaggca aggccccaca ggctgtccct ctggagaaaa 1621 gccaagcttc catggttatc aacagcgata cagatgacga ggaagaagtc tcagcagcgc 1681 tgactttggc acatctgaaa gagagccagc ctgctatatg gaacagagat gcagaagagg 1741 acatgcccca acgtgtggtc cttctgcagc gaagccaaac caccactgag agagacagtg 1801 acacagacgt ggaggaggaa gagctcccag tggaaaatag agaagctgtc ctcaaggatc 1861 acacaaagat tagagccctt gttagagcac attcagaaaa ggaccaacct ccttttgggg 1921 acagtgatga cagtgtggaa gcagataaga gctcacctgg gatccacctg gagagaagcc 1981 aagcctccac cacagtggac atcaacacac aagtggagaa ggaagtcccg ccagggtcag 2041 ccattataca tataaagaag catcaggtgt ctgtggaggg gacaaatcaa acagatgtga 2101 aagcagttgg gggaccagca aagctgcttg tggtatctct agaggaagcc tggcctctgc 2161 atggggactg tgaaacagat gcagaggagg gcacctccct aacagcctca gtagttgcag 2221 atgtaagaaa gagccagctt ccagcagaag gggatgctgg ggcagagtgg gctgcagctg 2281 ttcttaagca ggagagagct catgaggtgg gggcccaggg tgggccacct gtggcacaag 2341 tggagcagga cctccctatc tcaagagaga acctcacaga tctggtggtg gacacagaca 2401 ctctagggga atccacccag ccacagagag agggagccca ggtccccaca ggaagggaga 2461 gagaacaaca tgtgggtggg accaaggact ctgaagacaa ctatggtgat tctgaagatc 2521 tggacctaca agctacccag tgctttctgg agaatcaggg cctggaagca gtccagagca 2581 tggaggatga acctacccag gccttcatgt tgactccacc ccaagagctt ggcccttccc 2641 attgcagctt ccagacaaca ggtaccctag atgaaccatg ggaggtcctg gctacacagc 2701 cattctgtct gagagagtct gaggactctg agacccagcc ttttgacacg caccttgagg 2761 cctatggacc ttgcctgtct ccacctaggg caataccagg agaccaacat ccagagagcc 2821 cagttcacac agagccaatg gggattcaag gcagagggag gcagactgtg gataaagtca
2881 tgggtatacc aaaagaaaca gcagagaggg tgggccctga gagagggcca ttggagagag
2941 aaactgagaa actgctacca gaaagacaga cagatgtgac aggagaggaa gaattaacca
3001 aggggaaaca ggacagagaa caaaaacagt tgttagctag agacacccag agacaagaat
3061 ctgacaaaaa tggggaaagt gcaagtcctg aaagagatag ggagagtttg aaggtagaaa
3121 ttgagacatc tgaggaaata caagagaaac aagtacagaa gcagaccctt ccaagcaaag
3181 catttgagag agaagtagag agaccagtag caaacagaga gtgcgatcca gccgagttag
3241 aagagaaggt gcccaaagtg atcctggaga gagatacaca gagaggggag ccagagggag
3301 ggagccagga ccagaaaggg caggcctcca gcccaacacc agagcctggg gtgggggcgg
3361 gggaccttcc gggacctacc tcagcccccg taccttctgg gagccagtca ggtggaaggg
3421 gatccccagt gagccccagg aggcatcaga aaggcctcct gaattgcaag atgccacctg
3481 ctgagaaggc ttccaggatc agagctgctg agaaggtttc caggggcgat caggaatctc
3541 cagatgcttg tctgcctcct acagtacctg aagccccagc cccaccccaa aagcccctta
3601 actctcagag ccagaaacat cttgcacctc cgccccttct ttctcccctt ttaccttcta
3661 tcaagccaac cgttcgtaag accaggcaag atgggagtca ggaagctcca gaggctccct
3721 tgtcctcaga gctggagcct ttccacccaa agcctaaaat tagaactcgg aagtcctcca
3781 gaatgacacc ctttccagct acctctgctg cccctgagcc ccacccttcc acctccacag
3841 cccagccagt cactcccaag cccacatctc aggccactag gagcaggaca aataggtcct
3901 ctgtcaagac ccctgaacca gttgtcccca cagcccctga gctccagcct tccacctcca
3961 cagaccagcc tgtcacctct gagcccacat ctcaggttac taggggaaga aaaagtagat
4021 cctctgtcaa gacccctgaa acagttgtgc ccacagccct tgagctccag ccttccacct
4081 ccaccgaccg acctgtcacc tctgaaccca cctctcaggc tactagggga agaaaaaata
4141 gatcctctgt caagacccct gaaccagttg tccccacagc ccctgagctc cagccttcca
4201 cctccacaga ccagcctgtc acttctgagc ccacatatca ggctactagg ggaagaaaaa
4261 atagatcctc tgtcaagacc cctgaaccag ttgtgcccac agcccctgag ctccggcctt
4321 ccacctccac agaccgacct gtcaccccca agcccacatc tcggaccact aggagcagga
4381 caaatatgtc ctctgtcaag acccctgaaa cagttgtccc cacagcccct gagctccaga
4441 tttccacctc cacagaccaa cctgtcaccc ctaagcccac atctcggacc actaggagca
4501 ggacaaatat gtcctctgtg aagaaccctg aatcaactgt ccctatagcc cctgagctcc
4561 caccttccac ctccacagag cagcctgtca cccctgagcc cacatctcgg gctactaggg
4621 gaagaaaaaa tagatcctct ggcaagaccc ctgaaacact tgtccccaca gcccctaagc
4681 tcgagccttc cacttccaca gaccaacctg tcactcctga gcccacatct caggccacca
4741 ggggcaggac aaataggtcc tctgtgaaga cccctgaaac agttgtcccc acagcccctg
4801 agctccagcc ttccacctcc acagaccagc ctgttacccc tgagcctacg tctcaggcta
4861 ctaggggaag aacagataga tcctctgtca agactcctga aacagttgtc cccacagccc
4921 ctgagctaca ggcttccgcc tccacagacc agcctgtcac ctctgagccc acatctcgga
4981 ccactagggg aagaaaaaat cggtcctctg tcaagacccc tgaaacagtt gtgcccgcag
5041 cccctgagct ccagccttcc acctccacag accaacctgt cacccctgag cccacatctc
5101 gggccactag gggcaggaca aataggtcct ctgtcaagac ccctgaatca attgtcccta
5161 tagcccctga gcttcagcct tccacctcca gaaaccagct tgtcacccct gagcccacat
5221 ctcgggccac taggtgcagg acaaataggt cctctgtcaa gacccctgag ccagttgtcc
5281 ccacagcccc tgagccccat cctaccacct ccacagacca gcctgtcacc cccaagctca
5341 catctagggc cactaggaga aagacaaata ggtcctctgt caagactccc aaaccagttg
5401 aaccagcagc ctctgatctt gagcctttta cccccacaga ccagtccgtc acccctgagg
5461 ccatagctca gggtggtcag agcaaaacac tgaggtcttc cacagtaaga gctatgccgg
5521 ttcctaccac ccctgaattc caatctcctg tcaccacaga ccagcctatt tcccctgagc
5581 ctattactca acccagttgc atcaagaggc agagagccgc tgggaaccct ggctccctcg
5641 cagctcccat tgaccataag ccttgctctg cacccttgga acctaaatcc caggcctcaa
5701 ggaaccaaag atggggagca gtgagagcag ctgaatccct tacagccatt cctgagcctg
5761 cctctcccca gcttcttgag acaccaattc atgcctccca gatccaaaag gtggaaccag
5821 caggtagatc taggttcacc ccggagctcc agcctaaggc ctctcaaagc cgcaagaggt
5881 ctttagctac catggattca ccaccacatc aaaaacagcc ccaaagaggg gaagtctccc
5941 agaagacagt gattatcaag gaagaggaag aagatactgc agagaagcca gggaaggaag 6001 aggatgtcgt gactccaaaa ccaggcaaga gaaagagaga ccaggcagag gaggagccca 6061 acagaatacc aagccgcagc ctccgacgga ccaaacttaa ccaagaatca acagccccca 6121 aagtgctctt cacaggagtg gtggatgctc ggggagagcg ggctgtgctg gcactggggg 6181 gaagtctggc tggttcagcg gcagaggctt cccacctggt cactgatcgc atccgccgga 6241 cagtcaagtt cctgtgtgcc ctggggcggg gaatccccat tctgtccctg gactggctgc
6301 atcagtcccg caaggctggt ttcttcttac ccccggatga atatgtggtg accgaccctg 6361 agcaagagaa gaactttggc tttagccttc aagacgcact gagcagggct cgggagcgaa 6421 ggctgctaga gggctatgag atctatgtga cccctggagt ccagccacca ccacctcaga 6481 tgggagagat tattagctgc tgtggaggca catacctacc cagcatgcct cggtcctata 6541 agcctcagag agttgtgatc acatgccctc aggacttccc tcattgctcc attccactac
6601 gggttgggct gcccctcctc tcgcctgagt tcctgctgac tggagtgctg aagcaggaag 6661 ccaagccaga ggcctttgtc ctctcccctt tggagatgtc atccacctga gaactccact 6721 acccttttcc ctcccagacc acgaattaga agatatgtgg aagaaagaac tcagggcgtt 6781 agaaaggatt ggggtatatt gatacaactt gtcctggaac atgggtggga ccagaaatct 6841 ttatgaataa atgaaaagat aagggatttg gaagccacag gttgtttttt gtttgtttgt
6901 ttgttttttt aatggccatt ttattttatt tgtatttata gttttttatt tgtatagatt 6961 taggggatac aagatttctt acatgcatgt attaaatggc cattttaaaa ttagctagtt 7021 tcatgctcag atgtcataag tggcagctat ctttagccag actgttgcag ttattgctcg 7081 atgccactca tggtgtccta cctcctattt ggaaaccatc tctatttttt tcttactgag 7141 attcttactt tggggtcagg aacttgaagg gatgcttgga gtgagtagat ttgagggtcc
7201 agttatggag tgctactaaa acattttctt ctctcctggc ctctggaagc atctttagct 7261 ttgactttgg gcaagtctct gtacttttct ggccagcttt tccaggattt ataaaattag 7321 agcttcggct tgacctctgt gataaataaa tattcactct gtgccttaaa aaaaaaaaaa 7381 aaaaa
By "DNA damaging agent" is meant a chemotherapeutic agent that introduce lesions in the DNA of a cell that lead to replication-associated DNA double-strand breaks (DSBs), which are toxic to the cell.
By "DNA double-stand breaks (DSBs) repair," or "DNA double-stand breaks (DSBs) repair" is meant a collection of processes in which a cell identifies and corrects damage to DNA encoding the cell's genome.
By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.
By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.
In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes,"
"including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
"Detect" refers to identifying the presence, absence or amount of the analyte to be detected.
By "disease" is meant any condition or disorder that damages, or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include any cancer, including but not limited to breast cancer, prostate cancer, or colon cancer.
By "effective amount" is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
The invention provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.
By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
"Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
By "inhibitory nucleic acid" is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.
The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder. Cancers of the invention are those characterized by a reduction in, or an alteration in, or the loss of markers Pten and p53.
As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent.
By "reduces" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or
100%.
By "reference" is meant a standard or control condition.
A "reference sequence" is a defined sequence used as a basis for sequence
comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
By "siRNA" is meant a double stranded RNA. Optimally, a siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3' end. These dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream. Such siRNAs are used to downregulate mRNA levels or promoter activity.
By "specifically binds" is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;
aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e"3 and e"100 indicating a closely related sequence.
By "subject" is meant a mammal, including, but not limited to, a human or non- human mammal, such as a bovine, equine, canine, ovine, or feline.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.
Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGs. 1 A-1K show ATM-mediated phosphorylation of PTEN is required for binding the BRCT domain of MDCl upon DNA damage signaling. FIGs. 1 A and IB: Immunoblot (IB) analysis of anti-PTEN immunoprecipitations (IPs) and whole cell lysates (WCL) derived from NIH3T3 cells treatment with 30 μΜ etoposide (FIG. 1A) or after irradiation (IR) (5 Gy; Gy refers the gray symbol, a derived unit of ionizing radiation does in the International System of Units (SI)) (FIG. IB), at indicated time points before harvesting. FIG. 1C: IB analysis of anti-PTEN IPs and WCL derived from NIH3T3 cells firstly treated using 1 μΜ Ku55933 (ATM inhibitor), ΙμΜ VE821 (ATR inhibitor) and 1 μΜ Νιι7026 (DNAPK inhibitor) for 90 minutes (min) followed by addition of 30 μΜ etoposide for 30 min before harvesting. FIG. ID: IB analysis of anti-HA IPs and WCL derived from 293T cells that ectopically express HA-tagged wild type (WT) or S398A mutant mouse PTEN (mPTEN). 36 hours (hr) after transfection, 30 μΜ etoposide was added and cells were harvested at indicated time points for IP analysis. FIG. IE: A schematic representation of the indicated domains of 53BP1, MDCl and PTEN. FIG. IF: IB analysis of GST pull-down precipitations and WCL derived from U20S cells treatment with/without 30 μΜ etoposide for 30 min before harvesting. FIG. 1G: IB analysis of anti-PTEN IPs and WCL derived from U20S cells treatment with 30 μΜ etoposide at indicated time points. FIG. 1H: IB analysis of anti- Flag-IPs and WCL derived from U20S cells co-transfected empty vector (EV), HA-tagged wild type (WT) or T398A human PTEN (hPTEN) with Flag-MDCl, respectively. 36 hr after transfection, cells were treated with/without 30 μΜ etoposide for 30 min and harvested for IP assays. FIGs. II and 1 J: IB analysis of anti-PTEN IPs and whole cell lysates (WCL) derived from U20S cells (FIG. II), or derived ΐτονη Mmset+/+ and Mmsef ' MEFs (FIG. 1J), treatment with 30 μΜ etoposide as indicated time points before harvesting. FIG. IK: IB analysis of anti-PTEN IPs and WCL derived from Mdcl+/+ and Mdcl''' MEFs treatment with 30 μΜ etoposide as indicated time points before harvesting.
FIGs. 2A-2K show DNA-damaging agent induced phosphorylation of PTEN is required for its interaction with the BRCT domain of MDCl . FIGs. 2A and 2B: Immunoblot (IB) analysis of anti-PTEN immunoprecipitations (IPs) and whole cell lysates (WCL) derived from MEFs cells treatment after irradiation (FIG. 2A) or with/without 30 μΜ etoposide (IR, 5 Gy) treatment (FIG. 2B) at indicated time points before harvesting. FIG. 2C: IB analysis of anti-HA IPs and WCL derived from U20S cell lines stably expressing shScramble (shScr), shATM, or shATF transfected with indicated constructs. 36 hours (h) after transfection, cells were treated with/without 30 μΜ etoposide for 30 min before harvesting. FIGs. 2D and 2F: IB analysis of GST pull-down precipitations and WCL derived from U20S cells treatment with/without 30 μΜ etoposide for 30 min before harvesting. FIG. 2E: Sequence alignment of PTEN C-tails between human and mouse. FIG. 2G: A schematic representation of the indicated domains of PTEN, including N-terminus (N-tail and phosphatase domain) and C- terminus (C2 and C-tail domain). FIG. 2H: IB analysis of GST pull-down precipitations and WCL derived from U20S cells treatment with 30 μΜ etoposide for 30 min before harvesting. FIG. 21: IB analysis of anti-Flag IPs and WCL derived from U20S transfected with indicated constructs. 36 hours after transfection, cells were treated with/without 30 μΜ etoposide for 30 min before harvesting. FIG. 2J: A schematic illustrate to demonstrate that DNA damages induce MDCl -BRCT domain binding with PTEN and MMSET, respectively, largely through a phosphorylation-dependent manner. FIG. 2K: A schematic representation of the indicated domains of 53BP1, MDCl and BRCAl .
FIGs. 3 A-3B show MMSET methyltransferase specifically interacts with, and promotes the methylati on of PTEN. FIG. 3 A: Immunoblot (IB) analysis of anti -Flag immunoprecipitations (IPs) and whole cell lysates (WCL) derived from HEK293T cells with indicated antibodies. FIG. 3B: IB analysis of anti -Flag IPs and WCL derived from
HEK293T cells transfected with indicated constructs.
FIGs. 4A-4M show DNA damage promotes MMSET-mediated di-methylation of PTEN at K349, which is subsequently recognized by the tudor domain of 53BP1. FIG. 4A: Immunoblot (IB) analysis of anti-PTEN immunoprecipitation (IPs) and whole cell lysates (WCL) derived from U20S cells treatment with 30 μΜ etoposide as indicated time points before harvesting. FIG. 4B: IB analysis of anti-PTEN IPs and WCL derived from MDC1+/+ and MDCf ' MEFs treatment with 30 μΜ etoposide as indicated time points before harvesting. FIG. 4C: IB analysis of anti-HA IPs and WCL derived from 293T cells transfected with the indicated constructs. FIG. 4D: IB analysis of anti-HA IP and WCL derived from U20S cells transfected with HA-PTEN WT or K349R mutant and treated with/without etoposide at indicated time points. FIG. 4E: IB analysis of anti-PTEN IPs and WCL derived from U20S cells stably expressing shScramble or shMMSET that were treated with irradiation (TR) (5 Gy) at indicated time points before harvesting. FIG. 4F: IB analysis of anti-PTEN IPs and WCL derived from U20S cells treated with 1 μΜ or 2 DZNep for 24 hours followed by addition of 30 μΜ etoposide for 30 min before harvesting. FIG. 4G: IB analysis of anti-PTEN IPs and WCL derived from U20S cells treated with 30 μΜ etoposide at indicated time points before harvesting. FIG. 4H and 4J: IB analysis of GST pull-down precipitates and WCL derived from U20S cells transfected with the indicated constructs. 36 hr after transfection, cells were treated with/without 30 μΜ etoposide for 30 min and harvested for GST pull-down assays. FIG. 41: IB analysis of GST pull-down precipitates and WCL derived from U20S cells transfected with the indicated constructs. 36 hr after transfection, cells were harvested for GST pull-down assays. FIG. 4K: 1 μg of indicated biotin-labeled synthetic PTEN peptides were incubated with 250 ng purified recombinant GST-tagged 53BP1 tudor domain, respectively. Streptavidin beads were added to perform pull-down assays and the precipitations were analyzed by IB. Dot blot assays were performed to show equal amount of biotinylated peptides were used for the pull-down assay. FIG. 4L: IB analysis of anti-PTEN IPs and WCL derived from U20S cells stably expressing shScramble or shMMSET that were treated with/without 30 μΜ etoposide for 30 min before harvesting. FIG. 4M: IB analysis of anti-PTEN IPs and WCL derived from WT or MMSET ' MEFs that were treated with/without 30 μΜ etoposide for 30 min before harvesting.
FIGs. 5A-5C show PTEN binding with MM SET is largely dependent on MDC1 and the T398 phosphorylation status of PTEN. FIG. 5A: Immunoblot (IB) analysis of anti-PTEN immunoprecipitations (IPs) and whole cell ly sates (WCL) derived from MDC1+/+ or MDC1" " MEFs treated with 30 μΜ etoposide at indicated time points before harvesting. FIG. 5B: IB analysis of anti-Flag IP and WCL derived from U20S cells co-transfected with HA-PTEN WT or T398A mutant with Flag-MMSET and treated with/without etoposide for 60 min before harvesting. FIG. 5C: IB analysis of anti-HA IP and WCL derived from U20S cells co-transfected with indicated constructs and treated with/without 30 μΜ etoposide for 60 min before harvesting. FIGs. 6A-6G show MMSET largely binds the C-terminal domain of PTEN to promote di -methyl ati on of PTEN at the K349 residue. FIG. 6A: A schematic representation of the indicated domains of PTEN, including N-terminus (N-tail and phosphatase domain) and C-terminus (C2 and C-tail domain), which is required for PTEN interaction with
MMSET. FIG. 6B: Immunoblot (IB) analysis of immunoprecipitations (IPs) and whole cell lysates (WCL) derived HEK293T cells transfected with the indicated constructs and treated with 30 μΜ etoposide for 30 min before harvesting. FIG. 6C: IB analysis of anti-HA IP and WCL derived from HEK293T cells transfected with the indicated constructs and treated with/without 30 μΜ etoposide for 30 min before harvesting. FIGs. 6D and 6E: IB analysis of anti-HA IP and WCL derived from HEK293T cells transfected with the indicated constructs, which were treated with/without 30 μΜ etoposide for 30 min before harvesting. FIG. 6E: Mass spectrometry analysis was performed with immunoprecipitated HA-PTEN derived from HEK293T cells co-transfected with HA-PTEN and Flag-MMSET. Different peptides derived from PTEN were enriched and the K349 di-methylation site was identified as a +28 Dolton increased. FIG. 6F: The sequence alignment of PTEN among different species to illustrate that the K349 di-methylation site (K349me2) is evolutionarily conserved. FIG. 6G: A surface diagram of the crystal structure of PTEN (PDB code: 1D5R). The N-terminal domain of PTEN is indicated in the left side of the figure, and C-terminal domain is indicated in the right side of the figure. The identified Lys349, is located on the upper right side of the figure, and is at the outer face of PTEN C-terminal domain. The structure model was analyzed using PyMOL software.
FIGs. 7A-7C shows the generation and validation of the antibodies that specifically recognize K349 di-methylation (K349me2) of PTEN. FIG. 7A: A schematic representation of the various biotinylated synthetic PTEN-derived peptides covering amino acids 340-358 of PTEN. FIG. 7B: Each indicated synthetic peptides in (FIG. 7 A) was diluted and used for the dot immunoblot analysis with the anti-PTEN-K349me2, anti-PTEN-K349me3 or anti-Biotin antibody, respectively. FIG. 7C: IB analysis of anti-PTEN IPs and WCL derived from U20S cells treated with 1 μΜ or 2 DZNep for 24 hours followed by addition of 30 μΜ etoposide for 30 min before harvesting.
FIGs. 8A-8D show 53BP1 recognizes the K349 di-methylation species of PTEN largely through its tudor domain. FIG. 8A: A schematic illustration of the indicated domains of 53BP1 to show its tudor domain binding with reported dimethylation lysine 20 of H4 (H4K20me2) and dimethylation lysine 810 of Rb (RbK810me2). FIG. 8B: IB analysis of anti-His IPs and WCL derived from U20S cells treatment with/without 30 μΜ etoposide for 60 min before harvesting. FIG. 8C: IB analysis of GST pull-down and WCL derived from U20S cells co-transfected with HA-PTEN WT or K349R mutant with Flag-MMSET and treated with 30 μΜ etoposide for 60 min before harvesting. FIG. 8D: A schematic model to illustrate that DNA damage induces the interaction between the MDC1-BRCT domain and PTEN or MMSET, respectively, through a phosphorylation-dependent manner.
Subsequently, MMSET promoted the K349 dimethylation of PTEN, which is recognized by the 53BP1 tudor domain.
FIGs. 9A-9P show protein phosphatase activity of PTEN is required for efficient complement of DSBs repair largely through dephosphorylating γΗ2ΑΧ. FIG. 9A:
Immunoblot (IB) analysis of whole cell lysates (WCL) derived from PTEN^/+ and ΡΤΕΚ ' HCT116 cells after treatment with IR (5 Gy) as indicated time points. FIG. 9B:
Quantification of protein intensity in FIG. 9A was performed using the ImageJ software. γΗ2ΑΧ immunoblot bands were normalized to Vinculin, then normalized to the control (no IR treatment). FIGs. 9C and 9D: IB analysis of WCL derived from ΡΊΕ ' HCT116 cells introducing PTEN WT, C124S, G129E, Y138L as well as EV, which were treated with IR (5 Gy) at indicated time points before harvesting. Quantification of protein intensity in FIG. 9C was performed using the ImageJ software FIG. 9D. γΗ2ΑΧ immunoblot bands were normalized to Vinculin, then normalized to the control (no IR treatment). FIGs. 9E and 9F: IB analysis of WCL derived from ΡΊΕ ' HCT 116 cells introducing PTEN WT, K349R as well as EV, which were treated with IR (5 Gy) at indicated time points before harvesting (FIG. 9D). Quantification of protein intensity in (FIG. 9E) was performed using the ImageJ software (FIG. 9F). FIGs. 9G and 9H: IB analysis of anti-HA IPs, anti-Flag IPs and WCL derived from U20S cells transfected with the indicated constructs. 36 hours after transfection, cells were harvested for IP assays after treatment with 30 μΜ etoposide for 30 min. FIG. 91: In vitro dephosphorylation assays with bacterially purified recombinant GST- tagged PTEN WT and the indicated PTEN mutants including C124S, G129E, and Y138L incubating with indicated H2AX synthetic peptides, then analyzed by dot immunoblot analyses. FIG. 9J: Immunoblot (IB) analysis of whole cell lysates (WCL) derived from PTE} /+ and ΡΤΕΚ ' HCT116 cells after treatment with IR (5 Gy) as indicated time points. FIG 9K and FIG. 9L: IB analysis of WCL derived from Pten+/+, PtenG129E/+ and Ptencl24S/+ MEFs, which were treated with IR (5 Gy) at indicated time points before harvesting.
Quantification of protein intensity in (FIG. 9K) was performed using the ImageJ software (FIG. 9L). γΗ2ΑΧ immunoblot bands were normalized to Vinculin, and then normalized to the control (no IR treatment). FIG 9M and FIG. 9N: Immunohistochemistry (IHC) analysis of spleen tissue derived from Pten+/+, PtenG129E/+ and Ptencl24S/+ mice, which were treated with IR (3 Gy) and sacrificed at 24 h after irradiation (FIG. 9M). Bar, 50 μπι. IB analysis of the sample was performed using indicated antibodies (FIG. 9N). Four mice each group. FIG. 90: IB analysis of anti-PTEN IPs and WCL derived from Mmset+/+ and Mmset_/" MEFs treatment with/without IR (5 Gy) for 60 min before harvesting. FIG. 9P: HAPTEN accumulates at sites of laser microirradiation in HCTl 16-PTEN~ ~ cells. Scale bar: 10 μΜ.
FIGs. 1 OA- 10E shows the K349R mutation in PTEN does not affect its lipid phosphatase activity. FIG. 10A: Immunoblot (IB) analysis of whole cell lysates (WCL) derived from HCTl 16 TEA^ cells reconstituted with the indicated PTEN WT and PTEN- mutant proteins. FIG. 10B: A table summary of the lipid versus protein phosphatase activity of reported PTEN WT and various well-characterized PTEN mutants. FIG. IOC: IB analysis of WCL derived from HCTl 16 PTEN'1' cells reconstituted with the PTEN WT, K349R mutant, as well as empty vector (EV) using indicated antibodies. FIG. 10D: IB analysis of anti-HA IPs, anti-Flag IPs and WCL derived from U20S cells transfected with the indicated constructs. 36 hr after transfection, cells were harvested for IP assays after 30 uM etoposide treatment for 60 min. FIG. 10E: The amino acid sequences of synthetic H2AX and γΗ2ΑΧ peptides used in the present invention.
FIGs. 11 A-l 1C show the protein phosphatase activity and K349 methylation of PTEN is required for completing DSB repair process. FIG. 11 A: Immunoblot (IB) analysis of whole cell lysates (WCL) derived from JEN-deficient HCTl 16 cells reconstituted with the indicated PTEN WT and the indicated PTEN mutant proteins. FIGs. 1 IB and 11C: PTEN- deficient U87MG cells reconstituted with indicated constructs were subjected to
immunofluorescence assays with indicated antibodies as described in Methods.
Representative immunofluorescence images of 53BP1 or H2AX foci are shown.
Quantification of 1 IB and 11C are shown in FIG. 12A and FIG. 12B.
FIGs. 12A-12W show MMSET-mediated methylation of PTEN at K349 dictates cellular sensitivity to DNA-damaging agents. FIGs. 12A and 12B: TEN-deficient U87MG cells reconstituted with the indicated PTEN constructs were treated with IR (5 Gy) and immunostained at the indicated times with anti-53BPl (FIG. 12A) or anti-yH2AX (FIG. 12B). Quantification of 53BP1 or γΗ2ΑΧ foci positive cells (foci > 5 per cell) are shown, respectively. FIGs. 12C and 12D: U20S cells stably expressing shScr or shMMSET were treated with IR (5 Gy) and immunostained at the indicated times with anti-53BPl (FIG. 12C) or anti-YH2AX (FIG. 12D). Quantification of 53BP1 or γΗ2ΑΧ foci positive cells (foci > 5 per cell) are shown, respectively. FIGs. 12E and l2F: TEN-deficient U87MG cells reconstituted with the indicated PTEN constructs were pre-treated with 1 μΜ BKM120 for 24 hours followed by an additional 10 IR treatments (5 Gy). 48 hours post-irradiation, cells were harvested for cell viability assay (FIG. 12E) or cell apoptosis assays (FIG. 12F). Data are shown as mean ± s.d. from three independent experiments. * p<0.05 (t-test). FIG. 12G: Immunoblot (IB) analysis of whole cell lysates (WCL) derived from the samples of (FIGs. 12E-12F) with indicated antibodies. FIGs. 12H-12I: U20S cells stably expressing shScr or shMMSET were pre-treated with 1 μΜ BKM120 for 24 hours followed by an additional 10 IR treatments (5 Gy). 48 hours post-irradiation, cells were harvested for cell viability assay (FIG. 12H) or cell apoptosis assays (FIG. 121). Data are shown as mean ± s.d. from three independent experiments. * p<0.05 (t-test). FIGs. 12J and 12K: U20S cells stably expressing shScr or shMMSET were pre-treated with 1 μΜ BKM120 and 2 μΜ DZNep for 24 hours followed by an additional 10 IR treatments (5 Gy). 48 hours post-irradiation, cells were harvested for cell viability assay (FIG. 12J) or cell apoptosis assays (FIG. 12K). Data are shown as mean ± s.d. from three independent experiments. * p<0.05 (t-test). FIG. 12L: IB analysis of WCL derived from the samples of (FIG. 12J-12K) with indicated antibodies. FIG. 12M: HCT116 cells were pre-treated with/without 1 μΜ BKM120 for 24 h followed by additional IR (0.5 Gy) and/or 2 μΜ DZNep treatment. After one week, cells were stained with crystal violet. FIG 12N: Ρ7ΈΝ*/+ and PTEN' ' HCT116 cells were pre-treated with/without 1 μΜ BKM120 24 h followed by additional etoposide (20 μΜ) and/or 2 μΜ DZNep as indicated. 48 h post treatment, cells were harvested for cell viability and apoptosis assays. Data are represented as mean ± S.D., N = 3, *p < 0.05 and NS indicates no significant difference (Student's t-test). FIG. 120-Q: Tumor xenograft mouse assays were performed by inplanting PTEN-/- HCT1 16 cells stably expressing PTEN WT, K349R and empty vector (EV). Tumor growth rate in nude mice treated every other day with
combination of etoposide (20 mg/kg) and BKM120 (25 mg/kg) was shown (FIG. 120). Tumors were dissected after euthanizing the mice and tumors were recorded at the time of sacrifice (FIG. 12P). IB analysis of the samples was performed using indicated antibodies (FIG. 12Q). Statistical analysis of tumor volumes showed significant differences in mean tumor volumes between the PTEN-WT and the PTEN-K349R groups. Four mice each group. *p < 0.05 (Student's t-test). FIG. 12R: PTE /+ and ΡΊΕ ' HCT1 16 cells stably depleting MMSET by shRNA (with shScr as a negative control) were pre-treated with 1 μΜ
BKM120 for 24 hours (h) followed by additional etoposide (20 μΜ) treatment. 48 h post- treatment, cells were harvested for cell apoptosis assays. Data were represented as mean ± S.D., N = 3, and *p < 0.05 (Student's t-test). FIG. 12S: Tumor xenograft mouse assays were performed by injection of PTE} /+ and PTEN~ ~ Ή£Τ\ \6 cells stably expressing shRNA against MMSET or shScr as a negative control. Tumor growth rate in nude mice treated every other day with a combination of etoposide (20 mg/kg) and BKM120 (25 mg/kg) was shown in FIG. 12S. Tumors were dissected after euthanizing the mice and were analyzed by IB with indicated antibodies (FIG 12T). Statistical analysis of tumor volumes showed significant differences in mean tumor volumes between the shMMSET and the shScr groups. Four mice each group. *p < 0.05, NS indicates no significant difference (Student's t-test). FIG. 12U: PTEN*/+and ΡΤΕ ' HCT1 16 cells were pre-treated with 1 μΜ BKM120 for 24 hours (h) followedby additional etoposide (20 μΜ) treatment. 48 h post- treatment, cells were harvested for cell apoptosis assays. Data were represented as mean ± S.D., N = 3. *p < 0.05, NS indicates no significant difference (Student' s t-test). FIG 12V and FIG. 12W: Tumor xenograft mouse assays were performed by subcutaneously implanting PTE} /+ and ΡΤΕΚ ' HCT1 16 cells. Tumor growth rate in nude mice treated every other day with a combination of etoposide (20 mg/kg) and BKM120 (25 mg/kg) with DZNep (1 mg/kg) (or with vehicle as a negative control) was shown (FIG. 12V). Tumors were dissected after euthanizing the mice and were performed by IB analysis using indicated antibodies (FIG. 12W). Four mice each group. *p < 0.05, NS indicates no significant difference (Student's t-test).
FIGs. 13A-13B shows depletion of MMSET causes DNA DSB repair defects. FIGs. 13A and 13B : U20S cells stably expressing shScr or shMMSET were treated with IR (5 Gy) and immunostained at the indicated times with anti-53BPl (FIG. 13 A) or anti-yH2AX (FIG. 13B). Representative immunofluorescence images of 53BP1 or H2AX foci are shown.
Quantification of FIG. 13A and FIG. 13B are shown in FIG. 12C and FIG. 12D.
FIGs. 14A-14D show loss of PTEN protein phosphatase activity or K349 methylation event sensitizes cells to IR and BKM120 combination treatment. FIGs. 14A-14C: PTEN- deficient HCT1 16 cells reconstituted with the PTEN WT (PTEN^I+) or empty vector
(PTEW'~) were treated as indicated. Cells were harvested for cell viability assay (FIG. 14A) or cell apoptosis assays (FIG. 14B-14C). FIG. 14D: JEN-deficient HCTl 16 cells reconstituted with the indicated PTEN constructs were pre-treated with 1 μΜ BKM120 for 24 hours followed by an additional 10 IR treatments (5 Gy). 48 hours post-irradiation, cells were harvested for cell apoptosis assays. Data are shown as mean ± s.d. from three independent experiments. * p<0.05 or ** p<0.001 (t-test)
FIGs. 15A-15D show loss of PTEN protein phosphatase activity or K349 methylation event sensitizes cells to etoposide and BKM120 combination treatment. FIGs. 15A and 15B: TEN-deficient U87MG cells reconstituted with the PTEN WT (PTEN+/+) or empty vector were treated as indicated. Cells were harvested for cell apoptosis assays. FIGs. 15C and 15D: PTEN-deficient U87MG cells reconstituted with the indicated PTEN constructs were pre-treated with 1 μΜ BKM120 for 24 hours followed by additional 20 uM etoposide treatment. 48 hours after etoposide treatment, cells were harvested for cell apoptosis assays. Data are shown as mean ± s.d. from three independent experiments. * p<0.05 (t-test).
FIGs. 16A-16B show inhibition of MMSET by shRNA or DZNep sensitizes cells to combination treatment DNA damaging agents and BKM120. FIG. 16 A: HCTl 16 cells stably expressing shScr or shMMSET were pre-treated with/without 1 μΜ BKM120 or 2 μΜ DZNep for 24 hours followed by additional IR (2 Gy) as indicated. After 48 hours post- IR, cells were harvested for cell apoptosis assays. FIG. 16B: HCTl 16 cells were pre-treated with/without 1 uM BKM120 or 2 uM DZNep for 24 hours followed by additional IR (2 Gy). After 48 hours post-IR, cells were harvested for cell apoptosis assays.
FIGs. 17A and 17B show U20S cells stably expressing shScr or shMMSET were pre- treated with/without 1 μΜ BKM120 for 24 hours followed by additional etoposide treatment as indicated for 48 hours. Cells were harvested for cell viability assay. Data are represented as mean ± S.D., N = 3, and **p < 0.01 (Student's t-test). FIGs. 17C-17D show U20S cells stably expressing shScr or shMMSET were pre-treated with/without 1 μΜ BKM120 for 24 hours followed by additional etoposide treatment as indicated for 24 hours. After one week, cells were stained with crystal violet (FIG. 17C) and the colony number was counted (FIG. 17D). Data are represented as mean ± S.D., N = 3, and **p < 0.01 (Student's t-test).
FIG. 18 shows a schematic representation of how PTEN methylation and protein phosphatase activity response to DNA damage signaling. Upon DNA damage, the MMSET methyltransferase promotes the methylation of PTEN, which was recruited into DNA damage sites to help complete DNA damage repair through dephosphorylating γ-Η2ΑΧ. FIGs. 19A-19K show ATM-mediated phosphorylation of PTEN is required for binding the BRCT domain of MDC1 upon DNA damage signaling. FIGs. 19A and 19B: Immunoblot (IB) analysis of anti-PTEN immunoprecipitations (IPs) and whole cell lysates (WCL) derived from NIH3T3 cells treatment after irradiation (IR) (5 Gy) (FIG. 19A) or with 30 μΜ etoposide (FIG. 19B) as indicated time points before harvesting. FIG. 19C: IB analysis of anti-PTEN IPs and WCL derived from NIH3T3 cells pre-treated with 1 μΜ Ku55933 (ATM inhibitor), ΙμΜ VE821 (ATR inhibitor) or 1 μΜ ι7026 (DNAPK inhibitor) for 90 minutes (min) followed by addition of 30 μΜ etoposide for 30 min before harvesting. FIG. 19D: IB analysis of anti-HA IPs and WCL derived from U20S cell lines stably expressing shScr or shATM transfected with indicated constructs. 36 hours after transfection, cells were treated with/without 30 μΜ etoposide for 30 min before harvesting. FIG. 19E: IB analysis of anti-HA IPs and WCL derived from 293T cells that ectopically express HA-tagged wild type (WT) or S398A mutant mouse PTEN (mPTEN). 36 hours (hr) after transfection, 30 μΜ etoposide was added and cells were harvested at indicated time points for IP analysis. FIG. 19F: IB analysis of GST pull-down precipitations and WCL derived from U20S cells treatment with/without 30 μΜ etoposide for 30 min before harvesting. FIG. 19G: IB analysis of GST-pull down and WCL derived from U20S cells co- transfected empty vector (EV), HA-tagged wild type (WT) or T398A human PTEN (hPTEN) with GST-MDC1-BRCT, respectively. 36 hr after transfection, cells were treated
with/without 30 μΜ etoposide for 30 min and harvested for IP assays. FIGs. 19H and 191: IB analysis of anti-PTEN IPs and whole cell lysates (WCL) derived from U20S cells treatment with 30 μΜ etoposide as indicated time points before harvesting. FIG. 19J: IB analysis of anti-PTEN IPs and WCL derived from U20S cell lines stably expressing shScr or shATM transfected with indicated constructs. 36 hours after transfection, cells were treated with/without 30 uM etoposide for 30 min before harvesting. FIG. 19K: IB analysis of anti- PTEN IPs and WCL derived from C7+/+ and MDCJ'A MEFs treatment with 30 μΜ etoposide as indicated time points before harvesting.
FIGs. 20A-20I show protein phosphatase activity of PTEN is required for efficient complement of DSBs repair largely through dephosphorylating γΗ2ΑΧ. FIG. 20A:
Immunoblot (IB) analysis of whole cell lysates (WCL) derived from PTEN^/+ and ΡΤΕΚ ' HCT116 cells after treatment with IR (5 Gy) as indicated time points. FIG. 20B:
Quantification of protein intensity in (FIG. 20A) was performed using the ImageJ software. γΗ2ΑΧ immunoblot bands were normalized to Vinculin, then normalized to the control (no IR treatment). FIGs. 20C and 20D: IB analysis of WCL derived from PTEK HCTl 16 cells introducing PTEN WT, C124S, G129E, Y138L as well as EV, which were treated with IR (5 Gy) at indicated time points before harvesting (FIG. 20C). Quantification of protein intensity in (FIG. 20C) was performed using the Image J software (FIG. 20D). γΗ2ΑΧ immunoblot bands were normalized to Vinculin, then normalized to the control (no IR treatment). FIGs. 20E and 20F: IB analysis of WCL derived from ΡΊΕ ' HCTl 16 cells introducing PTEN WT, K349R as well as EV, which were treated with IR (5 Gy) at indicated time points before harvesting (FIG. 20D). Quantification of protein intensity in (FIG. 20E) was performed using the ImageJ software (FIG. 20F). γΗ2ΑΧ immunoblot bands were normalized to Vinculin, then normalized to the control (no IR treatment). FIG. 20G: IB analysis of anti -Flag IPs and WCL derived from U20S cells transfected with the indicated constructs. 36 hr after transfection, cells were harvested for IP assays after treatment with IR (5 Gy) for 60 min. FIG. 20H: IB analysis of anti-PTEN IPs and WCL derived from MMSET^/+ and A4MSETA MEFs treatment with/without IR (5 Gy) for 60 min before harvesting. FIG. 201: In vitro dephosphorylation assays with bacterially purified recombinant GST-tagged PTEN WT and the indicated PTEN mutants including C124S, G129E, and Y138L incubating with indicated H2AX synthetic peptides, then analyzed by dot immunoblot analyses.
FIGs. 21 A-21N show MMSET-mediated methylation of PTEN at K349 dictates cellular sensitivity to DNA-damaging agents. FIGs. 21 A and 21B: TEN-deficient U87MG cells reconstituted with the indicated PTEN constructs were treated with IR (5 Gy) and immunostained at the indicated times with anti-53BPl (FIG. 21 A) or anti-yH2AX (FIG. 2 IB). Quantification of 53BP1 or γΗ2ΑΧ foci positive cells (foci > 5 per cell) are shown, respectively. Data are represented as mean ± S.D., N = 3, and **p < 0.01 (Student's t-test). FIGs. 21C and 21D: U20S cells stably expressing shScr or shMMSET were treated with IR (5 Gy) and immunostained at the indicated times with anti-53BPl (FIG. 21C) or anti-yH2AX (FIG. 2 ID). Quantification of 53BP1 or γΗ2ΑΧ foci positive cells (foci > 5 per cell) are shown, respectively. Data are represented as mean ± S.D., N = 3, and **p < 0.01 (Student's t- test). FIGs. 2 IE and 2 IF: TEN-deficient U87MG cells reconstituted with the indicated PTEN constructs were pre-treated with 1 μΜ BKM120 for 24 hours followed by an additional 10 IR treatments (5 Gy). 48 hours post-irradiation, cells were harvested for cell viability assay (FIG. 21E) or cell apoptosis assays (FIG. 21F). Data are represented as mean ± S.D., N = 3, and *p < 0.05 (Student's t-test). FIG. 21G: Immunoblot (IB) analysis of whole cell lysates (WCL) derived from the samples of (FIG. 21E-21F) with indicated antibodies. FIGs. 21H and 211: U20S cells stably expressing shScr or shMMSET were pre-treated with/without 1 μΜ BKM120 for 24 hours followed by additional IR (10 Gy) treatment as indicated. 48 hours post-irradiation, cells were harvested for cell viability assay (FIG. 21H) or cell apoptosis assays (FIG. 211). Data are represented as mean ± S.D., N = 3, and *p < 0.05 (Student's t-test). FIGs. 21J and 21K: U20S cells stably expressing shScr or shMMSET were pre-treated with/without 1 μΜ BKM120 for 24 hours followed by additional IR (0.5 Gy) treatment as indicated. After one week, cells were stained with crystal violet (FIG. 21 J) and the colony number was counted (FIG. 21K). Data are represented as mean ± S.D., N = 3, and **p < 0.01 (Student's t-test). FIG. 21L: HCT116 cells were pre-treated with/without 1 μΜ BKM120 or 2 μΜ DZNep for 24 hours followed by additional IR (2 Gy) treatment. After 48 hours post-IR, cells were harvested for cell viability assay. Data are represented as mean ± S.D., N = 3, and **p < 0.01 (Student's t-test). FIGs. 21M: U20S cells stably were pre- treated with/without 1 μΜ BKM120 or 2 μΜ DZNep for 24 hours followed by additional IR (0.5 Gy) treatment. After one week, cells were stained with crystal violet (FIG. 21M). FIG. 21N: HCT116 cells were pre-treated with/without 1 μΜ BKM120 for 24 h followed by additional IR (0.5 Gy) and/or 2 μΜ DZNep treatment. After one week, the colony number was counted . Data are represented as mean ± S.D., N = 3, and **p < 0.01 (Student's t-test).
FIG. 22 shows a schematic model to illustrate that PTEN has lipid phosphatase and protein phosphatase activity, which involves in PI3K/Akt signaling pathway and DNA damage/yH2AX pathway, respectively.
FIG. 23 shows depletion of MMSET by shRNAs sensitizes cells to IR or etoposide with BKM120 combinational treatment. FIG. 23A-FIG.23C: HCT116 cells stably expressing shScramble (shScr) or shMMSET were pre-treated with/without Ι μΜ BKM120 for 24 hours (h) followed by additional IR (2 Gy) treatment as indicated. After 48 h post-IR treatment, cells were harvested for cell viability or apoptosis assays. Data were represented as mean ± S.D., N = 3, and *p < 0.05 (Student's t-test). FIG. 23D and FIG. 23E: HCT116 cells stably expressing shScr or shMMSET were pre-treated with/without 1 μΜ BKM120 for 24 h followed by additional IR (0.5 Gy) treatment as indicated. After one week, cells were stained with crystal violet (FIG. 23D) and the colony number was counted (FIG. 23E). Data were represented as mean ± S.D., N = 3, and **p < 0.01 (Student's t-test). FIG. 23F and FIG. 23G: HCT116 cells stably expressing shScr or shMMSET were pre-treated with/without 1 μΜ BKM120 for 24 h followed by additional etoposide (20 μΜ) treatment as indicated. After 48 h etoposide treatment, cells were harvested for cell apoptosis assays. Data were represented as mean ± S.D., N = 3, and **p < 0.01 (Student's t-test). FIG. 23H and FIG. 231: HCT116 cells stably expressing shScr or shMMSET were pre-treated with/without 1 μΜ BKM120 for 24 h followed by additional etoposide (20 μΜ) treatment as indicated. 24 h after etoposide treatment, cells were replaced with fresh medium. After one week, cells were stained with crystal violet (FIG. 23H) and the colony number was counted (FIG. 231). Data were represented as mean ± S.D., N = 3, and **p < 0.01 (Student's t-test). FIG. 23 J and FIG. 23K: PTE? /+ and PTEN~A HCT\ \6 cells were pre-treated with 1 μΜ BKM120 for 24 h followed by additional etoposide (20 μΜ) treatment. 48 h post-treatment, cells were harvested for cell viability and apoptosis assays. Data were represented as mean ± S.D., N = 3. *p < 0.05, NS indicates no significant difference (Student's t-test). FIG. 23L and FIG. 23M: PTE? /+ and ΡΤΕ^'ΉΟ,ΎΙ Ιβ cells were pre-treated with 1 μΜ BKM120 for 24 h followed by additional etoposide (20 μΜ) treatment as indicated. 24 h after etoposide treatment, cells were replaced with fresh medium. After one week, cells were stained with crystal violet (FIG. 23L) and the colony number was counted (FIG. 23M). Data were represented as mean ± S.D., N = 3. *p < 0.05, NS indicates no significant difference (Student's t-test). FIG. 23N: Xenografted tumors in FIG. 12S were dissected after euthanizing the mice and tumors were recorded at the time of sacrifice. Four mice each group.
FIG. 24 shows inhibition of MMSET by DZNep (a pan-inhibitor of S- adenosylmethionine-dependent methyltransferase including MMSET) sensitizes cells to IR or etoposide with BKM120 combinational treatment. FIG. 24 A: ΡΤΕΝ+/+ and ΡΊΕ ' HCT116 cells were pre-treated with/without 1 μΜ BKM120 24 h followed by additional etoposide (20 μΜ) and/or 2 μΜ DZNep as indicated. 48 h post treatment, cells were harvested for cell viability and apoptosis assays. Data are represented as mean ± S.D., N = 3, *p < 0.05 and NS indicates no significant difference (Student's t-test). FIG. 24B and FIG. 24C:
PTE} /+ and ΡΊΕ ' HCT116 cells were pre-treated with/without 1 μΜ BKM120 for 24 h followed by additional etoposide (20 μΜ) or 2 μΜ DZNep as indicated. 24 h post treatment, cells were replaced with fresh medium. After one week, cells were stained with crystal violet (FIG. 24B) and the colony number was counted (FIG. 24C). Data were represented as mean ± S.D., N = 3, *p < 0.05 and NS indicates no significant difference (Student's t-test). FIG. 24D: Xenografted tumors in FIG. 12V were dissected after euthanizing the mice and tumors were recorded at the time of sacrifice. Four mice each group. DETAILED DESCRIPTION OF THE INVENTION
As described below, the present invention features compositions and methods of treating cancers by inhibiting the activity of MMSET methyltransferase. In some
embodiments, the inhibition of MMSET methyltransferase sensitizes cancer cells to chemotherapeutic drugs.
This invention is based, at least in part on the discovery of a novel molecular mechanism for PTEN regulation of DSBs repair through its methylation modification event and protein phosphatase activity: Wherein, DNA DSBs promote ATM-dependent phosphorylation of T398-PTEN, which is specifically recognized by the BRCA1 C
Terminus (BRCT) domain of mediator of DNA damage checkpoint 1 (MDC1). MDC1 is pivotal for PTEN and MMSET interaction following DNA DSBs, which subsequently leads to MMSET -mediated di-methylation of K349 on PTEN. Furthermore, the tudor domain of 53BP1 (FIG. IE) recognizes the methylated PTEN and recruits PTEN to DSB sites to govern the timely repair of DSBs in part through dephosphorylation of γΗ2ΑΧ. As a result, compared to PTEN^/+ cells, the magnitude and duration of γΗ2ΑΧ in PTEN' ' cells are dramatically elevated, especially at the late stage post γ-irradiation (8 and 24 hour time points), which can be largely rescued by re-introducing WT, but not protein-phosphastase deficient mutants (C 124S and Y138L) or a methylation-deficient mutant (K349R) form of PTEN. Consistent with these findings, the levels of γΗ2ΑΧ are upregulated in Pten mice at 24 hours post γ-irradiation compared with Pten+/+ micQ. More importantly, inhibiting MMSET-mediated methylation of PTEN, either through expressing
methylation-deficient PTEN mutants (e.g., the protein-phosphatase dead mutant C124S or Y138L, or methylation-deficient mutant K349R or T398A), or through inhibiting MMSET methyltansferase, sensitizes cancer cells to chemotherapeutic drugs.
In some embodiments, inhibition of MMSET-mediated di-methylation of K349-
PTEN sensitizes cancer cells to chemotherapeutic drugs.
In some embodiments, the present invention provides a method of treating cancer, the method comprising administering (1) an effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein; and (ii) an effective amount of a chemotherapeutic agent to a subject having a cancer.
In some embodiments, the agent that inhibits the expression or activity of MMSET protein sensitizes the cancer to the chemotherapeutic agent. In some embodiments, the method reduces tumor growth, and/or increases subject survival.
In some embodiments, the method inhibits the growth of cancer cells.
In some embodiments, the agent that inhibits the expression or activity of MMSET protein reduces the effective amount of the chemotherapeutic agent necessary to treat the cancer.
In some embodiments, the cancer is prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, or other types of epithelium-derived carcinomas where PTEN inactivation is frequently observed.
Referring to FIG. 18, without intending to be limited to any particular theory, DNA damaging agents result in the death of cancer cells by reducing the integrity of the cancer cell's DNA. DNA integrity is critical for proper cellular function and proliferation. High levels of DNA damage are detected by cell-cycle checkpoint proteins, whose activation induces cell-cycle arrest to prevent the transmission of damaged DNA during mitosis. DNA lesions that occur during the S phase of the cell cycle block replication fork progression and can lead to replication-associated DNA double-strand breaks (DSBs), which are among the most toxic of all DNA lesions. If the damaged DNA cannot be properly repaired, cell death may result.
In support of a role for PTEN in DSBs repair, both large-scale proteomic analyses and in vitro specific biochemical kinase assays identified PTEN to be phosphorylated at T398 in human PTEN (S398 in mouse PTEN, thereafter referred as T/S398) by ataxia telangiectasia mutated (ATM), in response to DNA damage. Moreover, phosphorylation of PTEN at endogenous levels could be readily detected using the phospho-(Ser/Thr) ATM/ATR substrate antibody after etoposide or irradiation (TR) treatment in NIH3T3 (FIGs. 1 A-1B) and mouse embryonic fibroblasts (MEFs) (FIGs. 2A-2B). Moreover, the results described in the Examples below demonstrate that MDC1 plays a pivotal role in connecting the MMSET methyltransferase with PTEN, presumably by recruiting both proteins to the damage sites through its BRCT domain(s) to recognize ATM-mediated phosphorylation of MMSET and PTEN, respectively (FIG. 2K).
Referring again to FIG. 18, in some embodiments, DNA DSBs promote the interaction of PTEN with MDC1, following ATM-dependent phosphorylation of T/S398- PTEN. Additionally, DNADSBs enhance MMSET-mediated di-methylation of K349-PTEN, which is recognized by the tudor domain of 53BP1, thereby enhancing the recruitment of PTEN to DSB sites, and governing the repair process and cellular sensitivity to DNA damage in part by dephosphorylating γ-Η2ΑΧ.
In some embodiments, DNA damaging agents induce MMSET-mediated di- methylation of PTEN at lysine 349. In some embodiments, the di-methylation of PTEN at lysine 349 promotes the integration of PTEN with 53BP1, to facilitate the recruitment of PTEN to sites of DNA damage. Without intending to be limited to any particular theory, the MMSET-mediated di-methylation of PTEN at lysine 349 promotes the repair of damaged DNA, thereby reducing the efficacy of chemotherapeutic agents.
In some embodiments, the agent that inhibits the expression or activity of MMSET protein is a polypeptide, polynucleotide, or a small molecule.
In some embodiments, the agent that inhibits the expression or activity of MMSET protein is an inhibitory nucleic acid molecule that inhibits the expression of a MMSET protein.
In some embodiments, the inhibitory nucleic acid molecule is an antisense molecule, siRNA, or shRNA.
Inhibitory Nucleic Acids
Inhibitory nucleic acid molecules are those oligonucleotides that inhibit the expression or activity of a MMSET polypeptide. Such oligonucleotides include single and double stranded nucleic acid molecules (e.g., DNA, RNA, and analogs thereof) that bind a nucleic acid molecule that encodes a MMSET polypeptide (e.g., antisense molecules, siRNA, shRNA), as well as nucleic acid molecules that bind directly to the polypeptide to modulate its biological activity (e.g., aptamers).
siRNA
Short twenty-one to twenty-five nucleotide double-stranded RNAs are effective at down-regulating gene expression (Zamore et al., Cell 101 : 25-33; Elbashir et al., Nature 411 : 494-498, 2001, hereby incorporated by reference). The therapeutic effectiveness of a siRNA approach in mammals was demonstrated in vivo by McCaffrey et al. (Nature 418: 38- 39.2002).
Given the sequence of a target gene, siRNAs may be designed to inactivate that gene.
Such siRNAs, for example, could be administered directly to an affected tissue, or administered systemically. The nucleic acid sequence of a gene can be used to design small interfering RNAs (siRNAs). The 21 to 25 nucleotide siRNAs may be used, for example, as therapeutics to treat cancer.
The inhibitory nucleic acid molecules of the present invention may be employed as double-stranded RNAs for RNA interference (RNAi)-mediated knock-down of expression. In one embodiment, expression of MMSET polypeptide is reduced in a subject having cancer. RNAi is a method for decreasing the cellular expression of specific proteins of interest (reviewed in Tuschl, Chembiochem 2:239-245, 2001; Sharp, Genes & Devel. 15:485- 490, 2000; Hutvagner and Zamore, Curr. Opin. Genet. Devel. 12:225-232, 2002; and
Hannon, Nature 418 :244-251 , 2002). The introduction of siRNAs into cells either by transfection of dsRNAs or through expression of siRNAs using a plasmid-based expression system is increasingly being used to create loss-of-function phenotypes in mammalian cells.
In one embodiment of the invention, a double-stranded RNA (dsRNA) molecule is made that includes between eight and nineteen consecutive nucleobases of a nucleobase oligomer of the invention. The dsRNA can be two distinct strands of RNA that have duplexed, or a single RNA strand that has self-duplexed (small hairpin (sh)RNA). Typically, dsRNAs are about 21 or 22 base pairs, but may be shorter or longer (up to about 29 nucleobases) if desired. dsRNA can be made using standard techniques (e.g., chemical synthesis or in vitro transcription). Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296:550-553, 2002; Paddison et al. Genes & Devel. 16:948-958, 2002. Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99:6047- 6052, 2002; Miyagishi et al. Nature Biotechnol. 20:497-500, 2002; and Lee et al. Nature Biotechnol. 20:500-505 2002, each of which is hereby incorporated by reference.
Small hairpin RNAs (shRNAs) comprise an RNA sequence having a stem-loop structure. A "stem-loop structure" refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand or duplex (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion). The term "hairpin" is also used herein to refer to stem-loop structures. Such structures are well known in the art and the term is used consistently with its known meaning in the art. As is known in the art, the secondary structure does not require exact base-pairing. Thus, the stem can include one or more base mismatches or bulges. Alternatively, the base-pairing can be exact, i.e. not include any mismatches. The multiple stem-loop structures can be linked to one another through a linker, such as, for example, a nucleic acid linker, a miRNA flanking sequence, other molecule, or some combination thereof.
As used herein, the term "small hairpin RNA" includes a conventional stem-loop shRNA, which forms a precursor miRNA (pre-miRNA). While there may be some variation in range, a conventional stem-loop shRNA can comprise a stem ranging from 19 to 29 bp, and a loop ranging from 4 to 30 bp. "shRNA" also includes micro-RNA embedded shRNAs (miRNA-based shRNAs), wherein the guide strand and the passenger strand of the miRNA duplex are incorporated into an existing (or natural) miRNA or into a modified or synthetic (designed) miRNA. In some instances the precursor miRNA molecule can include more than one stem-loop structure. MicroRNAs are endogenously encoded RNA molecules that are about 22-nucleotides long and generally expressed in a highly tissue- or developmental - stage-specific fashion and that post-transcriptionally regulate target genes. More than 200 distinct miRNAs have been identified in plants and animals. These small regulatory RNAs are believed to serve important biological functions by two prevailing modes of action: (1) by repressing the translation of target mRNAs, and (2) through RNA interference (RNAi), that is, cleavage and degradation of mRNAs. In the latter case, miRNAs function
analogously to small interfering RNAs (siRNAs). Thus, one can design and express artificial miRNAs based on the features of existing miRNA genes.
shRNAs can be expressed from DNA vectors to provide sustained silencing and high yield delivery into almost any cell type. In some embodiments, the vector is a viral vector. Exemplary viral vectors include retroviral, including lentiviral, adenoviral, baculoviral and avian viral vectors, and including such vectors allowing for stable, single-copy genomic integrations. Retroviruses from which the retroviral plasmid vectors can be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. A retroviral plasmid vector can be employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which can be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14x, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy 1 :5-14 (1990), which is incorporated herein by reference in its entirety. The vector can transduce the packaging cells through any means known in the art. A producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a DNA replication protein. Such retroviral vector particles then can be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a DNA replication protein.
Examples of delivery methods suitable to deliver siRNA and shRNA molecules of the present invention are disclosed in Nature Materials Vol 12, 2013, pages 967-977, incorporated by reference in its entirety.
Catalytic RNA molecules or ribozymes that include an antisense sequence of the present invention can be used to inhibit expression of a nucleic acid molecule in vivo (e.g., a nucleic acid encoding MMSET). The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334:585-591. 1988, and U.S. Patent Application Publication No. 2003/0003469 Al, each of which is incorporated by reference.
Accordingly, the invention also features a catalytic RNA molecule that includes, in the binding arm, an antisense RNA having between eight and nineteen consecutive nucleobases. In preferred embodiments of this invention, the catalytic nucleic acid molecule is formed in a hammerhead or hairpin motif. Examples of such hammerhead motifs are described by Rossi et al., Aids Research and Human Retroviruses, 8: 183, 1992. Example of hairpin motifs are described by Hampel et al., "RNA Catalyst for Cleaving Specific RNA
Sequences," filed Sep. 20, 1989, which is a continuation-in-part of U.S. Ser. No. 07/247, 100 filed Sep. 20, 1988, Hampel and Tritz, Biochemistry, 28:4929, 1989, and Hampel et al., Nucleic Acids Research, 18: 299, 1990. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
Essentially any method for introducing a nucleic acid construct into cells can be employed. Physical methods of introducing nucleic acids include injection of a solution containing the construct, bombardment by particles covered by the construct, soaking a cell, tissue sample or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the construct. A viral construct packaged into a viral particle can be used to accomplish both efficient introduction of an expression construct into the cell and transcription of the encoded shRNA. Other methods known in the art for introducing nucleic acids to cells can be used, such as lipid-mediated carrier transport, chemical mediated transport, such as calcium phosphate, and the like. Thus the shRNA-encoding nucleic acid construct can be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, promote annealing of the duplex strands, stabilize the annealed strands, or otherwise increase inhibition of the target gene.
For expression within cells, DNA vectors, for example plasmid vectors comprising either an RNA polymerase II or RNA polymerase III promoter can be employed. Expression of endogenous miRNAs is controlled by RNA polymerase II (Pol II) promoters and in some cases, shRNAs are most efficiently driven by Pol II promoters, as compared to RNA polymerase III promoters (Dickins et al., 2005, Nat. Genet. 39: 914-921). In some embodiments, expression of the shRNA can be controlled by an inducible promoter or a conditional expression system, including, without limitation, RNA polymerase type II promoters. Examples of useful promoters in the context of the invention are tetracycline- inducible promoters (including TRE-tight), IPTG-inducible promoters, tetracycline transactivator systems, and reverse tetracycline transactivator (rtTA) systems. Constitutive promoters can also be used, as can cell- or tissue-specific promoters. Many promoters will be ubiquitous, such that they are expressed in all cell and tissue types. A certain embodiment uses tetracycline-responsive promoters, one of the most effective conditional gene expression systems in in vitro and in vivo studies. See International Patent Application
PCT/US2003/030901 (Publication No. WO 2004-029219 A2) and Fewell et al., 2006, Drug Discovery Today 11 : 975-982, for a description of inducible shRNA. Delivery of Polynucleotides
Naked polynucleotides, or analogs thereof, are capable of entering mammalian cells and inhibiting expression of a gene of interest. Nonetheless, it may be desirable to utilize a formulation that aids in the delivery of oligonucleotides or other nucleobase oligomers to cells (see, e.g., U.S. Pat. Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference).
In one embodiment, an inhibitory nucleic acid molecule described herein is delivered using a nanoparticle. Nanoparticle compositions suitable for use with inhibitory nucleic acid molecules are known in the art and described for example by Kanasty et al., Nature materials 12: 967-977, 2013, which is incorporated herein by reference. Such nanoparticle delivery compositions include cyclodextrin polymer (CDP)-based nanoparticles, lipid nanoparticles, cationic or ionizable lipid, lipid-anchored PEG, PEGylated nanoparticles, oligonucleotide nanoparticles (ONPs), and siRNA-polymer conjugate delivery systems (e.g., Dynamic Poly Conjugate, Triantennary GalNAc-siRNA).
Small Molecule MMSET Methyltransferase Inhibitors
Examples of compounds suitable as MMSET methyltransferase inhibitors include the pan-histone methyltransferase inhibitor (l S,2R,5R)-5-(4-Amino-lH-imidazo[4,5-c]pyridin-l- yl)-3-(hydroxymethyl)-3-cyclopentene-l,2-diol hydrochloride (3-Deazaneplanocin A hydrochloride).
Another example of a compound suitable as a MMSET methyltransferase inhibitor is the inhibitor 3-hydrazinylquinoxaline-2-thiol, disclosed in U.S. Patent No. 8,697,407, incorporated by reference in its entirety.
Another example of a compound suitable as a MMSET methyltransferase inhibitor is the inhibitor LEM-06, disclosed in Journal of Cancer Prevention Vol. 20, No. 2, 2015. pp. 113-120, incorporated by reference in its entirety. LEM-06 has the structure shown in the formula below:
Figure imgf000043_0001
In some embodiments, the agent that inhibits the expression or activity of MMSET protein inhibits the activity of ataxia telangiectasia mutated (ATM).
In some embodiments, the agent that inhibits the expression or activity of MMSET protein is the specific ATM kinase inhibitor Ku55933.
Ku55933 has the structure shown in the formula below:
Figure imgf000044_0001
In some embodiments, the agent that inhibits the expression or activity of MMSET protein is the specific ATM kinase inhibitor AZD0156, which is currently under clinical trial to treat advanced solid tumors.
AZD0156 has the structure shown in the formula below:
Figure imgf000044_0002
Chemotherapeutic Agents
Chemotherapeutic agents suitable for use in the methods of the present invention include, but are not limited to alkylating agents. Without intending to be limited to any particular theory, alkylating agents directly damage DNA to keep the cell from reproducing. Alkylating agents work in all phases of the cell cycle and are used to treat many different cancers, including leukemia, lymphoma, Hodgkin disease, multiple myeloma, and sarcoma, as well as cancers of the lung, breast, and ovary.
Alkylating agents are divided into different classes, including, but not limited to: (i) nitrogen mustards, such as, for example mechlorethamine (nitrogen mustard), chlorambucil, cyclophosphamide (Cytoxan®), ifosfamide, and melphalan; (ii) nitrosoureas, such as, for example, streptozocin, carmustine (BCNU), and lomustine; (iii) alkyl sulfonates, such as, for example, busulfan; (iv) riazines, such as, for example, dacarbazine (DTIC) and temozolomide (Temodar®); (v) ethylenimines, such as, for example, thiotepa and altretamine
(hexamethylmelamine); and (v) platinum drugs, such as, for example, cisplatin, carboplatin, and oxalaplatin. Therapeutic Methods
The methods and compositions provided herein can be used to treat or prevent progression of a cancer. In general, (i) the effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein; and (ii) the effective amount of a chemotherapeutic agent to a subject having a cancer can be
administered therapeutically and/or prophylactically.
The effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein can be administered concurrently with the effective amount of a chemotherapeutic agent. Alternatively, the effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein can be administered before the effective amount of a chemotherapeutic agent. Alternatively, the effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein can be administered after the effective amount of a
chemotherapeutic agent.
Treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk of developing such cancer. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, family history, and the like). Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
In some aspects, the effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein; and the effective amount of a chemotherapeutic agent may be administered in combination with one or more of any other standard anti-cancer therapies. For example, an MMSET inhibitor as described herein may be administered in combination with standard chemotherapeutics. Methods for administering combination therapies (e.g., concurrently or otherwise) are known to the skilled artisan and are described for example in Remington's Pharmaceutical Sciences by E. W. Martin.
Pharmaceutical Compositions
The present invention features compositions useful for treating cancer. The methods include administering an effective amount of (i) an effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein; and (ii) an effective amount of a chemotherapeutic agent to a subject having a cancer in a physiologically acceptable carrier.
Typically, the carrier or excipient for the composition provided herein is a
pharmaceutically acceptable carrier or excipient, such as sterile water, aqueous saline solution, aqueous buffered saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, ethanol, or combinations thereof. The preparation of such solutions ensuring sterility, pH, isotonicity, and stability is effected according to protocols established in the art. Generally, a carrier or excipient is selected to minimize allergic and other undesirable effects, and to suit the particular route of administration, e.g., subcutaneous, intramuscular, intranasal, and the like.
The administration may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing the disease symptoms in a subject. The composition may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, intrathecal, or intradermal injections that provide continuous, sustained levels of the agent in the patient. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the clinical symptoms of the cancer. Generally, amounts will be in the range of those used for other agents used in the treatment of cancer, although in certain instances lower amounts will be needed because of the increased specificity of the agent. A composition is administered at a dosage that ameliorates or decreases effects of the cancer as determined by a method known to one skilled in the art.
The therapeutic or prophylactic composition may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, intrathecally, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). Pharmaceutical compositions according to the invention may be formulated to release the active agent substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release
composition adjacent to or in contact with an organ, such as the heart; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target a disease using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type. For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.
Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the agent in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.
The pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra. Compositions for parenteral use may be provided in unit dosage forms (e.g., in single- dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active agent that reduces or ameliorates a cardiac dysfunction or disease, the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic agent(s) (e.g., an MMSET inhibitor described herein) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.
In some embodiments, the composition comprising the active therapeutic is formulated for intravenous delivery. As indicated above, the pharmaceutical compositions according to the invention may be in the form suitable for sterile injection. To prepare such a composition, the suitable therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where one of the agents is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like. Kits
The invention provides kits for the treatment or prevention of cancer. In some embodiments, the kit includes a therapeutic or prophylactic composition containing (i) an effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein; and (ii) an effective amount of a chemotherapeutic agent in unit dosage form. In other embodiments, the kit includes (i) an effective amount of an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein; and (ii) an effective amount of a chemotherapeutic agent in unit dosage form in a sterile container. Such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
If desired a pharmaceutical composition of the invention is provided together with instructions for administering the pharmaceutical composition to a subject having or at risk of contracting or developing cancer. The instructions will generally include information about the use of the composition for the treatment or prevention of cancer. In other embodiments, the instructions include at least one of the following: description of the
therapeutic/prophylactic agent; dosage schedule and administration for treatment or prevention of cancer or symptoms thereof; precautions; warnings; indications; counter- indications; over dosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
The practice of the present invention employs, unless otherwise indicated,
conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as,
"Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989);
"Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987);
"Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Example 1- DNA Damage Induced Endogenous Phosphorylated PTEN in Cell Lines.
Phosphorylation of PTEN at endogenous levels could be readily detected using the phospho-(Ser/Thr) ATM/ATR substrate antibody upon etoposide or irradiation (IR) treatment in Nffl3T3 (FIGs. 1A-1B) and mouse embryonic fibroblasts (MEFs) (FIGs. 2A-2B).
Moreover, the studies of this example demonstrate that ATM inhibitor (Ku55933), but not ATR inhibitor (VE821) or DNA dependent protein kinase (DNA-PK) inhibitors, could dramatically inhibit etoposide-induced phosphorylation of PTEN (FIG. 1C). Furthermore, depletion of ATM, but not ATR, largely abolished etoposide-induced phosphorylation of PTEN in cells (FIG. 2C), suggesting that ATM is the major physiological kinase to phosphorylate PTEN at T398 in response to DNA damage. Importantly, compared to wild type (WT) PTEN, the T398A-PTEN mutant is deficient in undergoing etoposide-induced phosphorylation (FIG. ID). Hence, consistent with previous reports using large-scale proteomic analyses and in vitro biochemical kinase assay, this example demonstrated that DNA damaging agents induced ATM-mediated phosphorylation of T398-PTEN at endogenous levels.
Example 2- DNA Damage Promoted the Specific Interaction Between PTEN and the BRCT domain of MDC2.
Recent studies revealed that pS/pTQ events, which are usually targets of DNA damage response kinases including ATM, ATR, and DNA-PK, could be recognized by the BRCA1 carboxy-terminal (BRCT) or forkhead-associated (FHA) domains in DNA damage repair (DDR) proteins to govern DNA damage repair process (FIG. 2K). Despite the subtle difference in the 398TQ399 phospho-motif in human PTEN (hPTEN) versus the 398SQ399 motif in mouse PTEN (mPTEN), they are both phospho acceptor and likely function in a similar manner (FIG 2E). Hence, in part, the studies in this example were intended to examine whether ATM-mediated phosphorylation of PTEN involves the DNA repair pathway through interaction with key DDR regulators with pS/pTQ-binding domains, such as MDCl, 53BP1, and BRCA1 (FIG. 2K). Interestingly, the studies in this example suggest that etoposide treatment enhanced both of hPTEN and mPTEN binding with the BRCT domain of MDCl, but not its FHA domain, nor the BRCT domains derived from 53BP1 or BRCA1 (FIGs. 1F-G and FIG. 2D, and FIG. 2F). Despite the subtle difference in 398TQ399 phospho-motif in human PTEN versus 398TQ399 motif in mouse PTEN (FIG. 2E), the studies in this example found that etoposide treatment was also capable of promoting the specific interaction between mouse PTEN and MDC1-BRCT domain, but not its FHA domain (FIG. 2F).
In support of 398pTQ399 motif as the primary site in human PTEN (hPTEN) to interact with MDC1, the studies presented demonstrate that MDC1 strongly binds to the C-terminal domain of hPTEN containing the ATM-meditated pT398 site, but not the N-terminal domains of hPTEN (FIGs. 2G-2H). Moreover, etoposide treatment could trigger MDC1-BRCT domain interaction with WT-hPTEN, but not the ATM-phosphorylati on-deficient T398A- hPTEN mutant (FIG. 1H). These results thus support a model that in response to DSBs, PTEN interacts with MDC1 largely through ATM-mediated phosphorylation of PTEN at the T398 residue (FIG. 2 J).
Furthermore, etoposide treatment also triggered the interaction between MDC1 and the MMSET methyltransferase in an ATM-dependent manner (FIG. 21), which indicated that MDC1, MMSET and PTEN might form a possible tertiary complex. In support of this notion, these studies in this example demonstrate that MDC1 and MMSET existed in immunoprecipitates of PTEN after etoposide treatment (FIG. II). Notably, ATM deficiency largely disrupted etoposide induced PTEN interaction with MMSET (FIG. 19 J), which might be due to loss of ATM-mediated phosphorylation of PTEN (FIG. 2C). Furthermore, the etoposide-induced interaction between PETN and MMSET was largely disrupted in Mdcl-/- MEFs (FIG. IK), which suggests that MDC1 plays a crucial role in mediating the interaction of PTEN and MMSET upon DNA damaging signaling (FIG. 19A-I, FIG. 19K). These findings prompted a further hypothesis that the binding of the MMSET methyltransferase and PTEN might induce methylation of PTEN (FIG. 2 J).
Example 3- DNA Damage Promoted the Specific Interaction Between PTEN and
MMSET.
The studies presented in this example demonstrated that only MMSET, but not other methyltransferases examined, including SET8 and EZH2, interacted with PTEN in cells (FIG. 3 A), further advocating for a specific interaction between MMSET and PTEN. Consistently, MMSET, but not SET8 nor EZH2, was capable of dramatically promoting the di-methylation of PTEN in cells, as revealed by the methyl-Lysine motif antibodies (FIG. 3B). Furthermore, the studies in this example showed a significant induction of di-methylation of PTEN at endogenous levels after etoposide treatment (FIG. 4A), which is largely correlative to the elevated interaction between MMSET and PTEN (FIG. 1 J). Importantly, compared to Mmset MEFs, etoposide treatment failed to induce the di-methylation of PTEN in Mmsef ' MEFs (FIG. 1J). Hence, these results support a possible role of MMSET in promoting PTEN methylation upon DNA damage.
Importantly, in keeping with the finding that MDC1 plays a crucial role in mediating the interaction of PTEN and MMSET (FIG. IK), these studies demonstrated that etoposide treatment failed to induce the di-methylation of PTEN MDCF ' MEFs as it did in MDC1+ MEFs (FIG. 4B). This is in part due to the fact that ATM-mediated phosphorylation of PTEN is impaired in MDCF ' MEFs in response to DNA damage (FIG. 5 A). Consistently, compared with WT-PTEN, ATM phosphorylation-deficient hPTEN-T398A mutant is also impaired in associating with MMSET in response to DNA damaging agent (FIG. 5B), thereby deficient in MMSET-mediated di-methylation in cells (FIG. 5C). Together, these results coherently demonstrate that MDCl plays a pivotal role in connecting the MMSET methyltransferase with PTEN, presumably by recruiting both proteins to the damage sites through its BRCT domain(s) to recognize ATM-mediated phosphorylation of MMSET and PTEN, respectively (FIG. 2J).
Example 4- Identification of the MMSET-Mediated Methylation Sites on PTEN.
Consistent with the observation that PTEN largely interacted with MMSET through the C-terminal domain, but not its N-terminal domain (FIGs. 6A-6B), major di- methylation sites were identified in PTEN C-terminal domain (FIG. 6C). Using a
mutagenesis approach to inactivate major lysine residues exposed at PTEN protein surface, the studies presented in this example demonstrated that the K349R mutant was largely deficient in undergoing MMSET mediated methylation in cells, but not other lysine mutants including K254R, K266R, K289R, K332R, and K402R (FIG. 4C). Consistently, DNA damaging signals such as etoposide treatment efficiently triggered MMSET-mediated di- methylation of WT-PTEN, but not the K349R-PTEN mutant (FIG. 6D, FIGs. 21 A-21N), further illustrating K349 as a major MMSET-mediated di-methylation site, at least in this experimental setting. Further tandem mass spectrometry analysis (LC-MS/MS) also confirmed lysine 349 (K349) in PTEN C-terminus to be di-methylated in cells (FIGs. 6E- 6G).
To further investigate the physiological role of di-methylation of K349-PTEN in
DSBs repairs, in collaboration with Cell Signaling Technology (CST), antibodies were generated that could specifically recognize PTEN K349 di-methylation (PTEN-K349me2) or K349 tri -methylation (PTEN-K349me3) status. Using dot blot assays with synthetic PTEN peptides with K349 non-, mono-, di- and tri-methylation (FIG. 7A), it was found that the PTEN-K349me2 antibody specifically recognized the di-methylated-K349-PTEN synthetic peptide, while the PTEN-K349me3 antibody specifically recognized the tri-methylated- K349-PTEN synthetic peptide (FIG. 7B). However, under physiological conditions, only di- methylation, but not tri-methylation, of PTEN at K349, upon etoposide treatment (FIG. 4D) was observed. More importantly, di-methylation of K349-PTEN was observed at
endogenous levels upon γ-irradiation, a process that can be largely abolished after depletion of endogenous MMSET (FIG. 4E and FIG. 4F). Furthermore, the results reported herein show that 3-deazaneplanocin A (DZNep), a pan-inhibitor of ^-adenosylmethionine-dependent methyltransferase that includes MMSET, could dramatically suppress K349 di-methylation of PTEN (FIG. 7C). These results revealed MMSET as the physiological upstream
methyltransferase that primarily promotes K349 di-methylation of PTEN in cells in response to DNA damaging signals.
Given that methylation signals at DNA damage sites can be recognized by the tudor domain containing proteins, such as 53BP1 or histone demethylases, to facilitate the recruitment of DDR regulators or effector proteins including p53 or pRb (FIG. 8 A), the next studies explored the physiological reader that can recognize MMSET-mediated K349 di- methylation of PTEN. To this end, it was found that etoposide treatment could dramatically enhance PTEN interaction with 53BP1 at both exogenous and endogenous levels (FIG. 4G, FIG. 4H, FIG. 8B, and FIG. 8C), indicating a possible role for the tudor domain of 53BP1 in mediating recruitment of PTEN to the damage sites (FIG. 41).
Interestingly, unlike the observed MDCl/PTEN interaction that is mediated through the BRCT domain (FIG. IF), the interaction between 53BP1 and PTEN is largely mediated through the tudor domain of 53BP1 (FIG. 4H). Specifically, etoposide treatment only enhanced PTEN interaction with the tudor domain derived from 53BP1, but neither SETDB 1 or KDM4 (FIG. 8C). Notably, the methyltransferase MMSET could promote the interaction between WT-PTEN and the tudor domain of 53BP1, indicating the involvement of methyl- K349-PTEN and its subsequent recognition by the 53BP1 -tudor domain (FIG. 41).
In keeping with this notion, the PTEN K349R mutant, but not K254R, K266R, K289R, K332R, and K402R mutants, was deficient in MMSET-induced (FIG. 41), or etoposide-triggered (FIG. 4J), interaction with the 53BP1 -tudor domain. In further support of a direct association between the 53BPl-tudor domain with methyl-K349-PTEN, using an in vitro pull-down assay with synthetic PTEN peptides with unmodified K349 (K349-me0), mono-methylated (K349-mel), di-methylated (K349-me2) and tri-methylated (K349-me3) (FIG. 7A), it was observed that purified recombinant tudor domain of 53BP1 specifically interacts with methylated (K349-me 1,2,3), but not with unmodified (K349-me0), synthetic PTEN peptides in vitro (FIG. 4K). Among all three methylated synthetic PTEN peptides, the di-methylated PTEN peptide (K349-me2) interacts with the 53BP1 tudor domain with the highest affinity (FIG. 4K). More importantly, depletion of MMSET could dramatically reduce endogenous PTEN binding with 53BP1 (FIG. 4J) in cells, which is largely correlative to the reduced PTEN K349 di -methyl ati on inTWMffi -depleted cells (FIG. 4L, and FIG. 4M). Taken together, these results demonstrate that MMSET-mediated K349 di -methyl ati on of PTEN is required for efficient recognition by the methyl-lysine reading tudor domain of 53BP1 upon DNA damage signals.
Upon DNA damage, 53BP1 can be recruited into DNA damage sites through different molecular mechanisms, such as tudor domain-mediated recognition of di-methylated K20 in H4, or through the ubiquitination dependent recruitment (UDR) motif of 53BP1 by recognizing ubiquitinated K15 in H2A, or BRCT domain-mediated interaction with γΗ2ΑΧ. On the other hand, 53BP1 also recruits the methylated tumor suppressor proteins including p53 and pRb into DNA damage sites through its tudor domain. Hence, the above observation is consistent with a model that DNA damaging-agents can promote MMSET-mediated K349 di -methyl ati on of PTEN, which is subsequently recognized by the 53BP1 tudor domain to facilitate the recruitment of PTEN to the DNA damage sites (FIG. 8D).
Example 5- The Role of PTEN at DNA Damage Sites.
To further examine this model by exploring the physiological function of PTEN at DNA damage sites, the DNA damage response in ΡΤΕΝ /+ and PTEN' ' HCT116 cells was investigated after exposure to γ-irradiation. Through screening a series of phosphorylated proteins involving in DNA damage repair pathway, it was discovered that the magnitude and duration of γΗ2ΑΧ was dramatically elevated especially at 4 hours, and to a lesser extent, 8 and 24 hours post-y-irradiation in PTEN' ' cells relative to ΡΤΕΝ /+ cells (FIG. 9A, FIG. 9B, and FIG. 9J). However, the levels of other markers including pS25/29-53BPl, pT68-Chk2, and pS1524-BRCAl were markedly decreased in PTEN' ' compared with ΡΤΕΝ /+ cells (FIG. 9A, FIG. 9B, and FIG. 9J). As PTEN also possesses the protein phosphatase activity to impact the biological functions of its several identified protein substrates, it was investigated whether the upregulation and duration of γΗ2ΑΧ after irradiation in PTEN' ' cells is relevant to PTEN protein phosphatase activity. Notably, re-introducing WT or G129E (lipid phosphatase dead mutant), but not Y138L (protein phosphastase dead mutant), nor C124S (lipid and protein phosphatase dead mutant), at comparable levels in ΡΤΕΚ ' HCT116 cells was capable of rescuing the elevated and prolonged γΗ2ΑΧ status upon γ-irradiation (FIGs. 9B-9D). On the other hand, consistent with previous reports, ectopic expression of PTEN WT and Y138L mutant, but not the lipid phosphatase deficient C124S nor G129E mutants, could reduce the levels of pS473-Akt in ΡΤΕΚ ' HCT116 cells by lipid phosphatase-dependent of antagonizing PI3K activity (FIGs. 10A-10B). More importantly, compared with Pten+/+ and PtenG129E/+ MEFs, the levels of γΗ2ΑΧ in Ptencl24S/+ MEFs during late stages of DSB repair post γ-irradiation (8 and 24 hour time points) were elevated (FIG. 9K, and FIG. 9L). Furthermore, Pten mice displayed high levels of γΗ2ΑΧ at 24 h post γ-irradiation in relative to Pten+/+ and PtenG129E/+ mice (FIG. 9M, and FIG. 9N). These results suggested that the protein phosphatase activity, but not lipid phosphatase activity, of PTEN plays a crucial role in regulation of γ-Η2ΑΧ status in response to DNA damage.
Interestingly, it was observed that even though the PTEN K349R mutant still possesses the lipid phosphatase activity at levels comparable to WT-PTEN (FIG. IOC), it is largely deficient in rescuing the prolonged γΗ2ΑΧ in ΡΤΕΚ ' HCT116 cells post-γ- irradiation (FIGs. 9E-9F). This was possibly due to the deficiency in di-methylation- dependent recruitment of PTEN to the DNA damage sites, which led to an impaired the interaction between γ-Η2ΑΧ and K349R-PTEN, compared to WT-PTEN, after IR or etoposide treatment (FIGs. 9G-9I, and FIG. 10D). In support of this notion, compared to Mmset+/+ MEFs, irradiation-induced interaction of γΗ2ΑΧ with PTEN was impaired in Mmsef ' MEFs (FIG. 90). These results indicated that DNA damaging agents could induce a portion of PTEN to colocalize with γΗ2ΑΧ. The studies presented in this example found that PTEN colocalized with γΗ2ΑΧ in DNA damage sites generated by laser micro-irradiation (FIG. 9P).
To further identify whether PTEN directly dephosphorylates γΗ2ΑΧ, an in vitro dephosphorylation assay using the purified recombinant PTEN WT, C124S, G129E and Y138L proteins incubating with biotin-labeled H2AX peptides with/without phosphorylation of Serl39 was performed (FIG. 10E). Notably, PTEN WT and G129E, but not the protein phosphatase dead mutants C124S and Y138L, could dramatically dephosphorylate the biotin- labeled γΗ2ΑΧ in vitro (FIG. 91). These results together suggest that in addition to its tumor suppressor role in the cytoplasm by antagonizing PI3K/Akt signaling, PTEN could directly dephosphorylate γΗ2ΑΧ through its protein phosphatase activity at the damage sites to possibly govern DSBs repair in the nucleus (see also FIGs. 20A-I, which show protein phosphatase activity of PTEN is required for efficient complement of DSBs repair largely through dephosphorylating γΗ2ΑΧ).
To further examine the PTEN protein phosphatase activity in regulating DSBs repair, PTEN-WT and various mutants including C124S, G129E, Y138L, K349R, T398A as well as the empty vector control, were retrovirrally introduced at comparable levels into PTEN- deficient U87MG cells. Consistent with results derived from ΡΤΕΚ ' HCT116 cells (FIGs. 8A-8B), ectopic expression of lipid phosphatase dead mutants C124S and G129E, but not Y138L, K349R, S398A mutants, failed to reduce pSer437-Akt (FIG. 11 A). The DNA damage response profiles of these cell lines were monitored by immuno-staining against DSBs markers 53BP1 and γ-Η2ΑΧ at indicated time points post γ-irradiation. Notably, 1 hour post-irradiation, the foci of 53BP1 could be observed in all cell lines (FIG. 12A and FIG. 1 IB), a faithful marker indicative of double strand breaks generated by IR. However, 24 hours after irradiation, 53BP1 foci were largely resolved in ΡΤΕΚ ' HCT116 cells stably expressing PTEN WT and G129E, but not in cells expressing C124S and Y138L that are deficient in protein phosphatase activity, or the K349R or S398A mutant that are deficient in undergoing MMSET-mediated di-methylation event (FIG. 12A and FIG. 11B). Consistently, cells expressing C124S, Y138L, K349R and S398A, but not WT nor G129E mutant form of PTEN, also exhibited deficiencies in resolving phosphorylated histone variant H2AX (γ- H2AX) (FIG. 12B and FIG. 11C). Furthermore, compared to control cells, depletion of the MMSET methyltransferase only had minimal effect on foci formation of 53BP1 (FIG. 12C and FIG. 13A) or γΗ2ΑΧ (FIG. 12D and FIG. 13B) at 1 hour post γ-irradiation. However, at 24 hours post γ-irradiation, MMSET deficiency dramatically inhibited the foci resolve of 53BP1 (FIG. 12C, FIG. 12E-FIG.12G, and FIG. 13A) or γΗ2ΑΧ (FIG. 12D, and FIG. 13B) at 24 hours post γ-irradiation, which suggests that like PTEN, MMSET deficiency also minimally affects DNA damage foci formation at the early stage (4 hour) of post irradiation and mainly inhibits the foci resolve at the late stage (24 hour) of post irradiation to govern DNA damage repair.
Given the critical role of DSBs repair in conferring cell growth and apoptosis signaling, it was further explored whether the di-methylation and protein phosphatase activity of PTEN play an important role in regulating cell survival upon DNA-damaging reagents. To exclude the effect of PTEN lipid phosphatase activity on DNA damage-induced cell survival and apoptosis, BKM120 was utilized, which is a pan-PI3K inhibitor to block the PTEN lipid phosphatase activity pathway (FIG. 22). To this end, it was observed that PTEN~ " HCTl 16 cells displayed more sensitive to a combination of IR and BKM120, a PI3K inhibitor (FIGs. 14A-14C). Furthermore, the radio- or chemo-sensitivity of PTEN' ' HCTl 16 cells with stably expressing WT, C124S, G129E, Y138L, K349R, S398A as well as empty vector as a negative control was monitored. Notably, in keeping with their deficiency to resolve DSBs foci (FIGs. 12A-12B), cells expressing the protein phosphatase mutant C124S, Y138L, or methylation deficient mutant K349R or S398A, the mutants were more sensitive to a combination treatment of IR or etoposide with BKM120 evidenced by both cell viability and apoptosis assays (FIGs. 14A -14C, and FIGs. 15A-15B). To further pinpoint the methylation status of PTEN as well as the PTEN protein-phosphatase activity involvement in this process, the radio- or chemo-sensitivity of PTEN-/- HCTl 16 cells stably expressing WT, C124S, G129E, Y138L, K349R, T398A were monitored, as well as EV as a negative control. Notably, consistent with their deficiency in resolving DSBs foci (FIG. 12A and FIG. 12B), when compared to cells with expression of WT -PTEN or a lipid-phosphatase dead G129E mutant, cells expressing the protein-phosphatase dead mutant C124S or Y138L, or methylation-deficient mutant K349R or T398A, were more sensitive to combination treatment of DNA damaging agents such as IR or etoposide with the pan-PI3K inhibitor, BKM120 (FIG. 14D, FIG. 15C, FIG. 15D, FIG. 21E, and FIG.21F).
More importantly, compared with PTEN-WT expressing cells, combination treatment with etoposide and BKM120 displayed a greater inhibition of xenografted tumor growth bearing PTEN-/- HCTl 16 cells stably expressing the methylation-deficient mutant K349R, accompanied with elevated γΗ2ΑΧ and cleaved caspase 3 levels in tumors with the PTEN- K349R status (FIGs. 120-12P). In further support for a critical role of MMSET-mediated PTEN di-methylation in dictating cellular sensitivity to DSB agents in this experimental setting, cells with depletion of the MMSET methyltransferase exhibited a marked decrease in cell viability, colony formation as well as elevated apoptosis following combination treatment of IR (FIGs 23A-FIG 23E) or etoposide (Fig. 12R and FIG. 23F-FIG.23I) with BKM120. Notably, it was observed that in PTEN+/+, but not PTEN-/- HCTl 16 cells, additional depletion of MMSET could greatly retarded in vivo xenograted tumor growth following combined treatment of etoposide with BKM120, coupled with increased γΗ2ΑΧ and caspase-3 cleavage (FIG. 12H-FIG.12T and FIG. 23J-FIG.23N). The studies of this example further demonstrated that inhibition of MMSET using the pan-methyltransferase inhibitor, DZNep 13, 14, also led to a marked elevation in cellular sensitivity to treatment with IR (FIG. 16A, FIG. 16B, FIG. 21L, FIG. 12M, and FIG. 21N) or etoposide (FIG.12U- FIG.12W and FIG. 12N, FIG. 24A, FIG. 24B, FIG. 24C, and FIG.24D) together with BKM120 in PTEN+/+, but not in PTEN-/- HCT116 cells. Taken together, these results demonstrate that MMSET-mediated PTEN methylation plays a critical role in dictating cellular sensitivity to combination treatment with DSB inducing agents and PI3K inhibitors.
As MMSET is a potential therapeutic target in cancer, developing MMSET specific inhibitor(s) and combining with DNA-damaging agents and PI3K inhibitors might be useful in treating cancers with AiMSEJoverexpressi on (FIG. 17A-FIG. 17D; FIG. 18). Such inhibitors could efficiently sensitize cancer cells with wild-type PTEN genetic status to chemo- or radio-therapeutics. Taken together, these studies uncover a critical role of PTEN methylation and its protein phosphatase activity in regulating DSBs repair and sensitivity to DNA damaging-agents, including both chemo- and radio-therapeutics, in part by governing DSB repair process via dephosphorylating γΗ2ΑΧ. These studies disclosed herein further extend knowledge about the precise molecular mechanism of how PTEN involve in DNA damage pathway.
Materials and Methods.
Cell Culture, Transfections, Viral Infections and Reagents: HEK293T, U20S,
U87MG and HCT116 PTEN"+ and PTEN"1' cells (as gifts from Dr. Todd Waldman in School of Medicine, Georgetown University) were cultured in DMEM medium supplemented with 10% FBS, 100 units of penicillin and 100 mg/ml streptomycin. Mouse embryonic fibroblasts (MEFs) MDC1+I+ and MDCF1' cells (a gift of Anyong Xie in the Sir Run Shaw Hospital and Institute of Translational Medicine, Zhejiang University School of Medicine, Hangzhou, China) were maintained in DMEM medium supplemented with 10% FBS.
Cell transfection was performed using lipofectamine and plus reagents according to standard protocols. Packaging of lentiviral and retroviral cDNA expressing viruses, as well as subsequent infection of various cell lines were performed according to standard protocols. Following viral infection, cells were selected in the presence of hygromycin (200 μg/mL) or puromycin (1 μg/mL) for 3 days. Etoposide was purchased from Sigma (E1383). KU-55933 (ATM Kinase Inhibitor, S1092), VE-821 (S8007), NU7026 (S2893), BKM120 (S2247), 3-deazaneplanocin A
(DZNeP), and HC1 (S7120) were purchased from Selleckchem.
Plasmid Construction and MMSET shRNAs: HA-hPTEN and HA-mPTEN were generated by inserting the corresponding cDNAs into pcDNA3-HA vector. Flag-MMSET, Flag-EZH2 and Flag-Set8 were constructed by cloning corresponding cDNAs into pFlag- CMV vector. pCMV-GST-MDCl-BRCT, pCMV-GST-53BPl-BRCT, pCMV-GST-BRCAl- BRCT and pCMV-GST-53BPl tudor domain were cloned into mammalian expression GST- fusion vectors. GST-PTEN was constructed by inserting the cDNA into pGEX-4T-l vector. pLenti-HA-PTEN and pBabe-Super-HA-PTEN were constructed by subcloning the PTEN cDNA into pLenti-HA-puro and pBabe-Super-HA-hygro vector, respectively. Various human PTEN mutants (hPTEN-T398A, K254R, K266R, K289R, K332R, K349R, C124S, G129E, Y138L) were generated using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. All mutants were generated using mutagenesis PCR and the sequences were verified by DNA sequencing.
pLentilox3.7-shRNA vectors to deplete endogenous MMSET were generously offered by Dr. Zhenkun Luo in the Department of Oncology, Mayo Clinic and previously described (34). The sequences for MMSET shRNA were listed below:
MMSET shRNA 1 : 5 ' -GC ACGCTAC AAC ACC AAGTTT (SEQ ID NO: 7);
MMSET shRNA 2: 5 ' -GC AC AGTCTTCGGAAGAGAGAC AC AATC A (SEQ ID
NO: 8)
Antibodies: All antibodies were used at a 1 : 1000 dilution in TBST buffer with 5% non-fat milk for western blot. Ant-phospho-ATM/ATR Substrate (S*Q) (9607), anti-pS15- p53 (9284), anti-pT68-Chk2 (2661), anti-Chk2 antibody (3440), anti -Mono-Methyl Lysine Motif (14679), anti-Di-Methyl Lysine Motif (14117), anti-Tri -Methyl Lysine Motif (14680), anti-His tag (2366), anti-yH2AX (9718), anti-pS1618-53BPl (6209), ant-pT543-53BPl (3428), anti-pS25/29-53BPl (2647), anti-pS428-ATR (2853), anti-pS1542-BRCAl (9009), anti-pS296-Chkl (2349), anti-pS473-Akt (4060), anti-Aktl antibody (2938) and anti-H2A (3636) were purchased from Cell Signaling Technology. Anti-PTEN (sc-7974), anti-MDCl (sc-27737), anti-p53 (sc-6243/126), anti-ATM (sc-23921), anti-ATR (sc-1887), anti-BRCAl (sc-6954/641), anti-Chkl (sc-8408), anti-HA (sc-805) and anti-GST (sc-459) were obtained from Santa Cruz. Anti-Flag (F-2425), anti-Flag (F-3165, clone M2), anti-Tubulin antibody (T-5168), anti-Vinculin (V9131), anti-Flag agarose beads (A-2220), anti-HA agarose beads (A-2095), peroxidase-conjugated anti -mouse secondary antibody (A-4416) and peroxidase- conjugated anti-rabbit secondary antibody (A-4914) were purchased from Sigma. Anti-HA (MMS-101P) and anti-MMSET/NSD2 (ab75359) was obtained from Covance and Abeam, respectively.
The polyclonal anti-PTEN-K349-me2 and anti-PTEN-K349-me3 antibodies generated by Cell Signaling Technology were derived from rabbit, with each antibody produced three clones. The antigen peptide sequence comes from 10 amino acids surrounding the
modification site (K349) of human PTEN. The antibodies were affinity purified using the antigen peptide column, but they were not counter selected on unmodified antigen.
Therefore, these antibodies were validated by different methods, including dot blotting assays with synthetic PTEN peptides with mono-methylation, di-methylation and tri-methylation modification at K349, PTEN K349R mutant, as well as reaction products derived from in vitro methylation assays.
Immunoblot and Immunoprecipitation Analysis: Cells with indicated treatments were lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail set I and II, Calbiochem). The protein concentrations of whole cell lysates were measured by the Beckman Coulter DU-800 spectrophotometer using the Bio-Rad protein assay reagent according to standard protocols. Equal amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For
immunoprecipitations analysis, 1000 μg lysates were incubated with the indicated antibody- conjugated beads for 3-4 h at 4 °C. The recovered immuno-complexes were washed four times with NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA and 0.5% NP- 40) before being resolved by SDS-PAGE and immunoblotted with indicated antibodies. For the in vivo PTEN methylation assays, EBC and NETN buffer containing 300 mM NaCl were used to disrupt the non-specific interacting proteins.
In Vitro Dephosphorylation Assays: For dephosphorylation of γ-Η2ΑΧ by PTEN, the GST-fusion PTEN WT, C124S, G129E and Y138L, as well as GST protein were purified from E. coli BL21 according to standard protocols. γ-Η2ΑΧ (Cat. No. : 12-0040) and H2AX (Cat. No. : 12-0039) peptides serving as substrate were purchased from EpiCypher. The dephosphorylation assays were performed in phosphatase assay buffer (20 mM HEPES, pH 7.2, 100 mM NaCl and 3 mM DTT). The reactions were incubated at 37 °C for 30 min with or without the addition of recombinant GST-fusion PTEN WT, C124S, G129E and Y138L, as well as GST protein as negative control, and were stopped by adding 3 x SDS loading buffer for western blot.
Peptide Synthesis: The PTEN peptides with/without methylation modification were synthesized at Tufts Medical School. Each contained an N-terminal biotin and free C- terminus and was synthesized in 0.1 mM scale. Peptides were diluted into lmg/ml for further biochemical assays. The sequences were listed below:
hPTEN WT:
Bi otin- FK VKL YF TKT EEP S PE (SEQ ID NO: 9)
hPTEN K349_mono-methylation (K-mel):
Biotin-NFKVKLYFTK(mel)TVEEPSNPE (SEQ ID NO: 10)
hPTEN K349_Di-methylation (K-me2):
Biotin-NFKVKLYFTK(me2)TVEEPSNPE (SEQ ID NO: 1 1)
hPTEN K349_Tri-methylation (K-me3):
Biotin-NFKVKLYFTK(me3)TVEEPSNPE (SEQ ID NO: 12)
Dot Immunoblot Assays: Peptides were spotted onto nitrocellulose membrane allowing solution to penetrates (usually 3-4 mm diameter) by applying it slowly as a volume 1 [iL once. The membrane was dried, and blocked in TBST buffer with 5% non-fat milk for immunoblot analysis with indicated antibodies according to standard protocols.
Peptide-binding assays: Peptides (2 μg) were incubated with 1 mg of whole cell lysates in a total volume of 500 μΐ^ EBC buffer. After incubation for 4 hr at 4°C, 10 μΐ^ Streptavidin agarose (Thermo Scientific 20353) was added in the sample for another 1 hr. The agarose was washed four times with NETN buffer. Bound proteins were added in 2 x SDS loading buffer and resolved by SDS-PAGE for immunoblot analysis.
Mass spectrometry analysis: For mass spectrometry analysis, anti-HA-PTEN immunoprecipitations (IP) were performed with the whole cell lysates derived from three 10 cm dishes of HEK293 cells co-transfected with Flag-MMSET and HA-PTEN. The cells were treated with 30 μΜ etoposide for 1 hr before harvesting for IP. The IP proteins were resolved by SDS-PAGE, and stained by Gelgold staining buffer. The band containing PTEN was reduced with 10 mM DTT for 30 min, alkylated with 55 mM iodoacetamide for 45 min, and in-gel-digested with trypsin enzymes. The resulting peptides were extracted from the gel and analyzed by microcapillary reversed-phase liquid chromatography -tandem mass spectrometry (LC-MS/MS) using a high resolution Orbitrap Elite (Thermo Fisher Scientific) in positive ion DDA mode via CID, according to standard protocols. MS/MS data were searched against the human protein database using Mascot (Matrix Science) and data analysis was performed using the Scaffold 4 software (Proteome Software). Peptides and modified peptides were accepted if they passed a 1% FDR threshold.
Immunofluorescence assays: Cells cultured on glass cover slips were fixed with 4% formaldehyde in PBS for 15 minutes at room temperature. The cells were permeabilized with 0.1% Triton X-100 in PBS for 5 minutes on ice. After washing 3 times in PBS, the samples were blocked for 30 minutes with 5% control goat serum. The samples were incubated with primary antibodies for 2 hours at room temperature. After rinsed 3 times using PBST containing 0.1% Tween-20, the coverslips incubated with Alexa-594-conjugated goat anti- mouse secondary antibody (Invitrogen) for 1 hour and washed 3 times with PBST, the nucleus was stained with 4, 6-diamidino-2-phenylindole (DAPI) for 10 minutes. Coverslips were rinsed 2 times with PBS and mounted onto slides.
Laser micro-irradiation and IF assays. HCT1 16-ΡΤΕΝ" cells stably expressing HA-PTEN were cultured for 48 hr prior to irradiation with 10 μΜ BrdU4 (Sigma- Aldrich cat. #B9285). UVA laser (50 mW) irradiation was conducted using an inverted microscope (Eclipse Ti; Nikon) with a Palm microbeam laser microdissection workstation. Following irradiation, cells were incubated at 37 °C for 5 minutes, washed once with cold PBS, and then fixed with PBS containing 4% paraformaldehyde (PFA) for 10 min at room temperature. Cells were then washed three times with cold PBS and permeabilized with PBS containing 0.5% Triton-XlOO for 30 min. After permeabilization, cells were washed with IF blocking buffer (PBS containing 1% BSA, 10% FBS, 0.25% Triton-XlOO, 0.02% NaN3), and then co- stained using antibodies for HA and γΗ2ΑΧ at 4°C overnight. Staining was conducted with fluorescent secondary and DAPI.
Murine models. The Beth Israel Deaconess Medical Center IACUC Committee on Animal Research approved all animal experiments. The transgenic mice used in this study
(Pten+/+, Pfe«cl24S/+, and Pten G129E/+) were reported previously. Four mice per genotype were randomly chosen and analyzed at the indicated age.
Xenograft tumor growth. For assaying tumor growth in the xenograft model, 6-week- old female nude mice housed in specific pathogen-free environments were injected s.c. with 1.0 x 106 HCT116 derivatives (n=5 for each group) mixed with DMEM medium and Matrigel
(vol/vol, 1 : 1). Treatments were initiated when the mean tumor volume in each randomized group reached around 100 mm . For BKM120, treatments were carried out orally, every other day, using an application volume 25 mg/kg. For etoposide, treatments were carried out by LP. injection with 20 mg/kg every other day. DZNep treatments were carried out by LP.
injection with 1 mg/kg body weight twice a week for 2 weeks. Treatments were stopped and animals sacrificed when the tumor size in the vehicle control group reached 500 to 600 mm . Tumor volumes were determined using calipers. All experimental procedures strictly complied with the IACUC guidelines.
Immunohistochemistry (IHC) assays. Individual tumors derived from nude mice were dissected and fixed in 4% paraformaldehyde for IHC analysis. For staining, tissues were fixed in 4% paraformaldehyde overnight, paraffin embedded, and then sectioned at 5μιη. After deparaffinization and rehydration, antigen retrieval was performed in a pressure cooker with sodium citrate buffer at 95°C for 25 minutes. Sections were incubated in a 0.3% H2O2 solution in lx PBS, and then a 10% serum solution in lx PBS for 30 minutes each solution was used to block endogenous peroxidase and background from the secondary antibody, respectively. The sections were stained with the γΗ2ΑΧ (Cell Signaling #9718, 1 :500) in lx PBS at 4°C overnight, and incubated in a biotinylated anti-rabbit secondary antibody in lx PBS (1 : 1000) at room temperature for 30 minutes. The Vectastain ABC Elite kit was used to enhance specific staining, and the staining was visualized using a 3 ' -diaminobenzidine (DAB) substrate. Stained sections were counterstained using hematoxylin and dehydrated before they were sealed with a coverslip with Richard-Allan Scientific® Cytoseal™ XYL Mounting Medium. Stained slides were visualized by a bright-field microscope.
IR treatments. Cells with 60% confluence were treated with irradiation (5 Gy). After
IR treatment, cells were left to recover at the incubator for the indicated times. Total-body irradiation (3 Gy) of 4 month-old mice (Pten l+, Ptencius/+, and Pten G129E/+) was performed, after which the mice were caged separately before being sacrificed at 24 h after treatment.
Moreover, these mice were carefully monitored during the recovery period.
Compound preparation. NVP-BKM120 (MedChem Express) was formulated in 0.5% methylcellulose/0.5% Tween 80) at 6 mg/ml. Etoposide (Selleckchem) was formulated in Saline at 10 mg/ml.
Cell viability and apoptosis assays: For cell viability assays, 2000 cells per well were plated in 96-well plates, and incubated with complete DMEM medium containing different treatments as indicated. Assays were performed with the Cell Titer-Glo
Luminescent Cell Viability Assay Kit according to the manufacturer's instructions
(Promega). For detection of apoptosis, cells treated with indicated treatment were co-stained with Annexin-V-PE and 7-AAD (Annexin V-PE Apoptosis Detection Kit I, BD Bioscience) and analyzed using BD FACSCanto flow cytometer according to the manufacturer's instructions.
Colony formation assays: Cells were seeded in 6-well plates (3000 cells/well) and pre-treated with/without BKM120 (ΙμΜ) for 24 hr hours followed by additional IR (0.5 Gy) or etoposide treatment as indicated for 24 hours. Cells were incubated for 6-10 days until formation of visible colonies. Colonies were fixed with 10% acetic acid/10% methanol for 20 min and stained with 0.4% crystal violet/20%) ethanol for 20 min. After staining, the plates were washed with distilled water and air-dried. The colony number was counted.
Statistics: Differences between control and test conditions were evaluated by Student's t test. These analyses were performed using the SPSS 11.5 Statistical Software and P < 0.05 was considered statistically significant.
Other Embodiments
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:
1. A method of sensitizing a neoplastic cell to chemotherapy, the method comprising contacting the cell with an agent that inhibits multiple myeloma SET domain protein
(MMSET) activity or expression and a chemotherapeutic agent, thereby sensitizing the cell to chemotherapy.
2. A method of sensitizing a neoplastic cell to radiation, the method comprising contacting the cell with an agent that inhibits MMSET activity or expression and exposing the cell to radiation, thereby sensitizing the cell to γ-irradiation.
3. A method of enhancing cell death or reducing proliferation in a neoplastic cell, the method comprising contacting the cell with an agent that inhibits MMSET activity or expression and a chemotherapeutic agent, thereby enhancing cell death or reducing proliferation in the cell.
4. A method of enhancing cell death or reducing proliferation in a neoplastic cell, the method comprising contacting the cell with an agent that inhibits MMSET activity or expression and exposing the cell to radiation, thereby enhancing cell death or reducing proliferation in the cell.
5. A method of enhancing chemotherapy sensitivity in a subject having a neoplasia, the method comprising administering to the subject an agent that inhibits MMSET activity or expression and a chemotherapeutic agent, thereby enhancing chemotherapy sensitivity in the subject.
6. A method of enhancing radiation sensitivity in a subject having a neoplasia, the method comprising administering to the subject radiation and an agent that inhibits MMSET activity or expression, thereby enhancing radiation sensitivity in the subject.
7. The method of any one of claims 1-6, wherein the agent that inhibits MMSET activity is a polypeptide, polynucleotide, or small molecule.
8. The method of claim 7, wherein the agent that inhibits MMSET activity is selected from the group consisting of: (l S,2R,5R)-5-(4-Amino-lH-imidazo[4,5-c]pyridin-l-yl)-3- (hydroxymethyl)-3-cyclopentene-l,2-diol hydrochloride, 3-hydrazinylquinoxaline-2-thiol, and LEM-06.
9. The method of claim 7, wherein the polynucleotide is an inhibitory nucleic acid molecule that inhibits the expression of MMSET.
10. The method of claim 9, wherein the inhibitory nucleic acid molecule is an antisense molecule, siRNA, or shRNA.
11. The method of claim 10, wherein the shRNA comprises or consists essentially of one of the following sequences:
MMSET shRNA 1 : 5 ' -GC ACGCTAC AAC ACC AAGTTT;
MMSET shRNA 2: 5 ' -GC AC AGTCTTCGGAAGAGAGAC AC AATC A:
12. The method of any one of claims 1-6, wherein the chemotherapeutic agent is selected from the group consisting of: doxorubicin and etoposide.
13. The method of any one of claims 1-6, wherein the chemotherapeutic agent is a PI3 kinase inhibitor.
14. The method of claim 13, wherein the PI3 kinase inhibitor is BKM120, BYL719 or RP6530.
15. The method of any one of claims 1-4, wherein the neoplastic cell is a mammalian cell.
16. The method of claim 5, wherein the mammalian cell is a murine, rat, or human cell.
17. The method of claim 5, wherein the cell is in vitro or in vivo.
18. The method of any one of claims 1-6, wherein the neoplastic cell or cancer comprises a mutation in PTEN or amplification of MMSET.
19. The method of any one of claims 1-6, wherein the method reduces neoplastic cell survival or proliferation.
20. The method of any one of claims 1-6, wherein the neoplastic cell is derived from prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, or other types of epithelium derived carcinomas where loss of function of PTEN is frequently observed.
21. The method of any one of claims 1-6, wherein the subject has prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, or other types of epithelium derived carcinomas where loss of function of PTEN is frequently observed.
22. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject an agent that inhibits the expression or activity of multiple myeloma SET domain (MMSET) protein; and a chemotherapeutic agent, thereby treating cancer in the subj ect.
23. The method of claim 22, wherein the method reduces tumor growth, and/or increases subject survival.
24. The method of claim 22, wherein the agent that inhibits the expression or activity of MMSET reduces the effective amount of the chemotherapeutic agent necessary to treat the cancer.
25. The method of claim 22, wherein the cancer is prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, or other types of epithelium derived carcinomas where loss of function of PTEN is frequently observed.
26. The method of claim 22, wherein the agent that inhibits the expression or activity of MMSET protein is a polypeptide, polynucleotide, or a small molecule.
27. The method of any one of claims 1-22, wherein the agent that inhibits MMSET activity is a pan-inhibitor of ^-adenosylmethionine-dependent methyltransferase.
28. The method of claim 27, wherein the agent that inhibits MMSET activity is selected from the group consisting of: (l S,2R,5R)-5-(4-Amino-lH-imidazo[4,5-c]pyridin-l-yl)-3- (hydroxymethyl)-3-cyclopentene-l,2-diol hydrochloride, 3-hydrazinylquinoxaline-2-thiol, and LEM-06.
29. The method of claim 26, wherein the agent that inhibits the expression or activity of MMSET protein is an inhibitory nucleic acid molecule that inhibits the expression of a MMSET protein.
30. The method of claim 27, wherein the inhibitory nucleic acid molecule is an antisense molecule, siRNA, or shRNA.
31. The method of claim 30, wherein the shRNA comprises or consists essentially of one of the following sequences:
MMSET shRNA 1 : 5' -GC ACGCTAC AAC ACC AAGTTT;
MMSET shRNA 2: 5 ' -GC AC AGTCTTCGGAAGAGAGAC AC AATC A.
32. The method of claim 22, wherein the subject has prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, gastric cancer, or other types of epithelium derived carcinomas where loss of function of PTEN is frequently observed.
33. A kit comprising a therapeutic or prophylactic composition containing (i) an effective amount of an agent of any one of claims 1-32; and (ii) an effective amount of a chemotherapeutic agent.
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