Markers Associated With Human Double Minute 2 Inhibitors

Abstract

The invention provides methods of monitoring differential gene expression of biomarkers to determine patient sensitivity to Human Double Minute inhibitors (MDM2i), methods of determining the sensitivity of a cell to an MDM2i by measuring biomarkers and methods of screening for candidate MDM2i.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. provisional application Ser. No. 61/677,859, filed Jul. 31, 2012, which is herein incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of pharmacogenomics, and the use of biomarkers useful in determining patient sensitivity prior to treatment, following patient response after treatment, cancer sensitivity and screening of compounds.

BACKGROUND

[0003] p53, also known as tumor protein 53, is a tumor suppressor gene involved in the prevention of cancer, often referred to as the gatekeeper or guardian of the genome (Levine, Cell 1997, 88:323-331). The p53 gene encodes for a transcription factor that is normally quiescent, and becoming activated when the cell is stressed or damaged, such as when DNA damage incurred from a mutagen. If the cell is stressed or damaged, p53 acts to limit the damage, or barring that, trigger the apoptotic pathway so the damaged cell is eliminated and no longer a threat to the organism (Vogelstein et al., Nature 2000, 408:307-310). An analysis of different cancers showed that p53 is mutated in about 50% of human cancers (Hollstein et al., Nucleic Acids Res. 1994, 22:3551-3555: Hollstein et al., Science 1991, 253(5015): 49-53). Humans who are heterozygous for p53, with only a single functional copy, will develop tumors early in adulthood, a disorder known as Li-Fraumeni syndrome (Varley et al., Hum. Mutat. 2003, 21(3):313-320). However, as much as p53 regulates the cell's fate, p53 is regulated by another protein known as MDM2.

[0004] Double minute 2 protein (MDM2) was discovered as a negative regulator of p53 (Fakharzadeh et al., EMBO J. 1991, 10(6):1565-1565). MDM2 encodes an E3 ligase containing a p53 binding domain and a nuclear export signal sequence, and upon complexing with p53, removes it from the nucleus and ubiquitinylates it, which promotes the degradation of the p53 protein via the ubiquitin-proteosome pathway (Haupt et al., Nature 1997, 387(6630):296-299; Piette et al., Oncogene 1997 15(9):1001-1010). In addition, MDM2 directly inhibits the activity of p53 by binding to the p53 transactivation domain, also preventing p53 mediated gene expression (Wu et a., Genes Dev. 1993, 7:1126-1132). Thus, MDM2 regulates p53 in multiple ways.

[0005] MDM2 is overexpressed in a number of cancers, for example, liposarcoma, glioblastoma, and leukemia (Momand et al., Nucleic Acids Res. 1998, 26(15):3453-3459). Overexpression of MDM2 can interfere with the activities of p53, preventing apoptosis and growth arrest of the tumor (de Rozieres et al., Oncogene 2000, 19(13):1691-1697). Overexpression of MDM2 correlates with poor prognosis in glioma, and acute lympocytic leukemia (Onel et al., Mol. Cancer Res. 2004, 2(1):1-8).

[0006] As MDM2 is an inhibitor of p53, therapeutics which prevent the binding of MDM2 to p53 would prevent the degradation of p53, allowing free p53 to bind and mediate gene expression in cancer cells, resulting in cell cycle arrest and apoptosis. There are previous reports of small molecule inhibitors of the p53-MDM2 interaction (Vassilev et al., Science, 2004, 303(5659):844-888; Zhang et al., Anticancer drugs, 2009 20(6):416-424; Vu et al., Curr. Topics Microbiol. Immuno., 2011, 348:151-172). The mode of binding of these compounds and a crystal structure of the human MDM2--Nutlin complex as well as a scaffold and pockets of the p53 binding site on MDM2 are also known (Vassilev, supra). The first of these MDM2 inhibitors, known as the Nutlins, bind MDM2 and occupy the p53 binding pocket, preventing the formation of the MDM2-p53 complex. This leads to less degradation of the p53 protein, and expression of p53 target genes. Cancer cell lines treated with Nutlins showed growth arrest and increased apoptosis. For example, the SJSA-1 osteosarcoma line contains amplified copies of the MDM2 gene. Treatment of this line with Nutlin-3 reduced proliferation and increased apoptosis (Vassilev et al., Science, 2004, 303(5659):844-888). The SJSA-1 cell line was used in creating xenographs in mouse. Administration of Nutlin-3 reduced xenograft growth by 90%. To investigate the effect the Nutlin compounds had on non-cancerous cells, human and mouse normal fibroblasts were treated with Nutlin-3 and while the proliferation of the cells was slowed, they retained their viability (Vassilev, supra).

[0007] Finding biomarkers which indicate which patient should receive a therapeutic is useful, especially with regard to cancer. This allows for more timely and aggressive treatment as opposed to a trial and error approach. In addition, the discovery of biomarkers which indicate that cells continue to be sensitive to the therapy after administration is also useful. These biomarkers can be used to monitor the response of those patients receiving the therapeutic. If biomarkers indicate that the patient has become insensitive to the treatment, then the dosage administered can be increased, decreased, completely discontinued or an additional therapeutic administered. As such, there is a need to develop biomarkers associated with MDM2 inhibitors. This approach ensures that the correct patients receive the appropriate treatment and during the course of the treatment the patient can be monitored for continued MDM2 inhibitor sensitivity.

[0008] In the development of MDM2 inhibitors, specific biomarkers will aid in understanding the mechanism of action upon administration. The mechanism of action may involve a complex cascade of regulatory mechanisms in the cell cycle and differential gene expression. This analysis is done at the pre-clinical stage of drug development in order to determine the particular sensitivity of cancer cells to the MDM2 inhibitor candidate and the activity of the candidate. Of particular interest in the pharmacodynamic investigation is the identification of specific markers of sensitivity and activity, such as the ones disclosed herein.

SUMMARY OF THE INVENTION

[0009] The invention relates to the analysis that a number of genes identified in Table 2 act as specific biomarkers in determining the sensitivity of cells to MDM2 inhibitors (henceforth "MDM2i"). The invention relates to the analysis that at least one of the biomarkers in Table 2 provides a "gene signature" for MDM2i that has increased accuracy and specificity in predicting which cancer cells are sensitive to MDM2i. The method analyzes the gene expression or protein level of at least one of the biomarkers in Table 2 in a cancer sample taken from a patient and compared to a baseline control predicts the sensitivity of the cancer sample to an MDM2i. The pattern of expression level changes may be indicative of a favorable response or an unfavorable one. In addition, the gene signature provided in Table 2 has increased predictive value because it also indicates that the p53 pathway is functional. This is an unexpected result as many tumors contain a mutated p53 and a non-functional pathway which provides the tumor with a growth advantage. The invention is an example of "personalized medicine" wherein patients are treated based on a functional genomic signature that is specific to that individual.

[0010] The predictive value of at least one biomarker in Table 2 can also be used after treatment with an MDM2i to determine if the patient remains sensitive to the treatment. Once the MDM2i therapeutic has been administered, the biomarkers are used to monitor the continued sensitivity of the patient to MDM2i treatment. The disclosure alsorelates to the up or down regulation of the expression of the identified genes after MDM2i treatment. This is useful in determining that patients receive the correct course of treatment. The invention comprises a method of predicting and monitoring the sensitivity of a patient to MDM2i treatment. The method includes the step of administration of an MDM2i to the patient and measurement of biomarker gene expression on a biological sample obtained from the patient. The response of the patient is evaluated based on the detection of gene expression of at least one biomarker from Table 2. Detection and/or alteration in the level of expression of at least one biomarker compared to baseline is indicative of the sensitivity of the patient to the treatment. The pattern of expression level changes can be indicative of a favorable patient response or an unfavorable one.

DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1A shows the in vitro potency of the MDM2i in disrupting p53-MDM2 interaction. FIG. 1B shows the in vitro potency of the MDM2i on the proliferation of cancer cells.

[0012] FIG. 2 shows the p53 status (mutant or wild type) and the sensitivity of the cancer cells to MDM2i(2). The X axis is the crossing point for sensitivity and the Y axis is the Amax value.

[0013] FIG. 3 are biomarkers showing the fold change in expression and the statistical significance of the overexpression value.

[0014] FIG. 4 is a Western blot of sensitive and insensitive representative cells treated with an MDM2i(1) for 4 hours at various concentrations and then probed for selected p53 target genes as pharmacodynamic biomarker representatives.

[0015] FIG. 5 is a graph demonstrating the increase in predictive value of the gene signature as opposed to a larger set of biomarkers.

[0016] FIG. 6 is a list of cell lines, each analyzed for the gene signature and the prediction of whether the cell line is sensitive or insensitive and the IC50 when treated with an MDM2i.

[0017] FIG. 7A-C shows the dose-dependent inhibition of tumor growth in the SJSA-1 xenograft model (predicted to be sensitive by the gene signature) following treatment with MDM2i(1), and the concomitant induction of p21(CDKN1A) expression as a representative pharmacodynamic biomarker.

[0018] FIG. 8 shows the expression of p21(CDKN1A) at the protein level after treatment of MDM2i(1) sensitive cells at efficacious doses.

[0019] FIG. 9 is a model of tumor samples predicted to be sensitive using the gene signature in the OncExpress database and the Primary Tumor Bank.

[0020] FIG. 10 represents the Positive Predictive Values (PPV) achieved by the MDM2i(2) sensitivity predictive models, built from multiple combinations of biomarkers described in Table 2.

[0021] FIG. 11 represents the Specificities achieved by the MDM2i sensitivity predictive models, built from multiple combinations of biomarkers described in Table 2.

[0022] FIG. 12 represents the Sensitivities achieved by the MDM2i sensitivity predictive models, built from multiple combinations of biomarkers described in Table 2.

[0023] FIG. 13 represents the PPV achieved by the MDM2i sensitivity predictive models, built from multiple combinations of biomarkers described in Table 2, together with p53 mutation status.

[0024] FIG. 14 represents the Specificities achieved by the MDM2i sensitivity predictive models, built from multiple combinations of biomarkers described in Table 2, together with p53 mutation status.

[0025] FIG. 15 represents the Sensitivities achieved by the MDM2i sensitivity predictive models, built from multiple combinations of biomarkers described in Table 2, together with p53 mutation status.

DESCRIPTION OF THE INVENTION

[0026] The aspects, features and embodiments of the present invention are summarized in the following items and can be used respectively alone or in combination:

[0027] 1. A method of predicting the sensitivity of a cancer patient for treatment with a Human Double Minute 2 inhibitor (MDM2i), the method comprising: a) measuring differential gene expression of at least one biomarker selected from Table 2 in a cancer sample obtained from the patient; and b) comparing the differential gene expression of the at least one biomarker with gene expression of said biomarker in a control sample, wherein the increase or decrease in gene expression comparison indicates that the patient is sensitive to treatment with an MDM2i.

[0028] 2. A method of treating a cancer patient comprising: a) measuring differential gene expression of at least one biomarker selected from Table 2 in a cancer sample obtained from the patient; b) comparing the differential gene expression of the at least one biomarker with gene expression of the biomarker in a control sample; c) determining sensitivity of the patient to an MDM2i; and d) administering to the patient an MDM2i.

[0029] 3. A method of predicting the sensitivity of a cancer cell to a Human Double Minute 2 inhibitor (MDM2i), the method comprising: a) measuring differential gene expression of at least one biomarker selected from Table 2 in the cell; b) comparing the differential gene expression of the at least one biomarker selected from Table 2 with gene expression from a normal or control cell.

[0030] 4. A method of determining the sensitivity of a cancer cell to a Human Double Minute 2 inhibitor (MDM2i), the method comprising: a) contacting a cancer cell with at least one MDM2i; b) measuring differential gene expression of at least one biomarker selected from Table 2 in the cell contacted with the MDM2i; c) comparing the differential gene expression of the at least one biomarker with gene expression of the biomarker from an untreated or placebo treated control cell; d) wherein there is an increase in the expression of at the least one biomarker when compared with the expression of the at least one biomarker from the untreated or placebo treated control cell.

[0031] 5. The method of any one of items 1 to 3, wherein more than one biomarker is selected from Table 2.

[0032] 6. The method of item 1 or 4, wherein at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve or all thirteen biomarkers are selected from Table 2.

[0033] 7. The method of any one of items 1 to 5, wherein p53 is selected as a biomarker in addition to any biomarker selected from Table 2.

[0034] 8. The method of any one of items 1 to 6, comprising the biomarkers MDM2, CDKN1A, ZMAT3, DDB2, FDXR, RPS27L, BAX, RRM2B, SESN1, CCNG1, XPC, TNFRSF10B and/or AEN.

[0035] 9. The method of any one of items 1 to 7, wherein comparing the differential gene expression of the at least one biomarker with gene expression of a control sample indicates a functional p53 gene pathway.

[0036] 10. The method of any one of items 1 to 8, wherein the cancer sample is selected from the group consisting of breast, lung, pancreas, ovary, central nervous system (CNS), endometrium, stomach, large intestine, colon, esophagus, bone, urinary tract, hematopoietic, lymphoid, liver, skin, melanoma, kidney, soft tissue sarcoma and pleura.

[0037] 11. The method of any one of items 1 to 9, wherein a nucleic acid or protein of at least one biomarker is measured.

[0038] 12. The method of any one of items 1, 2 or 5 to 11, wherein the expression of the at least one biomarker is increased in the cancer sample when compared to a control sample.

[0039] 13. The method of any one of items 1 to 11, wherein the MDM2i is selected from Table 1.

[0040] 14. The method of any one of items 1 to 12, wherein the MDM2i is a compound that binds to a p53 binding pocket of MDM2.

[0041] 15. The method of any one of items 1 to 13, wherein the MDM2i is a compound that binds to substantially the same p53 binding pocket of MDM2 as Nutlin-3a or the MDM2i from the Table 1.

[0042] 16. The method of any one of items 1 to 14, wherein the MDM2i is a compound that prevents the protein-protein interaction between p53 and MDM2.

[0043] 17. The method of any one of items 1 to 15, wherein the MDM2i is a compound that inhibits cell proliferation by inducing the p53 pathway activity.

[0044] 18. The method of any one of items 2 to 16 further comprising obtaining a biological sample from the patient prior to the administration of the MDM2i.

[0045] 19. The method of any one of items 2 to 17, wherein the MDM2i is administered in a therapeutically effective amount.

[0046] 20. The method of any one of items 3 to 11, or 13 to 19, wherein the gene expression of the at least one biomarker is increased in the cancer cell.

[0047] 21. The method of any one of items 4 to 11, or 13 to 20, wherein the IC50 of the cancer cell contacted with at least one MDM2i is less than 1 .mu.M

[0048] 22. The method of any one of items 4 to 11, or 13 to 21, wherein the cell is contacted by the MDM2i at least at two different time points.

[0049] 23. The method of any one of items 4 to 11, or 13 to 22, wherein the cell is contacted by two different MDM2i at step a).

[0050] 24. The method of item 23, wherein the cell is contacted by the two different MDM2i at the same time.

[0051] 25. The method of item 23, wherein the cell is contacted by two different MDM2i at different time points.

[0052] 26. The method of any one of items 4 to 11, or 13 to 25, wherein the steps b) and c) are repeated at a time points selected from the group consisting of: 4 hours, 8 hours, 16 hours, 24 hours, 48 hours, 3 days, 1 week, 1 month and several months after administration of each dose of MDM2i.

[0053] 27. A method of screening for MDM2i candidates the method comprising: a) contacting a cell with a MDM2i candidate; b) measuring gene expression of at least one biomarker selected from Table 2 in the cell contacted with the MDM2i candidate; and c) comparing the gene expression of the at least one biomarker selected from Table 2 from the cell contacted with the MDM2i candidate with gene expression of the at least one biomarker selected from Table 2 from a cell contacted with an MDM2i taken from Table 1 and the gene expression of at least one biomarker of an untreated or placebo treated cell.

[0054] 28. The method of item 27, wherein the differential gene expression of the MDM2i candidate is compared with the differential gene expression of an MDM2i selected from Table 1.

[0055] 29. The method of item 27 or 28, wherein the MDM2i candidate increases gene expression of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve or all thirteen biomarkers from Table 2.

[0056] 30. The method of any one of items 27 to 29, wherein the cell is a cancer cell selected from the group consisting of breast, lung, pancreas, ovary, central nervous system (CNS), endometrium, stomach, large intestine, colon, esophagus, bone, urinary tract, hematopoietic, lymphoid, liver, skin, melanoma, kidney, soft tissue sarcoma and pleura.

[0057] 31. The method of any one of items 27 to 30, wherein the expression of nucleic acid or protein of at least one biomarker of Table 2 is measured.

[0058] 32. The method of any one of items 27 to 31, comprising the biomarkers MDM2, CDKN1A, ZMAT3, DDB2, FDXR, RPS27L, BAX, RRM2B, SESN1, CCNG1, XPC, TNFRSF10B and AEN.

[0059] 33. Composition comprising an MDM2i for use in treatment of cancer in a selected cancer patient population, wherein the cancer patient population is selected on the basis of showing an increased gene expression rate of at least one biomarker selected from Table 2 in a cancer cell sample obtained from said patients compared to a normal control cell sample.

[0060] 34. The composition of item 33, wherein at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve or all thirteen biomarkers are selected from Table 2.

[0061] 35. The composition of items 33 or 34, wherein p53 is selected as a biomarker in addition to any biomarker selected from Table 2.

[0062] 36. The composition of any one of items 33 to 35, wherein the biomarker is MDM2, CDKN1A, ZMAT3, DDB2, FDXR, RPS27L, BAX, RRM2B, SESN1, CCNG1, XPC, TNFRSF10B and/or AEN.

[0063] 37. The composition of any one of items 33 to 36, wherein the MDM2i is selected from Table 1.

[0064] 38. The composition of any one of items 33 to 37, wherein the MDM2i is a compound that binds to a p53 binding pocket of MDM2.

[0065] 39. The composition of any one of items 33 to 38, wherein the MDM2i is a compound that binds to substantially the same p53 binding pocket of MDM2 as Nutlin-3a or the MDM2i from the Table 1.

[0066] 40. The composition of any one of items 33 to 39, wherein the MDM2i is a compound that prevents the protein-protein interaction between p53 and MDM2.

[0067] 41. The composition of any one of items 33 to 40, wherein the MDM2i is a compound that inhibits cell proliferation by inducing the p53 pathway activity.

[0068] 42. The composition of any one of items 33 to 41, wherein the cancer cell sample is selected from the group consisting of breast, lung, pancreas, ovary, central nervous system (CNS), endometrium, stomach, large intestine, colon, esophagus, bone, urinary tract, hematopoietic, lymphoid, liver, skin, melanoma, kidney, soft tissue sarcoma and pleura.

[0069] 43. The composition of any one of items 33 to 42, wherein the patients are selected on the basis of an increased gene expression of the biomarkers MDM2, CDKN1A, ZMAT3, DDB2, FDXR, RPS27L, BAX, RRM2B, SESN1, CCNG1, XPC, TNFRSF10B and AEN.

[0070] 44. A kit for predicting the sensitivity of a cancer patient for treatment with a Human Double Minute 2 inhibitor (MDM2i) comprising: i) means for detecting the expression of any one of the biomarkers from the table 2, preferably more than one, particularly at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve or all thirteen biomarkers selected from Table 2; and ii) instructions how to use said kit.

[0071] 45. The kit of item 44, wherein the biomarkers are MDM2, CDKN1A, ZMAT3, DDB2, FDXR, RPS27L, BAX, RRM2B, SESN1, CCNG1, XPC, TNFRSF10B or/and AEN.

[0072] 46. The kit of item 45 further comprising means for detecting the expression of p53.

[0073] 47. Use of the kit according to item 45 or 46 for any of the methods of items 1 to 32.

[0074] Further aspects describe the invention:

[0075] In one aspect, a disclosed invention relates to methods of analyzing at least one of the biomarkers identified in Table 2 in a sample containing cancer cells wherein increased or decreased expression of at least one biomarker when compared to a baseline indicates if the cancer cell will be sensitive to MDM2i treatment. The pattern of expression level changes can be indicative of a favorable patient response or of an unfavorable one and patients can be selected or rejected based on the increased or decreased expression of at least one biomarker from Table 2. Alternatively, all of the biomarkers in Table 2 can be assayed for as a single set.

[0076] After treatment with an MDM2i, the invention relates to methods of analyzing at least one of the biomarkers identified in Table 2 in a sample containing cancer cells wherein increased or decreased expression of the biomarker when compared to a baseline control after MDM2i treatment indicates that the patient is still sensitive to MDM2i treatment. Detection and/or alteration in the level of expression of at least one biomarker compared to a baseline is indicative of the MDM2i sensitivity, and this correlates with a response of the patient to the treatment. Alternatively, all of the biomarkers in Table 2 can be assayed for as a single set. The pattern of expression level changes can be indicative of a favorable patient response or of an unfavorable one.

[0077] Accordingly, the invention provides for a method of predicting the sensitivity of a cancer patient for treatment with a Human Double Minute 2 inhibitor (MDM2i), the method comprising: a) measuring differential gene expression of at least one biomarker selected from Table 2 in a cancer sample obtained from the patient; and b) comparing the differential gene expression of the at least one biomarker with gene expression of a control sample, wherein the increase or decrease in gene expression comparison indicates that the patient is sensitive to treatment with an MDM2i.

[0078] The method wherein more than one biomarker is selected from Table 2.

[0079] The method comprising the biomarkers MDM2, CDKN1A, ZMAT3, DDB2, FDXR, RPS27L, BAX, RRM2B, SESN1, CCNG1, XPC, TNFRSF10B and AEN.

[0080] The method wherein comparing the differential gene expression of the at least one biomarker with gene expression of a control sample indicates a functional p53 gene pathway.

[0081] The method wherein the cancer sample is selected from the group consisting of: breast, lung, pancreas, ovary, central nervous system (CNS), endometrium, stomach, large intestine, colon, esophagus, bone, urinary tract, hematopoietic, lymphoid, liver, skin, melanoma, kidney, soft tissue sarcoma and pleura.

[0082] The method wherein a nucleic acid or protein of at least one biomarker is measured.

[0083] The method wherein the gene expression of the at least one biomarker is increased.

[0084] The method wherein the MDM2i is selected from Table 1.

[0085] The method wherein the MDM2i is administered in a therapeutically effective amount.

[0086] A method of treating a cancer patient comprising: a) measuring differential gene expression of at least one biomarker selected from Table 2 in a cancer sample obtained from the patient; b) comparing the differential gene expression of the at least one biomarker with gene expression of a control sample; c) determining sensitivity of the patient to an MDM2i; and d) administering to the patient an MDM2i.

[0087] The method wherein more than one biomarker is selected from Table 2.

[0088] The method of comprising the biomarkers MDM2, CDKN1A, ZMAT3, DDB2, FDXR, RPS27L, BAX, RRM2B, SESN1, CCNG1, XPC, TNFRSF10B and AEN.

[0089] The method wherein the at least one biomarker indicates a functional p53 gene pathway.

[0090] The method further comprising obtaining a biological sample from the patient prior to the administration of the MDM2i.

[0091] The method wherein the cancer sample is selected from the group consisting of: breast, lung, pancreas, ovary, central nervous system (CNS), endometrium, stomach, large intestine, colon, esophagus, bone, urinary tract, hematopoietic, lymphoid, liver, skin, melanoma, kidney, soft tissue sarcoma and pleura.

[0092] The method wherein the MDM2i is selected from Table 1.

[0093] The method wherein the MDM2i is administered in a therapeutically effective amount.

[0094] A method of predicting the sensitivity of a cancer cell to a Human Double Minute 2 inhibitor (MDM2i), the method comprising: a) measuring differential gene expression of at least one biomarker selected from Table 2 in the cell b) comparing the differential gene expression of the at least on biomarker selected from Table 2 with gene expression from a normal or control cell.

[0095] The method wherein the MDM2i is selected from Table 1.

[0096] The method wherein more than one biomarker is selected from Table 2.

[0097] The method comprising the biomarkers MDM2, CDKN1A, ZMAT3, DDB2, FDXR, RPS27L, BAX, RRM2B, SESN1, CCNG1, XPC, TNFRSF10B and AEN.

[0098] The method wherein comparing the differential gene expression of the at least one biomarker with gene expression of a control sample indicates a functional p53 gene pathway.

[0099] The method wherein the cancer sample is selected from the group consisting of: breast, lung, pancreas, ovary, central nervous system (CNS), endometrium, stomach, large intestine, colon, esophagus, bone, urinary tract, hematopoietic, lymphoid, liver, skin, melanoma, kidney, soft tissue sarcoma and pleura.

[0100] The method wherein a nucleic acid or protein of at least one biomarker is measured.

[0101] The method wherein the gene expression of the at least one biomarker is increased.

[0102] The method wherein the MDM2i is selected from Table 1.

[0103] The method wherein the MDM2i is administered in a therapeutically effective amount.

[0104] A method of assaying for the sensitivity of a cancer cell to a Human Double Minute 2 inhibitor (MDM2i), the method comprising: a) contacting a cancer cell with at least one MDM2i; b) measuring differential gene expression of at least one biomarker selected from Table 2 in the cancer cell contacted with the MDM2i; c) comparing the differential gene expression with gene expression from an untreated or placebo treated control cell; d) wherein the IC50 of the cancer cell contacted with at least one MDM2i is less than 3 .mu.M.

[0105] The method wherein the cancer cell is contacted by the MDM2i at least two different time points.

[0106] The method wherein the cancer cell is contacted by two different MDM2i at step a).

[0107] The method wherein the cancer cell is contacted by the two different MDM2i at the same time.

[0108] The method wherein the cancer cell is contacted by two different MDM2i at different time points.

[0109] The method wherein the cancer cell is selected from the group consisting of breast, lung, pancreas, ovary, central nervous system (CNS), endometrium, stomach, large intestine, colon, esophagus, bone, urinary tract, hematopoietic, lymphoid, liver, skin, melanoma, kidney, soft tissue sarcoma and pleura.

[0110] The method wherein a nucleic acid or protein of at least one biomarker is measured.

[0111] The method wherein the gene expression of the at least one biomarker is increased.

[0112] The method comprising the biomarkers: MDM2, CDKN1A, ZMAT3, DDB2, FDXR, RPS27L, BAX, RRM2B, SESN1, CCNG1, XPC, TNFRSF10B and AEN.

[0113] The method wherein the steps b) and c) are repeated at time points of: 4 hours, 8 hours, 16 hours, 24 hours, 48 hours, 3 days, 1 week, 1 month and 2 months after contact with an MDM2i.

[0114] A method of screening for MDM2i candidates the method comprising: a) contacting a cell with a MDM2i candidate; b) measuring differential gene expression of at least one biomarker selected from Table 2 in the cell contacted with the MDM2i candidate; and c) comparing the differential gene expression of at least one biomarker selected from Table 2 from the cell contacted with the MDM2i candidate with differential gene expression of at least one biomarker selected from Table 2 from a cell contacted with an MDM2i taken from Table 1 and the differential gene expression of at least one biomarker of an untreated or placebo treated cell.

[0115] The method wherein the differential gene expression of the MDM2i candidate is compared with the differential gene expression of an MDM2i selected from Table 1. The method wherein the MDM2i candidate increases gene expression of at least one biomarker of Table 2.

[0116] The method wherein the cancer cell is selected from the group consisting of breast, lung, pancreas, ovary, central nervous system (CNS), endometrium, stomach, large intestine, colon, esophagus, bone, urinary tract, hematopoietic, lymphoid, liver, skin, melanoma, kidney, soft tissue sarcoma and pleura.

[0117] The method wherein the expression of nucleic acid or protein of at least one biomarker of Table 2 is measured.

[0118] The method comprising the biomarkers MDM2, CDKN1A, ZMAT3, DDB2, FDXR, RPS27L, BAX, RRM2B, SESN1, CCNG1, XPC, TNFRSF10B and AEN.

[0119] Composition comprising an MDM2i for use in treatment of cancer in a selected cancer patient population, wherein the cancer patient population is selected on the basis of showing an increased gene expression rate of at least one biomarker selected from Table 2 in a cancer cell sample obtained from said patients compared to a normal control cell sample. The composition wherein the cancer sample is selected from the group consisting of breast, lung, pancreas, ovary, central nervous system (CNS), endometrium, stomach, large intestine, colon, esophagus, bone, urinary tract, hematopoietic, lymphoid, liver, skin, melanoma, kidney, soft tissue sarcoma and pleura.

[0120] The composition wherein the patients are selected on the basis of an increased gene expression of the biomarkers MDM2, CDKN1A, ZMAT3, DDB2, FDXR, RPS27L, BAX, RRM2B, SESN1, CCNG1, XPC, TNFRSF10B and AEN.

[0121] A kit for predicting the sensitivity of a cancer patient for treatment with a Human Double Minute 2 inhibitor (MDM2i) comprising: i) means for detecting the expression of the biomarkers MDM2, CDKN1A, ZMAT3, DDB2, FDXR, RPS27L, BAX, RRM2B, SESN1, CCNG1, XPC, TNFRSF10B and AEN; and ii) instructions how to use said kit.

DEFINITIONS

[0122] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0123] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about." It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0124] The terms "marker" or "biomarker" are used interchangeably herein. A biomarker is a nucleic acid or polypeptide and the presence, absence or differential expression of the nucleic acid or polypeptide is used to determine sensitivity to any MDM2i. For example, CDKN1A is a biomarker and the mRNA expression of CDKN1A in a cancer cell is increased when compared to CDKN1A expression in normal (non-cancerous) tissue or control tissue.

[0125] "MDM2" refers to an E3 ubiquitin-protein ligase that mediates the ubiquitination of p53, permits the nuclear export of p53 and triggers p53 degradation. Unless specifically stated otherwise, MDM2 as used herein, refers to human MDM2-accession numbers NM_002392/NP_002383 (SEQ ID NO. 1/SEQ ID NO. 2).

[0126] A cell is "sensitive" or displays "sensitivity" for inhibition with an MDM2i when at least one of the biomarkers disclosed in Table 2 is differentially expressed. Alternatively, a cell is "sensitive" for inhibition with an MDM2i when all of the biomarkers disclosed in Table 2 as a set are differentially expressed.

[0127] A "control cell" or "normal cell" refers to non-cancerous tissue or cell.

[0128] A "control tissue" or "normal tissue" refers to non-cancerous tissue or cell.

[0129] A "control sample" or "normal sample" refers to non-cancerous tissue or cell.

[0130] The terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0131] A "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. A polynucleotide sequence can be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.

[0132] "Gene expression" or alternatively a "gene product" refers to the nucleic acids or amino acids (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.

[0133] The term "polypeptide" is used interchangeably with the term "protein" and in its broadest sense refers to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits can be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc.

[0134] As used herein the term "amino acid" refers to either natural and/or unnatural or synthetic amino acids, and both the D and L optical isomers, amino acid analogs, and peptidomimetics. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.

[0135] The term "isolated" means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, are normally associated with in nature. For example, an isolated polynucleotide is separated from the 3' and 5' contiguous nucleotides with which it is normally associated within its native or natural environment, e.g., on the chromosome. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof, does not require "isolation" to distinguish it from its naturally occurring counterpart. In addition, a "concentrated," "separated" or "diluted" polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater in a "concentrated" version or less than in a "separated" version than that of its naturally occurring counterpart. A polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof, which differs from the naturally occurring counterpart in its primary sequence or, for example, by its glycosylation pattern, need not be present in its isolated form since it is distinguishable from its naturally occurring counterpart by its primary sequence or, alternatively, by another characteristic such as glycosylation pattern. Thus, a non-naturally occurring polynucleotide is provided as a separate embodiment from the isolated naturally occurring polynucleotide. A protein produced in a bacterial cell is provided as a separate embodiment from the naturally occurring protein isolated from a eukaryotic cell in which it is produced in nature.

[0136] A "probe" when used in the context of polynucleotide manipulation refers to an oligonucleotide that is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target. Usually, a probe will comprise a label or a means by which a label can be attached, either before or subsequent to the hybridization reaction. Suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes.

[0137] A "primer" is a short polynucleotide, generally with a free 3'--OH group that binds to a target or "template" potentially present in a sample of interest by hybridizing with the target, and thereafter promoting polymerization of a polynucleotide complementary to the target. A "polymerase chain reaction" ("PCR") is a reaction in which replicate copies are made of a target polynucleotide using a "pair of primers" or a "set of primers" consisting of an "upstream" and a "downstream" primer, and a catalyst of polymerization, such as a DNA polymerase, and typically a thermally-stable polymerase enzyme. Methods for PCR are well known in the art, and taught, for example in PCR: A Practical Approach, M. MacPherson et al., IRL Press at Oxford University Press (1991). All processes of producing replicate copies of a polynucleotide, such as PCR or gene cloning, are collectively referred to herein as "replication." A primer can also be used as a probe in hybridization reactions, such as Southern or Northern blot analyses (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (1989)).

[0138] As used herein, "expression" refers to the process by which DNA is transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0139] "Differentially expressed" as applied to a gene, refers to the differential production of the mRNA transcribed and/or translated from the gene or the protein product encoded by the gene. A differentially expressed gene may be overexpressed or underexpressed as compared to the expression level of a normal or control cell. However, as used herein, overexpression is an increase in gene expression and generally is at least 1.25 fold or, alternatively, at least 1.5 fold or, alternatively, at least 2 fold, or alternatively, at least 3 fold or alternatively, at least 4 fold expression over that detected in a normal or control counterpart cell or tissue. As used herein, underexpression, is a reduction of gene expression and generally is at least 1.25 fold, or alternatively, at least 1.5 fold, or alternatively, at least 2 fold or alternatively, at least 3 fold or alternatively, at least 4 fold expression under that detected in a normal or control counterpart cell or tissue. The term "differentially expressed" also refers to where expression in a cancer cell or cancerous tissue is detected but expression in a control cell or normal tissue (e.g. non-cancerous cell or tissue) is undetectable.

[0140] A high expression level of the gene may occur because of over expression of the gene or an increase in gene copy number. The gene may also be translated into increased protein levels because of deregulation or absence of a negative regulator.

[0141] A "gene expression profile" refers to a pattern of expression of at least one biomarker that recurs in multiple samples and reflects a property shared by those samples, such as tissue type, response to a particular treatment, or activation of a particular biological process or pathway in the cells. Furthermore, a gene expression profile differentiates between samples that share that common property and those that do not with better accuracy than would likely be achieved by assigning the samples to the two groups at random. A gene expression profile may be used to predict whether samples of unknown status share that common property or not. Some variation between the levels of at least one biomarker and the typical profile is to be expected, but the overall similarity of the expression levels to the typical profile is such that it is statistically unlikely that the similarity would be observed by chance in samples not sharing the common property that the expression profile reflects.

[0142] The term "cDNA" refers to complementary DNA, i.e. mRNA molecules present in a cell or organism made into cDNA with an enzyme such as reverse transcriptase. A "cDNA library" is a collection of all of the mRNA molecules present in a cell or organism, all turned into cDNA molecules with the enzyme reverse transcriptase, then inserted into "vectors" (other DNA molecules that can continue to replicate after addition of foreign DNA). Exemplary vectors for libraries include bacteriophage (also known as "phage"), viruses that infect bacteria, for example, lambda phage. The library can then be probed for the specific cDNA (and thus mRNA) of interest.

[0143] As used herein, "solid phase support" or "solid support", used interchangeably, is not limited to a specific type of support. Rather a large number of supports are available and are known to one of ordinary skill in the art. Solid phase supports include silica gels, resins, derivatized plastic films, glass beads, plastic beads, alumina gels, microarrays, and chips. As used herein, "solid support" also includes synthetic antigen-presenting matrices, cells, and liposomes. A suitable solid phase support may be selected on the basis of desired end use and suitability for various protocols. For example, for peptide synthesis, solid phase support may refer to resins such as polystyrene (e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories), polyHIPE(R).TM. resin (obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TentaGelR.TM., Rapp Polymere, Tubingen, Germany), or polydimethylacrylamide resin (obtained from Milligen/Biosearch, California).

[0144] A polynucleotide also can be attached to a solid support for use in high throughput screening assays. PCT WO 97/10365, for example, discloses the construction of high density oligonucleotide chips. See also, U.S. Pat. Nos. 5,405,783; 5,412,087 and 5,445,934. Using this method, the probes are synthesized on a derivatized glass surface to form chip arrays. Photoprotected nucleoside phosphoramidites are coupled to the glass surface, selectively deprotected by photolysis through a photolithographic mask and reacted with a second protected nucleoside phosphoramidite. The coupling/deprotection process is repeated until the desired probe is complete.

[0145] As an example, transcriptional activity can be assessed by measuring levels of messenger RNA using a gene chip such as the Affymetrix.RTM. HG-U133-Plus-2 GeneChips. High-throughput, real-time quantitation of RNA of a large number of genes of interest thus becomes possible in a reproducible system.

[0146] The terms "stringent hybridization conditions" refers to conditions under which a nucleic acid probe will specifically hybridize to its target subsequence, and to no other sequences. The conditions determining the stringency of hybridization include: temperature, ionic strength, and the concentration of denaturing agents such as formamide. Varying one of these factors may influence another factor and one of skill in the art will appreciate changes in the conditions to maintain the desired level of stringency. An example of a highly stringent hybridization is: 0.015M sodium chloride, 0.0015M sodium citrate at 65-68.degree. C. or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42.degree. C. (see Sambrook, supra). An example of a "moderately stringent" hybridization is the conditions of: 0.015M sodium chloride, 0.0015M sodium citrate at 50-65.degree. C. or 0.015M sodium chloride, 0.0015M sodium citrate, and 20% formamide at 37-50.degree. C. The moderately stringent conditions are used when a moderate amount of nucleic acid mismatch is desired. One of skill in the art will appreciate that washing is part of the hybridization conditions. For example, washing conditions can include 02.X-0.1 X SSC/0.1% SDS and temperatures from 42-68.degree. C., wherein increasing temperature increases the stringency of the wash conditions.

[0147] When hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides, the reaction is called "annealing" and those polynucleotides are described as "complementary." A double-stranded polynucleotide can be "complementary" or "homologous" to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second. "Complementarity" or "homology" (the degree that one polynucleotide is complementary with another) is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules.

[0148] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology, Ausubel et al., eds., (1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant.

[0149] The term "cell proliferative disorders" shall include dysregulation of normal physiological function characterized by abnormal cell growth and/or division or loss of function. Examples of "cell proliferative disorders" include but are not limited to hyperplasia, neoplasia, metaplasia, and various autoimmune disorders, e.g., those characterized by the dysregulation of T cell apoptosis.

[0150] As used herein, the terms "neoplastic cells," "neoplastic disease," "neoplasia," "tumor," "tumor cells," "cancer," and "cancer cells," (used interchangeably) refer to cells which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation (i.e., de-regulated cell division). Neoplastic cells can be malignant or benign. A metastatic cell or tissue means that the cell can invade and destroy neighboring body structures.

[0151] The term "cancer" refers to cancer diseases including, for example, breast, lung, pancreas, ovary, central nervous system (CNS), endometrium, stomach, large intestine, colon, esophagus, bone, urinary tract, hematopoietic, lymphoid, liver, skin, melanoma, kidney, soft tissue sarcoma and pleura.

[0152] The term "PBMC" refers to peripheral blood mononuclear cells and includes "PBL"--peripheral blood lymphocytes.

[0153] "Suppressing" tumor growth indicates a reduction in tumor cell growth when contacted with an MDM2i compared to tumor growth without contact with an MDM2i compound. Tumor cell growth can be assessed by any means known in the art, including, but not limited to, measuring tumor size, determining whether tumor cells are proliferating using a 3H-thymidine incorporation assay, measuring glucose uptake by FDG-PET (fluorodeoxyglucose positron emission tomography) imaging, or counting tumor cells. "Suppressing" tumor cell growth means any or all of the following states: slowing, delaying and stopping tumor growth, as well as tumor shrinkage.

[0154] A "composition" is a combination of active agent and another carrier, e.g., compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like. Carriers also include pharmaceutical excipients and additives, for example; proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Carbohydrate excipients include, for example; monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.

[0155] Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/antibody components, which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.

[0156] The term "carrier" further includes a buffer or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. Additional carriers include polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-quadrature-cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as TWEEN 20.TM. and TWEEN 80.TM.), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0157] As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives and any of the above noted carriers with the additional provisio that they be acceptable for use in vivo. For examples of carriers, stabilizers and adjuvants, see Remington's Pharmaceutical Science., 15th Ed. (Mack Publ. Co., Easton (1975) and in the Physician's Desk Reference, 52nd ed., Medical Economics, Montvale, N.J. (1998).

[0158] An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages.

[0159] A "subject," "individual" or "patient" is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, simians, humans, farm animals, sport animals, and pets.

[0160] An "inhibitor" of MDM2 as used herein reduces the association of p53 and MDM2. This inhibition may include, for example, reducing the association of p53 and MDM2 before they are bound together, or reducing the association of p53 and MDM2 after they are bound together, thus freeing both molecules.

[0161] A number of genes have now been identified as biomarkers for MDM2i. The decrease or increase of gene expression of one or more of the biomarkers identified herein and in Table 2 can be used to determine patient sensitivity to any MDM2i, for example, the increase or overexpression of a biomarker indicates that a cancer patient is sensitive to and would favorably respond to administration of an MDM2i. As another example, after treatment with a MDM2i, a patient sample can be obtained and the sample assayed for sensitivity to discover if the patient is still sensitive to the MDM2i treatment. Alternatively, all of the biomarkers in Table 2 can be assayed for as a single set.

[0162] MDM2 inhibitors (MDM2i) are compounds which are inhibitors of the p53-MDM2 association, and are useful in conjunction with the methods or uses of the invention. MDM2i are useful in pharmaceutical compositions for human or veterinary use where inhibition of the p53-MDM2 association is indicated, e.g., in the treatment of tumors and/or cancerous cell growth. In particular, such compounds are useful in the treatment of human cancer, since the progression of these cancers may be at least partially dependent upon overriding the "gatekeeper" function of p53, for example the overexpression of MDM2. MDM2i compounds are useful in treating, for example, carcinomas (e.g., breast, lung, pancreas, ovary, central nervous system (CNS), endometrium, stomach, large intestine, colon, esophagus, bone, urinary tract, hematopoietic, lymphoid, liver, skin, melanoma, kidney, soft tissue sarcoma and pleura A listing of exemplary MDM2i compounds is found in Table 1 (see WO 2011076786). Other MDM2i that bind to a p53 binding pocket of MDM2, particularly to substantially the same p53 binding pocket of MDM2 as Nutlin-3a or substantially where the exemplary MDM2i from the Table 1 binds, can also be applied in the methods or uses of the invention. MDM2i used according to present embodiments can be structurally related to the one described in Table 1 (i.e. MDM2i(1) and MDM2i(2)) or to Nutlin 3a, such as, for example, substituted isoquinolinones, or quinazolinones. The methods included herein can also be used with other compounds such as the spiro-oxindoles, imidazolyl indole and cis-imidazoline (see Shangary et al., Mol. Cancer Ther. 2008 7(6): 1533-1542: Furet et al., BioOrg. Med. Chem. Let. 2012 22:3498-3502 and Carol et al., Pediatr. Blood Cancer 2012 pages 1-9, published online Jul. 2, 2012, prior to inclusion into journal). MDM2i as used herein prevents the protein-protein interaction between p53 and MDM2 or inhibits cell proliferation by inducing the p53 pathway activity.

TABLE-US-00001 TABLE 1 MDM2i compounds ##STR00001## MDM2i(1) ##STR00002## MDM2i(2)

[0163] Measurement of Gene Expression

[0164] Detection of gene expression can be by any appropriate method, including for example, detecting the quantity of mRNA transcribed from the gene or the quantity of cDNA produced from the reverse transcription of the mRNA transcribed from the gene or the quantity of the polypeptide or protein encoded by the gene. These methods can be performed on a sample by sample basis or modified for high throughput analysis. For example, using Affymetrix.TM. U133 microarray chips.

[0165] In one aspect, gene expression is detected and quantitated by hybridization to a probe that specifically hybridizes to the appropriate probe for that biomarker. The probes also can be attached to a solid support for use in high throughput screening assays using methods known in the art. WO 97/10365 and U.S. Pat. Nos. 5,405,783, 5,412,087 and 5,445,934, for example, disclose the construction of high density oligonucleotide chips which can contain one or more of the sequences disclosed herein. Using the methods disclosed in U.S. Pat. Nos. 5,405,783, 5,412,087 and 5,445,934, the probes of this invention are synthesized on a derivatized glass surface. Photoprotected nucleoside phosphoramidites are coupled to the glass surface, selectively deprotected by photolysis through a photolithographic mask, and reacted with a second protected nucleoside phosphoramidite. The coupling/deprotection process is repeated until the desired probe is complete.

[0166] In one aspect, the expression level of a gene is determined through exposure of a nucleic acid sample to the probe-modified chip. Extracted nucleic acid is labeled, for example, with a fluorescent tag, preferably during an amplification step. Hybridization of the labeled sample is performed at an appropriate stringency level. The degree of probe-nucleic acid hybridization is quantitatively measured using a detection device. See U.S. Pat. Nos. 5,578,832 and 5,631,734.

[0167] Alternatively any one of gene copy number, transcription, or translation can be determined using known techniques. For example, an amplification method such as PCR may be useful. General procedures for PCR are taught in MacPherson et al., PCR: A Practical Approach, (IRL Press at Oxford University Press (1991)). However, PCR conditions used for each application reaction are empirically determined. A number of parameters influence the success of a reaction. Among them are annealing temperature and time, extension time, Mg 2+ and/or ATP concentration, pH, and the relative concentration of primers, templates, and deoxyribonucleotides. After amplification, the resulting DNA fragments can be detected by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet illumination.

[0168] In one embodiment, the hybridized nucleic acids are detected by detecting one or more labels attached to the sample nucleic acids. The labels can be incorporated by any of a number of means well known to those of skill in the art. However, in one aspect, the label is simultaneously incorporated during the amplification step in the preparation of the sample nucleic acid. Thus, for example, polymerase chain reaction (PCR) with labeled primers or labeled nucleotides will provide a labeled amplification product. In a separate embodiment, transcription amplification, as described above, using a labeled nucleotide (e.g. fluorescein-labeled UTP and/or CTP) incorporates a label in to the transcribed nucleic acids.

[0169] Alternatively, a label may be added directly to the original nucleic acid sample (e.g., mRNA, polyA, mRNA, cDNA, etc.) or to the amplification product after the amplification is completed. Means of attaching labels to nucleic acids are well known to those of skill in the art and include, for example nick translation or end-labeling (e.g. with a labeled RNA) by kinasing of the nucleic acid and subsequent attachment (ligation) of a nucleic acid linker joining the sample nucleic acid to a label (e.g., a fluorophore).

[0170] Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads.TM.), fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3H, 125I, 35S, 14C, or 32P) enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

[0171] Detection of labels is well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label.

[0172] The detectable label may be added to the target (sample) nucleic acid(s) prior to, or after the hybridization, such as described in WO 97/10365. These detectable labels are directly attached to or incorporated into the target (sample) nucleic acid prior to hybridization. In contrast, "indirect labels" are joined to the hybrid duplex after hybridization. Generally, the indirect label is attached to a binding moiety that has been attached to the target nucleic acid prior to the hybridization. For example, the target nucleic acid may be biotinylated before the hybridization. After hybridization, an avidin-conjugated fluorophore will bind the biotin bearing hybrid duplexes providing a label that is easily detected. For a detailed review of methods of labeling nucleic acids and detecting labeled hybridized nucleic acids see Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization with Nucleic Acid Probes, P. Tijssen, ed. Elsevier, N.Y. (1993).

[0173] Detection of Polypeptides

[0174] Expression level of the biomarker can also be determined by examining protein expression or the protein product at least one of the biomarkers listed in Table 2. Determining the protein level involves measuring the amount of any immunospecific binding that occurs between an antibody that selectively recognizes and binds to the polypeptide of the biomarker in a sample obtained from a patient and comparing this to the amount of immunospecific binding of at least one biomarker in a control sample. The amount of protein expression of the biomarker can be increased or reduced when compared with control expression. Alternatively, all of the biomarkers in Table 2 can be assayed for as a single set.

[0175] A variety of techniques are available in the art for protein analysis. They include but are not limited to radioimmunoassays, ELISA (enzyme linked immunosorbent assays), "sandwich" immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), western blot analysis, immunoprecipitation assays, immunofluorescent assays, flow cytometry, immunohistochemistry, confocal microscopy, enzymatic assays, surface plasmon resonance and PAGE-SDS.

[0176] Assaying for Biomarkers and MDM2i Treatment

[0177] Once a patient has been predicted to be sensitive to an MDM2i, administration of any MDM2i to a patient can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents may be empirically adjusted.

[0178] At least one of the biomarkers provided in Table 2 can be assayed for after MDM2i administration in order to determine if the patient remains sensitive to the MDM2i treatment. In addition, at least one biomarker can be assayed for in multiple time points after a single MDM2i administration. For example, an initial bolus of an MDM2i is administered, at least one biomarker from Table 2 is assayed for at 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 16 hours, 24 hours, 48 hours, 3 days, 1 week or 1 month or several months after the first treatment. Alternatively, all of the biomarkers in Table 2 can be assayed for as a single set.

[0179] The at least one biomarker in Table 2 can be assayed for after each MDM2i administration, so if there are multiple MDM2i administrations, then at least one biomarker can be assayed for after each administration to determine continued patient sensitivity. The patient could undergo multiple MDM2i administrations and the biomarkers then assayed at different time points. For example, a course of treatment can require administration of an initial dose of MDM2i, a second dose a specified time period later, and still a third dose hours after the second dose. At least one biomarker of Table 2 could be assayed for at 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 16 hours, 24 hours, 48 hours, 3 days, 1 week or 1 month or several months after administration of each dose of MDM2i. Alternatively, all of the biomarkers in Table 2 can be assayed for as a single set.

[0180] It is also within the scope of the invention that different biomarkers are assayed for at different time points. Without being bound to any one theory, due to mechanism of action of the MDM2i or of the biomarker, the response to the MDM2i is delayed and at least one biomarker from Table 2 is assayed for at any time after administration to determine if the patient remains sensitive to MDM2i administration. An assay for at least one biomarker in Table 2 after each administration of MDM2i will provide guidance as to the means, dosage and course of treatment. Alternatively, all of the biomarkers in Table 2 can be assayed for as a single set.

[0181] Finally, there is administration of different MDM2 is and followed by assaying for at least one biomarker in Table 2. In this embodiment, more than one MDM2i is chosen and administered to the patient. At least one biomarker from Table 2 can then be assayed for after administration of each different MDM2i. This assay can also be done at multiple time points after administration of the different MDM2i. For example, a first MDM2i could be administered to the patient and at least one biomarker assayed at 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 16 hours, 24 hours, 48 hours, 3 days, 1 week or 1 month or several months after administration. A second MDM2i could then be administered and at least one biomarker could be assayed for again at 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 16 hours, 24 hours, 48 hours, 3 days, 1 week or 1 month or several months after administration of the second MDM2i. In each case, all of the biomarkers in Table 2 can be assayed for as a single set.

[0182] Another aspect of the invention provides for a method of assessing for suitable dose levels of an MDM2i, comprising monitoring the differential expression of at least one of the genes identified in Table 2 after administration of the MDM2i. For example, after administration of a first bolus of MDM2i, at least one biomarker of Table 2 is analyzed and based on this result, an increase or decrease in MDM2i dosage is recommended. After administration of the adjusted dosage of MDM2i the analysis of at least one biomarker will determine whether the patient is still sensitive to the adjusted dose and that the adjusted dose is providing the expected benefit, e.g., suppressing tumor growth. Alternatively, all of the biomarkers in Table 2 can be assayed for as a single set for assessing sensitivity to the dose of the MDM2i.

[0183] Kits for assessing the activity of any MDM2i can be made. For example, a kit comprising nucleic acid primers for PCR or for microarray hybridization for the biomarkers listed in Table 2 can be used for assessing MDM2i sensitivity. Alternatively, a kit supplied with antibodies for at least one of the biomarkers listed in Table 2 would be useful in assaying for MDM2i sensitivity.

[0184] It is well known in the art that cancers can become resistant to chemotherapeutic treatment, especially when that treatment is prolonged. Assaying for differential expression of at least one of the biomarkers in Table 2 can be done after prolonged treatment with any chemotherapeutic to determine if the cancer is sensitive to the MDM2i. For example, kinase inhibitors such as Gleevec.RTM. will strongly inhibit a specific kinase, but may also weakly inhibit other kinases. There are also other MDM2i, for example, the Nutlin family of compounds. If the patient has been previously treated with another chemotherapeutic or another MDM2i, it is useful information for the patient to assay for at least one of the biomarkers in Table 2 to determine if the tumor is sensitive to an MDM2i. This assay can be especially beneficial to the patient if the cancer goes into remission and then re-grows or has metastasized to a different site.

[0185] Screening for MDM2 Inhibitors

[0186] It is possible to assay for at least one biomarker listed in Table 2 to screen for other MDM2i. This method comprises assaying a cell with at least one biomarker from Table 2, which predicts if the cell is sensitive to an MDM2i candidate inhibitor, the cell is then contacted with the candidate MDM2i and the 1050 of the treated cell is compared with a known MDM2i contacting a sensitive cell. For example, for cells predicted to be sensitive to any MDM2i as determined by the differential expression of at least one biomarker in Table 2, the candidate MDM2i will have an IC50.ltoreq.3 .mu.M. The measurement of at least one biomarker from Table 2 expression can be done by methods described previously, for example, PCR or microarray analysis. Alternatively, all of the biomarkers in Table 2 can be assayed for as a single set.

TABLE-US-00002 TABLE 2 SEQ ID NO. Gene Name Accession number (nucleotide/protein) MDM2 NM_002392/NP_002383 SEQ ID NO. 1/SEQ ID NO. 2 CDKN1A NM_000389/NP_000380 SEQ ID NO. 3/SEQ ID NO. 4 ZMAT3 NM_022470/NP_071915 SEQ ID NO. 5/SEQ ID NO. 6 DDB2 NM_000107/NP_000098 SEQ ID NO. 7/SEQ ID NO. 8 FDXR NM_004110/NP_004101 SEQ ID NO. 9/SEQ ID NO. 10 RPS27L NM_015920/NP_057004 SEQ ID NO. 11/SEQ ID NO. 12 BAX NM_004324/NP_004315 SEQ ID NO. 13/SEQ ID NO. 14 RRM2B NM_015713/NP_056528 SEQ ID NO. 15/SEQ ID NO. 16 SESN1 NM_014454/NP_055269 SEQ ID NO. 17/SEQ ID NO. 18 CCNG1 NM_004060/NP_004051 SEQ ID NO. 19/SEQ ID NO. 20 XPC NM_004628/NP_004619 SEQ ID NO. 21/SEQ ID NO. 22 TNFRSF10B NM_003842/NP_003833 SEQ ID NO. 23/SEQ ID NO. 24 AEN NM_022767/NP_073604 SEQ ID NO. 25/SEQ ID NO. 26

EXAMPLES

Example 1

Both MDM2i(1) and MDM2i(2) are Equally Potent p53-MDM2 Inhibitors in Biochemical and Cellular Assays

[0187] TR-FRET Assay for IC.sub.50 determination: standard assay conditions consisted of 60 .mu.L total volume in white 384-well plates (Greiner Bio-One: Frickenhausen, Germany), in PBS buffer containing 125 mM NaCl, 0.001% Novexin, 0.01% Gelatin, 0.2% Pluronic F-127, 1 mM DTT and 1.7% final DMSO). Both MDM2i(1) and MDM2i(2) were added at different concentrations to 0.1 nM biotinylated MDM2 (human MDM2 amino acids 2-188, internal preparations), 0.1 nM Europium-labeled streptavidin (Perkin Elmer: Waltham, Mass., USA) and 10 nM Cy5-p53 peptide (Cy5-p53 aa18-26, internal preparation). After incubation at room temperature for 15 minutes, samples were measured on a GeniosPro reader (Tecan: Mannedorf, Germany). FRET assay readout was calculated from the raw data of the two distinct fluorescence signals measured in time resolved mode (fluorescence 665 nm/fluorescence 620 nm.times.1000). IC.sub.50 values are calculated by curve fitting using XLfit.RTM. (Fit Model #205). This data is shown in FIG. 1A.

[0188] Determination of binding rate constants (K.sub.on, K.sub.off): the rapid mixing tool of GeniosPro reader (Tecan: Mannedorf, Germany) was used to study fast binding kinetics (single well mode). Microplates containing the inhibitor and 20 nM Cy5-labeled p53 peptide in 50 .mu.l assay buffer were placed in the reader. After 10 min equilibration at 25.degree. C., binding reactions were initiated by injecting 50 .mu.l of buffer containing 0.2 nM biotinylated MDM2 and 0.2 nM europium-streptavidin at 475 .mu.l/s. Fluorescence was measured at 665 nM and at various time intervals, the first one 0.6 s after injection. In the absence of inhibitor, Cy5 fluorescence was maximal already at 0.6 s and remained stable for at least 15 min. In the presence of MDM2i(1) and MDM2i(2) fluorescence decreased slowly and measurements were made until steady-state was achieved. Control fluorescence was taken as the difference between wells containing 1% DMSO and wells containing 10 .mu.M Nutlin-3 as a control. The inhibitory effect at each time point was calculated as percent of the corresponding control. Progress curves obtained in the presence of different concentrations of inhibitor were combined and fitted as a whole. Nonlinear regression was performed with XLfit.RTM. using a novel fit methodology that was designed to obtain precise K.sub.on and K.sub.off values, based on the following respective equations: Fit=[Imin+((Imax-(((Ki nH)*Imax)/((y nH)+(Ki nH))))*(1-exp(((-1)*(koff+((y*koff)/Ki)))*X)))] and Fit=[Imin+((Imax-(((Ki nH)*Imax)/((y nH)+(Ki nH))))*(1-exp(((-1)*(kon*(Ki+y)))*X)))], where Ki represents the constant of inhibition, Imin represents the minimum inhibition (in %), Imax represents the maximum inhibition (in %), nH represents the Hill coefficient, x represents the time and y represents the inhibitor concentration). This data is shown in FIG. 1A.

[0189] Cell proliferation inhibition and GRIP p53 translocation assay: Effects of MDM2i(1) and MDM2i(2) on cellular growth and loss of viability is measured in both p53 wild-type (SJSA-1 and HCT116 p53.sup.wt/wt cells) and p53 mutant cell lines (SAOS2 and HCT116 p53.sup.-/- cells) using a standard proliferation assay based on the DNA-interacting fluorescent dye YOPRO (Invitrogen: Lucern, Switzerland). Briefly, cells are plated in 96-well plates overnight at 37.degree. C. and are treated with increasing concentrations of MDM2i(1) or MDM2i(2) for 72 hours. Cell concentration in each well is then determined using the DNA-interacting fluorescent dye YOPRO according to the manufacturer's instructions and the fluorescent signal is measured using a Gemini-EM standard plate reader (Molecular Devices:Sunnyvale, Calif., USA). IC.sub.50 values are calculated by curve fitting using XLfit.RTM. (Fit Model #201) and this data is shown in FIG. 1A.

[0190] The mechanistic p53-MDM2 Redistribution assay (GRIP assay) is used to directly monitor in cells the ability of compounds to modulate the p53-MDM2 protein-protein interaction. In this fully engineered assay, the p53 protein is tagged with a fluorescent GFP-label and is bound to MDM2 protein which is anchored in the cytoplasm of the cells. The treatment of the cells with specific compounds causes the dissociation of the interaction between the two proteins and the translocation of the released p53-GFP protein from the cytoplasm to the nuclei. This effect is detected and quantified using a high content imaging platform using the ArrayScan-VTi (Cellomics), following the fluorescent signal over time (see FIG. 1B, GRIP p53 translocation assay).

[0191] Altogether, using both in vitro and cellular assays, the results presented in FIGS. 1A and 1B show that both MDM2i(1) and MDM2i(2) are comparable potent p53-MDM2 protein-protein interaction inhibitors in vitro, inhibiting the p53-MDM2 protein-protein interaction, hampering tumor cell proliferation in a p53-dependent manner, and inducing p53 accumulation and translocation to the nucleus. This data is shown in FIG. 1B; note that there are large discrepancies in the IC50 between the cell lines. This is also an indication that there are differences in the sensitivity between the two cell lines to an MDM2i, and thus a determination of sensitivity can be useful in determining which patients receive the therapeutic.

Example 2

The p53 Mutational Status is Associated with MDM2i Chemical Sensitivity in Cell Lines

[0192] The association of p53 mutation to MDM2i(2) chemical sensitivity in a panel of cancer-relevant cell lines was tested by Fisher's Exact test. The cell line panel is the one covered by the Cancer Cell Line Encyclopedia (CCLE) initiative (Barretina J., Caponigro G., Stransky N., Venkatesan K., et al. The Cancer Cell Line Encyclopedia enables predictive modeling of anticancer drug sensitivity. Nature 483:603-7, 2012). A detailed genomic, genetic and pharmacologic characterization was conducted on the CCLE cell lines.

[0193] p53 mutation status in CCLE cell lines is taken from a data source of gene-level genetic alterations, for example, point-mutations, insertions, deletions and complex genetic alterations, compiled from the Sanger center COSMIC data and internal sources including Exome Capture Sequencing. This comprises data from approximately 1,600 cancer-related genes over the CCLE cell lines. Analysis of the CCLE panel revealed 244 cell lines containing a mutant p53, and 112 cell lines that expressed wild type p53.

[0194] The MDM2i(2) chemical sensitivity was determined from the pharmacologic characterization of the CCLE cell lines. The cell lines were separated in two groups according to MDM2i(2) sensitivity. One group contains the cell lines sensitive to MDM2i(2) compound, while the other group encompasses those being chemically insensitive to the MDM2i(2) compound. Such stratification resulted in two groups of 47 sensitive and 309 insensitive cell lines, respectively. From such in vitro chemical sensitivity data, the prediction that a cell would be sensitive to an MDM2i treatment was estimated to be 13%.

The statistical testing of the p53 mutation to sensitivity groups association shows an association between p53 mutation (mt) and the chemical sensitivity to MDM2i(2) (FIG. 2). The mt panel in FIG. 2 displays the MDM2i(2) sensitivity profiles for p53 mutated CCLE cell lines, the wild type (wt) panel displays the MDM2i(2) sensitivity profiles for p53 wild-type CCLE cell lines. Amax is defined as the maximal effect level (the inhibition at the highest tested MDM2i(2) concentration, calibrated to MG132, a proteaseome inhibitor used as a reference, as described in the CCLE publication referenced above, and IC50 is defined as of the .mu.M concentration at which MDM2i(2) response reached an absolute inhibition of -50 with respect to the reference inhibitor. Cell line count broken down by MDM2i(2) chemical sensitivity and p53 mutation status, and associated statistics: Data is also displayed as a contingency table with associated statistics.

[0195] This data indicates it is more likely for a cell line to show sensitivity to MDM2i(2) if its p53 mutation status is wild type. Indeed, the majority of p53 mutated cell lines are found insensitive to the compound, whereas more than two-third of p53 wild type cell lines are sensitive.

[0196] From this data we can conclude a p53 wild type genotype is the first indication of MDM2i sensitivity, and therefore it is the first stratification biomarker to be considered for selecting cancer patients responsive to an MDM2i.

Example 3

Prediction of Cell Line Chemical Sensitivity to MDM2i from Genomic Data and Clinical Implication

[0197] The two cell line sensitivity groups, given by MDM2i(2) treatment, are compared with the aim of identifying the biomarkers differentiating the sensitive cell lines from the insensitive cell lines, prior to any MDM2i treatment. Such biomarkers are used to predict the sensitivity of any MDM2i treatment. The biomarkers analyzed are the following types: 1) gene-level expression values generated by the Affymetrix GeneChip.TM. technology with the HG-U133 plus 2 array, summarized according to the RMA normalization method; 2) gene-level chromosome copy number values, obtained with the Affymetrix SNP6.0 technology (Affymetrix Santa Clara, Calif., USA) and processed using the Affymetrix apt software, and expressed as log 2 transformed ratios to a collection of HapMap reference normal samples; 3) gene-level genetic alterations or mutations, as described above in Example 2; 4) pathway-level expression values, summarizing pathway expression levels by a standardized average approach over the genes contributing to the pathways, as referenced in the GeneGo Metacore.RTM. knowledge base; 5) cell line lineage (cell line tissue of origin); 6) gene-level Tumor suppressor status, summarizing the activation status of a selection of tumor suppressor genes, by integrating the genetic alteration, copy number and expression information. Such genomic data was generated in the context of the CCLE cell line genomic and genetic characterization, and covers a total of about 45,000 genomic features.

[0198] Wilcoxon signed-rank tests or Fisher's exact tests are used to compare the two cell line group genomic features, depending on the feature type. The features having continuous values (gene expression and copy number, pathway expression features) are subjected to Wilcoxon signed-ranked test, those having discrete values (genetic alteration, tumor suppressor status and lineage features), to Fisher's exact test, for differential profile evaluation between sensitive and insensitive cell line groups. The significant features, discriminating the sensitive cell line group from the insensitive one, are the ones passing a false discovery rate-controlled p-value cutoff. Irrespective of the p-value limit, a minimum or maximum number of features per feature type are also required. To minimize the impact of the high degree of correlation among the features on the feature selection step, the feature data is clustered before the statistical tests as a pre-processing step. This step is performed at the feature type-level using the Frey's and Dueck's Affinity Propagation method (Clustering by Passing Messages Between Data Points. Frey B. and Dueck D. Science 315:972-6, 2007), and retrieves a set of features representing the most variability.

[0199] The cell line sensitivity groups or classes are defined as follows for this two-class comparison aiming at biomarker identification: a sensitive group of 47 sensitive cell lines, and an insensitive group of 204 insensitive cell lines. The 204 cell lines making this insensitive group are the most insensitive ones from the 309 insensitive cell line set mentioned in Example 2. This feature selection step yielded a total of about 200 significant features, having a significant differential profile to differentiate the sensitive cell line group from the insensitive one, and thus having the required properties to be considered as markers to predict the chemical sensitivity of samples to an MDM2i. As described above in Example 2, a relevant biomarker is the p53 mutation status itself. The statistics of the feature selection step (p-value 1.17E-21) confirmed its role in predicting sensitivity to an MDM2i. Furthermore, the odds-ratio associated with p53 status (0.024) indicates p53 mutation is more represented in MDM2i insensitive cell lines. Still noteworthy and still according to the statistics of the feature selection step, most of the predictive biomarkers are found to be a subset of p53 transcriptional target genes. These are shown in Table 2/FIG. 3. Their fold-changes indicate the transcripts of these biomarkers are more expressed in the MDM2i sensitive cell line population, as shown in FIG. 3. This is likely indicative of a level of p53 functional activity pre-existing before any treatment in cell lines that are sensitive to an MDM2i.

[0200] That the biomarkers in Table 2/FIG. 3 are reflective of a functional p53 pathway in MDM2i sensitive cells is verified in FIG. 4. In FIG. 4, cells have been treated with increasing concentrations of MDM2i(1) for 4 hours, prior to cell lysis. Whole cell lysates were prepared using a cell lysis buffer containing 50 mM Tris-HCl pH 7.5, 120 mM NaCl, 1 mM EDTA, 6 mM EGTA pH 8.5, 1% NP-40, 20 mM NaF, 1 mM PMSF and 0.5 mM Na-Vanadat, proteins were separated on NuPAGE 4-12% Bis-Tris Gel (Invitrogen # NP0322BOX, Lucerne; Switzerland), transferred onto Nitrocellulose Protran.RTM. BA 85 membranes (Whatman #10 401 261: Piscataway, N.J., USA) at 1.5 mA/cm.sup.2 membrane for 2 h using a semi-dry blotting system, and immunoblotted with either an anti-phospho-p53 (Ser.sup.15) (1/1000; Cell Signaling Technology #9284: Beverly, Mass., USA) rabbit polyclonal antibody, or an anti-p53 (Ab-6) (Pantropic, clone DO-1) (1/1000; Calbiochem # OP43 San Diego, Calif., USA), an anti-MDM2 (Ab-1, clone IF2) (1/1000; Calbiochem # OP46), an anti-p21.sup.wAF1(Ab-1, clone EA10) (1/500; Calbiochem # OP64: San Diego, Calif., USA) or an anti-.alpha.-Tubulin (Sigma # T5168: St. Louis, Mo., USA) mouse monoclonal antibodies, as indicated. As shown in FIG. 4, increasing concentrations of MDM2i(1) induces stabilization of p53 protein levels, with no significant increase of phospho-p53 in both C3A and COLO 792 cells. Interestingly, treatment with MDM2i(1) also induces a strong de novo expression of both p53 target genes p21(CDKN1A) and MDM2 in C3A sensitive cells, but not in COLO-792 insensitive cell line. Altogether, this data indicates that sensitivity to an MDM2i inhibitor is directly related to the presence of an intrinsic functional p53 pathway, that the biomarkers in Table2/FIG. 3, taken together as a set or alone, point directly to p53 pathway functionality before treatment, and indicates these biomarkers have a strong ability to predict patient sensitivity that correlates with the mechanism of action of p53.

[0201] The significant genomics features are used as the basis features for naive Bayes probabilistic modeling of the two MDM2i chemical sensitivity groups, or classes. The goal of the modeling step is to derive a classification scheme or classifier that predicts the patient's response (either sensitive or insensitive) of an unknown sample with a certain confidence. The predictive model is defined by training a naive Bayes algorithm over the entire chemically characterized COLE cell line population stratified into the two above-mentioned sensitivity classes. The performance of the classifier is evaluated through 5 repeats of 5-fold cross-validations of the data used to train the model. The model performance is summarized with the following class-level measures: sensitivity, specificity, positive predictive value, and negative predictive value. The sensitive class is used as the reference for the sensitivity and positive predicted value calculations. The default output of the naive Bayes algorithm is a score or probability, for each predicted sample to be assigned to one class or the other. A probability threshold is defined to transform the probability scores into a sensitive or insensitive class-level prediction. The probability threshold is defined as the probability maximizing the sensitivity and specificity calculated over all predicted samples. The entire and nearly-identical procedure is described in more details in the COLE publication referenced in Example 2.

[0202] To demonstrate a better predictive power can be achieved from at least one biomarker found in Table 2, and alternatively, the biomarkers in Table 2 as a set, than from p53 mutation status alone, or from the entire predictive feature set of about 200 genes, naive Bayes models from each of these feature sets were trained, and their performance assessed by cross-validation, as above mentioned, and compared (FIG. 5). FIG. 5 demonstrates the selected biomarkers found in Table 2 outperform both the p53 mutation status and the larger group of about 200 significant features, and provide a substantial improvement in predicting patient responsiveness to an MDM2i. This is particularly striking when performances are evaluated by positive predictive value (PPV).

[0203] A positive predicted value (PPV) of 76% suggests that 76% of the predicted sensitive cell lines will be sensitive to MDM2i treatment. As an extrapolation to a clinical setting, a PPV of 76% also suggests that 76% of cancer patients, predicted as sensitive to MDM2i(2) from tumor biopsies, would show clinical response upon MDM2i(2) treatment. This enrichment of clinical response by patient stratification, using the biomarkers in Table 2 and associated with the naive Bayes predictive model, was compared to the baseline clinical response rate without prior patient stratification. The baseline clinical response rate estimated from the chemical sensitivity data is 19% Thus, in a clinical perspective; the biomarkers of Table 2 have a clear increase of the clinical response in the predicted sensitive patient population. This increase in prediction is greater and more specific than assaying for p53 status alone or all of the approximately 200 genomic features initially selected. This can be seen in FIG. 5, where the "All 200 biomarkers" feature bar is the PPV of the approximately 200 genomic features initially selected. The PPV reported for "All 200 biomarkers" feature is 59%. The PPV for using p53 alone is 56% (FIG. 5). The PPV of the set of biomarkers disclosed in Table 2 is 76% ("Table 2 selected biomarkers"). This is a surprising result, as in general, the larger data set of 200 genomic features would provide more data points and more insight into prediction of MDM2i sensitivity. However, this is not the case, as the biomarkers in Table 2, when taken as a set, provide a 17% increase in predictive value. This is important in the clinic, as now 17 additional patients out of 100 would be predicted to receive the correct treatment.

[0204] In order to test the predictive value of the biomarkers of Table 2, 52 cell lines that were not previously examined in the pharmacologic characterization as part of the COLE project, were assayed for their sensitivity to MDM2i in proliferation assays. Briefly, cells are plated in 96-well plates overnight at 37.degree. C. and are treated with increasing concentrations of an MDM2i for 72 hours. Cell concentration in each well is then determined using the CellTiter-Glo Luminescent Cell Viability Assay.RTM. (Promega Cat. # G7571/2/3: Madison Wis., USA), according to the manufacturer's instructions and the luminescent signal is measured using a SYNERGY HT plate reader (BioTek: Winooski, Vt., USA). IC.sub.50 values are calculated by curve fitting using XLfit.RTM. (FIG. 6). Sensitivity of cell lines to an MDM2i is determined by comparing the observed IC.sub.50 of all cells that were tested in cell proliferation inhibition assay as described in Example 1. The cut-off for sensitivity was determined at IC.sub.50.ltoreq.3 .mu.M for both MDM2i(1) and MDM2i(2). Predictions of sensitivity for every cell lines are performed using the predictive model as described above, to confirm the values disclosed in FIG. 5. The cell lines are from a variety of tumors. For example; melanoma (COLO-829, COLO-849, IGR-1, MEL-JUSO,SK-MEL-1, SK-MEL-31, UACC-62, UACC-257), leukemia (BV173, EOL-1, GDM-1,HuNS1, L-540, MV-4-11,OCI-LY3, RS4:11, SUP-B15, HDLM2, JM1), breast cancer (CAL51, EFM-192, HCC202), pancreatic cancer (DAN-G), hepatic cancer (JHH-5) and lung cancer (RERF-LCK-KJ). This assay was done with the all of the biomarkers disclosed in Table 2 as a single set. Overall, 36/52 cell lines were predicted to be sensitive to an MDM2i, and of these 36 cell lines 24 were sensitive, resulting in a positive predictive value of 66%, again a significant increase in predictive value (PPV) over assaying for p53 alone. With regard to screening out the non-responding cells, 16/52 cell lines were predicted to be insensitive to a p53-MDM2 inhibitor, and 13/16 were found to indeed be insensitive, leading to a significant negative predictive value (NPV) of 81%. Overall, these data are similar to the predictive model performances described in FIG. 5. The actual in vitro testing of unrelated cell lines allowed testing of the MDM2i chemical sensitivity predictive model, hence validating the biomarkers disclosed in Table 2.

Example 4

MDM2i Treatment Inhibits Tumor Growth Inhibition In Vivo which is Correlated to a Dose-Dependent Increase of p21(CDKN1A) mRNA and Protein Levels in Tumors

[0205] To further validate the predictive biomarkers disclosed in FIG. 3, in vivo human xenograft models either from human primary samples or from cell lines were directly injected and grown in tumors subcutaneously in mice and then assessed for MDM2i sensitivity. All the animals were allowed to adapt for 4 days and housed in a pathogen-controlled environment (5 mice/Type III cage) with access to food and water ad libitum. Animals were identified with transponders. Studies were performed according to procedures covered by permit number 1975 issued by the Kantonales Veterinaramt Basel-Stadt and strictly adhered to the Eidgenossisches Tierschutzgesetz and the Eidgenossische Tierschutzverordnung. Subcutaneous tumors were induced by concentrating 3.0.times.10.sup.6 SJSA-1 osteosarcoma cells in 100 .mu.l of PBS (without Ca.sup.2+ and Mg.sup.2+) and injecting in the right flank of Harlan nude mice. The administration of MDM2i began 12-14 days post cell injection. MDM2i was prepared immediately before each administration. MDM2is were dissolved in 0.5% HPMC (hydroxypropylmethylcellulose) and were injected daily (q24 h) at 25, 50 or 100 mg/kg. Tumor volumes (TVol), determined from caliper measurements (using the formula l.times.w.times.h.times..pi./6) were measured three times per week. Tumor response was quantified by the change in tumor volume (endpoint minus starting value in mm.sup.3) as the T/C, i.e.

( .DELTA. TVol drug .DELTA. TVol vehicle .times. 100 ) . ##EQU00001##

In the case of a tumor regression, the tumor response was quantified by the percentage of regression of the starting TVol, i.e.

( .DELTA. TVol drug .DELTA. TVol Day 0 .times. 100 ) . ##EQU00002##

The body-weight (BW) of the mice was measured three times per week allowing calculation at any particular time-point relative to the day of initiation of treatment (day 0) of both the percentage change in BW (.DELTA.% BW). As shown in FIG. 7A, a 10-day treatment of SJSA-1 xenografted tumors with MDM2i(1) led to a dose-dependent tumor growth inhibition with a significant T/C of 50% at 25 mg/kg q24 h and of 3% (stasis) at 50 mg/kg q24 h. At 100 mg/kg q24 h for 10 days, MDM2i(1) treatment induced a significant tumor regression of 65% (FIG. 7B). All doses were well tolerated at q24 h schedule, as indicated by the mean body weight curves over time.

[0206] Anti-tumor activity of MDM2i(1) was correlated with a significant dose-dependent induction of p21(CDKN1A) mRNA levels in tumors (FIG. 7C). Briefly, total RNA was purified from cell pellets using the QIAshredder.RTM. (79654, Qiagen:Valencia Calif., USA) and RNeasy Mini Kite (74106, Qiagen: Valencia Calif., USA) according to the manufacturer's instructions, with the exception that no DNA digestion was performed. Total RNA was eluted with 50 .mu.L of RNase-free water. Total RNA was quantitated using the spectrophotometer ND-1000 Nanodrop.RTM. (Wilmington Del., USA). The qRT-PCR (Quantitative Reverse Transcriptase Polymerase Chain Reaction) was set up in triplicate per sample using the One-Step RT qPCR Master Mix Plus (RT-QPRT-032X, Eurogentec: Seraing, Belgium), with either control primers and primers for human p21(CDKN1A) (Hs00355782_m1, Applied Biosystems: Carlsbad Calif., USA) or mouse p21(CDKN1A) (Mm00432448_m1, Applied Biosystems: Carlsbad Calif., USA), namely TaqMan Gene Expression kit assays (20.times. probe dye FAM.TM. (or VIC)-TAMRA (or MGB); Applied Biosystems: Carlsbad Calif., USA). More specifically, a master mix was prepared on ice for a final concentration of: 1.times. Master Mix buffer, 1.times. primer solution, and 1.times. Euroscript reverse transcriptase, combined with H.sub.2O, total volume: 8 .mu.L/well. A MicroAmp Optical 384-well Reaction Plate (4309849, Applied Biosystems) was fixed on the bench, and 2 .mu.L of mRNA (concentration: 10 or 20 ng/.mu.l) (or water for negative control) were pipetted in triplicate, followed by addition of 8 .mu.L/well of master mix. The plate was then covered with a MicroAmp Optical Adhesive film kit (4313663, Applied Biosystems: Carlsbad Calif., USA), centrifuged for 5 min at 1000 rpm at 4.degree. C. and placed in a 7900 HT Fast Real-Time PCR System (Applied Biosystems: Carlsbad Calif., USA). The program was run with one cycle of 48.degree. C. for 30 min, one cycle of 95.degree. C. for 10 min, and finally 40 cycles of alternating 95.degree. C. for 15 sec and 60.degree. C. for 1 min. The number of cycles (CT) was determined, 2.sup.-CT values were calculated, and the value normalized by dividing with the 2.sup.-CT value obtained from the Gapdh control. Fold increase over control (i.e. DMSO- or vehicle-treated animals) was calculated and plotted in the bar graph.

[0207] In addition, the anti-tumor activity of MDM2i(1) was correlated with a significant dose-dependent induction of p21(CDKN1A) protein levels in tumors, as judged by immunohistochemistry (FIG. 8). SJSA-1 xenograft tumors were collected and a 3-4 mm slice out of the middle of the tumor was removed, transferred into pre-labelled histo-cassettes and immersion-fixed in neutral buffered formalin (NBF) 10% (v/v) (pH 6.8-7.2) (J. T. Baker, Winter Garden, Fla., USA), pre-cooled at 4.degree. C. Tumors were then fixed at room temperature for 24 hours, followed by processing in the TPC 15Duo (Tissue Processing Center, Medite) for paraffinization. Subsequently, the tumor slices were embedded in paraffin and from each paraffin block several 3 .mu.m thick sections were cut on a rotary microtome (Mikrom International AG, Switzerland), spread in a 48.degree. C. water-bath, mounted on glass slides (SuperFrost Plus, Thermo Scientific:Waltham Mass., USA), and dried in an oven either at 37.degree. C. overnight or at 60.degree. C. for 30 min. Dry tissue section were processed for immunohistochemistry (IHC) staining. p21(CDKN1A) immunohistochemistry has been performed using the mouse monoclonal antibody clone SX118 from Dako (Cat. No. M7202 Dako: Carpenteria Calif., USA) at a dilution of 1:50. Immunohistochemistry has been performed on a Ventana Discovery XT automated immunostainer using the N-Histofine Mousestain Kit (Nichirei Bioscience Inc, Japan) in combination with the DABMap Kit chromogen system, omitting the SA-HRP solution (Ventana/Roche Diagnostics GmbH, Mannheim, Germany). Antigen retrieval was done by using Cell Conditioning ULTRA.RTM. (Ventana/Roche Diagnostics GmbH, Mannheim, Germany) at mild (95.degree. C. for 8 min+100.degree. C. for 20 min) conditions. Mouse cross reactivities were blocked by using Blocking Reagents A and B from the N-Histofine Mousestain Kite (Nichirei Bioscience Inc, Japan) before and after primary antibody incubation, following the manufacturer instructions. The primary antibody was applied manually at the desired dilution in Dako antibody diluent (AbD), followed by incubation for 1 hour at ambient temperature. Corresponding negative controls were incubated with AbD only. Sections were subsequently stained using the labeled polymer system Simple Stain Mouse MAX PO (M) from the N-Histofine Mousestain Kite (Nichirei) and DAB substrate from the DABMap Kit (Ventana/Roche Diagnostics). Counterstaining of sections was done using hematoxylin (Ventana/Roche Diagnostics). After the automated staining run, slides were dehydrated in a graded series of ethanol, cleared in xylene and mounted with Pertex.RTM. mounting medium.

Example 5

Prediction of MDM2i(2) Sensitivity in Human Primary Tumor Mouse Xenograft Models and in Human Primary Tumors

[0208] The biomarkers in Table 2, were used in association with a naive Bayes predictive model to predict MDM2i sensitivity in a collection of human primary tumor samples and xenograft models, to demonstrate whether the biomarkers, and their associated predictive power, exists outside of in vitro cell line systems.

[0209] The gene-level expression values of all the biomarkers of Table 2 were used as the feature basis for naive Bayes probabilistic modeling. They were generated as described in Example 3, the only difference being in the RMA summarization step where the normalization was targeted to a reference set of normal & tumor samples. The naive Bayes modeling is conducted as described in Example 3.

[0210] The human primary tumor samples and xenograft models submitted for sensitivity prediction were a collection of about 18,000 and 503 samples, respectively, for which gene expression profiles, generated with Affymetrix technology (Human Genome U133 plus 2.0 array), are available. The samples of the collection were internally annotated with controlled vocabulary for sample ontology including pathology, histology and primary site. The associated gene chip data was gathered from both public and internal sources, and normalized as described above to the same reference sample set for consistency.

[0211] Ratios of MDM2i predicted sensitive samples from the collection were compared to the proportions of sensitive cell lines, as given by the MDM2i(2) chemical sensitivity data described in Example 3 above. A good correlation is expected to demonstrate the ability of the biomarkers disclosed in FIG. 3 to be predictive for MDM2i sensitivity in human primary tumor samples. For clarity, as well as to potentially identify lineages in which sensitive cell line proportions are underestimated in the cell line chemical sensitivity data, sensitive prediction ratio to sensitive cell line ratio comparison is broken down by tissue of origin.

[0212] FIG. 9 (left panel) shows a correlation between predicted sensitive human primary tumor samples from the collection and the sensitive cell lines from MDM2i chemical sensitivity data. It indicates the biomarkers disclosed in Table 2 and its use for predicting sensitivity outside of in vitro cell line samples is valid. It also indicates that the biomarkers disclosed in Table 2 can be used to predict MDM2i chemical sensitivity in human primary tumor samples. It reveals new tumor indications which have not been investigated previously and confirms results found in the current study The new indications, for example, liver (hepatocellular carcinoma) and kidney (renal cell carcinoma), represent potentially new disease indications to be pre-clinically and clinically evaluated with the biomarkers disclosed in Table 2 for treatment with an MDM2i. FIG. 9A also indicates that the biomarkers disclosed in Table 2 can be used to predict MDM2i chemical sensitivity in primary melanoma tumors, consistent with the results found in the current study.

[0213] FIG. 9 (right panel) shows a correlation between the fractions of predicted sensitive human primary tumor samples and the predicted sensitive ratios in the primary tumor xenograft collection. The tumor samples/xenografts/cell lines are organized by lineage. The dashed line in both panels is the identity line. It shows the data generated from the in vivo mouse xenograft models, in which the exemplified signature and associated predictive classifier can be studied and validated, is in line with the data from the rest of the in vivo collection samples. It confirms the mouse xenograft models as a source of material to validate the p53 downstream target gene based classifier approach to predict clinical outcome of cancer patients and diseases indications, in an in vivo pre-clinical setting.

Example 6

Single Biomarkers and any Combinations of the Identified Thirteen Biomarkers Predict Chemical Sensitivity to MDM2i

[0214] The thirteen biomarkers depicted in Table 2, when used in association with a naive Bayes predictive modeling framework, predict MDM2i sensitivity in both in vitro systems and in vivo, as exemplified in Examples 3 and 5. To investigate whether subsets of these thirteen biomarkers would also predict for MDM2i sensitivity, single biomarkers and multiple combinations of them are employed as feature basis for predictive modeling. Their prediction performances are then compared to the ones achieved with either the full thirteen biomarkers or with p53 mutation status when used as a predictive feature for MDM2i sensitivity prediction.

[0215] Two instances of p53 mutation status are considered in Example 6. These two instances are defined from the Exome Capture Sequencing data of the CCLE cell lines, as mentioned in Example 2, and are meant to be surrogates of clinical settings where the p53 gene is sequenced for stratification or clinical annotation of patients.

[0216] The first instance of p53 mutation status is defined from the mutations spanning exons 5 to 8 of p53. Exons 5 to 8 encompass the DNA binding domain of p53, which contains the majority of described p53 mutations, and are the p53 exons commonly targeted for sequencing in clinical settings (for example, Rapid sequencing of the p53 gene with a new automated DNA sequencer. Bharaj B., Angelopoulou K., and Diamandis E., Clinical Chemistry 44:7 1397-1403, 1998). The second instance considers the complete open reading frame of the main p53 transcript, and is therefore defined from all coding exon mutations.

[0217] Multiple biomarker combinations can be generated from the list of 13 biomarkers disclosed in Table 2. All combination types from 2 to 12 biomarkers are evaluated as feature basis for predictive modeling of MDM2i chemical sensitivity. When more than 50 different combinations exist for a given combination type, the number of evaluated combinations is restricted to 50. All 50 combinations in each combination type were randomly picked.

[0218] All predictive models associated to the above described feature sets (single biomarkers, 2-to-12 biomarker combinations, p53 mutation status instances) were trained and evaluated mostly as described in Example 3. What differs from Example 3 is as follows: The training data was slightly larger than the one used is Example 3 and encompasses 264 cell lines (47 from the sensitive class, and 217 from the insensitive class); A p-value threshold of 0.5 was used upon the naive Bayes probabilistic modeling to call a cell line either sensitive or insensitive in the 5-fold cross-validation scheme. Moreover, all sample strata generated by the cross-validation processes were randomly selected and independent from one-another. The performances of the combinatorial predictive models and their comparisons to the p53 mutation status instance and 13 biomarker models are shown in FIGS. 10 to 12.

[0219] FIG. 10 depicts the positive predicted values (PPV) achieved by the single biomarker, the combinatorial, the thirteen biomarker and the p53 mutation models. The PPV is an estimate of the clinical efficacy one would expect in a clinical trial upon patient selection with the considered modeling process. For convenience, the data is depicted as box-and-whisker plots when there are more than five data points to plot per feature set.

[0220] FIG. 10 shows that combinations from as few as two and three biomarkers outperform exon 5-to-8 p53 mutation ("ex5to8mt") and all-exon p53 mutation features ("allExMt"), respectively. Indeed, the upper and lower boundaries defined by the ends of the whiskers encompass about 99% of the data points, assuming a normal distribution of the data. Therefore, the majority of the evaluated 2- and 3-biomarker combinations show a higher PPV than the ones achieved by the p53 mutation status instances. Moreover, even if all single gene models do not outperform the two p53 mutations instances, a majority of them (around 75%, the box plus the upper whisker) outperforms the p53 all-exon mutations. Noteworthy, all single gene models give rise to PPVs that are higher than the sensitive cell line ratio (-18%) in the considered sample population, indicating that MDM2i sensitivity prediction models, built from as few as one biomarker, are capable of enriching the selected samples in sensitive ones.

[0221] Additionally, under this modeling exercise, as plotted in FIG. 10, the PPV given by the p53 exons 5-to-8 mutation status model, averaged over the 5 cross-validation repeats, is 48%. It is significantly lower than the p53 mutation PPV disclosed in Example 3 (56%). This indicates that, in a clinical setting where p53 exon 5-to-8 sequencing is employed for patient selection, which is common practice, the exemplified 13 biomarker-based patient selection has even higher added value than anticipated from Example 3.

[0222] FIG. 11 shows the specificities achieved by the several evaluated models. As for PPV in FIG. 10, every combination made from as few as 2 biomarkers is sufficient to achieve specificity higher than the ones obtained from the mutations instances only. All single biomarker models outperform the mutations, when specificity is used to monitor the model performances.

[0223] FIG. 12 shows the sensitivities. Sensitivity is also called recall, and is an estimate of the truly sensitive patient population retained upon patient selection. Combinations of 9 biomarkers as the feature basis for MDM2i sensitivity prediction models are sufficient to obtain sensitivities comparable to the one achieved the full 13 biomarker list. However, only a few 9-biomarker combinations would achieve sensitivities higher than the ones given by the 2 p53 mutation status predictive models. But noteworthy, all evaluated combinations, from as few as 2 biomarkers, and a majority of single biomarker models, display sensitivities higher than the one which is expected by chance upon random classification (.about.18%).

[0224] In conclusion from FIGS. 10, 11 and 12, single biomarkers, when used as feature basis in models predicting chemical sensitivity to MDM2i, are sufficient to achieve sample sensitivity predictions that would result in a significant enrichment of potentially MDM2i responding patient in a clinical setting. Furthermore, combinations made from any 2 biomarkers, or more, increase the expected clinical efficacy with respect to the one obtained with p53 mutation-based patient selection. Assembling 8 biomarkers, from any of the Table 2 thirteen ones, are sufficient to achieve a patient recall equivalent to the one given by the 13 biomarker model. The predictive model performance metrics, obtained for the 13 gene signature and associated combinations, can be further optimized by optimizing the class assignment p-value threshold used in the naive Bayes probabilistic step of the model, as it was done in Example 3.

[0225] In a further embodiment, it is investigated whether the biomarkers depicted in Table 2 could predict MDM2i chemical sensitivity in collaboration with p53 mutation status. Single biomarkers and combinations of 2 biomarkers and above are combined with p53 mutation status in feature lists. The feature lists are then utilized as basis for sensitivity predictive modeling, as previously and described above. The performances of the multiple resulting models are evaluated as described above.

[0226] The p53 mutation status instance which is used as an example is the p53 exon 5-to-8 mutations. FIGS. 13, 14 and 15 depict the PPVs, specificities and sensitivities of those models combining p53 mutation with biomarkers, respectively, and are compared to the results given by mutation only models and the full 13-biomarker model.

[0227] FIG. 13 shows that at least a single biomarker from the list of 13, in collaboration with p53 mutation status, is sufficient to achieve a PPV higher that the basal sensitivity rate (18%) in the data. It also shows that a single biomarker at minimum, still in combination with p53 mutation status, achieves a higher PPV than the ones obtained with the two above mentioned p53 mutation status instances, when employed as features in a predictive model. And finally, any 5 biomarkers in combination with p53 mutation recapitulate the PPV which is achieved by the 13 biomarker model.

[0228] The same conclusions are drawn from FIGS. 14 and 15 when specificities and sensitivities are taken into account as model performance metrics. Noteworthy from FIG. 15, a high sensitivity can be obtained from as few as one biomarker, when modeled along with p53 mutation.

[0229] In conclusion, combining a single or multiple biomarkers from Table 2 with p53 mutation status enables the prediction of MDM2i sensitivity, and would result, when applied for patient selection in a therapeutic or clinical setting, in a significant enrichment with a limited loss of potential MDM2i responding patients.

Sequence CWU 1

1

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tttaatgggt acagagttaa atttgaagga ataagttcta gctgaagtat 5160tatgaactcc aaataatgct ttgaggacct ccaaaggtaa aagtactaat ccctttggcc 5220atttattgag agagagagag agagagagta gggtgactat agttaatgta ttgaatgttc 5280ttgctacaaa taaatgatat ttgagctgat gggtgtgcta attacactga tttgatcaat 5340acccattgta tgtgaaacag tacatacacc atatttacaa ttatgtattt aacatttaaa 5400atttctaata taagtatctc tcaaactgtg gattaacttc ttgatttata tttaaatatg 5460aatcttaagc aaaacagtga aaataaccat cttgatttag tgtttttctc ccatatgtga 5520attgtatata cttaggtgaa gacaataaaa tcaactgaac tgtaagctta gaataggact 5580gaggtaattc tgcacagcaa ctttactaat ggtacattgt tgcttcaaaa ctctctctct 5640ctctctctgt ctgtctcaat aaatggccaa agggattagt agtttacctg tggaggtcct 5700ccaagcatta tttggagttg ataatacttc agctacaacc aagcagaatc tctttttttt 5760ggaggtcctc gaagcattat ttggagttga taatacttca gcttcaattt ggagttgata 5820atatttcagc tagaacctag tagaatctgt ttttttcctt tggaggtcct caaagcatta 5880ttggagttca taatactgaa gctagaacca agcagaatct gtttttttct gaggagtatc 5940ggtagcataa atgtgattat aaacatagta cacttgatat atggaggcag tgacagctat 6000ttttacaaaa tttaaatctg caaatggatt caacatgttt atgggttatt aaaattgtct 6060gatttcttag gttctttata gtacacgtgt tgaaaataaa tgattaagaa ttgtttcaag 6120aatgcaatta tttgatctta aatttttatg agttgttaaa atagaaatta tttgaatatc 6180atatatttgg gtaacaaaag gcacaagtct gaatgtgttt ctttttctgg aatggccatg 6240cctgcccact ttagaaatac aaatatcact gggcagcttg aagcagttgg gagcctccaa 6300tgagagcaac ttgagagaat gatgttgcaa gttagtagga gtaagaaatg ctgtgttctc 6360cctgtcttct cttaggtcac atggcagcct ggcctaagtg atcgtgaatg gtctataagg 6420gaggtagctg ggacagggag gggagtttgg gctagccacc gtaccacttg tcagcgtgaa 6480aagtaagatt gtaattgcct gtttagtttt ctgcctcatc tttgaaagtt ccaccaagct 6540gggaacctct tgattgtgag gcacaaatgt aagtacatca gaaaaaaaca aaaaaactgg 6600ctttaaagca ggagcttgtg ggcccctaag ccagacgggg actagctttt ggcattatat 6660aattaagatt ttttaaatcc ttaataaggg ttttatttta tttttattta ttttttgaga 6720cggagtcttg ctctgtggct caggctggag tacagtggtg caatcttggc tcactgcaac 6780ctctgcctcc tggctgtgtt caagtggttc tgcttcagcc tcccaagtag ctggggttag 6840agcaccctgt caccacgccc cgctaatttt tgtatttcta gcagagatga agtttcacta 6900tgttggccag gctgggctca aactcctgac ctcaagtgat ctgcccgcct tggcccccca 6960aagtgctgtg attacaggcg tgagccgcca cgcccagcct aataagggtt ttaaagataa 7020ttagtgtgta ggtctgtagg cttatgatgg taaccacaag ttgttaatgg cattgtgaaa 7080agtttttagt tgcgctttat gggtggatgc tgaattacat tttgatttga tacttataaa 7140aagaaaaagt atttcttcag cttaaaaaat tgtttaaaag tttgtgatca tattgtctac 7200catgtagcca gctttcaatt atatgtaaga gggacttttt gacatttaca aataatactt 7260tgaggtagat atctgaaagc accagcactt ggaaggtgtt cagaagtaac aaattataaa 7320atgagctaac aaacgaaagg caaaataaaa ccgtaaagca agcagatggg aggcgtgttc 7380agtaacttat tcataatgca tctgaaatga ttgctgtact caaatattta acgttagagt 7440aatagtattt tgaatgaaaa ccatagttga tt 74722497PRTHomo sapiens 2Met Val Arg Ser Arg Gln Met Cys Asn Thr Asn Met Ser Val Pro Thr 1 5 10 15 Asp Gly Ala Val Thr Thr Ser Gln Ile Pro Ala Ser Glu Gln Glu Thr 20 25 30 Leu Val Arg Pro Lys Pro Leu Leu Leu Lys Leu Leu Lys Ser Val Gly 35 40 45 Ala Gln Lys Asp Thr Tyr Thr Met Lys Glu Val Leu Phe Tyr Leu Gly 50 55 60 Gln Tyr Ile Met Thr Lys Arg Leu Tyr Asp Glu Lys Gln Gln His Ile 65 70 75 80 Val Tyr Cys Ser Asn Asp Leu Leu Gly Asp Leu Phe Gly Val Pro Ser 85 90 95 Phe Ser Val Lys Glu His Arg Lys Ile Tyr Thr Met Ile Tyr Arg Asn 100 105 110 Leu Val Val Val Asn Gln Gln Glu Ser Ser Asp Ser Gly Thr Ser Val 115 120 125 Ser Glu Asn Arg Cys His Leu Glu Gly Gly Ser Asp Gln Lys Asp Leu 130 135 140 Val Gln Glu Leu Gln Glu Glu Lys Pro Ser Ser Ser His Leu Val Ser 145 150 155 160 Arg Pro Ser Thr Ser Ser Arg Arg Arg Ala Ile Ser Glu Thr Glu Glu 165 170 175 Asn Ser Asp Glu Leu Ser Gly Glu Arg Gln Arg Lys Arg His Lys Ser 180 185 190 Asp Ser Ile Ser Leu Ser Phe Asp Glu Ser Leu Ala Leu Cys Val Ile 195 200 205 Arg Glu Ile Cys Cys Glu Arg Ser Ser Ser Ser Glu Ser Thr Gly Thr 210 215 220 Pro Ser Asn Pro Asp Leu Asp Ala Gly Val Ser Glu His Ser Gly Asp 225 230 235 240 Trp Leu Asp Gln Asp Ser Val Ser Asp Gln Phe Ser Val Glu Phe Glu 245 250 255 Val Glu Ser Leu Asp Ser Glu Asp Tyr Ser Leu Ser Glu Glu Gly Gln 260 265 270 Glu Leu Ser Asp Glu Asp Asp Glu Val Tyr Gln Val Thr Val Tyr Gln 275 280 285 Ala Gly Glu Ser Asp Thr Asp Ser Phe Glu Glu Asp Pro Glu Ile Ser 290 295 300 Leu Ala Asp Tyr Trp Lys Cys Thr Ser Cys Asn Glu Met Asn Pro Pro 305 310 315 320 Leu Pro Ser His Cys Asn Arg Cys Trp Ala Leu Arg Glu Asn Trp Leu 325 330 335 Pro Glu Asp Lys Gly Lys Asp Lys Gly Glu Ile Ser Glu Lys Ala Lys 340 345 350 Leu Glu Asn Ser Thr Gln Ala Glu Glu Gly Phe Asp Val Pro Asp Cys 355 360 365 Lys Lys Thr Ile Val Asn Asp Ser Arg Glu Ser Cys Val Glu Glu Asn 370 375 380 Asp Asp Lys Ile Thr Gln Ala Ser Gln Ser Gln Glu Ser Glu Asp Tyr 385 390 395 400 Ser Gln Pro Ser Thr Ser Ser Ser Ile Ile Tyr Ser Ser Gln Glu Asp 405 410 415 Val Lys Glu Phe Glu Arg Glu Glu Thr Gln Asp Lys Glu Glu Ser Val 420 425 430 Glu Ser Ser Leu Pro Leu Asn Ala Ile Glu Pro Cys Val Ile Cys Gln 435 440 445 Gly Arg Pro Lys Asn Gly Cys Ile Val His Gly Lys Thr Gly His Leu 450 455 460 Met Ala Cys Phe Thr Cys Ala Lys Lys Leu Lys Lys Arg Asn Lys Pro 465 470 475 480 Cys Pro Val Cys Arg Gln Pro Ile Gln Met Ile Val Leu Thr Tyr Phe 485 490 495 Pro 32175DNAHomo sapiens 3gttgtatatc agggccgcgc tgagctgcgc cagctgaggt gtgagcagct gccgaagtca 60gttccttgtg gagccggagc tgggcgcgga ttcgccgagg caccgaggca ctcagaggag 120gcgccatgtc agaaccggct ggggatgtcc gtcagaaccc atgcggcagc aaggcctgcc 180gccgcctctt cggcccagtg gacagcgagc agctgagccg cgactgtgat gcgctaatgg 240cgggctgcat ccaggaggcc cgtgagcgat ggaacttcga ctttgtcacc gagacaccac 300tggagggtga cttcgcctgg gagcgtgtgc ggggccttgg cctgcccaag ctctaccttc 360ccacggggcc ccggcgaggc cgggatgagt tgggaggagg caggcggcct ggcacctcac 420ctgctctgct gcaggggaca gcagaggaag accatgtgga cctgtcactg tcttgtaccc 480ttgtgcctcg ctcaggggag caggctgaag ggtccccagg tggacctgga gactctcagg 540gtcgaaaacg gcggcagacc agcatgacag atttctacca ctccaaacgc cggctgatct 600tctccaagag gaagccctaa tccgcccaca ggaagcctgc agtcctggaa gcgcgagggc 660ctcaaaggcc cgctctacat cttctgcctt agtctcagtt tgtgtgtctt aattattatt 720tgtgttttaa tttaaacacc tcctcatgta cataccctgg ccgccccctg ccccccagcc 780tctggcatta gaattattta aacaaaaact aggcggttga atgagaggtt cctaagagtg 840ctgggcattt ttattttatg aaatactatt taaagcctcc tcatcccgtg ttctcctttt 900cctctctccc ggaggttggg tgggccggct tcatgccagc tacttcctcc tccccacttg 960tccgctgggt ggtaccctct ggaggggtgt ggctccttcc catcgctgtc acaggcggtt 1020atgaaattca ccccctttcc tggacactca gacctgaatt ctttttcatt tgagaagtaa 1080acagatggca ctttgaaggg gcctcaccga gtgggggcat catcaaaaac tttggagtcc 1140cctcacctcc tctaaggttg ggcagggtga ccctgaagtg agcacagcct agggctgagc 1200tggggacctg gtaccctcct ggctcttgat acccccctct gtcttgtgaa ggcaggggga 1260aggtggggtc ctggagcaga ccaccccgcc tgccctcatg gcccctctga cctgcactgg 1320ggagcccgtc tcagtgttga gccttttccc tctttggctc ccctgtacct tttgaggagc 1380cccagctacc cttcttctcc agctgggctc tgcaattccc ctctgctgct gtccctcccc 1440cttgtccttt cccttcagta ccctctcagc tccaggtggc tctgaggtgc ctgtcccacc 1500cccaccccca gctcaatgga ctggaagggg aagggacaca caagaagaag ggcaccctag 1560ttctacctca ggcagctcaa gcagcgaccg ccccctcctc tagctgtggg ggtgagggtc 1620ccatgtggtg gcacaggccc ccttgagtgg ggttatctct gtgttagggg tatatgatgg 1680gggagtagat ctttctagga gggagacact ggcccctcaa atcgtccagc gaccttcctc 1740atccacccca tccctcccca gttcattgca ctttgattag cagcggaaca aggagtcaga 1800cattttaaga tggtggcagt agaggctatg gacagggcat gccacgtggg ctcatatggg 1860gctgggagta gttgtctttc ctggcactaa cgttgagccc ctggaggcac tgaagtgctt 1920agtgtacttg gagtattggg gtctgacccc aaacaccttc cagctcctgt aacatactgg 1980cctggactgt tttctctcgg ctccccatgt gtcctggttc ccgtttctcc acctagactg 2040taaacctctc gagggcaggg accacaccct gtactgttct gtgtctttca cagctcctcc 2100cacaatgctg aatatacagc aggtgctcaa taaatgattc ttagtgactt tacttgtaaa 2160aaaaaaaaaa aaaaa 21754164PRTHomo sapiens 4Met Ser Glu Pro Ala Gly Asp Val Arg Gln Asn Pro Cys Gly Ser Lys 1 5 10 15 Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Ser Glu Gln Leu Ser Arg 20 25 30 Asp Cys Asp Ala Leu Met Ala Gly Cys Ile Gln Glu Ala Arg Glu Arg 35 40 45 Trp Asn Phe Asp Phe Val Thr Glu Thr Pro Leu Glu Gly Asp Phe Ala 50 55 60 Trp Glu Arg Val Arg Gly Leu Gly Leu Pro Lys Leu Tyr Leu Pro Thr 65 70 75 80 Gly Pro Arg Arg Gly Arg Asp Glu Leu Gly Gly Gly Arg Arg Pro Gly 85 90 95 Thr Ser Pro Ala Leu Leu Gln Gly Thr Ala Glu Glu Asp His Val Asp 100 105 110 Leu Ser Leu Ser Cys Thr Leu Val Pro Arg Ser Gly Glu Gln Ala Glu 115 120 125 Gly Ser Pro Gly Gly Pro Gly Asp Ser Gln Gly Arg Lys Arg Arg Gln 130 135 140 Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 145 150 155 160 Lys Arg Lys Pro 58995DNAHomo sapiens 5gcagtggcga cgccgagccg gcgccctcag tctcctcctc caccgcctcc cggttccgca 60gtcacttcct gcagctgttt ccctgtgggt cgggttggac tgacttttga cagtcagcct 120tcggctgcgg agggggctcg gcggcggccg gcggagaaag ttgctccgag aagaggctgg 180gtcgagctgg gccgagccgg gcgcgcaggg cgggcgtcgc gggcgtcccg ggcggacgcg 240gcgcggagac tgccggcgcg tcccgggggt tccgatttga agaccttgct tctcatcacc 300cactggatta tgccccaggc ttccctaccc aatgatcctc ttgcaacacg ccgtgcttcc 360tccacctaag cagccctcac cctcgcctcc tatgtcagtg gccaccaggt ctacaggaac 420cttgcagctt ccaccacaga agccttttgg gcaggaggct tccttgcctc ttgcagggga 480agaagagtta tcgaagggag gggagcaaga ctgtgccctg gaggagctat gtaagcccct 540gtactgcaaa ctctgcaatg tcaccttgaa ctctgcacag caagcccagg ctcattatca 600gggtaaaaat catggtaaga aactccgaaa ttactatgca gcaaatagct gtcctcctcc 660tgctagaatg agcaatgtgg tcgagcctgc agctactcca gttgttccag tccctccgca 720gatgggctcc tttaagccag gaggccgagt gatcctggcc acggagaatg attactgtaa 780gctctgtgat gcctccttca gttccccagc tgtggctcag gctcactatc aagggaagaa 840tcatgccaag aggctgcggc tggcggaagc tcagagtaac tcattctcgg aatcctcaga 900gctgggtcaa cggcgggcca ggaaagaagg gaatgagttt

aagatgatgc ctaacaggag 960aaatatgtat acagtacaga ataattcagc aggtccttac ttcaatcccc gctctcggca 1020gagaattcca cgtgatctgg ccatgtgtgt tactccaagt ggccagtttt actgctcaat 1080gtgtaatgtt ggagctggcg aagagatgga attccggcag catttagaga gcaagcaaca 1140taagagcaag gtgtctgaac agcggtacag gaatgagatg gagaatctgg gatatgtata 1200gtgattatca tattaagata gagcagcttt tcctgcctgt tgtttgcctt ttgtcaactt 1260gccctgcttt gtggtctttt tgatatgagt acattcctct gcttaatgtt aatacatgta 1320acccacagtg gtaccatgag atgtcaaaac ctgggggccc gggggcgggg cggggggagg 1380tgggtgtgaa gaacgtgctt cttaggtcat aacgcttttg cagggtcaat ggtgttgagc 1440cgctcatagc atgtgaccta cctaccccat cagaaataac tttttatctt gctcaagttc 1500tggtcaacta gtagcctgac ggcttagaac tttgactatt taaaagtttc attttctttt 1560gcaattttag ttttatgtac tgttaaagaa ttgtactgaa ttctttttag atcacagtaa 1620aaataggttg gcagagattt cagtttccca gggcttaacc agaaccgcca cctcaatgca 1680ttgtcagtag aatacattat tagaaactgt taaggtcttt cccgggacat ttttttctgc 1740cattttcttt tgcaattgta gttttatgta ctgttaaaga attgtattga attcttttta 1800gatcaaagta aaaataggtc agcagagatt tcagtttccc agggcttaac cagaaccgcc 1860acctcaatgc attgtcagta ggatacatta ttagaaactg ttagggtctt tcccaggaca 1920tttttttttc tgtatcatgt ctccccatca ttgaagcgca aattttcttg aattcaaata 1980ctcccaatga gcttgtatac ttccaaacag ctaaacttga tttccagttg tggatttcac 2040acatataatt gccgccttct tccctcctct tttttccccc ctagttgaat cagcttgtct 2100aacaagccca ttttcatgcc ccagctgtgc tgtgggtttt ccaagcctca tatttgaata 2160ttcaatgagt ttaagatgga tatgatttca aaaaataggg ccgggcgcgg tggctcacgc 2220ctgtaatccc agcactttgg gaggccgaag caggcggatc acgaggtcag gaggtcgaga 2280tcatcctggc taacacagtg aaaccccatc tctactaaaa atacaaaaca ttagccgggc 2340gtggtggtgg gtgcctgtag tcccagctac tctggaggct gaggcaggag aatggcgtga 2400acccgggagg cggagcttgc agtgagccga gatcgcgcca ctgcactcca gcctgggtga 2460cagagcgaga ctccgtctca aaaaaaaaaa aaaaaaaaaa aaaaaaatat atatatatat 2520agatatattt tccagcagtt tttaggaaac aaaggtgtgt ttgattttcc aatttagagt 2580tacttcattg atagtaacat gcgcttatat catgatctaa accataagtt cagattgctt 2640gatattttgt tttgccagac ttttttgata atctgattct tacggtttac ttacattttc 2700ctctgtcttg ccagtctggt ggccatcctg aaaaatatca ccacctgctg ttttatacac 2760agaggaattt tttaaaaagc aaataaagtt tcactatctt cgtttccagt agaaatatac 2820acgaactatg gttttattct ttgtaattcg gctttcttga agatactaac aagtatatga 2880tagatcactt tggcatctga taattatatc agtattatta tttttgtttt tttgcgttta 2940aaataagaag ttgtatgaac ccaggaggtg gaggttgcag tcagccaaga atcgtgccac 3000tgcactccag tctgggcgac agagcaatac tccgtctcca aaaaaaaaaa aaaagaagct 3060ataatatata tctagattaa ctaatggagt gctctgtgaa tattcattaa ctacatttta 3120tctatttact atattttata tgggtaagac cagtggtcat ccgtatttct tttgaaagcc 3180cacttgatca ggaagtttat attttaagca gtatgtttgc acaatggtca gttcacattt 3240gattgcttgt attactaatt agaggtaaaa cttctatttt ttcttgataa ttctgtaggc 3300aaaaaaaatt ggggaaggca aattacaatc attcttcttc tgtaatactt tgggacaatt 3360tttaaacaac tcagacccat gtttaaaaac gagttttgtg tgattaaccc ttgtatagta 3420tccagttact gtcttctctt gaagtggtgg ttctttgaga agtagaatta ttttgtaact 3480tttcttctcc cagaaacaga agaatcaaat actattctgt taggattcta tctagacctg 3540tgactcttag tataataacc cttttattat tattattatt attatttttt cagtaatcta 3600taaagtagag ttgcttagtt ttgagcaatt tagggctttt cttattggcc agtaggttat 3660tcattttgct gctaaatata attttttggt taattttaac attatagtgc tggccgcttg 3720gttctatgat tacctaaaaa tctagttttt ttcctccata ggtagatgtg tgtgtatgca 3780tacccccaca cacacacgta catatatgtt gtcatagaga ggtatttcaa cccttatttc 3840aaataacagt agaagatacg aacatgaatt tatattttgt aaaaatgtca ttcatccact 3900ttgttttcca ttggaaatag ttttataaga agggttcccc ttgctctctc cacttaacaa 3960tttcattata tacgtagaaa aagcagccga cttaagggct tgatgttttt tcaggccttg 4020tggattcagg tttccagttt cccagtgccc ttaatggatg ttatgaatgc ataagcacat 4080tttcttttaa agaaagaagt tagatttata gtgttatttc ttacttgcta tatttctttg 4140cactaaaaaa gagctatgtg tttgttttat aggacacttt agtaccgtat tgcctacaat 4200aactcagttt gctccttaaa ttccaaactc tgggaagttg ataaataact tccatgatca 4260cttgtaaaag ttacatgcac atctgaaaaa aaatcacaaa tctctatgac agtaggttat 4320gttttgagct tgttaccttg cagtattctt ctctgttcca taggtgaaaa caaagggtga 4380cagaggttat caggcaggaa atgcctaaat agatacatct ctgcatggta caatttctca 4440tattcatgta ttccataaac agtgtttttt ctctgtttaa gcagactgtt gtttcttctg 4500agtcagtgat atgtgatctt cgttggtttc attttgtgaa gctcagtctg tctctgtaac 4560atactttagg atttgcacct tgtgctcccc aggaattatg ttagtgtcct tttctatgct 4620gtcttcaagg atggacagag gcctaaacca caacaacaac aaaaattaga aaaaaaaata 4680cttagatttc ggtaatcatc cataggaact catagcatca gtctcttttc tctgtgaata 4740atatctttgt gtaagttgtc ataaaaaatc aacgttagaa gatgacaatt agaattcttc 4800ttaggtattg agggtaagaa gttgtaaaga aagaaaattc gtgtttcaac taaaagattg 4860gattgcatat acctttgaag tggttttgta aaaaaagttc agttactaaa ctgagtgtgc 4920cctgtaatcc ttttgagtgc actgaagatg ctttgaaaat actttgttgg tcttcacatt 4980gtgcatcatg tcctgcaact tgtaaatatg tgcatgttgt ttatgtttgt ctgcctgtct 5040cttattgcac taaattctgc aaggtgaata atttttttat accatttatt tggaaaaagt 5100tcctcctcca actcttcttc actgaccttt taattagttg gtaaacttag aaaaccaggt 5160aggatttttg aagccctgag tttaaaagaa gaatcgtggc tatttgacat catccttaca 5220ttcccctgac ttaaaaagct aacaagaagc acgagatctt ccacctcatt ttagaaagct 5280tttctacaga actatatgct tgcttatagc tacctatcta cagtttgaac agggagaaag 5340ggagatatgt atgtctggtt acatcttcct tcactcttga attggctggg acagcaagca 5400acaacagttt gaggtccagt gacctacgta aaatgagtgt cgtttgtgtc aggggacaca 5460tgtaactgtg caacatggta ttttctacag ggagggctag gaagggtggt tttggaacct 5520aacctacttt gtgtcttcat tgtcagaact aaggttggaa atgcccttta aagaacttgg 5580tgaggcatat gctaggacaa tagagctgct ttagaaagaa attgagacat gttttgtcag 5640gacagataaa agtttaatgc aggcttttga acagatgttt taaaacaaat ttgaccttac 5700caactatttt tttcttctag ccaaatcatt gtacaggagt ttcatatatg ataaaaagtg 5760ttttgttatt gtttttgctt tcttggcagg ggagaacccc taccattgtg agcagttact 5820gtcactctgg ctgaatggac aaatagctgt gtaaatagct tctcataaaa cgcttctact 5880gatagctgtt tgactttttc ttcccgttat gaatgggagg aacatttgta aacttccttt 5940ctgggtagct accaaaaaac gtactggctt atggtcccat gtgaccttgt ctccgttaag 6000cccagaatct gagtgccttt agcaagtgtt agcatttcac agagagaaga gagacagaat 6060tattgctatt aatcaccatt cataaatagg agcttggaat aacaaaggtc tgtgtgaatt 6120ctgttctacg tctttttttt ccctttttta aataaatgtc aatttgatat caagtaaaac 6180tattgtcatt attgatcaga aaccatacat tctgaaaatg aatccagttt tcccaagctg 6240gcagaagtca gagatcattt ttcaccttga tctgtgttgc gtttgtactt agtgaatggc 6300agtgtggttg attggaagag cttgacactg atgtttggaa aaattcttta ttctagtggt 6360cattttcaaa cttgtcttct gtagagaggg gaaaaattat gtatttgcag ctaaagcgaa 6420gagactggca caataattat taattgtgtt agttcatttc tatattaaat gaagcatctt 6480cacaaatcag gtttgaagcc attaaaagca aaattttccc tagtttaatt aatttaaaat 6540tctaaattgc ctgactactt agaatcttag aaacgccctg ctagactgat tttattatag 6600aaatgttaac atgttcctca acattttctc aagaaaattt tcagacatat atcaaagttg 6660aaaaaacttt gcagtataac cactgctaga ttctgccatc aacatttgac tatacttgta 6720ttgccatgta tctgttcata ttctctatcc ctcttttcat ccatcgatcc atcagatttt 6780ttttttaaag aacattccaa agtaaatttt aggcaataca ctgccctaaa ttcttagcat 6840gggtgtcatt aattaaggtt tagtattcgt ttaaatatct attttgaaag tggtgtatga 6900agttgataat ctgagccaat taacatactt tttttttttt cctcctgtgg acttttgtct 6960tccagtttct ctttttgtgt gtacttaggg tgagggagga caattccttt caaacacaag 7020tttattttag aattggtcat ttttaagtgc ttgtcataaa ttttaaagtt tcaatataat 7080ttttcttttc cccacttctg ataatgtatt tttgccaaac tatgcagctt cttcaaaaaa 7140ctgtaaatta ctgattgggg tttatgaaat cagcgaatac ctcatttcaa tctgttgctg 7200cttttctccc tcttgtcact tctcctttca ctactttcag ctgctctacc agaaggtaga 7260ggggagtaaa gggtcaaacc cctatataat tagctgtttt aaacaactct aggagagcag 7320attaagtagc ttagtaagtt gaaactatta atttttctaa agaatttgat ttgaattcct 7380tgagaattgt aattatccac acttcctcca gctataatta gcagaattaa aaatgattgt 7440actgtacaat gagttgttga aattgtaagc cttagtaacc atctttagct ttattgtagt 7500catgtttaga aaaaattatt tttacatatg cctttatttt tatgcccact ttttgtggat 7560aagattcttt agataaaatc taaagaattt taagtgactt tctccaggtc atgaagattc 7620aatgggtaga attgaatcag aattgaaatg ttccagattc atattcttgt gtgtgtttga 7680taaaattcat ggcttccaaa gtaactgaac acttcctttg ggcccttgga gggaaaatcc 7740atatttttac taattacact ttttttttta gacatctggc agttctttga acttaaacat 7800attctcatgg ccatagttcc aaattaagcc cgacgcagtt gctaaaaatc ttgctgcact 7860gttgaatact aataatgcaa catttattgg atgtttttgc attttgatga ccttcatgat 7920tcatttataa gtctttgtaa gtgcttaagt gaccccctca ctagtgaaaa taataaatgt 7980tctatatcat ttattattat ttgtgtattc tctacatgat atattttttt aaggaagagt 8040aactccacat gtcagaatga gtgatattat cctagggcaa agcgcaaata gggcagtttg 8100tttctactct gcaaatatgg catgtatagg aacaaaactc ttttggagtg gctggtcatt 8160gttctgccct cttttggtac ctgagtacct ttctggggtt ttgtaaatcg tgtgtcattt 8220gtaagaattt cacgttaact ctgcgttact tggtgttcac ctgtggtatc cttgactgac 8280cataatgatt tagtttgggt atgatgtgtc tgctttgaaa tgccttactg gagtatgtca 8340gatcctgctt taaagcattc catatatctg ctgggacaat aagttgcttc tccttggaaa 8400tatgctctag attcagaagc aaaaccgatt ttgctttcac cattaaggtt gcattttaat 8460gcagttattg ttttaaatta gagaataaaa tgtaaaacca agggaggctt tagaaccctt 8520tattgaatgg catggcaaac ttttaaaact gcttttgcta tttcactaga actatctttg 8580ataaaggata tagctaaaaa atgtcagccc aaactgtgtg taattagggt tgtttattaa 8640aattttctct aaatgtcata cagaggctta agatctgtgt atgctgttgg gtcggagtgc 8700cagtcactgc tttggaagtc tgtgttctgg ggctgcagaa tgacaaacgt gtcatgggat 8760taaaaccaat caactgtgaa ttgtgaaatt gaaactactc tttcggtttt attttcttta 8820gcatattgag tatagaaatc tgaaacttat ttaaaattta tactgctttt gttgatggct 8880cattttggct gtgtatcctc acttatgtac tgatttctga taaaggcttg acattattat 8940aacacgcatt ttgtgttcca gtttaataaa acggtttctg agtcttgtct gaaaa 89956289PRTHomo sapiens 6Met Ile Leu Leu Gln His Ala Val Leu Pro Pro Pro Lys Gln Pro Ser 1 5 10 15 Pro Ser Pro Pro Met Ser Val Ala Thr Arg Ser Thr Gly Thr Leu Gln 20 25 30 Leu Pro Pro Gln Lys Pro Phe Gly Gln Glu Ala Ser Leu Pro Leu Ala 35 40 45 Gly Glu Glu Glu Leu Ser Lys Gly Gly Glu Gln Asp Cys Ala Leu Glu 50 55 60 Glu Leu Cys Lys Pro Leu Tyr Cys Lys Leu Cys Asn Val Thr Leu Asn 65 70 75 80 Ser Ala Gln Gln Ala Gln Ala His Tyr Gln Gly Lys Asn His Gly Lys 85 90 95 Lys Leu Arg Asn Tyr Tyr Ala Ala Asn Ser Cys Pro Pro Pro Ala Arg 100 105 110 Met Ser Asn Val Val Glu Pro Ala Ala Thr Pro Val Val Pro Val Pro 115 120 125 Pro Gln Met Gly Ser Phe Lys Pro Gly Gly Arg Val Ile Leu Ala Thr 130 135 140 Glu Asn Asp Tyr Cys Lys Leu Cys Asp Ala Ser Phe Ser Ser Pro Ala 145 150 155 160 Val Ala Gln Ala His Tyr Gln Gly Lys Asn His Ala Lys Arg Leu Arg 165 170 175 Leu Ala Glu Ala Gln Ser Asn Ser Phe Ser Glu Ser Ser Glu Leu Gly 180 185 190 Gln Arg Arg Ala Arg Lys Glu Gly Asn Glu Phe Lys Met Met Pro Asn 195 200 205 Arg Arg Asn Met Tyr Thr Val Gln Asn Asn Ser Ala Gly Pro Tyr Phe 210 215 220 Asn Pro Arg Ser Arg Gln Arg Ile Pro Arg Asp Leu Ala Met Cys Val 225 230 235 240 Thr Pro Ser Gly Gln Phe Tyr Cys Ser Met Cys Asn Val Gly Ala Gly 245 250 255 Glu Glu Met Glu Phe Arg Gln His Leu Glu Ser Lys Gln His Lys Ser 260 265 270 Lys Val Ser Glu Gln Arg Tyr Arg Asn Glu Met Glu Asn Leu Gly Tyr 275 280 285 Val 71870DNAHomo sapiens 7ctccgagacg ggtggggccg gagctccaag ctggtttgaa caagccctgg gcatgtttgg 60cgggaagttg gcttagctcg gctacctgtg gccccgcagt tttgtagtcc ccgccttgtt 120tctccccaga ggcctctcaa tcctccctcc atgatcttcg catagagcac agtacccctt 180cacacggagg acgcgatggc tcccaagaaa cgcccagaaa cccagaagac ctccgagatt 240gtattacgcc ccaggaacaa gaggagcagg agtcccctgg agctggagcc cgaggccaag 300aagctctgtg cgaagggctc cggtcctagc agaagatgtg actcagactg cctctgggtg 360gggctggctg gcccacagat cctgccacca tgccgcagca tcgtcaggac cctccaccag 420cataagctgg gcagagcttc ctggccatct gtccagcagg ggctccagca gtcctttttg 480cacactctgg attcttaccg gatattacaa aaggctgccc cctttgacag gagggctaca 540tccttggcgt ggcacccaac tcaccccagc accgtggctg tgggttccaa agggggagat 600atcatgctct ggaattttgg catcaaggac aaacccacct tcatcaaagg gattggagct 660ggagggagca tcactgggct gaagtttaac cctctcaata ccaaccagtt ttacgcctcc 720tcaatggagg gaacaactag gctgcaagac tttaaaggca acattctacg agtttttgcc 780agctcagaca ccatcaacat ctggttttgt agcctggatg tgtctgctag tagccgaatg 840gtggtcacag gagacaacgt ggggaacgtg atcctgctga acatggacgg caaagagctt 900tggaatctca gaatgcacaa aaagaaagtg acgcatgtgg ccctgaaccc atgctgtgat 960tggttcctgg ccacagcctc cgtagatcaa acagtgaaaa tttgggacct gcgccaggtt 1020agagggaaag ccagcttcct ctactcgctg ccgcacaggc atcctgtcaa cgcagcttgt 1080ttcagtcccg atggagcccg gctcctgacc acggaccaga agagcgagat ccgagtttac 1140tctgcttccc agtgggactg ccccctgggc ctgatcccgc accctcaccg tcacttccag 1200cacctcacac ccatcaaggc agcctggcat cctcgctaca acctcattgt tgtgggccga 1260tacccagatc ctaatttcaa aagttgtacc ccttatgaat tgaggacgat cgacgtgttc 1320gatggaaact cagggaagat gatgtgtcag ctctatgacc cagaatcttc tggcatcagt 1380tcgcttaatg aattcaatcc catgggggac acgctggcct ctgcaatggg ttaccacatt 1440ctcatctgga gccaggagga agccaggaca cggaagtgag agacactaaa gaaggtgtgg 1500gccagacaag gccttggagc ccacacatgg gatcaagtcc tgcaagcaga ggtggcgatt 1560tgttaaaggg ccaaaagtat ccaaggttag ggttggagca ggggtgctgg gacctggggc 1620actgtgggac tgggacactt ttatgttaat gctctggact tgcctccaga gactgctcca 1680gagttggtga cacagctgtc ccaagggccc ctctgtatct agcctggaac caaggttatc 1740ttggaactaa atgacttttc tcctctcagt gggtggtagc agagggatca agcagttatt 1800tgatttgtgc tcacttttga tatggccaat aaaaccatac cgactgagaa aaaaaaaaaa 1860aaaaaaaaaa 18708427PRTHomo sapiens 8Met Ala Pro Lys Lys Arg Pro Glu Thr Gln Lys Thr Ser Glu Ile Val 1 5 10 15 Leu Arg Pro Arg Asn Lys Arg Ser Arg Ser Pro Leu Glu Leu Glu Pro 20 25 30 Glu Ala Lys Lys Leu Cys Ala Lys Gly Ser Gly Pro Ser Arg Arg Cys 35 40 45 Asp Ser Asp Cys Leu Trp Val Gly Leu Ala Gly Pro Gln Ile Leu Pro 50 55 60 Pro Cys Arg Ser Ile Val Arg Thr Leu His Gln His Lys Leu Gly Arg 65 70 75 80 Ala Ser Trp Pro Ser Val Gln Gln Gly Leu Gln Gln Ser Phe Leu His 85 90 95 Thr Leu Asp Ser Tyr Arg Ile Leu Gln Lys Ala Ala Pro Phe Asp Arg 100 105 110 Arg Ala Thr Ser Leu Ala Trp His Pro Thr His Pro Ser Thr Val Ala 115 120 125 Val Gly Ser Lys Gly Gly Asp Ile Met Leu Trp Asn Phe Gly Ile Lys 130 135 140 Asp Lys Pro Thr Phe Ile Lys Gly Ile Gly Ala Gly Gly Ser Ile Thr 145 150 155 160 Gly Leu Lys Phe Asn Pro Leu Asn Thr Asn Gln Phe Tyr Ala Ser Ser 165 170 175 Met Glu Gly Thr Thr Arg Leu Gln Asp Phe Lys Gly Asn Ile Leu Arg 180 185 190 Val Phe Ala Ser Ser Asp Thr Ile Asn Ile Trp Phe Cys Ser Leu Asp 195 200 205 Val Ser Ala Ser Ser Arg Met Val Val Thr Gly Asp Asn Val Gly Asn 210 215 220 Val Ile Leu Leu Asn Met Asp Gly Lys Glu Leu Trp Asn Leu Arg Met 225 230 235 240 His Lys Lys Lys Val Thr His Val Ala Leu Asn Pro Cys Cys Asp Trp 245 250 255 Phe Leu Ala Thr Ala Ser Val Asp Gln Thr Val Lys Ile Trp Asp Leu 260 265 270 Arg Gln Val Arg Gly Lys Ala Ser Phe Leu Tyr Ser Leu Pro His Arg 275 280 285 His Pro Val Asn Ala Ala Cys Phe Ser Pro Asp Gly Ala Arg Leu Leu 290 295 300 Thr Thr Asp Gln Lys Ser Glu Ile Arg Val Tyr Ser Ala Ser Gln Trp 305 310 315 320 Asp Cys Pro Leu Gly Leu Ile Pro His Pro His Arg His Phe Gln His 325 330 335 Leu Thr Pro Ile Lys Ala Ala Trp His Pro Arg Tyr Asn Leu Ile Val 340 345 350 Val Gly Arg Tyr Pro Asp Pro Asn Phe Lys Ser Cys Thr Pro Tyr Glu 355 360 365 Leu Arg Thr Ile Asp Val Phe Asp Gly Asn Ser Gly Lys Met Met Cys 370 375 380 Gln Leu Tyr Asp Pro Glu Ser Ser Gly Ile Ser Ser Leu Asn Glu Phe 385 390 395 400 Asn Pro Met Gly Asp Thr Leu Ala Ser Ala Met Gly Tyr His Ile Leu 405 410 415 Ile Trp Ser Gln Glu Glu Ala Arg Thr Arg Lys 420 425 91936DNAHomo sapiens 9gcttgtgggc gggcccgggc aggagcgggc ttgccctgcg gagcagtagc taggaacaga 60tccacttgca ggttgctgtt cccagccatg gcttcgcgct gctggcgctg gtggggctgg 120tcggcgtggc ctcggacccg gctgcctccc gccgggagca ccccgagctt ctgccaccat 180ttctccacac aggagaagac cccccagatc tgtgtggtgg gcagtggccc agctggcttc 240tacacggccc aacacctgct aaagcacccc caggcccacg tggacatcta cgagaaacag 300cctgtgccct ttggcctggt gcgctttggt gtggcgcctg

atcaccccga ggtgaagaat 360gtcatcaaca catttaccca gacggcccat tctggccgct gtgccttctg gggcaacgtg 420gaggtgggca gggacgtgac ggtgccggag ctgcgggagg cctaccacgc tgtggtgctg 480agctacgggg cagaggacca tcgggccctg gaaattcctg gtgaggagct gccaggtgtg 540tgctccgccc gggccttcgt gggctggtac aacgggcttc ctgagaacca ggagctggag 600ccagacctga gctgtgacac agccgtgatt ctggggcagg ggaacgtggc tctggacgtg 660gcccgcatcc tactgacccc acctgagcac ctggaggccc tccttttgtg ccagagaacg 720gacatcacga aggcagccct gggtgtactg aggcagagtc gagtgaagac agtgtggcta 780gtgggccggc gtggacccct gcaagtggcc ttcaccatta aggagcttcg ggagatgatt 840cagttaccgg gagcccggcc cattttggat cctgtggatt tcttgggtct ccaggacaag 900atcaaggagg tcccccgccc gaggaagcgg ctgacggaac tgctgcttcg aacggccaca 960gagaagccag ggccggcgga agctgcccgc caggcatcgg cctcccgtgc ctggggcctc 1020cgctttttcc gaagccccca gcaggtgctg ccctcaccag atgggcggcg ggcagcaggt 1080gtccgcctag cagtcactag actggagggt gtcgatgagg ccacccgtgc agtgcccacg 1140ggagacatgg aagacctccc ttgtgggctg gtgctcagca gcattgggta taagagccgc 1200cctgtcgacc caagcgtgcc ctttgactcc aagcttgggg tcatccccaa tgtggagggc 1260cgggttatgg atgtgccagg cctctactgc agcggctggg tgaagagagg acctacaggt 1320gtcatagcca caaccatgac tgacagcttc ctcaccggcc agatgctgct gcaggacctg 1380aaggctgggt tgctcccctc tggccccagg cctggctacg cagccatcca ggccctgctc 1440agcagccgag gggtccggcc agtctctttc tcagactggg agaagctgga tgccgaggag 1500gtggcccggg gccagggcac ggggaagccc agggagaagc tggtggatcc tcaggagatg 1560ctgcgcctcc tgggccactg agcccagccc cagccccggc ccccagcagg gaagggatga 1620gtgttgggag gggaagggct gggtccgtct gagtgggact ttgcacctct gctgatcccg 1680gccggccctg gcttggaggc ttggctgctc ttccagcgtc tctcctccct cctggggaag 1740gtcgcccttg cgcgcaaggt tttagctttc agcaactgag gtaaccttag ggacaggtgg 1800aggtgtgggc cgatctaacc ccttacccat ctctctactg ctggactgtg gagggtcacc 1860aggttgggaa catgctggaa ataaaacagc tgcaaccaag aaaaaaaaaa aaaaaaaaaa 1920aaaaaaaaaa aaaaaa 193610497PRTHomo sapiens 10Met Ala Ser Arg Cys Trp Arg Trp Trp Gly Trp Ser Ala Trp Pro Arg 1 5 10 15 Thr Arg Leu Pro Pro Ala Gly Ser Thr Pro Ser Phe Cys His His Phe 20 25 30 Ser Thr Gln Glu Lys Thr Pro Gln Ile Cys Val Val Gly Ser Gly Pro 35 40 45 Ala Gly Phe Tyr Thr Ala Gln His Leu Leu Lys His Pro Gln Ala His 50 55 60 Val Asp Ile Tyr Glu Lys Gln Pro Val Pro Phe Gly Leu Val Arg Phe 65 70 75 80 Gly Val Ala Pro Asp His Pro Glu Val Lys Asn Val Ile Asn Thr Phe 85 90 95 Thr Gln Thr Ala His Ser Gly Arg Cys Ala Phe Trp Gly Asn Val Glu 100 105 110 Val Gly Arg Asp Val Thr Val Pro Glu Leu Arg Glu Ala Tyr His Ala 115 120 125 Val Val Leu Ser Tyr Gly Ala Glu Asp His Arg Ala Leu Glu Ile Pro 130 135 140 Gly Glu Glu Leu Pro Gly Val Cys Ser Ala Arg Ala Phe Val Gly Trp 145 150 155 160 Tyr Asn Gly Leu Pro Glu Asn Gln Glu Leu Glu Pro Asp Leu Ser Cys 165 170 175 Asp Thr Ala Val Ile Leu Gly Gln Gly Asn Val Ala Leu Asp Val Ala 180 185 190 Arg Ile Leu Leu Thr Pro Pro Glu His Leu Glu Ala Leu Leu Leu Cys 195 200 205 Gln Arg Thr Asp Ile Thr Lys Ala Ala Leu Gly Val Leu Arg Gln Ser 210 215 220 Arg Val Lys Thr Val Trp Leu Val Gly Arg Arg Gly Pro Leu Gln Val 225 230 235 240 Ala Phe Thr Ile Lys Glu Leu Arg Glu Met Ile Gln Leu Pro Gly Ala 245 250 255 Arg Pro Ile Leu Asp Pro Val Asp Phe Leu Gly Leu Gln Asp Lys Ile 260 265 270 Lys Glu Val Pro Arg Pro Arg Lys Arg Leu Thr Glu Leu Leu Leu Arg 275 280 285 Thr Ala Thr Glu Lys Pro Gly Pro Ala Glu Ala Ala Arg Gln Ala Ser 290 295 300 Ala Ser Arg Ala Trp Gly Leu Arg Phe Phe Arg Ser Pro Gln Gln Val 305 310 315 320 Leu Pro Ser Pro Asp Gly Arg Arg Ala Ala Gly Val Arg Leu Ala Val 325 330 335 Thr Arg Leu Glu Gly Val Asp Glu Ala Thr Arg Ala Val Pro Thr Gly 340 345 350 Asp Met Glu Asp Leu Pro Cys Gly Leu Val Leu Ser Ser Ile Gly Tyr 355 360 365 Lys Ser Arg Pro Val Asp Pro Ser Val Pro Phe Asp Ser Lys Leu Gly 370 375 380 Val Ile Pro Asn Val Glu Gly Arg Val Met Asp Val Pro Gly Leu Tyr 385 390 395 400 Cys Ser Gly Trp Val Lys Arg Gly Pro Thr Gly Val Ile Ala Thr Thr 405 410 415 Met Thr Asp Ser Phe Leu Thr Gly Gln Met Leu Leu Gln Asp Leu Lys 420 425 430 Ala Gly Leu Leu Pro Ser Gly Pro Arg Pro Gly Tyr Ala Ala Ile Gln 435 440 445 Ala Leu Leu Ser Ser Arg Gly Val Arg Pro Val Ser Phe Ser Asp Trp 450 455 460 Glu Lys Leu Asp Ala Glu Glu Val Ala Arg Gly Gln Gly Thr Gly Lys 465 470 475 480 Pro Arg Glu Lys Leu Val Asp Pro Gln Glu Met Leu Arg Leu Leu Gly 485 490 495 His 111084DNAHomo sapiens 11tagagcagac aagcccgccc cgccgctcct cccagcagct tctatcccgg aagttgatgc 60cgagcgcaga tcgcttgcag cttgctagct gtgtgggctg ggaggtctgg tagggctgag 120cttgcaagag gatcaacatg cctttggcta gagatttact acatccgtcc ttggaagagg 180aaaagaaaaa acataaaaag aaacgcctag tacaaagtcc aaattcttac tttatggatg 240taaaatgtcc aggttgctac aagatcacca cggttttcag ccatgctcag acagtggttc 300tttgtgtagg ttgttcaaca gtgttgtgcc agcctacagg aggaaaggcc agactcacag 360aagggtgttc atttagaaga aagcaacact aatgattcaa acagcttcct gaattttaat 420tttgtgttgt ctcacagaaa gccttatcat aaattccata attctaatta atttaccaag 480ataatgtaat tacatttggt tttgtaaggt atacagcagt aatctcctat tttggtgtca 540gtttttcaat aaagttttga ttatgggcaa atcccctctt tttctttttt taaaatatat 600ttgagtatgc catacattta tatatatggt gtatatgaat ttggtttaaa cattttaaaa 660tttattctga ttagtttgtg tctttttttt tttttttgag agagagagtc ctgctctgtc 720actcaagctg gagtgcagtg gtgcgatctc ggctcactgc aacctccgcc tcccaggtcc 780aagcaattct cttgccttgt cctcccaagt agctgggatt ataggcacac accaccatgc 840ctggctaatt tgtgtctcat tttcaagagt agaaacccta aatattttat tttcattcct 900tttccaaatt gctatgaatg ggattaaagg attacagatg taaagtctat tatttgtgaa 960ttctaaatgt agttctgctg ttgtacctgt ggaaacatct taaagaagta catattttgc 1020acgtcctgca cgtgtacccc agaacttaaa ctataattaa aaagaatagt ttcaaaaaaa 1080taca 10841284PRTHomo sapiens 12Met Pro Leu Ala Arg Asp Leu Leu His Pro Ser Leu Glu Glu Glu Lys 1 5 10 15 Lys Lys His Lys Lys Lys Arg Leu Val Gln Ser Pro Asn Ser Tyr Phe 20 25 30 Met Asp Val Lys Cys Pro Gly Cys Tyr Lys Ile Thr Thr Val Phe Ser 35 40 45 His Ala Gln Thr Val Val Leu Cys Val Gly Cys Ser Thr Val Leu Cys 50 55 60 Gln Pro Thr Gly Gly Lys Ala Arg Leu Thr Glu Gly Cys Ser Phe Arg 65 70 75 80 Arg Lys Gln His 13891DNAHomo sapiens 13tcacgtgacc cgggcgcgct gcggccgccc gcgcggaccc ggcgagaggc ggcggcggga 60gcggcggtga tggacgggtc cggggagcag cccagaggcg gggggcccac cagctctgag 120cagatcatga agacaggggc ccttttgctt cagggtttca tccaggatcg agcagggcga 180atgggggggg aggcacccga gctggccctg gacccggtgc ctcaggatgc gtccaccaag 240aagctgagcg agtgtctcaa gcgcatcggg gacgaactgg acagtaacat ggagctgcag 300aggatgattg ccgccgtgga cacagactcc ccccgagagg tctttttccg agtggcagct 360gacatgtttt ctgacggcaa cttcaactgg ggccgggttg tcgccctttt ctactttgcc 420agcaaactgg tgctcaaggc cctgtgcacc aaggtgccgg aactgatcag aaccatcatg 480ggctggacat tggacttcct ccgggagcgg ctgttgggct ggatccaaga ccagggtggt 540tgggtgagac tcctcaagcc tcctcacccc caccaccgcg ccctcaccac cgcccctgcc 600ccaccgtccc tgccccccgc cactcctctg ggaccctggg ccttctggag caggtcacag 660tggtgccctc tccccatctt cagatcatca gatgtggtct ataatgcgtt ttccttacgt 720gtctgatcaa tccccgattc atctaccctg ctgacctccc agtgacccct gacctcactg 780tgaccttgac ttgattagtg ccttctgccc tccctggagc ctccactgcc tctggaattg 840ctcaagttca ttgatgaccc tctgacccta gctctttcct tttttttttt t 89114218PRTHomo sapiens 14Met Asp Gly Ser Gly Glu Gln Pro Arg Gly Gly Gly Pro Thr Ser Ser 1 5 10 15 Glu Gln Ile Met Lys Thr Gly Ala Leu Leu Leu Gln Gly Phe Ile Gln 20 25 30 Asp Arg Ala Gly Arg Met Gly Gly Glu Ala Pro Glu Leu Ala Leu Asp 35 40 45 Pro Val Pro Gln Asp Ala Ser Thr Lys Lys Leu Ser Glu Cys Leu Lys 50 55 60 Arg Ile Gly Asp Glu Leu Asp Ser Asn Met Glu Leu Gln Arg Met Ile 65 70 75 80 Ala Ala Val Asp Thr Asp Ser Pro Arg Glu Val Phe Phe Arg Val Ala 85 90 95 Ala Asp Met Phe Ser Asp Gly Asn Phe Asn Trp Gly Arg Val Val Ala 100 105 110 Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu Lys Ala Leu Cys Thr Lys 115 120 125 Val Pro Glu Leu Ile Arg Thr Ile Met Gly Trp Thr Leu Asp Phe Leu 130 135 140 Arg Glu Arg Leu Leu Gly Trp Ile Gln Asp Gln Gly Gly Trp Val Arg 145 150 155 160 Leu Leu Lys Pro Pro His Pro His His Arg Ala Leu Thr Thr Ala Pro 165 170 175 Ala Pro Pro Ser Leu Pro Pro Ala Thr Pro Leu Gly Pro Trp Ala Phe 180 185 190 Trp Ser Arg Ser Gln Trp Cys Pro Leu Pro Ile Phe Arg Ser Ser Asp 195 200 205 Val Val Tyr Asn Ala Phe Ser Leu Arg Val 210 215 154932DNAHomo sapiens 15ggctggccga agttaggcgg agccccgagg cgggggaggc ggggccgggc cggcgcaggg 60agagtcactc aatggacagg cgagaaagca ggaccggcgc ggcggggcgg ggccggccga 120gtccctagag ctgggggcgg ggcggaccca gcggaccagc ggaccacctg ggtgctgtcg 180tagttggagg tggcctgagg agctcagttc cctcagcgcc cgtagcttcg gcggagtctg 240cgcgatgggc gacccggaaa ggccggaagc ggccgggctg gatcaggatg agagatcatc 300ttcagacacc aacgaaagtg aaataaagtc aaatgaagag ccactcctaa gaaagagttc 360tcgccggttt gtcatctttc caatccagta ccctgatatt tggaaaatgt ataaacaggc 420acaggcttcc ttctggacag cagaagaggt cgacttatca aaggatctcc ctcactggaa 480caagcttaaa gcagatgaga agtacttcat ctctcacatc ttagcctttt ttgcagccag 540tgatggaatt gtaaatgaaa atttggtgga gcgctttagt caggaggtgc aggttccaga 600ggctcgctgt ttctatggct ttcaaattct catcgagaat gttcactcag agatgtacag 660tttgctgata gacacttaca tcagagatcc caagaaaagg gaatttttat ttaatgcaat 720tgaaaccatg ccctatgtta agaaaaaagc agattgggcc ttgcgatgga tagcagatag 780aaaatctact tttggggaaa gagtggtggc ctttgctgct gtagaaggag ttttcttctc 840aggatctttt gctgctatat tctggctaaa gaagagaggt cttatgccag gactcacttt 900ttccaatgaa ctcatcagca gagatgaagg acttcactgt gactttgctt gcctgatgtt 960ccaatactta gtaaataagc cttcagaaga aagggtcagg gagatcattg ttgatgctgt 1020caaaattgag caggagtttt taacagaagc cttgccagtt ggcctcattg gaatgaattg 1080cattttgatg aaacagtaca ttgagtttgt agctgacaga ttacttgtgg aacttggatt 1140ctcaaaggtt tttcaggcag aaaatccttt tgattttatg gaaaacattt ctttagaagg 1200aaaaacaaat ttctttgaga aacgagtttc agagtatcag cgttttgcag ttatggcaga 1260aaccacagat aacgtcttca ccttggatgc agatttttaa aaaacctctc gttttaaaac 1320tctataaact tgtcattggt aaatagtagt ctattttcct ctgcttaaaa aaaattttaa 1380gtatatcctt taaaggactg ggggtttgct caaaaggaaa tccaaaacct attctaaaca 1440atttgcattt atataatttt cctgtttaac aacaagagtg tgacctaaat gcttttgtct 1500tgtcactgaa ataaaagatg gcattatgtg gttaagagca tggggcgagg ggtcagacat 1560gagtctaagg ttctgccctt actccagtgt gtgacccttg gcaagtcagt taatcttggt 1620aaacctcggt gtacttatct ttaaaatggg agtaatagta ggtcctaaat tcatagagtg 1680gatattagga ttaggatgca aaaataaatg cttaaccaac actactactg ttagcaccac 1740tactaattat cattcattga taatattaat tgcaatgatg ttgtaataaa atactctcat 1800ttccttaaaa taattgtgat tctaggtcct aggatctaga attagatctt tgtattttta 1860atgcttaggg gaagaatata agtatctcct taaaaagaac ataattctca ttcacgcaag 1920aataagttct ttgaattcct tagtatgtag tgaagaaaat ttagttgtta gttgctttgg 1980gaagcctact tatggagtgg aaaccaggag gttatcatgg tagttgacct tataagaaaa 2040atgattcttc ttcagaaatt aaaaacataa ctattgccag atttagctct ggaatgttta 2100gaatcaggct agaatagcat tttccaaaga atattctaag agctattagc tcctctagat 2160atttttttgg gggaaaaagg ggattctgtg gtcagatgag tttgggaaat gctgaacact 2220tcattcttct ttagcaagta cagtcagtac atcaaagact gagcagttca gtggtacata 2280aatttatctc gccctgcata ttcccaacat acttaacaca gatgtttttt acctgttaac 2340atctcaccca gctagtgttc ctcagaacaa agattggaaa aagctggccg agaaccattt 2400atacatagag gaagggctta tggactgaga aagggagaac atggtaggga ttattgaatc 2460atttcaaatt tataccagcc tgaatagtgt accagcaatt gacttaggct gtgtttcttt 2520atggttttaa aactcttgag ctgttataag agatagttct tttaatgtga ctatgcaaca 2580tgatagccaa tggtgaggga aaaggaggtt tctctagaag agtctgatga aaggccggga 2640accaaggttt ttgagaagtc tgcccctatt tatttttagt aagtatcaag aggtagcctg 2700agcctagtta gagttagacc tgtctttgga tgaagaagtc ttaatactga aatactgaat 2760ttttaataca ttattatttg gtattctgta taccccttca agcagttgtt tcccattccc 2820aacaaactgt actttataca attctggatg ctaaaactta gagattttct ctttgcataa 2880attttggctc cattctttcc ataacaatct aatcaaaact gggagttctc aagtgaatgc 2940aaaaggagca ggccataact ttatttgtta gatacactgt cagaaacttg agatcttttg 3000gcctatgata ataccattaa tttttgcatt gcttcagttt gccaagtgtt tttacatcat 3060ctcatttgat ctcaaaacag cttgacagag caactgttat tgaaatatta cagatggaaa 3120gaatgaggct cagggaagtt aaatgacttg gccaagatct gctcatcgtc actgtctgta 3180cagtattttt ttttagaggt tgtaatgtct cagatttagt cctttaccat ctatgttgat 3240ttgcttttgt ctatttcctc attaattgaa tatactttaa atatatatat taaagtatca 3300aaatatagag agacatttga actgtattca ggtaatatgt ttaaagatat ttatatattg 3360ccatacaaaa acttaacatt taaaactgat aatatctgta atgacatcag aatgaaagaa 3420aaaaaattgt acagtgtata ttcctttgtt ttgaatccaa atctttttca taggtaatga 3480cagatgcctt aatgtgaagc ttatttataa tagcaataaa cctaactgga tttggatgaa 3540gaagtcttaa tactgacata ctggattttt aatgcactgg tttgttattt ggtattctat 3600ctctttttcc aggcctccag gttgcacatt tatttattat gttcaatact ttggttctta 3660gttcttaaag aatcaagaag ttgtgtaatc ttttaaaaat attatcttgc agataaagaa 3720aaaaattaag agtgtgttta caactgtttt ctctttttta cagtacatgt atttaaatca 3780ttgctataat aaagttaagt tcattaggaa tataaaaact tgcagttcta tgatagattg 3840catttattaa aaatgtttca ttgtatcaca tagaaatatg gccaggaagg acttgagaag 3900acagtttgat ccattgcttt tagacaggac tgggttttgc tgtccaatta tatacaataa 3960tagtttttct tacaactaag ctggccccag ccttgtcttg atattaatac atgaaatttt 4020tataattgtc tcattgtctc atttagaaac atccatattt ttctgctttt tctattgcca 4080ttttttattt gtgcatgaat tgattattga gaaaatgtag cagtttgcat atttaaaaat 4140taatcatttt gcattttaca tttaaatatg ctaacatcac tgtcatagaa ttcccaaatt 4200tcatttgtag atactgaact aagggctaat gtcaggagct gatttttaat gataaagctg 4260cagatgggct aaataaaagc caaattaatc ctacaatcag gtattatgtt tttaaaccaa 4320gttgagtgaa ttggtagtgg acttgggaaa tcttccccag cagaatctgg atgaatggca 4380cagaattgaa atctctttgt ttcccaccat ttccctttaa gtgctctgct cctttgtaaa 4440aagttaaaga tttgaaagag aatctcatat tcccgaggca ttaggaagaa aggatttaat 4500cccttcaatt tggggcttaa tcttgtttaa aaaaatgtaa gtgaagatgg aaggctggag 4560agaatgattg ctttttgtac agttaaataa ggtcacaata ttcttacata ctttgtttta 4620caactgtgtt ttcatttttt caaatgtctg gccatttagc aaagttattt actatttact 4680gtgtacatag aaagctttat tatgtgtggt gtatctaaat tttttttgct gaaatacatt 4740atggtcaatc aagccaagcc tgcatgtaca gaatttgttt ttttttcaaa taaattagtt 4800gttttcttat ttttttggct tagtatgttg aaataaacta tggtatcttc atcattttgt 4860acatttcctt tttgaggaag gtttctttat aagtgcaagg gctaccctaa taaaggaatg 4920tatatactta ct 493216351PRTHomo sapiens 16Met Gly Asp Pro Glu Arg Pro Glu Ala Ala Gly Leu Asp Gln Asp Glu 1 5 10 15 Arg Ser Ser Ser Asp Thr Asn Glu Ser Glu Ile Lys Ser Asn Glu Glu 20 25 30 Pro Leu Leu Arg Lys Ser Ser Arg Arg Phe Val Ile Phe Pro Ile Gln 35 40 45 Tyr Pro Asp Ile Trp Lys Met Tyr Lys Gln Ala Gln Ala Ser Phe Trp 50 55 60 Thr Ala Glu Glu Val Asp Leu Ser Lys Asp Leu Pro His Trp Asn Lys 65 70 75 80 Leu Lys Ala Asp Glu Lys Tyr Phe Ile Ser His Ile Leu Ala Phe Phe 85 90 95 Ala Ala Ser Asp Gly Ile Val Asn Glu Asn Leu Val Glu Arg Phe Ser 100 105 110 Gln Glu Val Gln Val Pro Glu Ala Arg Cys Phe Tyr Gly Phe Gln Ile 115 120 125 Leu Ile Glu Asn Val His Ser Glu Met Tyr Ser Leu Leu Ile Asp Thr 130 135 140 Tyr Ile Arg Asp Pro Lys Lys Arg Glu Phe Leu Phe Asn Ala Ile Glu 145 150 155 160 Thr Met Pro Tyr

Val Lys Lys Lys Ala Asp Trp Ala Leu Arg Trp Ile 165 170 175 Ala Asp Arg Lys Ser Thr Phe Gly Glu Arg Val Val Ala Phe Ala Ala 180 185 190 Val Glu Gly Val Phe Phe Ser Gly Ser Phe Ala Ala Ile Phe Trp Leu 195 200 205 Lys Lys Arg Gly Leu Met Pro Gly Leu Thr Phe Ser Asn Glu Leu Ile 210 215 220 Ser Arg Asp Glu Gly Leu His Cys Asp Phe Ala Cys Leu Met Phe Gln 225 230 235 240 Tyr Leu Val Asn Lys Pro Ser Glu Glu Arg Val Arg Glu Ile Ile Val 245 250 255 Asp Ala Val Lys Ile Glu Gln Glu Phe Leu Thr Glu Ala Leu Pro Val 260 265 270 Gly Leu Ile Gly Met Asn Cys Ile Leu Met Lys Gln Tyr Ile Glu Phe 275 280 285 Val Ala Asp Arg Leu Leu Val Glu Leu Gly Phe Ser Lys Val Phe Gln 290 295 300 Ala Glu Asn Pro Phe Asp Phe Met Glu Asn Ile Ser Leu Glu Gly Lys 305 310 315 320 Thr Asn Phe Phe Glu Lys Arg Val Ser Glu Tyr Gln Arg Phe Ala Val 325 330 335 Met Ala Glu Thr Thr Asp Asn Val Phe Thr Leu Asp Ala Asp Phe 340 345 350 173212DNAHomo sapiens 17gaacccggtg gctgcacaga caaaaaagcc ccgaatggct ggagggcgtt cagctgttaa 60cagccttttg gggcagagca cggatttgac agctccacaa cgtgaggata tccactgacc 120ccgcgagacg gaggagaacg cttccccgaa attctctgcc caccaaagcc agcgctgcaa 180ggttgcaact ttcaaacttt gtttttccag aaagaagact gccctttcgt gtacaaggag 240agggtgagag ggtgacctag cttgtagatc ggctgaaggc accagtggtt ccaaatgtca 300cccagatgtg tgttttcatg acgatttgat ttctctgatt ttatttttac atttttcatt 360ttaaaaatac aaagcaattt ttttggggca tgctgaaagg taactgaaga ccgcaaagga 420aaaactattg tcatggctga aggagagaat gaagtgagat gggatggact ctgcagcaga 480gattcaacta ctagggagac agcattggaa aacattaggc aaaccatttt gaggaaaacc 540gagtatcttc gttcggtgaa agaaacacct catcgtccat cagacgggct ttcaaatacc 600gagtcttcgg atgggttgaa taagctactt gctcatctgc ttatgctttc taagaggtgt 660cccttcaaag atgtgagaga gaaaagtgag tttattctga agagcatcca ggaacttggc 720attagaattc ctcgaccact aggacaggga ccaagcagat tcatcccaga aaaggagatc 780ctccaagtgg ggagtgaaga cgcacagatg catgctttat ttgcagattc ttttgctgct 840ttgggccgtt tggataacat tacgttagtg atggttttcc acccacaata tttagaaagt 900ttcttaaaaa ctcagcacta tctactgcaa atggatgggc cgttacccct acattatcgt 960cactacattg gaataatggc tgcggcaaga catcagtgct cctacttagt gaacctgcat 1020gtaaatgatt tccttcatgt tggtggggac cccaagtggc tcaatggttt agagaatgct 1080cctcaaaaac tacagaattt aggagaactt aacaaagtgt tagcccatag accttggctt 1140attaccaaag aacacattga gggactttta aaagctgaag agcacagctg gtcccttgcg 1200gaattggtac atgcagtagt tttactcaca cactatcatt ctcttgcctc attcacattc 1260ggctgtggaa tcagtccaga aattcattgt gatggtggcc acacattcag acctccttct 1320gttagcaact actgcatctg tgacattaca aatggcaatc acagtgtgga tgagatgccg 1380gtcaactcag cagaaaatgt ttctgtaagt gattctttct ttgaggttga agccctcatg 1440gaaaagatga ggcagttaca ggaatgtcga gatgaagaag aggcaagtca ggaagagatg 1500gcttcacgtt ttgaaataga aaaaagagag agtatgtttg tcttctcttc agatgatgaa 1560gaagttacac cagcaagagc tgtatctcgt cattttgagg atactagtta tggctataaa 1620gatttctcta gacatgggat gcatgttcca acatttcgtg tccaggacta ttgctgggaa 1680gatcatggtt attctttggt aaatcgcctt tatccagatg tgggacagtt gattgatgaa 1740aaatttcaca ttgcttacaa tcttacttat aatacaatgg caatgcacaa agatgttgat 1800acctcaatgc ttagacgggc aatttggaac tatattcact gcatgtttgg aataagatat 1860gatgattatg actatggtga aattaaccag ctattggatc gtagctttaa agtttatatc 1920aaaactgttg tttgcactcc tgaaaaggtt accaaaagaa tgtatgatag cttctggagg 1980cagttcaagc actctgagaa ggttcatgtt aatctgcttc ttatagaagc taggatgcaa 2040gcagaactcc tttatgctct gagagccatt acccgctata tgacctgatg cctttccttc 2100attaaagatg attctggaat gatcagcaga tatagtctac aagggggaag gtactaagcc 2160ccaggaccaa tggtagacaa aataattcag aaatccattg tgccatgatt cctttagttt 2220ctgctatttt tctgtggaaa accactgctg gcacaagcag tgactgtttg gcagcttcaa 2280gtttagagct gtgaagacag gctgccattc acagtatttt gctttttgac agtacaagat 2340gctgtgtaac tgttttaata cagcaaatag taactctcca aatcctgttg cttttatgtt 2400aaataagata acaagaattg gagcatgcaa agaatgggac ttggataatg acttaagctt 2460tatatgtaaa gaattttaga agatcttggt gctgctattc ctgctggagg aatgaataga 2520tggctgtttc agttaagcta ttagtaataa aagtgaacat tgctactatc tgagcctaca 2580tacataactt gtgtgatttc aaattaaact tgcattatgt gttaattttc ttgcatctaa 2640aaaagcatag aattcctact cacacagctc agcaacaacc attttgatgg taacagttaa 2700tttctttcat tagtttttta aattcagggt tctggatatt aaattaaaat ggcattctta 2760aagattttct tcaaaaagca atcctaaatg aaagtgtgta aattataaga agctggcgat 2820cttttgatat gctgtttcac aggatcctga cactggaggg cagctgtctt gtgcattact 2880tgtgtttcca gcaccaaagt tgtgggacat gttgctgtag actgctgcgc agtcctgggt 2940gcattcagtc tctctgcctc tgcctgcctc ctggtcccca ctttaaaggc tgtgcagctc 3000cttaaataat aaagctggaa aatattttta gtcgggttat caaatttgat ttacaaaaac 3060gctaactttg tttgaaatgc aaacaggttt gaaaatatgt attaagtact ttgtattctg 3120gaagcgtgaa ttgcttttga agtctgtcag tattactggt atttttaaat aaagaagaat 3180ttttctccaa ttttaaaaaa aaaaaaaaaa aa 321218551PRTHomo sapiens 18Met Ala Glu Gly Glu Asn Glu Val Arg Trp Asp Gly Leu Cys Ser Arg 1 5 10 15 Asp Ser Thr Thr Arg Glu Thr Ala Leu Glu Asn Ile Arg Gln Thr Ile 20 25 30 Leu Arg Lys Thr Glu Tyr Leu Arg Ser Val Lys Glu Thr Pro His Arg 35 40 45 Pro Ser Asp Gly Leu Ser Asn Thr Glu Ser Ser Asp Gly Leu Asn Lys 50 55 60 Leu Leu Ala His Leu Leu Met Leu Ser Lys Arg Cys Pro Phe Lys Asp 65 70 75 80 Val Arg Glu Lys Ser Glu Phe Ile Leu Lys Ser Ile Gln Glu Leu Gly 85 90 95 Ile Arg Ile Pro Arg Pro Leu Gly Gln Gly Pro Ser Arg Phe Ile Pro 100 105 110 Glu Lys Glu Ile Leu Gln Val Gly Ser Glu Asp Ala Gln Met His Ala 115 120 125 Leu Phe Ala Asp Ser Phe Ala Ala Leu Gly Arg Leu Asp Asn Ile Thr 130 135 140 Leu Val Met Val Phe His Pro Gln Tyr Leu Glu Ser Phe Leu Lys Thr 145 150 155 160 Gln His Tyr Leu Leu Gln Met Asp Gly Pro Leu Pro Leu His Tyr Arg 165 170 175 His Tyr Ile Gly Ile Met Ala Ala Ala Arg His Gln Cys Ser Tyr Leu 180 185 190 Val Asn Leu His Val Asn Asp Phe Leu His Val Gly Gly Asp Pro Lys 195 200 205 Trp Leu Asn Gly Leu Glu Asn Ala Pro Gln Lys Leu Gln Asn Leu Gly 210 215 220 Glu Leu Asn Lys Val Leu Ala His Arg Pro Trp Leu Ile Thr Lys Glu 225 230 235 240 His Ile Glu Gly Leu Leu Lys Ala Glu Glu His Ser Trp Ser Leu Ala 245 250 255 Glu Leu Val His Ala Val Val Leu Leu Thr His Tyr His Ser Leu Ala 260 265 270 Ser Phe Thr Phe Gly Cys Gly Ile Ser Pro Glu Ile His Cys Asp Gly 275 280 285 Gly His Thr Phe Arg Pro Pro Ser Val Ser Asn Tyr Cys Ile Cys Asp 290 295 300 Ile Thr Asn Gly Asn His Ser Val Asp Glu Met Pro Val Asn Ser Ala 305 310 315 320 Glu Asn Val Ser Val Ser Asp Ser Phe Phe Glu Val Glu Ala Leu Met 325 330 335 Glu Lys Met Arg Gln Leu Gln Glu Cys Arg Asp Glu Glu Glu Ala Ser 340 345 350 Gln Glu Glu Met Ala Ser Arg Phe Glu Ile Glu Lys Arg Glu Ser Met 355 360 365 Phe Val Phe Ser Ser Asp Asp Glu Glu Val Thr Pro Ala Arg Ala Val 370 375 380 Ser Arg His Phe Glu Asp Thr Ser Tyr Gly Tyr Lys Asp Phe Ser Arg 385 390 395 400 His Gly Met His Val Pro Thr Phe Arg Val Gln Asp Tyr Cys Trp Glu 405 410 415 Asp His Gly Tyr Ser Leu Val Asn Arg Leu Tyr Pro Asp Val Gly Gln 420 425 430 Leu Ile Asp Glu Lys Phe His Ile Ala Tyr Asn Leu Thr Tyr Asn Thr 435 440 445 Met Ala Met His Lys Asp Val Asp Thr Ser Met Leu Arg Arg Ala Ile 450 455 460 Trp Asn Tyr Ile His Cys Met Phe Gly Ile Arg Tyr Asp Asp Tyr Asp 465 470 475 480 Tyr Gly Glu Ile Asn Gln Leu Leu Asp Arg Ser Phe Lys Val Tyr Ile 485 490 495 Lys Thr Val Val Cys Thr Pro Glu Lys Val Thr Lys Arg Met Tyr Asp 500 505 510 Ser Phe Trp Arg Gln Phe Lys His Ser Glu Lys Val His Val Asn Leu 515 520 525 Leu Leu Ile Glu Ala Arg Met Gln Ala Glu Leu Leu Tyr Ala Leu Arg 530 535 540 Ala Ile Thr Arg Tyr Met Thr 545 550 192484DNAHomo sapiens 19attcctcgtt agggcaggcg cggccccttc ggctccgagc tgaccctgat cagggccgag 60ttgtctcggc ggcgctgccg aggcctccac ccaggacagt ccccctcccc gggcctctct 120cctcttgcct acgagtcccc tctcctcgta ggcctctcgg atctgatatc gtggggtgag 180gtgagcaggc ccggggaggg tggttaccgc tgaggagctg cagtctctgt caagatgata 240gaggtactga caacaactga ctctcagaaa ctgctacacc agctgaatgc cctgttggaa 300caggagtcta gatgtcagcc aaaggtctgt ggtttgagac taattgagtc tgcacacgat 360aatggcctca gaatgactgc aagactaagg gactttgaag taaaagatct tcttagtcta 420actcagttct ttggctttga cacagagaca ttttctctag ctgtgaattt actggacaga 480ttcctgtcta aaatgaaggt acagcccaag caccttgggt gtgttggact gagctgcttt 540tatttggctg taaaatcaat agaagaggaa aggaatgtcc cattggcaac tgacttgatc 600cgaataagtc aatataggtt tacggtttca gacttgatga gaatggaaaa gattgtattg 660gagaaggtgt gttggaaagt caaagctact actgcctttc aatttctgca actgtattat 720tcactccttc aagagaactt gccacttgaa aggagaaata gcattaattt tgaaagacta 780gaagctcaac tgaaggcatg tcattgcagg atcatatttt ctaaagcaaa gccttctgtg 840ttggcattgt ctatcattgc attagagatc caagcacaga agtgtgtaga gttaacagaa 900ggaatagaat gtcttcagaa acattccaag ataaatggca gagatctgac cttctggcaa 960gagcttgtat ccaaatgttt aactgaatat tcatcaaata agtgttccaa accaaatgtt 1020cagaagttga aatggattgt ttctgggcgt actgcacggc aattgaagca tagctactac 1080agaataactc accttccaac aattcctgaa atggtccctt aactggatta ttacagcacc 1140aaaaaacttc tctgaagcct ttctccacaa ccttgttcta tggattccat aatgttacaa 1200tggatttaag ctatgaagcc tcaaaacatc acgagataag catgatggtc tcagacttgg 1260gaaaactgcc taatattatg ctgtagtgga attatgttta gatttgaatt catctgtgaa 1320gcattcaaat caaagctaaa agcctaaatg tgaaatgcta atgacaagcc tgagaaggta 1380aactgtgaat cttcatttct atcattgatc taactttaga tattggatca atatatttag 1440gtggtattga aaatgctatt ggaggagtca cactaatact atcaactatc agtcttccca 1500cagcttcaat cactgtcatt attctaatcc tactcctact taaattttaa gttatgaggt 1560ttatgtcaaa agcaacattt cacaaatgta cttttaaggc ataataaggg ttaacattct 1620aggcagtata aacacacccc ataatgcaag taataggtaa tctagagatg tggactttat 1680tgctatatgg gaattacatt taaatttgag ggcattttat ataaagaaaa atacagacct 1740ataaagtttg gcatattcat taagttatct tttaatattt ttttctagaa aacaggtgac 1800atttgtatct acgataaaaa tttttataca gaacctactg cctcaaactg aatcccatca 1860agaaaactag tttctattgt attagtaact caaaataaat tatcacttcg aaaacttgct 1920ttcccacact aaggtaagtt cagactagat tgaacactcc agaatttttt actacagact 1980gtttttaagt tagaagtgat ggcaatttta taaatagaga atatacttcc actgatgccc 2040ttactgtgcc aaaacaaaaa tcttaagaaa agcaagtaga caccttcata actatgaatg 2100aagctgctga agtagtgttt aggatcctcc atggcagtta gtgaatgtaa gaagtacagt 2160gttaaagtgt tgtaaacagt tactcagtgc aatgtatagc ctgagtctat ccatgatggc 2220tatatccaat ttgacatcac gttatggatc agtacacaat gaaaaaccaa agaaccacag 2280tatatcttat tcttaacttt tgtaaaccat gttttatggg taacttttta gttttcccaa 2340aaggctgata aatttcaata ttttgaatac atcattgtta attttgagtt ggcagaggta 2400aactaaccaa ctaccattat gttttagtac taagggatat acctttcaat aaagttaatg 2460aaattcaaaa aaaaaaaaaa aaaa 248420295PRTHomo sapiens 20Met Ile Glu Val Leu Thr Thr Thr Asp Ser Gln Lys Leu Leu His Gln 1 5 10 15 Leu Asn Ala Leu Leu Glu Gln Glu Ser Arg Cys Gln Pro Lys Val Cys 20 25 30 Gly Leu Arg Leu Ile Glu Ser Ala His Asp Asn Gly Leu Arg Met Thr 35 40 45 Ala Arg Leu Arg Asp Phe Glu Val Lys Asp Leu Leu Ser Leu Thr Gln 50 55 60 Phe Phe Gly Phe Asp Thr Glu Thr Phe Ser Leu Ala Val Asn Leu Leu 65 70 75 80 Asp Arg Phe Leu Ser Lys Met Lys Val Gln Pro Lys His Leu Gly Cys 85 90 95 Val Gly Leu Ser Cys Phe Tyr Leu Ala Val Lys Ser Ile Glu Glu Glu 100 105 110 Arg Asn Val Pro Leu Ala Thr Asp Leu Ile Arg Ile Ser Gln Tyr Arg 115 120 125 Phe Thr Val Ser Asp Leu Met Arg Met Glu Lys Ile Val Leu Glu Lys 130 135 140 Val Cys Trp Lys Val Lys Ala Thr Thr Ala Phe Gln Phe Leu Gln Leu 145 150 155 160 Tyr Tyr Ser Leu Leu Gln Glu Asn Leu Pro Leu Glu Arg Arg Asn Ser 165 170 175 Ile Asn Phe Glu Arg Leu Glu Ala Gln Leu Lys Ala Cys His Cys Arg 180 185 190 Ile Ile Phe Ser Lys Ala Lys Pro Ser Val Leu Ala Leu Ser Ile Ile 195 200 205 Ala Leu Glu Ile Gln Ala Gln Lys Cys Val Glu Leu Thr Glu Gly Ile 210 215 220 Glu Cys Leu Gln Lys His Ser Lys Ile Asn Gly Arg Asp Leu Thr Phe 225 230 235 240 Trp Gln Glu Leu Val Ser Lys Cys Leu Thr Glu Tyr Ser Ser Asn Lys 245 250 255 Cys Ser Lys Pro Asn Val Gln Lys Leu Lys Trp Ile Val Ser Gly Arg 260 265 270 Thr Ala Arg Gln Leu Lys His Ser Tyr Tyr Arg Ile Thr His Leu Pro 275 280 285 Thr Ile Pro Glu Met Val Pro 290 295 213729DNAHomo sapiens 21cgaaggggcg tggccaagcg caccgcctcg gggcggggcc ggcgttctag cgcatcgcgg 60ccgggtgcgt cactcgcgaa gtggaatttg cccagacaag caacatggct cggaaacgcg 120cggccggcgg ggagccgcgg ggacgcgaac tgcgcagcca gaaatccaag gccaagagca 180aggcccggcg tgaggaggag gaggaggatg cctttgaaga tgagaaaccc ccaaagaaga 240gccttctctc caaagtttca caaggaaaga ggaaaagagg ctgcagtcat cctgggggtt 300cagcagatgg tccagcaaaa aagaaagtgg ccaaggtgac tgttaaatct gaaaacctca 360aggttataaa ggatgaagcc ctcagcgatg gggatgacct cagggacttt ccaagtgacc 420tcaagaaggc acaccatctg aagagagggg ctaccatgaa tgaagacagc aatgaagaag 480aggaagaaag tgaaaatgat tgggaagagg ttgaagaact tagtgagcct gtgctgggtg 540acgtgagaga aagtacagcc ttctctcgat ctcttctgcc tgtgaagcca gtggagatag 600agattgaaac gccagagcag gcgaagacaa gagaaagaag tgaaaagata aaactggagt 660ttgagacata tcttcggagg gcgatgaaac gtttcaataa aggggtccat gaggacacac 720acaaggttca ccttctctgc ctgctagcaa atggcttcta tcgaaataac atctgcagcc 780agccagatct gcatgctatt ggcctgtcca tcatcccagc ccgctttacc agagtgctgc 840ctcgagatgt ggacacctac tacctctcaa acctggtgaa gtggttcatt ggaacattta 900cagttaatgc agaactttca gccagtgaac aagataacct gcagactaca ttggaaagga 960gatttgctat ttactctgct cgagatgatg aggaattggt ccatatattc ttactgattc 1020tccgggctct gcagctcttg acccggctgg tattgtctct acagccaatt cctctgaagt 1080cagcaacagc aaagggaaag aaaccttcca aggaaagatt gactgcggat ccaggaggct 1140cctcagaaac ttccagccaa gttctagaaa accacaccaa accaaagacc agcaaaggaa 1200ccaaacaaga ggaaaccttt gctaagggca cctgcaggcc aagtgccaaa gggaagagga 1260acaagggagg cagaaagaaa cggagcaagc cctcctccag cgaggaagat gagggcccag 1320gagacaagca ggagaaggca acccagcgac gtccgcatgg ccgggagcgg cgggtggcct 1380ccagggtgtc ttataaagag gagagtggga gtgatgaggc tggcagcggc tctgattttg 1440agctctccag tggagaagcc tctgatccct ctgatgagga ttccgaacct ggccctccaa 1500agcagaggaa agcccccgct cctcagagga caaaggctgg gtccaagagt gcctccagga 1560cccatcgtgg gagccatcgt aaggacccaa gcttgccagc ggcatcctca agctcttcaa 1620gcagtaaaag aggcaagaaa atgtgcagcg atggtgagaa ggcagaaaaa agaagcatag 1680ctggtataga ccagtggcta gaggtgttct gtgagcagga ggaaaagtgg gtatgtgtag 1740actgtgtgca cggtgtggtg ggccagcctc tgacctgtta caagtacgcc accaagccca 1800tgacctatgt ggtgggcatt gacagtgacg gctgggtccg agatgtcaca cagaggtacg 1860acccagtctg gatgacagtg acccgcaagt gccgggttga tgctgagtgg tgggccgaga 1920ccttgagacc ataccagagc ccatttatgg acagggagaa gaaagaagac ttggagtttc 1980aggcaaaaca catggaccag cctttgccca ctgccattgg cttatataag aaccaccctc 2040tgtatgccct gaagcggcat ctcctgaaat atgaggccat ctatcccgag acagctgcca 2100tccttgggta ttgtcgtgga gaagcggtct actccaggga ttgtgtgcac actctgcatt 2160ccagggacac gtggctgaag aaagcaagag tggtgaggct tggagaagta ccctacaaga 2220tggtgaaagg cttttctaac cgtgctcgga aagcccgact tgctgagccc cagctgcggg 2280aagaaaatga cctgggcctg tttggctact ggcagacaga ggagtatcag cccccagtgg 2340ccgtggacgg gaaggtgccc cggaacgagt ttgggaatgt gtacctcttc ctgcccagca 2400tgatgcctat tggctgtgtc cagctgaacc tgcccaatct

acaccgcgtg gcccgcaagc 2460tggacatcga ctgtgtccag gccatcactg gctttgattt ccatggcggc tactcccatc 2520ccgtgactga tggatacatc gtctgcgagg aattcaaaga cgtgctcctg actgcctggg 2580aaaatgagca ggcagtcatt gaaaggaagg agaaggagaa aaaggagaag cgggctctag 2640ggaactggaa gttgctggcc aaaggtctgc tcatcaggga gaggctgaag cgtcgctacg 2700ggcccaagag tgaggcagca gctccccaca cagatgcagg aggtggactc tcttctgatg 2760aagaggaggg gaccagctct caagcagaag cggccaggat actggctgcc tcctggcctc 2820aaaaccgaga agatgaagaa aagcagaagc tgaagggtgg gcccaagaag accaaaaggg 2880aaaagaaagc agcagcttcc cacctgttcc catttgagca gctgtgagct gagcgcccac 2940tagaggggca cccaccagtt gctgctgccc cactacaggc cccacacctg ccctgggcat 3000gcccagcccc tggtggtggg ggcttctctg ctgagaaggc aaactgaggc agcatgcacg 3060gaggcggggt caggggagac gaggccaagc tgaggaggtg ctgcaggtcc cgtctggctc 3120cagcccttgt cagattcacc cagggtgaag ccttcaaagc tttttgctac caaagcccac 3180tcaccctttg agctacagaa cactttgcta ggagatactc ttctgcctcc tagacctgtt 3240ctttccatct ttagaaacat cagtttttgt atggaagcca ccgggagatt tctggatggt 3300ggtgcatccg tgaatgcgct gatcgtttct tccagttaga gtcttcatct gtccgacaag 3360ttcactcgcc tcggttgcgg acctaggacc atttctctgc aggccactta ccttcccctg 3420agtcaggctt actaatgctg ccctcactgc ctctttgcag taggggagag agcagagaag 3480tacaggtcat ctgctgggat ctagttttcc aagtaacatt ttgtggtgac agaagcctaa 3540aaaaagctaa aatcaggaaa gaaaaggaaa aatacgaatt gaaaattaag gaaatgttag 3600taaaatagat gagtgttaaa ctagattgta ttcattacta gataaaatgt ataaagctct 3660ctgtactaag gagaaatgac ttttataaca ttttgagaaa ataataaagc atttatctaa 3720aaaaaaaaa 372922940PRTHomo sapiens 22Met Ala Arg Lys Arg Ala Ala Gly Gly Glu Pro Arg Gly Arg Glu Leu 1 5 10 15 Arg Ser Gln Lys Ser Lys Ala Lys Ser Lys Ala Arg Arg Glu Glu Glu 20 25 30 Glu Glu Asp Ala Phe Glu Asp Glu Lys Pro Pro Lys Lys Ser Leu Leu 35 40 45 Ser Lys Val Ser Gln Gly Lys Arg Lys Arg Gly Cys Ser His Pro Gly 50 55 60 Gly Ser Ala Asp Gly Pro Ala Lys Lys Lys Val Ala Lys Val Thr Val 65 70 75 80 Lys Ser Glu Asn Leu Lys Val Ile Lys Asp Glu Ala Leu Ser Asp Gly 85 90 95 Asp Asp Leu Arg Asp Phe Pro Ser Asp Leu Lys Lys Ala His His Leu 100 105 110 Lys Arg Gly Ala Thr Met Asn Glu Asp Ser Asn Glu Glu Glu Glu Glu 115 120 125 Ser Glu Asn Asp Trp Glu Glu Val Glu Glu Leu Ser Glu Pro Val Leu 130 135 140 Gly Asp Val Arg Glu Ser Thr Ala Phe Ser Arg Ser Leu Leu Pro Val 145 150 155 160 Lys Pro Val Glu Ile Glu Ile Glu Thr Pro Glu Gln Ala Lys Thr Arg 165 170 175 Glu Arg Ser Glu Lys Ile Lys Leu Glu Phe Glu Thr Tyr Leu Arg Arg 180 185 190 Ala Met Lys Arg Phe Asn Lys Gly Val His Glu Asp Thr His Lys Val 195 200 205 His Leu Leu Cys Leu Leu Ala Asn Gly Phe Tyr Arg Asn Asn Ile Cys 210 215 220 Ser Gln Pro Asp Leu His Ala Ile Gly Leu Ser Ile Ile Pro Ala Arg 225 230 235 240 Phe Thr Arg Val Leu Pro Arg Asp Val Asp Thr Tyr Tyr Leu Ser Asn 245 250 255 Leu Val Lys Trp Phe Ile Gly Thr Phe Thr Val Asn Ala Glu Leu Ser 260 265 270 Ala Ser Glu Gln Asp Asn Leu Gln Thr Thr Leu Glu Arg Arg Phe Ala 275 280 285 Ile Tyr Ser Ala Arg Asp Asp Glu Glu Leu Val His Ile Phe Leu Leu 290 295 300 Ile Leu Arg Ala Leu Gln Leu Leu Thr Arg Leu Val Leu Ser Leu Gln 305 310 315 320 Pro Ile Pro Leu Lys Ser Ala Thr Ala Lys Gly Lys Lys Pro Ser Lys 325 330 335 Glu Arg Leu Thr Ala Asp Pro Gly Gly Ser Ser Glu Thr Ser Ser Gln 340 345 350 Val Leu Glu Asn His Thr Lys Pro Lys Thr Ser Lys Gly Thr Lys Gln 355 360 365 Glu Glu Thr Phe Ala Lys Gly Thr Cys Arg Pro Ser Ala Lys Gly Lys 370 375 380 Arg Asn Lys Gly Gly Arg Lys Lys Arg Ser Lys Pro Ser Ser Ser Glu 385 390 395 400 Glu Asp Glu Gly Pro Gly Asp Lys Gln Glu Lys Ala Thr Gln Arg Arg 405 410 415 Pro His Gly Arg Glu Arg Arg Val Ala Ser Arg Val Ser Tyr Lys Glu 420 425 430 Glu Ser Gly Ser Asp Glu Ala Gly Ser Gly Ser Asp Phe Glu Leu Ser 435 440 445 Ser Gly Glu Ala Ser Asp Pro Ser Asp Glu Asp Ser Glu Pro Gly Pro 450 455 460 Pro Lys Gln Arg Lys Ala Pro Ala Pro Gln Arg Thr Lys Ala Gly Ser 465 470 475 480 Lys Ser Ala Ser Arg Thr His Arg Gly Ser His Arg Lys Asp Pro Ser 485 490 495 Leu Pro Ala Ala Ser Ser Ser Ser Ser Ser Ser Lys Arg Gly Lys Lys 500 505 510 Met Cys Ser Asp Gly Glu Lys Ala Glu Lys Arg Ser Ile Ala Gly Ile 515 520 525 Asp Gln Trp Leu Glu Val Phe Cys Glu Gln Glu Glu Lys Trp Val Cys 530 535 540 Val Asp Cys Val His Gly Val Val Gly Gln Pro Leu Thr Cys Tyr Lys 545 550 555 560 Tyr Ala Thr Lys Pro Met Thr Tyr Val Val Gly Ile Asp Ser Asp Gly 565 570 575 Trp Val Arg Asp Val Thr Gln Arg Tyr Asp Pro Val Trp Met Thr Val 580 585 590 Thr Arg Lys Cys Arg Val Asp Ala Glu Trp Trp Ala Glu Thr Leu Arg 595 600 605 Pro Tyr Gln Ser Pro Phe Met Asp Arg Glu Lys Lys Glu Asp Leu Glu 610 615 620 Phe Gln Ala Lys His Met Asp Gln Pro Leu Pro Thr Ala Ile Gly Leu 625 630 635 640 Tyr Lys Asn His Pro Leu Tyr Ala Leu Lys Arg His Leu Leu Lys Tyr 645 650 655 Glu Ala Ile Tyr Pro Glu Thr Ala Ala Ile Leu Gly Tyr Cys Arg Gly 660 665 670 Glu Ala Val Tyr Ser Arg Asp Cys Val His Thr Leu His Ser Arg Asp 675 680 685 Thr Trp Leu Lys Lys Ala Arg Val Val Arg Leu Gly Glu Val Pro Tyr 690 695 700 Lys Met Val Lys Gly Phe Ser Asn Arg Ala Arg Lys Ala Arg Leu Ala 705 710 715 720 Glu Pro Gln Leu Arg Glu Glu Asn Asp Leu Gly Leu Phe Gly Tyr Trp 725 730 735 Gln Thr Glu Glu Tyr Gln Pro Pro Val Ala Val Asp Gly Lys Val Pro 740 745 750 Arg Asn Glu Phe Gly Asn Val Tyr Leu Phe Leu Pro Ser Met Met Pro 755 760 765 Ile Gly Cys Val Gln Leu Asn Leu Pro Asn Leu His Arg Val Ala Arg 770 775 780 Lys Leu Asp Ile Asp Cys Val Gln Ala Ile Thr Gly Phe Asp Phe His 785 790 795 800 Gly Gly Tyr Ser His Pro Val Thr Asp Gly Tyr Ile Val Cys Glu Glu 805 810 815 Phe Lys Asp Val Leu Leu Thr Ala Trp Glu Asn Glu Gln Ala Val Ile 820 825 830 Glu Arg Lys Glu Lys Glu Lys Lys Glu Lys Arg Ala Leu Gly Asn Trp 835 840 845 Lys Leu Leu Ala Lys Gly Leu Leu Ile Arg Glu Arg Leu Lys Arg Arg 850 855 860 Tyr Gly Pro Lys Ser Glu Ala Ala Ala Pro His Thr Asp Ala Gly Gly 865 870 875 880 Gly Leu Ser Ser Asp Glu Glu Glu Gly Thr Ser Ser Gln Ala Glu Ala 885 890 895 Ala Arg Ile Leu Ala Ala Ser Trp Pro Gln Asn Arg Glu Asp Glu Glu 900 905 910 Lys Gln Lys Leu Lys Gly Gly Pro Lys Lys Thr Lys Arg Glu Lys Lys 915 920 925 Ala Ala Ala Ser His Leu Phe Pro Phe Glu Gln Leu 930 935 940 234154DNAHomo sapiens 23acttggacgc gcttgcggag gattgcgttg acgagactct tatttattgt caccaacctg 60tggtggaatt tgcagttgca cattggatct gattcgcccc gccccgaatg acgcctgccc 120ggaggcagtg aaagtacagc cgcgccgccc caagtcagcc tggacacata aatcagcacg 180cggccggaga accccgcaat ctctgcgccc acaaaataca ccgacgatgc ccgatctact 240ttaagggctg aaacccacgg gcctgagaga ctataagagc gttccctacc gccatggaac 300aacggggaca gaacgccccg gccgcttcgg gggcccggaa aaggcacggc ccaggaccca 360gggaggcgcg gggagccagg cctgggcccc gggtccccaa gacccttgtg ctcgttgtcg 420ccgcggtcct gctgttggtc tcagctgagt ctgctctgat cacccaacaa gacctagctc 480cccagcagag agcggcccca caacaaaaga ggtccagccc ctcagaggga ttgtgtccac 540ctggacacca tatctcagaa gacggtagag attgcatctc ctgcaaatat ggacaggact 600atagcactca ctggaatgac ctccttttct gcttgcgctg caccaggtgt gattcaggtg 660aagtggagct aagtccctgc accacgacca gaaacacagt gtgtcagtgc gaagaaggca 720ccttccggga agaagattct cctgagatgt gccggaagtg ccgcacaggg tgtcccagag 780ggatggtcaa ggtcggtgat tgtacaccct ggagtgacat cgaatgtgtc cacaaagaat 840caggtacaaa gcacagtggg gaagtcccag ctgtggagga gacggtgacc tccagcccag 900ggactcctgc ctctccctgt tctctctcag gcatcatcat aggagtcaca gttgcagccg 960tagtcttgat tgtggctgtg tttgtttgca agtctttact gtggaagaaa gtccttcctt 1020acctgaaagg catctgctca ggtggtggtg gggaccctga gcgtgtggac agaagctcac 1080aacgacctgg ggctgaggac aatgtcctca atgagatcgt gagtatcttg cagcccaccc 1140aggtccctga gcaggaaatg gaagtccagg agccagcaga gccaacaggt gtcaacatgt 1200tgtcccccgg ggagtcagag catctgctgg aaccggcaga agctgaaagg tctcagagga 1260ggaggctgct ggttccagca aatgaaggtg atcccactga gactctgaga cagtgcttcg 1320atgactttgc agacttggtg ccctttgact cctgggagcc gctcatgagg aagttgggcc 1380tcatggacaa tgagataaag gtggctaaag ctgaggcagc gggccacagg gacaccttgt 1440acacgatgct gataaagtgg gtcaacaaaa ccgggcgaga tgcctctgtc cacaccctgc 1500tggatgcctt ggagacgctg ggagagagac ttgccaagca gaagattgag gaccacttgt 1560tgagctctgg aaagttcatg tatctagaag gtaatgcaga ctctgccatg tcctaagtgt 1620gattctcttc aggaagtcag accttccctg gtttaccttt tttctggaaa aagcccaact 1680ggactccagt cagtaggaaa gtgccacaat tgtcacatga ccggtactgg aagaaactct 1740cccatccaac atcacccagt ggatggaaca tcctgtaact tttcactgca cttggcatta 1800tttttataag ctgaatgtga taataaggac actatggaaa tgtctggatc attccgtttg 1860tgcgtacttt gagatttggt ttgggatgtc attgttttca cagcactttt ttatcctaat 1920gtaaatgctt tatttattta tttgggctac attgtaagat ccatctacac agtcgttgtc 1980cgacttcact tgatactata tgatatgaac cttttttggg tggggggtgc ggggcagttc 2040actctgtctc ccaggctgga gtgcaatggt gcaatcttgg ctcactatag ccttgacctc 2100tcaggctcaa gcgattctcc cacctcagcc atccaaatag ctgggaccac aggtgtgcac 2160caccacgccc ggctaatttt ttgtattttg tctagatata ggggctctct atgttgctca 2220gggtggtctc gaattcctgg actcaagcag tctgcccacc tcagactccc aaagcggtgg 2280aattagaggc gtgagccccc atgcttggcc ttacctttct acttttataa ttctgtatgt 2340tattatttta tgaacatgaa gaaactttag taaatgtact tgtttacata gttatgtgaa 2400tagattagat aaacataaaa ggaggagaca tacaatgggg gaagaagaag aagtcccctg 2460taagatgtca ctgtctgggt tccagccctc cctcagatgt actttggctt caatgattgg 2520caacttctac aggggccagt cttttgaact ggacaacctt acaagtatat gagtattatt 2580tataggtagt tgtttacata tgagtcggga ccaaagagaa ctggatccac gtgaagtcct 2640gtgtgtggct ggtccctacc tgggcagtct catttgcacc catagccccc atctatggac 2700aggctgggac agaggcagat gggttagatc acacataaca atagggtcta tgtcatatcc 2760caagtgaact tgagccctgt ttgggctcag gagatagaag acaaaatctg tctcccacgt 2820ctgccatggc atcaaggggg aagagtagat ggtgcttgag aatggtgtga aatggttgcc 2880atctcaggag tagatggccc ggctcacttc tggttatctg tcaccctgag cccatgagct 2940gccttttagg gtacagattg cctacttgag gaccttggcc gctctgtaag catctgactc 3000atctcagaaa tgtcaattct taaacactgt ggcaacagga cctagaatgg ctgacgcatt 3060aaggttttct tcttgtgtcc tgttctatta ttgttttaag acctcagtaa ccatttcagc 3120ctctttccag caaacccttc tccatagtat ttcagtcatg gaaggatcat ttatgcaggt 3180agtcattcca ggagtttttg gtcttttctg tctcaaggca ttgtgtgttt tgttccggga 3240ctggtttggg tgggacaaag ttagaattgc ctgaagatca cacattcaga ctgttgtgtc 3300tgtggagttt taggagtggg gggtgacctt tctggtcttt gcacttccat cctctcccac 3360ttccatctgg catcccacgc gttgtcccct gcacttctgg aaggcacagg gtgctgctgc 3420ctcctggtct ttgcctttgc tgggccttct gtgcaggacg ctcagcctca gggctcagaa 3480ggtgccagtc cggtcccagg tcccttgtcc cttccacaga ggccttccta gaagatgcat 3540ctagagtgtc agccttatca gtgtttaaga tttttctttt atttttaatt tttttgagac 3600agaatctcac tctctcgccc aggctggagt gcaacggtac gatcttggct cagtgcaacc 3660tccgcctcct gggttcaagc gattctcgtg cctcagcctc cggagtagct gggattgcag 3720gcacccgcca ccacgcctgg ctaatttttg tatttttagt agagacgggg tttcaccatg 3780ttggtcaggc tggtctcgaa ctcctgacct caggtgatcc accttggcct ccgaaagtgc 3840tgggattaca ggcgtgagcc accagccagg ccaagctatt cttttaaagt aagcttcctg 3900acgacatgaa ataattgggg gttttgttgt ttagttacat taggctttgc tatatcccca 3960ggccaaatag catgtgacac aggacagcca tagtatagtg tgtcactcgt ggttggtgtc 4020ctttcatgct tctgccctgt caaaggtccc tatttgaaat gtgttataat acaaacaagg 4080aagcacattg tgtacaaaat acttatgtat ttatgaatcc atgaccaaat taaatatgaa 4140accttatata aaaa 415424440PRTHomo sapiens 24Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys 1 5 10 15 Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly Pro 20 25 30 Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu 35 40 45 Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro Gln 50 55 60 Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu 65 70 75 80 Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser 85 90 95 Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe 100 105 110 Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro 115 120 125 Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe 130 135 140 Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys 145 150 155 160 Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile 165 170 175 Glu Cys Val His Lys Glu Ser Gly Thr Lys His Ser Gly Glu Val Pro 180 185 190 Ala Val Glu Glu Thr Val Thr Ser Ser Pro Gly Thr Pro Ala Ser Pro 195 200 205 Cys Ser Leu Ser Gly Ile Ile Ile Gly Val Thr Val Ala Ala Val Val 210 215 220 Leu Ile Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp Lys Lys Val 225 230 235 240 Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly Asp Pro Glu 245 250 255 Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp Asn Val Leu 260 265 270 Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gln Val Pro Glu Gln Glu 275 280 285 Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly Val Asn Met Leu Ser 290 295 300 Pro Gly Glu Ser Glu His Leu Leu Glu Pro Ala Glu Ala Glu Arg Ser 305 310 315 320 Gln Arg Arg Arg Leu Leu Val Pro Ala Asn Glu Gly Asp Pro Thr Glu 325 330 335 Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala Asp Leu Val Pro Phe Asp 340 345 350 Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp Asn Glu Ile 355 360 365 Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr Leu Tyr Thr 370 375 380 Met Leu Ile Lys Trp Val Asn Lys Thr Gly Arg Asp Ala Ser Val His 385 390 395 400 Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly Glu Arg Leu Ala Lys Gln 405 410 415 Lys Ile Glu Asp His Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu 420 425 430 Gly Asn Ala Asp Ser Ala Met Ser 435 440 253134DNAHomo sapiens 25agtcggagcc gggcttgccc gggcatgtgg gagctgccgg ctttccggac gccacgtgca 60gaccggaaga gacacgcggg gcttcaggct gctgccccat tggaagatta ctccccaggc 120ttcccttgcc ccaagcagtg agctgactgg aatggtaccc cgggaggccc ctgagtctgc 180tcagtgcctg tgcccttccc tcaccatccc aaatgccaag gatgtgcttc ggaagaggca 240caagagaagg agccgacagc accagcggtt catggcccgg aaggccttgc tgcaggagca 300ggggctgctg agcatgcctc cagaaccagg gtcctcccca ctgcccaccc ctttcggggc 360agcgacagca actgaagctg ccagcagtgg gaagcagtgt ctgagggctg gatctggcag 420tgccccatgc

agcagaaggc ctgctcccgg gaaagcctca gggcccttgc ccagcaagtg 480tgtggctatc gactgtgaga tggtgggcac gggaccccga gggcgggtaa gcgagctggc 540ccgctgttcc attgtgagct accatggcaa tgtcctctat gacaagtaca tcaggcctga 600gatgcccatc gctgactacc gtacccgctg gagtggcatc actcggcagc acatgcgcaa 660ggctgtcccc ttccaggtgg cccagaaaga gatccttaag ctcctgaagg gcaaggtggt 720ggtggggcac gcgctgcaca acgacttcca ggcgctcaag tatgtccacc ctcggagcca 780gacccgggat acgacctatg tcccaaactt cctcagcgag cccggcctcc acacccgggc 840ccgggtctct ctaaaggacc tggccctgca gctgctgcac aagaagatcc aggtgggcca 900gcacgggcac tcatcagtag aagatgccac gacagccatg gagctctacc ggctggtgga 960ggtgcagtgg gaacagcagg aggcccgcag cctctggacc tgccccgagg acagagaacc 1020tgacagcagc acagacatgg aacagtacat ggaggaccag tactggcccg atgacctggc 1080ccacggcagc agaggaggag ccagggaggc acaggacaga aggaattgag aagggggcgg 1140ggctccctgg ctgggcttcc ggtgtggccg gtaggaagtg ggggccagga gagcagcggg 1200cactccttcc tgggcagggt ggggcaggat gcagtgagcc agccccaggg ccagaggagt 1260aggggtcatc tgttaccttg acaccctctg cacacagcat agccctctct ctctccaggg 1320ctgttggttc tttctcctga ctcctgtggt tttgctaatg gcactttaca gactccatgg 1380agatgtcagg tggaccatct tctagggccc agcaggagta gggaatgtgc caacagactg 1440cccaggttgc cgtggccttc cccacccccc agatctcctg agtcatcatg ctgtgctaat 1500gaaagggatc atatcatcct ctctggggat ggtgggtggg ggtgtcaata tcctggagct 1560cccttacccc aactcaatga cttgggggta aagttctctt ccttttgttg cctacctctt 1620cctccactca tttgggttca gaataaacat gccctgaagt taaagaggag ttaagtccta 1680aaggaggcat ttcttcccca cctccctgac ctggaactct ggctacagcc attgtaggaa 1740ctccgtgcct ggcctgtcag ctccctgcta ggctacagtg gaatagcaga gcccacaggc 1800ttctcgtggg gagttggctc cttaacattt cttggcaaca gaaagcccca ggcacagctc 1860agggaggagg gaaggcaggt aagctttgga cgagaactgg catatttatt ttgacccaaa 1920tcagggattt ccccagtcca cccagtactg ggctcttaag caaaagtctg agaaacaaga 1980cagtggtttg aattctgggg cctttgtgta ggattgtgcc tgacctttat ttatttatta 2040acagcagggc cactcgtcta gggcagtgga gtctgcgtgt ctcctggggc tggggcaggg 2100cattggcagt tacgcagtgg ccctgaacct ggtctggtgc ccccgaacct ggtctgatgc 2160cccctcagct ctttgacaat cactgtggct gttgggtttt ctcctatttc taaaaatgtt 2220ctcttctttc ctaagtgaca gttttgaagt attggataac caagagctca ggtcacacag 2280accttggagc cccatctttg cttgcagctt agctttgaga tactaagcaa gcgagggact 2340tcactcttga ggctgttttt tcctcatctg taaagtgggg atagtggtac ggcctacctc 2400atagggttgt aatgagcact aaatgtgaag tgcttggcac agtgcctggc acatggtgag 2460ccacagctac tgtgagtttc tgtttatccc cctttttttt ttttaagccc attgtttatt 2520ctttgagaat ttgttgtaac ttcttcagga taacacctga gtccacaggc tgagcagctg 2580tggcccagac agaactgctc cggcttggct gttccagcag gtggggcgct ggcctcggtg 2640agggcacagc agcaaggttc acggatatcc gtgtgtcttg tctgtggcca ccaggcacag 2700gtttggcttc cggtcagtgt cccgacactg tgcgggaggt gacaacagag caaagcagcg 2760caggggtcag ggaggtacag acactgctga aatcacacta ccccaccctc agctgaagcc 2820ccacgttcca caaacttggg gtcatagatt gtccagtcac tggctccctc cctgtcagca 2880cagcacagag gaaggggcta actgaatctt ttaccacttc tggcctggct ccagaacttt 2940gttctagatt ccttaaaagt cggtagctga tgtcaaactc aattgagcag tagctttgat 3000cccttggtct gggggtcgaa ggaagatggt gctgttatca gcggggaaat gtactattta 3060agatcagctt tgttgtaaaa ccatttgttc tagaataaaa ctcaattgga aacgtgaaaa 3120aaaaaaaaaa aaaa 313426325PRTHomo sapiens 26Met Val Pro Arg Glu Ala Pro Glu Ser Ala Gln Cys Leu Cys Pro Ser 1 5 10 15 Leu Thr Ile Pro Asn Ala Lys Asp Val Leu Arg Lys Arg His Lys Arg 20 25 30 Arg Ser Arg Gln His Gln Arg Phe Met Ala Arg Lys Ala Leu Leu Gln 35 40 45 Glu Gln Gly Leu Leu Ser Met Pro Pro Glu Pro Gly Ser Ser Pro Leu 50 55 60 Pro Thr Pro Phe Gly Ala Ala Thr Ala Thr Glu Ala Ala Ser Ser Gly 65 70 75 80 Lys Gln Cys Leu Arg Ala Gly Ser Gly Ser Ala Pro Cys Ser Arg Arg 85 90 95 Pro Ala Pro Gly Lys Ala Ser Gly Pro Leu Pro Ser Lys Cys Val Ala 100 105 110 Ile Asp Cys Glu Met Val Gly Thr Gly Pro Arg Gly Arg Val Ser Glu 115 120 125 Leu Ala Arg Cys Ser Ile Val Ser Tyr His Gly Asn Val Leu Tyr Asp 130 135 140 Lys Tyr Ile Arg Pro Glu Met Pro Ile Ala Asp Tyr Arg Thr Arg Trp 145 150 155 160 Ser Gly Ile Thr Arg Gln His Met Arg Lys Ala Val Pro Phe Gln Val 165 170 175 Ala Gln Lys Glu Ile Leu Lys Leu Leu Lys Gly Lys Val Val Val Gly 180 185 190 His Ala Leu His Asn Asp Phe Gln Ala Leu Lys Tyr Val His Pro Arg 195 200 205 Ser Gln Thr Arg Asp Thr Thr Tyr Val Pro Asn Phe Leu Ser Glu Pro 210 215 220 Gly Leu His Thr Arg Ala Arg Val Ser Leu Lys Asp Leu Ala Leu Gln 225 230 235 240 Leu Leu His Lys Lys Ile Gln Val Gly Gln His Gly His Ser Ser Val 245 250 255 Glu Asp Ala Thr Thr Ala Met Glu Leu Tyr Arg Leu Val Glu Val Gln 260 265 270 Trp Glu Gln Gln Glu Ala Arg Ser Leu Trp Thr Cys Pro Glu Asp Arg 275 280 285 Glu Pro Asp Ser Ser Thr Asp Met Glu Gln Tyr Met Glu Asp Gln Tyr 290 295 300 Trp Pro Asp Asp Leu Ala His Gly Ser Arg Gly Gly Ala Arg Glu Ala 305 310 315 320 Gln Asp Arg Arg Asn 325

Claims

1. A method of predicting the sensitivity of a cancer patient for treatment with a Human Double Minute 2 inhibitor (MDM2i), the method comprising: a) measuring differential gene expression of at least one biomarker selected from Table 2 in a cancer sample obtained from the patient; and b) comparing the differential gene expression of the at least one biomarker with gene expression of said biomarker in a control sample, wherein the increase or decrease in gene expression comparison indicates that the patient is sensitive to treatment with an MDM2i.

2. The method of claim 1, wherein more than one biomarker is selected from Table 2.

3. The method of claim 1, comprising the biomarkers MDM2, CDKN1A, ZMAT3, DDB2, FDXR, RPS27L, BAX, RRM2B, SESN1, CCNG1, XPC, TNFRSF10B and AEN.

4. The method of claim 1, wherein comparing the differential gene expression of the at least one biomarker with gene expression of a control sample indicates a functional p53 gene pathway.

5. The method of claim 1, wherein the cancer sample is selected from the group consisting of breast, lung, pancreas, ovary, central nervous system (CNS), endometrium, stomach, large intestine, colon, esophagus, bone, urinary tract, hematopoietic, lymphoid, liver, skin, melanoma, kidney, soft tissue sarcoma and pleura.

6. The method of claim 1, wherein a nucleic acid or protein of at least one biomarker is measured.

7. The method of claim 1, wherein the expression of the at least one biomarker is increased in the cancer sample when compared to a control sample.

8. The method of claim 1, wherein the MDM2i is selected from Table 1.

9.-16. (canceled)

17. A method of predicting the sensitivity of a cancer cell to a Human Double Minute 2 inhibitor (MDM2i), the method comprising: a) obtaining a cancer sample from a cancer patient, b) measuring differential gene expression of at least two biomarkers selected from Table 2 in the cell; and c) comparing the differential gene expression of the at least two biomarkers selected from Table 2 with gene expression of the at least two biomarkers from a normal or control cell.

18. The method of claim 17, wherein the MDM2i is selected from Table 1.

19. The method of claim 17, wherein more than three biomarkers are selected from Table 2.

20. The method of claim 17, comprising the biomarkers MDM2, CDKN1A, ZMAT3, DDB2, FDXR, RPS27L, BAX, RRM2B, SESN1, CCNG1, XPC, TNFRSF10B and AEN.

21. The method of claim 17, wherein comparing the differential gene expression of the at least two biomarkers with gene expression of a control sample indicates a functional p53 gene pathway.

22. The method of claim 17, wherein the cancer sample is selected from the group consisting of breast, lung, pancreas, ovary, central nervous system (CNS), endometrium, stomach, large intestine, colon, esophagus, bone, urinary tract, hematopoietic, lymphoid, liver, skin, melanoma, kidney, soft tissue sarcoma and pleura.

23. The method of claim 17, wherein a nucleic acid or protein of at least two biomarkers is measured.

24. The method of claim 17, wherein the gene expression of the at least two biomarkers is increased in the cancer cell.

25.-48. (canceled)