Methods for determining resistance to checkpoint inhibitor therapy

Abstract

The present disclosure relates to animal models, methods for screening and testing anti-cancer drug candidates, and methods for treatment, evaluation of efficacy of cancer treatment, and selection of patients for cancer therapy.

Description

[Technical field]

[0001] Priority This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 121,083, filed December 3, 2020, No. 63 / 173,090, filed April 9, 2021, No. 63 / 215,735, filed June 28, 2021, and No. 63 / 276,066, filed November 5, 2021, the contents of each of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates, in part, to methods useful for the detection and treatment of drug-resistant cancers, and to methods for developing novel therapies for drug-resistant cancers.

[0003] Instructions for electronically submitted text files The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: Copy of Sequence Listing in Computer Readable Format (Filename: SHK-038PC1_ST25; Creation Date: December 1, 2021; Size: 1,571 bytes). [Background technology]

[0004] Drug resistance remains one of the greatest challenges in cancer treatment. Drug resistance is found across all types of cancer and all treatment modalities, including molecular targeted therapy, immunotherapy, and chemotherapy. Furthermore, cancer treatment is typically approached empirically, and many patients with chemotherapy-resistant disease often undergo multiple cycles of toxic treatment before a lack of efficacy becomes evident. The inability to predict the response to a particular treatment is a major obstacle to improving outcomes for cancer patients. In some patients, the initiation of effective treatment is delayed by the inability to predict the response. Furthermore, it is also common for patients with advanced cancer to be administered drugs that help shrink the tumor, only for the cancer to recur weeks or months later and the drug no longer works. Unfortunately, there are few effective treatment options available for some patients with cancers that are resistant to anti-checkpoint therapy. Thus, drug resistance, either present before treatment (intrinsic or primary resistance) or developing after treatment (acquired resistance), is the cause of most recurrences of cancer, one of the leading causes of death. Therefore, a better understanding of the mechanisms of drug resistance is required to provide guidance for future cancer treatments. Furthermore, new treatments for patients suffering from drug-resistant cancers and methods for developing appropriate drug selection methods for drug-resistant cancer patients are needed to improve the outcomes of cancer patients. Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, the present disclosure provides, in part, methods for selecting patients for and treating cancer based on gene expression profiles of, for example, anti-PD-1 resistant cancers. The present disclosure also provides animal models suitable for testing anti-cancer drug candidates, and methods for producing pharmaceutical compositions for treating cancer. [Means for solving the problem]

[0006] In one aspect, the disclosure provides a method of determining a cancer treatment for a patient, comprising: (a) obtaining a biological sample from a subject; (b) (i) positive regulation of cell cycle processes, regulation of the G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of a defense response, positive regulation of IFNβ production, regulation of an inflammatory response, regulation of an innate immune response, negative regulation of antigen processing / presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or (ii) phospholipid export, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid export, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes evaluating the sample for the presence, absence, or level of one or more genes associated with a Gene Ontology (GO) pathway selected from: (c) selecting a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2 based on the evaluation of step (b); and In one embodiment, the mouse belongs to the BALB / c or C57BL / 6 strain. In one embodiment, the cancer therapy capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2 is an antibody. In one embodiment, the antibody is selected from nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS936559, MPDL328OA (ROCHE), cemiplimab (LIBTAYO), atezolizumab (TECENTRIQ), avelumab (BAVENCIO), and durvalumab (imfinzi).

[0007] In one aspect, the disclosure provides a method of selecting a patient for cancer treatment, comprising: (a) obtaining a biological sample from a subject; (b) (i) positive regulation of cell cycle processes, regulation of the G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of a defense response, positive regulation of IFNβ production, regulation of an inflammatory response, regulation of an innate immune response, negative regulation of antigen processing / presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or (ii) phospholipid export, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid export, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes evaluating the sample for the presence, absence, or level of one or more genes associated with a Gene Ontology (GO) pathway selected from: (c) selecting a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; and In one embodiment, the mouse belongs to the BALB / c or C57BL / 6 strain. In one embodiment, the cancer therapy capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2 is an antibody. In one embodiment, the antibody is selected from nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS936559, MPDL328OA (ROCHE), cemiplimab (LIBTAYO), atezolizumab (TECENTRIQ), avelumab (BAVENCIO), and durvalumab (imfinzi).

[0008] In one aspect, the present disclosure provides a method of treating cancer, comprising: (a) obtaining a biological sample from a subject; (b) (i) positive regulation of cell cycle processes, regulation of the G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of a defense response, positive regulation of IFNβ production, regulation of an inflammatory response, regulation of an innate immune response, negative regulation of antigen processing / presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or (ii) phospholipid export, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid export, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes evaluating the sample for the presence, absence, or level of one or more genes associated with a Gene Ontology (GO) pathway selected from: (c) (i) an agent capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2; (ii) antimetabolite chemotherapeutic agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine, and acivicin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine and ribozynoindole), inhibitors of rRNA and / or tRNA synthesis (e.g., calfluxin (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitors inhibitor NCT-503), amino acid uptake inhibitors (e.g., the SLC7A11 inhibitors sulfasalazine, erastin, or sorafenib), modulators of post-translational modifications (e.g., the glycosylation inhibitors tunicamycin, ppGalNAc-T3), modulators of protein degradation, and modulators of protein transport (e.g., cyclosporine A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin, etc.) selecting a cancer therapy selected from the group consisting of: (d) administering said cancer therapy using the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2, and optionally administering said therapy selected in steps (c)(ii) and (c)(iii); In one embodiment, the mouse belongs to the BALB / c or C57BL / 6 strain. In one embodiment, the cancer therapy capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2 is an antibody. In one embodiment, the antibody is selected from nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS936559, MPDL328OA (ROCHE), cemiplimab (LIBTAYO), atezolizumab (TECENTRIQ), avelumab (BAVENCIO), and durvalumab (imfinzi).

[0009] In embodiments, the lack of upregulation of one or more genes associated with a cellular response to IFNγ, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with a cellular response to IFNγ, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with a cellular response to IFNγ, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0010] In embodiments, upregulation of one or more genes associated with cellular response to IFNγ, as compared to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with cellular response to IFNγ, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with cellular response to IFNγ, as compared to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0011] In embodiments, upregulation of one or more genes associated with the type I IFN signaling pathway, as compared to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with the type I IFN signaling pathway, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with the type I IFN signaling pathway, as compared to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0012] In embodiments, the lack of upregulation of one or more genes associated with the type I IFN signaling pathway, as compared to a previous biological sample from the subject, is indicative of a response to a cancer treatment that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with the type I IFN signaling pathway, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with the type I IFN signaling pathway, as compared to a standard, is indicative of a response to a cancer treatment that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0013] In embodiments, the biological sample is a fresh tissue sample, a frozen tumor tissue specimen, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue specimen. In embodiments, the biological sample is a biopsy sample. In embodiments, the biological sample comprises a body fluid selected from blood, plasma, serum, tears, tears, bone marrow, blood, blood cells, ascites, tissue or fine needle biopsy samples, cell-containing body fluids, free floating nucleic acids, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluid, skin swabs, vaginal swabs, oral swabs, nasal swabs, lavages or irrigations such as ductal washings or bronchoalveolar lavages, aspirates, scrapes, bone marrow specimens, tissue biopsy specimens, surgical specimens, feces, other body fluids, secretions and / or excretions, and / or cells therefrom.

[0014] In embodiments, the biological sample comprises at least one tumor cell.

[0015] In embodiments, the evaluating is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS), or a combination thereof.

[0016] In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a Gene Ontology (GO) pathway identified herein.

[0017] In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more of the nucleic acids of one or more genes associated with a Gene Ontology (GO) pathway identified herein.

[0018] In embodiments, the assessing provides information to classify the patient into a high-risk or low-risk group. In embodiments, the high-risk classification includes a high level of tumor cells that are resistant to cancer therapies that utilize the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the low-risk classification includes a low level of tumor cells that are resistant to cancer therapies that utilize the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0019] In one aspect, the disclosure provides a transgenic non-human animal comprising one or more tumor cells, the tumor cells comprising: (a) upregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from positive regulation of cell cycle processes, regulation of G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of defense response, positive regulation of IFNβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing / presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or (b) downregulation of one or more genes associated with a gene ontology (GO) pathway selected from phospholipid export, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid export, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes. In one embodiment, the transgenic non-human animal is transgenic. In one embodiment, the tumor cells are resistant to cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In one embodiment, the transgenic non-human animal is a rodent. In one embodiment, the rodent is a mouse. In one embodiment, the mouse belongs to the BALB / c or C57BL / 6 strain.

[0020] In one aspect, the present disclosure provides a method for testing anti-cancer drug candidates, comprising: (a) providing a transgenic non-human animal according to any one of the embodiments of the present disclosure; (b) administering the anticancer drug candidate to the transgenic non-human animal; and (c) evaluating whether the anti-cancer drug candidate is effective in slowing or inhibiting cancer growth in the transgenic non-human animal; and In one aspect, the present disclosure relates to a method for testing a candidate anti-cancer drug, comprising:

[0021] In one aspect, the disclosure provides a method for manufacturing a pharmaceutical composition for treating cancer, comprising: (a) providing a transgenic non-human animal according to any one of the embodiments of the present disclosure; (b) administering the anti-cancer drug candidate to the transgenic non-human animal; and (c) selecting an anti-cancer drug effective to slow or inhibit cancer growth in said transgenic non-human animal; and (d) formulating the anticancer drug or candidate for administration to a human patient; and In one aspect, the disclosure relates to said anti-cancer drug candidate selected from a chemotherapeutic agent, a cytotoxic agent, and a checkpoint inhibitor.

[0022] In embodiments, the upregulation is in comparison to healthy tissue. In embodiments, the upregulation is in comparison to another biological sample from a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the upregulation is in comparison to a previous biological sample obtained from the subject. In embodiments, the downregulation is in comparison to healthy tissue. In embodiments, the downregulation is in comparison to another biological sample from a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the downregulation is in comparison to a previous biological sample obtained from the subject.

[0023] In some embodiments, the antibody is selected from nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS936559, MPDL328OA (ROCHE), cemiplimab (LIBTAYO), atezolizumab (TECENTRIQ), avelumab (BAVENCIO), and durvalumab (imfinzi).

[0024] In one aspect, the disclosure relates to a method of determining a cancer treatment for a patient, the method comprising: (a) obtaining a biological sample from the subject; and (b) analyzing the biological sample for: (i) detection of a gene selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP1, TAP2, CASP1, IRF, LTBR, ​​PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1. and / or (ii) assessing expression of a gene selected from RPL41, RPS15, RPS8, TRIM7, and LRG1; and (c) selecting a cancer therapy based on the assessment of step (b), wherein the cancer therapy is selected from (i) an agent capable of inhibiting the function and / or activity of PD-1, PD-L1, and / or PD-L2; (ii) an antimetabolite chemotherapeutic agent (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6- diazo-5-oxo-L-norleucine (DON), azaserine, and acivicin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozynoindoles), inhibitors of rRNA and / or tRNA synthesis (e.g., calfluxine (CX-3 543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7A11 inhibitors sulfasalazine, erastin, or sorafenib), regulators of post-translational modifications (e.g., glycosylation inhibitors (tunicamycin, ppGalNAc-T3), regulators of protein degradation and regulators of protein transport (e.g., cyclosporine A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin, etc.), or topoisomerase inhibitors.

[0025] In one aspect, the disclosure relates to a method of selecting a patient for cancer treatment, the method comprising: (a) obtaining a biological sample from the subject; and (b) examining the biological sample for a gene selected from: (i) CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP1, TAP2, CASP1, IRF, LTBR, ​​PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1; and / or (ii) RPL41, RPS15, RPS8, TRIM7, and LRG1. and (c) selecting a cancer therapy based on the assessment of step (b), wherein the cancer therapy is selected from the group consisting of: (i) an agent capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2; (ii) antimetabolite chemotherapeutic agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine and acivicin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.);and (iii) protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozynoindole), inhibitors of rRNA and / or tRNA synthesis (e.g., calfluxine (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., the SLC7A11 inhibitors sulfasalazine, erastin, or sorafenib), modulators of post-translational modifications (e.g., the glycosylation inhibitors tunicamycin, ppGalNAc-T3), modulators of protein degradation and modulators of protein transport (e.g., cyclosporine A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin, etc.), or topoisomerase inhibitors;

[0026] In one aspect, the disclosure relates to a method of treating cancer, the method comprising: (a) obtaining a biological sample from a subject; and (b) examining the biological sample for a gene selected from: (i) CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP1, TAP2, CASP1, IRF, LTBR, ​​PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1; and / or (ii) RPL41, RPS15, RPS8, TRIM 7 and LRG1; and (c) (i) agents capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2; (ii) antimetabolite chemotherapeutic agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine and acivicin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.);and (iii) protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozynoindole), inhibitors of rRNA and / or tRNA synthesis (e.g., calfluxine (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7A11 inhibitors sulfasalazine, erastin, or sorafenib), post-translational modification inhibitors. (c) selecting a cancer therapy selected from a glycosylation inhibitor (e.g., tunicamycin, ppGalNAc-T3), a modulator of protein degradation, and a modulator of protein trafficking (e.g., cyclosporine A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin, etc.) or a topoisomerase inhibitor; (d) administering a cancer therapy that utilizes its ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2, and optionally administering the treatment selected in steps (c)(ii) and (c)(iii);

[0027] In one aspect, the disclosure relates to a method of determining a cancer treatment for a patient, the method comprising: (a) obtaining a biological sample from the subject; (b) evaluating the biological sample for activation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras; and (c) selecting a cancer therapy based on the evaluation of step (b), wherein the cancer therapy is selected from: (i) an agent capable of inhibiting a function and / or activity of PD-1, PD-L1, and / or PD-L2; (ii) an agent capable of inhibiting a function and / or activity of PD-1, PD-L1, and / or PD-L2; antagonist chemotherapeutic agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine, and acivicin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozynoindole), rR Inhibitors of NA and / or tRNA synthesis (e.g., calfluxin (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7A11 inhibitor sulfasalazine, erastin or sorafenib). and wherein the inhibitor is selected from among inhibitors of cyclosporine A, cyclosporine B, cyclosporine C, cyclosporine D, cyclosporine E, cyclosporine F, cyclosporine F, cyclosporine F, cyclosporine F, cyclosporine B, cyclosporine F, cyclosporine C, cyclosporine D ...

[0028] In one aspect, the disclosure relates to a method of selecting a patient for cancer treatment, the method comprising: (a) obtaining a biological sample from the subject; (b) assessing the biological sample for activation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras; and (c) selecting a cancer therapy based on the assessment of step (b), wherein the cancer therapy is selected from: (i) an agent capable of inhibiting a function and / or activity of PD-1, PD-L1, and / or PD-L2; (ii) an agent capable of inhibiting a function and / or activity of PD-1, PD-L1, and / or PD-L2; antagonist chemotherapeutic agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine, and acivicin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozynoindole), rR Inhibitors of NA and / or tRNA synthesis (e.g., calfluxin (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7A11 inhibitor sulfasalazine, erastin or sorafenib). and wherein the inhibitor is selected from among inhibitors of cyclosporine A, cyclosporine B, cyclosporine C, cyclosporine D, cyclosporine E, cyclosporine F, cyclosporine F, cyclosporine F, cyclosporine F, cyclosporine B, cyclosporine F, cyclosporine C, cyclosporine D ...

[0029] In one aspect, the disclosure relates to a method of treating cancer, the method comprising: (a) obtaining a biological sample from a subject; (b) assessing the biological sample for activation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras; and (c) administering to a subject an antibody capable of inhibiting the function and / or activity of PD-1, PD-L1, and / or PD-L2; (i) an agent capable of inhibiting the function and / or activity of PD-1, PD-L1, and / or PD-L2; (ii) an antimetabolite chemotherapeutic agent (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-1, 5-methyl ... (iii) protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine and ribozynoindole), inhibitors of rRNA and / or tRNA synthesis (e.g., calfluxin (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitors R1 62, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503, amino acid uptake inhibitors (e.g., SLC7A11 inhibitors sulfasalazine, erastin, or sorafenib), regulators of post-translational modifications (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), regulators of protein degradation, and regulators of protein transport (e.g., cyclosporine A, fendi, (c) administering a cancer therapy selected from the group consisting of cyclosporine, cyclosporine, riboflavin, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin) or a topoisomerase inhibitor; and (d) administering a cancer therapy that utilizes its ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2, and optionally administering the treatment selected in steps (c)(ii) and (c)(iii).

[0030] In one aspect, the disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising administering a cancer treatment selected from antimetabolite chemotherapeutic agents, topoisomerase inhibitors, protein translation inhibitors, ribosome biogenesis inhibitors, inhibitors of rRNA and / or tRNA synthesis, inhibitors of amino acid synthesis, inhibitors of amino acid uptake, regulators of post-translational modification, regulators of protein degradation, regulators of protein trafficking, topoisomerase inhibitors, wherein the subject has received or is receiving anti-cancer drug treatment with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2, and wherein the subject has experienced a lack of response, resistance or refractory to the cancer therapy with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0031] In one aspect, the disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising: (a) administering an anti-cancer treatment capable of inhibiting a function and / or activity of PD-1, PD-L1 and / or PD-L2; (b) assessing the anti-tumor response to the anti-cancer treatment capable of inhibiting a function and / or activity of PD-1, PD-L1 and / or PD-L2 by monitoring tumor size reduction in the subject; (c) if a lack of tumor size reduction is observed, administering a Trim7 modulator and / or a proteasome inhibitor; (d) re-assessing the anti-tumor response to the anti-cancer treatment capable of inhibiting a function and / or activity of PD-1, PD-L1 and / or PD-L2 by monitoring tumor size reduction in the subject; and (e) discontinuing administration of the Trim7 modulator if a reduction in tumor size is observed. In embodiments, the proteasome inhibitor is selected from bortezomib, carfilzomib, ixazomib, oprozomib, delanzomib, and marizomib.

[0032] In one aspect, the present invention relates to a method of treating cancer in a subject in need thereof, the method comprising: (a) administering an anti-cancer treatment capable of inhibiting a function and / or activity of PD-1, PD-L1 and / or PD-L2; (b) assessing an anti-tumor response to the anti-cancer treatment capable of inhibiting a function and / or activity of PD-1, PD-L1 and / or PD-L2 using: (i) obtaining a biological sample from the subject; and (ii) assessing the biological sample for overexpression and / or activation of TRIM7; and (c) assessing the overexpression and / or activation of TRIM7. If overexpression and / or activation is observed, administering a Trim7 modulator and / or a proteasome inhibitor, (d) reassessing the anti-tumor response from an anti-cancer treatment capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2 using (i) obtaining a biological sample from the subject; (ii) evaluating the biological sample for TRIM7 overexpression and / or activation; and (e) withdrawing administration of the Trim7 modulator if TRIM7 overexpression and / or activation is not observed. In an embodiment, the proteasome inhibitor is selected from bortezomib, carfilzomib, ixazomib, oprozomib, delanzomib and marizomib.

[0033] In one aspect, the disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising: (a) administering an anti-cancer treatment capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2; (b) assessing overexpression and / or activation of TRIM7 using: (i) obtaining a biological sample from the subject; and (ii) evaluating the biological sample for overexpression and / or activation of TRIM7; (c) if overexpression and / or activation of TRIM7 is observed, administering a Trim7 modulator and / or a proteasome inhibitor; (d) re-assessing overexpression and / or activation of TRIM7 using: (i) obtaining a biological sample from the subject; (ii) evaluating the biological sample for overexpression and / or activation of TRIM7; and (e) discontinuing administration of the Trim7 modulator if overexpression and / or activation of TRIM7 is not observed. In embodiments, the proteasome inhibitor is selected from bortezomib, carfilzomib, ixazomib, oprozomib, delanzomib, and marizomib.

[0034] In embodiments, the Trim7 modulator is a Trim7 inhibitor. In embodiments, the Trim7 modulator is selected from small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antisense RNA, guide RNA (gRNA), small molecule, antibody, peptide, and peptidomimetic. In embodiments, the small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antisense RNA, or guide RNA (gRNA) inhibits the production of Trim7 protein. In embodiments, the peptidomimetic mimics the target of Trim7, thereby inhibiting the activity of Trim7.

[0035] In an embodiment, the Trim7 regulator is a mitogen-activated and stress-activated kinase 1 (MSK1) inhibitor, where the MSK1 inhibitor regulates Trim7 via downstream effects of inhibition of MSK1. In an embodiment, the MSK1 inhibitor is selected from Ro31-8220, SB-747651A and H89. In an embodiment, the MSK1 inhibitor is SB-747651A.

[0036] Any aspect disclosed herein may be combined with any other aspect.

[0037] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0038] [Figure 1A] Generation of anti-PD-1 resistant CT26 tumors. Figure 1A shows a schematic of the method used to generate anti-PD-1 resistant CT26 tumors. Briefly, BALB / C mice were inoculated with 500,000 murine colon cancer CT26 cells, and when the average tumor volume reached 80-100 mm3 (day 0 is shown), the mice were treated with anti-PD-1 (clone RMP1-14; BioXcell) antibody. Tumors were excised, and viable tumor cells were dissociated and cultured in vitro. These cells were referred to as "first round", "first generation" or "F1 generation" cells. First generation cells were inoculated into BALB / C mice and after another treatment passage of anti-PD-1 "second round", "second generation" or "F2 generation" cells were isolated. After two more rounds (total of four rounds) of anti-PD-1 selection, "fourth round", "fourth generation" or "F4 generation" cells were isolated. [Figure 1B]Development of anti-PD-1 resistant CT26 tumors. Figure 1B shows a graph comparing the efficacy of anti-PD-1 antibody (100 μg of anti-PD-1 clone RMP1-14; BioXcell) in BALB / C mice with CT26 parental cells and PD-1 resistant cells. BALB / C mice were inoculated with CT26 parental cells and PD-1 resistant 4th generation cells in the hind flank. When the starting tumor volume (STV) reached 80-100 mm3, mice were randomized into two treatment groups: (1) vehicle (PBS), and (2) anti-PD-1 antibody. Mice received a series of intraperitoneal injections of vehicle or 100 μg of anti-PD-1 (clone RMP1-14; BioXcell) on days 0, 3, and 6. Tumor volumes were measured on the indicated days. [Figure 2A] Transcriptome profiling of anti-PD-1 resistant cell lines using RNA-seq. Figure 2A (top panel) shows principal component analysis (PCA) that spatially separated samples based on transcriptome expression. Figure 2A (bottom panel) shows differentially expressed genes (DEGs) determined between groups (parental vs. 2nd gen, parental vs. 4th gen, 2nd gen vs. 4th gen) and plotted in a heatmap. Hierarchical clustering was performed to rank order genes in each row, where a subset of gene expression was lower (blue) in one dataset and higher (red) in the other, separating genes into two major clusters in each comparison. [Figure 2B] Figure 2B shows transcriptome profiling of anti-PD-1 resistant cell lines using RNA-seq. Figure 2B shows up- or down-regulated genes in each dataset. Genes were entered into PANTHER to identify gene ontology (GO) related to each gene set. Gene sets are shown with associated p-values. [Figure 2C]Figure 2C shows transcriptome profiling of anti-PD-1 resistant cell lines using RNA-seq. Figure 2C (top panel) shows a Venn diagram of gene expression overlap between all datasets. Figure 2C (bottom panel) shows transcripts per million (TPM; normalized expression data) for selected genes, demonstrating higher baseline expression of genes related to PD-L1, antigen processing / presentation, protein translation, and ER trafficking in some datasets than others. [Figure 2D] Transcriptome profiling of anti-PD-1 resistant cell lines using RNA-seq. Figure 2D shows transcripts per million (TPM; normalized expression data) of selected genes, demonstrating higher baseline expression of genes related to PD-L1, antigen processing / presentation, protein translation, and ER trafficking in some datasets than others. [Diagram 3] Figure 1 shows discordance in gene expression and cell surface protein expression for PD-L1 / 2, MHC class I, and B2M. Parental and 4th generation anti-PD-1 resistant cells were harvested from culture and analyzed by flow cytometry for surface expression of PD-L1, PD-L2, MHC class I, and beta2 microglobulin (B2M). Gates were drawn as shown, and above each plot is shown the percentage of cells in each gate, and to the right of each percentage is shown the MFI (mean fluorescence intensity) for each marker. [Figure 4A]Transcriptome profiling of anti-PD-1 resistant cell lines is shown. Figure 4A shows a comparison of second and fourth generation anti-PD-1 resistant cell lines with B16.F10, a murine melanoma tumor, which served as a model of anti-PD-1 "primary resistance" since these tumors do not respond to anti-PD-1 therapy. Cells were cultured in IFNγ for 24 hours to assess in vitro responsiveness. This mimics how tumor cells respond in vivo as immune cells infiltrate and secrete effector cytokines such as IFNγ. Figure 4A (left panel) shows the DEGs identified between untreated parental CT26 and IFNγ-treated parental CT26. Of these, 338 genes had data available from other datasets and their values ​​are shown in the other columns. Log2 fold changes were plotted in a heatmap and genes were hierarchically clustered based on parental CT26. Genes were separated into three major clusters. Figure 4A (right panel) shows the GO pathways associated with the DEGs. Associated genes were entered into PANTHER to identify pathways associated with the dysregulated genes. [Figure 4B] Transcriptome profiling of anti-PD-1 resistant cell lines. Figure 4B shows transcripts per million (TPM; normalized expression data) for selected genes, demonstrating that CT26 anti-PD-1 resistant cells have baseline hyperactivation of type I and type II interferons, yet when the cells are challenged with IFNγ, they downregulate these genes. [Figure 5A]Figures 5A-5D show the paradoxical dysregulation of certain genes associated with acquired resistance to anti-PD-1. Genes encoding CD274 (Figure 5A) and B2m (Figure 5B), which are overexpressed in fourth-generation anti-PD-1 resistant cells, are overexpressed in wild-type CT26 cells but are suppressed in fourth-generation anti-PD-1 resistant cells in the presence of IFNγ. Meanwhile, genes encoding Trim7 (Figure 5C) and Lrg1 (Figure 5D), which are suppressed in fourth-generation anti-PD-1 resistant cells, are suppressed in wild-type CT26 cells but are overexpressed in fourth-generation anti-PD-1 resistant cells in response to IFNγ. Second-generation anti-PD-1 resistant cells display an intermediate phenotype. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 6A] Identification of driver genes involved in acquired resistance to anti-PD-1 and functional pathways affected by driver genes is shown. Figure 6A, panels 1-4 show the methodology used to identify driver genes. Figure 6A (panel 1) shows 1,999 genes are downregulated and 3607 genes are upregulated in 4th generation anti-PD-1 resistant cells in the presence of IFNγ compared to CT26 cells. Figure 6A (panel 2) shows 1,060 genes that downregulate 4th generation anti-PD-1 resistant cells but upregulate CT26 cells. Figure 6A (panel 3) shows 688 genes that downregulate 4th generation anti-PD-1 resistant cells compared to CT26 cells as revealed by sorting according to responsiveness to IFNγ. Figure 6A (panel 4) shows 70 genes that are upregulated in vivo in 4th generation anti-PD-1 resistant cells compared to CT26 cells. [Figure 6B] Figure 6B shows the identification of driver genes involved in acquired resistance to anti-PD-1 and functional pathways affected by the driver genes. Figure 6B shows the Gene Ontology (GO) pathways associated with genes identified using the methodology of Figure 6A. [Figure 6C]Figure 6C shows the identification of driver genes involved in acquired resistance to anti-PD-1 and functional pathways affected by the driver genes. Figure 6C shows functional pathways affected by the TRIM family of proteins. [Figure 6D] Figure 6 shows the identification of driver genes involved in acquired resistance to anti-PD-1 and functional pathways affected by the driver genes. Figure 6D shows the functional pathways in which Elk1 and c-Jun play a role. [Figure 6E] Figure 6 shows the identification of driver genes involved in acquired resistance to anti-PD-1 and functional pathways affected by the driver genes. Figure 6E shows the functional connections between Lrg1, B2m, and Arg1 and other genes. [Figure 6F] Figure 6F shows the identification of driver genes involved in acquired resistance to anti-PD-1 and functional pathways affected by driver genes. Figure 6F shows the expression levels of Elk1 in tumors and surrounding normal tissues in the Cancer Genome Atlas (TCGA) Cancer Genomics Program. [Figure 7A] We show that the Stat1 gene, which is overexpressed in response to IFNγ, is overexpressed in fourth-generation anti-PD-1 resistant cells and is suppressed in fourth-generation anti-PD-1 resistant cells in the presence of IFNγ. Second-generation anti-PD-1 resistant cells display an intermediate phenotype. [Figure 7B] We show that the Stat2 gene, which is overexpressed in response to IFNγ, is overexpressed in fourth generation anti-PD-1 resistant cells and is suppressed in fourth generation anti-PD-1 resistant cells in the presence of IFNγ. Second generation anti-PD-1 resistant cells display an intermediate phenotype. [Figure 7C] We show that the Irf1 gene, which is overexpressed in response to IFNγ, is overexpressed in fourth-generation anti-PD-1 resistant cells and suppressed in fourth-generation anti-PD-1 resistant cells in the presence of IFNγ. Second-generation anti-PD-1 resistant cells display an intermediate phenotype. [Figure 7D]We show that the Tap1 gene, which is overexpressed in response to IFNγ, is overexpressed in fourth-generation anti-PD-1 resistant cells and suppressed in fourth-generation anti-PD-1 resistant cells in the presence of IFNγ. Second-generation anti-PD-1 resistant cells display an intermediate phenotype. [Figure 8A] Pathway analysis of differentially expressed genes led to the identification of Ras and Rap1 signaling pathways. Figure 8A shows pathway identification using the web-based GENe SeT AnaLysis Toolkit (WebGestalt) using the top 1,000 genes from Figure 6A (panel 2). [Figure 8B] Figure 8B shows a volcano plot of the data presented in Figure 8A. [Figure 8C] Pathway analysis of differentially expressed genes led to the identification of Ras and Rap1 signaling pathways. Figure 8C shows the RAS signaling pathway. Convergence with Raf / Mek / Erk signaling is indicated using an ellipse. [Figure 8D] Pathway analysis of differentially expressed genes led to the identification of Ras and Rap1 signaling pathways. Figure 8D shows the RAP1 signaling pathway. Convergence with Raf / Mek / Erk signaling is indicated using ellipses. [Figure 9A] We show that the Ccl5 (RANTES) gene, which is overexpressed in response to IFNγ, is overexpressed in fourth generation anti-PD-1 resistant cells and suppressed in fourth generation anti-PD-1 resistant cells in the presence of IFNγ. Second generation anti-PD-1 resistant cells display an intermediate phenotype. [Figure 9B] We show that the Cxcl10 (IP-10) gene, which is overexpressed in response to IFNγ, is overexpressed in fourth-generation anti-PD-1 resistant cells and suppressed in fourth-generation anti-PD-1 resistant cells in the presence of IFNγ. Second-generation anti-PD-1 resistant cells display an intermediate phenotype. [Figure 9C]We show that the Ifnb1 gene, which is overexpressed in response to IFNγ, is overexpressed in fourth-generation anti-PD-1 resistant cells and suppressed in fourth-generation anti-PD-1 resistant cells in the presence of IFNγ. Second-generation anti-PD-1 resistant cells display an intermediate phenotype. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The present disclosure is based, in part, on the surprising discovery that upregulation of one or more genes associated with the following Gene Ontology (GO) functions: positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, positive regulation of IFNα production, positive regulation of defense response, positive regulation of IFNβ production, and regulation of inflammatory response is associated with resistance (acquired or primary resistance) to anti-PD-1 therapy. This is surprising, especially since these pathways are all known to be involved in the sensitivity of cancer cells to anti-PD-1 antibodies. Karachaliou et al.,Interferon gamma,an important marker of response to immune checkpoint blockade in non-small cell lung cancer and melanoma patients,Ther Adv Med Oncol.10:1758834017749748(2018);Rafique et al.,Immune Checkpoint Blockade and Interferon-α in Melanoma,Semin Oncol.42(3):436-447(2015);Uehara et al.,Intratumoral injection of IFN-β induces chemokine production in melanoma and augments the therapeutic efficacy of anti-PD-L1 mAb,Biochemical and Biophysical Research Communications 490(2)521-527(2017). Thus, these results establish biomarkers associated with acquired and primary resistance to anti-PD-1 therapy.

[0040] In embodiments, these results establish biomarkers associated with acquired and primary resistance to anti-PD-1 therapy. Thus, based on these biomarkers, in embodiments disclosed herein, patients may be selected for treatment with anti-PD-1 therapy based on evaluating a biological sample from the patient for the presence, absence, or levels of genes associated with one or more Gene Ontology (GO) pathways disclosed herein. For example, in embodiments, observed upregulation or downregulation of one or more genes associated with various GO functions disclosed herein may be used to diagnose resistance (acquired or primary resistance) to anti-PD-1 therapy.

[0041] Single-cell RNA sequencing of tumors that have developed acquired resistance to the PD-1 inhibitory antibodies disclosed herein demonstrated the progressive acquisition of a transcriptionally hyperactive phenotype. Specifically, more transcripts were upregulated than downregulated in PD-1 acquired resistance tumors than in parental PD-1 antibody sensitive tumors. This observation, without wishing to be bound by theory, suggests that acquired resistance is an active process in which tumor cells that upregulate genes in specific pathways gain a survival advantage over cells that do not upregulate or that downregulate overall transcriptional activity. Thus, in embodiments, tumors that have acquired resistance to checkpoint inhibitors, including PD-1 or PD-L1 blocking agents, may be more sensitive to certain classes of chemotherapy that target transcriptionally active cells. Thus, in some aspects, tumors that have acquired resistance to checkpoint inhibitors may be treated with certain classes of chemotherapy that target transcriptionally active cells. In embodiments, the chemotherapy classes include antimetabolite chemotherapy agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, etc.) or topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.) In embodiments, the chemotherapy classes may be used as neoadjuvant and / or adjuvant therapies.

[0042] Furthermore, the data presented herein demonstrate that acquired resistance tumors are characterized by a transcriptionally hyperactive phenotype, but many of the upregulated transcripts are not accompanied by a corresponding increase in protein expression. For example, genes associated with CD274 (PD-L1), beta2 macroglobulin, and other transcripts associated with interferon sensitivity and antigen presentation are upregulated in acquired resistance tumors, but there is no corresponding increase in the amount of PD-L1 or beta2 macroglobulin protein expression in acquired resistance tumors. Taken together, these findings suggest, among other things, that acquired resistance tumors attempt to upregulate many of the key genes that would drive increased sensitivity to antitumor immune responses, but that acquired defects in one or more post-transcriptional proteins perturb the response. For example, an increase in PD-L1 mRNA levels would be expected to lead to increased levels of PD-L1 protein. Thus, in embodiments, modulators of one or more processes including protein translation (e.g., assembly and / or function of ribosomal complexes, proper expression and / or function of tRNAs, proper synthesis and / or incorporation of amino acids, etc.), post-translational modification (e.g., modification of translated proteins with carbohydrates important for function or protection from degradation), or trafficking mechanisms (e.g., post-translational peptide processing, signal peptide recognition and cleavage, trafficking through the ER / Golgi network, etc.) may serve to combine defects in post-translational processes to create a synthetic lethal phenotype in cancer cell populations that develop resistance to PD-1 or PD-L1 blockade. In embodiments, the class of chemotherapy may be used as a neoadjuvant therapy or as an adjuvant therapy.

[0043] Methods for determining a patient's cancer treatment; methods for selecting patients for cancer treatment; and methods of treatment Accordingly, in one aspect, the disclosure relates to a method of determining a cancer treatment for a patient, the method comprising: (a) obtaining a biological sample from a subject; and (b) determining whether or not a patient is at risk for cancer. The method further comprises determining whether or not a patient is at risk for cancer. The method further comprises determining whether or not a patient is at risk for cancer. The method further comprises determining whether or not a patient is at risk for cancer. , negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid export, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis process; and (c) selecting a cancer therapy with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2 based on the evaluation of step (b).

[0044] In one aspect, the disclosure relates to a method of treating cancer, the method comprising: (a) obtaining a biological sample from a subject; and (b) detecting a signaling molecule that is involved in (i) positive regulation of cell cycle processes, regulation of G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of defense response, positive regulation of IFNβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or (ii) phospholipid excretion, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit regulation, and / or (iii) regulation of cellular functions, such as ... assessing the sample for the presence, absence, or level of one or more genes associated with a Gene Ontology (GO) pathway selected from mitochondrial assembly, phospholipid export, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis process; and (c) evaluating the sample for the presence, absence, or level of one or more genes associated with a Gene Ontology (GO) pathway selected from: ...and (iii) protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine and ribozynoindoles), inhibitors of rRNA and / or tRNA synthesis (e.g., calfluxine (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7A11 inhibitors sulfasalazine, erastin or sorafenib), modulation of post-translational modifications. (c) selecting a cancer therapy to use selected from a glycosylation inhibitor (e.g., tunicamycin, ppGalNAc-T3), a modulator of protein degradation and a modulator of protein trafficking (e.g., cyclosporine A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin, etc.) or a topoisomerase inhibitor; and (d) administering said cancer therapy using its ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2, and optionally administering said therapy selected in steps (c)(ii) and (c)(iii);

[0045] In non-limiting embodiments, upregulation of genes associated with a GO pathway selected from positive regulation of cell cycle processes, regulation of G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of defense response, positive regulation of IFNβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing, presentation of endogenous peptides via MHC class I is not observed. If no downregulation of genes associated with a GO pathway selected from phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid efflux, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis process is observed, then cancer therapy using its ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2 is continued.

[0046] In non-limiting embodiments, upregulation of genes associated with a GO pathway selected from positive regulation of cell cycle processes, regulation of G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of defense response, positive regulation of IFNβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or upregulation of genes associated with a GO pathway selected from positive regulation of phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid efflux, regulation of translation, mitochondrial function, and / or upregulation of genes associated with a GO pathway selected from positive regulation of IFNα production, positive regulation of defense response, positive regulation of IFNβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing / presentation of endogenous peptides via MHC class I; If downregulation of genes related to GO pathways selected from endodorial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication and ATP biosynthesis process is observed, then continue to administer cancer therapy with the ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity, where supplementation of administration of cancer therapy includes (i) agents with the ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity; (ii) antimetabolite chemotherapeutic agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine and acivicin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.);and (iii) protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozynoindole), inhibitors of rRNA and / or tRNA synthesis (e.g., calfluxine (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., cyclosporine ... cancer therapies selected from: intracellular transport inhibitors (e.g., the SLC7A11 inhibitors sulfasalazine, erastin, or sorafenib), modulators of post-translational modifications (e.g., the glycosylation inhibitors tunicamycin, ppGalNAc-T3), modulators of protein degradation and modulators of protein transport (e.g., cyclosporine A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin, etc.) or topoisomerase inhibitors;

[0047] In non-limiting embodiments, upregulation of genes associated with a GO pathway selected from positive regulation of cell cycle processes, regulation of G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of defense response, positive regulation of IFNβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or upregulation of genes associated with a GO pathway selected from phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid efflux, regulation of translation, mitochondrial function, and / or upregulation of genes associated with a GO pathway selected from positive regulation of IFNα production, positive regulation of defense response, positive regulation of IFNβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing / presentation of endogenous peptides via MHC class I; If downregulation of genes related to GO pathways selected from chondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication and ATP biosynthesis process is observed, then the administration of cancer therapy with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2 is not continued, where the administration of cancer therapy is selected from (i) agents with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; (ii) antimetabolite chemotherapeutic agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine and acivicin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.);and (iii) protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozynoindole), inhibitors of rRNA and / or tRNA synthesis (e.g., calfluxine (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., cyclosporine ... cancer therapies selected from: intracellular transport inhibitors (e.g., the SLC7A11 inhibitors sulfasalazine, erastin, or sorafenib), modulators of post-translational modifications (e.g., the glycosylation inhibitors tunicamycin, ppGalNAc-T3), modulators of protein degradation and modulators of protein transport (e.g., cyclosporine A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin, etc.) or topoisomerase inhibitors;

[0048] In embodiments, upregulation of one or more genes associated with the GO pathways listed in (i) relative to healthy tissue indicates a lack of response, resistance or refractory to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, upregulation of one or more genes associated with the GO pathways listed in (i) relative to another biological sample from a patient known to be sensitive to anti-PD-1 therapy indicates a lack of response, resistance or refractory to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, upregulation of one or more genes associated with the GO pathways listed in (i) relative to a previous biological sample obtained from the subject indicates a lack of response, resistance or refractory to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0049] In embodiments, the lack of upregulation of one or more genes associated with the GO pathways listed in (i) compared to healthy tissue indicates a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with the GO pathways listed in (i) compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy indicates a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with the GO pathways listed in (i) compared to a previous biological sample obtained from the subject indicates a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0050] In embodiments, downregulation of one or more genes associated with the GO pathways listed in (ii) relative to healthy tissue indicates a lack of response, resistance or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, downregulation of one or more genes associated with the GO pathways listed in (ii) relative to another biological sample from a patient known to be sensitive to anti-PD-1 therapy indicates a lack of response, resistance or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, downregulation of one or more genes associated with the GO pathways listed in (ii) relative to a previous biological sample obtained from the subject indicates resistance, lack of response or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0051] In embodiments, the lack of downregulation of one or more genes associated with the GO pathways listed in (ii) compared to healthy tissue indicates a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of downregulation of one or more genes associated with the GO pathways listed in (ii) compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy indicates a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of downregulation of one or more genes associated with the GO pathways listed in (ii) compared to a previous biological sample obtained from the subject indicates a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0052] In embodiments, lack of upregulation of one or more genes associated with a GO pathway compared to a previous biological sample from the subject indicates a response to a cancer therapy with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2, where the GO pathway is selected from (a) cellular response to IFNγ, (b) negative regulation of antigen processing and presentation, (c) type I IFN signaling pathway, (d) positive regulation of IκB kinase / NFκB signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I. In embodiments, lack of upregulation of one or more genes associated with a GO pathway compared to patients known to be sensitive to anti-PD-1 therapy indicates a response to cancer therapy with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2, where the GO pathway is selected from (a) cellular response to IFNγ, (b) negative regulation of antigen processing and presentation, (c) type I IFN signaling pathway, (d) positive regulation of IκB kinase / NFκB signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I. In embodiments, lack of upregulation of one or more genes associated with a GO pathway compared to the standard indicates a response to cancer therapy with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2, where the GO pathway is selected from (a) cellular response to IFNγ, (b) negative regulation of antigen processing and presentation, (c) type I IFN signaling pathway, (d) positive regulation of IκB kinase / NFκB signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I.

[0053] In embodiments, upregulation of one or more genes associated with a GO pathway compared to a previous biological sample from the subject indicates resistance, lack of response, or refractoryness to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2, and the GO pathway is selected from (a) cellular response to IFNγ, (b) negative regulation of antigen processing and presentation, (c) type I IFN signaling pathway, (d) positive regulation of IκB kinase / NFκB signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I. In embodiments, upregulation of one or more genes associated with a GO pathway relative to patients known to be sensitive to anti-PD-1 therapy is indicative of resistance, lack of response, or refractoryness to cancer therapy that utilizes the ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity, and the GO pathway is selected from (a) cellular response to IFNγ, (b) negative regulation of antigen processing and presentation, (c) type I IFN signaling pathway, (d) positive regulation of IκB kinase / NFκB signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I. In embodiments, upregulation of one or more genes associated with a GO pathway relative to a standard indicates resistance, lack of response, or refractoryness to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2, and the GO pathway is selected from (a) cellular response to IFNγ, (b) negative regulation of antigen processing and presentation, (c) type I IFN signaling pathway, (d) positive regulation of IκB kinase / NFκB signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I.

[0054] In embodiments, the lack of upregulation of one or more genes associated with a cellular response to IFNγ, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with a cellular response to IFNγ, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with a cellular response to IFNγ, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0055] In embodiments, upregulation of one or more genes associated with cellular response to IFNγ, as compared to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with cellular response to IFNγ, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with cellular response to IFNγ, as compared to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0056] In embodiments, upregulation of one or more genes associated with the type I IFN signaling pathway, as compared to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with the type I IFN signaling pathway, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with the type I IFN signaling pathway, as compared to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0057] In embodiments, the lack of upregulation of one or more genes associated with the type I IFN signaling pathway, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with the type I IFN signaling pathway, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with the type I IFN signaling pathway, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0058] In embodiments, upregulation of one or more genes associated with positive regulation of cell cycle processes, as compared to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with positive regulation of cell cycle processes, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with positive regulation of cell cycle processes, as compared to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0059] In embodiments, the lack of upregulation of one or more genes associated with positive regulation of cell cycle processes, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with positive regulation of cell cycle processes, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with positive regulation of cell cycle processes, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0060] In embodiments, upregulation of one or more genes associated with regulating G1 / S transition, relative to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with regulating G1 / S transition, relative to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with regulating G1 / S transition, relative to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0061] In embodiments, the lack of upregulation of one or more genes associated with regulating G1 / S transition, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of upregulation of one or more genes associated with regulating G1 / S transition, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of upregulation of one or more genes associated with regulating G1 / S transition, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity.

[0062] In embodiments, upregulation of one or more genes associated with regulating cell division, as compared to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with regulating cell division, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with regulating cell division, as compared to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0063] In embodiments, the lack of upregulation of one or more genes associated with regulating cell division, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with regulating cell division, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with regulating cell division, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0064] In embodiments, upregulation of one or more genes associated with regulating cell proliferation, as compared to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with regulating cell proliferation, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with regulating cell proliferation, as compared to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0065] In embodiments, the lack of upregulation of one or more genes associated with modulating cell proliferation, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with modulating cell proliferation, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with modulating cell proliferation, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0066] In embodiments, upregulation of one or more genes associated with positive regulation of IκB kinase / NFκB signaling, relative to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with positive regulation of IκB kinase / NFκB signaling, relative to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with positive regulation of IκB kinase / NFκB signaling, relative to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0067] In embodiments, the lack of upregulation of one or more genes associated with positive regulation of IκB kinase / NFκB signaling, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with positive regulation of IκB kinase / NFκB signaling, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with positive regulation of IκB kinase / NFκB signaling, as compared to a standard, is indicative of a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0068] In embodiments, upregulation of one or more genes associated with modulating the innate immune response, as compared to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with modulating the innate immune response, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with modulating the innate immune response, as compared to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0069] In embodiments, the lack of upregulation of one or more genes associated with modulating the innate immune response, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with modulating the innate immune response, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with modulating the innate immune response, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0070] In embodiments, upregulation of one or more genes associated with negative regulation of antigen processing / presentation, relative to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with negative regulation of antigen processing / presentation, relative to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with negative regulation of antigen processing / presentation, relative to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0071] In embodiments, the lack of upregulation of one or more genes associated with negative regulation of antigen processing / presentation compared to a previous biological sample from the subject indicates a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of upregulation of one or more genes associated with negative regulation of antigen processing / presentation compared to patients known to be sensitive to anti-PD-1 therapy indicates a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of upregulation of one or more genes associated with negative regulation of antigen processing / presentation compared to a standard indicates a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity.

[0072] In embodiments, upregulation of one or more genes associated with antigen processing / presentation of endogenous peptides via MHC class I, relative to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with antigen processing / presentation of endogenous peptides via MHC class I, relative to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with antigen processing / presentation of endogenous peptides via MHC class I, relative to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0073] In embodiments, the lack of upregulation of one or more genes associated with antigen processing / presentation of endogenous peptides via MHC class I, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with antigen processing / presentation of endogenous peptides via MHC class I, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with antigen processing / presentation of endogenous peptides via MHC class I, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0074] In embodiments, upregulation of one or more genes associated with positive regulation of IFNα production, relative to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with positive regulation of IFNα production, relative to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with positive regulation of IFNα production, relative to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0075] In embodiments, the lack of upregulation of one or more genes associated with positive regulation of IFNα production, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of upregulation of one or more genes associated with positive regulation of IFNα production, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of upregulation of one or more genes associated with positive regulation of IFNα production, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity.

[0076] In embodiments, upregulation of one or more genes associated with positive regulation of a protective response, as compared to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with positive regulation of a protective response, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with positive regulation of a protective response, as compared to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0077] In embodiments, the lack of upregulation of one or more genes associated with positive regulation of a protective response, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with positive regulation of a protective response, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with positive regulation of a protective response, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0078] In embodiments, upregulation of one or more genes associated with positive regulation of IFNβ production, as compared to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with positive regulation of IFNβ production, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with positive regulation of IFNβ production, as compared to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0079] In embodiments, the lack of upregulation of one or more genes associated with positive regulation of IFNβ production, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of upregulation of one or more genes associated with positive regulation of IFNβ production, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of upregulation of one or more genes associated with positive regulation of IFNβ production, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity.

[0080] In embodiments, upregulation of one or more genes associated with modulating inflammatory response compared to a previous biological sample from the subject indicates resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with modulating inflammatory response compared to a patient known to be sensitive to anti-PD-1 therapy indicates resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, upregulation of one or more genes associated with modulating inflammatory response compared to a standard indicates resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0081] In embodiments, the lack of upregulation of one or more genes associated with modulating an inflammatory response, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with modulating an inflammatory response, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of one or more genes associated with modulating an inflammatory response, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0082] In embodiments, downregulation of one or more genes associated with phospholipid efflux, relative to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with phospholipid efflux, relative to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with phospholipid efflux, relative to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0083] In embodiments, the lack of downregulation of one or more genes associated with phospholipid efflux, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of downregulation of one or more genes associated with phospholipid efflux, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of downregulation of one or more genes associated with phospholipid efflux, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity.

[0084] In embodiments, downregulation of one or more genes associated with negative regulation of fibrinolysis, as compared to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with negative regulation of fibrinolysis, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with negative regulation of fibrinolysis, as compared to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0085] In embodiments, the lack of downregulation of one or more genes associated with negative regulation of fibrinolysis compared to a previous biological sample from the subject indicates a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of downregulation of one or more genes associated with negative regulation of fibrinolysis compared to patients known to be sensitive to anti-PD-1 therapy indicates a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of downregulation of one or more genes associated with negative regulation of fibrinolysis compared to a standard indicates a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity.

[0086] In embodiments, downregulation of one or more genes associated with chylomicron assembly, relative to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with chylomicron assembly, relative to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with chylomicron assembly, relative to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0087] In embodiments, the lack of downregulation of one or more genes associated with chylomicron assembly compared to a previous biological sample from the subject indicates a response to a cancer therapy that utilizes an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of downregulation of one or more genes associated with chylomicron assembly compared to patients known to be sensitive to anti-PD-1 therapy indicates a response to a cancer therapy that utilizes an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of downregulation of one or more genes associated with chylomicron assembly compared to a standard indicates a response to a cancer therapy that utilizes an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity.

[0088] In embodiments, downregulation of one or more genes associated with plasma membrane repair, relative to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with plasma membrane repair, relative to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with plasma membrane repair, relative to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0089] In embodiments, the lack of downregulation of one or more genes associated with plasma membrane repair, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of downregulation of one or more genes associated with plasma membrane repair, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of downregulation of one or more genes associated with plasma membrane repair, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity.

[0090] In embodiments, downregulation of one or more genes associated with SRP-dependent cotranslational protein targeting to the membrane, as compared to a previous biological sample from the subject, indicates resistance, lack of response, or refractory to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with SRP-dependent cotranslational protein targeting to the membrane, as compared to a patient known to be sensitive to anti-PD-1 therapy, indicates resistance, lack of response, or refractory to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with SRP-dependent cotranslational protein targeting to the membrane, as compared to a standard, indicates resistance, lack of response, or refractory to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0091] In embodiments, the lack of downregulation of one or more genes associated with SRP-dependent cotranslational protein targeting to the membrane, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of downregulation of one or more genes associated with SRP-dependent cotranslational protein targeting to the membrane, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of downregulation of one or more genes associated with SRP-dependent cotranslational protein targeting to the membrane, as compared to a standard, is indicative of a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0092] In embodiments, downregulation of one or more genes associated with small ribosomal subunit assembly, as compared to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with small ribosomal subunit assembly, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with small ribosomal subunit assembly, as compared to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0093] In embodiments, the lack of downregulation of one or more genes associated with small ribosomal subunit assembly compared to a previous biological sample from the subject indicates a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of downregulation of one or more genes associated with small ribosomal subunit assembly compared to patients known to be sensitive to anti-PD-1 therapy indicates a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of downregulation of one or more genes associated with small ribosomal subunit assembly compared to a standard indicates a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0094] In embodiments, downregulation of one or more genes associated with phospholipid efflux relative to a previous biological sample from the subject indicates resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with phospholipid efflux relative to a patient known to be sensitive to anti-PD-1 therapy indicates resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with phospholipid efflux relative to a standard indicates resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0095] In embodiments, the lack of downregulation of one or more genes associated with phospholipid efflux, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of downregulation of one or more genes associated with phospholipid efflux, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of downregulation of one or more genes associated with phospholipid efflux, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity.

[0096] In embodiments, downregulation of one or more genes associated with translational regulation, relative to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with translational regulation, relative to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with translational regulation, relative to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0097] In embodiments, the lack of downregulation of one or more genes associated with translational regulation compared to a previous biological sample from the subject indicates a response to a cancer therapy that utilizes an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of downregulation of one or more genes associated with translational regulation compared to patients known to be sensitive to anti-PD-1 therapy indicates a response to a cancer therapy that utilizes an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of downregulation of one or more genes associated with translational regulation compared to a standard indicates a response to a cancer therapy that utilizes an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity.

[0098] In embodiments, downregulation of one or more genes associated with mitochondrial respiratory chain complex I, as compared to a previous biological sample from the subject, indicates resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with mitochondrial respiratory chain complex I, as compared to a patient known to be sensitive to anti-PD-1 therapy, indicates resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with mitochondrial respiratory chain complex I, as compared to a standard, indicates resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0099] In embodiments, the lack of downregulation of one or more genes associated with mitochondrial respiratory chain complex I, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of downregulation of one or more genes associated with mitochondrial respiratory chain complex I, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of downregulation of one or more genes associated with mitochondrial respiratory chain complex I, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity.

[0100] In embodiments, downregulation of one or more genes associated with mitochondrial translation elongation, as compared to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with mitochondrial translation elongation, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with mitochondrial translation elongation, as compared to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0101] In embodiments, the lack of downregulation of one or more genes associated with mitochondrial translation elongation compared to a previous biological sample from the subject indicates a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of downregulation of one or more genes associated with mitochondrial translation elongation compared to patients known to be sensitive to anti-PD-1 therapy indicates a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity. In embodiments, the lack of downregulation of one or more genes associated with mitochondrial translation elongation compared to a standard indicates a response to a cancer therapy that employs an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity.

[0102] In embodiments, downregulation of one or more genes associated with DNA-dependent DNA replication, as compared to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with DNA-dependent DNA replication, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with DNA-dependent DNA replication, as compared to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0103] In embodiments, the lack of downregulation of one or more genes associated with DNA-dependent DNA replication, as compared to a previous biological sample from the subject, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of downregulation of one or more genes associated with DNA-dependent DNA replication, as compared to patients known to be sensitive to anti-PD-1 therapy, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of downregulation of one or more genes associated with DNA-dependent DNA replication, as compared to a standard, is indicative of a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0104] In embodiments, downregulation of one or more genes associated with the ATP biosynthesis process, as compared to a previous biological sample from the subject, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with the ATP biosynthesis process, as compared to a patient known to be sensitive to anti-PD-1 therapy, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2. In embodiments, downregulation of one or more genes associated with the ATP biosynthesis process, as compared to a standard, is indicative of resistance, lack of response, or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0105] In embodiments, the lack of downregulation of one or more genes associated with the ATP biosynthesis process compared to a previous biological sample from the subject indicates a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of downregulation of one or more genes associated with the ATP biosynthesis process compared to patients known to be sensitive to anti-PD-1 therapy indicates a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of downregulation of one or more genes associated with the ATP biosynthesis process compared to a standard indicates a response to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0106] In embodiments, cancer therapy using the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2 may be indicated, for example, when expression of one or more of Rpl41, Rps15 and Rps8 is high, and / or expression of one or more of Cd274, B2M, Tap1, Tap2, Casp1 and Gasta3 is low. In embodiments, cancer therapy using the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2 may not be indicated, for example, when expression of one or more of Rpl41, Rps15 and Rps8 is low, and / or expression of one or more of Cd274, B2M, Tap1, Tap2, Casp1 and Gasta3 is high. In embodiments, patients characterized by low expression of one or more of Rpl41, Rps15, and Rps8 and / or high expression of one or more of Cd274, B2M, Tap1, Tap2, Casp1, and Gasta3 are likely to benefit from adjuvant or neoadjuvant therapy that eliminates PD-1 non-responsive cells.

[0107] In an embodiment, the biological sample is a fresh tissue sample, a frozen tumor tissue specimen, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue specimen. In an embodiment, the biological sample is a biopsy sample. In an embodiment, the biopsy sample is selected from endoscopic biopsy, bone marrow biopsy, endoscopic biopsy (e.g., cystoscopy, bronchoscopy, and colonoscopy), needle biopsy (e.g., fine needle aspiration, core needle biopsy, vacuum-assisted biopsy, X-ray-assisted biopsy, computed tomography (CT)-assisted biopsy, magnetic resonance imaging (MRI)-assisted biopsy, and ultrasound-assisted biopsy), skin biopsy (e.g., shave biopsy, punch biopsy, and incision biopsy), and surgical biopsy.

[0108] In an embodiment, the biological sample comprises a body fluid selected from blood, plasma, serum, tears, tears, bone marrow, blood, blood cells, peritoneal fluid, tissue or fine needle biopsy sample, cell-containing body fluid, free floating nucleic acid, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, lavage or irrigation fluid such as ductal washings or bronchoalveolar lavage, aspirate, scrape, bone marrow specimen, tissue biopsy specimen, surgical specimen, feces, other body fluid, secretion and / or excretion, and / or cells therefrom. In an embodiment, the biological sample is obtained by a technique selected from scrape, swab, and biopsy. In an embodiment, the biological sample is obtained by using a brush, (cotton) swab, spatula, rinse / wash, punch biopsy device, puncture of a cavity with a needle or surgical instrument.

[0109] In an embodiment, the biological sample comprises at least one tumor cell. In an embodiment, the tumor is selected from lymphoma and non-Hodgkin's lymphoma, B-cell lymphoma (including low-grade / follicular non-Hodgkin's lymphoma (NHL)), small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; or chronic myeloblastic leukemia.In some embodiments, the cancer is selected from the group consisting of basal cell carcinoma, biliary tract cancer, bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatocellular carcinoma; intraepithelial neoplasia; kidney or renal cancer; laryngeal cancer; leukemia; liver cancer. cancer);lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma);melanoma;myeloma;neuroblastoma;oral cancer (lips, tongue, mouth, and pharynx);ovarian cancer;pancreatic cancer;prostate cancer;retinoblastoma;rhabdomyosarcoma;rectal cancer;respiratory system cancer;salivary gland cancer;sarcoma;skin cancer;squamous cell carcinoma;stomach cancer;testicular cancer;thyroid cancer;uterine or endometrial cancer;urinary system cancer;vulvar cancer;lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, and B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL));small lymphocytic (SL) NHL;intermediate-grade / follicular NHL;intermediate-grade diffuse NHL;high-grade immunoblastic NHL;high-grade lymphoblastic NHL;high-grade small non-cleaving cell NHL;bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with nevus, edema (e.g., associated with brain tumors), and Meigs syndrome cancer; renal carcinoma; colorectal cancer; and adrenal cancer.

[0110] In embodiments, the evaluating is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS), or a combination thereof.

[0111] In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a Gene Ontology (GO) pathway selected from: (i) positive regulation of cell cycle processes, regulation of G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of defense response, positive regulation of IFNβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or (ii) phospholipid export, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid export, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis process.

[0112] In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a Gene Ontology (GO) pathway selected from (a) cellular response to IFNγ, (b) negative regulation of antigen processing and presentation, (c) type I IFN signaling pathway, (d) positive regulation of IκB kinase / NFκB signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I. In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with cellular response to IFNγ. In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with type I IFN signaling pathway. In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with positive regulation of IFNα production. In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with positive regulation of a defensive response. In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with positive regulation of IFNβ production. In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with regulation of an inflammatory response.

[0113] In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more of the nucleic acids of one or more genes associated with a Gene Ontology (GO) pathway selected from: (i) positive regulation of cell cycle processes, regulation of G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of defense response, positive regulation of IFNβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or (ii) phospholipid export, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid export, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis process.

[0114] In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a Gene Ontology (GO) pathway selected from (a) cellular response to IFNγ, (b) negative regulation of antigen processing and presentation, (c) type I IFN signaling pathway, (d) positive regulation of IκB kinase / NFκB signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I. In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with cellular response to IFNγ. In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with type I IFN signaling pathway. In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with positive regulation of IFNα production. In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with positive regulation of a defensive response. In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with positive regulation of IFNβ production. In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with regulation of an inflammatory response. In an embodiment, the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or a probe.

[0115] In embodiments, the assessing provides information to classify the patient into a high-risk or low-risk group. In embodiments, the high-risk classification includes a high level of tumor cells that are resistant to cancer therapies that utilize the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the low-risk classification includes a low level of tumor cells that are resistant to cancer therapies that utilize the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0116] In embodiments, the low or high risk classification indicates that neoadjuvant therapy is withheld. In embodiments, the low or high risk classification indicates that adjuvant therapy is withheld. In embodiments, the assessing predicts a positive response to and / or a positive benefit from cancer treatment. In embodiments, the assessing predicts a negative or neutral response to and / or a negative or neutral benefit from cancer treatment. In embodiments, the assessing predicts a positive response to and / or a positive benefit from neoadjuvant chemotherapy, or non-response to and / or a lack of benefit from neoadjuvant chemotherapy. In embodiments, the assessing predicts a positive response to and / or a benefit from adjuvant chemotherapy, or non-response to and / or a lack of benefit from adjuvant chemotherapy. In embodiments, the evaluating predicts a negative or neutral response to neoadjuvant chemotherapy and / or a negative or neutral benefit from neoadjuvant chemotherapy, or non-responsiveness to neoadjuvant chemotherapy and / or a lack of benefit from neoadjuvant chemotherapy. In embodiments, the evaluating predicts a negative or neutral response to adjuvant chemotherapy and / or a negative or neutral benefit from adjuvant chemotherapy, or non-responsiveness to adjuvant chemotherapy and / or a lack of benefit from adjuvant chemotherapy. In embodiments, the evaluating informs administration of neoadjuvant therapy. In embodiments, the evaluating informs administration of adjuvant therapy. In embodiments, the evaluating informs withholding of neoadjuvant therapy. In embodiments, the evaluating informs withholding of adjuvant therapy. In embodiments, the neoadjuvant therapy and / or adjuvant therapy is a chemotherapeutic agent. In embodiments, the neoadjuvant therapy and / or adjuvant therapy is a cytotoxic agent. In embodiments, the neoadjuvant and / or adjuvant therapy is a checkpoint inhibitor.

[0117] In embodiments, the candidate anti-cancer drug is selected from a chemotherapeutic agent, a cytotoxic agent, and a checkpoint inhibitor.

[0118] In embodiments, the chemotherapy class includes antimetabolite chemotherapy agents (eg, 5-fluorouracil, methotrexate, capecitabine, azacitidine, etc.) or topoisomerase inhibitors (eg, irinotecan, topotecan, etoposide, doxorubicin, etc.).

[0119] In embodiments, chemotherapy is selected from protein translation (e.g., assembly and / or function of ribosomal complexes, proper expression and / or function of tRNAs, proper synthesis and / or incorporation of amino acids, etc.), post-translational modification (e.g., modification of translated proteins with carbohydrates important for function or protection from degradation), or trafficking mechanisms (e.g., post-translational peptide processing, signal peptide recognition and cleavage, trafficking through the ER / Golgi network, etc.), and may serve to combine defects in post-translational processes to create a synthetic lethal phenotype in cancer cell populations that develop resistance to PD-1 or PD-L1 blocking agents. Classes of chemotherapy include antimetabolite chemotherapy agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, etc.) or topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.). In embodiments, the neoadjuvant and / or adjuvant therapy is selected from chemotherapeutic agents, cytotoxic agents, checkpoint inhibitors, antimetabolite chemotherapeutic agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.). In embodiments, the neoadjuvant and / or adjuvant therapy is selected from protein translation inhibitors (e.g., regulators of ribosomal complex assembly and / or function, regulators of tRNA expression and / or function, regulators of amino acid synthesis and / or uptake, regulators of post-translational modifications (e.g., modification of translated proteins with carbohydrates), regulators of protein degradation, and regulators of protein trafficking (e.g., post-translational peptide processing, signal peptide recognition and cleavage, transport through the ER / Golgi network, etc.), etc.) or topoisomerase inhibitors.

[0120] In embodiments, the neoadjuvant and / or adjuvant therapy includes protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborines, lamotrigine, and ribozynoindoles), inhibitors of rRNA and / or tRNA synthesis (e.g., calfluxine (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) , PAGDH inhibitor NCT-503), inhibitors of amino acid uptake (e.g., SLC7A11 inhibitors sulfasalazine, erastin, or sorafenib), regulators of post-translational modifications (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), regulators of protein degradation, and regulators of protein transport (e.g., cyclosporine A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin).

[0121] In embodiments, the upregulation is in comparison to healthy tissue. In embodiments, the upregulation is in comparison to another biological sample from a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the upregulation is in comparison to a previous biological sample obtained from the subject. In embodiments, the downregulation is in comparison to healthy tissue. In embodiments, the downregulation is in comparison to another biological sample from a patient known to be sensitive to anti-PD-1 therapy. In embodiments, the downregulation is in comparison to a previous biological sample obtained from the subject.

[0122] In an embodiment, the biological sample is a fresh tissue sample, a frozen tumor tissue specimen, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue specimen. In an embodiment, the biological sample is a biopsy sample. In an embodiment, the biopsy sample is selected from endoscopic biopsy, bone marrow biopsy, endoscopic biopsy (e.g., cystoscopy, bronchoscopy, and colonoscopy), needle biopsy (e.g., fine needle aspiration, core needle biopsy, vacuum-assisted biopsy, X-ray-assisted biopsy, computed tomography (CT)-assisted biopsy, magnetic resonance imaging (MRI)-assisted biopsy, and ultrasound-assisted biopsy), skin biopsy (e.g., shave biopsy, punch biopsy, and incision biopsy), and surgical biopsy. In an embodiment, the biological sample comprises a body fluid selected from blood, plasma, serum, tears, tears, bone marrow, blood, blood cells, ascites, tissue or fine needle biopsy sample, cell-containing body fluid, free floating nucleic acid, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, lavage or irrigation fluid such as ductal washing or bronchoalveolar lavage, aspirate, scrape, bone marrow specimen, tissue biopsy specimen, surgical specimen, feces, other body fluid, secretion and / or excretion, and / or cells therefrom. In an embodiment, the biological sample is obtained by a technique selected from scrape, swab, and biopsy. In an embodiment, the biological sample is obtained by using a brush, (cotton) swab, spatula, rinse / wash, punch biopsy device, puncture of a cavity with a needle or surgical instrument. In an embodiment, the biological sample comprises at least one tumor cell.

[0123] In embodiments, the tumor is Hodgkin's lymphoma and non-Hodgkin's lymphoma, B-cell lymphoma (including low-grade / follicular non-Hodgkin's lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell lymphoma. myeloblastic leukemia;or chronic myeloblastic leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer;bone cancer;brain and central nervous system cancer;breast cancer;peritoneal cancer;cervical cancer;choriocarcinoma;colorectal cancer;connective tissue cancer;digestive system cancer;endometrial cancer;esophageal cancer;eye cancer;head and neck cancer;gastric cancer (including gastrointestinal cancer);glioblastoma;liver cancer;hepatocellular carcinoma;intraepithelial neoplasia;kidney or renal cancer;laryngeal cancer;leukemia;liver cancer;lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma);melanoma;myeloma;neuroblastoma;oral cancer (lip, tongue , mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphomas, including Hodgkin lymphoma and non-Hodgkin lymphoma, and B-cell lymphomas, including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoma and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with nevus, edema (e.g., associated with brain tumors), and Meigs syndrome cancer; renal cancer; colorectal cancer; and adrenal cancer.

[0124] In embodiments, the evaluating is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS), or a combination thereof. In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a Gene Ontology (GO) pathway selected from: (i) positive regulation of cell cycle processes, regulation of G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of defense response, positive regulation of IFNβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing / presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or (ii) phospholipid export, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid export, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis process.

[0125] In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a Gene Ontology (GO) pathway selected from (a) cellular response to IFNγ, (b) negative regulation of antigen processing / presentation, (c) type I IFN signaling pathway, (d) positive regulation of IκB kinase / NFκB signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I. In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with the cellular response to IFNγ. In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with the type I IFN signaling pathway.

[0126] In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more of the nucleic acids of one or more genes associated with a Gene Ontology (GO) pathway selected from: (i) positive regulation of cell cycle processes, regulation of G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of defense response, positive regulation of IFNβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing / presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or (ii) phospholipid export, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid export, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis process. In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a Gene Ontology (GO) pathway selected from (a) cellular response to IFNγ, (b) negative regulation of antigen processing / presentation, (c) type I IFN signaling pathway, (d) positive regulation of IκB kinase / NFκB signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I. In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a cellular response to IFNγ. In an embodiment, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with the type I IFN signaling pathway. In an embodiment, the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or a probe.

[0127] In embodiments, the assessing provides information to classify the patient into a high or low risk group. In embodiments, a high risk classification includes a high level of inclusion of tumor cells that are resistant to cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, a low risk classification includes a low level of inclusion of tumor cells that are resistant to cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, a low risk classification indicates that the cancer therapy is withheld. In embodiments, a high risk classification indicates that the cancer therapy is administered.

[0128] In one aspect, the disclosure relates to a method of determining a cancer treatment for a patient, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the biological sample for expression of a gene selected from (i) CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP1, TAP2, CASP1, IRF, LTBR, ​​PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1; and / or (ii) a gene selected from RPL41, RPS15, RPS8, TRIM7, and LRG1; and (c) selecting a cancer therapy based on the evaluation of step (b).

[0129] In one aspect, the disclosure relates to a method of determining a cancer treatment for a patient, the method comprising: (a) obtaining a biological sample from the subject; and (b) analyzing the biological sample for a gene selected from (i) CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP1, TAP2, CASP1, IRF, LTBR, ​​PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1; and / or (ii) a gene selected from RPL41, RPS15, RPS8, TRIM7, and LRG1. and (c) selecting a cancer therapy based on the assessment of step (b), wherein the cancer therapy is selected from the group consisting of: (i) an agent capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2; (ii) antimetabolite chemotherapeutic agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine and acivicin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.);and (iii) protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozynoindole), inhibitors of rRNA and / or tRNA synthesis (e.g., calfluxine (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., the SLC7A11 inhibitors sulfasalazine, erastin, or sorafenib), modulators of post-translational modifications (e.g., the glycosylation inhibitors tunicamycin, ppGalNAc-T3), modulators of protein degradation and modulators of protein transport (e.g., cyclosporine A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin, etc.), or topoisomerase inhibitors;

[0130] In one aspect, the disclosure relates to a method of selecting a patient for cancer treatment, the method comprising: (a) obtaining a biological sample from a subject; (b) evaluating the biological sample for expression of a gene selected from (i) CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP1, TAP2, CASP1, IRF, LTBR, ​​PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1; and / or (ii) a gene selected from RPL41, RPS15, RPS8, TRIM7, and LRG1; and (c) selecting a cancer therapy based on the evaluation of step (b).

[0131] In one aspect, the disclosure relates to a method of selecting a patient for cancer treatment, the method comprising: (a) obtaining a biological sample from the subject; and (b) examining the biological sample for a gene selected from: (i) CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP1, TAP2, CASP1, IRF, LTBR, ​​PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1; and / or (ii) RPL41, RPS15, RPS8, TRIM7, and LRG1. and (c) selecting a cancer therapy based on the assessment of step (b), wherein the cancer therapy is selected from the group consisting of: (i) an agent capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2; (ii) antimetabolite chemotherapeutic agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine and acivicin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.);and (iii) protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozynoindole), inhibitors of rRNA and / or tRNA synthesis (e.g., calfluxine (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., the SLC7A11 inhibitors sulfasalazine, erastin, or sorafenib), modulators of post-translational modifications (e.g., the glycosylation inhibitors tunicamycin, ppGalNAc-T3), modulators of protein degradation and modulators of protein transport (e.g., cyclosporine A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin, etc.), or topoisomerase inhibitors;

[0132] In one aspect, the disclosure relates to a method of treating cancer, the method comprising: (a) obtaining a biological sample from a subject; and (b) examining the biological sample for a gene selected from: (i) CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP1, TAP2, CASP1, IRF, LTBR, ​​PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1; and / or (ii) RPL41, RPS15, RPS8, TRIM 7 and LRG1; and (c) (i) agents capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2; (ii) antimetabolite chemotherapeutic agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine and acivicin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.);and (iii) protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozynoindoles), inhibitors of rRNA and / or tRNA synthesis (e.g., calfluxine (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., the GLUD1 inhibitor R162, the BCAT1 inhibitor gabapentin, the glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), the PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., the SLC7A11 inhibitor sulfasalazine, ethoxyquinone ... (d) selecting a cancer therapy selected from a cancer therapy capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2; (e) administering a cancer therapy capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2;

[0133] In non-limiting embodiments, if upregulation of overexpression of genes selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP1, TAP2, CASP1, IRF, LTBR, ​​PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1 is not observed, and / or downregulation of genes selected from RPL41, RPS15, RPS8, TRIM7, and LRG1 is not observed, administration of a cancer treatment capable of inhibiting the function and / or activity of PD-1, PD-L1, and / or PD-L2 is continued.

[0134] In non-limiting embodiments, upregulation of overexpression of a gene selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP1, TAP2, CASP1, IRF, LTBR, ​​PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1 is observed, and / or downregulation of a gene selected from RPL41, RPS15, RPS8, TRIM7, and LRG1 is observed, indicating a decrease in the function and / or expression of PD-1, PD-L1, and / or PD-L2. The administration of cancer therapy continues, which may include (i) agents capable of inhibiting the function and / or activity of PD-1, PD-L1, and / or PD-L2; (ii) antimetabolite chemotherapeutic agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine, and acivicin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., Silvestro, ol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine and ribozynoindol), inhibitors of rRNA and / or tRNA synthesis (e.g., calfluxine (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., Supplemental administration of a cancer therapy selected from among the SLC7A11 inhibitors sulfasalazine, erastin, or sorafenib), regulators of post-translational modifications (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), regulators of protein degradation, and regulators of protein transport (e.g., cyclosporine A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin) is performed.

[0135] In embodiments, a cancer treatment capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2 is selected when the biological sample comprises at least one tumor cell, and a gene selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP1, TAP2, CASP1, IRF, LTBR, ​​PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1 is not upregulated in the at least one tumor cell compared to healthy tissue, a previous biological sample obtained from the subject, or another biological sample from a patient known to be sensitive to anti-PD-1 therapy, and / or a gene selected from RPL41, RPS15, RPS8, TRIM7, and LRG1 is not downregulated in the at least one tumor cell compared to healthy tissue, a previous biological sample obtained from the subject, or another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

[0136] In embodiments, the biological sample comprises at least one tumor cell, and a gene selected from AP2, CASP1, IRF, LTBR, ​​PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1 is upregulated in the at least one tumor cell compared to healthy tissue, a previous biological sample obtained from the subject, or another biological sample from a patient known to be sensitive to anti-PD-1 therapy, and / or or another biological sample from the patient known to be sensitive to anti-PD-1 therapy, then the cancer therapy is selected from antimetabolite chemotherapeutic agents, topoisomerase inhibitors, ribosome biogenesis inhibitors, inhibitors of rRNA and / or tRNA synthesis, inhibitors of amino acid synthesis, amino acid uptake inhibitors, regulators of post-translational modifications, regulators of protein degradation, regulators of protein transport, or topoisomerase inhibitors.

[0137] In embodiments, upregulation of one or more genes listed in (b)(i) relative to healthy tissue indicates a lack of response, resistance or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, downregulation of one or more genes listed in (b)(ii) relative to healthy tissue indicates a lack of response, resistance or refractory to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0138] In embodiments, upregulation of one or more genes listed in (b)(i) relative to another biological sample from a patient known to be sensitive to anti-PD-1 therapy indicates a lack of response, resistance or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, downregulation of one or more genes listed in (b)(ii) relative to another biological sample from a patient known to be sensitive to anti-PD-1 therapy indicates a lack of response, resistance or refractory to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0139] In embodiments, upregulation of one or more genes listed in (b)(i) relative to a previous biological sample obtained from the subject indicates a lack of response, resistance or refractoriness to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, downregulation of one or more genes listed in (b)(ii) relative to a previous biological sample obtained from the subject indicates a lack of response, resistance or refractoriness to a cancer treatment that employs the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0140] In embodiments, a lack of upregulation of one or more genes listed in (b)(i) relative to healthy tissue indicates a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, a lack of downregulation of one or more genes listed in (b)(ii) relative to healthy tissue indicates a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0141] In embodiments, the lack of upregulation of one or more genes listed in (b)(i) relative to another biological sample from a patient known to be sensitive to anti-PD-1 therapy indicates a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of downregulation of one or more genes listed in (b)(ii) relative to another biological sample from a patient known to be sensitive to anti-PD-1 therapy indicates a response to a cancer therapy that employs an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0142] In embodiments, a lack of upregulation of one or more genes listed in (b)(i) relative to a previous biological sample obtained from the subject indicates that a lack of response to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2 will occur. In embodiments, a lack of downregulation of one or more genes listed in (b)(ii) relative to a previous biological sample obtained from the subject indicates that a lack of response to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2 will occur.

[0143] In an embodiment, the biological sample is a fresh tissue sample, a frozen tumor tissue specimen, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue specimen. In an embodiment, the biological sample is a biopsy sample. In an embodiment, the biopsy sample is selected from endoscopic biopsy, bone marrow biopsy, endoscopic biopsy (e.g., cystoscopy, bronchoscopy, and colonoscopy), needle biopsy (e.g., fine needle aspiration, core needle biopsy, vacuum-assisted biopsy, X-ray-assisted biopsy, computed tomography (CT)-assisted biopsy, magnetic resonance imaging (MRI)-assisted biopsy, and ultrasound-assisted biopsy), skin biopsy (e.g., shave biopsy, punch biopsy, and incision biopsy), and surgical biopsy. In an embodiment, the biological sample comprises a body fluid selected from blood, plasma, serum, tears, tears, bone marrow, blood, blood cells, peritoneal fluid, tissue or fine needle biopsy sample, cell-containing body fluid, free floating nucleic acid, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, lavage or irrigation fluid such as ductal washings or bronchoalveolar lavage, aspirate, scrape, bone marrow specimen, tissue biopsy specimen, surgical specimen, feces, other body fluid, secretion and / or excretion, and / or cells therefrom. In an embodiment, the biological sample is obtained by a technique selected from scrape, swab, and biopsy. In an embodiment, the biological sample is obtained by using a brush, (cotton) swab, spatula, rinse / wash, punch biopsy device, puncture of a cavity with a needle or surgical instrument.

[0144] In an embodiment, the biological sample comprises at least one tumor cell. In an embodiment, the tumor is selected from the group consisting of Hodgkin's and non-Hodgkin's lymphomas, B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell lymphoma. myeloblastic leukemia;or chronic myeloblastic leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer;bone cancer;brain and central nervous system cancer;breast cancer;peritoneal cancer;cervical cancer;choriocarcinoma;colorectal cancer;connective tissue cancer;digestive system cancer;endometrial cancer;esophageal cancer;eye cancer;head and neck cancer;gastric cancer (including gastrointestinal cancer);glioblastoma;liver cancer;hepatocellular carcinoma;intraepithelial neoplasia;kidney or renal cancer;laryngeal cancer;leukemia;liver cancer;lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma);melanoma;myeloma;neuroblastoma;oral cancer (lip, tongue , mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphomas, including Hodgkin lymphoma and non-Hodgkin lymphoma, and B-cell lymphomas, including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoma and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with nevus, edema (e.g., associated with brain tumors), and Meigs syndrome cancer; renal cancer; colorectal cancer; and adrenal cancer.

[0145] In embodiments, the evaluating is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, Western blotting, in cell western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS), or a combination thereof. In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by one or more genes listed in (b)(i) and / or (b)(ii). In embodiments, the agent that specifically binds to one or more proteins comprises an antibody, an antibody-like molecule, or a binding fragment thereof. In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more of the nucleic acids of one or more genes associated with the genes listed in (b)(i) and / or (b)(ii). In embodiments, the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or a probe.

[0146] In one aspect, the disclosure relates to a method of determining a cancer treatment for a patient, the method comprising: (a) obtaining a biological sample from the subject; (b) assessing the biological sample for activation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras; and (c) selecting a cancer therapy based on the assessment of step (b), wherein the cancer therapy is selected from: (i) an agent capable of inhibiting the function and / or activity of PD-1, PD-L1, and / or PD-L2; (ii) an anti-metabolite. Anti-substance chemotherapy agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine, and acivicin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozynoindole), rRNA and / or inhibitors of tRNA synthesis (e.g., calfluxin (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7A11 inhibitor sulfasalazine, erastin or sorafenib). and wherein the inhibitor is selected from among inhibitors of cyclosporine A, cyclosporine B, cyclosporine C, cyclosporine D, cyclosporine E, cyclosporine F, cyclosporine F, cyclosporine F, cyclosporine F, cyclosporine B, cyclosporine F, cyclosporine C, cyclosporine D ...

[0147] In one aspect, the disclosure relates to a method of selecting a patient for cancer treatment, the method comprising: (a) obtaining a biological sample from the subject; (b) assessing the biological sample for activation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras; and (c) selecting a cancer therapy based on the assessment of step (b), wherein the cancer therapy is selected from: (i) an agent capable of inhibiting a function and / or activity of PD-1, PD-L1, and / or PD-L2; (ii) an agent capable of inhibiting a function and / or activity of PD-1, PD-L1, and / or PD-L2; antagonist chemotherapeutic agents (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-L-norleucine (DON), azaserine, and acivicin), topoisomerase inhibitors (e.g., irinotecan, topotecan, etoposide, doxorubicin, etc.); and (iii) protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine, and ribozynoindole), rR Inhibitors of NA and / or tRNA synthesis (e.g., calfluxine (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitor R162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7A11 inhibitor sulfasalazine, erastin or sorafe nibs), regulators of post-translational modifications (e.g., the glycosylation inhibitors tunicamycin, ppGalNAc-T3), regulators of protein degradation and regulators of protein transport (e.g., cyclosporine A, fendiline, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin, etc.), or topoisomerase inhibitors.

[0148] In one aspect, the disclosure relates to a method of treating cancer, the method comprising: (a) obtaining a biological sample from a subject; (b) assessing the biological sample for activation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras; and (c) administering to a subject an antibody capable of inhibiting the function and / or activity of PD-1, PD-L1, and / or PD-L2; (i) an agent capable of inhibiting the function and / or activity of PD-1, PD-L1, and / or PD-L2; (ii) an antimetabolite chemotherapeutic agent (e.g., 5-fluorouracil, methotrexate, capecitabine, azacitidine, 6-diazo-5-oxo-1, 5-methyl ... (iii) protein translation inhibitors (e.g., silvestrol and omacetaxine), ribosome biogenesis inhibitors (e.g., diazaborine, lamotrigine and ribozynoindole), inhibitors of rRNA and / or tRNA synthesis (e.g., calfluxin (CX-3543) and CX-5461), inhibitors of amino acid synthesis (e.g., GLUD1 inhibitors R 162, BCAT1 inhibitor gabapentin, glutaminase inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES), PAGDH inhibitor NCT-503), amino acid uptake inhibitors (e.g., SLC7A11 inhibitors sulfasalazine, erastin, or sorafenib), regulators of post-translational modifications (e.g., glycosylation inhibitors tunicamycin, ppGalNAc-T3), regulators of protein degradation, and regulators of protein transport (e.g., cyclosporine A, fendi, (c) administering a cancer therapy selected from the group consisting of cyclosporine, cyclosporine, riboflavin, parbendazole, paroxetine, parthenolide, quinacrine, sertraline, spiperone, thimerosal, astemizole, perhexiline, HUN-7293, CAM741, CK147, and cotransin) or a topoisomerase inhibitor; and (d) administering a cancer therapy that utilizes its ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2, and optionally administering the treatment selected in steps (c)(ii) and (c)(iii).

[0149] In embodiments, a cancer treatment capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2 is selected when the biological sample comprises at least one tumor cell, and a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1 and Ras is not upregulated in the at least one tumor cell when compared to healthy tissue, a previous biological sample obtained from the subject, or another biological sample from a patient known to be sensitive to anti-PD-1 therapy.

[0150] In embodiments, if the biological sample comprises at least one tumor cell and a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras is upregulated in the at least one tumor cell compared to healthy tissue, a previous biological sample obtained from the subject, or another biological sample from a patient known to be sensitive to anti-PD-1 therapy, the cancer therapy is selected from antimetabolite chemotherapeutic agents, topoisomerase inhibitors, protein translation inhibitors, ribosome biogenesis inhibitors, inhibitors of rRNA and / or tRNA synthesis, inhibitors of amino acid synthesis, amino acid uptake inhibitors, regulators of post-translational modifications, regulators of protein degradation, regulators of protein transport, topoisomerase inhibitors.

[0151] In embodiments, upregulation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1 and Ras relative to healthy tissue indicates lack of response, resistance or refractory to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, upregulation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1 and Ras relative to another biological sample from a patient known to be sensitive to anti-PD-1 therapy indicates lack of response, resistance or refractory to a cancer therapy that utilizes an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, upregulation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras, as compared to a previous biological sample obtained from the subject, is indicative of the development of lack of response, resistance, or refractoryness to a cancer therapy that employs the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2.

[0152] In embodiments, the lack of upregulation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1 and Ras compared to healthy tissue indicates a response to a cancer therapy with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1 and Ras compared to another biological sample from a patient known to be sensitive to anti-PD-1 therapy indicates a response to a cancer therapy with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the lack of upregulation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1 and Ras compared to a previous biological sample obtained from the subject indicates a lack of response to a cancer treatment with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2 occurs.

[0153] In an embodiment, the biological sample is a fresh tissue sample, a frozen tumor tissue specimen, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue specimen. In an embodiment, the biological sample is a biopsy sample. In an embodiment, the biopsy sample is selected from endoscopic biopsy, bone marrow biopsy, endoscopic biopsy (e.g., cystoscopy, bronchoscopy, and colonoscopy), needle biopsy (e.g., fine needle aspiration, core needle biopsy, vacuum-assisted biopsy, X-ray-assisted biopsy, computed tomography (CT)-assisted biopsy, magnetic resonance imaging (MRI)-assisted biopsy, and ultrasound-assisted biopsy), skin biopsy (e.g., shave biopsy, punch biopsy, and incision biopsy), and surgical biopsy. In an embodiment, the biological sample comprises a body fluid selected from blood, plasma, serum, tears, tears, bone marrow, blood, blood cells, peritoneal fluid, tissue or fine needle biopsy sample, cell-containing body fluid, free floating nucleic acid, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, feces, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, lavage or irrigation fluid such as ductal washings or bronchoalveolar lavage, aspirate, scrape, bone marrow specimen, tissue biopsy specimen, surgical specimen, feces, other body fluid, secretion and / or excretion, and / or cells therefrom. In an embodiment, the biological sample is obtained by a technique selected from scrape, swab, and biopsy. In an embodiment, the biological sample is obtained by using a brush, (cotton) swab, spatula, rinse / wash, punch biopsy device, puncture of a cavity with a needle or surgical instrument.

[0154] In an embodiment, the biological sample comprises at least one tumor cell. In an embodiment, the tumor is selected from the group consisting of Hodgkin's and non-Hodgkin's lymphomas, B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphocytic leukemia (ALL); hairy cell lymphoma. myeloblastic leukemia;or chronic myeloblastic leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer;bone cancer;brain and central nervous system cancer;breast cancer;peritoneal cancer;cervical cancer;choriocarcinoma;colorectal cancer;connective tissue cancer;digestive system cancer;endometrial cancer;esophageal cancer;eye cancer;head and neck cancer;gastric cancer (including gastrointestinal cancer);glioblastoma;liver cancer;hepatocellular carcinoma;intraepithelial neoplasia;kidney or renal cancer;laryngeal cancer;leukemia;liver cancer;lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma);melanoma;myeloma;neuroblastoma;oral cancer (lip, tongue , mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphomas, including Hodgkin lymphoma and non-Hodgkin lymphoma, and B-cell lymphomas, including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoma and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with nevus, edema (e.g., associated with brain tumors), and Meigs syndrome cancer; renal cancer; colorectal cancer; and adrenal cancer.

[0155] In embodiments, the evaluating is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, Western blotting, in cell western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS), or a combination thereof. In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras. In embodiments, the agent that specifically binds to the one or more proteins comprises an antibody, an antibody-like molecule, or a binding fragment thereof. In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more of the nucleic acids of one or more genes associated with a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras. In embodiments, the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or a probe.

[0156] Trim7 is a member of a large family that includes approximately 80 different tripartite motif proteins (tripartite motif containing (TRIM)). The protein family includes 80 TRIM protein members in humans. In embodiments, Trim family activation can be assayed by modulation of the innate immune response through control of IFN-responsive genes (i.e., IRF1 / IRF3 / IRF7, JAK / STAT and NFκB). In embodiments, Trim family members have a C-terminal SPRY domain (e.g., in Trim6 and Trim7), and in embodiments, Trim7 is assayed based on induction of proliferation, EMT, and acquisition of a chemoresistance phenotype.

[0157] In embodiments, Trim7 pathway activation can be assayed using E3 ubiquitin ligase activity. In embodiments, Trim7 pathway activation can be assayed by c-Jun / AP1 activation by Ras-Raf-MEK-ERK signaling. In embodiments, Trim7 pathway activation can be assayed by assaying protein ubiquitination.

[0158] In embodiments, Trim7 pathway activation can be assayed using ubiquitination and stabilization of the AP1 coactivator RACO-1 and / or increased AP1-mediated gene expression. AP1-mediated gene expression signatures are well known in the art.

[0159] In embodiments, Trim7 pathway activation may be assayed based on K63-linked ubiquitination of target proteins, such as proteins involved in cell proliferation and innate immune response. In embodiments, Trim7 pathway activation may be assayed based on Trim7 phosphorylation, K63-linked ubiquitination and / or protein levels of the AP-1 coactivator known as RACO-1. ​​In embodiments, Trim7 pathway activation may be assayed based on the levels or activity of STING (Stimulator of Interferon Genes, MITA, ERIS, MPYS, TMEM173). In embodiments, Trim7 pathway activation may be assayed via K48-linked ubiquitination of STING. In embodiments, Trim7 pathway activation may be assayed for upregulation of IFNb, IP-10 and Rantes. In embodiments, Trim7 pathway activation may be assayed based on activation of proteins shown in FIG. 6C.

[0160] Mitogen-activated protein kinase 8 interacting protein 1 (Mapk8ip1) is also known as c-Jun-amino-terminal kinase interacting protein 1. In embodiments, Mapk8ip1 pathway activation may be assayed based on assaying functional multiprotein complexes in different components of the JNK pathway, including RAC1 or RAC2, MAP3K11 / MLK3 or MAP3K7 / TAK1, MAP2K7 / MKK7, MAPK8 / JNK1 and / or MAPK9 / JNK2. In embodiments, Mapk8ip1 pathway activation may be assayed based on the level and / or sensitivity to programmed cell death by apoptosis.

[0161] In embodiments, ETS-like-1 protein (Elk1) pathway activation can be assayed based on the activation of Ras-Raf-MEK-ERK signaling, which is known in the art. In embodiments, Elk1 pathway activation can be assayed based on Elk1 phosphorylation. In embodiments, Elk1 pathway activation can be assayed based on the activation of Elk1 target genes, which is known in the art (see, for example, Odrowaz and Sharrocks, ELK1 Uses Different DNA Binding Modes to Regulate Functionally Distinct Classes of Target Genes, PLOS Genetics 8(5):e1002694(2012)).

[0162] In embodiments, leucine-rich alpha2-glycoprotein 1 (Lrg1) pathway activation may be assayed based on induction of TGFβ and / or SMAD1 / 5 / 8 signaling.

[0163] In embodiments, Ras pathway activation can be assayed based on the activation of the proteins shown in FIG. 8C.

[0164] In an embodiment, activation of the Rap1 pathway can be assayed based on the activation of the proteins shown in FIG. 8D.

[0165] In embodiments, Arginase 1 (Arg1) pathway activation can be assayed based on the assay hydrolysis of arginine to ornithine and urea. In embodiments, Arginase 1 (Arg1) pathway activation can be assayed based on the inhibition of tumor infiltrating lymphocytes (TIL). In embodiments, Arginase 1 (Arg1) pathway activation can be assayed based on polyamine levels or synthesis (via L-ornithine).

[0166] In one aspect, the disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising administering a cancer treatment selected from antimetabolite chemotherapeutic agents, topoisomerase inhibitors, protein translation inhibitors, ribosome biogenesis inhibitors, inhibitors of rRNA and / or tRNA synthesis, inhibitors of amino acid synthesis, inhibitors of amino acid uptake, regulators of post-translational modification, regulators of protein degradation, regulators of protein trafficking, topoisomerase inhibitors, wherein the subject has received or is receiving anti-cancer drug treatment with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2, and wherein the subject has experienced a lack of response, resistance or refractory to the cancer therapy with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

[0167] In one aspect, the disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising: (a) administering an anti-cancer treatment capable of inhibiting a function and / or activity of PD-1, PD-L1 and / or PD-L2; (b) assessing the anti-tumor response to the anti-cancer treatment capable of inhibiting a function and / or activity of PD-1, PD-L1 and / or PD-L2 by monitoring tumor size reduction in the subject; (c) if a lack of tumor size reduction is observed, administering a Trim7 modulator and / or a proteasome inhibitor; (d) re-assessing the anti-tumor response to the anti-cancer treatment capable of inhibiting a function and / or activity of PD-1, PD-L1 and / or PD-L2 by monitoring tumor size reduction in the subject; and (e) discontinuing administration of the Trim7 modulator if a reduction in tumor size is observed. In embodiments, the proteasome inhibitor is selected from bortezomib, carfilzomib, ixazomib, oprozomib, delanzomib, and marizomib.

[0168] In one aspect, the present invention relates to a method of treating cancer in a subject in need thereof, the method comprising: (a) administering an anti-cancer treatment capable of inhibiting a function and / or activity of PD-1, PD-L1 and / or PD-L2; (b) assessing an anti-tumor response to the anti-cancer treatment capable of inhibiting a function and / or activity of PD-1, PD-L1 and / or PD-L2 using: (i) obtaining a biological sample from the subject; and (ii) assessing the biological sample for overexpression and / or activation of TRIM7; and (c) assessing the overexpression and / or activation of TRIM7. If overexpression and / or activation is observed, administering a Trim7 modulator and / or a proteasome inhibitor, (d) reassessing the anti-tumor response from an anti-cancer treatment capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2 using (i) obtaining a biological sample from the subject; (ii) evaluating the biological sample for TRIM7 overexpression and / or activation; and (e) withdrawing administration of the Trim7 modulator if TRIM7 overexpression and / or activation is not observed. In an embodiment, the proteasome inhibitor is selected from bortezomib, carfilzomib, ixazomib, oprozomib, delanzomib and marizomib.

[0169] In one aspect, the disclosure relates to a method of treating cancer in a subject in need thereof, the method comprising: (a) administering an anti-cancer treatment capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2; (b) assessing overexpression and / or activation of TRIM7 using: (i) obtaining a biological sample from the subject; and (ii) evaluating the biological sample for overexpression and / or activation of TRIM7; (c) if overexpression and / or activation of TRIM7 is observed, administering a Trim7 modulator and / or a proteasome inhibitor; (d) re-assessing overexpression and / or activation of TRIM7 using: (i) obtaining a biological sample from the subject; (ii) evaluating the biological sample for overexpression and / or activation of TRIM7; and (e) discontinuing administration of the Trim7 modulator if overexpression and / or activation of TRIM7 is not observed. In embodiments, the proteasome inhibitor is selected from bortezomib, carfilzomib, ixazomib, oprozomib, delanzomib, and marizomib.

[0170] In embodiments, the Trim7 modulator is a Trim7 inhibitor. In embodiments, the Trim7 modulator is selected from small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antisense RNA, guide RNA (gRNA), small molecule, antibody, peptide, and peptidomimetic. In embodiments, the small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antisense RNA, or guide RNA (gRNA) inhibits the production of Trim7 protein. In embodiments, the peptidomimetic mimics the target of Trim7, thereby inhibiting the activity of Trim7.

[0171] In embodiments, the Trim7 inhibitor is a small molecule or peptide inhibitor that binds to Trim7 protein at or near a protein segment selected from MAAVGPRTGPGTGAEALALAAEL (SEQ ID NO: 1), AATRPFPLPCP (SEQ ID NO: 2), HGSQAAAARAAAARCG (SEQ ID NO: 3), and NVSLKTFVLKGMLKKFKEDLRGELEKEEKV (SEQ ID NO: 4).

[0172] In an embodiment, the Trim7 regulator is a mitogen-activated and stress-activated kinase 1 (MSK1) inhibitor, where the MSK1 inhibitor regulates Trim7 via downstream effects of inhibition of MSK1. In an embodiment, the MSK1 inhibitor is selected from Ro31-8220, SB-747651A and H89. In an embodiment, the MSK1 inhibitor is SB-747651A.

[0173] In embodiments, the proteasome inhibitor is selected from bortezomib, carfilzomib, ixazomib, oprozomib, delanzomib, and marizomib.

[0174] In embodiments, the evaluating is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, Western blotting, in cell western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS), or a combination thereof. In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more proteins encoded by the Trim7 pathway. In embodiments, the agent that specifically binds to one or more proteins comprises an antibody, an antibody-like molecule, or a binding fragment thereof. In embodiments, the evaluating is performed by contacting the sample with an agent that specifically binds to one or more of the nucleic acids of one or more genes associated with the Trim7 pathway. In embodiments, the agent that specifically binds to one or more of the nucleic acids is a nucleic acid primer or a probe.

[0175] In an embodiment, the evaluating is performed by assaying E3 ubiquitin ligase activity. In an embodiment, the evaluating is performed by assaying protein ubiquitination and / or K48-linked ubiquitination of Stimulator of Interferon Genes (STING) and / or AP-1 coactivator RACO-1. ​​In an embodiment, the evaluating is performed by assaying c-Jun / AP1 activation and / or AP1-mediated increase in gene expression by Ras-Raf-MEK-ERK signaling. In an embodiment, the evaluating is performed by assaying ubiquitination and stabilization of AP1 coactivator RACO-1. ​​In an embodiment, the evaluating is performed by assaying K63-linked ubiquitination of a target protein, such as a protein involved in cell proliferation and innate immune response. In an embodiment, the evaluating is performed by assaying Trim7 phosphorylation, K63-linked ubiquitination and / or protein levels of AP-1 coactivator RACO-1. In an embodiment, the evaluating is performed by assaying upregulation of IFNβ, IP-10 and / or Rantes.

[0176] In embodiments, the method further comprises administration of an anti-checkpoint agent. In embodiments, the anti-checkpoint agent is an antibody selected from nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS936559, MPDL328OA (ROCHE), cemiplimab (LIBTAYO), atezolizumab (TECENTRIQ), avelumab (BAVENCIO), and durvalumab (imfinzi). In embodiments, the pharmaceutical composition comprising the cancer therapy and the anti-checkpoint agent are administered simultaneously or contemporaneously. In embodiments, the pharmaceutical composition comprising the cancer therapy is administered after the anti-checkpoint agent is administered. In embodiments, the pharmaceutical composition comprising the cancer therapy is administered before the anti-checkpoint agent is administered.

[0177] In embodiments, the anti-checkpoint agent is an antibody selected from nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS936559, MPDL328OA (ROCHE), cemiplimab (LIBTAYO), atezolizumab (TECENTRIQ), avelumab (BAVENCIO), and durvalumab (imfinzi).

[0178] Transgenic non-human animal models for testing cancer therapies In one aspect, the disclosure relates to a transgenic non-human animal comprising one or more tumor cells, the tumor cells exhibiting (a) positive regulation of cell cycle processes, regulation of the G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of a defense response, positive regulation of IFNβ production, regulation of an inflammatory response, regulation of an innate immune response, negative regulation of antigen processing and presentation, and negative regulation of antigen processing of endogenous peptides via MHC class I. / upregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from the listing; and / or (b) downregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid efflux, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes. In an embodiment, the transgenic non-human animal is transgenic.

[0179] In embodiments, the tumor cells are resistant to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. In embodiments, the cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2 is an antibody. In embodiments, the antibody is an antibody selected from nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559, MPDL328OA (ROCHE), cemiplimab (LIBTAYO), atezolizumab (TECENTRIQ), avelumab (BAVENCIO), and durvalumab (imfinzi). In embodiments, the one or more tumor cells have (a) upregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from positive regulation of cell cycle processes, regulation of G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of defense response, positive regulation of IFNβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or (b) downregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from phospholipid export, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid export, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis process. In embodiments, the one or more tumor cells have upregulation of one or more genes associated with a cellular response to IFNγ.In embodiments, the one or more tumor cells have upregulation of one or more genes associated with the type I IFN signaling pathway.

[0180] The transgenic non-human animal may be any animal known to be useful for mimicking human cancer. In embodiments, the transgenic non-human animal may be a pig, cow, dog, cat, horse, donkey, goat, sheep, llama, or non-human primate (e.g., chimpanzee). In embodiments, the transgenic non-human animal is a mammal. In embodiments, the transgenic non-human animal may be a rodent, such as a rat, mouse, hamster, rabbit, or guinea pig. In a preferred embodiment, the transgenic non-human animal is a mouse. In a preferred embodiment, the transgenic non-human animal is a rat. In embodiments, the mouse belongs to the BALB / c or C57BL / 6 strain. Such mice may be purchased from different suppliers, for example, Charles River Laboratories. In embodiments, the transgenic non-human animal is a transgenic transgenic non-human animal, such as, but not limited to, a transgenic mouse. In embodiments, the transgenic non-human animal is a transgenic transgenic mouse. In embodiments, one or more genetic alterations that cause spontaneous and / or induced tumors.

[0181] In embodiments, the formation of tumors in transgenic non-human animals (e.g., mice) is caused by gene knockout of one or more genes, optionally the gene knockout of one or more genes is inducible. In embodiments, the knockout of one or more genes is performed using cre-loxP, CRISPR / Cas9, etc., or a combination thereof. In embodiments, the knockout of one or more genes is associated with upregulation of one or more genes in cells having the knockout of one or more genes. In embodiments, the knockout of one or more genes is associated with downregulation of one or more genes in cells having the knockout of one or more genes. In embodiments, the upregulation and downregulation of one or more genes are independent and optionally inducible. In embodiments, the upregulation and / or downregulation is caused by placing one or more sequences (e.g., but not limited to, RNAi constructs, Cre recombinase constructs, and gene knock-in constructs) under the control of a promoter that controls the expression of one or more genes.

[0182] In embodiments, the formation of tumors in transgenic non-human animals (e.g., mice) is caused by knock-in of one or more tumor-causing genes, optionally inducible knock-in of one or more tumor-causing genes. Such tumor-causing genes are known in the art, and in some embodiments, include c-Myc, HRAS, G12V Or Kras G12Dand dominant negative p53 mutants. In an embodiment, the knock-in of one or more tumor-causing genes is performed using cre-loxP, CRISPR / Cas9, etc., or a combination thereof. In an embodiment, the knock-in of one or more tumor-causing genes is associated with upregulation of one or more genes in cells with the knock-in of one or more tumor-causing genes. In an embodiment, the knock-in of one or more tumor-causing genes is associated with downregulation of one or more genes in cells with the knock-in of one or more tumor-causing genes. In an embodiment, the upregulation and downregulation of one or more genes are independently and optionally inducible. In an embodiment, the upregulation and / or downregulation is caused by placing one or more sequences (such as, but not limited to, an RNAi construct, a Cre recombinase construct, and a further gene knock-in construct) under the control of a promoter that controls the expression of one or more genes.

[0183] In embodiments, the formation of tumors in transgenic non-human animals (e.g., mice) is caused by chromosomal translocation, optionally the chromosomal translocation is inducible. In embodiments, the chromosomal translocation is performed using cre-loxP, CRISPR / Cas9, etc., or a combination thereof. In embodiments, the chromosomal translocation is associated with upregulation of one or more genes in cells with the chromosomal translocation. In embodiments, the chromosomal translocation is associated with downregulation of one or more genes in cells with the chromosomal translocation. In embodiments, the upregulation and downregulation of one or more genes are independent and optionally inducible. In embodiments, the upregulation and / or downregulation is caused by placing one or more sequences (e.g., but not limited to, RNAi constructs, Cre recombinase constructs, and gene knock-in constructs) under the control of a promoter that controls the expression of one or more genes.

[0184] In embodiments, the formation of tumors in transgenic non-human animals (e.g., mice) is caused by chromosomal inversion, optionally the chromosomal inversion is inducible. In embodiments, the chromosomal inversion is performed using cre-loxP, CRISPR / Cas9, etc., or a combination thereof. In embodiments, the chromosomal inversion is associated with upregulation of one or more genes in cells with the chromosomal inversion. In embodiments, the chromosomal inversion is associated with downregulation of one or more genes in cells with the chromosomal inversion. In embodiments, the upregulation and downregulation of one or more genes are independent and optionally inducible. In embodiments, the upregulation and / or downregulation is caused by placing one or more sequences (e.g., but not limited to, RNAi constructs, Cre recombinase constructs, and gene knock-in constructs) under the control of a promoter that controls the expression of one or more genes.

[0185] In any of the embodiments disclosed herein, the transgenic mouse has a knock-in construct(s) that causes upregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from positive regulation of cell cycle processes, regulation of G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of defense response, positive regulation of IFNβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing and presentation, and antigen processing / presentation of endogenous peptides via MHC class I. In any of the embodiments disclosed herein, the transgenic mice have a knock-in construct(s) that cause downregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid efflux, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes.

[0186] In any of the embodiments disclosed herein, the transgenic mouse has a promoter that controls the expression of one or more genes, causing upregulation of one or more genes and / or downregulation of one or more genes, for example, by placing an RNAi construct, Cre recombinase and / or a gene knock-in construct under the control of the promoter. In an embodiment, the promoter is a strong promoter. In an embodiment, the promoter is an inducible promoter that allows for controlled and independent upregulation and / or downregulation of one or more genes. In an embodiment, the promoter is selected from a doxycycline-inducible promoter, a tamoxifen-inducible promoter and a coumarate-inducible promoter. In an embodiment, the promoter can be a tissue-specific promoter. In embodiments, tissue specific promoters include mouse mammary tumor virus (MMTV) or whey acidic protein (WAP) promoters for expression in the mammary gland, mouse proopiomelanocortin-alpha (POMC) promoter for central nervous system (hippocampus) specific expression, human caudal homeobox 2 (CDX2) promoter for colonic epithelium specific expression, mouse SRY box-containing gene 2 (Sox2) promoter for epiblast cell specific expression, gamma-glutamyltransferase 1 (Ggt1) promoter for renal epithelium specific expression, hepatic the albumin promoter / enhancer (Alb) for liver (hepatocyte)-specific expression, the human pulmonary surfactant-related protein C (SFTPC) promoter for lung (endoderm)-specific expression, the pancreatic and duodenal homeobox 1 (pdx1) promoter for pancreatic epithelium-specific expression, the rat insulin II promoter for pancreatic β-cell-specific expression, and the leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5), fatty acid-binding protein (Fabp) or mouse ribulin 1 promoter for small intestine and large intestine (crypt)-specific expression.

[0187] Methods for generating transgenic mouse models in cancer research are widely known in the art (Walrath et al., Genetically Engineered Mouse Models in Cancer Research, Adv Cancer Res. 106:113-164 (2010)). These methods usually involve the construction of a targeting vector with a modified sequence that is introduced into mouse embryonic stem cells. The genetically modified embryonic stem cells are then inserted into mice. The blastocyst is implanted into the uterus of a female mouse, ultimately resulting in chimeric offspring. Heterozygous mice are then mated to obtain mice that are homozygous for the knock-out gene and / or knock-in. Alternative methods for gene inactivation include the CRISPR / Cas9 system (Beil-Wagner et al. (2016) Sci Rep., 6:21377). Methods for incorporating modified promoter sequences are known in the art and include the use of targeting vectors that are introduced into animal embryonic stem cells, as described above. The knocked-out embryonic stem cells are then inserted into animal blastocysts to generate offspring, which are then crossed to obtain homozygous mice.Alternatively, the method for promoter modification in genome can include CRISPR / Cas9 system.Apart from modifying the genome of animals, it may also be possible to achieve the production of proteins in transgenic non-human animals by relying on viral or non-viral expression vectors.

[0188] Some methods include injecting a vector carrying a nucleic acid construct that directs the desired change in the mouse. In embodiments, the vector is a DNA vector. In embodiments, the vector is a viral vector. The vector then reaches the desired tissue and induces the desired genetic change in the mouse.

[0189] Expression vectors are known that allow the expression of transgenes in transgenic non-human animals, such as mice. Useful mammalian expression vectors are described in the prior art and such expression systems are commercially available from different manufacturers. Useful expression systems include plasmid or viral vector-based systems, such as pLIVE (Minis Bio), LENTI-Smart (InvivoGen), GenScript expression vectors, pAdVAntage (Promega), ViraPower Lentiviral, Adenoviral Expression Systems (Invitrogen) and adeno-associated virus expression systems (Cell Biolabs).

[0190] A suitable mammalian expression vector usually comprises a promoter operably linked to the nucleic acid encoding the up-regulated and / or down-regulated gene(s). The promoter sequence must be compact and ensure strong expression. Suitable promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, the spleen focus forming virus (SFFV) U3 promoter and the adenovirus major late promoter (Ad MLP) and promoters mentioned above. Optionally, the mammalian expression vector may comprise a suitable enhancer element to increase the expression level. Examples include the SV40 early gene enhancer Dijkema et al (1985) EMBO J. 4:761 and the enhancer of the long terminal repeat (LTR) of Rous sarcoma virus (Gorman et al. (1982b) Proc. Natl. Acad. Sci. 79:6777). The expression vector also optionally comprises a transcription termination sequence and a polyadenylation sequence to improve the expression of the gene encoding the up-regulated and / or down-regulated gene(s). Suitable transcription terminators and polyadenylation signals may be derived, for example, from SV40 (Sambrook et al (1989), Molecular Cloning: A Laboratory Manual). Any other elements known in the art to aid in efficient expression may be added to the expression vector, such as the woodchuck hepatitis posttranscriptional regulatory element (wPRE). In an embodiment, the vector may be one that can be administered to an animal by injection. In an embodiment, the mammalian expression vector is a pLIVE expression vector (Minis Bio).

[0191] In an embodiment, the vector is an expression vector, optionally a viral expression vector. Viral vectors typically contain a viral genome with a portion of the native sequence deleted in order to introduce a heterologous polynucleotide without destroying the infectivity of the virus. Due to the specific interaction between the viral components and the host cell receptor, viral vectors are highly suitable for efficient transfer of genes into target cells. Suitable viral vectors for facilitating gene transfer into mammalian cells or organisms are well known in the art and can be derived from various types of viruses, such as retroviruses, adenoviruses, adeno-associated viruses (AAV), orthomyxoviruses, paramyxoviruses, papovaviruses, picornaviruses, lentiviruses, herpes simplex viruses, vaccinia viruses, poxviruses or alphaviruses. For an overview of various viral vector systems, see Nienhuis et al., Hematology, Vol. 16: Viruses and Bone Marrow, NS Young (ed.), 353-414 (1993).

[0192] In an embodiment, the vector is an adeno-associated virus (AVV) vector, such as an AAV vector selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, or a chimeric AAV derived therefrom, which is better suited for high-efficiency transduction in tissues of interest (Wu et al, 2006, Mol Therapy 14:316-27; Bowles et al, 2012, Mol Therapy 20:443-455). Upon transfection, AAV induces little, if any, immune response in the host. Furthermore, in contrast to other vector systems, AAV vectors can also efficiently pass from blood to terminally differentiated cells. Thus, in an embodiment, AAV is highly suitable for gene transfer approaches. In an embodiment, AAV serotype 6 and AAV serotype 9 are used for transduction in mice.

[0193] Recombinant viral vectors can be produced according to standard techniques. For example, recombinant adenoviral or adeno-associated viral vectors can be grown in human 293 cells (providing E1A and E1B functions in trans) to titers in the range of 107-1013 viral particles / mL. Prior to their in vivo application, viral vectors can be desalted by gel filtration, e.g., a Sepharose column, and purified by subsequent filtration. Purification reduces potential adverse effects in subjects to whom the vector is administered. The administered virus is substantially free of wild-type and replication-competent virus. The purity of the virus can be demonstrated by suitable methods, e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by silver staining. This is applicable to both AAV and adenoviral vectors.

[0194] Transduction of the vector into the transgenic non-human animal can be accomplished by systemic application, for example by intravenous (including hydrodynamic tail vein injection), intraarterial or intraperitoneal delivery of the vector. In a preferred embodiment, the vector is administered systemically.

[0195] Methods for generating transgenic non-human animal models for testing cancer therapies - Patents.com In one aspect, the disclosure relates to a method of making a transgenic non-human animal comprising one or more cancer cells that are unresponsive, resistant or refractory to a cancer therapy capable of inhibiting a function and / or activity of PD-1, PD-L1 and / or PD-L2, the method comprising: (a) injecting one or more parental cancer cells that are responsive to the cancer therapy in the non-human animal; (b) administering the cancer therapy to the non-human animal; (c) isolating the cancer cells that survive the cancer therapy; (d) injecting the cancer cells that survive the cancer therapy into a different non-human animal of the same species; and (e) repeating steps (b)-(d) two to ten additional times.

[0196] In an embodiment, steps (b)-(d) are repeated at least one additional time. In an embodiment, steps (b)-(d) are repeated at least two additional times. In an embodiment, steps (b)-(d) are repeated at least three additional times. In an embodiment, steps (b)-(d) are repeated less than five times. In an embodiment, the transgenic non-human animal is a rodent. In an embodiment, the rodent is a mouse.

[0197] In embodiments, the mouse belongs to the BALB / c or C57BL / 6 strain. In embodiments, the cancer therapy capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2 is an antibody. In embodiments, the antibody is an antibody selected from nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559, MPDL328OA (ROCHE), cemiplimab (LIBTAYO), atezolizumab (TECENTRIQ), avelumab (BAVENCIO), and durvalumab (imfinzi).

[0198] In embodiments, the cancer therapy is capable of inhibiting tumor growth when administered to a transgenic non-human animal bearing a parental cancer cell tumor, as compared to an untreated transgenic non-human animal bearing the parental cancer cell tumor. In embodiments, cancer cells surviving the cancer therapy are characterized by the presence or absence of a gene(s) that is / are associated with a positive regulation of cell cycle processes, regulation of the G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of a defense response, positive regulation of IFNβ production, regulation of an inflammatory response, regulation of an innate immune response, negative regulation of antigen processing and presentation, and antigen processing / presentation of endogenous peptides via MHC class I. and / or (b) upregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid efflux, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis process.

[0199] In embodiments, the one or more tumor cells have upregulation of one or more genes associated with a cellular response to IFNγ.In embodiments, the one or more tumor cells have upregulation of one or more genes associated with the type I IFN signaling pathway.

[0200] In one aspect, the present disclosure relates to a transgenic animal produced according to the method of any of the embodiments disclosed herein.

[0201] Methods for testing cancer therapies and making pharmaceutical compositions for treating cancer In one aspect, the present disclosure relates to a method of testing a candidate anti-cancer drug comprising: (a) providing a transgenic non-human animal of any of the embodiments disclosed herein or produced according to any of the embodiments disclosed herein; (b) administering the candidate anti-cancer drug to the transgenic non-human animal; and (c) evaluating whether the candidate anti-cancer drug is effective in slowing or inhibiting cancer growth in the transgenic non-human animal. In one aspect, the present disclosure relates to a candidate anti-cancer drug selected from a chemotherapeutic agent, a cytotoxic agent, and a checkpoint inhibitor.

[0202] In one aspect, the disclosure relates to a method of manufacturing a pharmaceutical composition for treating cancer, comprising: (a) providing a transgenic non-human animal of any of the embodiments disclosed herein or produced according to any of the embodiments disclosed herein, (b) administering a candidate anti-cancer drug to the transgenic non-human animal, (c) selecting an anti-cancer drug effective to slow or inhibit cancer growth in the transgenic non-human animal, and (d) formulating the anti-cancer drug or candidate for administration to a human patient. In embodiments, the candidate anti-cancer drug is selected from a chemotherapeutic agent, a cytotoxic agent, and a checkpoint inhibitor.

[0203] formulation The cancer therapy (and / or additional agent) described herein may have a sufficiently basic functional group that can react with inorganic or organic acids, or a carboxyl group that can react with inorganic or organic bases to form pharma- ceutically acceptable salts. Pharmaceutically acceptable acid addition salts are formed from pharma- ceutically acceptable acids as is well known in the art. Such salts include, for example, the pharma- ceutically acceptable salts listed in Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P.H. Stahl and C.G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are incorporated herein by reference in their entirety.

[0204] In embodiments, the compositions described herein are in the form of a pharma- ceutically acceptable salt.

[0205] Furthermore, any cancer therapy (and / or additional agent) described herein may be administered to a subject as a component of a composition that includes a pharma- ceutically acceptable carrier or vehicle. Such compositions may optionally include a suitable amount of a pharma- ceutical acceptable excipient to provide a suitable form for administration. The pharmaceutical excipient may be a liquid, such as water and an oil, such as of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. The pharmaceutical excipient may be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. Additionally, auxiliary agents, stabilizers, thickeners, lubricants, and coloring agents may be used. In one embodiment, the pharma- ceutical acceptable excipient is sterile when administered to a subject. Water is a useful excipient when any agent described herein is administered intravenously. Saline and aqueous dextrose and glycerol solutions may also be used as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. Any of the agents described herein, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0206] In embodiments, the compositions described herein are resuspended in a saline buffer (including but not limited to TBS, PBS, etc.).

[0207] Administration, Dosing and Treatment Regimens The present disclosure includes the described cancer therapy (and / or additional agent) in various formulations. Any cancer therapy (and / or additional agent) described herein can be in the form of a solution, suspension, emulsion, droplets, tablets, pills, pellets, capsules, capsules containing liquid, powders, sustained release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. DNA or RNA constructs encoding protein sequences may also be used. In one embodiment, the composition is in the form of a capsule (see, for example, U.S. Pat. No. 5,698,155). Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), which is incorporated herein by reference.

[0208] If necessary, the formulation containing the cancer therapy (and / or additional agent) can also contain a solubilizing agent. The agent can also be delivered using a suitable vehicle or delivery device known in the art. The combination therapy outlined herein can be co-delivered in a single delivery vehicle or delivery device. The composition for administration can optionally contain a local anesthetic, such as lignocaine, to reduce pain at the injection site.

[0209] The formulations containing the cancer therapy (and / or additional agent) of the present disclosure can be conveniently provided in unit dosage form and can be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agent into association with the carrier, which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired formulation (e.g., wet or dry granulation, powder blending, etc., followed by tableting using conventional methods known in the art).

[0210] In one embodiment, any cancer therapy (and / or additional agent) described herein is formulated in accordance with routine procedures into a composition compatible with the modes of administration described herein.

[0211] Routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, by inhalation, or topically, particularly to the ear, nose, eye, or skin. In some embodiments, administration is by oral or parenteral injection. In most cases, administration results in the release of any agent described herein into the bloodstream.

[0212] Any cancer therapy (and / or additional agent) described herein may be administered orally. Such cancer therapy (and / or additional agent) may also be administered by any other convenient route, for example, by intravenous infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with another biologically active agent. Administration may be systemic or local. Various delivery systems are known, for example, encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and may be used for administration.

[0213] In certain embodiments, it may be desirable to administer locally to the area requiring treatment. In one embodiment, for example, in the treatment of cancer, the cancer therapy (and / or additional agent) is administered to and / or targeted to the tumor microenvironment (e.g., cells, molecules, extracellular matrix and / or blood vessels surrounding and / or supplying tumor cells, including tumor vasculature; tumor-infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPCs); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen-presenting cells; T cells; regulatory T cells; macrophages; neutrophils; and other immune cells located in the vicinity of the tumor) or lymph nodes. In an embodiment, for example, in the treatment of cancer, the cancer therapy (and / or additional agent) is administered intratumorally.

[0214] Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous and intraarticular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, etc. They may also be prepared in the form of sterile solid compositions (e.g., lyophilized compositions) and dissolved or suspended in a sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.

[0215] The dosage and administration schedule of any cancer therapy (and / or additional agent) described herein may depend on a variety of parameters, including, but not limited to, the disease being treated, the general health of the subject, and the discretion of the administrating physician. Any of the cancer therapies described herein may be administered to a subject in need thereof prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of the additional agent. In embodiments, any cancer therapy described herein and additional agents are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 to 2 hours apart, 2 to 3 hours apart, 3 to 4 hours apart, 4 to 5 hours apart, 5 to 6 hours apart, 6 to 7 hours apart, 7 to 8 hours apart, 8 to 9 hours apart, 9 to 10 hours apart, 10 to 11 hours apart, 11 to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart.

[0216] The dosage of any cancer therapy (and / or additional agent) described herein may depend on several factors, including the severity of the condition, whether the condition is being treated or prevented, and the age, weight, and health of the subject being treated. In addition, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic, or efficacy profile of a therapeutic agent) information on a particular subject may affect the dosage used. Furthermore, the exact individual dosage may be adjusted somewhat depending on a variety of factors, including the specific combination of agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variation in dosage may be expected.

[0217] When administering any of the cancer therapies (and / or additional agents) described herein by parenteral injection, the dosage can be about 0.1 mg to about 250 mg / day, about 1 mg to about 20 mg / day, or about 3 mg to about 5 mg / day. In general, when administered orally or parenterally, the dosage of any of the agents described herein can be about 0.1 mg to about 1500 mg / day, or about 0.5 mg to about 10 mg / day, or about 0.5 mg to about 5 mg / day, or about 200 to about 1,200 mg / day (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1,000 mg, about 1,100 mg, about 1,200 mg / day).

[0218] In embodiments, administration of the cancer therapies (and / or additional agents) described herein is by parenteral injection at a dose of about 0.1 mg to about 1500 mg per treatment, or about 0.5 mg to about 10 mg per treatment, or about 0.5 mg to about 5 mg per treatment, or about 200 to about 1,200 mg per treatment (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1,000 mg, about 1,100 mg, about 1,200 mg per treatment).

[0219] In embodiments, a suitable dosage of the cancer therapy (and / or additional agent) is from about 0.01 mg / kg to about 100 mg / kg body weight of the subject, or from about 0.01 mg / kg to about 10 mg / kg body weight of the subject, for example, about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.06 mg / kg, about 0.07 mg / kg, about 0.08 mg / kg, about 0.09 mg / kg, about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.6 mg / kg, about 0.7 mg / kg, about 0.8 mg / kg, about 0.9 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, about 20 mg / kg, about 21 mg / kg, about 22 mg / kg, about 23 mg / kg, about 24 mg / kg, about 25 mg / kg, about 26 mg / kg, about 27 mg / kg, about 28 mg / kg, about 29 mg / kg, about 30 mg / kg, about 31 mg / kg, about 32 mg / kg, about 33 mg / kg, about 34 mg / kg, about 35 mg / kg, about 36 mg / kg, about 37 mg / kg, about 38 mg / kg, about 39 mg / about 1.1 mg / kg, about 1.2 mg / kg, about 1.3 mg / kg, about 1.4 mg / kg, about 1.5 mg / kg, about 1.6 mg / kg, about 1.7 mg / kg, about 1.8 mg / kg, 1.9 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg body weight (including all values ​​and ranges therebetween).

[0220] In another embodiment, delivery may be in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989)).

[0221] Any cancer therapy (and / or additional agent) described herein may be administered by controlled or sustained release means or by delivery devices known to those of skill in the art. Examples include, but are not limited to, U.S. Patent Nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms may be useful, for example, to provide controlled or sustained release of one or more active ingredients using hydropropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multi-layer coatings, microparticles, liposomes, microspheres, or combinations thereof to provide desired release profiles at various rates. Controlled or sustained release of active ingredients may be stimulated by a variety of conditions, including, but not limited to, changes in pH, changes in temperature, stimulation with light of appropriate wavelengths, enzyme concentration or availability, water concentration or availability, or other physiological conditions or compounds.

[0222] In another embodiment, polymeric materials may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).

[0223] In another embodiment, a controlled release system can be placed near the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems discussed in the review by Langer, 1990, Science 249:1527-1533 may also be used.

[0224] Administration of any cancer therapy (and / or additional agent) described herein can be, independently, 1 to 4 times per day, or 1 to 4 times per month, or 1 to 6 times per year, or once every 2, 3, 4 or 5 years. Administration can be daily or for a period of 1, 2, 3, 6 months, 1, 2, 3 years, or for the life of the subject.

[0225] The dosing regimen utilizing any of the cancer therapies (and / or additional agents) described herein may be selected according to a variety of factors, including the type, species, age, weight, sex, and medical condition of the subject; the severity of the condition being treated; the route of administration; the renal or hepatic function of the subject; the pharmacogenomic makeup of the individual; and the particular compound of the present invention being used. Any of the cancer therapies (and / or additional agents) described herein may be administered in a single dose per day, or the total daily dosage may be administered in divided doses two, three, or four times per day. Additionally, any of the cancer therapies (and / or additional agents) described herein may be administered continuously, rather than intermittently, throughout the dosing regimen. EXAMPLES

[0226] The examples herein are provided to illustrate the benefits and advantages of the present disclosure and to further assist those skilled in the art in preparing or using cells resistant to anti-PD-1, anti-PD-L1 and / or anti-PD-L2 therapy. The examples herein are also presented to more fully describe preferred aspects of the present disclosure. The examples should not be construed in any way as limiting the scope of the present disclosure, which is defined by the appended claims. The examples may include or incorporate any of the variations, aspects or embodiments of the present disclosure described above. Each of the variations, aspects or embodiments described above may also further include or incorporate any or all other variations, aspects or embodiments of the present disclosure.

[0227] Example 1: Generation of anti-PD-1 resistant CT26 tumors Mouse colon cancer CT26 cells were subjected to selection to survive anti-PD-1 treatment and generate anti-PD-1 antibody-resistant CT26 cells. The method used to generate anti-PD-1 resistant CT26 tumor cells is shown in Figure 1A. Briefly, BALB / C mice were obtained from Jackson Laboratory, and after several days of acclimation, the mice were inoculated with 500,000 mouse colon cancer CT26 cells in the hind flank. The average tumor volume was 80-100 mm. 3Once tumor size reached 100 μg (indicated day 0), mice were given a series of intraperitoneal injections of 100 μg anti-PD-1 (clone RMP1-14; BioXcell) on days 0, 3, and 6. Approximately 10–14 days after the first treatment, tumors were excised from mice that did not respond to anti-PD-1 therapy. Tumors were dissociated using collagenase (StemCell Technologies), washed with 1× PBS, and seeded in IMDM culture medium supplemented with 10% fetal bovine serum, 1% GLUTiMAX, and 1% antibiotic-antimycotic (all GIBCO). Cells were passaged at least five times and then inoculated into new recipient mice as described above (round 2). Again, tumors were cultured until tumor size reached 80–100 mm 3 Once the F2 generation of anti-PD-1 was reached, another course of treatment with anti-PD-1 was initiated: intraperitoneal injections of 100 μg anti-PD-1 (clone RMP1-14; BioXcell) were administered on days 0, 3, and 6. This process was repeated for a total of four rounds, at which point none of the treated mice responded to anti-PD-1 therapy. Cell lines generated after two rounds of anti-PD-1 selection are referred to as "round two," "generation two," or "F2 generation" throughout this disclosure. Cell lines generated after four rounds of anti-PD-1 selection are referred to as "round four," "generation four," or "F4 generation."

[0228] We compared the efficacy of anti-PD-1 antibodies in CT26 cell or anti-PD-1 antibody-resistant CT26 cell allografts. Briefly, BALB / C mice were inoculated with parental CT26 cells and PD-1 resistant fourth-generation cells in the hind flank. Starting tumor volumes (STVs) of 80-100 mm were 3Upon reaching 10 days, mice were randomized into two treatment groups: (1) vehicle (PBS) and (2) anti-PD-1 antibody. Mice received a series of intraperitoneal injections of vehicle or 100 μg of anti-PD-1 (clone RMP1-14; BioXcell) on days 0, 3, and 6. Tumor volumes were measured on the indicated days and plotted as a function of time. As shown in FIG. 1B, the growth of CT26 parental cell tumors was significantly delayed when treated with anti-PD-1 antibody compared to vehicle-only controls (compare the solid gray line with the solid black line in FIG. 1B). In contrast, PD-1-resistant cells showed little, if any, tumor retardation. These results demonstrate, inter alia, that anti-PD-1 resistance of anti-PD-1-resistant cells developed after treatment (acquired resistance) in immunocompetent mice.

[0229] Thus, anti-PD-1 antibody resistant cells have been developed. Also, a mouse model of cells with anti-PD-1 resistant cells has been developed. Thus, the mouse model disclosed herein is useful for testing anti-cancer drug candidates by administering the anti-cancer drug candidates to mice with anti-PD-1 antibody resistant CT26 cell allografts and evaluating whether the anti-cancer drug candidates are effective in slowing or inhibiting cancer growth. Anti-cancer drugs or candidates that are effective in slowing or inhibiting cancer growth can be formulated for administration to human patients.

[0230] Example 2: Transcriptome profiling of anti-PD-1 resistant cell lines using RNA-seq Three separate vials of parental CT26 cells (ATCC; “experimental replicates”), two independently isolated tumors from “round 2” mice, and four independently isolated tumors from “round 4” mice (both “biological replicates”) were cultured for 24 hours at 37°C / 5% CO2 with and without 20ng / mL mouse IFNγ (Biolegend). The next day, RNA was isolated from the cells using Qiagen RNeasy reagent according to the manufacturer’s instructions, including QIASHREDDER homogenization and on-column DNaseI digestion. The isolated RNA was subjected to RNA-seq and data analysis. Briefly, sequencing libraries were generated and sequenced on the ILLUMINA HISEQ (2×150 paired end reads), with 20×10 6 Reads greater than 1 were targeted. Sequences were trimmed using TRIMMOMATIC v.0.36 and mapped to the Mus musculus GRCm38 reference genome using STAR ALIGNER v.2.5.2b. Unique gene hit counts were calculated by using FEATURECOUNTS from the SUBREAD package v.1.5.2. Only unique reads falling within exonic regions were counted. Differential gene expression was determined using DESeq2 and p-values ​​and log2 fold changes were generated using Wald tests. Significance cutoffs were log2 fold change >1 and adjusted p-value <0.05.

[0231] As shown in Figure 2A (top panel), principal component analysis (PCA) demonstrated that samples could be spatially separated based on transcriptome expression. Differentially expressed genes (DEGs) were determined between groups (parent vs. 2nd generation, parent vs. 4th generation, 2nd generation vs. 4th generation) and plotted in a heatmap. As shown in Figure 2A (bottom panel), hierarchical clustering was performed to rank order genes in each row and separate genes into two major clusters in each comparison, with a subset of genes whose expression was lower in one dataset (blue) and higher in the other (red). Genes were then analyzed using the PANTHER application to identify gene ontology (GO) associated with each gene set. Gene sets are shown with associated p-values. Up- or down-regulated genes were identified in each dataset. As shown in Figure 2B, genes associated with the following GOs were up-regulated in 2nd generation cells compared to parental CT26 cells: positive regulation of cell cycle process, regulation of G1 / S transition, regulation of cell division, and regulation of cell proliferation. The following GO-associated genes were downregulated in second-generation cells compared to parental CT26 cells: phospholipid efflux, negative regulation of fibrinolysis, chylomicron assembly, and plasma membrane repair (Figure 2B). Furthermore, as shown in Figure 2B, the following GO-associated genes were upregulated in fourth-generation cells compared to parental CT26 cells: positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, and regulation of innate immune response. This was surprising, especially since cellular response to IFNγ is widely known to be involved in sensitivity to anti-PD-1 and other immunotherapeutic drugs. The following GO-associated genes were downregulated in fourth-generation cells compared to parental CT26 cells: SRP-dependent cotranslational protein targeting to membrane, ribosomal small subunit assembly, phospholipid efflux, and control of translation (Figure 2B). Interestingly, genes associated with the following GOs were upregulated in fourth generation cells compared to second generation cells: cellular response to IFNγ, positive regulation of IκB kinase / NFκB signaling, negative regulation of antigen processing / presentation, and antigen processing / presentation of endogenous peptides via MHC class I ( Figure 2B ).Genes associated with the following GOs were downregulated in 4th generation cells compared to 2nd generation cells: mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis process (Figure 2B). A Venn diagram of gene expression overlap between all datasets was generated. As shown in Figure 2C (upper panel), the DEGs showed significant overlap between 4th generation cells and 2nd generation cells. Figure 2A (lower panel) shows the transcripts per million (TPM; normalized expression data) in selected genes, demonstrating higher baseline expression of genes related to PD-L1, antigen processing / presentation, protein translation, ER trafficking, in some datasets than others. As shown in Figure 2C (lower panel), genes Cd274, B2M, Tap1, Tap2, Casp1, and Gasta3 showed progressively increasing expression from CT26 parental cells to 2nd and 4th generation cells. Furthermore, genes Rpl41, Rps15, and Rps8 showed a gradual decrease in expression from CT26 parental cells to second and fourth generation cells (Figure 2C (bottom panel)). As shown in Figure 2D, genes Stat1, Stat2, Irf, Ltbr, and Pvr showed a gradual increase in expression from CT26 parental cells to second and fourth generation cells.

[0232] These results establish, among other things, biomarkers associated with acquired resistance to anti-PD-1 therapy. Such biomarkers can be used to identify patients who may benefit from anti-PD-1 therapy or who will not benefit from anti-PD-1 therapy. Thus, based on these biomarkers, patients can be selected for treatment with anti-PD-1 therapy based on evaluating a biological sample from the patient for the presence, absence, or level of genes associated with one or more Gene Ontology (GO) pathways disclosed herein. Cancer therapy using the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2 can be indicated, for example, when expression of one or more of Rpl41, Rps15, and Rps8 is high, and / or expression of one or more of Cd274, B2m, Tap1, Tap2, Casp1, and Gasta3 is low. In contrast, cancer therapy using the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2 may not be indicated when, for example, expression of one or more of Rpl41, Rps15 and Rps8 is low and / or expression of one or more of Cd274, B2m, Tap1, Tap2, Casp1 and Gasta3 is high. Patients characterized by low expression of one or more of Rpl41, Rps15 and Rps8 and / or high expression of one or more of Cd274, B2m, Tap1, Tap2, Casp1 and Gasta3 are likely to benefit from adjuvant or neoadjuvant therapy that eliminates PD-1 non-responsive cells.

[0233] Example 3: Cell surface expression of PD-L1, PD-L2, MHC class I, and β2 microglobulin in anti-PD-1 resistant CT26 Because Cd274 (PD-L1), β2 microglobulin (B2m), and Tap1 and Tap2 genes (potentially involved in processing and transport of major histocompatibility complex class I-related antigen to the endoplasmic reticulum) were gradually downregulated from CT26 parental cells to second-generation and fourth-generation cells, surface expression of PD-L1, PD-L2, MHC class I, and β2 microglobulin (B2m) was examined. Briefly, parents were harvested from culture and analyzed by flow cytometry for surface expression of PD-L1, PD-L2, MHC class I, and β2 microglobulin (B2M). Gates were drawn as shown, and the percentage of cells in each gate is shown above each plot, and the MFI (mean fluorescence intensity) of each marker is shown to the right of each percentage. As shown in Figure 3, surface expression of PD-L1, MHC class I, and β2 microglobulin (B2M) was not downregulated in fourth-generation anti-PD-1 resistant cells compared to parental cells. On the other hand, surface expression of PD-L2 was downregulated in 4th generation anti-PD-1 resistant cells compared to parental cells.These data indicate that discrepancies in gene expression and cell surface protein expression were observed for PD-L1 / 2 MHC class I and B2M surface expression compared to RNA expression.

[0234] Example 4: Transcriptome profiling of anti-PD-1 resistant and primary resistant anti-PD-1 resistant cell lines Next, the transcriptome profiles of second and fourth generation anti-PD-1 resistant cell lines were studied by comparing them with each other and with primary anti-PD-1 resistant cell lines. The B16.F10 murine melanoma tumor cell line was used as a model of anti-PD-1 "primary resistance" since these tumors do not respond to anti-PD-1 therapy. Three separate vials of parental CT26 cells (ATCC; "experimental replicates"), two independently isolated tumors from "second round" mice, four independently isolated tumors from "fourth round" mice (both "biological replicates"), and two separate vials of parental B16.F10 cells (ATCC; "experimental replicates") were cultured for 24 h at 37 °C / 5% CO2 with or without 20 ng / mL murine IFNγ (Biolegend) to assess in vitro responsiveness. This mimics how tumor cells would respond in vivo, as immune cells infiltrate and secrete effector cytokines such as IFNγ. The following day, RNA was isolated from the cells using Qiagen RNeasy reagent according to the manufacturer's instructions, including QIAGEN homogenization and on-column DNaseI digestion. Isolated RNA was subjected to RNA-seq and data analysis. Briefly, sequencing libraries were generated and analyzed using the ILLUMINA Sequenced with HISEQ (2 × 150 paired end reads), 20 × 10 per sample 6More than 100 reads were targeted. Sequences were trimmed using TRIMMOMATIC v.0.36 and mapped to the Mus musculus GRCm38 reference genome using STAR ALIGNER v.2.5.2b. Unique gene hit counts were calculated by using FEATURECOUNTS from the SUBREAD package v.1.5.2. Only unique reads falling within exonic regions were counted. Differential gene expression was determined using DESeq2 and p-values ​​and log2 fold changes were generated using Wald tests. A log2 fold change >1 and adjusted p-value <0.05 were used as cutoffs for significance. DEGs were identified between untreated and IFNγ-treated parental CT26. Log2 fold changes were plotted in a heatmap and genes were hierarchically clustered based on parental CT26, as shown in Figure 4A (left panel). Of these, 338 genes had data available from other datasets and their values ​​are shown in other columns (Figure 4A (left panel)). Genes were segregated into three major clusters (Figure 4A (left panel)). The associated genes were entered into PANTHER to identify pathways associated with the dysregulated genes and to identify GO pathways associated with the DEGs. As shown in Figure 4A (right panel), upregulation of expression of genes associated with the following GO pathways was enriched in second and fourth generation cells compared to both parental CT26 and B16.F10 cells: L-phenylalanine catabolic process, phospholipid efflux, tyrosine catabolic process, positive regulation of transcription from RNA polymerase II promoters in response to acidic pH, and drug transport. Thus, upregulation of genes associated with one or more of these GO pathways is likely associated with acquired resistance to anti-PD-1 therapy. Furthermore, downregulation of expression of genes related to the following GO pathways was observed in 4th generation cells compared to both parental CT26 and B16.F10 cells: protection of NK cell-mediated cytotoxicity, IGS15-protein conjugation, MHC class I-mediated antigen processing / presentation, MHC protein complex assembly, and cytosol to ER trafficking ( Figure 4A (right panel)).Therefore, downregulation of one or more of these GO pathways is likely to be associated with acquired resistance to anti-PD-1 therapy. As shown in Figure 4A (right panel), downregulation of expression of genes related to the following GO pathways was enriched in 4th generation cells and B16.F10 cells compared to both parental CT26 cells: positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, positive regulation of IFNα production, positive regulation of defense response, positive regulation of IFNβ production, and regulation of inflammatory response. Therefore, downregulation of one or more of these GO pathways is likely to be associated with resistance to anti-PD-1 therapy. Figure 4B shows the transcripts per million (TPM; normalized expression data) of selected genes. As shown in Figure 4B, CT26 anti-PD-1 resistant cells have baseline hyperactivation of type I and type II interferons, but when the cells are challenged with IFNγ, they downregulate these genes. This was surprising because, among other things, positive regulation of IκB kinase / NFκB signaling, the type I IFN signaling pathway, positive regulation of IFNα production, positive regulation of the defensive response, positive regulation of IFNβ production, and regulation of the inflammatory response are all widely known to be involved in sensitivity to anti-PD-1 and other immunotherapeutics.

[0235] These results establish, inter alia, biomarkers associated with acquired resistance to anti-PD-1 therapy and biomarkers associated with resistance (acquired or primary resistance) to anti-PD-1 therapy. Such biomarkers can be used to identify patients who may benefit from anti-PD-1 therapy or who will not benefit from anti-PD-1 therapy. Thus, based on these biomarkers, patients can be selected for treatment with anti-PD-1 therapy based on evaluating a biological sample from the patient for the presence, absence, or level of genes associated with one or more Gene Ontology (GO) pathways disclosed herein. Cancer therapy using the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2 can be indicated, for example, when Trim7 and / or Ank3 expression is high and / or Tap2 and / or Casp1 expression is low. In contrast, cancer therapy using the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2 may not be indicated, for example, when expression of Trim7 and / or Ank3 is high and / or expression of Tap2 and / or Casp1 is low. Patients characterized by high expression of Trim7 and / or Ank3, and / or low expression of Tap2 and / or Casp1 are likely to benefit from adjuvant or neoadjuvant therapy that eliminates PD-1 non-responsive cells.

[0236] Example 5: Paradoxical dysregulation of differentially regulated genes in anti-PD-1 resistant cells compared to parental cells Next, we tested the effect of IFNγ on the expression of specific genes that are differentially expressed in 4th generation anti-PD-1 resistant cells compared to wild-type CT26 cells. Briefly, parental CT26 cells, 2nd generation anti-PD-1 resistant cells, and 4th generation anti-PD-1 resistant cells were cultured with and without 20ng / mL mouse IFNγ (Biolegend) at 37°C / 5%CO2 for 24 hours. The next day, RNA was isolated from the cells using Qiagen RNeasy reagent according to the manufacturer's instructions, including QIASHREDDER homogenization and on-column DNaseI digestion. The isolated RNA was subjected to RNA-seq and data analysis. Briefly, sequencing libraries were generated and sequenced on ILLUMINA HISEQ (2x150 paired end reads), and 20x10 6 Reads greater than 1 were targeted. Sequences were trimmed using TRIMMOMATIC v.0.36 and mapped to the Mus musculus GRCm38 reference genome using STAR ALIGNER v.2.5.2b. Unique gene hit counts were calculated by using FEATURECOUNTS from the SUBREAD package v.1.5.2. Only unique reads falling within exonic regions were counted. Differential gene expression was determined using DESeq2 and p-values ​​and log2 fold changes were generated using Wald tests. Significance cutoffs were log2 fold change >1 and adjusted p-value <0.05.

[0237] To understand the effect of IFNγ, we analyzed the transcripts per million (TPM; normalized expression data) of representative genes that were either overexpressed (Cd274 and B2m) or suppressed (Trim7 and Lrg1). As expected, the expression of Cd274 (PD-L1) increased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells (Figure 5A (left panel)). The expression of Cd274, which was increased in wild-type CT26 cells, was increased when wild-type CT26 cells were induced with IFNγ (compare the closed circle data in Figure 5A (left and right panels)). Paradoxically, the expression of Cd274 (PD-L1) decreased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells in response to IFNγ (Figure 5A (right panel)).

[0238] Expression of B2M also increased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells, as expected (Figure 5B, left panel). Expression of B2M in wild-type CT26 cells increased when wild-type CT26 cells were induced with IFNγ (compare closed circle data in Figure 5B, left and right panels). Paradoxically, expression of B2M decreased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells in response to IFNγ (Figure 5B, right panel).

[0239] As expected, Trim7 expression was decreased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells (Figure 5C, left panel). Trim7 expression, which was increased in wild-type CT26 cells, was increased when wild-type CT26 cells were induced with IFNγ (compare closed circle data in Figure 5C, left and right panels). Paradoxically, Trim7 expression was decreased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells in response to IFNγ (Figure 5C, right panel).

[0240] Similarly, Lrg1 expression was decreased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells (Figure 5D, left panel). Lrg1 expression in wild-type CT26 cells was increased when wild-type CT26 cells were induced with IFNγ (compare closed circle data in Figure 5D, left and right panels). Paradoxically, Lrg1 expression was decreased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells in response to IFNγ (Figure 5D, right panel).

[0241] These results suggest paradoxical dysregulation of genes such as Cd274, B2m, Trim7 and Lrg1, among others, when grown in the presence or absence of IFNγ, compared to the parental cancer cells.

[0242] Example 6: Identification of driver genes of resistance Without being bound by theory, since dysregulation was observed in multiple genes, it was hypothesized that the paradoxical dysregulation is a functional consequence of acquired resistance to anti-PD-1 agents. To identify the driver genes involved in the observed paradoxical dysregulation, the analysis disclosed in FIG. 6A was performed. Briefly, differentially expressed genes (DEGs) were identified in 4th generation anti-PD-1 resistant cells compared to parental CT26 cells when grown in the presence of IFNγ. As shown in FIG. 6A (panel 1), 1,999 genes are downregulated and 3607 genes are upregulated in 4th generation anti-PD-1 resistant cells in the presence of IFNγ compared to CT26 cells. From these genes, those that downregulated 4th generation anti-PD-1 resistant cells but upregulated CT26 cells were identified. As shown in FIG. 6A (panel 2), 1,060 genes were narrowed down using this criterion (4th generation anti-PD-1 resistant cells were downregulated, but CT26 cells were upregulated). These genes were further narrowed down based on their responsiveness to IFNγ. As shown in Figure 6A (panel 3), 688 genes down-regulate 4th generation anti-PD-1 resistant cells compared to CT26 cells, as revealed by sorting according to their responsiveness to IFNγ. These genes include Lrg1, Spry2, Arg1, Trim8, Trim2, Mapk8ip1, Trim7, Trim6, etc. (Figure 6A (panel 3)).

[0243] To further narrow these genes, parental CT26 cells, second-generation anti-PD-1 resistant cells, and fourth-generation anti-PD-1 resistant cells were then expanded in vivo in mice. RNA was isolated from tumors and subjected to RNA-seq and data analysis. (From the 688 genes from Figure 6A (panel 3), we identified those genes that were also upregulated in vivo. As shown in Figure 6A (panel 4), 70 genes were upregulated in vivo in 4th generation anti-PD-1 resistant cells compared to CT26 cells. These genes include Krt8, Mapk8ip1, Arg1, and Lrg1. Figure 6B shows the Gene Ontology (GO) pathways enriched in at least two steps in Figure 6A. This analysis identified the TRIM protein family (particularly Trim7), Mapk8ip1, Elk1, Lrg1, Arg1 as some of the drivers. Figure 6C shows the functional pathways influenced by the TRIM family of proteins. Figure 6D shows the functional pathways in which Elk1 and c-Jun play a role. Figure 6E shows the functional connections between Lrg1, B2m, and Arg1 and other genes. Figure 6F shows the functional connections between the Cancer Genome 1 shows the expression levels of Elk1 in tumors and surrounding normal tissues in the Cancer Genomics Atlas (TCGA) Program.

[0244] Example 7: Paradoxical dysregulation of additional differentially regulated genes Transcripts per million (TPM; normalized expression data) for additional representative overexpressed genes (Stat1, Stat2, Irf1, and Tap1) were analyzed. Expression of Stat1 increased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells (Figure 7A (left panel)). Expression of Stat1 in wild-type CT26 cells increased when wild-type CT26 cells were induced with IFNγ (compare closed circle data in Figure 7A (left and right panels)). Paradoxically, expression of Stat1 decreased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells in response to IFNγ (Figure 7A (right panel)).

[0245] Stat2 expression was also increased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells (Figure 7B, left panel). Stat2 expression in wild-type CT26 cells was increased when wild-type CT26 cells were induced with IFNγ (compare closed circle data in Figure 7B, left and right panels). Paradoxically, Stat2 expression was decreased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells in response to IFNγ (Figure 7B, right panel).

[0246] Similarly, Irf1 expression increased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells (Figure 7C, left panel). Irf1 expression in wild-type CT26 cells increased when wild-type CT26 cells were induced with IFNγ (compare closed circle data in Figure 7C, left and right panels). Paradoxically, Irf1 expression decreased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells in response to IFNγ (Figure 7C, right panel).

[0247] As expected, Tap1 expression increased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells (Figure 7D, left panel). Tap1 expression in wild-type CT26 cells increased when wild-type CT26 cells were induced with IFNγ (compare closed circle data in Figure 7D, left and right panels). Paradoxically, Tap1 expression decreased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells in response to IFNγ (Figure 7D, right panel).

[0248] Example 8: Pathway analysis of differentially regulated genes To perform pathway analysis, the WEB-based GEne SeT AnaLysis Toolkit (WebGestalt) was used. Briefly, the top 1,000 genes that downregulated 4th generation anti-PD-1 resistant cells but upregulated CT26 cells (see Figure 6A (panel 2)) were ranked based on the fold difference in expression between 4th generation anti-PD-1 resistant cells and the parental CT-26 sample. These genes and rankings were input into WebGestalt to identify enriched / de-enriched pathways associated with these genes using gene set enrichment analysis (GSEA) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway function database. Benjamini-Hochberg false positive rate (FDR) was used for statistical significance. This analysis showed that the following pathways were overactive in 4th generation anti-PD-1 resistant cells compared to parental CT-26 cells: Circardian entrainment, cAMP signaling pathway, cholinergic synapse, melanogenesis, Rap1 signaling pathway, human cytomegalovirus infection, human immunodeficiency 1 virus infection, glutamatergic synapse, pathway in cancer, and Ras signaling pathway (Figure 8A). The following pathways were suppressed in 4th generation anti-PD-1 resistant cells compared to parental CT-26 cells: systemic lupus erythematosus, microRNA in cancer, hepatocellular carcinoma, tuberculosis, and protein processing in the endoplasmic reticulum. To visualize the enrichment scores and significance, a volcano plat was prepared based on the data shown in Figure 8A. Figure 8B shows the volcano plot. As shown in Figure 8B, circadian entrainment was the furthest "point" to the right, while pathways in cancer and Ras signaling were the highest (i.e., with the most significant FDR value). Although there was no strong significance (all FDR>.05, which is often the case with noise in genomics), this unbiased analysis identified the Ras / Rap1 signaling pathway. Figure 8C shows the RAS signaling pathway, showing convergence with Raf / Mek / Erk signaling. Figure 8D shows the RAP1 signaling pathway, showing convergence with Raf / Mek / Erk signaling.

[0249] Example 9: Paradoxical dysregulation of Ccl5 (RANTES), Cxcl10 (IP-10) and Ifnb1 We analyzed nest transcripts per million (TPM; normalized expression data) for Ccl5 (RANTES), Cxcl10 (IP-10), and Ifnb1, genes that are induced by Trim7 activation.

[0250] Ccl5 (RANTES) and Cxcl10 (IP-10) are genes regulated by IFNγ. The promoters regulating Ccl5 (RANTES) and Cxcl10 (IP-10) contain binding sites for STAT1 dimers and / or ISGF3. As expected, expression of Ccl5 (RANTES) increased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells (Figure 9A (left panel)). Expression of Ccl5 (RANTES), which was increased in wild-type CT26 cells, increased when wild-type CT26 cells were induced with IFNγ (compare the closed circle data in Figure 9A (left and right panels)). Paradoxically, expression of Ccl5 (RANTES) decreased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells in response to IFNγ (Figure 9A (right panel)).

[0251] Expression of Cxcl10 (IP-10) increased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells, as expected (Figure 9B, left panel). Expression of Cxcl10 (IP-10) in wild-type CT26 cells increased when wild-type CT26 cells were induced with IFNγ (compare closed circle data in Figure 9B, left and right panels). Paradoxically, expression of Cxcl10 (IP-10) decreased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells in response to IFNγ (Figure 9B, right panel).

[0252] Expression of Ifnb1 also increased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells (Figure 9C, left panel). Expression of Ifnb1 in wild-type CT26 cells increased when wild-type CT26 cells were induced with IFNγ (compare closed circle data in Figure 9C, left and right panels). Paradoxically, expression of Ifnb1 decreased from wild-type CT26 cells to second-generation anti-PD-1-resistant cells and fourth-generation anti-PD-1-resistant cells in response to IFNγ (Figure 9C, right panel).

[0253] These results, among others, indicate that acquired resistance to anti-PD-1 is associated with the paradoxical regulation of Trim7-regulated genes, such as Ccl5 (RANTES), Cxcl10 (IP-10), and Ifnb1. Thus, these three results suggest that anti-PD-1-resistant tumors may be treated with Trim7-modulating agents, such as Trim7 inhibitors.

[0254] Incorporation by Reference All patents and publications referenced herein are incorporated by reference in their entirety. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior invention. As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The contents of any individual section may be equally applicable to all sections.

[0255] Equivalent While the invention has been disclosed in relation to specific embodiments thereof, it will be understood that further modifications are possible, and this application is intended to cover any variations, uses, or adaptations of the invention in accordance with the principles of the invention, including departures from the present disclosure as come within known or customary practice within the art to which the invention pertains, and as may be applied to the following essential features as set forth in the appended claims.

[0256] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments specifically disclosed herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A pharmaceutical for treating cancer in a subject in need thereof, comprising: (a) the subject has been administered an anti-cancer treatment that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; (b) the anti-tumor response due to said anti-cancer treatment with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2, or overexpression and / or activation of TRIM7 has been evaluated using a step of evaluating a biological sample obtained from said subject for overexpression and / or activation of TRIM7, wherein: (I) if TRIM7 overexpression and / or activation is observed, a Trim7 regulator and / or a proteasome inhibitor is administered; (II) The anti-tumor response of the anti-cancer treatment with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2, or the overexpression and / or activation of TRIM7 is re-evaluated using a step of evaluating the overexpression and / or activation of TRIM7 using a second biological sample obtained from the subject; and (III) if TRIM7 overexpression and / or activation is not observed, administration of the Trim7 regulator is withdrawn; Medicine.

2. A pharmaceutical for treating cancer in a subject in need thereof, comprising: (a) the subject has been administered an anti-cancer treatment capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2; (b) an anti-tumor response to said anti-cancer treatment with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2 has been evaluated by monitoring the reduction in tumor size in said subject, wherein: (i) if a lack of tumor size reduction is observed, a Trim7 modulator and / or a proteasome inhibitor is administered to the subject; (ii) the anti-tumor response to the anti-cancer treatment with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2 by monitoring the tumor shrinkage in the subject is re-evaluated; (iii) if a reduction in tumor size is observed, administration of the Trim7 modulating agent is withdrawn; Medicine.

3. The Trim7 modulator is a Trim7 inhibitor; the Trim7 modulating agent is selected from small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antisense RNA, or guide RNA (gRNA) that inhibits Trim7 protein; or small molecules, antibodies, peptides, and peptidomimetics that inhibit the activity of Trim7 by mimicking the target of Trim7; and / or the Trim7 modulator is a mitogen and stress activated kinase 1 (MSK1) inhibitor, the MSK1 inhibitor modulating Trim7 via the downstream effects of inhibition of MSK1; The pharmaceutical composition according to claim 1 or 2.

4. The evaluating is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescence staining, ELISA, fluorescence activated cell sorting (FACS), or a combination thereof; said evaluating is effected by contacting said sample with an agent that specifically binds to one or more proteins encoded by the Trim7 pathway; said evaluating is effected by contacting said sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with said Trim7 pathway; said evaluating being effected by assaying E3 ubiquitin ligase activity; said evaluating being performed by assaying protein ubiquitination and / or K48-linked ubiquitination of Stimulator of Interferon Genes (STING) and / or AP-1 coactivator RACO-1; said evaluating being effected by assaying c-Jun / AP1 activation and / or increase in AP1-mediated gene expression via Ras-Raf-MEK-ERK signaling; said evaluating being effected by assaying ubiquitination and stabilization of the AP1 coactivator RACO-1; said evaluating being effected by assaying K63-linked ubiquitination of the target protein; said evaluating is performed by assaying Trim7 phosphorylation, K63-linked ubiquitination and / or protein levels of said AP-1 coactivator RACO-1; and / or The evaluating is performed by assaying upregulation of IFNβ, IP-10 and / or RANTES. The pharmaceutical composition according to claim 1 or 3.

5. A method for determining a cancer treatment for a subject or selecting a subject for cancer treatment, comprising: (a) (i) positive regulation of cell cycle processes, regulation of the G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of defense response, positive regulation of IFNβ production, regulation of inflammatory response, regulation of innate immune response, negative regulation of antigen processing / presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or (ii) Phospholipid export, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid export, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes. evaluating a biological sample obtained from the subject for the presence, absence, or level of one or more genes associated with a Gene Ontology (GO) pathway selected from: (b) selecting a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2 based on the evaluation of step (a); and The method includes:

6. Upregulation of one or more genes associated with the GO pathways described in (i) relative to healthy tissue indicates lack of response, resistance, or refractoryness to said cancer therapy utilizing the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; upregulation of one or more genes associated with the GO pathways described in (i) relative to another biological sample from a subject known to be sensitive to anti-PD-1 therapy indicates lack of response, resistance, or refractoryness to said cancer therapy utilizing the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; upregulation of one or more genes associated with a GO pathway as described in (i) relative to a previous biological sample obtained from the subject indicates the development of resistance, lack of response, or refractory to the cancer therapy utilizing the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; Lack of upregulation of one or more genes associated with the GO pathways described in (i) relative to healthy tissue indicates a response to said cancer therapy using the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; Lack of upregulation of one or more genes associated with a GO pathway as described in (i), compared to another biological sample from a subject known to be sensitive to anti-PD-1 therapy, indicates a response to said cancer therapy using an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; A lack of upregulation of one or more genes associated with a GO pathway as described in (i) compared to a previous biological sample obtained from the subject indicates a response to the cancer therapy using an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; Downregulation of one or more genes associated with the GO pathways described in (ii) relative to healthy tissue indicates lack of response, resistance, or refractoryness to said cancer therapy utilizing the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; Downregulation of one or more genes associated with the GO pathways described in (ii) relative to another biological sample from a subject known to be sensitive to anti-PD-1 therapy indicates lack of response, resistance, or refractoryness to said cancer therapy utilizing the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; downregulation of one or more genes associated with a GO pathway as described in (ii) relative to a previous biological sample obtained from the subject indicates progression of resistance, lack of response, or refractory disease to the cancer therapy utilizing the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; Lack of downregulation of one or more genes associated with the GO pathways described in (ii) compared to healthy tissue indicates a response to said cancer therapy using the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; Lack of downregulation of one or more genes associated with a GO pathway as described in (ii) compared to another biological sample from a subject known to be sensitive to anti-PD-1 therapy indicates a response to said cancer therapy using an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; A lack of downregulation of one or more genes associated with a GO pathway as described in (ii) compared to a previous biological sample obtained from the subject indicates a response to the cancer therapy using an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; Lack of upregulation of one or more genes associated with a GO pathway, as compared to a previous biological sample from the subject, indicates a response to the cancer therapy using an ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity, wherein the GO pathway is selected from (a) cellular response to IFNγ, (b) negative regulation of antigen processing / presentation, (c) type I IFN signaling pathway, (d) positive regulation of IκB kinase / NFκB signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I; Lack of upregulation of one or more genes associated with a GO pathway, relative to subjects known to be sensitive to anti-PD-1 therapy, indicates a response to said cancer therapy with the ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity, wherein said GO pathway is selected from (a) cellular response to IFNγ, (b) negative regulation of antigen processing / presentation, (c) type I IFN signaling pathway, (d) positive regulation of IκB kinase / NFκB signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I; Lack of upregulation of one or more genes associated with a GO pathway, relative to a standard, indicates a response to said cancer therapy with the ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity, wherein said GO pathway is selected from (a) cellular response to IFNγ, (b) negative regulation of antigen processing / presentation, (c) type I IFN signaling pathway, (d) positive regulation of IκB kinase / NFκB signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I; upregulation of one or more genes associated with a GO pathway, as compared to a previous biological sample from said subject, indicates resistance, lack of response, or refractory to said cancer therapy utilizing the ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity, wherein said GO pathway is selected from (a) cellular response to IFNγ, (b) negative regulation of antigen processing / presentation, (c) type I IFN signaling pathway, (d) positive regulation of IκB kinase / NFκB signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I; upregulation of one or more genes associated with a GO pathway, relative to subjects known to be sensitive to anti-PD-1 therapy, indicates resistance, lack of response, or refractoryness to said cancer therapy utilizing the ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity, said GO pathway being selected from (a) cellular response to IFNγ, (b) negative regulation of antigen processing / presentation, (c) type I IFN signaling pathway, (d) positive regulation of IκB kinase / NFκB signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I; upregulation of one or more genes associated with a GO pathway relative to a standard indicates resistance, lack of response, or refractory to said cancer therapy with the ability to inhibit PD-1, PD-L1 and / or PD-L2 function and / or activity, said GO pathway being selected from (a) cellular response to IFNγ, (b) negative regulation of antigen processing / presentation, (c) type I IFN signaling pathway, (d) positive regulation of IκB kinase / NFκB signaling and antigen processing, and (e) presentation of endogenous peptides via MHC class I; A lack of upregulation of one or more genes associated with cellular response to IFNγ, as compared to a previous biological sample from the subject, indicates a response to the cancer therapy using an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; A lack of upregulation of one or more genes associated with cellular response to IFNγ, as compared to subjects known to be sensitive to anti-PD-1 therapy, indicates a response to said cancer therapy using the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; A lack of upregulation of one or more genes associated with cellular response to IFNγ, relative to the standard, indicates a response to said cancer therapy using the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; upregulation of one or more genes associated with cellular response to IFNγ, as compared to a previous biological sample from said subject, indicates resistance, lack of response, or refractory to said cancer therapy utilizing the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; upregulation of one or more genes associated with cellular response to IFNγ, relative to subjects known to be sensitive to anti-PD-1 therapy, indicates resistance, lack of response, or refractoryness to said cancer therapy utilizing the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; upregulation of one or more genes associated with cellular response to IFNγ, relative to the standard, indicates resistance, lack of response, or refractory to said cancer therapy utilizing the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; upregulation of one or more genes associated with the type I IFN signaling pathway, as compared to a previous biological sample from said subject, indicates resistance, lack of response, or refractory to said cancer therapy utilizing the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; upregulation of one or more genes associated with the type I IFN signaling pathway relative to subjects known to be sensitive to anti-PD-1 therapy indicates resistance, lack of response, or refractoryness to said cancer therapy utilizing the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; upregulation of one or more genes associated with the type I IFN signaling pathway relative to the standard indicates resistance, lack of response, or refractory to said cancer therapy utilizing the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; A lack of upregulation of one or more genes associated with the type I IFN signaling pathway, as compared to a previous biological sample from the subject, indicates a response to the cancer therapy using an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; Lack of upregulation of one or more genes associated with the type I IFN signaling pathway compared to subjects known to be sensitive to anti-PD-1 therapy indicates a response to said cancer therapy using the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; and / or Lack of upregulation of one or more genes associated with the type I IFN signaling pathway, relative to the control, indicates a response to said cancer therapy using an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2. The method according to claim 5.

7. The evaluating is performed by DNA sequencing, RNA sequencing, immunohistochemical staining, Western blotting, in cell western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS), or a combination thereof; The evaluating comprises: (i) positive regulation of cell cycle processes, regulation of the G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of a defense response, positive regulation of IFNβ production, regulation of an inflammatory response, regulation of an innate immune response, negative regulation of antigen processing / presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or (ii) Phospholipid export, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid export, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes. with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a Gene Ontology (GO) pathway selected from: said assessing being effected by contacting said sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a Gene Ontology (GO) pathway selected from: (a) cellular response to IFNγ; (b) negative regulation of antigen processing / presentation; (c) type I IFN signaling pathway; (d) positive regulation of IκB kinase / NFκB signaling and antigen processing; and (e) presentation of endogenous peptides via MHC class I. said assessing is performed by contacting said sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with a cellular response to IFNγ; and / or said assessing is effected by contacting said sample with an agent that specifically binds to one or more proteins encoded by one or more genes associated with the type I IFN signaling pathway; The evaluating comprises: (i) positive regulation of cell cycle processes, regulation of the G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of a defense response, positive regulation of IFNβ production, regulation of an inflammatory response, regulation of an innate immune response, negative regulation of antigen processing / presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or (ii) Phospholipid export, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid export, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes. with an agent that specifically binds to one or more of the nucleic acids of one or more genes associated with a Gene Ontology (GO) pathway selected from said assessing being effected by contacting said sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a Gene Ontology (GO) pathway selected from: (a) cellular response to IFNγ; (b) negative regulation of antigen processing / presentation; (c) type I IFN signaling pathway; (d) positive regulation of IκB kinase / NFκB signaling and antigen processing; and (e) presentation of endogenous peptides via MHC class I. said assessing is performed by contacting said sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a cellular response to IFNγ; and / or said assessing being effected by contacting said sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with the type I IFN signaling pathway; The method according to claim 5 or 6.

8. said evaluating providing information classifying said subject into a high-risk or low-risk group; wherein the high risk classification includes a high level of tumor cells resistant to the cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2, and the low risk classification includes a low level of tumor cells resistant to the cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; said low-risk or high-risk classification indicating that neoadjuvant therapy should be withheld; said low-risk or high-risk classification indicating forgoing adjuvant therapy; said assessing is predictive of a positive response to and / or benefit from said cancer treatment; said evaluating is predictive of a negative or neutral response to said cancer treatment and / or a negative or neutral benefit from said cancer treatment; said evaluating being predictive of a positive response to and / or benefit from neoadjuvant chemotherapy, or non-responsiveness to and / or lack of benefit from neoadjuvant chemotherapy; said evaluating being predictive of a positive response to and / or benefit from adjuvant chemotherapy, or non-responsiveness to and / or lack of benefit from adjuvant chemotherapy; said evaluating being predictive of a negative or neutral response to and / or a negative or neutral benefit from neoadjuvant chemotherapy, or non-responsiveness to and / or lack of benefit from neoadjuvant chemotherapy; said evaluating being predictive of a negative or neutral response to and / or a negative or neutral benefit from adjuvant chemotherapy, or non-responsiveness to and / or lack of benefit from adjuvant chemotherapy; said evaluating informs administering or withholding said cancer treatment; said evaluating informs administering neoadjuvant therapy; said evaluating informs administering adjuvant therapy; said evaluating informs withholding neoadjuvant therapy; said evaluating informs withholding adjuvant therapy; wherein said neoadjuvant therapy and / or said adjuvant therapy is selected from chemotherapeutic agents, cytotoxic agents, checkpoint inhibitors, antimetabolite chemotherapeutic agents, topoisomerase inhibitors; and / or the neoadjuvant therapy and / or the adjuvant therapy is selected from protein translation inhibitors, ribosome integrity inhibitors, regulators of tRNA expression and / or function, inhibitors of rRNA and / or tRNA synthesis, regulators of amino acid synthesis and / or uptake, regulators of post-translational modifications, regulators of protein degradation and regulators of protein transport, or topoisomerase inhibitors, The method according to any one of claims 5 to 7.

9. 1. A transgenic non-human animal comprising one or more tumor cells, said tumor cells comprising: (a) upregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from positive regulation of cell cycle processes, regulation of the G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of a defense response, positive regulation of IFNβ production, regulation of an inflammatory response, regulation of an innate immune response, negative regulation of antigen processing / presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or (b) downregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from phospholipid export, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid export, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes. A transgenic non-human animal comprising:

10. the tumor cells are resistant to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; the cancer therapy is selected from nivolumab, pembrolizumab, pidilizumab (CT-011), MK-3475, BMS 936559, MPDL328OA, cemiplimab, atezolizumab, avelumab, and durvalumab; the one or more tumor cells (a) upregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from positive regulation of cell cycle processes, regulation of the G1 / S transition, regulation of cell division, regulation of cell proliferation, positive regulation of IκB kinase / NFκB signaling, type I IFN signaling pathway, cellular response to IFNγ, positive regulation of IFNα production, positive regulation of a defense response, positive regulation of IFNβ production, regulation of an inflammatory response, regulation of an innate immune response, negative regulation of antigen processing / presentation, and antigen processing / presentation of endogenous peptides via MHC class I; and / or (b) downregulation of one or more genes associated with a Gene Ontology (GO) pathway selected from phospholipid export, negative regulation of fibrinolysis, chylomicron assembly, plasma membrane repair, SRP-dependent cotranslational protein targeting to membranes, small ribosomal subunit assembly, phospholipid export, regulation of translation, mitochondrial respiratory chain complex I, mitochondrial translation elongation, DNA-dependent DNA replication, and ATP biosynthesis processes. and / or The transgenic non-human animal is a rodent. The transgenic non-human animal of claim 9.

11. 11. A method for testing a candidate anti-cancer drug, comprising evaluating whether the candidate anti-cancer drug is effective in slowing or inhibiting cancer growth in the transgenic non-human animal, wherein the candidate anti-cancer drug is administered to the transgenic non-human animal of claim 9 or 10.

12. 1. A method for manufacturing a pharmaceutical composition for treating cancer, comprising: (a) selecting an anti-cancer drug effective to slow or inhibit cancer growth in the transgenic non-human animal of claim 9 or 10, wherein the anti-cancer drug is administered to the transgenic non-human animal of claim 9 or 10; (b) formulating the anti-cancer drug or candidate for administration to a human subject; and The method includes:

13. 13. The method of claim 11 or 12, wherein the anti-cancer drug is selected from a chemotherapeutic agent, a cytotoxic agent, and a checkpoint inhibitor.

14. A method for determining a cancer treatment for a subject or selecting a subject for cancer treatment, comprising: (a) detecting a biological sample obtained from said subject; (i) a gene selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP1, TAP2, CASP1, IRF, LTBR, ​​PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1; and / or (ii) a gene selected from RPL41, RPS15, RPS8, TRIM7, and LRG1 and assessing the expression of (b) selecting said cancer therapy based on the assessment of step (a), said cancer therapy comprising: (i) an agent capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2; (ii) an antimetabolite chemotherapeutic agent, or a topoisomerase inhibitor; and (iii) selected from protein translation inhibitors, ribosome biogenesis inhibitors, inhibitors of rRNA and / or tRNA synthesis, inhibitors of amino acid synthesis, amino acid uptake inhibitors, regulators of post-translational modification, regulators of protein degradation, and regulators of protein transport; The method includes:

15. (A) The cancer therapy is selected using an ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2 when the biological sample contains at least one tumor cell, and / or a gene selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP1, TAP2, CASP1, IRF, LTBR, ​​PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1 is not upregulated in said at least one tumor cell when compared to healthy tissue, a previous biological sample obtained from said subject, or another biological sample from a subject known to be sensitive to anti-PD-1 therapy; and / or a gene selected from RPL41, RPS15, RPS8, TRIM7, and LRG1 is not downregulated in said at least one tumor cell when compared to healthy tissue, a previous biological sample obtained from said subject, or another biological sample from a subject known to be sensitive to anti-PD-1 therapy; (B) the biological sample contains at least one tumor cell; and and / or a gene selected from CD274, B2M, STAT1, STAT2, TRIM7, IRF1, TAP1, TAP2, CASP1, IRF, LTBR, ​​PVR, GASTA3, LRG1, SPRY2, ARG1, TRIM8, TRIM2, MAPK8IP1, TRIM6, and KRT1 is upregulated in said at least one tumor cell compared to healthy tissue, a previous biological sample obtained from said subject, or another biological sample from a subject known to be sensitive to anti-PD-1 therapy; and / or if a gene selected from RPL41, RPS15, RPS8, TRIM7, and LRG1 is downregulated in the at least one tumor cell when compared to healthy tissue, a previous biological sample obtained from the subject, or another biological sample from a subject known to be sensitive to anti-PD-1 therapy; the cancer therapy is selected from antimetabolite chemotherapeutic agents, topoisomerase inhibitors, protein translation inhibitors, ribosome biogenesis inhibitors, inhibitors of rRNA and / or tRNA synthesis, inhibitors of amino acid synthesis, amino acid uptake inhibitors, regulators of post-translational modifications, regulators of protein degradation, regulators of protein transport, or topoisomerase inhibitors; (C) upregulation of one or more of the genes listed in (b)(i) relative to healthy tissue indicates lack of response, resistance or refractoryness to cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; (D) downregulation of one or more genes listed in (b)(ii) relative to healthy tissue indicates lack of response, resistance or refractoryness to cancer therapy utilizing the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; (E) upregulation of one or more of the genes listed in (b)(i), relative to another biological sample from a subject known to be sensitive to anti-PD-1 therapy, indicates lack of response, resistance, or refractoryness to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; (F) downregulation of one or more of the genes listed in (b)(ii) relative to another biological sample from a subject known to be sensitive to anti-PD-1 therapy indicates lack of response, resistance or refractoryness to a cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; (G) upregulation of one or more of the genes listed in (b)(i), relative to a previous biological sample obtained from the subject, indicates a lack of response, resistance, or progression of refractory disease to the cancer therapy utilizing the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; (H) downregulation of one or more of the genes listed in (b)(ii) relative to a previous biological sample obtained from the subject indicates a lack of response, resistance or progression of refractory disease to the cancer therapy employing the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; (I) lack of upregulation of one or more genes listed in (b)(i) relative to healthy tissue indicates a response to said cancer therapy using an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; (J) lack of downregulation of one or more genes listed in (b)(ii) relative to healthy tissue indicates a response to said cancer therapy using an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; (K) a lack of upregulation of one or more genes listed in (b)(i), as compared to another biological sample from the subject known to be sensitive to anti-PD-1 therapy, indicates responsiveness to cancer therapy using the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; (L) a lack of downregulation of one or more genes listed in (b)(ii), as compared to another biological sample from the subject known to be sensitive to anti-PD-1 therapy, indicates responsiveness to cancer therapy using the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; (M) a lack of upregulation of one or more genes listed in (b)(i), as compared to a previous biological sample obtained from the subject, indicates a progression of a lack of response to said cancer therapy employing the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; (N) a lack of downregulation of one or more genes listed in (b)(ii) compared to a previous biological sample obtained from the subject indicates a progression of a lack of response to the cancer therapy using the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; and / or (O) the biological sample is a fresh tissue sample, a frozen tumor tissue specimen, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue specimen; The method of claim 14.

16. said evaluating being performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS), or a combination thereof; said assessing is effected by contacting said sample with an agent that specifically binds to one or more proteins encoded by one or more genes listed in (b)(i) and / or (b)(ii), optionally wherein said agent that specifically binds to one or more proteins comprises an antibody, an antibody-like molecule, or a binding fragment thereof; the assessing is effected by contacting the sample with an agent that specifically binds to one or more of the nucleic acids of one or more genes related to the genes listed in (b)(i) and / or (b)(ii); 16. The method according to claim 14 or 15.

17. A method for determining a cancer treatment for a subject or selecting a subject for cancer treatment, comprising: (a) assessing a biological sample obtained from the subject for activation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras; (b) selecting said cancer therapy based on the assessment of step (a), the cancer therapy comprising: (i) an agent capable of inhibiting the function and / or activity of PD-1, PD-L1 and / or PD-L2; (ii) antimetabolite chemotherapeutic agents, and topoisomerase inhibitors; and (iii) selected from protein translation inhibitors, ribosome biogenesis inhibitors, inhibitors of rRNA and / or tRNA synthesis, inhibitors of amino acid synthesis, amino acid uptake inhibitors, regulators of post-translational modification, regulators of protein degradation, and regulators of protein transport; The method includes:

18. (A) the cancer therapy having the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2 is selected when the biological sample contains at least one tumor cell; a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras is not upregulated in said at least one tumor cell when compared to healthy tissue, a previous biological sample obtained from said subject, or another biological sample from a subject known to be sensitive to anti-PD-1 therapy; (B) the biological sample contains at least one tumor cell; and a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras is upregulated in the at least one tumor cell compared to healthy tissue, a previous biological sample obtained from the subject, or another biological sample from a subject known to be sensitive to anti-PD-1 therapy; The cancer therapy is selected from antimetabolite chemotherapeutic agents, topoisomerase inhibitors, protein translation inhibitors, ribosome biogenesis inhibitors, inhibitors of rRNA and / or tRNA synthesis, inhibitors of amino acid synthesis, amino acid uptake inhibitors, regulators of post-translational modifications, regulators of protein degradation, regulators of protein transport, topoisomerase inhibitors; (C) upregulation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras relative to healthy tissue indicates lack of response, resistance, or refractory to said cancer therapy employing the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; (D) upregulation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras as compared to another biological sample from a subject known to be sensitive to anti-PD-1 therapy indicates lack of response, resistance, or refractory to said cancer therapy employing the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; (E) upregulation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras, relative to a previous biological sample obtained from the subject, indicates a lack of response, resistance, or progression of refractory disease to the cancer therapy that utilizes the ability to inhibit the function and / or activity of PD-1, PD-L1, and / or PD-L2; (F) Lack of upregulation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras compared to healthy tissue indicates a response to said cancer therapy with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; (G) lack of upregulation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras, as compared to another biological sample from a subject known to be sensitive to anti-PD-1 therapy, indicates a response to said cancer therapy with the ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2; and / or (H) a lack of upregulation of a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras, as compared to a previous biological sample obtained from the subject, indicates a progression of a lack of response to the cancer therapy employing an ability to inhibit the function and / or activity of PD-1, PD-L1 and / or PD-L2.

20. The method of claim 17.

19. the biological sample is a fresh tissue sample, a frozen tumor tissue specimen, cultured cells, circulating tumor cells, or a formalin-fixed paraffin-embedded tumor tissue specimen; the biological sample is a biopsy sample; the biological sample comprises a sample selected from blood, plasma, serum, tears, tears, bone marrow, blood, blood cells, peritoneal fluid, tissue or fine needle biopsy sample, cell-containing bodily fluids, free floating nucleic acid, sputum, saliva, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, faeces, lymph, gynecological fluid, skin swab, vaginal swab, oral swab, nasal swab, lavage or irrigation fluid, aspirate, scraping, bone marrow specimen, tissue biopsy specimen, surgical specimen, other bodily fluids, secretions and / or excretions, and / or cells therefrom; The biological sample is obtained by a technique selected from a scrape, a swab, and a biopsy; The biological sample comprises at least one tumor cell, wherein the tumor is selected from the group consisting of basal cell carcinoma, biliary tract cancer, bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer; glioblastoma; hepatic carcinoma; hepatocellular carcinoma; intraepithelial neoplasia; kidney or renal cancer; laryngeal cancer; leukemia; liver cancer; lung cancer; melanoma; myeloma; neuroblastoma; oral cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer. cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma; B-cell lymphoma; low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and other carcinomas and sarcomas; and Meigs syndrome cancer; renal cancer colorectal cancer; and adrenal cancer, The method according to any one of claims 5 to 8 and 14 to 18.

20. said evaluating being performed by DNA sequencing, RNA sequencing, immunohistochemical staining, western blotting, in cell western, immunofluorescence staining, ELISA, and fluorescence activated cell sorting (FACS), or a combination thereof; said assessing is effected by contacting said sample with an agent that specifically binds to one or more proteins encoded by a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras; and / or wherein said assessing is effected by contacting said sample with an agent that specifically binds to one or more nucleic acids of one or more genes associated with a pathway selected from Mapk8ip1, Trim7, Elk1, Lrg1, Arg1, Rap1, and Ras.

20. The method according to any one of claims 17 to 19.