WO2013059320A1 - Treatment of cancers with mutant npm1 with mek inhibitors - Google Patents
Treatment of cancers with mutant npm1 with mek inhibitors Download PDFInfo
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- WO2013059320A1 WO2013059320A1 PCT/US2012/060614 US2012060614W WO2013059320A1 WO 2013059320 A1 WO2013059320 A1 WO 2013059320A1 US 2012060614 W US2012060614 W US 2012060614W WO 2013059320 A1 WO2013059320 A1 WO 2013059320A1
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Classifications
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/203—Retinoic acids ; Salts thereof
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- A—HUMAN NECESSITIES
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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- A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
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- A61P35/02—Antineoplastic agents specific for leukemia
Definitions
- the field includes molecular biology, cell biology, and medicine, including cancer medicine.
- the field of the invention concerns compositions for treatment of acute myeloid leukemia (AML).
- AML acute myeloid leukemia
- AML Acute myeloid leukemia
- Ras gene point mutations have been detected.
- the efficacy of these agents is variable, especially in patients with high- risk AML (relapsed, resistant/refractory, or adverse cytogenetics).
- the persistence of CD34 + /CD387CD123 + leukemia stem cells (LSC) results in relapse.
- the BM has a microenvironment characterized by hypoxia and mesenchymal stem cells (MSC) in sub- endosteal niches, which generates a sanctuary for subpopulations of LSC to evade chemotherapy- and signal transduction inhibitor-induced death and allows acquisition of a drug resistant phenotype (Konopleva et ah, 2002; Tabe et ah, 2004; Zeng et ah, 2008; Fiegl et al. , 2008). Therefore, more effective treatment regimens are needed.
- MSC mesenchymal stem cells
- the present invention is directed to one or more systems, methods and/or compositions related to cancer treatment and/or prevention.
- the present invention concerns compositions and uses thereof for treatment of particular types of cancer, including those that harbor one or more NPM1 mutations; in specific cases, the cancer is leukemia or lymphoma, including acute myeloid leukemia (AML) or chronic myelomonocytic leukemia (CML), for example.
- the individual has myelodysplasia syndrome.
- the individual has a solid tumor, such as in the lung, breast, colon, prostate, pancreas, kidney, liver, skin, bone, gall bladder, spleen, uterus, cervix, ovary, testes, rectum, stomach, bladder, nasopharynx, esophagus, and so forth.
- a solid tumor such as in the lung, breast, colon, prostate, pancreas, kidney, liver, skin, bone, gall bladder, spleen, uterus, cervix, ovary, testes, rectum, stomach, bladder, nasopharynx, esophagus, and so forth.
- At least some aspects of the invention provide combination strategies that allow the concomitant suppression of multiple critical signaling pathways in cancer, including leukemias.
- the activation of certain signaling pathways confers resistance to apoptosis and to chemotherapeutic agents in AML, for example, both in vivo and in vitro, resulting in synergistic "cytotoxic" effects in AML, including in the hypoxic BM microenvironment.
- Embodiments of the invention exert significant anti-leukemia efficacy and lowers the dose required of each single agent in AML.
- Embodiments of the present invention concern MEK inhibitors, including small molecule MEK inhibitors (for example, (N-((S)-2,3-Dihydroxy-propyl)-3-(2- fluoro-4-iodo-phenylamino)-isonicotinamide), hereafter called Compound 1) that are capable of decreasing mutant NPMl protein levels in addition to the suppression of MEK activity.
- MEK inhibitors for example, (N-((S)-2,3-Dihydroxy-propyl)-3-(2- fluoro-4-iodo-phenylamino)-isonicotinamide
- the effect on mutant NPMl downregulation results from inhibition of MEK activity, indicating for the first time a role of MAPK kinase signaling in the regulation of mutant NPMl levels.
- Particular embodiments of the invention encompass a dual-inhibition function of mutant NPMl and MEK/ERK signaling, which are both related to leukemogenesis.
- NPMl mutation includes selecting an individual that has been determined to have a NPMl mutation and treating the individual with an effective amount of a MEK inhibitor.
- the individual may be selected as having a NPMl mutation following assaying a biological sample from the individual, including a sample from bone marrow, blood, cerebrospinal fluid, urine, stool, semen, nipple aspirate, and so forth. Biopsies may be taken from the individual.
- An assay to determine whether or not an individual has cancer having NPMl mutation may be of any suitable kind, including an assay for nucleic acid or polypeptide, for example.
- Exemplary assays include polymerase chain reaction, sequencing, immunohistochemistry, immunoblot, and so forth.
- the present invention includes compounds that exert a cytotoxic effect on cells harboring mutant NPMl .
- proteasome activation is involved in the degradation of mutant NPM protein, and in particular cases, this activation is enhanced for treatment.
- the demonstration that knocking down MEK using siRNA also decreased mutant NPMl levels indicates that ERK activation is associated with modulation of mutant NPMl .
- combination therapy with the invention is employed, and such therapy may be additive in effect, or it may be synergistic in at least certain cases.
- Some exemplary additional anti-cancer agents include CCI-779, RADOOl, Sorafenib, Ara-C, all-trans retinoic acid ABT737, LAP inhibitor such as XIAP or survivin antisense, or SMAC mimetics inhibiting, among other targets, cIAP l and cIAP2, rapamycin, cytarabine, HDAC inhibitor, or demethylating agent.
- there is a method of treating an individual identified as having a cancer having a NPMl mutation comprising the step of administering to the individual a MEK inhibitor.
- a method of downregulating mutant NPMl in an individual comprising the steps of: a) identifying an individual with a NPMl mutation; b) administering to the individual a NPMl inhibitor, and in at least certain cases the NPMl inhibitor is further defined as a MEK inhibitor.
- methods and compositions of the invention target a particular population of individuals with cancer or suspected of having cancer.
- populations include those with constitutively activated mitogen-activated protein kinase (MAPK) or Ras/Raf/MEK/ERK signaling pathway and/or that have nucleophosmin- 1 (NPMl) mutations; additional characteristics may include those individuals with cancer cells having normal karyotype, with cancer cells that are CD34/CD133 negative, individuals having high bone marrow blasts, individuals having high white blood cell count, individuals having cytoplasmically located NPMl (particularly in the cytoplasm of leukaemic blasts, for example), and/or has individuals with cancer cells having one or more FLT3-ITD or FLT3 point mutations.
- MPK mitogen-activated protein kinase
- NPMl nucleophosmin- 1
- the individual has high- risk AML (for example, relapsed, resistant/refractory, or having adverse cytogenetics, including, but not restricted to, abnormalities of chromosomes 5, 7, 8, and 1 1).
- the individual has mutations in their leukemic or bone marrow stroma cells, including IHD1, IDH2, p53, and others.
- the individual has CD34 + /CD387CD123 + and/or ALDH+ leukemia stem cells (LSC).
- LSC ALDH+ leukemia stem cells
- Certain embodiments of the invention encompass targeting leukemias with NPMl mutations (including with NPM1/FLT3-ITD mutations, for example).
- nucleophosmin- 1 (NPMl) mutation there are methods for treating an individual having cancer cells with a nucleophosmin- 1 (NPMl) mutation, comprising selecting an individual that has been determined to have a NPMl mutation and treating the individual with an effective amount of a MEK inhibitor.
- a method for determining a treatment regimen for an individual comprising the steps of (a) obtaining a biological sample comprising genetic material of the individual, wherein the subject is undergoing or is to undergo cancer therapy; and (b) determining the presence of a NPMl mutation in the biological sample, wherein when a NPMl mutation is present, the individual is administered an effective amount of a MEK inhibitor.
- the cancer being treated is leukemia (such as acute myeloid leukemia or chronic myelomonocytic leukemia), lymphoma, or myelodysplasia syndrome.
- leukemia such as acute myeloid leukemia or chronic myelomonocytic leukemia
- lymphoma lymphoma
- myelodysplasia syndrome myelodysplasia syndrome.
- Exemplary MEK inhibitors may be selected from the group consisting of N-((S)-2,3-Dihydroxy-propyl)-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide, CI- 1040, PD035901, AZD6244, GSK1 120212, GDC-0973, U0126, XL-518, ARRY-162, ARRY-300, PD184161, PD184352, PD0325901, ARRY-142886 (AZD6244), RO4927350, PD 0325901, CIP-1374, TAK-733, CH4987655, RDEA1 19, and a combination thereof.
- an individual is treated with an additional anti-cancer agent, such as one selected from the group consisting of chemotherapy, hormone therapy, surgery, radiation, immunotherapy, and a combination thereof.
- the additional anti-cancer agent is CCI-779, RAD001, Sorafenib, Ara-C, all- trans retinoic acid, ABT737, IAP inhibitor, SMAC mimetic, rapamycin, cytarabine, HDAC inhibitor, or a demethylating agent, for example.
- the method step of determining whether there is a NPMl mutation in cancer cells of the individual comprises the step of obtaining a biological sample having genetic material from the individual.
- the sample comprises bone marrow, biopsy, blood, urine, cerebrospinal fluid, cheek scrapings, stool, semen, serum, or biopsy material from leukemic tumors or tissue.
- Genetic material may comprise DNA, RNA, or both, for example.
- determination of the NPMl mutation in a biological sample from the individual comprises assaying nucleic acid from the sample, such as DNA or RNA.
- the assay comprises polymerase chain reaction, sequencing, immunohistochemistry, flow cytometry, or a combination thereof.
- determination of the NPM1 mutation in a biological sample from the individual comprises assaying polypeptide from the sample, such as by immunohistochemistry, immunoblot, flow cytometry, or a combination thereof.
- the methods further comprise the step of determining whether the individual has cancer cells comprising a FLT 3 mutation.
- the methods comprise the step of treating the individual with an effective amount of a mTOR inhibitor, a FLT3-ITD inhibitor, an aurora kinase inhibitor, or a combination thereof.
- FIG. 1 shows human leukemia cells (OCI/AML3) that were treated with combined sorafenib and Ara-C for 48 hours, resulting in synergistically enhanced cell killing.
- the average combination index (CI) at ED50, ED75 and ED90 was 0.279 ⁇ 0.077.
- FIG. 2 demonstrates targeting MEK/mTOR signaling simultaneously resulted in highly synergistic proapoptotic effect on human leukemia cell line U937.
- the average combination index (CI) at ED50, ED75 and ED90 was 0.014 ⁇ 0.008.
- FIG. 3 shows that ATRA enhances MEK inhibitor induced cytotoxic effects in OCT/AML3 leukemic cells.
- the average combination index (CI) at ED50, ED75 and ED90 was 0.26 ⁇ 0.11.
- FIG. 4 Murine mesenchymal stem cells (MS5) protect from sorafenib- induced cell death in vitro in co-culture systems. Sorafenib sensitive leukemia cells (Ba/F3- FLT3-ITD) were exposed to indicated concentrations of sorafenib for 72 hours, and apoptosis was measured using flow cytometry.
- FIG. 5 Murine leukemia model was established by injecting Ba/F-FLT3- ITD cells i.v.
- the BM leukemia cells (GFP; green immunofluorescence) were less sensitive to 2-week sorafenib administration compared with the efficacy in spleen or blood.
- FIG. 6 Murine Leukemia modal was established by directly injecting Ba/F3FLT3- ITD cells into BM cavity of tibia in SCID mice. Two weeks after injection, the leukemia cells are distributed throughout the BM, liver, spleen, and blood.
- FIG. 7 demonstrates that Compound 1 shows a predominant cytostatic effect in AML ells that have constitutive ERK activation.
- FIG. 8 demonstrates that Compound 1 shows lower (nanomolar level) IC5 0 S of cell growth in sensitive AML cell lines.
- FIG. 9 shows that Compound 1 induces inhibition of cell growth by delaying cell cycle Progression into S phase.
- FIG. 10 demonstrates that the sensitivity of Compound 1 to induce apoptosis in AML cell lines is dependent upon threshold activity of ERK, and relates to upregulation of Bim and downregulation of Mcl-1 proteins.
- FIG. 11 illustrates that Compound 1 induces loss of mitochondrial membrane potential in sensitive cell lines.
- FIG. 12 shows that p53 level plays a minor effect on Compound 1- induced AML cell death.
- FIG. 13 demonstrates that Compound 1 suppresses phospho-ERK and - S6K levels and decreases Mut-NPMl level in OCI/AML3 cells.
- FIG. 14 provides that NIH-3T3-NPMl-mut cell is sensitive to
- FIG. 15 shows that the level of mutant NPMl is downregulated by Compound 1 and partially abrogated by proteasome inhibitor MG132.
- FIG. 16 illustrates localization of wild-type and mutant NPMl proteins in human AML cells.
- FIG. 17 shows that Compound 1 treatment decreases Mut-NPMl level in OCLA.ML3 leukemic cells.
- FIG. 18 illustrates that targeting MEK signaling by inhibitors results in downregulation of mutant-NPMl, which is not Compound 1 specificity, and also accompanies with decrease of phospho-S6K, phospho-Rb and cdk2.
- FIG. 19 provides that suppressing phospho-ERK by MEK siRNA is associated with decrease of mutant NPMl level in OCLA.ML3 cells.
- FIG. 20 demonstrates that Compound 1 shows moderate sensitivity in NPM1/FLT3-ITD dual mutations in AML patient samples ex vivo.
- FIG. 21 provides combination of Compound 1 with mTOR inhibition that shows synergistic pro-apoptotic effects in human AML cell lines.
- FIG. 22 shows that combination of Compound 1 with ABT737 exerts variable pro-apoptotic effects in human AML cell lines with high basal Bcl-2 levels.
- FIG. 23 demonstrates that combination of Compound 1 with Ara-C shows additive pro-apoptotic effects in human AML cell lines.
- FIG. 24 shows moderate synergistic effect after combination treatment of Compound 1 and Doxorubicin in human AML cells.
- FIG. 25 demonstrates that combined Compound 1 with ATRA synergists pro-apoptotic effect in human APL cells NB4 and AML cells OCI/AML3.
- FIG. 26 provides evidences that combined Compound 1 with Nutlin-3a only exert pro-apoptotic effect in p53 wild type AML cells, but not in p53 mutant cells U937.
- FIG. 27 illustrates that combination of Compound 1 with sorafenib performs synergistic pro-apoptotic effect in human AML cells OCLA.ML3 and MOLM13, the latter harbor FLT3-ITD mutations and only require quite lower concentration of sorafenib.
- FIG. 28 shows combination treatment of Compound 1 with mTOR Inhibitor CCI-779 in human AML cell line MOLM13.
- FIG. 29 demonstrates that mesenchymal stromal cells protect OCI/AML3 leukemic cells from Compound 1 -induced apoptosis.
- an effective amount means that amount which, when administered to a subject or patient for treating cancer, for example, is sufficient to effect such treatment for the disease, including alleviation of at least one symptom of the disease.
- an individual has cancer that has a mutant NPM1 and the individual is treated therefor.
- Cancers that have mutant NPM1 may be of any kind, but in specific embodiments the cancer is leukemia, lymphoma, or non- small-cell lung carcinomas al.
- the cancer includes acute myeloid leukemia (AML) or chronic myelomonocytic leukemia (CML).
- AML is a frequent hematopoietic malignancy with complex genetic disorders. A number of aberrant molecular events have been identified in AML that could promote leukemogenesis.
- NPMl nucleophosmin-1
- compositions that decrease mutant NPMl protein in addition to suppressing MEK activity, and this results in a cytotoxic effect in cancer cells that harbor NPMl mutation(s).
- the Examples herein demonstrate a decrease of mutant NPMl, but not of wildtype NPMl, after 2 hours of Compound 1 treatment, and this occurred parallel to the downregulation of phospho-ERK and -S6K.
- the decrease in mutant NPMl protein is partially abrogated by proteasome inhibitor MG132, indicating that in at least some embodiments proteasome activation is involved in the degradation of mutant NPM protein.
- mutant NPMl which exclusively localizes to the cytoplasm, decreased significantly after 4 hours of Compound 1 treatment, as detected by immunoblotting and immunofluorescence staining.
- Different MEK inhibitors were tested for such dual activity in exemplary OCLA.ML3 cells. The results showed significantly decreased mutant but not wild- type NPMl levels after 24 hours with all tested MEK inhibitors.
- MEK siRNA was used for knock-down of MEK and suppression of ERK activation. Suppression of phospho-ERK resulted in decrease of mutant NPMl levels, but not of wt-NPMl, indicating that ERK activation is associated with modulation of mutant NPMl.
- the exemplary small molecule MEK inhibitor Compound 1 affects the downregulation of mutant-NPMl and is accompanied by decreased phospho-ERK, -S6K, - Rb and cdk2, in certain embodiments.
- the suppression results from inhibition of MEK activity in at least certain embodiments of the invention.
- NPM1 is a phosphoprotein normally localized in the nucleolus, having high expression in proliferating cells. NPM1 protein binds to nucleic acids, regulates centrosome duplication, and also regulates ribosomal function. In addition, NPM1 binds to several proteins, like p53 and proteins that react to and regulate p53 (like Rb, 17 pl9ARF 18 and HDM2). In certain aspects, mutations in NPM1 are a primary event in leukemogenesis, given the high frequency of NPM1 mutations in normal-karyotype AMLs and the fact that cytoplasmic NPM1 fails to carry out its normal functions such as binding to proteins and transferring them. NPM1 mutations often occur in association with FLT3/ITD mutations; FLT3 is a receptor tyrosine kinase having important roles in hematopoietic stem/progenitor cell survival and proliferation.
- an individual is treated for a cancer that has cells that harbor one or more mutations in nucleophosmin (which may also be referred to as also known as N038, nucleolar phosphoprotein B23, numatrin, or NPM1).
- the cancer having at least one NPM1 mutation may be of origin in any tissue, but in specific embodiments the cancer is leukemia or lymphoma, for example.
- the mutation may be present in coding region or non-coding region.
- the mutation may be present in a regulatory sequence. In particular, the mutation may be in an exon, intron, 5'UTR, or 3'UTR.
- a mutation may be identified in any NPM1 polynucleotide sequence, including genomic or mRNA sequences.
- Exemplary NPM1 polynucleotide are obtainable by standard means in the art, including from the National Center for Biotechnology Information's GenBank® database, and exemplary sequences are provided at least in the following, all of which are incorporated herein by reference: NM_001037738.2;
- GenBank® genomic sequences include at least the following, all of which are incorporated herein by reference: NG_016018.1; M28699.1; EU418625.1 (containing exon 12 and partial coding sequence).
- an exon is mutated in NPM1. Although any exon may be mutated, in certain cases the mutation is in exon 4, 5, 6, or 12, for example.
- any NPM1 mutation is encompassed by the invention, including point mutations, inversion, translocations, deletions, frame shifts, and so forth, for example.
- the mutation is t(5;17)(q35;ql2), leading to NPM-RARa as a mutated product, which is associated with acute promyelocytic leukaemia (APL) (Redner et al, 1996).
- APL acute promyelocytic leukaemia
- the mutation is t(2;5)(p23;q35), leading to NPM-ALK, which is associated with anaplastic large cell lymphoma (ALCL) (Morris et al, 1994).
- the mutation is t(3;5)(q25;q35) that results in NPM-MLF1, leading to myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) (Yoneda-Kato et al, 1996).
- MDS myelodysplastic syndrome
- AML acute myeloid leukaemia
- Exemplary mutations are described in the literature and are encompassed in the invention. For example, deletion (-5q35, -5) leads to MDS (de novo, therapy-related), AML (French-American-British classification of leukaemia (FAB-M6)), and non-small-cell lung carcinoma (Olney et al, 2002; Mendes-da-Silva et al, 2000).
- NPM1 compared to the wildtype sequence (Pazhakh et al, 2011); the sequences were identified by using primers NPM1-F (5 '-TTAACTCTCTGGTGGTAGAATGAA-3 ' ; SEQ ID NO:l) and NPM1-R (5'- CAAGACTATTTGCCATTCCTAAC-3 ' ; SEQ ID NO:2), as described in previous studies (Falini et al, 2005).
- Allele B forward seq
- ACGGTACGTCTCTAGA SEQ ID NO: 1
- Mutations in NPM1 may be identified by any suitable method in the art, but in certain embodiments the mutations are identified by one or more of polymerase chain reaction, sequencing, histochemical stain for NPM1 localization, as well as immunostaining method using anti-mutant-NPMl antibody.
- mononucleated cells may be isolated by standard Ficoll-Hypaque density gradient centrifugation. Nucleic acid isolation, cDNA synthesis and screening for NPM1 gene mutations may be performed using a melting curve-based LightCycler assay (Roche Diagnostics, Mannheim, Germany). AML samples with an aberrant melting curve may undergo nucleotide sequence analysis, for example.
- qualitative assays for NPM1 mutations are employed for detecting NPM1 mutations in R A or DNA extracted from fresh bone marrow or peripheral blood leukemic cells, although plasma and paraffin-embedded samples may also be employed.
- the artisan may use synthetic transcripts and total RNA from leukemic cell lines in an assay that can specifically detect NPM1 wild-type and mutants A, B, D, or J transcripts (for example) in the same reaction (Hafez et ah, 2010).
- a mutation in NPM1 is identified in a polypeptide.
- Exemplary GenBank® sequences include at least the following, all of which are incorporated by reference herein in their entirety: AAH03670.2; AAH09623.1 ; NP 002511.1; NP_001032827.1 ; NP_954654.1 ; BAG70175.1 ; BAG70050.1 ; AAH50628.1 ; and AAH08495.1.
- the mutation may be located in any region of the NPM1 protein, but in specific embodiments it is located in a hydrophobic segment, such as one near the N-terminus that is involved in oligomerization as well as chaperone activity.
- the mutation may be found in one of two acidic stretches that are employed for binding to histones, or it may be located in a central portion between the two acidic domains that is required for ribonuclease activity.
- the mutation may be located in the C-terminal domain that contains basic regions involved in nucleic-acid binding; the basic clusters are followed by an aromatic stretch, which contains two tryptophan residues (288 and 290) that are required for nucleolar localization of the protein.
- NPM nuclear-localization signal
- NES nuclear-export signal
- the NPM1 mutation results in a truncated protein.
- Molecular methods and their alternatives are currently available for identifying mutated NPMl (Falini et ah, 2010), which also includes exemplary mutations.
- the mutation identified in a polypeptide may be identified by any means standard in the art, but in specific embodiments the mutation is identified by immunohistochemistry, western blot, and/or PCR.
- Western blot assay may employ antibodies that recognize NPMl mutants but not wild-type NPMl protein in lysates from AML samples, for example (Martelli et ah, 2008).
- the exemplary antibodies employed therein identify a specific band (37kDa) of mutated NPMl protein only in NPMl-mutated AML cases and recognizes over 95% of NPMl mutations.
- one or more MEK inhibitors are utilized in the treatment of an individual that has a NPMl mutation in cancer cells.
- the MEK inhibitor encompassed by the invention inhibits MEK but also downregulates mutant NPMl in cancer cells.
- the MEK inhibitor may be of any kind of molecule, but in certain embodiments the inhibitor is a small molecule, protein, or nucleic acid.
- the MEK inhibitor is selected from the group consisting of Compound 1, CI-1040, PD035901, AZD6244, GSK1 120212, GDC-0973, U0126, XL-518, ARRY-162, ARRY-300, PD184161, PD184352, PD0325901, ARRY-142886 (AZD6244), RO4927350, PD 0325901, CIP-1374, TAK-733, CH4987655, RDEA1 19, and combination thereof.
- the MEK inhibitor is a non-ATP-competitive small- molecule inhibitor (e.g. PD 098059, U0126, PD 184352 and its derivatives) or a biological inhibitor (e.g. anthrax lethal toxin and Yersinia outer protein J).
- the MEK inhibitor is a pyrrole derivative.
- MEK inhibitors are described that are pyrrole inhibitors of MEK kinase using structure-based drug design, for example (Wallace et ah, 2010).
- the MEK inhibitor is a 4-anilino-3-cyano-6,7-dialkoxyquinoline, including 4-anilino-3-cyano-6,7-dialkoxyquinolines with different anilino groups at the 4-position (Zhang et al, 2001).
- an "anti-cancer” agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.
- these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell.
- This process may involve contacting the cells with the expression construct and the agent(s) or multiple factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the second agent(s).
- MEK inhibitor therapy could be used similarly in conjunction with chemotherapeutic, radiotherapeutic, or immunotherapeutic intervention, in addition to pro-apoptotic or cell cycle regulating agents, for example.
- the MEK inhibitors can be employed with an aurora kinase inhibitor or an inhibitor of MDM2, for example.
- the additional agent is one that allows penetration of the bone marrow microenvironment, for example, characterized by hypoxia and mesenchymal stem cells (MSC) in sub-endosteal niches.
- the additional agent targets mTOR signaling.
- the invention includes combinations of MEK inhibitors with rapamycin analogs (CCI-779 or RAD001, for example); a Raf/FLT3-ITD inhibitor, such as Sorafenib or AC- 220; a conventional chemotherapy agent, such as Ara-C; and/or a differentiation inducing agent, such as all-trans retinoic acid (ATRA).
- the MEK inhibitor and the additional anti-cancer agent provides a synergistic therapeutic effect.
- Specific inhibitors include a BH3 mimetic (ABT737), an HDM2 inhibitor, such as nutlin and its derivatives, SMAC mimetics, Ara-C, rapamycin, HDAC inhibitors and hypomethylating agents, for example.
- the MEK inhibitor therapy may be used simultaneously with the other agent, or it may precede or follow the other agent treatment by intervals ranging from minutes to weeks, for example.
- the other agent and MEK inhibitor are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and MEK inhibitor would still be able to exert an advantageously combined effect on the cell.
- MEK inhibitor therapy is "A” and the secondary agent, such as radio- or chemotherapy, for example, is "B":
- Cancer therapies also include a variety of combination therapies with both chemical-and radiation-based treatments.
- Combination chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing.
- CDDP cisplatin
- carboplatin carboplatin
- the combination chemotherapy may depend on the type of leukemia.
- Acute lymphoblastic leukemia (ALL) medicines may comprise prednisone, methotrexate, 1- asparaginase, vincristine, and doxorubicin or daunorubicin.
- Acute myelogenous leukemia (AML) medicines may include daunorubicin with cytarabine (and idarubicin or mitoxantrone may be used instead of daunorubicin).
- Acute promyelocytic leukemia (APL) medicines may comprise all-iraws-retinoic acid (ATRA) and chemotherapy with arsenic trioxide, idarubicin, or daunorubicin, for example.
- ATRA all-iraws-retinoic acid
- Chemotherapy for chronic lymphocytic leukemia may include one medicine or a combination of medicines, including, for example, cyclophosphamide, vincristine, prednisone, fludarabine or chlorambucil, or the monoclonal antibodies rituximab and alemtuzumab.
- Tyrosine kinase inhibitors may be employed for chronic myelogenous leukemia (CML), such as imatinib, dasatinib, or nilotinib, or interferon alfa (with or without cytarabine), hydroxyurea, or busulfan.
- Radiation treatment is used sometimes in cancer.
- leukemia for example, it may be used for cells in the brain and spinal fluid or testicles. It is also used, though rarely, in an emergency to treat compression of the trachea (windpipe).
- ⁇ -rays X-rays
- X-rays X-rays
- UV-irradiation UV-irradiation
- Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
- Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
- contacted and “exposed,” when applied to a cell are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell.
- both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.
- Immunotherapeutics generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
- the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
- the antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing.
- the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
- the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
- Various effector cells include cytotoxic T cells and NK cells.
- Immunotherapy thus, could be used as part of a combined therapy, in conjunction with MEK inhibitors.
- the general approach for combined therapy is discussed below.
- the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
- Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55.
- the secondary treatment is a gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the MEK inhibitor.
- Delivery of a MEK inhibitor in conjuction with a vector encoding one of the following gene products, for example, will have a combined anti-hyperproliferative effect on target tissues.
- proteins are encompassed within the invention, including inducers of cellular proliferation, inhibitors of cellular proliferation, or regulators of programmed cell death.
- Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.
- a venous access device large plastic tube may be placed into a large vein.
- Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed.
- Tumor resection refers to physical removal of at least part of a tumor.
- treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
- a cavity may be formed in the body.
- Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, or 12 months. These treatments may be of varying dosages as well.
- Other agents may be of varying dosages as well.
- agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment.
- additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adehesion, or agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers.
- Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, ⁇ - lbeta, MCP-1, RANTES, and other chemokines.
- cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL would potentiate the apoptotic inducing abililties of the present invention by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti- hyperproliferative effects on the neighboring hyperproliferative cell population.
- cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyerproliferative efficacy of the treatments. Inhibitors of cell adehesion are contemplated to improve the efficacy of the present invention.
- cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.
- FAKs focal adhesion kinase
- Lovastatin Lovastatin
- Hormonal therapy may also be used in conjunction with the present invention or in combination with any other cancer therapy previously described.
- the use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.
- compositions of the present invention comprise an effective amount of one or more MEK inhibitors dissolved or dispersed in a pharmaceutically acceptable carrier.
- pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
- the preparation of an pharmaceutical composition that contains at least one MEK inhibitor or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference.
- preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
- pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
- the MEK inhibitor may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
- the present invention can be administered intravenously, intradermally, trans dermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneous ly, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
- the MEK inhibitor may be formulated into a composition in a free base, neutral or salt form.
- Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
- compositions of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent.
- the carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers.
- compositions may also comprise various antioxidants to retard oxidation of one or more component.
- microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
- preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
- the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.
- the composition is combined or mixed thoroughly with a semi-solid or solid carrier.
- the mixing can be carried out in any convenient manner such as grinding.
- Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach.
- stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
- the present invention may concern the use of a pharmaceutical lipid vehicle compositions that include a MEK inhibitor, one or more lipids, and an aqueous solvent.
- lipid will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term "lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance.
- Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof.
- neutral fats phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof.
- lipids are also encompassed by the compositions and methods of the present invention.
- the MEK inhibitor may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art.
- the dispersion may or may not result in the formation of liposomes.
- the actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
- compositions may comprise, for example, at least about 0.1% of an active compound.
- the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
- the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
- a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
- a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
- the MEK inhibitor are formulated to be administered via an alimentary route.
- Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract.
- the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually.
- these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
- the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et ah, 1997; Hwang et ah, 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety).
- the tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.
- a binder such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof
- an excipient such as, for
- the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001.
- the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells.
- a syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
- any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
- the active compounds may be incorporated into sustained-release preparation and formulations.
- compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally- administered formulation.
- a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
- the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically- effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
- the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
- suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids.
- traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof.
- suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
- the MEK inhibitor may be administered via a parenteral route.
- parenteral includes routes that bypass the alimentary tract.
- the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543, 158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).
- Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
- the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety).
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
- a coating such as lecithin
- surfactants for example
- the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
- isotonic agents for example, sugars or sodium chloride.
- Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
- the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
- aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
- sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
- one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035- 1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
- preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
- Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
- the preferred methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
- a powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
- the active compound MEK inhibitor may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.
- topical i.e., transdermal
- mucosal administration intranasal, vaginal, etc.
- inhalation inhalation
- compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder.
- Ointments include all oleaginous, adsorption, emulsion and water-solubly based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only.
- Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram.
- compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base.
- Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture.
- Transdermal administration of the present invention may also comprise the use of a "patch".
- the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.
- the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
- Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety).
- the delivery of drugs using intranasal microparticle resins Takenaga et ah, 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725, 871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts.
- aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant.
- the typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent.
- Suitable propellants include hydrocarbons and hydrocarbon ethers.
- Suitable containers will vary according to the pressure requirements of the propellant.
- Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms. Kits of the Invention
- compositions described herein may be comprised in a kit.
- one or more MEK inhibitors may be comprised in a kit.
- the kits will thus comprise, in suitable container means, a MEK inhibitor and, optionally, an additional agent of the present invention.
- kits may be packaged either in aqueous media or in lyophilized form.
- the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.
- the kits of the present invention also will typically include a means for containing the MEK inhibitor, optional lipid, optional additional agent, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.
- the kit may have a single container means, and/or it may have distinct container means for each compound.
- the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
- the MEK inhibitor compositions may also be formulated into a syringeable composition.
- the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit.
- the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
- the kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
- compositions for identifying a NPM1 mutation and/or a FLT3 mutation such as PCR primers and appropriate reagents for PCR and/or a microassay chip for detection of the mutation(s).
- sequencing, hybridization, and/or restriction enzyme reagents are provided in a kit.
- an additional agent such as an mTOR inhibitor, aurora kinase inhibitor, and/or FLT3-ITD inhibitor is included in a kit.
- aspects of the present invention employ a strategy of combining a MEK inhibitor with rapamycin analogues (CCI779 or RAD001) (Zeng et al, 2007), the Raf/FLT3-ITD inhibitor Sorafenib, the conventional chemotherapy agent Ara-C or differentiation inducing agents, such as all- trans retinoic acid (ATRA) (Milella et al, 2007).
- rapamycin analogues CCI779 or RAD001
- the Raf/FLT3-ITD inhibitor Sorafenib the conventional chemotherapy agent Ara-C or differentiation inducing agents, such as all- trans retinoic acid (ATRA) (Milella et al, 2007).
- ATRA all- trans retinoic acid
- Other embodiments include AK inhibitors combined with the same or related compounds.
- aspects of the invention provide that activation of multiple signaling pathways is associated with chemo-resistance in acute myeloid leukemia (AML). Therefore, targeting MEK and Aurora kinase (AK), well-established, prognostically relevant targets in AML, and other critical survival pathways, identifies specific biomarkers of synergistic effects in leukemic cells and stem cells in vitro under the physiologically relevant condition of a hypoxic microenvironment, and this is then applied in vivo.
- AML acute myeloid leukemia
- Embodiments of the invention include the definition of molecular and functional effects of MEK and AK blockade in AML. Other embodiments include the development of optimized combinations of MEK inhibitor Compound 1, AK inhibitor R763, and other targeted agents against leukemic cells and stem cells and the identification of biomarkers of synergistic effects. In some embodiments, the influence of the bone marrow microenvironment on the efficacy of MEK and AK inhibition in vitro. In certain embodiments, one can assess in vivo efficacy of effective combination regimen in murine leukemia models. Exemplary Results
- MEK inhibitors other than Compound 1 have been tested. While CI- 1040 alone resulted in dephosphorylation of MEK/ERK, the resulting effects were predominantly cytostatic (Milella et al, 2001). Combination with ATRA converted the cytostatic into a cytotoxic response (Milella et al, 2007) . Excellent synergism was also observed with Bcl2 inhibitors, from Bcl-2 anti-sense to small molecule inhibitors such as HA 141 and ABT_737 (Milella et al, 2002; Konopleva et al, 2006).
- hypoxic BM niches are considered sanctuaries for leukemia cells evading chemotherapy, it is useful to characterize anti-leukemia effects in the BM microenvironment.
- Co-culture systems of leukemia and MSC in normoxic and hypoxic conditions are established, in addition to a live confocal microscopy imaging system that allows live imaging under normoxic and hypoxic conditions of leukemia cells, alone or in co-culture with MSC.
- MSC protects leukemia cells from kinase inhibitor (e.g. Imatinib or Sorafenib) and chemotherapy (e.g. Ara-C) induced cell death in vitro (Fig. 4).
- kinase inhibitor e.g. Imatinib or Sorafenib
- chemotherapy e.g. Ara-C
- Raf/MEK inhibitors such as sorafenib or AZD6244 can elicit upregulation of pro-apoptotic protein Bim32 and a flow cytometric assay may be employed to allow determination of phospho-ERK in bulk leukemia and in AML progenitor/stem cells.
- AK inhibitors have focused on apoptosis induction, cell cycle effects, and polyploidization of target cells.
- AML cell lines are exposed to increasing concentrations of Compound 1 and R763 for different periods of time.
- R763 is tested in MoLml3, OCI-AML3, U937 and KG-1 cells.
- Cell viability and apoptosis induction are determined by quantitative flow cytometry techniques (pERK activity gated on leukemic bulk or progenitor/stem cells).
- DNA ploidy, MMP, Annexin V/PI, absolute cell counts are determined.
- Compound land R763 are combined with Ara-C, daunorubicin, etoposide, Nutlin-3a, ABT737, sorafenib, ATRA and 5-Aza or DAC for assessing synergistic effects in human AML cells, especially in cells that are resistant to single agents, by determining effects on cell growth, cell cycle progression, mitochondrial membrane potential and apoptosis using the methods mentioned herein, for example.
- the initial experiments are performed with 2-3 AML cell lines with apoptosis induction as endpoint.
- Combination indices are determined using the Chou-Talalay method. Furthermore, combination effects are assessed in 10-20 freshly-isolated primary AML samples by determining induction of apoptosis (Annexin V positivity, loss of MMP) in bulk cells and in LSC defined as CD34 + /CD387CD 123 + cells (Konopleva et al, 2006).
- CD34 + /CD38 " or CD34 + cells, which represent leukemic progenitors are analyzed. Optimized combinations are also tested in colony-forming assays that determines differential effects on normal vs. leukemic colony- forming cells.
- AML cells or primary AML samples are treated with combinations in the absence or presence of BM mesenchymal stem cells (MS5 or primary human MSC) feeder layers, and apoptosis are measured by annexinV positivity in CD34 + /CD387CD123 + cells by 4-color flow cytometry assay (Konopleva et al, 2006). Effects on protein phosphorylation, cell proliferation and cell death of bulk tumor or tumor stem cells can be determined by 10-color flow cytometry, for example.
- hypoxia 1 % oxygen tension
- IC5 0 S and ED 50 s are derived as appropriate and correlated with constitutive MAPK and AK activation status.
- hypoxia is useful, as there is striking induction of pERK in AML cells in hypoxia, in a specific embodiment of the invention. Therefore, the ability of AS703026 to suppress this effect in hypoxia, which is the physiological pC>2 in the bone marrow, is further characterized.
- the inventors have established an imageable in vivo leukemia model by injecting i.v. murine leukemia Baf3/FLT3-ITD and other human leukemia cells (stably transduced with firefly luciferase fused to GFP) into SCID mice, resulting in engraftment in BM, spleen and liver (Zhang et ah, 2008; Zeng et ah, 2008). Growth and topical dissemination of the leukemic cells is quantified by bioluminescence imaging (BU).
- BU bioluminescence imaging
- Colentazine is injected i.p., for example, and the animals are imaged by Xenogen IVIS; bioluminescence is quantified as the total sum integrated signal in a standard period of time (i.e., 2 min).
- Leukemia tissue infiltration is determined by GFP-positivity and quantified by spectral image analysis (CRI system). Survival is calculated using the method of Kaplan- Meier.
- NOD/Scid/IL2Ry _/" mice are considered the optimal model of human leukemia.
- FACS-sorted LSC CD34 + /CD387CD123 + and normal human HSC are injected Lv. into NaG (NOD/Scid/lL2Ry "/ ) mice.
- the efficacy of apoptosis induction in LSC and patho- morphological changes will be determined by immunohistochemistry (IHC) and flow cytometry and effects of selected drug combinations will be determined by Kaplan-Meier analysis.
- IHC immunohistochemistry
- AML Acute myeloid leukemia
- AML is a hematopoietic malignancy with complex genetic disorders.
- a number of aberrant molecular events have been identified in AML that are able to promote leukemogenesis.
- constitutive activation of the mitogen-activated protein kinase (MAPK) or Ras/Raf/MEK/ERK signaling pathway are detectable in about 70% of adult AML patients and associated with a strikingly negative impact on patient survival (Kornblau SM et al, Blood 2001).
- Nucleophosmin- 1 (NPM1) gene mutations are primary genetic lesions that can be detected in 35% of primary AML cases with higher incidence in normal karyotype and CD34/CD133 negative AML (Falini B et al, N Engl J Med 2005). The specific cytoplasmic dislocation of the mutant nucleophosmin might exert oncogenic action by binding and interacting with other protein partners, for example, K-Ras (Inder KL et al, Commun Integr Biol. 2010). NPM1 mutations have been significantly associated with high bone marrow blasts and white blood cell count (Thiede C et al, Blood. 2006).
- Fms-like tyrosine kinase 3 (FLT3) gene are another frequent molecular abnormalities which account for 25-40 % of AML and are associated with poor prognosis (Gilliland DG et al, Blood 2002).
- FLT3-ITD mutations can be found in more than 40% of the NPM1 -mutated patients, and are associated with poor prognosis (Thiede C et al, Blood. 2006).
- all of these mutations are associated with high/constitutive activation of Ras/Raf/MEK/Erk signaling. Therefore, targeting MEK-ERK signaling by suppressing MEK activation is beneficial for leukemia therapy, in specific embodiments of the invention.
- the present invention discloses the effects of the novel MEK inhibitor Compound 1 on human AML, as an exemplary cancer.
- cytostatic effect was associated with suppression of phosphorylated-ERK and down-regulation of cdk2/4 levels (Fig. 10).
- Compound 1 showed cytotoxic effects in OCI/AML3 and MOLM13 cells, which have NPM1 and FLT3-YTO mutations, respectively; both of these cell lines contain wildtype p53 (Fig. 7).
- Apoptosis induction in these cells was mediated by the mitochondria-associated intrinsic cell death pathway with loss of mitochondrial membrane potential (Fig. 1 1) and by modulating pro-apoptotic protein Bim and anti-apoptotic protein Mcl-1 (Fig. 10).
- OCI/AML3-p53- shRNA knockdown cells were compared with parental cells in a dose-response fashion. Results indicate that loss of p53 has a marginal effect on Compound 1 -induced apoptosis (Fig. 12).
- mutant NPM1 was exclusively localized in the cytoplasm and that levels decreased significantly after 4 hours of Compound 1 treatment (Figs. 16 and 17), indicating that proteasome activation is involved in the degradation of mutant NPM protein, in at least certain aspects of the invention.
- mutant-NPMl is exclusive to Compound 1
- different MEK inhibitors were tested on OCI/AML3 cells. The results showed significantly decrease in mutant but not wild-type NPM1 levels after 24 hours with all tested MEK inhibitors. Furthermore, inhibition of mutant NPM1 was accompanied by downregulation of phospho-ERK, -S6K, -Rb and cdk2, suggesting that downregulation of mutant-NPMl is not exclusive for Compound 1, but rather results from inhibition of MEK activity (Fig. 18).
- MEK siRNA was used for knocking down MEK and suppressing ERK activation. Result showed that suppression of phospho-ERK resulted in decrease of mutant NPMl, but not wt-NPMl (Fig. 19), indicating that ERK activation is associated with modulation of mutant NPMl .
- Combination of Compound 1 and ABT737 was also active in inducing apoptosis in OCI/AML3 and KG-1 cells, which have high Bcl-2 basal levels (Fig. 22).
- MEK inhibition results in complete downregulation of Mcl-1, a major anti-apoptotic protein which is not targeted by ABT737 (Konopleva M et ah, Cancer Cell. 2006).
- Synergistic suppression of Mcl-1 by Compound 1 and Bcl-2 by ABT737 may greatly enhance the pro-apoptotic effects (Konopleva et al. Cancer Cell, 2006).
- combination of Compound 1 with cytarabine also known as Ara-C
- these combination strategies sensitized U937 and KG-lcells to Compound 1, which showed more resistance to treatment with Compound 1 alone.
- OCI/AML3 cells were treated with Compound 1 in the presence or absence of a feeder layer of mesenchymal stroma cell (MSC). Apoptosis induction and correlated signaling proteins were measured by flow cytometry and Western blot, respectively. The results showed that MSC protected the leukemic cells from Compound 1 -induced apoptosis (FIG. 29).
- OCLAML3 cells display higher basal levels of phospho-ERK in the presence of MSC feeder layer (FIG. 19), in certain aspects of the invention.
- the novel small molecule MEK inhibitor Compound 1 displayed effective anti-leukemic activity in AML cells with constitutive activation of ERK.
- the drug preferentially induces inhibition of cell proliferation rather than apoptosis.
- Compound 1 also showed an impressive cytotoxic effect in OCI/AML3 cells that is related to downregulation of mutant but not wild-type NPMl protein, in embodiments of the invention.
- the decrease of mutant NPMl is associated with suppression of MEK activation.
- synergistic effects of combining Compound 1 with, for example, an mTOR inhibitor or ABT-737 were observed. These results indicate that combining Compound 1 with other pro-apoptotic drugs or signal transduction inhibitors is effective in the treatment of resistant AML.
- the findings provide therapeutic guidance for utilization of Compound 1 in AML, including for AML patients harboring NPMl or dual NPM1/FLT3-ITD mutations, for example.
- FIG. 24 demonstrates the treatment of combined Compound 1 and Doxorubicin synergizes with pro-apoptotic effects in human leukemic cells.
- MOLM13 and KG-1 were treated with indicated concentrations of the drugs for 48 hours.
- the cells were stained with Annexin V-Fluo and apoptosis induction was measured by flow cytometry. Error bars were generated from triplicate experiments and combination index (CI) values were determined using CalcuSyn software (BioSoft).
- FIG. 25 shows that the combination of Compound 1 and ATRA treatment significantly enhances apoptosis induction of leukemias.
- APL cells NB4 and AML cells OCI/AML3 were treated with Compound 1 and ATRA simultaneously for 72 hours.
- Apoptosis induction was measured by counting percentage of annexin V positive population using flow cytometry.
- FIG. 26 shows that targeting MEK/ERK and MDM2/p53 signaling pathways enhances cell death in p53 wild type leukemic cells.
- p53 wild type leukemic cells MOLM13 and p53 mutant cells U937 cells were treated with Compound 1 and Nutlin3a, a small-molecule antagonist of MDM2, for 48 hours.
- Cell apoptosis was analyzed with flow cytometry after annexin V staining. Synergistic apoptosis induction was observed only in p53 wild type cells MOLM13, but not in p53 mutant cells U937.
- FIG. 27 demonstrates blockade of Raf/MEK/ERK and FLT3 signaling pathways synergizes with cell apoptosis induction in AML cells.
- MEK inhibitor Compound 1 and multiple kinase inhibitor sorafenib which targets FLT3-ITD mutations and Raf/MEK signaling as well, were used for treating human AML cells OCI/AML3 and MOLM13, which also harbors FLT3-ITD mutations, for 48 hours.
- FIG. 28 shows combination treatment of Compound 1 with exemplary mTOR inhibitor CCI-779 in an exemplary human AML cell line MOLM13.
- MEK/ERK Constitutive activation of MEK/ERK is consistently found in 50-70% of acute myeloid leukemia (AML) patients, with a striking negative impact on response to chemotherapy and patient survival (Kornblau et ah, 2001 ; Ricciardi et ah, 2005). Therefore, targeting MEK/ERK signaling is a useful strategy for leukemia therapy.
- Studies using MEK inhibitor Compound 1 against AML have shown a predominant cytostatic effect on AML cells with constitutive ERK activation.
- IC5 0 was determined as 7 and 83 nM in OCI/AM13 (with nucleophosmin-1 (NPM1) mutation, p53 wt) and MOLM13 (FLT3-ITD mutation, p53 wt) cells, respectively. It also demonstrated pro-apoptotic activity in these cells. Further analysis demonstrated that the cytotoxic effect was not associated to p53 or FLT3-ITD status. Impressively, OCI/AML3 cells were highly sensitive to Compound 1 -induced apoptosis, accompanied with downregulation of phospho-S6K and NPM1 protein levels.
- NPM1 downregulation showed a similar pattern as phospho-S6K in a dose-dependent manner, but did not show a correlation with downregulation of phospho-ERK, indicating that an ERK- inhibiton-independent mechanism is involved in apoptosis induction in NPM1 mutant leukemic cells treated with Compound 1, in certain embodiments of the invention.
- Mutations of NPMl gene are present in 50-60% of adult acute myeloid leukemias with normal karyotype (AML-NK) (Ralini et ah, 2007), and have been given a new provisional entity in the 2008 WHO classification of myeloid neoplasms (Vardiman et ah, 2009).
- NPMl mutations have been implicated in promoting cell growth, as expression increases in response to mitogenic stimuli and above-normal levels are detected in highly proliferating malignant cells (Grisendi et ah, 2006). NPMl mutations, are not significantly associated with KRAS mutation and P53 mutations (Dohner et ah, 2005; Suzuki et ah, 2005), but a functional link between NPMl and p53 stability (Colombo et ah, 2002), which may involve control of cell proliferation and apoptosis (Ye, 2005), has been postulated. Therefore, targeting MEK/ERK signaling in NPMl mutant cells is beneficial for anti-leukemia therapy, in certain aspects of the invention.
- OCI/AML3 cells are used for these studies. OCI/AML3 cells are exposed to increasing concentrations of Compound 1 for different periods of time. Correlative signaling and proteins (such as NPMl, phospho-ERK, -S6K, -AKT, pi 9, MDM2 and p53 as well as cell cycle and apoptosis-related proteins are investigated by Western blot and multi-parametric phospho flow cytometry, for example. Further, cells (such as OCI/AML2 cells) are infected with lentiviral-NPMl mutant or wt genes.
- Compound 1 in the engineered cells are investigated by assessing cell viability (trypan blue exclusion cell count), cell cycle distribution (PI DNA staining, BrdU incorporation), and induction of apoptosis (assessment of mitochondrial membrane potential - ⁇ - by CMX-Ros/Mitotracker Green double staining, Annexin V/PI staining, flow cytometry).
- IC5 0 S are also determined by measuring cell viability and apoptosis.
- effects of MSC-69B in relevant genes and proteins are characterized using real-time PCR and immunoblotting.
- MEK inhibitors predominant effects are the induction of cytostatic rather than cytotoxic effects in AML, different combination strategies are investigated for enhancing pro-apoptotic effects.
- Compound 1 is combined with Ara-C, daunorubicin, etoposide, Nutlin-3a, ABT737, sorafenib, and ATRA, for example, for assessing synergistic effects in human AML cells, especially in cells that are resistant to
- Compound 1 by determining effects on cell growth, cell cycle progression, mitochondrial membrane potential and apoptosis using the exemplary methods mentioned above.
- the initial studies are performed with 2-3 AML cell lines with apoptosis induction as endpoint.
- Combination indices are determined using the Chou-Talalay method. Combination effects are assessed in 10-20 freshly-isolated primary AML samples by determining induction of apoptosis (Annexin V positivity, loss of MMP) in bulk cells and in LSC defined as CD34+/CD38-/CD 123+ cells (Konopleva et al, 2006). Considering that some samples with low blast percentage may not have sufficient LSC for analysis, in certain aspects, either CD34 + /CD38 " or CD34 + cells, which represent leukemic progenitors, are analyzed.
- AML cells or primary AML samples are treated with combinations in the absence or presence of BM mesenchymal stromal cells (MS5 or primary human MSC) cultured on MSC feeder layers.
- Apoptosis is measured by annexinV positivity in CD34+/CD38-/CD123+ cells by a 4- color flow cytometry assay (Konopleva et al,. 2006).
- hypoxic (1% oxygen tension) conditions In order to better model the physiologic BM microenvironment, these studies may be performed in hypoxic (1% oxygen tension) conditions. IC5 0 S and ED 50 s are derived and correlated with constitutive MAPK activation status. Studies in hypoxia are useful, as there is striking induction of pERK in AML cells in hypoxia (Fiegl et al, 2010). Therefore, the ability of Compound 1 to suppress this effect in hypoxia, which is the physiological ⁇ ( 3 ⁇ 4 in the bone marrow, is considered. A number of optimized combinations are tested in stromal co-culture systems in hypoxia. EXAMPLE 4
- mice Female SCID mice (8-10 weeks old) are allowed to acclimate to their new environment for 1 week prior to the study.
- Leukemia models are established by injecting MOLM13-NPMl-mut cells, which is a human acute myeloid leukemia cell and stably transduced with lentiviral vectors expressing mutant NPMl and firefly luciferase-GFP. These cells are FACS-sorted and expanded in vitro for approximately 1 month prior to injection into animals; these genetically modified cells do not produce virus. Briefly, the AML cells (at 1- 2* 10 6 cells/mouse) are suspended in 200 ⁇ of PBS and be injected iv. in the tail vein of recipient mice.
- the animals are then distributed to five groups randomly for Compound 1 (30mg/kg q.d., gavage), rapamycin (4 mg/kg q.d., i.p.) , combination treatment, vehicle treatment and blank control groups.
- Each group consists of eight tumor-bearing mice.
- mice receive Compound 1, rapamycin or combination treatment on day 7 of the inoculation of leukemic cells.
- the drugs are given using QDx5/week schedule at above indicated doses for 3 weeks, for example. These dose ranges are based on the efficacy studies in tumor xenograft animal models.
- Treated animals are monitored daily for signs of morbidity and mortality and sacrificed when paralyzed or terminally ill.
- Body weights and tumor volumes are determined twice weekly. Histological and immunohistochemical analyses are performed on bone marrow, lung, liver, spleen and brain tissue samples for detection of human cells for all long-term survivors.
- mice for each group are sacrificed at 5 weeks of inoculation using CO 2 .
- Bone marrow cells are collected by flushing both femurs with PBS.
- Single cell suspensions of bone marrow and spleen are analyzed by flow cytometry to determine engraftment level (using GFP and anti-human CD45 antibody).
- a two-sample t-test is performed to test whether the percentage of engrafting leukemic cells in the mouse bone marrow or other organs is the same in the control and the treatment groups. Effects on target inhibition and on selected biomarkers are evaluated in leukemic cells isolated from mouse bone marrow and spleen by FACS-sorting of CD45(+) human cells. Spleen, lungs, liver, and brain are collected for histological analyses and GFP immunohistochemistry. The remaining five animals are sacrificed upon observation of any signs of morbidity.
- mice are anesthetized using isoflurane.
- Colerterazine Native; Biotium, Hayward, CA; 1 mg/kg in ⁇ volume
- Xenogen IVIS imager 1, 2 or 5 min increments of exposure and light capture; during imaging the animals are continuously anesthetized via inhalation of isoflurane vapors.
- Serial bioluminescence is quantified as the total sum integrated signal in a standard period of time (i.e., 2 min).
- a two-sample t-test is performed to test whether tumor volume as defined by the bioluminescence integrity signal is the same in the control and the treatment groups.
- mice Phase I Number of mice required: For the first set of studies, Compound 1 is tested on 8 animals/group using 15mg/kg and 30gm/kg doses, and plus 4 mice for vehicle treatment and 4 mice for control group. The optimal doses are used on combination treatment.
- NOG mice restrained in acrylic box, given whole body irradiation (nonlethal, cesium source, 250 cGy, irradiator is located in SB.8044)
- mice will be injected with 5xl0 6 peripheral blood mononuclear cells (PBMC) from acute myeloid leukemia patient with NPMl mutation after Ficoll-Hypaque purification. All injections are done via the tail vein while the mouse is in the restraint device for 15 seconds (maximum). The cells are injected in 0.2 ml sterile PBS, 27- gauge needle.
- PBMC peripheral blood mononuclear cells
- mice are treated with inhibitors (Compound 1 and rapamycin, for example) or vehicle control, for 4 weeks.
- the most effective dose is chosen based on the results of the studies in cell lines (Phase I) (defined as lack of toxicity and most effective anti-leukemia control).
- mice observed daily including the weekends for signs of disease. Animals that exhibit excessive tumor burden, hunched posture, lack of eating and drinking, difficulty reaching food, loss of >20% weight, lateral recumbence, respiratory distress are euthanized immediately. Twice (at 4 and 6 weeks post leukemic cell injection) blood samples are collected via tail vein bleed (lOOuL) into tubes containing heparine. Single cell suspensions are analyzed by flow cytometry to determine engraftment level (using anti- human CD45) and lineage expression (based on antigen expression defined in Phase II). [0194] At 6-8 weeks animals will be sacrificed using CO 2 .
- Bone marrow, blood, liver and spleen are sampled for morphology, flow cytometry, PCR and FISH to assess for evidence of leukemia.
- a biological marker one can also evaluate effects on target proteins by protein array technique in human leukemic cells isolated from mouse bone marrow by FACS-sorting of CD45(+) cells. If the mice develop lymphoma (immunodeficiency/radiotherapy) or infection they will show the same signs and therefore are sacrificed for evaluation. However, lymphoma usually occurs well beyond age 6 months when mice are allowed to age.
- Reagents preparation [0197] 1. Compound 1 is supplied as dry compound synthesized by EMD Serono Research Institute, Boston, MA. It can be solved in CremophorEL/Ethanol/Water (12.5/12.5/75). The drug is administrated in a volume of 200 by oral gavage.
- Rapamycin is obtained from Biovision Research Products (Mountain View, CA). Rapamycin is dissolved in 100% ethanol (10 mg/mL stock), and further diluted in an aqueous solution of 5.2% Tween-80 and 5.2% polyethylene glycol (PEG-400) (final ethanol concentration, 2%) immediately before use. The drug will be administrated i.p. in a volume of 100 by i.p.
- Doggrell SA Dawn of Aurora kinase inhibitors as anticancer drugs.
- Falini B Falini B, Nicoletti I, Martelli MF, Mecucci C. Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): biologic and clinical features. Blood 2007; 109:874-885.
- Fiegl M Samudio I, Clise-Dwyer K et al. CXCR4 expression and biological activity in acute myeloid leukemia are dependent on oxygen partial pressure. Blood. 2008. [0208] Fiegl M, Samudio I, Mnojan Z, Korchin B, Fritsche H, Andreeff M. : Physiological hypoxia promotes lipid raft and PI3K-dependent activation of MAPK 42/44 in leukemia cells Leukemia, e-publ. 5/2010
- Giles FJ, Cortes J, Jones D et al. MK-0457 a novel kinase inhibitor, is active in patients with chronic myeloid leukemia or acute lymphocytic leukemia with the T3151 BCR-ABL mutation. Blood. 2007; 109:500-502.
- Konoplev S Rassidakis GZ, Estey E et al. Overexpression of CXCR4 predicts adverse overall and event-free survival in patients with unmutated FLT3 acute myeloid leukemia with normal karyotype. Cancer. 2007; 109: 1 152-1 156.
- Konopleva M Contractor R, Tsao T et al. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell. 2006; 10:375-388.
- Martelli MP Manes N, Liso A, Pettirossi V, Verducci Galletti B, Bigerna B, et al.
- Ye K. Nucleophosmin/B23 a multifunctional protein that can regulate apoptosis. Cancer Biol.Ther. 2005;4:918-923.
- Yee KW, Zeng Z Konopleva M et al. Phase 1/11 study of the mammalian target of rapamycin inhibitor everolimus (RADOOl) in patients with relapsed or refractory hematologic malignancies. Clin Cancer Res. 2006; 12:5165-5173.
- Zhao S Konopleva M, Cabreira-Hansen M et al. Inhibition of phosphatidylinositol 3kinase dephosphorylates BAD and promotes apoptosis in myeloid leukemias. Leukemia. 2004; 18:267-275.
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Abstract
The present invention generally concerns treatment of cancer in individuals having at least a mutant NPM1 protein, wherein the treatment includes a compound that downregulates level or inhibits activity of mutant NPM1, including, for example, a MEK inhibitor. In certain aspects, the invention concerns combination therapy with MEK inhibitors and other anti -cancer therapies in individuals having cancer with mutant NPM1.
Description
DESCRIPTION
TREATMENT OF CANCERS WITH MUTANT NPM1 PROTEIN WITH MEK
INHIBITORS
The application claims priority to U. S. provisional patent application No. 61/547,886 filed October 17, 2011 , which is incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0001] In embodiments of the present invention, the field includes molecular biology, cell biology, and medicine, including cancer medicine. In particular aspects the field of the invention concerns compositions for treatment of acute myeloid leukemia (AML).
BACKGROUND OF THE INVENTION
[0002] Acute myeloid leukemia (AML), which accounts for approximately 80% of all adult acute leukemias (Smith et al , 2004), is a clonal disorder affecting hematopoietic stem cells, characterized by an accumulation of immature leukemic cells in the bone marrow (8M) subsequently leading to marrow failure. In about 15-25% of AML cases, Ras gene point mutations have been detected.
[0003] In addition, mutations of FLT3 are found in approximate 30% of AML. These mutations lead to constitutive Ras and downstream Raf/MEK/ERK activation with concomitant activating effects on the P13KlAkt pathway (Birkenkamp et al, 2004). The dysregulated activity of either or both of these signal transduction pathways critically contribute to hematopoietic cell survival and proliferation and thereby to leukemogenesis (Kornblau et al, 2006; Min et al, 2003). Constitutive activation of the MEK/ERK or PI3K/Akt/mTOR signaling modules is consistently detectable in over 70% of AML patients, with a striking negative impact on response to chemotherapy and patient survival (Kornblau et al, 2001). Many small molecule inhibitors targeting Raf/MEK/ERK or mTOR signaling have been developed and are being evaluated in clinical trials (Milella et al, 2001 ; Yee et al, 2006). Sorafenib is a highly effective inhibitor of FLT3-ITD mutations and has encouraging clinical results (Zhang et al, 2008). Likewise, Aurora kinases have been identified as compelling targets for leukemia therapies (Doggrell , 2004). Aur A was over-expressed and
AML samples expressing high levels of Aur A were induced to undergo apoptosis when treated with VX-6S0, a specific inhibitor. Concomitant inhibition of Aurora kinases and MDM2 (p53 interaction activates the p53dependent checkpoint and synergistically induced apoptosis in AML) (Kojima et ah, 2008). In general, however, the clinical efficacy of MEK and AK inhibitors in AML has not yet been established, although promising responses have been reported (Giles et ah, 2007).
[0004] The efficacy of these agents is variable, especially in patients with high- risk AML (relapsed, resistant/refractory, or adverse cytogenetics). The persistence of CD34+/CD387CD123+ leukemia stem cells (LSC) results in relapse. The BM has a microenvironment characterized by hypoxia and mesenchymal stem cells (MSC) in sub- endosteal niches, which generates a sanctuary for subpopulations of LSC to evade chemotherapy- and signal transduction inhibitor-induced death and allows acquisition of a drug resistant phenotype (Konopleva et ah, 2002; Tabe et ah, 2004; Zeng et ah, 2008; Fiegl et al. , 2008). Therefore, more effective treatment regimens are needed. BRIEF SUMMARY OF THE INVENTION
[0005] The present invention is directed to one or more systems, methods and/or compositions related to cancer treatment and/or prevention. In particular embodiments, the present invention concerns compositions and uses thereof for treatment of particular types of cancer, including those that harbor one or more NPM1 mutations; in specific cases, the cancer is leukemia or lymphoma, including acute myeloid leukemia (AML) or chronic myelomonocytic leukemia (CML), for example. In alternative embodiments the individual has myelodysplasia syndrome. In some embodiments, the individual has a solid tumor, such as in the lung, breast, colon, prostate, pancreas, kidney, liver, skin, bone, gall bladder, spleen, uterus, cervix, ovary, testes, rectum, stomach, bladder, nasopharynx, esophagus, and so forth.
[0006] At least some aspects of the invention provide combination strategies that allow the concomitant suppression of multiple critical signaling pathways in cancer, including leukemias. In particular, the activation of certain signaling pathways confers resistance to apoptosis and to chemotherapeutic agents in AML, for example, both in vivo and in vitro, resulting in synergistic "cytotoxic" effects in AML, including in the hypoxic BM microenvironment. Embodiments of the invention exert significant anti-leukemia efficacy
and lowers the dose required of each single agent in AML. In addition, the present invention is of general significance, because it contributes to a better understanding of relevant molecular events related to chemo-resistance of malignant cells in their bone marrow (BM) microenvironment. [0007] Embodiments of the present invention concern MEK inhibitors, including small molecule MEK inhibitors (for example, (N-((S)-2,3-Dihydroxy-propyl)-3-(2- fluoro-4-iodo-phenylamino)-isonicotinamide), hereafter called Compound 1) that are capable of decreasing mutant NPMl protein levels in addition to the suppression of MEK activity. In specific embodiments of the invention, the effect on mutant NPMl downregulation results from inhibition of MEK activity, indicating for the first time a role of MAPK kinase signaling in the regulation of mutant NPMl levels. Particular embodiments of the invention encompass a dual-inhibition function of mutant NPMl and MEK/ERK signaling, which are both related to leukemogenesis. In certain embodiments of the invention, there is reduction of leukemic blasts following utilization of the MEK inhibitors. [0008] In particular embodiments, the individual having cancer cells with a
NPMl mutation includes selecting an individual that has been determined to have a NPMl mutation and treating the individual with an effective amount of a MEK inhibitor. The individual may be selected as having a NPMl mutation following assaying a biological sample from the individual, including a sample from bone marrow, blood, cerebrospinal fluid, urine, stool, semen, nipple aspirate, and so forth. Biopsies may be taken from the individual.
[0009] An assay to determine whether or not an individual has cancer having NPMl mutation may be of any suitable kind, including an assay for nucleic acid or polypeptide, for example. Exemplary assays include polymerase chain reaction, sequencing, immunohistochemistry, immunoblot, and so forth.
[0010] In particular embodiments, the present invention includes compounds that exert a cytotoxic effect on cells harboring mutant NPMl . In specific cases of the invention, proteasome activation is involved in the degradation of mutant NPM protein, and in particular cases, this activation is enhanced for treatment. The demonstration that knocking down MEK using siRNA also decreased mutant NPMl levels indicates that ERK activation is associated with modulation of mutant NPMl .
[0011] In embodiments of the invention, combination therapy with the invention is employed, and such therapy may be additive in effect, or it may be synergistic in at least certain cases. Some exemplary additional anti-cancer agents include CCI-779, RADOOl, Sorafenib, Ara-C, all-trans retinoic acid ABT737, LAP inhibitor such as XIAP or survivin antisense, or SMAC mimetics inhibiting, among other targets, cIAP l and cIAP2, rapamycin, cytarabine, HDAC inhibitor, or demethylating agent.
[0012] In some embodiments, there is a method of treating an individual identified as having a cancer having a NPMl mutation, comprising the step of administering to the individual a MEK inhibitor. In specific embodiments of the invention, there is a method of downregulating mutant NPMl in an individual, comprising the steps of: a) identifying an individual with a NPMl mutation; b) administering to the individual a NPMl inhibitor, and in at least certain cases the NPMl inhibitor is further defined as a MEK inhibitor.
[0013] In particular aspects of the invention, methods and compositions of the invention target a particular population of individuals with cancer or suspected of having cancer. Such populations include those with constitutively activated mitogen-activated protein kinase (MAPK) or Ras/Raf/MEK/ERK signaling pathway and/or that have nucleophosmin- 1 (NPMl) mutations; additional characteristics may include those individuals with cancer cells having normal karyotype, with cancer cells that are CD34/CD133 negative, individuals having high bone marrow blasts, individuals having high white blood cell count, individuals having cytoplasmically located NPMl (particularly in the cytoplasm of leukaemic blasts, for example), and/or has individuals with cancer cells having one or more FLT3-ITD or FLT3 point mutations. In certain embodiments of the invention, the individual has high- risk AML (for example, relapsed, resistant/refractory, or having adverse cytogenetics, including, but not restricted to, abnormalities of chromosomes 5, 7, 8, and 1 1). In certain embodiments the individual has mutations in their leukemic or bone marrow stroma cells, including IHD1, IDH2, p53, and others.. In certain embodiments, the individual has CD34+/CD387CD123+ and/or ALDH+ leukemia stem cells (LSC). Certain embodiments of the invention encompass targeting leukemias with NPMl mutations (including with NPM1/FLT3-ITD mutations, for example).
[0014] In one embodiment of the invention, there are methods for treating an individual having cancer cells with a nucleophosmin- 1 (NPMl) mutation, comprising
selecting an individual that has been determined to have a NPMl mutation and treating the individual with an effective amount of a MEK inhibitor.
[0015] In some embodiments of the invention, there is a method for determining a treatment regimen for an individual, comprising the steps of (a) obtaining a biological sample comprising genetic material of the individual, wherein the subject is undergoing or is to undergo cancer therapy; and (b) determining the presence of a NPMl mutation in the biological sample, wherein when a NPMl mutation is present, the individual is administered an effective amount of a MEK inhibitor.
[0016] In certain embodiments, the cancer being treated is leukemia (such as acute myeloid leukemia or chronic myelomonocytic leukemia), lymphoma, or myelodysplasia syndrome.
[0017] Exemplary MEK inhibitors may be selected from the group consisting of N-((S)-2,3-Dihydroxy-propyl)-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide, CI- 1040, PD035901, AZD6244, GSK1 120212, GDC-0973, U0126, XL-518, ARRY-162, ARRY-300, PD184161, PD184352, PD0325901, ARRY-142886 (AZD6244), RO4927350, PD 0325901, CIP-1374, TAK-733, CH4987655, RDEA1 19, and a combination thereof.
[0018] In some embodiments of the invention, an individual is treated with an additional anti-cancer agent, such as one selected from the group consisting of chemotherapy, hormone therapy, surgery, radiation, immunotherapy, and a combination thereof. In specific embodiments, the additional anti-cancer agent is CCI-779, RAD001, Sorafenib, Ara-C, all- trans retinoic acid, ABT737, IAP inhibitor, SMAC mimetic, rapamycin, cytarabine, HDAC inhibitor, or a demethylating agent, for example.
[0019] In particular aspects of the invention, the method step of determining whether there is a NPMl mutation in cancer cells of the individual comprises the step of obtaining a biological sample having genetic material from the individual. In specific embodiments, the sample comprises bone marrow, biopsy, blood, urine, cerebrospinal fluid, cheek scrapings, stool, semen, serum, or biopsy material from leukemic tumors or tissue. Genetic material may comprise DNA, RNA, or both, for example.
[0020] In some embodiments, determination of the NPMl mutation in a biological sample from the individual comprises assaying nucleic acid from the sample, such
as DNA or RNA. In particular aspects, the assay comprises polymerase chain reaction, sequencing, immunohistochemistry, flow cytometry, or a combination thereof. In some embodiments, determination of the NPM1 mutation in a biological sample from the individual comprises assaying polypeptide from the sample, such as by immunohistochemistry, immunoblot, flow cytometry, or a combination thereof.
[0021] In certain embodiments of the invention, the methods further comprise the step of determining whether the individual has cancer cells comprising a FLT 3 mutation. In some aspects, the methods comprise the step of treating the individual with an effective amount of a mTOR inhibitor, a FLT3-ITD inhibitor, an aurora kinase inhibitor, or a combination thereof.
[0022] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawings.
[0024] FIG. 1 shows human leukemia cells (OCI/AML3) that were treated with combined sorafenib and Ara-C for 48 hours, resulting in synergistically enhanced cell killing. The average combination index (CI) at ED50, ED75 and ED90 was 0.279 ± 0.077.
[0025] FIG. 2 demonstrates targeting MEK/mTOR signaling simultaneously resulted in highly synergistic proapoptotic effect on human leukemia cell line U937. The average combination index (CI) at ED50, ED75 and ED90 was 0.014 ± 0.008.
[0026] FIG. 3 shows that ATRA enhances MEK inhibitor induced cytotoxic effects in OCT/AML3 leukemic cells. The average combination index (CI) at ED50, ED75 and ED90 was 0.26 ± 0.11.
[0027] FIG. 4 Murine mesenchymal stem cells (MS5) protect from sorafenib- induced cell death in vitro in co-culture systems. Sorafenib sensitive leukemia cells (Ba/F3- FLT3-ITD) were exposed to indicated concentrations of sorafenib for 72 hours, and apoptosis was measured using flow cytometry.
[0028] FIG. 5 Murine leukemia model was established by injecting Ba/F-FLT3- ITD cells i.v. The BM leukemia cells (GFP; green immunofluorescence) were less sensitive to 2-week sorafenib administration compared with the efficacy in spleen or blood.
[0029] FIG. 6 Murine Leukemia modal was established by directly injecting Ba/F3FLT3- ITD cells into BM cavity of tibia in SCID mice. Two weeks after injection, the leukemia cells are distributed throughout the BM, liver, spleen, and blood.
[0030] FIG. 7 demonstrates that Compound 1 shows a predominant cytostatic effect in AML ells that have constitutive ERK activation.
[0031] FIG. 8 demonstrates that Compound 1 shows lower (nanomolar level) IC50S of cell growth in sensitive AML cell lines.
[0032] FIG. 9 shows that Compound 1 induces inhibition of cell growth by delaying cell cycle Progression into S phase.
[0033] FIG. 10 demonstrates that the sensitivity of Compound 1 to induce apoptosis in AML cell lines is dependent upon threshold activity of ERK, and relates to upregulation of Bim and downregulation of Mcl-1 proteins.
[0034] FIG. 11 illustrates that Compound 1 induces loss of mitochondrial membrane potential in sensitive cell lines.
[0035] FIG. 12 shows that p53 level plays a minor effect on Compound 1- induced AML cell death.
[0036] FIG. 13 demonstrates that Compound 1 suppresses phospho-ERK and - S6K levels and decreases Mut-NPMl level in OCI/AML3 cells. [0037] FIG. 14 provides that NIH-3T3-NPMl-mut cell is sensitive to
Compound 1 -induced apoptosis compared with its parent cell.
[0038] FIG. 15 shows that the level of mutant NPMl is downregulated by Compound 1 and partially abrogated by proteasome inhibitor MG132.
[0039] FIG. 16 illustrates localization of wild-type and mutant NPMl proteins in human AML cells.
[0040] FIG. 17 shows that Compound 1 treatment decreases Mut-NPMl level in OCLA.ML3 leukemic cells.
[0041] FIG. 18 illustrates that targeting MEK signaling by inhibitors results in downregulation of mutant-NPMl, which is not Compound 1 specificity, and also accompanies with decrease of phospho-S6K, phospho-Rb and cdk2.
[0042] FIG. 19 provides that suppressing phospho-ERK by MEK siRNA is associated with decrease of mutant NPMl level in OCLA.ML3 cells.
[0043] FIG. 20 demonstrates that Compound 1 shows moderate sensitivity in NPM1/FLT3-ITD dual mutations in AML patient samples ex vivo. [0044] FIG. 21 provides combination of Compound 1 with mTOR inhibition that shows synergistic pro-apoptotic effects in human AML cell lines.
[0045] FIG. 22 shows that combination of Compound 1 with ABT737 exerts variable pro-apoptotic effects in human AML cell lines with high basal Bcl-2 levels.
[0046] FIG. 23 demonstrates that combination of Compound 1 with Ara-C shows additive pro-apoptotic effects in human AML cell lines.
[0047] FIG. 24 shows moderate synergistic effect after combination treatment of Compound 1 and Doxorubicin in human AML cells.
[0048] FIG. 25 demonstrates that combined Compound 1 with ATRA synergists pro-apoptotic effect in human APL cells NB4 and AML cells OCI/AML3.
[0049] FIG. 26 provides evidences that combined Compound 1 with Nutlin-3a only exert pro-apoptotic effect in p53 wild type AML cells, but not in p53 mutant cells U937. [0050] FIG. 27 illustrates that combination of Compound 1 with sorafenib performs synergistic pro-apoptotic effect in human AML cells OCLA.ML3 and MOLM13, the latter harbor FLT3-ITD mutations and only require quite lower concentration of sorafenib.
[0051] FIG. 28 shows combination treatment of Compound 1 with mTOR Inhibitor CCI-779 in human AML cell line MOLM13.
[0052] FIG. 29 demonstrates that mesenchymal stromal cells protect OCI/AML3 leukemic cells from Compound 1 -induced apoptosis.
DETAILED DESCRIPTION OF THE INVENTION
[0053] As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more. Furthermore, as used herein, the terms "including", "containing", and "having" are open-ended in interpretation and interchangeable with the term "comprising".
[0054] As used herein the specification, "effective amount," "therapeutically effective amount" or "pharmaceutically effective amount" means that amount which, when administered to a subject or patient for treating cancer, for example, is sufficient to effect such treatment for the disease, including alleviation of at least one symptom of the disease.
[0055] In general embodiments of the invention, there are methods and compositions for treating cancer. In particular embodiments, an individual has cancer that has a mutant NPM1 and the individual is treated therefor. Cancers that have mutant NPM1 may be of any kind, but in specific embodiments the cancer is leukemia, lymphoma, or non- small-cell lung carcinomas al. In particular aspects, the cancer includes acute myeloid leukemia (AML) or chronic myelomonocytic leukemia (CML). In certain embodiments the individual has myelodysplastic syndrome.
[0056] AML is a frequent hematopoietic malignancy with complex genetic disorders. A number of aberrant molecular events have been identified in AML that could promote leukemogenesis. For example, constitutive activation of the mitogen-activated protein kinase (MAPK) or Ras/Raf/MEK/ERK signaling pathway is detectable in about 70% of adult AML patients and associated with a striking negative impact on patient survival (Kornblau SM et al, Blood 2001). Nucleophosmin-1 (NPMl) gene mutations is another primary genetic lesion that is detectable in 35% of primary AML cases with higher incidence in normal karyotype and CD34/CD 133 negative AML (Falini B et al, N Engl J Med 2005) and has been significantly associated with high bone marrow blasts and white blood cell count (Thiede C et al, Blood. 2006). Therefore, targeting MEK-ERK signaling by suppressing MEK activation and, simultaneously, downregulating mutant NPMl is beneficial for cancer therapy, including for the one-third of adult AML patients harboring NPMl mutation.
[0057] In certain embodiments there are compositions that decrease mutant NPMl protein in addition to suppressing MEK activity, and this results in a cytotoxic effect in cancer cells that harbor NPMl mutation(s). The Examples herein demonstrate a decrease of mutant NPMl, but not of wildtype NPMl, after 2 hours of Compound 1 treatment, and this occurred parallel to the downregulation of phospho-ERK and -S6K. The decrease in mutant NPMl protein is partially abrogated by proteasome inhibitor MG132, indicating that in at least some embodiments proteasome activation is involved in the degradation of mutant NPM protein. Furthermore, mutant NPMl, which exclusively localizes to the cytoplasm, decreased significantly after 4 hours of Compound 1 treatment, as detected by immunoblotting and immunofluorescence staining. Different MEK inhibitors were tested for such dual activity in exemplary OCLA.ML3 cells. The results showed significantly decreased mutant but not wild- type NPMl levels after 24 hours with all tested MEK inhibitors. To further confirm the role of suppression of phospho-ERK in the regulation of mutant NPMl, MEK siRNA was used for knock-down of MEK and suppression of ERK activation. Suppression of phospho-ERK resulted in decrease of mutant NPMl levels, but not of wt-NPMl, indicating that ERK activation is associated with modulation of mutant NPMl. [0058] The exemplary small molecule MEK inhibitor Compound 1 affects the downregulation of mutant-NPMl and is accompanied by decreased phospho-ERK, -S6K, -
Rb and cdk2, in certain embodiments. The suppression results from inhibition of MEK activity in at least certain embodiments of the invention.
I. NPM1 mutations and Assays Therefor
[0059] NPM1 is a phosphoprotein normally localized in the nucleolus, having high expression in proliferating cells. NPM1 protein binds to nucleic acids, regulates centrosome duplication, and also regulates ribosomal function. In addition, NPM1 binds to several proteins, like p53 and proteins that react to and regulate p53 (like Rb, 17 pl9ARF 18 and HDM2). In certain aspects, mutations in NPM1 are a primary event in leukemogenesis, given the high frequency of NPM1 mutations in normal-karyotype AMLs and the fact that cytoplasmic NPM1 fails to carry out its normal functions such as binding to proteins and transferring them. NPM1 mutations often occur in association with FLT3/ITD mutations; FLT3 is a receptor tyrosine kinase having important roles in hematopoietic stem/progenitor cell survival and proliferation.
A. NPM1 Mutations
[0060] In embodiments of the invention, an individual is treated for a cancer that has cells that harbor one or more mutations in nucleophosmin (which may also be referred to as also known as N038, nucleolar phosphoprotein B23, numatrin, or NPM1). The cancer having at least one NPM1 mutation may be of origin in any tissue, but in specific embodiments the cancer is leukemia or lymphoma, for example. The mutation may be present in coding region or non-coding region. The mutation may be present in a regulatory sequence. In particular, the mutation may be in an exon, intron, 5'UTR, or 3'UTR.
[0061] A mutation may be identified in any NPM1 polynucleotide sequence, including genomic or mRNA sequences. Exemplary NPM1 polynucleotide are obtainable by standard means in the art, including from the National Center for Biotechnology Information's GenBank® database, and exemplary sequences are provided at least in the following, all of which are incorporated herein by reference: NM_001037738.2;
NMJ99185.3; NM_002520.6; BC107754.1 ; AY740640.1 ; BC009623.2; BC002398.2;
BC050628.1; BC003670.1 ; BC021983.1; BC021668.1 ; BC016824.1; BC016768.1 ;
BC016716.1; BC014349.1; BC012566.1 ; BC008495.1; AB451361.1; AB451236.1; EF429251.1; DQ303464.1; AY740639.1; AY740638.1 ; AY740637.1 ; AY740636.1 ;
AY740635.1; AY740634.1. Exemplary GenBank® genomic sequences include at least the
following, all of which are incorporated herein by reference: NG_016018.1; M28699.1; EU418625.1 (containing exon 12 and partial coding sequence).
[0062] In certain embodiments of the invention, an exon is mutated in NPM1. Although any exon may be mutated, in certain cases the mutation is in exon 4, 5, 6, or 12, for example.
[0063] Any NPM1 mutation is encompassed by the invention, including point mutations, inversion, translocations, deletions, frame shifts, and so forth, for example. In specific embodiments, the mutation is t(5;17)(q35;ql2), leading to NPM-RARa as a mutated product, which is associated with acute promyelocytic leukaemia (APL) (Redner et al, 1996). In some cases, the mutation is t(2;5)(p23;q35), leading to NPM-ALK, which is associated with anaplastic large cell lymphoma (ALCL) (Morris et al, 1994). In particular aspects, the mutation is t(3;5)(q25;q35) that results in NPM-MLF1, leading to myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) (Yoneda-Kato et al, 1996). [0064] Exemplary mutations are described in the literature and are encompassed in the invention. For example, deletion (-5q35, -5) leads to MDS (de novo, therapy-related), AML (French-American-British classification of leukaemia (FAB-M6)), and non-small-cell lung carcinoma (Olney et al, 2002; Mendes-da-Silva et al, 2000).
[0065] The following provides exemplary mutations in NPM1 compared to the wildtype sequence (Pazhakh et al, 2011); the sequences were identified by using primers NPM1-F (5 '-TTAACTCTCTGGTGGTAGAATGAA-3 ' ; SEQ ID NO:l) and NPM1-R (5'- CAAGACTATTTGCCATTCCTAAC-3 ' ; SEQ ID NO:2), as described in previous studies (Falini et al, 2005).
[0066] Wild type (forward seq)^- ACGGTCTCTAGAACTT (SEQ ID NO:3)
[0067] Wild type (reverse seq)→TGCCAGAGATCTTGAA (SEQ
ID NO:4)
[0068] Allele A (forward seq) — ACGGTCTGTCTCTAGA (SEQ ID
NO:5)
[0069] Allele A (reverse seq)→TGCCAGACAGAGATCT (SEQ ID
NO:6)
[0070] Allele B (forward seq) — ACGGTACGTCTCTAGA (SEQ ID
NO:7)
[0071] Allele B (reverse seq)→TGCCATGCAGAGATCT (SEQ ID
[0072] Allele D (forward seq) — ACGGTCCGTCTCTAGA (SEQ ID
NO:9)
[0073] Allele D (reverse seq)→TGCCAGGCAGAGATC T(SEQ ID NO: 10)
[0074] In addition, Mardis et al. (2009) report the exemplary NPM1 mutation W288fs. Furthermore, mutations as reviewed in Ahmad et al. (2009) are encompassed in the invention (see below).
B. Assays for NPM1 Mutations in Polynucleotides
[0076] Mutations in NPM1 may be identified by any suitable method in the art, but in certain embodiments the mutations are identified by one or more of polymerase chain reaction, sequencing, histochemical stain for NPM1 localization, as well as immunostaining method using anti-mutant-NPMl antibody. To obtain genetic material, mononucleated cells may be isolated by standard Ficoll-Hypaque density gradient centrifugation. Nucleic acid
isolation, cDNA synthesis and screening for NPM1 gene mutations may be performed using a melting curve-based LightCycler assay (Roche Diagnostics, Mannheim, Germany). AML samples with an aberrant melting curve may undergo nucleotide sequence analysis, for example. [0077] In specific embodiments, qualitative assays for NPM1 mutations are employed for detecting NPM1 mutations in R A or DNA extracted from fresh bone marrow or peripheral blood leukemic cells, although plasma and paraffin-embedded samples may also be employed.
[0078] In some cases, the artisan may use synthetic transcripts and total RNA from leukemic cell lines in an assay that can specifically detect NPM1 wild-type and mutants A, B, D, or J transcripts (for example) in the same reaction (Hafez et ah, 2010).
C. Assays for NPM1 Mutations in Polypeptides
[0079] In some embodiments of the invention, a mutation in NPM1 is identified in a polypeptide. Exemplary GenBank® sequences include at least the following, all of which are incorporated by reference herein in their entirety: AAH03670.2; AAH09623.1 ; NP 002511.1; NP_001032827.1 ; NP_954654.1 ; BAG70175.1 ; BAG70050.1 ; AAH50628.1 ; and AAH08495.1.
[0080] The mutation may be located in any region of the NPM1 protein, but in specific embodiments it is located in a hydrophobic segment, such as one near the N-terminus that is involved in oligomerization as well as chaperone activity. The mutation may be found in one of two acidic stretches that are employed for binding to histones, or it may be located in a central portion between the two acidic domains that is required for ribonuclease activity. In some cases, the mutation may be located in the C-terminal domain that contains basic regions involved in nucleic-acid binding; the basic clusters are followed by an aromatic stretch, which contains two tryptophan residues (288 and 290) that are required for nucleolar localization of the protein. The mutation in certain embodiments is located in the basic clusters or the aromatic stretch, including in tryptophan 288 or 290. In addition, NPM includes a nuclear-localization signal (NLS) and a nuclear-export signal (NES), either or which the mutation may be located. [0081] In some embodiments of the invention, the NPM1 mutation results in a truncated protein.
[0082] Molecular methods and their alternatives are currently available for identifying mutated NPMl (Falini et ah, 2010), which also includes exemplary mutations. The mutation identified in a polypeptide may be identified by any means standard in the art, but in specific embodiments the mutation is identified by immunohistochemistry, western blot, and/or PCR.
[0083] Western blot assay may employ antibodies that recognize NPMl mutants but not wild-type NPMl protein in lysates from AML samples, for example (Martelli et ah, 2008). The exemplary antibodies employed therein identify a specific band (37kDa) of mutated NPMl protein only in NPMl-mutated AML cases and recognizes over 95% of NPMl mutations. MEK Inhibitors
[0084] In embodiments of the invention, one or more MEK inhibitors are utilized in the treatment of an individual that has a NPMl mutation in cancer cells. The MEK inhibitor encompassed by the invention inhibits MEK but also downregulates mutant NPMl in cancer cells. In at least some embodiments, there is downregulation of phospho-ERK and -S6K following use of the MEK inhibitor of the invention.
[0085] The MEK inhibitor may be of any kind of molecule, but in certain embodiments the inhibitor is a small molecule, protein, or nucleic acid. In specific embodiments of the invention, the MEK inhibitor is selected from the group consisting of Compound 1, CI-1040, PD035901, AZD6244, GSK1 120212, GDC-0973, U0126, XL-518, ARRY-162, ARRY-300, PD184161, PD184352, PD0325901, ARRY-142886 (AZD6244), RO4927350, PD 0325901, CIP-1374, TAK-733, CH4987655, RDEA1 19, and combination thereof. In specific embodiments, the MEK inhibitor is a non-ATP-competitive small- molecule inhibitor (e.g. PD 098059, U0126, PD 184352 and its derivatives) or a biological inhibitor (e.g. anthrax lethal toxin and Yersinia outer protein J). In some cases, the MEK inhibitor is a pyrrole derivative. MEK inhibitors are described that are pyrrole inhibitors of MEK kinase using structure-based drug design, for example (Wallace et ah, 2010). In particular cases, the MEK inhibitor is a 4-anilino-3-cyano-6,7-dialkoxyquinoline, including 4-anilino-3-cyano-6,7-dialkoxyquinolines with different anilino groups at the 4-position (Zhang et al, 2001).
III. Combination Therapies
[0086] In order to increase the effectiveness of a one or more MEK inhibitors in the treatment of cancers having NPM1 mutations, it may be desirable to combine these compositions with other agents effective in the treatment of hyperproliferative disease, such as anti-cancer agents. An "anti-cancer" agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with the expression construct and the agent(s) or multiple factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the second agent(s).
[0087] Tumor cell resistance to chemotherapy and radiotherapy agents represents a major problem in clinical oncology. One goal of current cancer research is to find ways to improve the efficacy of one therapy by combining it with another therapy. In the context of the present invention, it is contemplated that MEK inhibitor therapy could be used similarly in conjunction with chemotherapeutic, radiotherapeutic, or immunotherapeutic intervention, in addition to pro-apoptotic or cell cycle regulating agents, for example. In specific embodiments, the MEK inhibitors can be employed with an aurora kinase inhibitor or an inhibitor of MDM2, for example. In certain embodiments, the additional agent is one that allows penetration of the bone marrow microenvironment, for example, characterized by hypoxia and mesenchymal stem cells (MSC) in sub-endosteal niches. In certain embodiments of the invention, the additional agent targets mTOR signaling. In certain aspects, the invention includes combinations of MEK inhibitors with rapamycin analogs (CCI-779 or RAD001, for example); a Raf/FLT3-ITD inhibitor, such as Sorafenib or AC- 220; a conventional chemotherapy agent, such as Ara-C; and/or a differentiation inducing agent, such as all-trans retinoic acid (ATRA). In particular embodiments, the MEK inhibitor and the additional anti-cancer agent provides a synergistic therapeutic effect. Specific
inhibitors include a BH3 mimetic (ABT737), an HDM2 inhibitor, such as nutlin and its derivatives, SMAC mimetics, Ara-C, rapamycin, HDAC inhibitors and hypomethylating agents, for example.
[0088] The MEK inhibitor therapy may be used simultaneously with the other agent, or it may precede or follow the other agent treatment by intervals ranging from minutes to weeks, for example. In embodiments where the other agent and MEK inhibitor are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and MEK inhibitor would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one may contact the cell with both modalities within about 12-24 h of each other and, in some cases, within about 6-12 h of each other, for example. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several d (2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. [0089] Various combinations may be employed, MEK inhibitor therapy is "A" and the secondary agent, such as radio- or chemotherapy, for example, is "B":
[0090] A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B
B/A/B/B
[0091] B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A
[0092] B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A
A/A/B/A
[0093] Administration of the therapeutic MEK inhibitor of the present invention to a patient will follow general protocols for the administration of chemotherapeutics, for example, taking into account the toxicity, if any, of the MEK inhibitor. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described hyperproliferative cell therapy.
A. Chemotherapy
[0094] Cancer therapies also include a variety of combination therapies with both chemical-and radiation-based treatments. Combination chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate, or any analog or derivative variant of the foregoing.
[0095] In embodiments wherein the cancer cells that have a NPM1 mutation are leukemia cells, the combination chemotherapy may depend on the type of leukemia. Acute lymphoblastic leukemia (ALL) medicines may comprise prednisone, methotrexate, 1- asparaginase, vincristine, and doxorubicin or daunorubicin. Acute myelogenous leukemia (AML) medicines may include daunorubicin with cytarabine (and idarubicin or mitoxantrone may be used instead of daunorubicin). Acute promyelocytic leukemia (APL) medicines may comprise all-iraws-retinoic acid (ATRA) and chemotherapy with arsenic trioxide, idarubicin, or daunorubicin, for example.
[0096] Chemotherapy for chronic lymphocytic leukemia (CLL) may include one medicine or a combination of medicines, including, for example, cyclophosphamide, vincristine, prednisone, fludarabine or chlorambucil, or the monoclonal antibodies rituximab and alemtuzumab. Tyrosine kinase inhibitors may be employed for chronic myelogenous leukemia (CML), such as imatinib, dasatinib, or nilotinib, or interferon alfa (with or without cytarabine), hydroxyurea, or busulfan.
B. Radiotherapy
[0097] Radiation treatment is used sometimes in cancer. In treating leukemia, for example, it may be used for cells in the brain and spinal fluid or testicles. It is also used, though rarely, in an emergency to treat compression of the trachea (windpipe).
[0098] Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a
broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
[0099] The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing or stasis, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.
C. Immunotherapy
[0100] Immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.
[0101] Immunotherapy, thus, could be used as part of a combined therapy, in conjunction with MEK inhibitors. The general approach for combined therapy is discussed below. Generally, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55.
D. Genes
[0102] In yet another embodiment, the secondary treatment is a gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the MEK inhibitor. Delivery of a MEK inhibitor in conjuction with a vector encoding one of the following gene products, for example, will have a combined anti-hyperproliferative effect on target tissues. A variety of proteins are encompassed within the invention, including inducers of cellular proliferation, inhibitors of cellular proliferation, or regulators of programmed cell death.
E. Surgery
[0103] Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies. Although in the exemplary case of leukemia surgery is not effective, a venous access device (large plastic tube) may be placed into a large vein.
[0104] Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
[0105] Upon excision of part of all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, or 12 months. These treatments may be of varying dosages as well.
F. Other agents
[0106] It is contemplated that other agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adehesion, or agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, ΜΓΡ- lbeta, MCP-1, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL would potentiate the apoptotic inducing abililties of the present invention by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti- hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyerproliferative efficacy of the treatments. Inhibitors of cell adehesion are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.
[0107] Hormonal therapy may also be used in conjunction with the present invention or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.
IV. Pharmaceutical Preparations
[0108] Pharmaceutical compositions of the present invention comprise an effective amount of one or more MEK inhibitors dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of an pharmaceutical composition that contains at least one MEK inhibitor or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
[0109] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
[0110] The MEK inhibitor may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, trans dermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneous ly, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
[0111] The MEK inhibitor may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such
organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like. [0112] Further in accordance with the present invention, the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
[0113] In accordance with the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.
[0114] In a specific embodiment of the present invention, the composition is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in an the composition include
buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
[0115] In further embodiments, the present invention may concern the use of a pharmaceutical lipid vehicle compositions that include a MEK inhibitor, one or more lipids, and an aqueous solvent. As used herein, the term "lipid" will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term "lipid" is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present invention.
[0116] One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the MEK inhibitor may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes. [0117] The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event,
determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
[0118] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
[0119] In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.
A. Alimentary Compositions and Formulations
[0120] In preferred embodiments of the present invention, the MEK inhibitor are formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed
in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
[0121] In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et ah, 1997; Hwang et ah, 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety). The tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Upon reaching the small intestines, the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells. A syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations. [0122] For oral administration the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally- administered formulation. For
example, a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically- effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
[0123] Additional formulations which are suitable for other modes of alimentary administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
B. Parenteral Compositions and Formulations
[0124] In further embodiments, the MEK inhibitor may be administered via a parenteral route. As used herein, the term "parenteral" includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543, 158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).. [0125] Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference
in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. [0126] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035- 1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
[0127] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients
from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
C. Miscellaneous Pharmaceutical Compositions and Formulations
[0128] In other preferred embodiments of the invention, the active compound MEK inhibitor may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.
[0129] Pharmaceutical compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder. Ointments include all oleaginous, adsorption, emulsion and water-solubly based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture. Transdermal administration of the present invention may also comprise the use of a "patch". For example, the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.
[0130] In certain embodiments, the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et ah, 1998) and lysophosphatidyl-glycerol compounds (U.S.
Pat. No. 5,725, 871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety). [0131] The term aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms. Kits of the Invention
[0132] Any of the compositions described herein may be comprised in a kit. In a non-limiting example, one or more MEK inhibitors may be comprised in a kit. The kits will thus comprise, in suitable container means, a MEK inhibitor and, optionally, an additional agent of the present invention.
[0133] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the MEK inhibitor, optional lipid, optional additional agent, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained. The kit may have a single container means, and/or it may have distinct container means for each compound.
[0134] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The MEK inhibitor compositions may also be formulated into a
syringeable composition. The container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit. However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
[0135] In some embodiments of the invention, there are compositions for identifying a NPM1 mutation and/or a FLT3 mutation, such as PCR primers and appropriate reagents for PCR and/or a microassay chip for detection of the mutation(s). In some embodiments, sequencing, hybridization, and/or restriction enzyme reagents are provided in a kit.
[0136] In specific embodiments, an additional agent such as an mTOR inhibitor, aurora kinase inhibitor, and/or FLT3-ITD inhibitor is included in a kit.
EXAMPLES
[0137] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1
DEVELOPMENT OF EFFECTIVE COMBINATION THERAPIES TARGETING MEK AND AURORA KINASE PATHWAYS IN ACUTE MYELOID LEUKEMIA
[0138] Most of the inhibitors targeting Raf/MEK/ERK or mTOR signaling are relatively "non-cytotoxic", i.e. cytostatic agents. To optimize therapeutic efficacy, aspects of
the present invention employ a strategy of combining a MEK inhibitor with rapamycin analogues (CCI779 or RAD001) (Zeng et al, 2007), the Raf/FLT3-ITD inhibitor Sorafenib, the conventional chemotherapy agent Ara-C or differentiation inducing agents, such as all- trans retinoic acid (ATRA) (Milella et al, 2007). Other embodiments include AK inhibitors combined with the same or related compounds.
[0139] Aspects of the invention provide that activation of multiple signaling pathways is associated with chemo-resistance in acute myeloid leukemia (AML). Therefore, targeting MEK and Aurora kinase (AK), well-established, prognostically relevant targets in AML, and other critical survival pathways, identifies specific biomarkers of synergistic effects in leukemic cells and stem cells in vitro under the physiologically relevant condition of a hypoxic microenvironment, and this is then applied in vivo.
[0140] Embodiments of the invention include the definition of molecular and functional effects of MEK and AK blockade in AML. Other embodiments include the development of optimized combinations of MEK inhibitor Compound 1, AK inhibitor R763, and other targeted agents against leukemic cells and stem cells and the identification of biomarkers of synergistic effects. In some embodiments, the influence of the bone marrow microenvironment on the efficacy of MEK and AK inhibition in vitro. In certain embodiments, one can assess in vivo efficacy of effective combination regimen in murine leukemia models. Exemplary Results
[0141] MEK inhibitors other than Compound 1 have been tested. While CI- 1040 alone resulted in dephosphorylation of MEK/ERK, the resulting effects were predominantly cytostatic (Milella et al, 2001). Combination with ATRA converted the cytostatic into a cytotoxic response (Milella et al, 2007) . Excellent synergism was also observed with Bcl2 inhibitors, from Bcl-2 anti-sense to small molecule inhibitors such as HA 141 and ABT_737 (Milella et al, 2002; Konopleva et al, 2006). This effect was related to the complete downregulation of Mcl-l in AML, and to de-phosphorylation (ERK is the kinase for Bcl-2) of Bcl-2 (Konopleva et al, 2006). High synergism was also observed with mTOR and MDM2 inhibitors (e.g. Nutlin 3a), that activate p53 signaling (Kojima et al, 2007). In addition, with the FLT3 inhibitor Sorafenib, up-stream inhibition does not guarantee complete inhibition of downstream targets such as MEK/ERK, presumably due to
crosstalk with other signaling pathways. This has resulted in an embodiment of the invention of dual (or triple) "intra-pathway" inhibition targeting upstream and downstream kinases.
[0142] Several combination regimes were considered for characterizing synergism of the indicated agents on human AML cell lines (Figs. 1-3). The results indicated that combination of MEK inhibitor with either Ara-C, ATRA or rapamycin analogue (CCI- 779) results in synergistic anti-leukemia effects in vitro. AK inhibitors have shown activity in preclinical models and in patients with AML and other leukemias (Doggrell, 2004; Kojima et al, 2008; Giles et al, 2007; Huang et al, 2008). This approach provides the basis for additional characterization of optimized combination strategies of MEK and AK inhibitors and other agents. On the other hand, since the hypoxic BM niches are considered sanctuaries for leukemia cells evading chemotherapy, it is useful to characterize anti-leukemia effects in the BM microenvironment. Co-culture systems of leukemia and MSC in normoxic and hypoxic conditions are established, in addition to a live confocal microscopy imaging system that allows live imaging under normoxic and hypoxic conditions of leukemia cells, alone or in co-culture with MSC. MSC protects leukemia cells from kinase inhibitor (e.g. Imatinib or Sorafenib) and chemotherapy (e.g. Ara-C) induced cell death in vitro (Fig. 4). Several leukemia models are established in vivo by Lv. (Fig. 5) or direct BM injection (Fig. 6) of leukemic cells into Scid and NOD/Scid mice. Most recently, we have successfully established the murine model for primary AML cells [NOG (NOD/Scid/IL2Ry"/_-)] (Zeng et al, 2008). [0143] Furthermore, the inventors have established that the human AML bone marrow is hypoxic and that this hypoxia upregulates the chemokine-receptor CXCR4, which conveys resistance to chemotherapeutics and kinase inhibitors (Zeng et al, 2008). Inhibition of CXCR4 by small molecules or peptides can partially reverse this effect. This has already resulted in clinical trials of CXCR4 inhibitors in combination with chemotherapy or kinase inhibitors, pointing to the potential clinical relevance of this concept (Pis Drs. Andreeff and Konopleva). Hypoxia effects have been mitigated by HIF-1 a (Enzon) or mTOR inhibitors (Frolova et al., ASH 2008).
[0144] Finally, Raf/MEK inhibitors such as sorafenib or AZD6244 can elicit upregulation of pro-apoptotic protein Bim32 and a flow cytometric assay may be employed to allow determination of phospho-ERK in bulk leukemia and in AML progenitor/stem cells. Our studies of AK inhibitors have focused on apoptosis induction, cell cycle effects, and polyploidization of target cells.
Research Design and Methods
[0145] Defining molecular and functional effects of MEK and AK blockade in AML
[0146] In order to assess the molecular and functional consequences of MEK blockade on AML cells, AML cell lines are exposed to increasing concentrations of Compound 1 and R763 for different periods of time. One can contrast effects of Compound lin OCI-AML3 and HL-60 cells with high baseline ERK phosphorylation and in U937 and KG-1 cells that do not exhibit activation of this signaling pathway. R763 is tested in MoLml3, OCI-AML3, U937 and KG-1 cells. Functional effects on cell growth, cell cycle progression, and induction of apoptosis are investigated, using either short-term liquid cultures to assess cell viability (trypan blue exclusion cell count), cell cycle distribution (PI DNA staining, BrdU incorporation), and induction of apoptosis (assessment of mitochondrial membrane potential -DDm - by CMXRos/ Mitotracker Green double staining, Annexin V/PI staining, flow cytometry). IC50S are also determined by measuring cell viability and apoptosis. Further, effects of Compound Ion pERK, pAKT473, pFLT3 and pSTAT5 phosphorylation are characterized using immunoblotting. R763 effects on polyploidy and pH3AX are also determined.
[0147] Furthermore, the effect of Compound land R763 on primary AML samples is investigated. Freshly-isolated AML samples (n=20) are enriched by density centrifugation over Ficoll-Paque, and are exposed to different concentrations for 24-72 hours. Cell viability and apoptosis induction are determined by quantitative flow cytometry techniques (pERK activity gated on leukemic bulk or progenitor/stem cells). Furthermore, DNA ploidy, MMP, Annexin V/PI, absolute cell counts are determined.
[0148] The effect of R763 on FLT3-ITD is determined using FLT3-ITD transfected cells and primary AML cells with FLT3-ITD. [0149] Develop optimized combinations ofMEK inhibitor Compound land AK inhibitor R763, and other targeted agents against leukemic cells and stem cells and identify biomarkers of synergistic effects
[0150] Based on the biological relevance of MEK and AK signaling and the observed effects of Compound land R763 on protein expression profiling in AML leukemia cells, different combination strategies are investigated. Compound land R763 are combined with Ara-C, daunorubicin, etoposide, Nutlin-3a, ABT737, sorafenib, ATRA and 5-Aza or
DAC for assessing synergistic effects in human AML cells, especially in cells that are resistant to single agents, by determining effects on cell growth, cell cycle progression, mitochondrial membrane potential and apoptosis using the methods mentioned herein, for example. The initial experiments are performed with 2-3 AML cell lines with apoptosis induction as endpoint. Combination indices are determined using the Chou-Talalay method. Furthermore, combination effects are assessed in 10-20 freshly-isolated primary AML samples by determining induction of apoptosis (Annexin V positivity, loss of MMP) in bulk cells and in LSC defined as CD34+/CD387CD 123+ cells (Konopleva et al, 2006).
[0151] Considering that some samples with low blast percentage may not have sufficient LSC for analysis, in specific embodiments, either CD34+/CD38" or CD34+ cells, which represent leukemic progenitors, are analyzed. Optimized combinations are also tested in colony-forming assays that determines differential effects on normal vs. leukemic colony- forming cells.
[0152] To further characterize the mechanisms involved, effects of the most potent combination therapies identified as described above on relevant signaling modules and BcI-2 family of proteins, intrinsic apoptotic pathway proteins, and other factors are investigated, which may be responsible for the pro-apoptotic effects expected of the combinations in at least some cases. To evaluate the impact of critical proteins found in the above mechanistic studies, relevant siR As (Dharmacon Research, Inc.) are used to block the expression of specific proteins and their effects on the efficacy of combinations are determined. These studies identify highly effective combination therapies, and also biomarkers for the expected synergistic effects.
[0153] Evaluate the influence of the bone marrow microenvironment on the efficacy ofMEK and AK inhibition in vitro
[0154] To further characterize the influence of the BM microenvironment, co- culture systems are used for performing combination experiments, i.e. AML cells or primary AML samples are treated with combinations in the absence or presence of BM mesenchymal stem cells (MS5 or primary human MSC) feeder layers, and apoptosis are measured by annexinV positivity in CD34+/CD387CD123+ cells by 4-color flow cytometry assay (Konopleva et al, 2006). Effects on protein phosphorylation, cell proliferation and cell death of bulk tumor or tumor stem cells can be determined by 10-color flow cytometry, for example.
[0155] In order to better mimic the influence of the physiologic BM microenvironment, these studies are also performed in hypoxic (1 % oxygen tension) conditions. IC50S and ED50s are derived as appropriate and correlated with constitutive MAPK and AK activation status. These studies in hypoxia are useful, as there is striking induction of pERK in AML cells in hypoxia, in a specific embodiment of the invention. Therefore, the ability of AS703026 to suppress this effect in hypoxia, which is the physiological pC>2 in the bone marrow, is further characterized.
[0156] Assess in vivo efficacy of effective combination regime in murine leukemia models
[0157] The inventors have established an imageable in vivo leukemia model by injecting i.v. murine leukemia Baf3/FLT3-ITD and other human leukemia cells (stably transduced with firefly luciferase fused to GFP) into SCID mice, resulting in engraftment in BM, spleen and liver (Zhang et ah, 2008; Zeng et ah, 2008). Growth and topical dissemination of the leukemic cells is quantified by bioluminescence imaging (BU). One can therefore evaluate the therapeutic efficacy of selected combination therapies by determining the leukemia-burden in mice, based on results obtained in in vitro studies, by non-invasive BU. Colentazine is injected i.p., for example, and the animals are imaged by Xenogen IVIS; bioluminescence is quantified as the total sum integrated signal in a standard period of time (i.e., 2 min). Leukemia tissue infiltration is determined by GFP-positivity and quantified by spectral image analysis (CRI system). Survival is calculated using the method of Kaplan- Meier.
[0158] NOD/Scid/IL2Ry_/" (NOG) mice are considered the optimal model of human leukemia. One can test the combination of selected agents in this model using primary AML blasts in vivo, and track and assess the interaction of LSC with MSC in mouse BM niches. FACS-sorted LSC CD34+/CD387CD123+ and normal human HSC are injected Lv. into NaG (NOD/Scid/lL2Ry"/ ) mice. The efficacy of apoptosis induction in LSC and patho- morphological changes will be determined by immunohistochemistry (IHC) and flow cytometry and effects of selected drug combinations will be determined by Kaplan-Meier analysis.
[0159] Sample sizes (estimated):
[0160] 1) Compound 1, R763, Compound 1+R763, controls at 10 mice/group = 40 mice x 2 dose levels = 80 mice; 2) MEK + best combination + control x 1 repeat = 60 mice; 3) AKI + best combination + control x 1 repeat = 60 mice; 4) For selected studies (IHC, IVIS): additional 30 mice; TOTAL: 230 mice (estimated). EXAMPLE 2
DEVELOPMENT OF EFFECTIVE COMBINATION THERAPIES BY TARGETING MEK AND MUTANT NPM1 IN ACUTE MYELOID LEUKEMIA
[0161] Acute myeloid leukemia (AML) is a hematopoietic malignancy with complex genetic disorders. A number of aberrant molecular events have been identified in AML that are able to promote leukemogenesis. For example, constitutive activation of the mitogen-activated protein kinase (MAPK) or Ras/Raf/MEK/ERK signaling pathway are detectable in about 70% of adult AML patients and associated with a strikingly negative impact on patient survival (Kornblau SM et al, Blood 2001). Nucleophosmin- 1 (NPM1) gene mutations are primary genetic lesions that can be detected in 35% of primary AML cases with higher incidence in normal karyotype and CD34/CD133 negative AML (Falini B et al, N Engl J Med 2005). The specific cytoplasmic dislocation of the mutant nucleophosmin might exert oncogenic action by binding and interacting with other protein partners, for example, K-Ras (Inder KL et al, Commun Integr Biol. 2010). NPM1 mutations have been significantly associated with high bone marrow blasts and white blood cell count (Thiede C et al, Blood. 2006). Internal tandem duplication (ITD) mutations of Fms-like tyrosine kinase 3 (FLT3) gene are another frequent molecular abnormalities which account for 25-40 % of AML and are associated with poor prognosis (Gilliland DG et al, Blood 2002). FLT3-ITD mutations can be found in more than 40% of the NPM1 -mutated patients, and are associated with poor prognosis (Thiede C et al, Blood. 2006). Importantly, all of these mutations are associated with high/constitutive activation of Ras/Raf/MEK/Erk signaling. Therefore, targeting MEK-ERK signaling by suppressing MEK activation is beneficial for leukemia therapy, in specific embodiments of the invention. The present invention discloses the effects of the novel MEK inhibitor Compound 1 on human AML, as an exemplary cancer.
Exemplary Results
[0162] Several human AML cell lines with different basal levels of ERK activation were tested in in vitro experiments (Table 1; see Fig. 7 panel). The studies
demonstrate that Compound 1 has a predominantly cytostatic effect on AML cells with constitutive ERK activation (OCI/AML3, MOLM13 and HL60) compared with cells with low levels of MEK/ERK activation (KG-1 and U937) (Fig. 7). IC50 was to 7nM in OCI/AML3 cells after 72 hours of MSC-69B treatment (Fig. 8). BrdUrd incorporation revealed that the agent induces a profound Gi-S block (Fig. 9). Mechanistically, the cytostatic effect was associated with suppression of phosphorylated-ERK and down-regulation of cdk2/4 levels (Fig. 10). However, Compound 1 showed cytotoxic effects in OCI/AML3 and MOLM13 cells, which have NPM1 and FLT3-YTO mutations, respectively; both of these cell lines contain wildtype p53 (Fig. 7). Apoptosis induction in these cells was mediated by the mitochondria-associated intrinsic cell death pathway with loss of mitochondrial membrane potential (Fig. 1 1) and by modulating pro-apoptotic protein Bim and anti-apoptotic protein Mcl-1 (Fig. 10). To address the role of p53 in drug-induced apoptosis, OCI/AML3-p53- shRNA knockdown cells were compared with parental cells in a dose-response fashion. Results indicate that loss of p53 has a marginal effect on Compound 1 -induced apoptosis (Fig. 12).
[0163] Since OCI/AML3 cells showed impressive sensitivity to Compound 1, in specific embodiments exclusive molecular alterations contribute to the effect. The studies demonstrated that mutant NPM1, but not wt-NPMl, level decreased in OCI/AML3 cells measured by Western blot after 2 hours of Compound 1 treatment, which occurred in parallel to the downregulation of phospho-ERK and -S6K (Fig. 13). These results were further confirmed in NIH-3T3 fibroblasts stably transfected with mutant NPM1 (Fig. 14). The decrease in mutant NPM1 protein could be partially abrogated by proteasome inhibitor MG132 (Fig. 15). To investigate the effects of Compound 1 on the intracellular location of mutant NPM1 protein, immunoblotting and immunostaining of mutant NPM1 were performed in different cellular fractions. Results demonstrated that mutant NPM1 was exclusively localized in the cytoplasm and that levels decreased significantly after 4 hours of Compound 1 treatment (Figs. 16 and 17), indicating that proteasome activation is involved in the degradation of mutant NPM protein, in at least certain aspects of the invention.
[0164] To determine if the observed downregulation of mutant-NPMl is exclusive to Compound 1, different MEK inhibitors were tested on OCI/AML3 cells. The results showed significantly decrease in mutant but not wild-type NPM1 levels after 24 hours with all tested MEK inhibitors. Furthermore, inhibition of mutant NPM1 was accompanied
by downregulation of phospho-ERK, -S6K, -Rb and cdk2, suggesting that downregulation of mutant-NPMl is not exclusive for Compound 1, but rather results from inhibition of MEK activity (Fig. 18). To further address the role of suppression of phospho-ERK in mutant NPMl decrease, MEK siRNA was used for knocking down MEK and suppressing ERK activation. Result showed that suppression of phospho-ERK resulted in decrease of mutant NPMl, but not wt-NPMl (Fig. 19), indicating that ERK activation is associated with modulation of mutant NPMl .
[0165] To investigate the effect of Compound 1 on AML patient samples, leukemic blasts were cultured ex vivo with Compound 1 for 48-72 hours and apoptosis induction was determined using multi-color flow cytometry. The results showed that MSC- 69B moderately induced apoptosis in a patient harboring NPMl and FLT3-ITD dual mutations, but only showed pro-apoptotic effects in one of four FLT3-ITD mutations patients. No effect was observed in NPMl and FLT3 wild-type patients (Fig. 20), indicating activity of Compound 1 in AML patient with NPM1/FLT3-ITD dual mutations, in certain embodiments of the invention. Studies with additional patient samples are performed.
[0166] To develop potential combination strategies, several combination treatments of Compound 1 were tested with conventional chemotherapeutic drugs, including cytarabine, mTOR inhibition and BH3 mimetic ABT-737. A significant synergistic pro- apoptotic effect was observed for the combination of Compound 1 with a mTOR inhibitor in MOLM13 and U937 cells that have high p-ERK and p-AKT basal levels, and the averaged combination indices (CIs) at ED50, ED75 and ED90 were 0.07 ± 0.01 and 0.09 ± 0.02, respectively (Fig. 21). Combination of Compound 1 and ABT737 was also active in inducing apoptosis in OCI/AML3 and KG-1 cells, which have high Bcl-2 basal levels (Fig. 22). MEK inhibition results in complete downregulation of Mcl-1, a major anti-apoptotic protein which is not targeted by ABT737 (Konopleva M et ah, Cancer Cell. 2006). Synergistic suppression of Mcl-1 by Compound 1 and Bcl-2 by ABT737 may greatly enhance the pro-apoptotic effects (Konopleva et al. Cancer Cell, 2006). In addition, combination of Compound 1 with cytarabine (also known as Ara-C) showed pro-apoptotic effects in human AML cells (Fig. 23). Importantly, these combination strategies sensitized U937 and KG-lcells to Compound 1, which showed more resistance to treatment with Compound 1 alone.
[0167] To characterize the influence of bone marrow stroma in AML sensitivity to MEK inhibition (which in certain aspects of the invention mimics physiologic interaction
of AML leukemic cells and bone marrow stroma), OCI/AML3 cells were treated with Compound 1 in the presence or absence of a feeder layer of mesenchymal stroma cell (MSC). Apoptosis induction and correlated signaling proteins were measured by flow cytometry and Western blot, respectively. The results showed that MSC protected the leukemic cells from Compound 1 -induced apoptosis (FIG. 29). Mechanistically, OCLAML3 cells display higher basal levels of phospho-ERK in the presence of MSC feeder layer (FIG. 19), in certain aspects of the invention.
[0168] The novel small molecule MEK inhibitor Compound 1 displayed effective anti-leukemic activity in AML cells with constitutive activation of ERK. The drug preferentially induces inhibition of cell proliferation rather than apoptosis. Compound 1 also showed an impressive cytotoxic effect in OCI/AML3 cells that is related to downregulation of mutant but not wild-type NPMl protein, in embodiments of the invention. The decrease of mutant NPMl is associated with suppression of MEK activation. Furthermore, synergistic effects of combining Compound 1 with, for example, an mTOR inhibitor or ABT-737 were observed. These results indicate that combining Compound 1 with other pro-apoptotic drugs or signal transduction inhibitors is effective in the treatment of resistant AML. The findings provide therapeutic guidance for utilization of Compound 1 in AML, including for AML patients harboring NPMl or dual NPM1/FLT3-ITD mutations, for example.
[0169] FIG. 24 demonstrates the treatment of combined Compound 1 and Doxorubicin synergizes with pro-apoptotic effects in human leukemic cells. MOLM13 and KG-1 were treated with indicated concentrations of the drugs for 48 hours. The cells were stained with Annexin V-Fluo and apoptosis induction was measured by flow cytometry. Error bars were generated from triplicate experiments and combination index (CI) values were determined using CalcuSyn software (BioSoft). FIG. 25 shows that the combination of Compound 1 and ATRA treatment significantly enhances apoptosis induction of leukemias. APL cells NB4 and AML cells OCI/AML3 were treated with Compound 1 and ATRA simultaneously for 72 hours. Apoptosis induction was measured by counting percentage of annexin V positive population using flow cytometry. CI values showed impressive synergistic effect (CI = 0.06) in APL cells NB4.
[0170] FIG. 26 shows that targeting MEK/ERK and MDM2/p53 signaling pathways enhances cell death in p53 wild type leukemic cells. p53 wild type leukemic cells MOLM13 and p53 mutant cells U937 cells were treated with Compound 1 and Nutlin3a, a
small-molecule antagonist of MDM2, for 48 hours. Cell apoptosis was analyzed with flow cytometry after annexin V staining. Synergistic apoptosis induction was observed only in p53 wild type cells MOLM13, but not in p53 mutant cells U937. FIG. 27 demonstrates blockade of Raf/MEK/ERK and FLT3 signaling pathways synergizes with cell apoptosis induction in AML cells. MEK inhibitor Compound 1 and multiple kinase inhibitor sorafenib, which targets FLT3-ITD mutations and Raf/MEK signaling as well, were used for treating human AML cells OCI/AML3 and MOLM13, which also harbors FLT3-ITD mutations, for 48 hours. Enhanced effect of apoptosis induction were shown in both cell lines, and much more impressive synergistic effect was observed in FLT3-ITD mutations harboring cells MOLM13 (CI = 0.06).
[0171] FIG. 28 shows combination treatment of Compound 1 with exemplary mTOR inhibitor CCI-779 in an exemplary human AML cell line MOLM13.
EXAMPLE 3
DEVELOPMENT OF EFFECTIVE THERAPEUTIC STRATEGY BY TARGETING MEK AND RELATIVE PATHWAYS IN ACUTE MYELOID LEUKEMIA
[0172] Constitutive activation of MEK/ERK is consistently found in 50-70% of acute myeloid leukemia (AML) patients, with a striking negative impact on response to chemotherapy and patient survival (Kornblau et ah, 2001 ; Ricciardi et ah, 2005). Therefore, targeting MEK/ERK signaling is a useful strategy for leukemia therapy. Studies using MEK inhibitor Compound 1 against AML have shown a predominant cytostatic effect on AML cells with constitutive ERK activation. IC50 was determined as 7 and 83 nM in OCI/AM13 (with nucleophosmin-1 (NPM1) mutation, p53 wt) and MOLM13 (FLT3-ITD mutation, p53 wt) cells, respectively. It also demonstrated pro-apoptotic activity in these cells. Further analysis demonstrated that the cytotoxic effect was not associated to p53 or FLT3-ITD status. Impressively, OCI/AML3 cells were highly sensitive to Compound 1 -induced apoptosis, accompanied with downregulation of phospho-S6K and NPM1 protein levels. NPM1 downregulation showed a similar pattern as phospho-S6K in a dose-dependent manner, but did not show a correlation with downregulation of phospho-ERK, indicating that an ERK- inhibiton-independent mechanism is involved in apoptosis induction in NPM1 mutant leukemic cells treated with Compound 1, in certain embodiments of the invention.
[0173] Mutations of NPMl gene are present in 50-60% of adult acute myeloid leukemias with normal karyotype (AML-NK) (Ralini et ah, 2007), and have been given a new provisional entity in the 2008 WHO classification of myeloid neoplasms (Vardiman et ah, 2009). NPMl mutations have been implicated in promoting cell growth, as expression increases in response to mitogenic stimuli and above-normal levels are detected in highly proliferating malignant cells (Grisendi et ah, 2006). NPMl mutations, are not significantly associated with KRAS mutation and P53 mutations (Dohner et ah, 2005; Suzuki et ah, 2005), but a functional link between NPMl and p53 stability (Colombo et ah, 2002), which may involve control of cell proliferation and apoptosis (Ye, 2005), has been postulated. Therefore, targeting MEK/ERK signaling in NPMl mutant cells is beneficial for anti-leukemia therapy, in certain aspects of the invention.
[0174] In the present invention, mechanisms of cytotoxicity in NPMl mutant AML are characterized using MEK inhibitor MSC-69B. In order to characterize the specificity of Compound 1 in NPMl mutant AML cells, studies are performed comparing Compound 1 signaling modulation to treatment with other MEK inhibitiors, such as CI- 1040, etc., for example. In addition, exemplary combinatorial strategies are also characterized by combining MSC-69B with conventional chemotherapeutics and other targeted agents in "normoxia" and in physiological hypoxic conditions. Mechanisms of Compound 1 apoptosis induction are considered, especially in NPMl mutant AML, and optimized combination therapies for AML patients with specific cytogenetic and molecular abnormalities are provided, in specific embodiments.
Molecular and functional effects of Compound 1 in NPMl mutant AML
[0175] In order to assess the molecular and functional effects of Compound 1 in AML with NPMl mutatons, OCI/AML3 cells are used for these studies. OCI/AML3 cells are exposed to increasing concentrations of Compound 1 for different periods of time. Correlative signaling and proteins (such as NPMl, phospho-ERK, -S6K, -AKT, pi 9, MDM2 and p53 as well as cell cycle and apoptosis-related proteins are investigated by Western blot and multi-parametric phospho flow cytometry, for example. Further, cells (such as OCI/AML2 cells) are infected with lentiviral-NPMl mutant or wt genes. The functional effects of Compound 1 in the engineered cells are investigated by assessing cell viability (trypan blue exclusion cell count), cell cycle distribution (PI DNA staining, BrdU incorporation), and induction of apoptosis (assessment of mitochondrial membrane potential
-ΔΨηι - by CMX-Ros/Mitotracker Green double staining, Annexin V/PI staining, flow cytometry). IC50S are also determined by measuring cell viability and apoptosis. Further, effects of MSC-69B in relevant genes and proteins are characterized using real-time PCR and immunoblotting. [0176] Further investigation for evaluating the specificity of Compound 1 in targeting NPMl mutant AML, other MEK inhibitors such as CI- 1040, AZD6244, GSKl 120212 etc. (for example) are used for testing their effects on NPMl wildtype or mutation cells. Cell viability, proliferation and apoptosis are assessed using the above- recorded methods, and the modulation of relevent proteins are characterized using immunoblotting, for example.
[0177] To evaluate the potential clinical relavance of Compound 1 in leukemias, the effect of Compound 1 on primary AML samples are investigated. Freshly- isolated AML samples (n=20) are enriched by density centrifugation over Ficoll-Paque, and is then exposed to different concentrations for 24-72 hours, for example. Cell viability and apoptosis induction are determined by quantitative flow cytometry techniques (considering the majority of NPMl mutaions are harbored in CD34 negative leukemic cells, CD34 gating is applied for defining CD34 positive and negative populations for the tests). Furthermore, DNA ploidy, MMP, Annexin V/PI, absolute cell counts are determined. NPMl status of the detected samples is determined by PCR. Optimized combinations of MEK inhibitor Compound 1 and conventional
chemotherapeutics and other targeted agents against leukemic cells and stem cells and identification of biomarkers of synergistic effects
[0178] Based on previous studies, MEK inhibitors predominant effects are the induction of cytostatic rather than cytotoxic effects in AML, different combination strategies are investigated for enhancing pro-apoptotic effects. Compound 1 is combined with Ara-C, daunorubicin, etoposide, Nutlin-3a, ABT737, sorafenib, and ATRA, for example, for assessing synergistic effects in human AML cells, especially in cells that are resistant to
Compound 1, by determining effects on cell growth, cell cycle progression, mitochondrial membrane potential and apoptosis using the exemplary methods mentioned above. The initial studies are performed with 2-3 AML cell lines with apoptosis induction as endpoint.
Combination indices are determined using the Chou-Talalay method. Combination effects are assessed in 10-20 freshly-isolated primary AML samples by determining induction of apoptosis (Annexin V positivity, loss of MMP) in bulk cells and in LSC defined as
CD34+/CD38-/CD 123+ cells (Konopleva et al, 2006). Considering that some samples with low blast percentage may not have sufficient LSC for analysis, in certain aspects, either CD34+/CD38" or CD34+ cells, which represent leukemic progenitors, are analyzed.
[0179] Effects of potent combination therapies identified as described above on relevant signaling modules and Bcl-2 family of proteins, intrinsic apoptotic pathway proteins, and other factors are investigated, which in certain embodiments of the invention is responsible for the pro-apoptotic effects of the combinations. To evaluate the impact of critical proteins found in the above mechanistic studies, relevant siRNAs (Dharmacon Research, Inc.) are used to block the expression of specific proteins and their effects on the efficacy of combinations are determined. These studies identify highly effective combination therapies and also biomarkers for synergistic effects.
Evaluate effects of the bone marrow microenvironment on the efficacy of MEK inhibitors and optimized combinations in vitro
[0180] To further characterize the influence of the BM microenvironment on drug sensitivity, co-culture systems are used for performing combination experiments, i.e.
AML cells or primary AML samples are treated with combinations in the absence or presence of BM mesenchymal stromal cells (MS5 or primary human MSC) cultured on MSC feeder layers. Apoptosis is measured by annexinV positivity in CD34+/CD38-/CD123+ cells by a 4- color flow cytometry assay (Konopleva et al,. 2006).
[0181] In order to better model the physiologic BM microenvironment, these studies may be performed in hypoxic (1% oxygen tension) conditions. IC50S and ED50s are derived and correlated with constitutive MAPK activation status. Studies in hypoxia are useful, as there is striking induction of pERK in AML cells in hypoxia (Fiegl et al, 2010). Therefore, the ability of Compound 1 to suppress this effect in hypoxia, which is the physiological ρ(¾ in the bone marrow, is considered. A number of optimized combinations are tested in stromal co-culture systems in hypoxia.
EXAMPLE 4
DEVELOPMENT OF EFFECTIVE COMBINATION STRATEGIES BY TARGETING MEK AND MTOR USING MSC-69B PLUS RAPAMYCIN IN ACUTE
MYELOID LEUKEMIA Phase I: Evaluation of Anti-leukemic Activities of Combined Compound 1 with
Rapamycin in the in vivo Leukemia Mouse Model by Transplanting NPMl/ FLT3-ITD Mutations Leukemic Cell Lines
[0182] Female SCID mice (8-10 weeks old) are allowed to acclimate to their new environment for 1 week prior to the study. Leukemia models are established by injecting MOLM13-NPMl-mut cells, which is a human acute myeloid leukemia cell and stably transduced with lentiviral vectors expressing mutant NPMl and firefly luciferase-GFP. These cells are FACS-sorted and expanded in vitro for approximately 1 month prior to injection into animals; these genetically modified cells do not produce virus. Briefly, the AML cells (at 1- 2* 106 cells/mouse) are suspended in 200 μΐ of PBS and be injected iv. in the tail vein of recipient mice. The animals are then distributed to five groups randomly for Compound 1 (30mg/kg q.d., gavage), rapamycin (4 mg/kg q.d., i.p.) , combination treatment, vehicle treatment and blank control groups. Each group consists of eight tumor-bearing mice.
[0183] Mice receive Compound 1, rapamycin or combination treatment on day 7 of the inoculation of leukemic cells. The drugs are given using QDx5/week schedule at above indicated doses for 3 weeks, for example. These dose ranges are based on the efficacy studies in tumor xenograft animal models.
[0184] Treated animals are monitored daily for signs of morbidity and mortality and sacrificed when paralyzed or terminally ill. Body weights and tumor volumes (if available) are determined twice weekly. Histological and immunohistochemical analyses are performed on bone marrow, lung, liver, spleen and brain tissue samples for detection of human cells for all long-term survivors.
[0185] Endpoints:
[0186] Three mice for each group are sacrificed at 5 weeks of inoculation using CO2. Bone marrow cells are collected by flushing both femurs with PBS. Single cell suspensions of bone marrow and spleen are analyzed by flow cytometry to determine engraftment level (using GFP and anti-human CD45 antibody). A two-sample t-test is
performed to test whether the percentage of engrafting leukemic cells in the mouse bone marrow or other organs is the same in the control and the treatment groups. Effects on target inhibition and on selected biomarkers are evaluated in leukemic cells isolated from mouse bone marrow and spleen by FACS-sorting of CD45(+) human cells. Spleen, lungs, liver, and brain are collected for histological analyses and GFP immunohistochemistry. The remaining five animals are sacrificed upon observation of any signs of morbidity.
[0187] In addition, one can utilize bioluminescence imaging to non-invasively monitor leukemia burden. At each time point, mice are anesthetized using isoflurane. Colerterazine (native; Biotium, Hayward, CA; 1 mg/kg in ΙΟΟμΙ volume) are injected IP to anesthetized animals, and the animals are then non-invasively imaged by Xenogen IVIS imager using 1, 2 or 5 min increments of exposure and light capture; during imaging the animals are continuously anesthetized via inhalation of isoflurane vapors. Serial bioluminescence is quantified as the total sum integrated signal in a standard period of time (i.e., 2 min). A two-sample t-test is performed to test whether tumor volume as defined by the bioluminescence integrity signal is the same in the control and the treatment groups.
[0188] Phase I Number of mice required: For the first set of studies, Compound 1 is tested on 8 animals/group using 15mg/kg and 30gm/kg doses, and plus 4 mice for vehicle treatment and 4 mice for control group. The optimal doses are used on combination treatment. Five groups including Compound 1, rapamycin, combination and vehicle/control grouops (8 mice/group) are requested, i.e., 5 cohorts (untreated mice, vehicle control, Compound 1, rapamycin and combination) x 8 animals/group x 2 (repeat the experiment) + priliminary experiment set (8 x 2 + 4 + 4 mice) + extra 6 mice =200 mice (total)
Phase II: Evaluation of Anti-leukemic Activities of Combined Compound 1 with
Rapamycin in the in vivo Leukemia Mouse Model by Transplanting Human Primary AML Leukemic Cells
[0189] NOD/Scid/IL2Rgamma KO Mice (NOG mice)
[0190] NOG mice restrained in acrylic box, given whole body irradiation (nonlethal, cesium source, 250 cGy, irradiator is located in SB.8044)
[0191] 24 hrs post-radiation, mice will be injected with 5xl06 peripheral blood mononuclear cells (PBMC) from acute myeloid leukemia patient with NPMl mutation after
Ficoll-Hypaque purification. All injections are done via the tail vein while the mouse is in the restraint device for 15 seconds (maximum). The cells are injected in 0.2 ml sterile PBS, 27- gauge needle.
[0192] Starting on day 14 after cells injection, mice are treated with inhibitors (Compound 1 and rapamycin, for example) or vehicle control, for 4 weeks. The most effective dose is chosen based on the results of the studies in cell lines (Phase I) (defined as lack of toxicity and most effective anti-leukemia control).
[0193] Mice observed daily including the weekends for signs of disease. Animals that exhibit excessive tumor burden, hunched posture, lack of eating and drinking, difficulty reaching food, loss of >20% weight, lateral recumbence, respiratory distress are euthanized immediately. Twice (at 4 and 6 weeks post leukemic cell injection) blood samples are collected via tail vein bleed (lOOuL) into tubes containing heparine. Single cell suspensions are analyzed by flow cytometry to determine engraftment level (using anti- human CD45) and lineage expression (based on antigen expression defined in Phase II). [0194] At 6-8 weeks animals will be sacrificed using CO2. Bone marrow, blood, liver and spleen are sampled for morphology, flow cytometry, PCR and FISH to assess for evidence of leukemia. As a biological marker, one can also evaluate effects on target proteins by protein array technique in human leukemic cells isolated from mouse bone marrow by FACS-sorting of CD45(+) cells. If the mice develop lymphoma (immunodeficiency/radiotherapy) or infection they will show the same signs and therefore are sacrificed for evaluation. However, lymphoma usually occurs well beyond age 6 months when mice are allowed to age.
[0195] Phase II number of mice required: One can use 20 AML samples, which includes 10 NPMl wildtype AML and 10 NPMl mutation patients, (fresh or cryopreserved), and with each sample one can inject 5 mice/treatment arm: Compound 1, rapamycin, combination, vehicle and control using QDx5/week schedule. 20 samples X 5 mice = 100 mice
[0196] Reagents preparation:
[0197] 1. Compound 1 is supplied as dry compound synthesized by EMD Serono Research Institute, Boston, MA. It can be solved in CremophorEL/Ethanol/Water (12.5/12.5/75). The drug is administrated in a volume of 200 by oral gavage.
[0198] 2. Rapamycin is obtained from Biovision Research Products (Mountain View, CA). Rapamycin is dissolved in 100% ethanol (10 mg/mL stock), and further diluted in an aqueous solution of 5.2% Tween-80 and 5.2% polyethylene glycol (PEG-400) (final ethanol concentration, 2%) immediately before use. The drug will be administrated i.p. in a volume of 100 by i.p.
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[0242] Zhang W, Konopleva M, Ruvolo VR et al. Sorafenib induces apoptosis of AML cells via Bim-mediated activation of the intrinsic apoptotic pathway. Leukemia. 2008;22:808-818.
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[0245] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines,
manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.
Claims
A method for treating an individual having cancer cells with a nucleophosmin- 1 (NPM1) mutation, comprising selecting an individual that has been determined to have a NPM1 mutation and treating the individual with an effective amount of a MEK inhibitor.
The method of claim 1, wherein the cancer is leukemia, lymphoma, or myelodysplasia syndrome.
The method of claim 2, wherein the leukemia is acute myeloid leukemia or chronic myelomonocytic leukemia.
The method of claim 1, wherein the MEK inhibitor is selected from the group consisting of N-((S)-2,3-Dihydroxy-propyl)-3- (2-fluoro-4-iodo-phenylamino)-isonicotinamide, CI- 1040, PD035901, AZD6244, GSK1120212, GDC-0973, U0126, XL- 518, ARRY-162, ARRY-300, PD184161, PD 184352, PD0325901, ARRY-142886 (AZD6244), RO4927350, PD 0325901, CIP-1374, TAK-733, CH4987655, RDEA119, and combination thereof.
The method of claim 1, wherein the individual is treated with an additional anti-cancer agent.
The method of claim 5, wherein the additional anti-cancer agent is selected from the group consisting of chemotherapy, hormone therapy, surgery, radiation, immunotherapy, and a combination thereof.
The method of claim 5, wherein the additional anti-cancer agent is CCI-779, RADOOl, Sorafenib, Ara-C, all-trans retinoic acid, ABT737, IAP inhibitor, SMAC mimetic, rapamycin, cytarabine, HDAC inhibitor, or a demethylating agent.
8. The method of claim 1, wherein determination of the NPM1 mutation in cancer cells of the individual comprises the step of obtaining a biological sample having genetic material from the individual.
9. The method of claim 8, wherein the sample comprises bone marrow, biopsy, blood, urine, cerebrospinal fluid, cheek scrapings, stool, semen, serum, or biopsy material from leukemic tumors or tissue.
10. The method of claim 8, wherein the genetic material comprises DNA, RNA, or both.
1 1. The method of claim 8, wherein determination of the NPM1 mutation in a biological sample from the individual comprises assaying nucleic acid from the sample.
12. The method of claim 11, wherein the nucleic acid is DNA or RNA.
13. The method of claim 1 1, wherein the assay comprises
polymerase chain reaction, sequencing, immunohistochemistry, flow cytometry, or a combination thereof.
14. The method of claim 1, wherein determination of the NPM1 mutation in a biological sample from the individual comprises assaying polypeptide from the sample.
15. The method of claim 14, wherein the assay comprises
immunohistochemistry, immunoblot, flow cytometry, or a combination thereof.
16. The method of claim 1, further comprising the step of
determining whether the individual has cancer cells comprising a FLT 3 mutation.
17. The method of claim 1, further comprising the step of treating the individual with an effective amount of a mTOR inhibitor, a FLT3-ITD inhibitor, an aurora kinase inhibitor, or a combination thereof.
18. An in vitro method for determining a treatment regimen for an individual, comprising the steps of:
(a) obtaining a biological sample comprising genetic material of the individual, wherein the subject is undergoing or is to undergo cancer therapy; and (b) determining the presence of a NPM1 mutation in the biological sample, wherein when a NPM1 mutation is present, the individual is administered an effective amount of a MEK inhibitor.
19. The method of claim 18, wherein the cancer is leukemia, lymphoma, or myelodysplasia; syndrome.
20. The method of claim 19, wherein the leukemia is acute myeloid leukemia or chronic myelomonocytic leukemia.
21. The method of claim 18, wherein the MEK inhibitor is selected from the group consisting of N-((S)-2,3-Dihydroxy-propyl)-3- (2-fluoro-4-iodo-phenylamino)-isonicotinamide, CI- 1040,
PD035901, AZD6244, GSK1120212, GDC-0973, U0126, XL- 518, ARRY-162, ARRY-300, PD184161, PD 184352, PD0325901, ARRY-142886 (AZD6244), RO4927350, PD 0325901, CIP-1374, TAK-733, CH4987655, RDEA119, and combination thereof.
22. The method of claim 18, wherein the individual is treated with an additional anti-cancer agent.
23. The method of claim 22, wherein the additional anti-cancer agent is selected from the group consisting of chemotherapy, hormone therapy, surgery, radiation, immunotherapy, and a combination thereof.
24. The method of claim 22, wherein the additional anti-cancer agent is CCI-779, RADOOl, Sorafenib, Ara-C, all-trans retinoic acid, ABT737, IAP inhibitor, SMAC mimetic, rapamycin, cytarabine, HDAC inhibitor, or a demethylating agent.
25. The method of claim 18, wherein the sample comprises bone marrow, biopsy, blood, urine, cerebrospinal fluid, cheek scrapings, stool, semen, serum, biopsy material from leukemic tumors or tissue.
26. The method of claim 18, wherein the genetic material
comprises DNA, RNA, or both.
27. The method of claim 18, wherein the determining step
comprises assaying nucleic acid from the sample.
28. The method of claim 27, wherein the nucleic acid is DNA or RNA.
29. The method of claim 27, wherein the assay comprises
polymerase chain reaction, sequencing, immunohistochemistry, flow cytometry, or a combination thereof.
30. The method of claim 18, wherein the determining step
comprises assaying polypeptide from the sample.
31. The method of claim 30, wherein the assay comprise
immunohistochemistry, immunoblot, flow cytometry, or a combination thereof.
32. The method of claim 18, further comprising the step of
determining whether the individual has cancer cells comprising a FLT 3 mutation.
33. A composition comprising an effective amount of a MEK inhibitor for use in treating an individual having cancer cells with a nucleophosmin- 1 ( PM1) mutation.
34. The composition of claim 33, wherein the cancer is leukemia, lymphoma, or myelodysplasia syndrome.
35. The composition of claim 34, wherein the leukemia is acute myeloid leukemia or chronic myelomonocytic leukemia.
36. The composition of claim 33, wherein the MEK inhibitor is selected from the group consisting of N-((S)-2,3-Dihydroxy- propyl)-3-(2-fluoro-4-iodo-phenylamino)-isonicotinamide, CI-
1040, PD035901, AZD6244, GSK1 120212, GDC-0973, U0126, XL-518, ARRY-162, ARRY-300, PD184161, PD 184352, PD0325901, ARRY-142886 (AZD6244),
RO4927350, PD 0325901, CIP-1374, TAK-733, CH4987655, RDEA 119, and combination thereof.
37. The composition of claim 33, wherein the individual is treated with an additional anti-cancer agent.
38. The composition of claim 37, wherein the additional anticancer agent is selected from the group consisting of chemotherapy, hormone therapy, surgery, radiation, immunotherapy, and a combination thereof.
39. The composition of claim 37, wherein the additional anticancer agent is CCI-779, RADOOl, Sorafenib, Ara-C, all-trans retinoic acid, ABT737, IAP inhibitor, SMAC mimetic, rapamycin, cytarabine, HDAC inhibitor, or a demethylating agent.
40. The composition of claim 33, further defined as a composition for use in treating an individual from whom a biological sample has been obtained and determined in vitro to comprise an NPM1 mutation in cancer cells of the individual.
41. The composition of claim 40, wherein the sample comprises bone marrow, biopsy, blood, urine, cerebrospinal fluid, cheek scrapings, stool, semen, serum, or biopsy material from leukemic tumors or tissue.
42. The composition of claim 40, wherein the genetic material comprises DNA, RNA, or both.
43. The composition of claim 33, wherein the cancer cells of the individual are also tested and determined to comprise a FLT 3 mutation.
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