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1. (WO2018226685) METHODS FOR SENSITIZING CANCER CELLS TO T CELL-MEDIATED KILLING BY MODULATING MOLECULAR PATHWAYS
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What is claimed is:

1. A method of sensitizing cancer cells in a subject afflicted with a cancer to cytotoxic T cell-mediated killing comprising administering to the subject a therapeutically effective amount of an agent that modulates the copy number, amount, and/or activity of at least one biomarker listed in Tables 1-9 in the cancer cells, thereby sensitizing the cancer cells in the subject to cytotoxic T cell-mediated killing.

2. The method of claim 1, wherein the agent downregulates the copy number, amount, and/or activity of at least one biomarker listed in Table 1, 5, 7 or 9.

3. The method of claim 2, wherein the agent is a small molecule inhibitor, CRISPR guide RNA (gRNA), RNA interfering agent, antisense oligonucleotide, peptide or peptidomimetic inhibitor, aptamer, antibody, or intrabody.

4. The method of claim 3, wherein the RNA interfering agent is a small interfering RNA (siRNA), CRISPR RNA (crRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwi-interacting RNA (piRNA).

5. The method of claim 3, wherein the RNA interfering agent is a CRISPR guide RNA (gRNA).

6. The method of claim 3, wherein the agent comprises an antibody and/or intrabody, or an antigen binding fragment thereof, which specifically binds to the at least one biomarker listed in Table 1, 5, 7 or 9.

7. The method of claim 6, wherein the antibody and/or intrabody, or antigen binding fragment thereof, is murine, chimeric, humanized, composite, or human.

8. The method of claim 6 or 7, wherein the antibody and/or intrabody, or antigen binding fragment thereof, is detectably labeled, comprises an effector domain, comprises an Fc domain, and/or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabodies fragments.

9. The method of any one of claims 6-8, wherein the antibody and/or intrabody, or antigen binding fragment thereof, is conjugated to a cytotoxic agent.

10. The method of claim 9, wherein the cytotoxic agent is selected from the group consisting of a chemotherapeutic agent, a biologic agent, a toxin, and a radioactive isotope.

11. The method of claim 1, wherein the agent upregulates the copy number, amount, and/or activity of at least one biomarker listed in Table 2, 4, 6 or 8.

12. The method of claim 11, wherein the agent is a nucleic acid molecule encoding the one or more biomarkers listed in Table 2, 4, 6 or 8 or fragment thereof, a polypeptide of the one or more biomarkers listed in Table 2, 4, 6 or 8 or fragment(s) thereof, or a small molecule that binds to the one or more biomarkers listed in Table 2, 4, 6 or 8.

13. The method of any one of claims 1-12, further comprising treating the cancer in the subject by administering to the subject at least one immunotherapy.

14. The method of claim 13, wherein the immunotherapy is cell-based.

15. The method of claim 13, wherein the immunotherapy comprises an cancer vaccine, adoptive T cell therapies, and/or virus.

16. The method of claim 13, wherein the immunotherapy inhibits an immune checkpoint and/or inhibits TNF alpha.

17. The method of claim 16, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, and A2aR.

18. The method of claim 17, wherein the immune checkpoint is selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4.

19. The method of claim 18, wherein the immune checkpoint is PD-1 or CTLA-4.

20. The method of any one of claims 1-19, wherein the agent reduces the number of proliferating cells in the cancer and/or reduces the volume or size of a tumor comprising the cancer cells.

21. The method of any one of claims 1-20, wherein the agent increases the amount of CD8+ T cells, CD4+ T cells, granzyme B+CD8+ T cells, and/or CD45+ immune cells infiltrating a tumor comprising the cancer cells.

22. The method of any one of claims 1-21, wherein the CD45+ immune cells express higher levels of genes associated with productive antitumor immunity.

23. The method of claim 22, wherein the genes are related to IFN-γ response, IFN-a response, and/or tumor necrosis factor a signaling via F-KB .

24. The method of any one of claims 1-22, wherein the agent increases the percentage of dendritic cells within the CD45+ immune cells.

25. The method of any one of claims 1-214, wherein the agent increases the ratio of tumor-inhibitory Ml -like macropahges to tumor-promoting M2-like macrophages within the CD45+immune cells.

26. The method of any one of claims 1-25, wherein the agent increases the responsiveness of cancer cells to IFN-γ and/or IFN-a.

27. The method of any one of claims 1-26, wherein the agent increases the production of chemokines in response to IFN-γ in cancer cells.

28. The method of claim 27, wherein the chemokine is CXCL9 and/or CXCL10.

29. The method of any one of claims 1-28, wherein the agent increases cancer cell surface levels of H2-Kb and/or PD-L1 in response to IFN-γ.

30. The method of any one of claims 1-29, wherein the agent increases chromatin accessibility for IFN-y-responsive genes in cancer cells.

31. The method of any one of claims 1-30, wherein the agent downregulates genes associated with mTORCl pathway and/or cholesterol homeostasis in cancer cells.

32. The method of any one of claims 1-31, wherein the agent sensitizes cancer cells to immune checkpoint blockade therapy.

33. The methof of claim 32, wherein the immune checkpoint is PD-1 and/or CTLA-4.

34. The method of any one of claims 1-33, wherein the agent is administered in a pharmaceutically acceptable.

35. The method of any one of claims 1-34, further comprising administering to the subject at least one additional therapeutic agent or regimen for treating the cancer.

36. A method of sensitizing cancer cells to cytotoxic T cell-mediated killing comprising contacting the cancer cells with 1) an agent that modulates the copy number, amount, and/or activity of at least one biomarker listed in Tables 1-9 in the cancer cells and 2) cytotoxic T cells, thereby sensitizing the cancer cells to cytotoxic T cell-mediated killing.

37. The method of claim 36, wherein the agent downregulates the copy number, amount, and/or activity of at least one biomarker listed in Table 1, 5, 7 or 9.

38. The method of claim 37, wherein the agent is a small molecule inhibitor, CRISPR guide RNA (gRNA), RNA interfering agent, antisense oligonucleotide, peptide or peptidomimetic inhibitor, aptamer, antibody, or intrabody.

39. The method of claim 38, wherein the RNA interfering agent is a small interfering RNA (siRNA), CRISPR RNA (crRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwi-interacting RNA (piRNA).

40. The method of claim 38, wherein the RNA interfering agent is a CRISPR guide RNA (gRNA).

41. The method of claim 38, wherein the agent comprises an antibody and/or intrabody, or an antigen binding fragment thereof, which specifically binds to the at least one biomarker listed in Table 1, 5, 7 or 9.

42. The method of claim 41, wherein the antibody and/or intrabody, or antigen binding fragment thereof, is murine, chimeric, humanized, composite, or human.

43. The method of claim 41 or 42, wherein the antibody and/or intrabody, or antigen binding fragment thereof, is detectably labeled, comprises an effector domain, comprises an Fc domain, and/or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabodies fragments.

44. The method of any one of claims 41-43, wherein the antibody and/or intrabody, or antigen binding fragment thereof, is conjugated to a cytotoxic agent.

45. The method of claim 44, wherein the cytotoxic agent is selected from the group consisting of a chemotherapeutic agent, a biologic agent, a toxin, and a radioactive isotope.

46. The method of claim 36, wherein the agent upregulates the copy number, amount, and/or activity of at least one biomarker listed in Table 2, 4, 6 or 8.

47. The method of claim 46, wherein the agent is a nucleic acid molecule encoding the one or more biomarkers listed in Table 2, 4, 6 or 8 or fragment thereof, a polypeptide of the one or more biomarkers listed in Table 2, 4, 6 or 8 or fragment(s) thereof, or a small molecule that binds to the one or more biomarkers listed in Table 2, 4, 6 or 8.

48. The method of any one of claims 36-47, further comprising contacting the cancer cells with at least one immunotherapy.

49. The method of claim 48, wherein the immunotherapy is cell-based.

50. The method of claim 48, wherein the immunotherapy comprises a cancer vaccine and/or virus.

51. The method of claim 48, wherein the immunotherapy inhibits an immune checkpoint and/or inhibits TNF alpha.

52. The method of claim 51, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TEVI-3, TEVI-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, and A2aR.

53. The method of claim 52, wherein the immune checkpoint is selected from the group consisting of PD-1, PD-L1, PD-L2, and CTLA-4.

54. The method of claim 53, wherein the immune checkpoint is PD-1 or CTLA-4.

55. The method of any one of claims 36-54, wherein the agent reduces the number of proliferating cells in the cancer and/or reduces the volume or size of a tumor comprising the cancer cells.

56. The method of any one of claims 36-55, wherein the agent increases the amount of CD8+ T cells, CD4+ T cells, granzyme B+CD8+ T cells, and/or CD45+ immune cells infiltrating a tumor comprising the cancer cells.

57. The method of any one of claims 36-56, wherein the CD45+ immune cells express higher levels of genes associated with productive antitumor immunity.

58. The method of claim 57, wherein the genes are related to IFN-γ response, IFN-a response, and/or tumor necrosis factor a signaling via F-KB .

59. The method of any one of claims 36-57, wherein the agent increases the percentage of dendritic cells within the CD45+ immune cells.

60. The method of any one of claims 36-59, wherein the agent increases the ratio of tumor-inhibitory Ml -like macropahges to tumor-promoting M2-like macrophages within the CD45+immune cells.

61. The method of any one of claims 36-60, wherein the agent increases the

responsiveness of cancer cells to IFN-γ and/or IFN-a.

62. The method of any one of claims 36-61, wherein the agent increases the production of chemokines in response to IFN-γ in cancer cells.

63. The method of claim 62, wherein the chemokine is CXCL9 and/or CXCL10.

64. The method of any one of claims 36-63, wherein the agent increases cancer cell surface levels of H2-Kb and/or PD-L1 in response to IFN-γ.

65. The method of any one of claims 36-64, wherein the agent increases chromatin accessibility for IFN-y-responsive genes in cancer cells.

66. The method of any one of claims 36-65, wherein the agent downregulates genes associated with mTORCl pathway and/or cholesterol homeostasis in cancer cells.

67. The method of any one of claims 36-66, wherein the agent sensitizes cancer cells to immune checkpoint blockade therapy.

68. The methof of claim 67, wherein the immune checkpoint is PD-1 and/or CTLA-4.

69. The method of any one of claims 36-68, wherein the agent is administered in a pharmaceutically acceptable formulation.

70. The method of any one of claims 36-69, further comprising contacting the cancer cells with at least one additional cancer therapeutic agent or regimen.

71. A method of determining whether a subject afflicted with a cancer or at risk for developing a cancer would benefit from increasing sensitivity of the cancer cells to cytotoxic T cell-mediate killing by modulating the copy number, amount, and/or activity of at least one biomarker listed in Tables 1-9, the method comprising:

a) obtaining a biological sample from the subject;

b) determining the copy number, amount, and/or activity of at least one biomarker listed in Tables 1-9;

c) determining the copy number, amount, and/or activity of the at least one biomarker in a control; and

d) comparing the copy number, amount, and/or activity of the at least one biomarker detected in steps b) and c);

wherein the presence of, or a significant increase in, the copy number, amount, and/or activity of, the at least one biomarker listed in Table 1, 5, 7 or 9 and/or the absence of, or a significant decrease in, the copy number, amount, and/or activity of, the at least one biomarker listed in Table 2, 4, 6 or 8, in the subject sample relative to the control copy number, amount, and/or activity of the at least one biomarker indicates that the subject afflicted with the cancer or at risk for developing the cancer would benefit from increasing sensitivity of the cancer cells to cytotoxic T cell-mediate killing by modulating the copy number, amount, and/or activity of the at least one biomarker listed in Tables 1-9.

72. The method of claim 71, further comprising recommending, prescribing, or administering an agent that modulates the at least one biomarker listed in Tables 1-9 if the cancer is determined to benefit from the agent, optionally further administering at least one additional cancer therapeutic agent or regimen.

73. The method of claim 72, further comprising recommending, prescribing, or administering cancer therapy other than an agent that modulates the at least one biomarker listed in Tables 1-9 if the cancer is determined to not benefit from the agent.

74. The method of claim 72, wherein the cancer therapy is selected from the group consisting of targeted therapy, chemotherapy, radiation therapy, and/or hormonal therapy.

75. The method of any one of claims 71-74, wherein the control sample is determined from a cancerous or non-cancerous sample from either the patient or a member of the same species to which the patient belongs.

76. The method of any one of claims 71-75, wherein the control sample comprises cells.

77. A method for predicting the clinical outcome of a subject afflicted with a cancer, the method comprising:

a) determining the copy number, amount, and/or activity of at least one biomarker listed in Tables 1-9;

b) determining the copy number, amount, and/or activity of the at least one biomarker in a sample from a control having a good clinical outcome; and

d) comparing the copy number, amount, and/or activity of the at least one biomarker in the subject sample and in the sample from the control subject;

wherein the presence of, or a significant increase in, the copy number, amount, and/or activity of, the at least one biomarker listed in Table 1, 5, 7 or 9 and/or the absence of, or a significant decrease in, the copy number, amount, and/or activity of, the at least one biomarker listed in Table 2, 4, 6 or 8, in the subject sample as compared to the copy number, amount and/or activity in the sample from the control subject, is an indication that the subject has a poor clinical outcome.

78. A method for monitoring the progression of a cancer in a subject, the method comprising:

a) detecting in a subject sample at a first point in time the amount or activity of at least one biomarker listed in Tables 1-9;

b) repeating step a) at a subsequent point in time; and

c) comparing the amount or activity of at least one biomarker listed in Tables 1-9 detected in steps a) and b) to monitor the progression of the cancer in the subject.

79. A method of assessing the efficacy of the agent for treating a cancer in a subject, comprising:

a) detecting in a subject sample at a first point in time the copy number, amount, and/or or activity of at least one biomarker listed in Tables 1-9;

b) repeating step a) during at least one subsequent point in time after administration of the agent; and

c) comparing the copy number, amount, and/or activity detected in steps a) and b), wherein the absence of, or a significant decrease in, the copy number, amount, and/or activity of, the at least one biomarker listed in Table 1, 5, 7 or 9, and/or the presence of, or a significant increase in, the copy number, amount, and/or activity of, the at least one biomarker listed in Table 2, 4, 6 or 8, in the subsequent sample as compared to the copy number, amount, and/or activity in the sample at the first point in time, indicates that the agent treats the cancer in the subject.

80. The method of claim 78 or 79, wherein between the first point in time and the subsequent point in time, the subject has undergone treatment, completed treatment, and/or is in remission for the cancer.

81. The method of any one of claims 78-80, wherein the first and/or at least one subsequent sample is selected from the group consisting of ex vivo and in vivo samples.

82. The method of any one of claims 78-81, wherein the first and/or at least one subsequent sample is obtained from an animal model of the cancer.

83. The method of any one of claims 78-82, wherein the first and/or at least one subsequent sample is a portion of a single sample or pooled samples obtained from the subject.

84. The method of any one of claims 71-83, wherein the sample comprises cells, serum, peritumoral tissue, and/or intratumoral tissue obtained from the subject.

85. A cell-based assay for screening for agents that sensitize a cancer cell to cytotoxic T cell-mediated killing comprising contacting the cancer cell with cytotoxic T cells and a test agent, and determining the ability of the test agent to decrease the copy number, amount, and/or activity of at least one biomarker listed in Table 1, 5, 7 or 9, and/or increase the copy number, amount, and/or activity of the at least one biomarker listed in Table 2, 4, 6 or 8.

86. The cell-based assay of claim 85, wherein the step of contacting occurs in vivo, ex vivo, or in vitro.

87. The method or assay of any one of claims 71-86, further comprising determining a reduction in the number of proliferating cells in the cancer and/or a reduction in the volume or size of a tumor comprising the cancer cells.

88. The method or assay of any one of claims 71-87, further comprising determining an increased number of CD8+ T cells, CD4+ T cells, granzyme B+CD8+ T cells, and/or CD45+ immune cells infiltrating a tumor comprising the cancer cells.

89. The method or assay of any one of claims 71-88, further comprising determining an increased responsiveness of cancer cells to IFN-γ and/or IFN-a.

90. The method or assay of any one of claims 71-89, further comprising determining decreased expression levels of genes related to mTORCl pathway and/or cholesterol hoeostasis.

91. The method or assay of any one of claims 71-90, further comprising determining responsiveness to the agent that modulates the at least one biomarker listed in Tables 1-9 measured by at least one criteria selected from the group consisting of clinical benefit rate, survival until mortality, pathological complete response, semi-quantitative measures of pathologic response, clinical complete remission, clinical partial remission, clinical stable disease, recurrence-free survival, metastasis free survival, disease free survival, circulating tumor cell decrease, circulating marker response, and RECIST criteria.

92. The method or assay of any one of claims 71-91, further comprising contacting the cancer cells with at least one additional cancer therapeutic agent or regimen.

93. The method or assay of any one of claims 1-92, wherein the at least one biomarker listed in Table 1, 5, 7 or 9 is selected from the group consisting of PD-Ll, Ptpn2, Serpinb9, Otulin, Rela, Ikbkg, Ikbkb, Rnf31, Sharpin, Rraga, Rragb, Rragc, Lamtorl, Tbkl, Fadd, Atg5, Nsdhl, Gne, Gale, Eroll, Cd44, Nadk, Nampt, Arid2, Pbrml, Sox4, Hdac5, Ptpnl l, Cflar, or Brd7, or a fragment thereof.

94. The method or assay of any one of claims 1-93, wherein the at least one biomarker listed in Table 1, 5, 7 or 9 is Arid2, Pbrml, or Brd7, or a fragment thereof.

95. The method or assay of any one of claims 1-92, wherein the at least one biomarker listed in Table 2, 4, 6 or 8 is selected from the group consisting of H2-D1, B2m, Tapl, Tap2, Nlrc5, Jakl, Jak2, Statl, Ifngrl, Ifngr2, Nfl, Dusp6, Spredl, Rasa2, or SPOP, or a fragment thereof.

96. The method or assay of any one of claims 1-95, wherein the cancer is selected from the group consisting of melanoma, head and neck squamous carcinoma, kidney cancer, colorectal cancer, gliomas, neuroblastoma, prostate cancer, breast cancer, pancreatic ductal carcinoma, epithelial ovarian cancer, B-CLL, leukemia, B cell lymphoma, renal cell carcinoma, lung adenocarcinoma and squamous carcinoma.

97. The method or assay of any one of claims 1-96, wherein the cancer is resistant to blockade of at least one immune checkpoint.

98. The method or assay of any one of claims 1-97, wherein the subject is an animal model of the cancer, optionally wherein the animal model is a mouse model.

99. The method or assay of any one of claims 1-98, wherein the subject is a mammal.

100. The method or assay of claim 99, wherein the mammal is a mouse or a human.

101. The method or assay of claim 100, wherein the mammal is a human.