Methods for diagnosing and treating cancer by means of expression status and mutational status of nrf2 and downstream target genes of said gene

Abstract

To provide methods of identifying a subject having cancer, such as lung cancer.SOLUTION: The present invention provides a method of identifying a subject having cancer, comprising: (a) determining an expression level of at least one gene selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in a sample obtained from the subject; and (b) comparing the expression level of the at least one gene to a reference expression level of the at least one gene, wherein an increase in the expression level of the at least one gene in the sample relative to the reference expression level of the at least one gene identifies the subject having the cancer.SELECTED DRAWING: None

Description

Technical Field

[0001] Sequence Listing This application includes a sequence listing that has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on July 10, 2017, is named 50474-127WO2_Sequence_Listing_7.10.17_ST25 and is 216,092 bytes in size.

[0002] The present invention generally relates to methods for diagnosing, treating, and prognosticating cancer, such as lung cancer.

Background Art

[0003] Cancer remains one of the most deadly threats to human health. In particular, lung cancer is the leading cause of cancer-related death in both men and women in the United States, despite recent advances in treatment. The majority of lung cancers are non-small cell lung cancers (NSCLCs), most commonly either adenocarcinomas or squamous subtypes. Recent studies have identified the patterns of point mutations that underlie these phenotypes (Imielinski et al. Cell. 150(6):1107-1120, 2012), but despite the increasing number of identified mutations related to various cellular pathways, a comprehensive understanding of the nature and impact of these mutations on these cellular pathways is lacking.

[0004] Thus, there is an unmet need in the field of developing effective diagnostic and treatment strategies for cancers such as lung cancer.

Summary of the Invention

[0005] The present invention provides compositions and methods for diagnosing, treating, and prognosticating cancer, such as lung cancer (e.g., non-small cell lung cancer (NSCLC)) and head and neck cancer.

[0006] In one aspect, the present invention features a method for diagnosing cancer in a subject, the method comprising: (a) determining the expression level of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) gene selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in a sample obtained from the subject; and (b) comparing the expression level of the at least one gene with a reference expression level of the at least one gene, wherein an increase in the expression level of the at least one gene in the sample relative to the reference expression level of the at least one gene identifies a subject having cancer.

[0007] In another aspect, the present invention features a method of identifying a subject having a cancer that is an NRF2-dependent cancer, the method comprising: (a) determining the expression level of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) gene selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in a sample obtained from the subject; (b) comparing the expression level of the at least one gene to a reference expression level of the at least one gene; and (c) determining whether the subject's cancer is an NRF2-dependent cancer, wherein an increase in the expression level of the at least one gene in the sample relative to the reference expression level of the at least one gene identifies a subject having an NRF2-dependent cancer. In some embodiments of any of the foregoing aspects, the expression levels of at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) genes selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in a sample obtained from the subject are determined.In some embodiments, the expression levels of at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) genes selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL are determined in a sample obtained from a subject. In some embodiments, the expression levels of at least four (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) genes selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL are determined in a sample obtained from a subject. In some embodiments, the expression levels of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in a sample obtained from a subject are determined.

[0008] In some embodiments, the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21) of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, or NQO1 is determined. In some embodiments, the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of AKR1B10, AKR1C2, ME1, KYNU, CABYR, TRIM16L, AKR1C4, CYP4F11, RSPO3, AKR1B15, NR0B1, and AKR1C3 is determined.

[0009] In some embodiments, (a) the expression level of at least two genes in a sample is the average (e.g., mean or median) of at least two genes of the sample, (b) the reference expression level of at least two genes is the average (e.g., mean or median) of at least two genes of a reference, and (c) the average (e.g., mean or median) of at least two genes of the sample is compared to the average of at least two genes of the reference.

[0010] In some embodiments, the reference expression level is the average expression level of at least one gene in a population of subjects. In some embodiments, the population of subjects is a population of subjects sharing a common ethnicity.

[0011] In some embodiments, the reference expression level is the average expression level of at least one gene in a population of subjects having cancer (e.g., lung cancer, e.g., non-small cell lung cancer (NSCLC), e.g., squamous NSCLC).

[0012] In some embodiments, the expression level is the mRNA expression level. In some embodiments, the mRNA expression level is determined by PCR, RT-PCR, RNA-seq, gene expression profiling, serial analysis of gene expression, or microarray analysis.

[0013] In other embodiments, the expression level is the protein expression level. In some embodiments, the protein expression level is determined by Western blot, immunohistochemistry, or mass spectrometry.

[0014] In some embodiments, any of the foregoing methods further comprises determining the DNA sequence of NRF2. In some embodiments, the DNA sequence is determined by PCR, exome-seq, microarray analysis, or whole-genome sequencing.

[0015] In another aspect, the present invention features a method of diagnosing cancer in a subject, the method comprising determining the DNA sequence in a sample obtained from the subject, and the presence of NRF2 DNA containing a deletion of all or part of exon 2 thereof identifies the subject as having cancer. In some embodiments, the DNA sequence is determined by PCR, exome-seq, microarray analysis, or whole-genome sequencing.

[0016] In another aspect, the present invention features a method of identifying a subject having cancer, the method comprising determining the mRNA expression level of NRF2 containing a deletion of all or part of exon 2 thereof in a sample obtained from the subject, and the presence of NRF2 containing a deletion of all or part of exon 2 thereof identifies the subject as having cancer. In some embodiments, the mRNA expression level is determined by PCR, RT-PCR, RNA-seq, gene expression profiling, serial analysis of gene expression, or microarray analysis. In some embodiments, the method further comprises determining the DNA sequence of NRF2. In some embodiments, the DNA sequence is determined by PCR, exome-seq, microarray analysis, or whole-genome sequencing.

[0017] In some embodiments of any of the foregoing aspects, NRF2 further includes a deletion of all or part of its exon 3.

[0018] In another aspect, the present invention features a method of diagnosing cancer in a subject, the method including determining the protein expression level of NRF2 comprising a deletion of all or part of its Neh2 domain in a sample obtained from the subject, and the presence of NRF2 comprising a deletion of all or part of its Neh2 domain identifies the subject as having cancer.

[0019] In another aspect, the present invention features a method of identifying a subject having cancer, the method including determining the protein expression level of NRF2 comprising a deletion of all or part of its Neh2 domain in a sample obtained from the subject, and the presence of NRF2 comprising a deletion of all or part of its Neh2 domain identifies the subject as having cancer.

[0020] In some embodiments of any of the foregoing aspects, NRF2 further includes a deletion of all or part of its Neh4 domain. In some embodiments, the protein expression is determined by Western blot, immunohistochemistry, or mass spectrometry.

[0021] In some embodiments, the method further comprises administering a therapeutically effective amount of an NRF2 pathway antagonist to the subject. In some embodiments, the method further comprises administering a therapeutically effective amount of an anti-cancer agent to the subject. In other embodiments, the method comprises administering an anti-cancer agent and an NRF2 pathway antagonist. In some embodiments, the anti-cancer agent and the NRF2 pathway antagonist are co-administered. In other embodiments, the anti-cancer agent and the NRF2 pathway antagonist are administered sequentially. In some embodiments, the anti-cancer agent is selected from the group consisting of an anti-angiogenic agent, a chemotherapeutic agent, a growth inhibitor, a cytotoxic agent, and an immunotherapy. In some embodiments, the anti-angiogenic agent is a VEGF antagonist. In some embodiments, the NRF2 pathway antagonist is selected from the group consisting of a CREB antagonist, a CREB binding protein (CBP) antagonist, a Maf antagonist, an activating transcription factor 4 (ATF4) antagonist, a protein kinase C (PKC) antagonist, a Jun antagonist, a glucocorticoid receptor antagonist, a UbcM2 antagonist, a HACE1 antagonist, a c-Myc agonist, a SUMO agonist, a KEAP1 agonist, a CUL3 agonist, or a retinoic acid receptor α (RARα) agonist.

[0022] In another aspect, the present invention features a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of an NRF2 pathway antagonist, wherein the expression level of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) of the following genes in a sample obtained from the subject: AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL, has been determined to be increased relative to a reference expression level of at least one of the genes. In other embodiments, the expression levels of at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) of the genes selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in a sample obtained from the subject are determined. In other embodiments, the expression levels of at least three (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) of the genes selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in a sample obtained from the subject are determined.In other embodiments, the expression levels of at least 4 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) genes selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL are determined in a sample obtained from a subject. In other embodiments, the expression levels of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in a sample obtained from a subject are determined.

[0023] In some embodiments, the expression levels of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21) of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, or NQO1 are determined. In other embodiments, the expression levels of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of AKR1B10, AKR1C2, ME1, KYNU, CABYR, TRIM16L, AKR1C4, CYP4F11, RSPO3, AKR1B15, NR0B1, and AKR1C3 are determined.

[0024] In some embodiments, (a) the expression levels of at least two genes in a sample are the average of at least two genes of the sample, (b) the reference expression levels of the at least two genes are the average of at least two genes of a reference, and (c) the average of the at least two genes of the sample is compared to the average of the at least two genes of the reference. In some embodiments, the reference expression level is the average expression level of at least one gene in a population of subjects. In some embodiments, the population of subjects is a population of subjects sharing a common ethnicity. In some embodiments, the reference expression level is the average expression level of at least one gene in a population of subjects having cancer.

[0025] In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC), e.g., squamous NSCLC.

[0026] In some embodiments, the expression level is an mRNA expression level. In some embodiments, the mRNA expression level is determined by PCR, RT-PCR, RNA-seq, gene expression profiling, serial analysis of gene expression, or microarray analysis. In some embodiments, the mRNA expression level is determined by RNA-seq.

[0027] In some embodiments, the method further comprises determining the DNA sequence of NRF2 (e.g., by PCR, exome-seq, microarray analysis, or whole genome sequencing).

[0028] In some embodiments, the expression level is a protein expression level. In some embodiments, the protein expression is determined by Western blot, immunohistochemistry, or mass spectrometry.

[0029] In another aspect, the present invention features a method of treating a subject having cancer, the method comprising: (a) determining the mRNA expression level of NRF2 comprising a deletion of all or a portion of exon 2 in a sample obtained from the subject, wherein the presence of NRF2 mRNA comprising a deletion of all or a portion of exon 2 identifies the subject as having cancer; and (b) administering to the subject a therapeutically effective amount of an NRF2 pathway antagonist.

[0030] In some embodiments, the mRNA expression is determined by PCR, RT-PCR, RNA-seq, gene expression profiling, serial analysis of gene expression, or microarray analysis. In some embodiments, the mRNA expression level is determined by RNA-seq. In some embodiments, the method further comprises determining the DNA sequence of NRF2 (e.g., by PCR, exome-seq, microarray analysis, or whole genome sequencing).

[0031] In another aspect, the present invention features a method of treating a subject having cancer, the method comprising: (a) determining the DNA sequence of NRF2 comprising a deletion of all or a portion of exon 2 in a sample obtained from the subject, wherein the presence of NRF2 DNA comprising a deletion of all or a portion of exon 2 identifies the subject as having cancer; and (b) administering to the subject a therapeutically effective amount of an NRF2 pathway antagonist. In some embodiments, the DNA sequence is determined by PCR, exome-seq, microarray analysis, or whole genome sequencing. In some embodiments, NRF2 (e.g., mRNA or DNA) further comprises a deletion of all or a portion of exon 3.

[0032] In another aspect, the present invention features a method of treating a subject having cancer, the method comprising: (a) determining the protein expression level of NRF2 in a sample obtained from the subject, the protein expression level including a deletion of all or a portion of its Neh2 domain, wherein the presence of the NRF2 protein including the deletion of all or a portion of Neh2 identifies the subject as having cancer; and (b) administering to the subject a therapeutically effective amount of an NRF2 pathway antagonist.

[0033] In some embodiments, the NRF2 protein further includes a deletion of all or a portion of its Neh4 domain. In some embodiments, protein expression is determined by Western blot, immunohistochemistry, or mass spectrometry. In some embodiments, the method further includes determining the DNA sequence of NRF2 (e.g., by PCR, exome-seq, microarray analysis, or whole genome sequencing).

[0034] In some embodiments, the method includes administering to the subject a therapeutically effective amount of an anti-cancer agent. In some embodiments, the anti-cancer agent and the NRF2 pathway antagonist are co-administered. In other embodiments, the anti-cancer agent and the NRF2 pathway antagonist are administered sequentially. In some embodiments, the anti-cancer agent is selected from the group consisting of an anti-angiogenic agent, a chemotherapeutic agent, a growth inhibitor, a cytotoxic agent, and an immunotherapy. In some embodiments, the anti-angiogenic agent is a VEGF antagonist. In some embodiments, the NRF2 pathway antagonist is selected from the group consisting of a CREB antagonist, a CREB-binding protein (CBP) antagonist, a Maf antagonist, an activating transcription factor 4 (ATF4) antagonist, a protein kinase C (PKC) antagonist, a Jun antagonist, a glucocorticoid receptor antagonist, a UbcM2 antagonist, a HACE1 antagonist, a c-Myc agonist, a SUMO agonist, a KEAP1 agonist, a CUL3 agonist, or a retinoic acid receptor α (RARα) agonist.

[0035] In some embodiments, the sample obtained from the subject is, for example, a tumor sample from a biopsy sample. In some embodiments, the sample is obtained from a subject who has not received prior treatment. In some embodiments, the subject has lung cancer (e.g., non-small cell lung cancer (NSCLC), e.g., squamous NSCLC) or head and neck cancer (e.g., squamous head and neck cancer).

Brief Description of the Drawings

[0036]

Figure 1-1

Figure 1-2

Figure 1-3

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14-1

Figure 14-2

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28A

Figure 28B

Figure 28C

Figure 29

Figure 30

Figure 31

Figure 32

Figure 33

Figure 34

Figure 35

Figure 36

Figure 37

Figure 38

Figure 39

Figure 40

Figure 41

Figure 42

Figure 43

Figure 44

Figure 45

Figure 46

Figure 47

Figure 48

Figure 49

Figure 50

Figure 51

Figure 52

Figure 53

Figure 54

Figure 55

Figure 56

Figure 57

Figure 58

Figure 59

Figure 60

Figure 61

Figure 62

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] I. INTRODUCTION The present invention provides a diagnostic method for cancer, such as lung cancer (e.g., NSCLC) or head and neck squamous cell carcinoma (e.g., HNSC), and an accompanying treatment method. The present invention is based, at least in part, on the discovery that splice variants in NRF2 that remove exon 2 or exon 2+3 result in an unexpected mechanism for NRF2 activation in cancer. NRF2 splice variants result in NRF2 activation by a mutually exclusive mechanism from mutations in KEAP1 or NRF2, and also result in similar NRF2 target gene expression profiles. Cell lines with microdeletions that result in these NRF2 splice variants have loss of NRF2-KEAP1 interaction, increased NRF2 stabilization, induction of NRF2 transcriptional response, and NRF2 pathway dependence. This occurs in 3-6% of squamous NSCLC and 1-2% of HNSC, resulting in activation of NRF2 target genes and pathway dependence similar to KEAP1 mutations.

[0038] This discovery is useful for diagnosing a subject suffering from cancer (e.g., by detecting NRF2 splice variants or by detecting a gene or protein expression profile consistent with the presence of NRF2 splice variants), and for treating a subject according to such a diagnosis (e.g., by administering a therapeutically effective amount of an NRF2 pathway antagonist, such as a cAMP response element-binding protein (CREB)-binding protein (CBP) inhibitor).

[0039] II. DEFINITIONS The terms "diagnose", "diagnosing", or "diagnosis" are used herein to refer to the identification or classification of a molecular or pathological condition, disease, or disorder (e.g., cancer). For example, "diagnosis" can refer to the identification of a specific type of cancer. "Diagnosis" can also refer to the classification of a specific subtype of cancer (e.g., a subtype characterized by the expression of one biomarker or a combination of biomarkers (e.g., a specific gene or the protein encoded by that gene)), for example, by histopathological criteria or by molecular features.

[0040] The terms "cancer" and "cancerous" typically refer to or describe a physiological state in mammals characterized by uncontrolled cell growth. This definition includes benign and malignant cancers, as well as latent tumors or micrometastases. Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, glioblastomas, sarcomas, and leukemias. Cancers include, for example, breast cancer, squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer (NSCLC), adenocarcinoma of the lung, squamous cell carcinoma of the lung (e.g., squamous NSCLC)), various types of head and neck cancers (e.g., HNSC), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancers), pancreatic cancer, ovarian cancer, cervical cancer, liver cancer, bladder cancer, hepatocellular carcinoma, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, and liver cancer, as well as 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 lymphoblastic NHL, high grade small non-cleaved cell NHL, large cell variant NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as nevus, edema (such as those associated with brain tumors), and abnormal vascular proliferation associated with Meigs syndrome.

[0041] As used herein, "patient" or "subject" refers to any single animal, such as a human (including mammals such as dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, non-human primates, and humans), that is eligible for treatment and has experienced or has ever experienced one or more signs, symptoms, or other indicators of a disease or disorder such as cancer. Intended to be included as patients are any patients participating in clinical studies showing no clinical signs of disease, patients participating in epidemiological studies, or patients who have been used as controls at one time. The patient may or may not have been previously treated with an NRF2 pathway antagonist or another drug. The patient may be naive to additional drug(s) used when treatment herein is initiated, i.e., the patient is at a set point "at baseline" (i.e., at a point in time prior to administration of the first dose of the NRF2 pathway antagonist in the treatment method herein, such as the day the subject is screened prior to the start of treatment), and may not have been previously treated with a therapy other than, for example, an NRF2 pathway antagonist (such as a VEGF antagonist or a PD-1 axis-binding antagonist). Such "naive" patients or subjects are generally considered candidates for treatment with such additional drug(s).

[0042] The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a biomarker in a biological sample. "Expression" generally refers to the process by which information (e.g., genetic code and / or epigenetic information) is converted into a structure that exists and functions in a cell. Thus, as used herein, "expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or further modification of a polynucleotide and / or polypeptide (e.g., post-translational modification of a polypeptide). Fragments of the transcribed polynucleotide, translated polypeptide, or polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide), whether they are derived from transcripts or degraded transcripts generated by alternative splicing, or, for example, from post-translational processing of a polypeptide by proteolysis, are also considered expressed. An "expressed gene" includes those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (e.g., transfer and ribosomal RNA).

[0043] The terms "biomarker" and "marker" are used interchangeably herein to refer to DNA, RNA, protein, carbohydrate, or glycolipid-based molecular markers, and the expression or presence of this in a sample from a subject or patient can be detected by standard methods (or methods disclosed herein). Such biomarkers include, but are not limited to, the mRNA sequences described in Table 1 and the proteins encoded thereby. The expression of such biomarkers can be higher or lower in samples obtained from patients sensitive or responsive to NRF2 pathway antagonists than a reference level (e.g., the average (e.g., mean or median) expression level of the biomarker in a sample from a group / population of patients, e.g., patients with cancer, being tested for responsiveness to an NRF2 pathway antagonist; the median expression level of the biomarker in a sample from a group / population of patients, e.g., patients with cancer, identified as non-responsive to an NRF2 pathway antagonist; the level in a sample previously obtained from an individual; or the level in a sample from a patient who has received prior treatment with an NRF2 pathway antagonist in the primary tumor setting and who may now be experiencing metastasis). Individuals having an expression level higher or lower than the reference expression level of at least one gene, such as those described in Table 1, can be identified as subjects / patients who may respond to treatment with an NRF2 pathway antagonist. For example, such subjects / patients showing an expression level that is most extremely 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% higher or lower (i.e., higher or lower than) a reference level (such as an average level) can be identified as subjects / patients (e.g., patients with cancer) who may respond to treatment with an NRF2 pathway antagonist. TIFF2025081326000001.tif198170

[0044] As used herein, the term "ABCC2" refers to any native ABCC2 (ATP-binding cassette subfamily C, member 2) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed ABCC2 and any form of ABCC2 obtained from intracellular processing. The term also encompasses naturally occurring variants of ABCC2, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human ABCC2 is set forth in SEQ ID NO: 1. The amino acid sequence of the exemplary protein encoded by human ABCC2 is shown in SEQ ID NO: 33.

[0045] As used herein, the term "AKR1B10" refers to any native AKR1B10 (aldo-keto reductase family 1, member B10) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed AKR1B10 and any form of AKR1B10 obtained from intracellular processing. The term also encompasses naturally occurring variants of AKR1B10, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human AKR1B10 is set forth in SEQ ID NO: 2. The amino acid sequence of the exemplary protein encoded by human AKR1B10 is shown in SEQ ID NO: 34.

[0046] As used herein, the term "AKR1B15" refers to any native AKR1B15 (aldo-keto reductase family 1, member B15) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed AKR1B15, and any form of AKR1B15 obtained from intracellular processing. The term also encompasses naturally occurring variants of AKR1B15, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human AKR1B15 is set forth in SEQ ID NO: 3. The amino acid sequence of the exemplary protein encoded by human AKR1B15 is shown in SEQ ID NO: 35.

[0047] As used herein, the term "AKR1C2" refers to any native AKR1C2 (aldo-keto reductase family 1, member C2) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed AKR1C2, and any form of AKR1C2 obtained from intracellular processing. The term also encompasses naturally occurring variants of AKR1C2, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human AKR1C2 is set forth in SEQ ID NO: 4. The amino acid sequence of the exemplary protein encoded by human AKR1C2 is shown in SEQ ID NO: 36.

[0048] As used herein, the term "AKR1C3" refers to any native AKR1C3 (aldo-keto reductase family 1, member C3) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed AKR1C3, and any form of AKR1C3 obtained from intracellular processing. The term also encompasses naturally occurring variants of AKR1C3, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human AKR1C3 is set forth in SEQ ID NO: 5. The amino acid sequence of the exemplary protein encoded by human AKR1C3 is shown in SEQ ID NO: 37.

[0049] As used herein, the term "AKR1C4" refers to any native AKR1C4 (aldo-keto reductase family 1, member C4) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed AKR1C4, and any form of AKR1C4 obtained from intracellular processing. The term also encompasses naturally occurring variants of AKR1C4, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human AKR1C4 is set forth in SEQ ID NO: 6. The amino acid sequence of the exemplary protein encoded by human AKR1C4 is shown in SEQ ID NO: 38.

[0050] As used herein, the term "CABYR" refers to any native CABYR (calcium-binding tyrosine-(Y)-phosphorylation regulated) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" non-processed CABYR, and any form of CABYR obtained from intracellular processing. The term also encompasses naturally occurring variants of CABYR, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human CABYR is set forth in SEQ ID NO: 7. The amino acid sequence of the exemplary protein encoded by human CABYR is shown in SEQ ID NO: 39.

[0051] As used herein, the term "CYP4F11" refers to any native CYP4F11 (chromosome P450, family 4, subfamily F, polypeptide 11) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" non-processed CYP4F11, and any form of CYP4F11 obtained from intracellular processing. The term also encompasses naturally occurring variants of CYP4F11, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human CYP4F11 is set forth in SEQ ID NO: 8. The amino acid sequence of the exemplary protein encoded by human CYP4F11 is shown in SEQ ID NO: 40.

[0052] As used herein, the term "FECH" refers to any native FECH (ferrochelatase) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" non-processing FECH and any form of FECH obtained from intracellular processing. The term also encompasses naturally occurring variants of FECH, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human FECH is set forth in SEQ ID NO: 9. The amino acid sequence of the exemplary protein encoded by human FECH is shown in SEQ ID NO: 41.

[0053] As used herein, the term "FTL" refers to any native FTL (ferritin, light chain polypeptide) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" non-processing FTL and any form of FTL obtained from intracellular processing. The term also encompasses naturally occurring variants of FTL, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human FTL is set forth in SEQ ID NO: 10. The amino acid sequence of the exemplary protein encoded by human FTL is shown in SEQ ID NO: 42.

[0054] As used herein, the term "GCLM" refers to any native GCLM (glutamate cysteine ligase, modifier subunit) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" non-processing GCLM and any form of GCLM obtained from intracellular processing. The term also encompasses naturally occurring variants of GCLM, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human GCLM is set forth in SEQ ID NO: 11. The amino acid sequence of the exemplary protein encoded by human GCLM is shown in SEQ ID NO: 43.

[0055] As used herein, the term "GSR" refers to any native GSR (glutathione reductase) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" non-processing GSR, and any form of GSR obtained from intracellular processing. The term also encompasses naturally occurring variants of GSR, such as splice variants or allelic variants. The nucleic acid sequence of an exemplary human GSR is set forth in SEQ ID NO: 12. The amino acid sequence of the exemplary protein encoded by human GSR is shown in SEQ ID NO: 44.

[0056] As used herein, the term "KYNU" refers to any native KYNU (kynureninase) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" non-processing KYNU, and any form of KYNU obtained from intracellular processing. The term also encompasses naturally occurring variants of KYNU, such as splice variants or allelic variants. The nucleic acid sequence of an exemplary human KYNU is set forth in SEQ ID NO: 13. The amino acid sequence of the exemplary protein encoded by human KYNU is shown in SEQ ID NO: 45.

[0057] As used herein, the term "ME1" refers to any native ME1 (malic enzyme 1, NADP(+)-dependent, cytoplasmic) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" non-processing ME1, and any form of ME1 obtained from intracellular processing. The term also encompasses naturally occurring variants of ME1, such as splice variants or allelic variants. The nucleic acid sequence of an exemplary human ME1 is set forth in SEQ ID NO: 14. The amino acid sequence of the exemplary protein encoded by human ME1 is shown in SEQ ID NO: 46.

[0058] As used herein, the term "NFE2L2" or "NRF2" refers to any native NFE2L2 or NRF2 (nuclear factor, erythroid 2-like 2) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed NFE2L2 and any form of NFE2L2 obtained from intracellular processing. The term also encompasses naturally occurring variants of NFE2L2, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human NFE2L2 is set forth in SEQ ID NO: 15. The amino acid sequence of the exemplary protein encoded by human NFE2L2 is shown in SEQ ID NO: 47.

[0059] As used herein, the term "NQO1" refers to any native NQO1 (NAD(P)H dehydrogenase, quinone 1) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed NQO1 and any form of NQO1 obtained from intracellular processing. The term also encompasses naturally occurring variants of NQO1, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human NQO1 is set forth in SEQ ID NO: 16. The amino acid sequence of the exemplary protein encoded by human NQO1 is shown in SEQ ID NO: 48.

[0060] As used herein, the term "NR0B1" refers to any native NR0B1 (nuclear receptor subfamily 0, group B, member 1) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" non-processed NR0B1 and any form of NR0B1 obtained from intracellular processing. The term also encompasses naturally occurring variants of NR0B1, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human NR0B1 is set forth in SEQ ID NO: 17. The amino acid sequence of the exemplary protein encoded by human NR0B1 is shown in SEQ ID NO: 49.

[0061] As used herein, the term "OSGIN1" refers to any native OSGIN1 (oxidative stress-induced growth inhibitor 1) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" non-processed OSGIN1 and any form of OSGIN1 obtained from intracellular processing. The term also encompasses naturally occurring variants of OSGIN1, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human OSGIN1 is set forth in SEQ ID NO: 18. The amino acid sequence of the exemplary protein encoded by human OSGIN1 is shown in SEQ ID NO: 50.

[0062] As used herein, the term "PGD" refers to any native PGD (phosphogluconate dehydrogenase) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" non-processed PGD and any form of PGD obtained from intracellular processing. The term also encompasses naturally occurring variants of PGD, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human PGD is set forth in SEQ ID NO: 19. The amino acid sequence of the exemplary protein encoded by human PGD is shown in SEQ ID NO: 51.

[0063] As used herein, the term "RSPO3" refers to any native RSPO3 (R-spondin 3) derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed RSPO3 and any form of RSPO3 obtained from intracellular processing. The term also encompasses naturally occurring variants of RSPO3, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human RSPO3 is set forth in SEQ ID NO: 20. The amino acid sequence of the exemplary protein encoded by human RSPO3 is shown in SEQ ID NO: 52.

[0064] As used herein, the term "SLC7A11" refers to any native SLC7A11 (solute family 7 (anionic amino acid transporter light chain, Xc- system), member 11) derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed SLC7A11 and any form of SLC7A11 obtained from intracellular processing. The term also encompasses naturally occurring variants of SLC7A11, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human SLC7A11 is set forth in SEQ ID NO: 21. The amino acid sequence of the exemplary protein encoded by human SLC7A11 is shown in SEQ ID NO: 53.

[0065] As used herein, the term "SRXN1" refers to any native SRXN1 (sulfiredoxin 1) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" non-processing SRXN1, and any form of SRXN1 obtained from intracellular processing. The term also encompasses naturally occurring variants of SRXN1, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human SRXN1 is set forth in SEQ ID NO: 22. The amino acid sequence of the exemplary protein encoded by human SRXN1 is shown in SEQ ID NO: 54.

[0066] As used herein, the term "TALDO1" refers to any native TALDO1 (transaldolase 1) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" non-processing TALDO1, and any form of TALDO1 obtained from intracellular processing. The term also encompasses naturally occurring variants of TALDO1, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human TALDO1 is set forth in SEQ ID NO: 23. The amino acid sequence of the exemplary protein encoded by human TALDO1 is shown in SEQ ID NO: 55.

[0067] As used herein, the term "TRIM16" refers to any native TRIM16 (tripartite motif containing 16) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" non-processing TRIM16, and any form of TRIM16 obtained from intracellular processing. The term also encompasses naturally occurring variants of TRIM16, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human TRIM16 is set forth in SEQ ID NO: 24. The amino acid sequence of the exemplary protein encoded by human TRIM16 is shown in SEQ ID NO: 56.

[0068] As used herein, the term "TRIM16L" refers to any native TRIM16L (tripartite motif-containing 16-like) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed TRIM16L and any form of TRIM16L obtained from intracellular processing. The term also encompasses naturally occurring variants of TRIM16L, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human TRIM16L is set forth in SEQ ID NO: 25. The amino acid sequence of the exemplary protein encoded by human TRIM16L is shown in SEQ ID NO: 57.

[0069] As used herein, the term "TXN" refers to any native TXN (thioredoxin) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" unprocessed TXN and any form of TXN obtained from intracellular processing. The term also encompasses naturally occurring variants of TXN, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human TXN is set forth in SEQ ID NO: 26. The amino acid sequence of the exemplary protein encoded by human TXN is shown in SEQ ID NO: 58.

[0070] As used herein, the term "TXNRD1" refers to any native TXNRD1 (thioredoxin reductase 1) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" non-processing TXNRD1 and any form of TXNRD1 obtained from intracellular processing. The term also encompasses naturally occurring variants of TXNRD1, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human TXNRD1 is set forth in SEQ ID NO: 27. The amino acid sequence of the exemplary protein encoded by human TXNRD1 is shown in SEQ ID NO: 59.

[0071] As used herein, the term "UGDH" refers to any native UGDH (uridine diphosphate (UDP)-glucose 6-dehydrogenase) from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length" non-processing UGDH and any form of UGDH obtained from intracellular processing. The term also encompasses naturally occurring variants of UGDH, such as splice variants or allelic variants. The nucleic acid sequence of exemplary human UGDH is set forth in SEQ ID NO: 28. The amino acid sequence of the exemplary protein encoded by human UGDH is shown in SEQ ID NO: 60.

[0072] The terms "sample" and "biological sample" are used interchangeably to refer to any biological sample obtained from an individual, including body fluids, body tissues (e.g., tumor tissue), cells, or other sources. Body fluids include, for example, lymph, serum, whole blood, peripheral blood mononuclear cells, frozen whole blood, plasma (including fresh or frozen), urine, saliva, semen, synovial fluid, and cerebrospinal fluid. Samples also include breast tissue, kidney tissue, colon tissue, brain tissue, muscle tissue, synovial tissue, skin, hair follicles, bone marrow, and tumor tissue. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.

[0073] As used herein, "tissue sample" or "cell sample" means a collection of similar cells obtained from the tissue of a subject or individual. Sources of tissue or cell samples can be solid tissues from fresh, frozen, and / or preserved organs, tissue samples, biopsies, and / or aspirates; any blood component such as blood or plasma; body fluids such as cerebrospinal fluid, amniotic fluid, ascites, or interstitial fluid; cells from any point during the pregnancy or development of a subject. A tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from diseased tissue / organs. A tissue sample may contain compounds that do not naturally mix with natural tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0074] As used herein, "reference sample", "reference cell", "reference tissue", "control sample", "control cell", or "control tissue" refers to a sample, cell, tissue, standard, or level used for comparison purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from healthy and / or non-affected parts (e.g., tissue or cells) of the body of the same subject or individual. For example, healthy and / or non-affected cells or tissues adjacent to diseased cells or tissues (e.g., cells or tissues adjacent to a tumor). In another embodiment, the reference sample is obtained from untreated tissue and / or cells of the body of the same subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from healthy and / or non-affected parts (e.g., tissue or cells) of the body of an individual who is not the subject or individual. In still another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from untreated tissue and / or cells of the body of an individual who is not the subject or individual. In another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from one or more cell lines (e.g., one or more normal cell lines).

[0075] As used herein, the terms "identifying a patient" or "identifying the patient" refer to using information or data generated with respect to at least one level of a gene set forth in Table 1, the presence of NRF2 mRNA having a deletion of all or part of exon 2 or exon 2+3 thereof, or the presence of NRF2 protein having a deletion of all or part of Neh2 or Neh2+4 thereof in a patient sample, to identify or select a patient as being likely or unlikely to benefit from a therapy comprising an NRF2 pathway antagonist. The information or data used or generated can be in any form, written, oral, or electronic. In some embodiments, the use of the information or data generated includes communication, presentation, reporting, storage, transmission, transfer, supply, dissemination, distribution, or combinations thereof. In some embodiments, the communication, presentation, reporting, storage, transmission, transfer, supply, dissemination, distribution, or combinations thereof are performed by a computer device, an analyzer unit, or combinations thereof. In some further embodiments, the communication, presentation, reporting, storage, transmission, transfer, supply, dissemination, distribution, or combinations thereof are performed by an experimenter or medical practitioner. In some embodiments, the information or data includes a comparison of at least one level of a gene set forth in Table 1 to a reference level. In some embodiments, the information or data includes an indication that at least one of the genes set forth in Table 1 is present or absent in the sample. In some embodiments, the information or data includes an indication that the NRF2 mRNA has a deletion of all or part of exon 2 or exon 2+3 thereof. In some embodiments, the information or data includes an indication that the NRF2 protein has a deletion of all or part of Neh2 or Neh2+4 thereof. In some embodiments, the information or data includes an indication that the patient is likely or unlikely to respond to a therapy comprising an NRF2 pathway antagonist.

[0076] The term "primer" generally refers to a single-stranded polynucleotide that can hybridize to a nucleic acid and enable polymerization of a complementary nucleic acid by providing a free 3'-OH group.

[0077] As used herein, the term "treatment" (and variations thereof such as "treating" or "treatment") refers to a clinical intervention aimed at altering the natural course of an individual being treated and can be performed for prophylaxis or during the course of a clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

[0078] As used herein, "administering" means a method of providing a dosage of a compound (e.g., an NRF2 pathway antagonist) to a subject. The compositions used in the methods described herein can be administered, for example, intravitreally (e.g., by intravitreal injection), by eye drops, intramuscularly, intravenously, intradermally, transdermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrathoracically, intratracheally, intrathecally, intranasally, intravaginally, rectally, topically, intratumorally, intraperitoneally, subcutaneously, subconjunctivally, intracellularly, mucosally, epicardially, intraumbilically, intraocularly, intraorbitally, orally, topically, transdermally, by inhalation, by injection, by transplantation, by infusion, by continuous infusion, by topical perfusion directly into target cells, by catheter, by lavage, in a cream, or in a lipid composition. The compositions utilized in the methods described herein can also be administered systemically or locally. The method of administration can vary depending on various factors, such as the compound or composition being administered and the severity of the condition, disease, or disorder being treated.

[0079] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective to achieve a desired therapeutic or prophylactic result over a required period of time at a required dosage.

[0080] As used herein, the term "antibody" is used in the broadest sense and includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, encompassing various antibody structures as long as they exhibit the desired antigen-binding activity.

[0081] The "percent amino acid sequence identity (%)" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity, introducing gaps if necessary, and without considering any conservative substitutions as part of the sequence identity. Alignments for determining percent amino acid sequence identity can be achieved using various methods within the scope of the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR). One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes herein, the percent amino acid sequence identity values are generated using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison computer program was described by Genentech, Inc., and the source code was submitted to the U.S. Copyright Office (Washington D.C., 20559) along with user documentation and is registered under U.S. Copyright No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0082] In the situation where ALIGN-2 is used for amino acid sequence comparison, the percentage of amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, can be expressed as a given amino acid sequence A having, or including, a specific percentage of amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 × fraction X / Y Wherein, X is the number of amino acid residues scored as exact matches in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, it is understood that the percentage of amino acid sequence identity of A to B is not equal to the percentage of amino acid sequence identity of B to A. Unless specifically indicated otherwise, all amino acid sequence identity % values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0083] The term "antineoplastic agent" refers to a composition useful in cancer treatment that includes at least one active therapeutic agent, such as an "anticancer agent". Examples of therapeutic agents (anticancer agents) include, for example, chemotherapeutic agents, growth inhibitors, cytotoxic agents, agents used in radiation therapy, anti-angiogenic agents, apoptosis agents, anti-tubulin agents, and other agents for treating cancer, such as anti-HER-2 antibodies, anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (TARCEVA™)), platelet-derived growth factor inhibitors (e.g., GLEEVEC™ (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-β, BlyS, APRIL, BCMA, or VEGF receptor(s), TRAIL / Apo2, and other bioactive and organic chemical agents, etc., but are not limited thereto. Combinations of these are also included in the present invention.

[0084] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or blocks cellular function and / or causes cell death or destruction. Cytotoxic agents include radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioisotopes of Lu); chemotherapeutic agents or chemotherapeutic drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; enzymes such as nuclease and their fragments, antibiotics, fragments and / or variants thereof, toxins such as small molecule toxins or enzyme active toxins derived from bacteria, fungi, plants, or animals, and various antitumor or anticancer agents disclosed below, but are not limited thereto.

[0085] "Chemotherapeutic agent" is a chemical compound useful for the treatment of cancer. Examples of "chemotherapeutic agents" are chemical compounds useful for the treatment of cancer. Examples of chemotherapeutic agents include, for example, alkylating agents such as temozolomide (TMZ), an imidazotetrazine derivative of the alkylating agent dacarbazine. Further examples of chemotherapeutic agents include, for example, paclitaxel or topotecan or pegylated liposomal doxorubicin (PLD). Other examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, and uredopa; ethyleneimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin; bryostatin; calistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); erythrobins; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1 (see, for example, Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)) and other antibiotics; dynemicin including dynemicin A;Bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), actinomycin, actinomycin, aclacinomycin, azaserine, bleomycin, calicheamicin, carabicin, caminomycin, cardinophilin, chromomycin, daunorubicin, daunomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, queramycin, rhodomycin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimethoprim; purine analogs such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, drostanolone propionate, epithioestanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; dexamethasone; diacodone; elfomithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;Pirarubicin; Losoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Razoxane; Rizoxin; Sizofiran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2’,2’’-Trichlorotriethylamine; Trichothecene (especially, T-2 toxin, Verrucarin A, Lolitrem A, and Anguizine); Urethane; Vinblastine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacitabine; Arabinoside (“Ara-C”); Cyclophosphamide; Thiotepa; Taxoids, e.g., TAXOL (registered trademark) paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE (registered trademark) Cremophor-free albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE (registered trademark) docetaxel (Rhone-Poulenc Rorer, Antony, France); Chlorambucil; GEMZAR (registered trademark) gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs such as cisplatin, oxaliplatin, and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine; NAVELBINE (registered trademark) vinorelbine; Novantrone; Teniposide; Edatrexate; Daunomycin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11) (including a treatment regimen of irinotecan with 5-FU and leucovorin); Topoisomerase inhibitor RFS2000; Difluoromethylornithine (DMFO); Retinoids such as retinoic acid; Capecitabine; Combretastatin; Leucovorin (LV); Oxaliplatin including an oxaliplatin treatment regimen (FOLFOX); Lapatinib (Tykerb.RTM.);Inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva.RTM.)), and VEGF that reduce cell proliferation, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing are included.;

[0086] The terms "programmed death ligand 1" and "PD-L1" as used herein refer to the native sequence PD-L1 polypeptide, polypeptide variants, and fragments of the native sequence polypeptide and polypeptide variants. The PD-L1 polypeptides described herein can be isolated from a variety of sources such as human tissue types or other sources, or prepared by recombinant or synthetic methods.

[0087] The term "PD-L1 axis-binding antagonist" refers to a molecule that inhibits the interaction of a PD-L1 axis-binding partner with one or more of its binding partners so as to remove T cell dysfunction resulting from signal transduction on the PD-1 signaling axis, and as a result, T cell function is restored or enhanced. As used herein, PD-L1 axis-binding antagonists include PD-L1 binding antagonists and PD-1 binding antagonists, and molecules that interfere with the interaction between PD-L1 and PD-1 (e.g., PD-L2-Fc fusions).

[0088] As used herein, a "PD-L1 binding antagonist" is a molecule that reduces, blocks, inhibits, suppresses, or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners such as PD-1 and / or B7-1. In some embodiments, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific embodiment, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, a PD-L1 binding antagonist includes anti-PD-L1 antibodies and antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, small molecule antagonists, polynucleotide antagonists, and other molecules that reduce, block, inhibit, suppress, or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners such as PD-1 and / or B7-1. In one embodiment, a PD-L1 binding antagonist reduces negative signals mediated by cell surface proteins expressed on T lymphocytes and other cells, either by or through signal transduction mediated by PD-L1 or PD-1, thereby reducing the dysfunctional state of dysfunctional T cells. In some embodiments, a PD-L1 binding antagonist is an anti-PD-L1 antibody. In a specific embodiment, the anti-PD-L1 antibody is YW243.55.S70. In another specific embodiment, the anti-PD-L1 antibody is MDX-1105. In yet another specific embodiment, the anti-PD-L1 antibody is atezolizumab (MPDL3280A). In yet another specific embodiment, the anti-PD-L1 antibody is MEDI4736 (durvalumab). In yet another specific embodiment, the anti-PD-L1 antibody is MSB0010718C (avelumab).

[0089] As used herein, a "PD-1 binding antagonist" is a molecule that reduces, blocks, inhibits, suppresses, or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners such as PD-L1 and / or PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its binding partner. In a specific embodiment, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies and antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, small molecule antagonists, polynucleotide antagonists, and other molecules that reduce, block, inhibit, suppress, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, a PD-1 binding antagonist reduces negative signals mediated by signal transduction via PD-1 or PD-L1 and mediated by or through cell surface proteins expressed on T lymphocytes and other cells, thereby reducing the dysfunctional state of dysfunctional T cells. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a specific embodiment, the PD-1 binding antagonist is MDX-1106 (nivolumab). In another specific embodiment, the PD-1 binding antagonist is MK-3475 (pembrolizumab). In another specific embodiment, the PD-1 binding antagonist is CT-011 (pidilizumab). In another specific embodiment, the PD-1 binding antagonist is MEDI-0680 (AMP-514). In another specific embodiment, the PD-1 binding antagonist is PDR001. In another specific embodiment, the PD-1 binding antagonist is REGN2810 described herein. In another specific embodiment, the PD-1 binding antagonist is BGB-108 described herein. In another specific embodiment, the PD-1 binding antagonist is AMP-224.

[0090] The term "vascular endothelial growth factor" or "VEGF" refers to vascular endothelial growth factor. The term "VEGF" encompasses its homologs and isoforms. The term "VEGF" also includes known isoforms of VEGF, such as splice isoforms, such as VEGF 111 VEGF 121 VEGF 145 VEGF 165 VEGF 189 and VEGF 206 and the 110 - amino - acid human vascular endothelial cell growth factor generated by plasmin cleavage of VEGF 165 described in Ferrara Mol.Biol.Cell.21:687(2010), Leung et al., Science,246:1306(1989), and Houck et al., Mol.Endocrin.,5:1806(1991), including these naturally - occurring alleles and processing forms. The term "VEGF" also refers to VEGF derived from non - human species such as mouse, rat, or primate. VEGF derived from a specific species may be denoted by terms such as hVEGF for human VEGF, mVEGF for mouse VEGF, etc. The term "VEGF" is also used to refer to cleavage forms of the polypeptide that include amino acids 8 - 109 or 1 - 109 of the 165 - amino - acid human vascular endothelial cell growth factor. Any reference to such a form of VEGF in this application is, for example, "VEGF 109 ", "VEGF(8 - 109)", "VEGF(1 - 109)", or "VEGF 165It can be identified by "". The amino acid positions of "cleaved" native VEGF are numbered as shown in the native VEGF sequence. For example, amino acid position 17 (methionine) in cleaved native VEGF is also position 17 (methionine) in native VEGF. Cleaved native VEGF has a binding affinity comparable to that of native VEGF for the KDR and Flt-1 receptors. As used herein, the term "VEGF variant" refers to a VEGF polypeptide that contains one or more amino acid mutations in the native VEGF sequence. Optionally, the one or more amino acid mutations include amino acid substitutions (plural possible). For the purpose of concisely denoting the VEGF variants described herein, numbers are known to refer to the positions of amino acid residues along the deduced native VEGF amino acid sequence (provided in Leung et al., supra and Houck et al., supra).

[0091] As used herein, the term "VEGF antagonist" refers to a molecule that can bind to VEGF, reduce VEGF expression levels, or neutralize, block, inhibit, suppress, reduce, or interfere with VEGF biological activities including, but not limited to, VEGF binding to one or more VEGF receptors, VEGF signal transduction, and VEGF-mediated angiogenesis and endothelial cell survival or proliferation. For example, a molecule that can neutralize, block, inhibit, suppress, reduce, or interfere with the biological activity of VEGF can exert its action by binding to one or more VEGF receptors (VEGFRs) (e.g., VEGFR1, VEGFR2, VEGFR3, membrane-bound VEGF receptor (mbVEGFR), or soluble VEGF receptor (sVEGFR)). A polypeptide that specifically binds to VEGF, an anti-VEGF antibody and its antigen-binding fragment, a receptor molecule and derivative that specifically binds to VEGF and thereby blocks its binding to one or more receptors, a fusion protein (e.g., VEGF-Trap (Regeneron)), and VEGF 121- Geronin (Peregrine) is included as a VEGF antagonist useful in the methods of the present invention. VEGF antagonists include antagonist variants of the VEGF polypeptide, antisense nucleic acid base oligomers complementary to at least one fragment of a nucleic acid molecule encoding the VEGF polypeptide, small RNAs complementary to at least one fragment of a nucleic acid molecule encoding the VEGF polypeptide, ribozymes targeting VEGF, peptibodies against VEGF, and VEGF aptamers. VEGF antagonists also include polypeptides that bind to VEGFR, anti-VEGFR antibodies, and antigen-binding fragments thereof, as well as derivatives or fusion proteins that bind to VEGFR and thereby block, inhibit, suppress, reduce, or interfere with the biological activity of VEGF (e.g., signal transduction of VEGF). VEGF antagonists include non-peptide small molecules that bind to VEGF or VEGFR and can block, inhibit, suppress, reduce, or interfere with the biological activity of VEGF. Thus, the term "VEGF activity" specifically includes the VEGF-mediated biological activity of VEGF. In certain embodiments, the VEGF antagonist reduces or inhibits the expression level or biological activity of VEGF by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, the VEGF inhibited by the VEGF-specific antagonist is VEGF(8-109), VEGF(1-109), or VEGF 165 is.

[0092] As used herein, VEGF antagonists include anti-VEGFR2 antibodies and related molecules (e.g., ramucirumab, tanibirumab, aflibercept), anti-VEGFR1 antibodies and related molecules (e.g., icrucumab, aflibercept (VEGF Trap-Eye, EYLEA®), and ziv-aflibercept (VEGF Trap, ZALTRAP®)), bispecific VEGF antibodies (e.g., MP-0250, vanucizumab (VEGF-ANG2), and the bispecific antibodies disclosed in US2001 / 0236388), bispecific antibodies comprising a combination of two of the anti-VEGF, anti-VEGFR1, and anti-VEGFR2 arms, anti-VEGF antibodies (e.g., bevacizumab, sevastizumab, and ranibizumab), and non-peptide small molecule VEGF antagonists (e.g., pazopanib, axitinib, vandetanib, sunitinib, cabozantinib, lenvatinib, nintedanib, orantinib, teratinib, dovitinib, cediranib, motesanib, sulfatinib, apatinib, foretinib, famitinib, and thiazovivin), but are not limited thereto.

[0093] The terms "anti-VEGF antibody", "antibody that binds VEGF", and "antibody that specifically binds VEGF" refer to an antibody that can bind VEGF with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeting VEGF. In one embodiment, the degree of binding of the anti-VEGF antibody to an irrelevant non-VEGF protein is less than about 10% of the binding of the antibody to VEGF, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds VEGF has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M). In certain embodiments, the anti-VEGF antibody binds to an epitope of VEGF that is conserved among VEGFs from different species.

[0094] In certain embodiments, the anti-VEGF antibody can be used as a therapeutic agent in targeting and interfering with a disease or condition in which VEGF activity is involved. Also, the antibody can be subjected to other biological activity assays, for example, to evaluate its effectiveness as a therapeutic agent. Such assays are known in the art and depend on the target antigen of the antibody and its intended use. Examples include HUVEC inhibition assays, tumor cell growth inhibition assays (e.g., those described in WO89 / 06692), antibody-dependent cell cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays (U.S. Patent No. 5,500,362), and agonist activity or hematopoietic assays (see WO95 / 27062). The anti-VEGF antibody typically does not bind to other VEGF homologs such as VEGF-B or VEGF-C, nor to other growth factors such as PIGF, PDGF, or bFGF. In one embodiment, the anti-VEGF antibody is a monoclonal antibody that binds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB10709. In another embodiment, the anti-VEGF antibody is a recombinant humanized anti-VEGF monoclonal antibody produced according to Presta et al. (1997) Cancer Res. 57:4593-4599, and examples include, but are not limited to, the antibody known as bevacizumab (BV, AVASTIN®).

[0095] The anti-VEGF antibody "ranibizumab", also known as "Lucentis®" or "rhuFab V2", is a humanized affinity mature anti-human VEGF Fab fragment. Ranibizumab is produced by standard recombinant technology methods in an Escherichia coli expression vector and by bacterial fermentation. Ranibizumab is not glycosylated and has a molecular weight of approximately 48,000 daltons. See WO98 / 45331 and US2003 / 0190317. Further preferred antibodies include the G6 or B20 series antibodies (e.g., G6-31, B20-4.1) described in PCT Publication Nos. WO2005 / 012359 and WO2005 / 044853, which are hereby incorporated by reference in their entireties. For further preferred antibodies, see U.S. Pat. Nos. 7,060,269, 6,582,959, 6,703,020, 6,054,297, WO98 / 45332, WO96 / 30046, WO94 / 10202, EP0666868B1, U.S. Patent Application Publication Nos. 2006 / 009360, 2005 / 0186208, 2003 / 0206899, 2003 / 0190317, 2003 / 0203409, and 2005 / 0112126, and Popkov et al., Journal of Immunological Methods 288:149-164 (2004). Other preferred antibodies include those that bind to functional epitopes on human VEGF that include the residues F17, M18, D19, Y21, Y25, Q89, 191, K101, E103, and C104, or alternatively the residues F17, Y21, Q22, Y25, D63, 183, and Q89. Further anti-VEGF antibodies include the anti-VEGF antibodies described in PCT Publication No. WO2009 / 155724.

[0096] The term "co-administration" is used herein to refer to the administration of two or more therapeutic agents where at least a portion of the administrations overlap in time. Thus, co-administration includes dosing regimens where the administration of one or more other agent(s) continues after the administration of one or more agent(s) has been discontinued.

[0097] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, and all pre-cancerous and cancerous cells and tissues, whether malignant or benign. "Cancer", "cancerous", "cell proliferative disorder", "proliferative disorder", and "tumor" are not mutually exclusive as referred to herein.

[0098] III. Methods A. Diagnostic Methods Provided herein are methods for diagnosing cancer (e.g., lung cancer (e.g., squamous NSCLC or non-squamous NSCLC) or head and neck cancer (e.g., HNSC)) in a subject. Also provided herein are methods for identifying a subject having a cancer that is NRF2-dependent (e.g., lung cancer, e.g., squamous non-small cell lung cancer or non-squamous non-small cell lung cancer, or head and neck cancer). Any of the methods can be obtained based on the expression level of a biomarker provided herein, e.g., a splice variant of NRF2 (e.g., NRF2 mRNA or NRF2 protein), or increased expression of one or more NRF2 target genes. Any of the methods can further comprise administering an NRF2 pathway antagonist to the subject. Any of the methods can further comprise administering to the subject an effective amount of a second therapeutic agent (e.g., one or more (e.g., 1, 2, 3, or 4 or more) additional NRF2 pathway antagonists or one or more (e.g., 1, 2, 3, or 4 or more) anti-cancer agents).

[0099] The present invention provides a method for diagnosing cancer in a subject, the method comprising determining the expression level of at least one gene selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 genes) in a sample obtained from the subject, and comparing the expression level of the at least one gene with a reference expression level of the at least one gene, wherein an increase in the expression level of the at least one gene in the sample relative to the reference expression level of the at least one gene identifies a subject having cancer.

[0100] The present invention further provides a method for identifying a subject having cancer that is an NRF2-dependent cancer, the method comprising determining the expression level of at least one gene selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 genes) in a sample obtained from the subject, comparing the expression level of the at least one gene with a reference expression level of the at least one gene, and determining whether the cancer of the subject is an NRF2-dependent cancer, wherein an increase in the expression level of the at least one gene in the sample relative to the reference expression level of the at least one gene identifies a subject having an NRF2-dependent cancer.

[0101] In any of the foregoing methods, the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21) of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, or NQO1 is determined.

[0102] In any of the foregoing methods, the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) newly identified NRF2 target genes is determined. Newly identified NRF2 target genes include AKR1B10, AKR1C2, ME1, KYNU, CABYR, TRIM16L, AKR1C4, CYP4F11, RSPO3, AKR1B15, NR0B1, and AKR1C3.

[0103] The present invention further provides a method for diagnosing cancer in a subject, the method comprising determining the mRNA expression level of NRF2 comprising deletion of all or part of its exon 2 in a sample obtained from the subject (e.g., a tumor sample), and the presence of NRF2 comprising deletion of all or part of its exon 2 identifies the subject as having cancer. In some embodiments, NRF2 further comprises deletion of all or part of its exon 3. The presence and / or expression level of a gene (e.g., NRF2, KEAP1, AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, or FTL), among others, can be determined qualitatively or quantitatively based on any suitable criteria known in the art, including but not limited to DNA, mRNA, cDNA, protein fragments, and / or gene copy number.

[0104] The present invention further provides a method for diagnosing cancer in a subject, the method comprising determining the protein expression level of NRF2 comprising deletion of all or part of its Neh2 domain in a sample obtained from the subject, and the presence of NRF2 comprising deletion of all or part of its Neh2 domain identifies the subject as having cancer. In some embodiments, NRF2 further comprises deletion of all or part of its Neh4 domain.

[0105] The present invention further provides a method for identifying a subject having cancer, the method comprising determining the mRNA expression level of NRF2 comprising deletion of all or part of its exon 2 in a sample (e.g., a tumor sample) obtained from the subject, and the presence of NRF2 comprising deletion of all or part of its exon 2 identifies the subject as having cancer. In some embodiments, NRF2 further comprises deletion of all or part of its exon 3. The presence and / or expression level of a gene (e.g., NRF2, KEAP1, AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, or FTL), including but not limited to, can be determined qualitatively or quantitatively based on any suitable criteria known in the art, including DNA, mRNA, cDNA, protein fragments, and / or gene copy number.

[0106] The present invention further provides a method for identifying a subject having cancer, the method comprising determining the protein expression level of NRF2 comprising deletion of all or part of its Neh2 domain in a sample obtained from the subject, and the presence of NRF2 comprising deletion of all or part of its Neh2 domain identifies the subject as having cancer. In some embodiments, NRF2 further comprises deletion of all or part of its Neh4 domain.

[0107] The presence and / or expression level / amount of the various biomarkers described herein in a sample can be analyzed by several methodologies, many of which are known in the art and understood by those of ordinary skill in the art, including immunohistochemistry (“IHC”), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence-activated cell sorting (“FACS”), MassARRAY, proteomics, quantitative blood-based assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization, fluorescence in situ hybridization (FISH), Southern analysis, Northern analysis, whole genome sequencing, massively parallel DNA sequencing (e.g., next generation sequencing), NANOSTRING® including, for example, branched DNA, SISBA, TMA, polymerase chain reaction (PCR) including quantitative real-time PCR (qRT-PCR) and other amplification-based detection methods, RNA-Seq, microarray analysis, gene expression profiling, and / or serial analysis of gene expression (“SAGE”), and / or any one of a variety of assays that can be performed by protein, gene, and / or tissue array analysis, but are not limited thereto. Typical protocols for assessing the status of genes and gene products can be found, for example, in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern blotting), 4 (Southern blotting), 15 (immunoblotting), and 18 (PCR analysis). Multiplex immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery (“MSD”) can also be used.

[0108] In some embodiments of any of the methods described herein, DNA from a clinical tumor sample can be sequenced using next-generation sequencing methods, such as the targeted gene pull-down sequencing method described in Frampton et al. (Nature Biotechnology. 31(11):1023-1033, 2013), which is incorporated herein by reference in its entirety. Such next-generation sequencing methods can be used in conjunction with any of the methods disclosed herein that enable the detection of various mutations (e.g., insertions, deletions, base substitutions, focal gene amplifications, and / or homozygous gene deletions) while allowing the use of small samples (e.g., from a small core needle biopsy, fine needle aspiration, and / or cell block) or fixed samples (e.g., formalin-fixed paraffin-embedded (FFPE) samples).

[0109] In any of the foregoing methods, the presence and / or expression level / amount of a biomarker (e.g., NRF2, KEAP1, AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, or FTL) is measured by determining the protein expression level of the biomarker. In certain embodiments, the method comprises contacting a biological sample with an antibody (e.g., an anti-NRF2 antibody) that specifically binds to the biomarker under conditions that permit binding of the biomarker, and detecting whether a complex is formed between the antibody and the biomarker. Such methods can be in vitro or in vivo methods. Any method known in the art or described herein for measuring protein expression levels may be used. For example, in some embodiments, the protein expression level of the biomarker is determined using a method selected from the group consisting of flow cytometry (e.g., fluorescence-activated cell sorting (FACS (trademark))), Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunohistochemistry (IHC), immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, and HPLC. In some embodiments, the protein expression level of the biomarker is determined in tumor cells.

[0110] In some embodiments, the presence and / or expression level / amount of a biomarker (e.g., NRF2, KEAP1, AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, or FTL) is measured by determining the mRNA expression level of the biomarker. In certain embodiments, the presence and / or expression level / amount of a gene is determined using a method comprising: (a) performing gene expression profiling, PCR (such as RT-PCR), RNA-seq, microarray analysis, SAGE, MassARRAY technology, or FISH on a sample (such as a subject cancer sample); and (b) determining the presence and / or expression level / amount of the biomarker in the sample. In one embodiment, the PCR method is qRT-PCR. In one embodiment, the PCR method is multiplex PCR. In some embodiments, gene expression is measured by microarray. In some embodiments, gene expression is measured by qRT-PCR. In some embodiments, expression is measured by multiplex PCR.

[0111] Methods for the evaluation of mRNA in cells are well known and include, for example, hybridization assays using complementary DNA probes (in situ hybridization using labeled riboprobes specific for one or more genes, Northern blot, and related techniques, etc.), and various nucleic acid amplification assays (RT-PCR using complementary primers specific for one or more of the genes, and other amplification-based detection methods, such as branched DNA, SISBA, TMA, etc.). Samples derived from mammals can be conveniently assayed for mRNA using Northern, dot blot, or PCR analysis. In addition, such methods can include one or more steps that enable determination of the level of target mRNA in a biological sample (e.g., by simultaneously examining the level of a comparative control mRNA sequence of a "housekeeping" gene such as an actin family member).

[0112] In some embodiments of any of the methods, the biomarker is NRF2 (e.g., exon 2 deletion NRF2 or exon 2+3 deletion NRF2). In one embodiment, the expression level of the biomarker is determined using a method that includes performing WGS analysis on a sample (such as a tumor sample obtained from a patient) and determining the expression level of the biomarker in the sample. In some embodiments, exon 2 deletion NRF2 or exon 2+3 deletion NRF2 is determined relative to a reference. In some embodiments, the reference is a reference value. In some embodiments, the reference is a reference sample (e.g., a control cell line sample, a tissue sample from a non-cancer patient, or a wild-type NRF2 tissue sample).

[0113] In addition to or alternatively to mRNA expression analysis, other biomarkers such as protein expression can be quantified according to the methods described above. For example, the methods of the present invention involve testing a sample for genomic biomarkers (e.g., the presence of exon 2 deletion NRF2 or exon 2+3 deletion NRF2, or upregulation of one or more NRF2 target genes, such as AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, or FTL), and additionally testing the sample for protein biomarkers (e.g., one or more protein transcripts of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, or FTL).

[0114] In some embodiments of any of the methods, a DNA sequence can serve as a biomarker. The DNA can be quantified according to any method known in the art, including but not limited to PCR, exome-seq (e.g., whole exome sequencing), DNA microarray analysis, NANOSTRING® technology, or whole genome sequencing.

[0115] In some cases, the expression level of a gene in a sample is the average of the gene (e.g., average expression or median expression), the reference expression level of the gene is the average of the reference gene (e.g., average expression or median expression), and the average of the gene in the sample is compared to the average of the reference gene.

[0116] In certain embodiments, the presence and / or expression level / amount of a biomarker in a first sample is increased or elevated compared to the presence / absence and / or expression level / amount in a second sample. In certain embodiments, the presence / absence and / or expression level / amount of a biomarker in a first sample is decreased or reduced compared to the presence and / or expression level / amount in a second sample. In certain embodiments, the second sample is a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. Further disclosure for determining the presence / absence and / or expression level / amount of a gene is described herein.

[0117] In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or a combined plurality of samples from the same subject or individual obtained at one or more time points different from when the test sample was obtained. For example, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from the same subject or individual at a time point earlier than when the test sample was obtained. Such reference samples, reference cells, reference tissues, control samples, control cells, or control tissues can be useful when the reference sample is obtained during an initial cancer diagnosis and the test sample is obtained later when the cancer has become metastatic.

[0118] In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined plurality of samples from one or more healthy individuals who are not patients. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined plurality of samples from one or more individuals who have a disease or disorder (e.g., cancer) and are not the subject or individual. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from normal tissue, or a pooled plasma or serum sample from one or more individuals who are not patients. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from tumor tissue, or a pooled plasma or serum sample from one or more individuals who are not patients and have a disease or disorder (e.g., cancer).

[0119] In some embodiments of any of the methods, increased or elevated expression refers to an overall increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the level of a biomarker (e.g., a protein or nucleic acid (e.g., a gene (DNA or mRNA)) as detected by methods known in the art of the standard such as those described herein, compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, elevated expression refers to an increase in the expression level / amount of a biomarker in a sample, where the increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold the expression level / amount of the respective biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, elevated expression refers to an overall increase of greater than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold compared to a reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., a housekeeping gene).

[0120] In some embodiments of any of the methods, reduced expression refers to a global reduction of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the level of a biomarker (e.g., a protein or nucleic acid (e.g., a gene (DNA or mRNA))), detected by methods known in the art of the standard such as those described herein, compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, reduced expression refers to a decrease in the expression level / amount of a biomarker in a sample, and the decrease is at least about 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, 0.1-fold, 0.05-fold, or 0.01-fold of the expression level / amount of the respective biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

[0121] B. Treatment methods The present invention provides a method for treating a patient suffering from cancer (e.g., lung cancer (e.g., squamous NSCLC or non-squamous NSCLC) or head and neck cancer (e.g., HNSC)). In some cases, the method of the present invention comprises administering to the patient an effective amount of an NRF2 pathway antagonist. Any of the NRF2 pathway antagonists described herein or otherwise known in the art can be used in the method. In some cases, the method comprises determining the presence and / or expression level of an NRF2 splice variant (e.g., exon 2-deleted NRF2 or exon 2+3-deleted NRF2) or an NRF2 target gene in a sample obtained from the patient, and administering to the patient an NRF2 pathway antagonist based on the presence and / or expression level of the NRF2 splice variant (e.g., exon 2-deleted NRF2 or exon 2+3-deleted NRF2) or the NRF2 target gene, using, for example, any of the methods described in the following examples herein or known in the art.

[0122] The present invention provides a method for treating a subject suffering from cancer (e.g., lung cancer (e.g., squamous NSCLC or non-squamous NSCLC) or head and neck cancer (e.g., HNSC)), the method comprising determining the expression level of at least one gene selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 genes) in a sample obtained from the subject, and comparing the expression level of the at least one gene with a reference expression level of the at least one gene, wherein an increase in the expression level of the at least one gene in the sample relative to the reference expression level of the at least one gene identifies a subject having cancer, and administering to the subject a therapeutically effective amount of one or more NRF2 pathway antagonists.

[0123] The present invention further provides a method for treating a subject suffering from cancer (e.g., lung cancer (e.g., squamous NSCLC or non-squamous carcinoma NSCLC) or head and neck cancer), wherein the expression level of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) newly identified NRF2 target genes is determined. Newly identified NRF2 target genes include AKR1B10, AKR1C2, ME1, KYNU, CABYR, TRIM16L, AKR1C4, CYP4F11, RSPO3, AKR1B15, NR0B1, and AKR1C3.

[0124] In some cases, the present invention provides a method of treating a subject having cancer (e.g., lung cancer (e.g., squamous NSCLC or non-squamous carcinoma NSCLC) or head and neck cancer), wherein the mRNA expression level of NRF2 comprises a deletion of all or part of exon 2 thereof in a sample obtained from the subject, and the presence of NRF2 comprising a deletion of all or part of exon 2 thereof identifies the subject as having cancer, and further provides a method of administering to the subject a therapeutically effective amount of one or more NRF2 pathway antagonists. In some embodiments, NRF2 further comprises a deletion of all or part of exon 3 thereof.

[0125] In some cases, the present invention provides a method of treating a subject having cancer (e.g., lung cancer (e.g., squamous NSCLC or non-squamous carcinoma NSCLC) or head and neck cancer (e.g., HNSC)), wherein the NRF2 protein comprises a deletion of all or part of its Neh2 domain in a sample obtained from the subject, and the presence of NRF2 comprising a deletion of all or part of its Neh2 domain identifies the subject as having cancer, and further provides a method of administering to the subject a therapeutically effective amount of one or more NRF2 pathway antagonists. In some embodiments, NRF2 further comprises a deletion of all or part of its Neh4 domain.

[0126] In any of the aforementioned methods, the NRF2 pathway antagonist can be any NRF2 pathway antagonist known in the art or described herein.

[0127] In some cases, the method further comprises administering to the subject an effective amount of a second therapeutic agent (e.g., one or more anti-cancer agents). In some cases, the second therapeutic agent is selected from the group consisting of angiogenesis inhibitors, chemotherapeutic agents, growth inhibitors, cytotoxic agents, immunotherapies, and combinations thereof. In some embodiments, the immunotherapy is a VEGF antagonist (e.g., anti-VEGFR2 antibodies and related molecules (e.g., ramucirumab, tanibirumab, aflibercept), anti-VEGFR1 antibodies and related molecules (e.g., icrucumab, aflibercept (VEGF Trap-Eye, EYLEA®), and ziv-aflibercept (VEGF Trap, ZALTRAP®)), bispecific VEGF antibodies (e.g., MP-0250, vanucizumab (VEGF-ANG2), and bispecific antibodies disclosed in US2001 / 0236388), bispecific antibodies comprising a combination of two of the anti-VEGF, anti-VEGFR1, and anti-VEGFR2 arms, anti-VEGF antibodies (e.g., bevacizumab, sevastizumab, and ranibizumab), and non-peptide small molecule VEGF antagonists (e.g., pazopanib, axitinib, vandetanib, sunitinib, cabozantinib, lenvatinib, nintedanib, orantinib, teratinib, dovitinib, cediranib, motesanib, sulfatinib, apatinib, foretinib, famitinib, and tivozanib). In other embodiments, the immunotherapy is a PD-1 axis-binding antagonist (e.g., YW243.55.S70, MDX-1105, MPDL3280A (atezolizumab), MEDI4736 (durvalumab), MSB0010718C (avelumab), MDX-1106 (nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, BGB-108, or AMP-224).

[0128] The compositions (e.g., NRF2 pathway antagonists) used in the methods described herein can be administered by any suitable method, including, for example, intravenous, intramuscular, subcutaneous, intradermal, transdermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intrathecal, intranasal, intravaginal, intrarectal, topical, intratumoral, peritoneal, subconjunctival, intracystic, mucosal, epicardial, intraumbilical, intraocular, intraorbital, oral, topical, transdermal, intravitreal (e.g., by intravitreal injection), by eye drops, by inhalation, by injection, by transplantation, by infusion, by continuous infusion, by topical perfusion directly immersing target cells, by catheter, by lavage, in creams, or in lipid compositions. The compositions utilized in the methods described herein can also be administered systemically or topically. The method of administration can vary depending on various factors, such as the compound or composition being administered and the severity of the condition, disease, or disorder being treated. In some embodiments, the NRF2 pathway antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by transplantation, by inhalation, intrathecally, intraventricularly, or intranasally. Dosage can be obtained by any suitable route, for example, by injection such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time points, bolus administration, and pulse infusion.

[0129] The NRF2 pathway antagonists (and additional anti-cancer agents) described herein can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the dosing schedule, and other factors known to the medical practitioner. The NRF2 pathway antagonists can, although not necessarily, optionally be formulated and / or administered with one or more agents currently being used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of the Nrd2 pathway inhibitor present in the formulation, the type of disorder or treatment, and the other factors discussed above. These are generally used at the same dosage and route of administration as those described herein, or at about 1 to 99% of the dosage described herein, or at any dosage and any route determined to be empirically / clinically appropriate.

[0130] In some embodiments, the method further comprises administering to the subject an effective amount of a second therapeutic agent (e.g., one or more anti-cancer agents). In some embodiments, the second therapeutic agent is selected from the group consisting of anti-angiogenic agents, chemotherapeutic agents, growth inhibitors, cytotoxic agents, immunotherapies, and combinations thereof.

[0131] Such combination therapies as described above include combined administration (where two or more therapeutic agents (e.g., an NRF2 pathway antagonist and an anti-cancer agent) are included in the same or separate formulations) and separate administrations where the administration of the NRF2 pathway antagonist can occur before, at the same time as, and / or following the administration of the additional anti-cancer agent(s). In one embodiment, the administration of the NRF2 pathway antagonist and the administration of the additional anti-cancer agent occur within about 1 month of each other, or within about 1, 2, or 3 weeks of each other, or within about 1, 2, 3, 4, 5, or 6 days of each other.

[0132] C. NRF2 Pathway Antagonists for Use in the Methods of the Invention Provided herein are methods for treating or delaying the progression of cancer (e.g., lung cancer (e.g., squamous NSCLC) or head and neck cancer) in a subject, including administering to the subject a therapeutically effective amount of an NRF2 pathway antagonist. Any of the foregoing methods can be based on the expression levels of the biomarkers provided herein, e.g., NRF2 expression or the expression of any protein or mRNA involved in the NRF2 pathway in a tumor sample, e.g., a biopsy containing tumor cells.

[0133] In some embodiments, the NRF2 pathway antagonist is a small molecule, e.g., a small molecule that can bind to NRF2 or a protein or gene that controls the expression, stability, or activity of NRF2.

[0134] In some embodiments, the NRF2 pathway antagonist is an antagonist of an NRF2 agonist. Examples of NRF2 agonists include, but are not limited to, cAMP response element-binding protein (CREB), CREB-binding protein (CBP), Maf, activating transcription factor 4 (ATF4), protein kinase C (PKC), Jun, glucocorticoid receptor, UbcM2, and those homologous to the ankyrin repeats containing E6-AP carboxyl terminus and E3 ubiquitin-protein ligase 1 (HACE1). Accordingly, examples of NRF2 pathway antagonists include, but are not limited to, CREB antagonists, CBP antagonists, Maf antagonists, ATF4 antagonists, PKC antagonists, Jun antagonists, glucocorticoid receptor antagonists, UbcM2 antagonists, and HACE1 antagonists, such as those described in Table 2.

[0135] In some embodiments, the NRF2 pathway antagonist is an agonist of the NRF2 antagonist. Examples of NRF2 antagonists include, but are not limited to, c-Myc, SUMO, KEAP1, CUL3, and retinoic acid receptor α (RARα). Thus, examples of NRF2 pathway antagonists include, but are not limited to, c-Myc agonists, SUMO, KEAP1 agonists, CUL3 agonists, and RARα agonists, such as those described in Table 3. TIFF2025081326000002.tif213170TIFF2025081326000003.tif95170

[0136] In some embodiments of the present invention, derivatives of the compounds listed in Table 2 or 3 can also be administered as NRF2 pathway antagonists. Derivatives of the compounds listed in Table 2 or 3 are small molecules that have a different structure from the parent compound but retain the ability to antagonize the NRF2 pathway. Derivatives of a compound can change its interaction with certain other molecules or proteins relative to the parent compound. Derivatives of a compound can also include salts, adducts, or other variants of the parent compound. In some embodiments of the present invention, any derivative of a compound described herein (e.g., any one of the compounds listed in Table 2 or 3) can be used in place of the parent compound. In some embodiments, any derivative of the compounds listed in Table 2 or 3 can be used in a method for treating a subject having cancer, such as lung cancer.

[0137] In some embodiments, the NRF2 pathway antagonist is an antibody (e.g., an anti-NRF2 antibody or an antibody against a protein or gene that controls NRF2 expression, stability, or activity, such as the targets listed in Table 2 or 3). In some embodiments, the anti-NRF2 antibody can inhibit the binding between NRF2 and the antioxidant response element. In some embodiments, the anti-NRF2 antibody can inhibit the binding between NRF2 and a cofactor (e.g., Maf, PKC, Jun, ATF4, or CBP). In some embodiments, the antibodies of the present invention are antibody fragments selected from the group consisting of Fab, Fab’-SH, Fv, scFv, and (Fab’) 2 fragments. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a derivative of a known antibody having any of the above characteristics. Derivatives of the antibody include antibody variants having about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% or less sequence identity to the parent antibody. The percent amino acid sequence identity (%) is determined according to methods known in the art, including those by ALIGN-2 as described above.

[0138] In some embodiments, the NRF2 pathway antagonist is an inhibitor of any downstream biomarker (e.g., a gene or protein, such as a gene or protein involved in iron sequestration (e.g., ferritin, light chain polypeptide (FTL), ferritin, heavy chain polypeptide 1 (FTH), or heme oxygenase 1 (HMOX1)), GSH utilization (e.g., glutathione peroxidase 2 (GPX2), glutathione S-transferase alpha 1 (GSTA1), glutathione S-transferase alpha 2 (GSTA2), glutathione S-transferase alpha 3 (GSTA3), glutathione S-transferase alpha 5 (GSTA5), glutathione S-transferase mu 1 (GSTM1), glutathione S-transferase mu 2 (GSTM2), glutathione S-transferase mu 3 (GSTM3), or glutathione S-transferase pi 1 (GSTP1)), quinone detoxification (e.g., NAD(P)H dehydrogenase, quinone 1 (NQO1)), GSH production and regeneration (e.g., glutamate-cysteine ligase, modifier subunit (GCLM), glutamate-cysteine ligase, catalytic subunit (GCLC), glutathione reductase (GSR), or solute carrier family 7 (anionic amino acid transporter light chain, Xc-system), member 11 (SLC7A11, or XCT)), thioredoxin (TXN) production, regeneration, and utilization (e.g., thioredoxin 1, (TXN1), thioredoxin reductase 1 (TXNRD1), or peroxiredoxin 1 (PRDX1)), NADPH production (e.g., glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (PGD), malic enzyme 1, NADP(+)-dependent cytoplasmic (ME1), isocitrate dehydrogenase 1 (NADP+), soluble (IDH1)), or any of these genes or proteins in Table 1).

[0139] In some embodiments, the NRF2 pathway antagonist comprises a compound that inhibits NRF2 from binding to the antioxidant response element (ARE), for example, by competitively binding to the ARE binding site on NRF2, by competitively binding to the ARE, or otherwise by interfering with transcriptional cofactors (e.g., small Maf proteins).

[0140] In some embodiments, the NRF2 pathway antagonist comprises an agonist or antagonist of an NRF2-related gene such that the pharmacological effect of the compound comprises downregulation of one or more pathways downstream of NRF2-mediated transcription. Such NRF2-related genes include, for example, Kelch-like ECH-associated protein 1 (KEAP1), Ectoderm-Neural Cortex 1 (ENC1) (including the BTB domain), Protein Kinase C delta (PRKCD), Protein Kinase C beta (PRKCB), Polyamine Modulating Factor 1 (PMF1), Cullin 3 (CUL3), Nuclear Factor, Erythroid 2 (NFE2), Activating Transcription Factor 4 (ATF4), Heme Oxygenase 1 (HMOX1), Heme Oxygenase 2 (HMOX2), Ubiquitin C (UBC), V-Maf tendinopathy fibrosarcoma oncogene homolog K (MAFK), UDP Glucuronosyltransferase 1 family, polypeptide A6 (UGT1A6), V-Maf tendinopathy fibrosarcoma oncogene homolog F (MAFF), CREB Binding Protein (CREBBP), V-Maf tendinopathy fibrosarcoma oncogene homolog G (MAFG), cAMP Response Element Binding Protein 1 (CREB1), FXYD Domain-Containing Ion Transport Regulator 2 (FXYD2), Jun Proto-Oncogene (JUN), Small Ubiquitin-like Modifier 2 (SUMO2), Small Ubiquitin-like Modifier 1 (SUMO1), V-Myc avian myelocytomatosis viral oncogene homolog (MYC), Zeta Crystallin (Quinone Reductase) (CRYZ), Aldo-Keto Reductase Family 7, Member A2 (Aflatoxin Aldehyde Reductase) (AKR7A2), and Glutathione S-Transferase Alpha 2 (GSTA2).

[0141] In some embodiments, a method of increasing ubiquitination of NRF2 in a cell is provided, the method comprising contacting the cell with an inhibitor of the NRF2 pathway under conditions that allow inhibition of the NRF2 pathway in the cell. The increased ubiquitination of NRF2 can be determined, for example, by enhancing the immunoaffinity of ubiquitinated NRF2 after trypsin digestion followed by mass spectrometry according to known methods. In some embodiments, the increase in ubiquitination can be determined by comparing the ubiquitination of wild-type NRF2 in a cell or cell population contacted with an NRF2 pathway antagonist with the ubiquitination of exon 2 or exon 2+3 deleted NRF2 in a cell or cell population contacted with an NRF2 pathway antagonist and / or the ubiquitination of exon 2 or exon 2+3 deleted NRF2 in a cell or cell population contacted with an NRF2 pathway antagonist.

[0142] In some embodiments of the invention, the NRF2 pathway antagonist is ascorbic acid, bursatol, luteolin, or ochratoxin A.

Example

[0143] Example 1: Materials and Experimental Methods A. Mutation and Copy Number Analysis For 99 NSCLC cell lines, non-synonymous mutation and copy number data for KRas, LKB1, KEAP1, and NRF2 were obtained from Klijn et al. (Nat Biotechnol. 33(3):306 - 312, 2015). 13 additional NSCLC cell lines were subjected to copy number analysis. In addition, exome sequencing was applied to 104 NSCLC cell lines. For The Cancer Genome Atlas (TCGA), tumor mutation and copy number data were retrieved from cBioPortal using the R software package CGDS-R (Cerami et al. Cancer Discovery. 2:401 - 404, 2012, Gao et al. Sci. Signal. 6:11, 2013).

[0144] B. RNA-seq Analysis and Induction of Mutant KEAP1 Gene Expression Signatures Raw RNA-seq data of 99 NSCLC cell lines were retrieved from the European Genome-phenome Archive (accession number EGAS00001000610) (PMID: 25485619). Mutations of KEAP1 and NRF2 in each of the NSCLC cell lines are provided in Table 4. Raw RNA-seq data were downloaded from TCGA and aligned to the human reference genome (GRCh37 / hg19) using GSNAP version 2013-10-10 (Wu and Nacu. Bioinformatics 26:873-881, 2010), allowing a maximum of two mismatches (parameters: "-M2 -n10 -B2 -i1 -N1 -w200000 -E1 --pairmax-rna=200000"). Gene expression levels were quantified as RPKM (reads per kilobase of target and per million reads sequenced) values derived from the number of reads mapped to each RefSeq gene. The DESeq R package (PMID: 20979621) was used to measure differential gene expression between KEAP1 mutant and KEAP1 wild-type cell lines and reported as fold change and associated adjusted p-values. Ward clustering of samples and genes in the variance (using Euclidean distance) was performed using the stabilized count data. The "NMF" R package was used to create the associated heatmap. TIFF2025081326000004.tif223170

[0145] C. Splice Variant Analysis Analysis of splice variants was performed using the SGSeq software package available from the Bioconductor project website (Gentleman et al. Genome Biol. 5:R80, 2004). Exons and splice junctions were predicted from BAM files for 7,384 TCGA samples at 54 genomic loci of known cancer genes using the parameters α = 2, psi = 0, β = 0.2, γ = 0.2. The predicted features were integrated across samples and processed into exon bins separating exons. Splice junctions and exon bins were combined into a genome-wide splice graph. Splice events consisting of two or more alternative splice variants were identified from the graph. Splice variants were quantified with respect to FPKM and relative usage Ψ. Briefly, local estimates of relative usage at the start and end of a variant were obtained as the fraction of fragments compatible with the variant. Estimates at the start and end of the event were combined using a weighted average, where the weights were proportional to the total number of fragments spanning the boundary. Relative usage estimates with a denominator less than 20 were set to NA. To obtain local estimates of absolute expression at the start and end of a variant, the compatibility count n was converted to FPKM as nI(NxL)x10 9 where N is the total number of aligned fragments and L is the effective length (the number of allowed positions for a compatible fragment). Splice variants detected in TCGA samples were also quantified in 2,958 genotype-tissue expression project (GTEx) samples from normal human tissues (Consortium. Science. 348:648 - 660, 2015).

[0146] D. Identification of cancer-specific splice variants Considering only internal splice variants (not including transcript start or end), the start and end of each splice variant were required to overlap or extend exons belonging to the annotated reference gene transcripts downloaded from the UCSC Genome Browser website (Pruitt et al. Nucleic Acids Res. 33:D501-504, 2005, Rosenbloom et al. Nucleic Acids Res. 43:D670-681, 2015). Retained introns were excluded. Considering TCGA signatures (a total of 6,359 cancer samples) containing at least 100 cancer samples, splice variants were selected that had (i) FPKM > 2 and relative usage Ψ > 0.2 in at least one cancer sample, (ii) FPKM < 1 in >99.9% of GTEx samples, and (iii) FPKM approximately 0 in >97.5% of GTEx samples. The FPKM-based criteria had to be met at both the start and end of the splice variant. Variants that met the FPKM-based criteria for which Ψ could not be estimated were included after manual inspection.

[0147] E. Analysis of targeted paired-end exome-seq data All samples within FoundationCORE were processed and sequenced as previously described (Frampton et al. Nat. Biotechnol. 31, 1023-1031, 2014). NRF2 exon 2 and exon 2+3 deletions were screened across the FoundationCORE dataset (n = 58,707) using two different approaches.

[0148] First, we investigated the rearrangement calls based on discordant read pairs and / or split reads for direct evidence of the loss of NRF2 exon 2 or exon 2+3. This approach provides direct evidence of the targeted deletion, but since the intron regions of NRF2 are not captured, the deletion can only be detected by this approach if the breakpoint is within the bait region. Thus, this approach identifies a limited subset of NRF2 exon 2 or exon 2+3 deletions where the breakpoint occurs near the intron-exon boundary or within the exon.

[0149] The second approach utilizes copy number log ratio data from individual bait regions. The copy number log ratio values were determined by an in-house algorithm trained on the specific tumor cellularity of each sample. Z-scores were calculated by comparing the log ratio for each exon in NRF2 to control polymorphic capture regions immediately adjacent to NRF2 (n = 15, equally spaced at approximately 1 MB intervals from approximately 3 MB upstream and approximately 12 MB downstream of NRF2). Exon 2 deletions with and without concomitant exon 3 deletions were specifically investigated. These are referred to herein as the exons of interest (EOI). An EOI deletion was called if (1) the z-score for the EOI but not the non-EOI in NRF2 was < -2 and (2) a 0.2 log ratio decrease from the non-EOI in NRF2 was calculated. The mutual exclusivity between NRF2 exon 2 or exon 2+3 deletions and short variants in NRF2 or KEAP1 was specifically investigated within lung squamous cell carcinomas (n = 1,218).

[0150] F. Cell Culture KMS-27 (RPMI-1640), JHH-6 (Williams Media E), HuCCT1 (RPMI-1640), and HUH-1 (DMEM) cells were from JCRB, and 293 (EMEM) cells were from ATCC. The cells were cultured in the designated medium in the presence of 2 mM glutamine and 10% FBS.

[0151] G. Western Blotting Cell lysates were prepared in RIPA buffer (Sigma) supplemented with complete EDTA-free protease inhibitor (Roche) and phoSTOP (Roche), Phosphatase Inhibitor Cocktail 2 (Sigma) and Phosphatase Inhibitor Cocktail 3 (Sigma) phosphatase inhibitors. Lysates were run on Novex Tris-Glycine 4–12% gradient gels (ThermoFisher) and transferred onto iBlot nitrocellulose (Invitrogen). Blots were pre-incubated with 5% non-fat dry milk (Merck) in TBST (10 mM Tris pH 8, 150 mM NaCl, 0.1% TWEEN-20) and subsequently incubated with 5% bovine serum albumin (Sigma) in TBST containing antibodies. The secondary antibodies used were ECL Anti-Rabbit HRP and ECL Anti-Mouse HRP (both from GE Healthcare). Blots were developed with Chemiluminescence Substrate Kit (Protein Simple) and visualized with a FluorChem HD2 imager (Protein Simple). The antibodies used in this study were against KEAP1 (Cell Signaling G1010), NRF2 (Abcam ab62352), HSP90 (Cell Signaling 4877), HDAC2 (Cell Signaling 5113), β-actin (Sigma A2228), HA (Roche 11815016001), and FLAG (Sigma F2426). λ phosphatase was from NEB (P0753L) and phosphatase inhibitors were omitted from the lysis buffer in these experiments.

[0152] H. Cell viability and DNA fragmentation analysis The cells were reverse transfected with siRNA using Dharmafect2 reagent (ThermoFisher) and OptiMEM (Gibco). Four days after transfection, the cells were measured for viability using CellTiter-Glo reagent (Promega), and luminescence was detected on an EnVision Multi-label Reader (Perkin Elmer). The cells were reverse transfected with siRNA using Dharmafect2 reagent (ThermoFisher) and OptiMEM (Gibco). Four days after transfection, the cells were measured for apoptosis using propidium iodide (PI) (LifeTechnologies) staining and flow cytometry according to the published protocol (Riccardi and Nicoletti Nat.Protoc. 1:1458-1461, 2006). Staurosporine, 1 μM, (Enzo) was added 24 hours before staining as a positive control. The siRNAs targeting NRF2 exon 2 had the sequences: 5'-TGGAGTAAGTCGAGAAGTA-3' (SEQ ID NO: 29) and 5'-ACAACTAGATGAAGAGACA-3' (SEQ ID NO: 30). The siRNAs targeting NRF2 exon 5 had the sequences: 5'-TGACAGAAGTTGACAATTA-3' (SEQ ID NO: 31) and 5'-GTAAGAAGCCAGATGTTAA-3' (SEQ ID NO: 32) and were used together with a non-targeting siRNA as a control siRNA. The stained cells were analyzed on a Becton Dickinson FACS Caliber instrument. The siRNA targeting KEAP1 was from Dhamacon (L012453-00).

[0153] DNA fragments were quantified by propidium iodide (PI) staining and measured by flow cytometry according to Riccardi et al. (Nature Protocols, 1:1458-1461 (2006)).

[0154] I. Taqman analysis Total cellular RNA was extracted using the RNeasy Kit (Qiagen). RNA was converted to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and cDNA was amplified using Taqman Gene Expression primer-probe sets (ThermoFisher) with Taqman Gene Expression Master Mix reagent (Applied Biosystems). Taqman amplification / detection was performed on a QuantStudio7Flex Real-Time PCR System. The primer-probe sets used were Hs00232352_ml and Hs00975961_gl (ThermoFisher) for detecting NRF2 exons 2 and 5, respectively. All of the NRF2 target gene Taqman primer-probe sets used were SLC7A11 (Hs00921938_ml), SGRN (Hs00921938_ml), NR0B1 (Hs03043658_ml), GCLC (HsOOI55249_ml), and GPX2 (Hs01591589_ml) from ThermoFisher.

[0155] J. 293 Transfection Plasmid DNA was transfected into cells using Lipofectamine2000 (ThermoFisher) and OptiMEM (Gibco) as recommended by the manufacturer protocol. Lysates were prepared 2 - 3 days after transfection. The expression plasmids used were pRK5.NRF2, pRK5.NRF2.delta.e2, pRK5.NRF2.delta.e2,3, pRK5.NRF2.FLAG, and pRK5.KEAP1.HA.

[0156] K. Tumor Xenograft Model On the right flank of 11- to 12-week-old female C.B-17 SCID beige mice (Charles River Laboratories), 10 x 10 6 A549 shRNA cells per mouse or 10 x 10 6 H441 shRNA cells in 100 μl of HBSS were inoculated. When the tumor volume reached approximately 150 - 250 mm 3 , the mice were randomized and allowed free access to drinking water containing 1 mg / ml of doxycycline in (5% sucrose) or no doxycycline (5% sucrose alone). Doxycycline was replaced three times a week and sucrose was replaced once a week. Tumor volume was determined using digital calipers (Fred V. Fowler Company, Inc.) using the formula (L x W x W) / 2 and plotted as mean tumor volume (mm 3 ) ± SEM. Tumor growth inhibition (TGI%) was calculated as the ratio of the area under the curve (AUC) for each dose group per day relative to the vehicle, such that TGI% = 100 x 1 - (AUC treatment / day) / (AUC vehicle / day). In another study, mice with tumors of 150 - 250 mm 3 were dosed with 1 mg / ml of doxycycline for 5 days prior to tumor resection and analyzed for NRF2 levels by Western blotting.

[0157] A549 xenografts treated with L. ErbB3 antibody Female nude mice (n = 10) with subcutaneous A549 tumors (75 - 144 mm3) on day 1 were treated with vehicle or 50 mg / kg of YW57.88.5 (100 mg / kg loading dose) administered intravenously once a week for 4 weeks (qwk x 4). Tumors were measured twice a week and each animal was euthanized at the endpoint when its tumor reached a volume of 1000 mm 3 or at the earliest of the last day of the treatment regimen.

[0158] Example 2: Identification of NSCLC cell lines with mutations in KEAP1 and NRF2 To identify mutations, copy number, and loss of heterozygosity (LOH) of KEAP1 and NRF2 in NSCLC, a panel of 113 NSCLC cell lines profiled by RNA-seq, exome-seq, or SNP array was documented (Figure 1A). KEAP1 mutations were seen in 29 / 113 cell lines (26%), and NRF2 mutations were detected in 4 / 113 cell lines (4%). Except for the NCI-H661 cell line, all KEAP1 mutant cell lines showed homozygous expression of the mutant allele, which was generally associated with copy-neutral LOH. In contrast, NRF2 mutations were heterozygous and not associated with LOH. Two additional cell lines (HCC1534 and NCI-HI437) did not show detectable KEAP1 mRNA due to biallelic loss of KEAP1 DNA. NRF2 mutations were in previously identified hotspots in the KEAP1 interface region (Figure 1B) (Shibata et al. Proc. Natl. Acad. Sci. U.S.A. 105:13568-13573, 2008) and included point mutations and in-frame 3-amino acid deletions. Mutations in KEAP1 were scattered throughout the primary sequence (Figure 1C), with few obvious hotspots. However, when mapped to the KEAP1 / NRF2 peptide crystal structure (Fukutomi et al. Mol. Cell. Biol. 34:832-846, 2014), the mutations clustered in loops extending from the KEAP1 core β-propeller near the interaction site with NRF2 (Figure 1D).

[0159] Example 3: Identification of the mutant KEAP1 gene signature To determine the transcriptional consequences of KEAP1 mutations in NSCLC cell lines, genes that were significantly differentially expressed (p<0.01, absolute mean fold change >2) in KEAP1 mutant cell lines compared to wild-type KEAP1 cell lines were identified. Overall, 27 genes were significantly upregulated in KEAP1 mutant cell lines (Figure 2A-2B), 15 of which have previously been identified as NRF2 target genes from ChIP-seq or RNA-seq studies (Chorley et al. Nucleic Acids Res. 4:7416-7429, 2012, Hirotsu et al. Nucleic Acids Res. 40:10228-10239, 2012, Malhotra et al. Nucleic Acids Res. 38:5718-5734, 2010). Only one gene, HSPB1, was identified as significantly downregulated using these cutoffs.

[0160] Unsupervised clustering of 230 TCGA lung adenocarcinomas based on the expression of these 27 genes resulted in the division of two major groups (Figure 3A). One group was mainly characterized by high expression of the 27 signature genes, containing 43 tumors, 32 of which (74%) were KEAP1 mutants. The other group, characterized by low expression, contained 187 tumors, 179 of which were KEAP1 wild-type. Notably, using the same genes to cluster lung squamous cell carcinomas, NRF2 and KEAP1 mutant tumors were distinguished from NRF2 / KEAP1 wild-type tumors (Figure 3B), indicating that NRF2 mediates most of the transcriptional consequences of KEAP1 loss / mutation. Interestingly, there were several squamous NSCLC tumors that showed high expression of KEAP1 mutant genes without any known mutations in either KEAP1 or NRF2. Proteomic data were available for 17 out of the 27 upregulated genes in the KEAP1 mutant cell line (37 wild-type KEAP1, 6 mutant KEAP1) in a smaller subset of cell lines. Consistent with the increased levels of mRNA of these genes in the mutant KEAP1 cell line, protein targets of all but one of these 17 genes (SLC7A11, which had low peptide coverage) also showed increased expression in the mutant KEAP1 cell line compared to the wild-type cell line (Figure 4).

[0161] Example 4: Identification of Aberrant Splicing of NRF2 in Tumor Samples For most tumors with high expression of 27 candidate NRF2 target genes, the elevated gene expression can be explained by mutations in KEAP1 or NRF2. However, there were several tumors that showed high expression of candidate NRF2 target genes in the absence of characterized mutations in either KEAP1 or NRF2. Cancer-related transcript changes are increasingly recognized as putative driver events. Thus, the hypothesis was put forward that NRF2 pathway activation in these tumors could be driven by splice changes not recognized by whole-exome sequencing. Fifty-four known cancer genes were analyzed to identify splice variants that are repeatedly observed in cancer cells from TCGA but rarely detected in normal samples from GTEx (see Example 1). Nineteen cancer types were selected, each containing at least 100 cancer samples (6,359 samples in total). Nine recurrent candidate cancer-specific splice variants were identified in the 54 cancer genes considered (≥2 samples and >1% of samples of a given cancer type). Using the same detection criteria as for cancer samples, none of these variants could be detected in normal controls (2,958 samples in total). Grouping of related variants with shared splice sites resulted in five independent changes in four cancer genes (Figure 5). These changes included several well-documented oncogenic splice variants, including EGFRvIII in brain cancer, MET exon 14 skipping in lung adenocarcinoma, and CTNNB1 exon 3 deletion in colorectal cancer (Cho et al. Cancer Res. 71(24):7587-7596, 2011; Kong-Beltran et al. Cancer Res. 66(1):283-289, 2006; Iwao et al. Cancer Res. 58(5):1021-1026, 1998). Interestingly, previously uncharacterized splice variants in NRF2 were frequently observed and occurred in patients with squamous NSCLC (3.3%, 16 / 481) and at a lower prevalence in patients with HNSC (1.5%, 6 / 403) (Figure 5A).A more detailed analysis of NRF2 splice variants in lung squamous cell carcinoma revealed two co-occurring splice variants in the same patient, corresponding to the skipping of NRF2 exon 2 in mRNA transcribed from either one of two alternative promoters (2.1%, 10 / 481) (Figure 6). Two additional splice variants co-occurred in a different set of patients (1.2%, 6 / 481) and corresponded to the skipping of both NRF2 exons 2 and 3 (exon 2+3) in mRNA having either one of two alternative transcript starts (Figure 6). All patients expressing NRF2 splice variants lacking exon 2 or exon 2+3 also showed expression of normal NRF2 transcripts as evidenced by split reads supporting the inclusion of exon 2. Both exons 2 and 3 are part of the NRF2 coding sequence, and skipping of exon 2 or exon 2+3 is predicted to result in protein isoforms having either an N-terminal truncation or an in-frame deletion (Figure 7). The high recurrence and conservation of coding capacity of NRF2 transcripts lacking exon 2 indicate that these splice variants may represent gain-of-function events conferring a selective advantage. This is supported by the finding that exon 2 encodes the Neh2 domain and is mutated in 15% of squamous cell lung cancers, enabling interaction with KEAP1 (Itoh et al. Genes Dev. 13(1):76-86, 1999).

[0162] To evaluate whether the observed NRF2 splice variants can explain NRF2 pathway activation in patients without mutations in KEAP1 or NRF2, we examined the co-occurrence of NRF2 splice variants and NRF2 pathway mutations. In the TCGA collection, 178 squamous lung tumors were profiled by exome-seq. In this subset, 10 tumors (6%) showing exon 2 or exon 2+3 deletions were mutually exclusive with 48 tumors (27%) showing mutations in either NRF2 or KEAP1 (Figure 8A). Also, all exon 2 deletion tumors showed high expression of 27 candidate NRF2 target genes (Figure 8B). Similar observations were made for head and neck cancers, where NRF2 exon deletions in 5 tumors (2%) were mutually exclusive with NRF2 or KEAP1 mutations in 26 tumors (9%) (Figures 9A-9B). These results indicate that exon 2 deletions represent an alternative mechanism for NRF2 activation in subsets of squamous NSCLC and head and neck tumors. Importantly, these results show that, in addition to exome sequencing, consideration of splice changes increased the percentage of patients identified as having putative NRF2 pathway activation from 27% (48 / 178) to 33% (58 / 178) in lung squamous carcinoma and from 9% (26 / 275) to 11% (31 / 275) in head and neck squamous carcinoma.

[0163] Example 5: Demonstration of NRF2 splicing deficiency in cell lines To identify cell line models for further study, we analyzed the lead evidence of the identified splice variants in RNA-seq data from a large panel of human cancer cell lines (described in Klijn et al. Nat. Biotechnol. 33(3):306-312, 2014). Among 611 cell lines, we identified one multiple myeloma cell line, KMS-27, and one hepatocellular carcinoma cell line, JHH-6, both of which showed evidence of heterozygous skipping of NRF2 exon 2 by junction reads (Figure 10). RT-PCR-based NRF2 exon 2 skipping in JHH-6 and KMS-27 mRNAs was demonstrated. Using a series of forward and reverse primers derived from exon 1 and exon 3 / 4, respectively (Figure 11A), exon 2 deletion (Δe2NRF2) in mRNAs isolated from JHH-6 and KMS-27 cells was confirmed (Figure 11B). Sequencing of the PCR products confirmed the expected deletion of exon 2 (Figure 12A-12C). Based on the RNA-seq data, point mutations were not detected in the coding sequences of NRF2 or KEAP1 in JHH-6 or KMS-27 (Klijn et al. Nat. Biotechnol. 33(3):306-312, 2014).

[0164] NRF2 / KEAP1 alterations are quite common in hepatocellular carcinoma (10%) but rare in multiple myeloma (0%), so JHH-6 cells were further tested. Specifically, the expression of the exon 2 deletion form of the NRF2 protein was tested. Western blotting of whole cell lysates from JHH-6 cells, as well as the KEAP1 mutant HUH-1 strain and the HuCCT1 cells as a representative wild-type KEAP1 hepatocellular carcinoma cell line was performed. The level of NRF2 in JHH-6 cells was equivalent to that seen in HUH-1 cells, and these were much higher than those in wild-type KEAP1 HuCCT1 cells (Figure 13). Also, a lower molecular weight species consistent with the deletion of exon 2 was detectable in JHH-6 and was reduced after NFE2L2 siRNA transfection, confirming that this actually represents a form of NRF2. Although the altered NRF2 isoform was visible, it was surprising that it was not more abundant considering the lack of the KEAP1 interaction motif. A hypothesis was put forward that the phosphorylated form of exon 2-deleted NRF2 could co-migrate with the non-phosphorylated form of wild-type NRF2 in the 4–12% gels used. Indeed, dephosphorylation of the JHH-6 lysate showed that the exon 2 deletion form of NRF2 was significantly more abundant than the wild-type form (Figure 14A, middle panel). Similarly, KMS-27 cells expressed the exon 2 deletion form of NRF2, which was the major species seen after dephosphorylation (Figure 15).

[0165] The stability of NRF2 in three hepatocellular carcinoma cell lines was tested using cycloheximide, which abrogates total protein synthesis. Dephosphorylated lysates were used to enable more accurate quantification of total NRF2. The experiment showed an increased stability of Δe2NRF2 in JHH-6 cells equivalent to that of NRF2 in HUH-1 cells, and both were more stable than NRF2 in HuCCT1 cells (Figures 14A–14B). The exon 2 deletion form of NRF2 in JHH-6 cells also showed significant nuclear localization when compared to HUH-1 cells. (Figure 16).

[0166] To determine whether the deletion of exon 2 in JHH-6 cells rendered NRF2 resistant to KEAP1-mediated control, the stability of NRF2 in response to KEAP1 knockdown was examined. Knockdown of KEAP1 in HuCCT1 cells resulted in increased steady-state levels of NRF2 due to increased stability (Figure 14C). However, knockdown of KEAP1 in JHH-6 cells did not affect the levels or stability of exon 2-deleted NRF2. As expected, knockdown of KEAP1 did not increase the stability of wild-type NRF2 in the KEAP1 mutant HUH-1 cell line (Figure 14D).

[0167] Example 6: Evaluation of exon 2 and / or exon 2+3 deletions on NRF2 Using the NRF2 / KEAP1 gene signature described in Example 3, among 16 hepatocellular carcinoma cell lines, JHH-6 cells were determined to exhibit one of the highest expressions of NRF2 target genes similar to those found in the mutant KEAP1-expressing strains from RNA-seq data (Figure 17A). Similarly, among 18 multiple myeloma cell lines investigated, KMS-27 cells exhibit one of the highest expressions of these genes (Figure 17B). The expression of these genes can be summarized by a "NRF2 target gene score" calculated as the average z-score of individual target genes across 611 cell lines investigated. This provides a single score per cell line that reflects the degree of overexpression of the signature genes in a given strain. The NRF2 target score shows that JHH-6 cells exhibit a score similar to that of hepatocellular carcinoma cell lines expressing KEAP1 mutations (Figure 18A), and confirms that KMS-27 cells exhibit the highest score among multiple myeloma cell lines, despite multiple myeloma showing a low overall NRF2 target gene score (indicated by negative values) (Figure 18B).

[0168] Next, the dependence of JHH-6 cells expressing exon 2-deleted NRF2 on NRF2 protein expression was compared with wild-type NRF2-expressing HuCCT1 cells. Knockdown of NRF2 in JHH-6 cells caused a marked decrease in cell viability similar to that seen in the mutant KEAP1 hepatocellular carcinoma cell line HUH-1. In contrast, NRF2 knockdown had a more modest effect on the viability of HuCCT1 cells (Figure 19). Since NRF2 knockdown was equally efficient in all three cell lines, this was not due to poor NRF2 knockdown in HuCCT1 cells (Figure 20). NRF2 knockdown also resulted in decreased expression of four well-characterized NRF2 target genes, which was slightly reduced in the wild-type KEAP1 HuCCT1 cell line (Figure 21). The decreased viability was likely due, at least in part, to apoptosis measured by an increase in fragmented DNA (Figure 22).

[0169] To explain how loss of NRF2 exon 2 affects the ability of NRF2 to be controlled by KEAP1, transient expression in 293 cells was used. KEAP1 decreased the expression of full-length NRF2 but had a smaller effect on the expression of NRF2 lacking exon 2 or exon 2+3 (Figure 23, upper panel). The inhibitory effect of KEAP1 on full-length NRF2 expression was almost abolished by the proteasome inhibitor MG132 as expected. Full-length NRF2 and KEAP1 interacted with each other, while deletion of exon 2 or exon 2+3 completely abolished the ability of KEAP1 to bind to NRF2 (Figure 23, lower panel). As a result, truncated NRF2 remained stable after KEAP1 expression, in contrast to wild-type NRF2 (Figure 24A-24B), although the truncated form of NRF2 appeared to have a slightly reduced intrinsic stability. However, the altered NRF2 isoforms were transcriptionally active as judged by their ability to increase NRF2 target gene expression (Figure 25). Most genes were similarly increased by NRF2 lacking exon 2 or exon 2+3 compared to full-length NRF2 and were resistant to the effect of KEAP1 overexpression. Interestingly, NRF2 lacking exon 2+3 was poor at increasing GPX2 expression, indicating that there may be subtle differences in the transcriptional activation of this form of NRF2. Consistent with this observation, 22 out of 27 target genes described in Example 3, in addition to GPX2, showed a lower median expression in exon 2+3-deleted squamous lung tumors compared to exon 2-deleted tumors (Figure 26).

[0170] Example 7: Mechanistic analysis of NRF2 exon 2 splicing changes Analysis of exome-seq data for KMS-27 and JHH-6 showed a decrease in reads mapping to exon 2, indicating that the observed transcript variants could be the result of genomic changes (Figure 27A). Whole-genome sequencing (WGS) of JHH-6 and KMS-27 showed that these cell lines harbored microdeletions surrounding NRF2 exon 2 that spanned 4,685 and 2,981 nucleotides, respectively (Figure 27B). To investigate the causal mechanism in patients, targeted paired-end exome-seq data from a large cohort (n = 1,218) of clinical squamous NSCLC tumors with high read coverage (>300x) were analyzed. In this dataset, 11 tumors showed a decrease in copy number for exon 2 or exon 2+3 compared to nearby control regions (Materials and Methods, Figure 27B). The focal nature of the deletions can be understood by examining the log ratio from defined genomic regions targeted for sequencing (Figure 28B). Seven tumors with discordant read pairs contained several kilobases of DNA and were consistent with structural variants affecting exon 2 or exon 2+3 (Figure 28A). In total, 16 patients showed evidence of genomic changes affecting NRF2 exon 2 or exon 2+3, and the identified events were mutually exclusive with point mutations or indels in NRF2 and KEAP1, which are known to activate this pathway. An additional cohort of 45 squamous NSCLC tumors was analyzed, for which both RNA and DNA were available. RT-PCR analysis identified a single patient with loss of exon 2 that was highly enriched in the tumor compared to adjacent normal tissue (Figure 29). RNA-seq analysis confirmed that the transcript variant was expressed in the tumor where it was identified but absent in adjacent normal tissue (Figure 30). Expression of NRF2 target genes was also elevated to a similar extent as TCGA tumors with known mutations in this pathway, while adjacent normal tissue showed low expression of these genes (Figure 31). Finally, whole-genome sequencing confirmed that the transcript variant was the result of a somatic genomic microdeletion of 5,233 nucleotides surrounding exon 2 (Figure 28C).These data indicate that genomic microdeletions are a clinically relevant mechanism for NRF2 pathway activation.

[0171] These data indicate that the set of genes controlled by NRF2 is conserved across different tissues and conditions. This has practical value in the use of a single gene signature to identify tumors with NRF2 activation in both NSCLC and HNSC (Figure 32). Interestingly, this NRF2 / KEAP1 signature is only activated in tumors. Matched normal samples for lung and head and neck tumors showed only low NRF2 target gene activity (Figure 33). This indicates that inhibition of the NRF2 pathway may have a selective benefit in tumors that show pathway deregulation compared to normal tissues.

[0172] Genomic deletions within genes that result in the activation of oncogenes have been previously reported for a number of genes, including EGFR and CTNNB1. Such variants are not routinely assayed, partly due to the limitations of current genomic technologies. In particular, small deviations, including small copy number changes that affect individual exons, are difficult to detect by single-exon seq alone. Thus, intragenic deletions remain relatively under-investigated and new variants are still being discovered. Recent studies of small cell lung cancer and adult T cell leukemia / lymphoma have identified recurrent microdeletions in TP73, IKZF2, and CARD11 using whole-genome sequencing (George et al. Nature 524, 47-53:2015, Kataoka et al. Nat. Genet. 47:1304-1315, 2015). In this study, we used publicly available RNA-seq data generated as part of the TCGA project to identify recurrent transcriptional changes in known cancer genes. It is difficult to assess the general prevalence of NRF2 exon deletions due to differences between patient cohorts. For example, when analyzing TCGA lung squamous cell carcinoma with available RNA-seq data (n = 481), we identified 3% (16 / 481) of patients with deletions of NRF2 exon 2 or exon 2+3. When analyzing a subset of patients with available exome-seq data (n = 178) that can perform somatic variant calling, the proportion of patients with NRF2 exon deletions was 6% (10 / 178). This explains that NRF2 exon deletions increased the proportion of patients with putative NRF2 pathway activation from 27% (48 / 178) to 33% (58 / 178) in lung squamous cell carcinoma and from 9% (26 / 275) to 11% (31 / 275) in head and neck squamous cell carcinoma compared to the assessment of mutations in NRF2 or KEAP1 by exome-seq alone (Figures 8A and 9A). Analysis of real-world clinical samples from patients who underwent genomic profiling showed a prevalence of NRF2 exon deletions of 1-2% in lung squamous cell carcinoma.However, since the optimization criteria for determining single exon deletions in samples with variable tumor content have not yet been established and only obvious deletions were considered, the latter analysis is insufficiently sensitive. Nevertheless, the results presented herein are consistent with the concept that the regulation of this pathway frequently varies in certain tumor signatures such as squamous NSCLC and head and neck cancers. Additional screening of known cancer genes can also be performed via sequencing of the complete locus including introns or by combining data from exome and RNA sequencing experiments.

[0173] Analysis of the structures of the three deletions identified by WGS showed different breakpoints, but in each case the genomic regions adjacent to the deletions showed 2 to 6 nucleotides of sequence homology (Figure 34). The DNA sequences of the 3'-end, 5'-end, and junction reads of JHH-6 cells are provided by SEQ ID NOs: 61 to 63, respectively. The DNA sequences of the 3'-end, 5'-end, and junction reads of KMS-27 cells are provided by SEQ ID NOs: 64 to 66, respectively. The DNA sequences of the 3'-end, 5'-end, and junction reads of primary tumor cells are provided by SEQ ID NOs: 67 to 69, respectively.

[0174] In addition to point mutations, NRF2 often shows genomic amplification. Interestingly, the intensity of the NRF2 deletion product in KMS-27 cells by RT-PCR analysis was similar to that of wild-type NRF2 and seemed to be more abundant in JHH-6 cells (Figure 13). This was also reflected in the WGS read counts, showing a higher abundance of the deletion form compared to the wild-type allele (Figure 27B). These results are consistent with the observation that JHH-6 cells have 5 copies of the NRF2 locus by SNP array while KMS-27 cells have 2 copies. The amplification of NRF2 is at a reasonable frequency in the analyzed TCGA samples, including squamous (4.5%) and adenomatous (2.6%) NSCLC, HNSC (12.2%), and liver cancer (3.6%), representing a mechanism to increase NRF2 transcriptional output. In the case of JHH-6 cells, these data indicate that the deletion allele is preferentially amplified, providing an additional mechanism to enhance NRF2 signaling in this cell line. However, preferential amplification of the truncated / spliced allele was not observed in primary tumors, indicating that exon 2 or exon 2+3 deletions alone can provide sufficient NRF2 activity for clonal selection.

[0175] The deletion of exon 2 provides an excellent mechanism for increasing NRF2 activity by removing the interaction site with KEAP1 while retaining the remainder of the gene that is functionally intact with respect to DNA binding and transcriptional activation functions. Indeed, our biochemical analysis confirmed almost complete loss of KEAP1 binding and the resulting stabilization of NRF2 when exon 2 was deleted (Figs. 23 and 24). Considering the NRF2 point mutations seen in tumors, mutations surrounding the ETGE high-affinity binding site result in complete loss of KEAP1 interaction, and mutations in the lower-affinity DLG motif vary in their ability to disrupt the NRF2 / KEAP1 complex (Fukutomi et al. Mol Cell Biol. 34(5):832-846, 2014, Shibata et al. Proc. Natl. Acad. Sci. USA. 105(36):13568-13573, 2008). However, even point mutations that do not disrupt the complex alter the nature of the interaction to prevent KEAP1-mediated ubiquitination of NRF2 (Shibata et al. Proc. Natl. Acad. Sci. USA. 105(36):13568-13573, 2008). In the case of deletions of both exons 2 and 3, the interaction with KEAP1 is similarly abrogated, but exon 3 contains the Neh4 domain that was previously associated with transcriptional activation by NRF2 through binding to the CREB (cAMP response element-binding protein) binding protein (CBP) (Katoh et al. Genes Cells. 6(10):857-868, 2001). Neh4 (contained in exon 3) and Neh5 (contained in exon 4) have been shown to act synergistically in the recruitment of CBP. Consistent with this, a reduced ability of Δe2+3NRF2 to induce several NRF2 target genes was observed compared to Δe2NRF2 or tumor-associated point mutations in NRF2 (Figs. 25 and 26).

[0176] Deletions seen in human tumors that remove the interaction domain with E3 ligase have also been observed in other genes. For example, 7 out of 222 colorectal tumors showed a small genomic deletion (234 - 677 bp) encompassing exon 3 of β-catenin (Iwao et al. Cancer Res. 58(5):1021 - 1026, 1998) that removes the interaction site with its E3 ligase β-TRCP (Hart et al. Curr. Biol. 9(4):207 - 210, 1999). Similarly, most of the TMPRSS-ERG fusion proteins seen in prostate cancer encode a truncated version of ERG that renders them resistant to ubiquitination and degradation mediated by SPOP (An et al. Mol. Cell. 59(6):904 - 916, 2015).

[0177] In addition, mutations that result in MET exon 14 skipping remove amino acid residue Y1003, which is required for Cbl requirement and subsequent ubiquitination and downregulation. Thus, deletions within small genes represent an effective mechanism by which oncogenes during tumorigenesis and progression escape normal degradation.

[0178] Example 8: NRF2 knockdown in mutant KEAP1 cells This example provides a characterization of the effect of KEAP1 mutations on the requirement for NRF2 activity under different growth environments, and shows that NRF2 activity is essential for growth under anchorage-independent conditions.

[0179] The results of NRF2 inhibition across wild-type and mutant KEAP1 and NRF2 cell lines were investigated. Stable cell lines expressing three independent NRF2 shRNAs under the control of doxycycline, and three independent non-targeting controls (NTCs), were constructed. These NRF2 shRNAs were effective in reducing NRF2 protein levels in five KEAP1 mutant cell lines, two NRF2 mutant cell lines, and five wild-type NSCLC cell lines, as well as in immortalized but non-transformed lung epithelial BEAS2B cells (Figure 35). After doxycycline addition, the viability of most cell lines decreased to varying degrees, with KEAP1 mutant cell lines generally showing a significantly greater decrease (Figures 36 and 37). Knockdown of NRF2 by siRNA in a larger panel of NSCLC cell lines confirmed a genotype-dependent effect on cell viability (Figure 38).

[0180] The results of NRF2 knockdown in tumor xenografts were characterized. KEAP1 mutant A549 cell lines and KEAP1 wild-type H441 cell lines expressing dox-inducible NRF2 shRNAs were transplanted into the flanks of female SCID mice. NRF2 was effectively knocked down in mice treated with doxycycline in both tumors (Figures 39 and 40). NRF2 knockdown in the KEAP1 mutant A549 cell line had a dramatic effect on tumor growth, resulting in complete tumor regression in 5 out of 10 tumors (Figure 41A). In contrast, the effect on KEAP1 wild-type H441 growth was more modest, resulting in a 37% reduction in tumor growth and all animals showing a maintained tumor burden (Figure 41B).

[0181] To understand the differential effects of NRF2 knockdown on tumor growth in xenografts versus 2D growth on plastic, several additional cell culture environments were tested. NRF2 knockdown in cells grown on low attachment plates and / or in low oxygen (0.5%) showed similar results to cells grown on plastic (Figure 42). In contrast, the growth of KEAP1 mutant cell lines was significantly impaired when cultured in soft agar (Figures 43 and 44), on micropatterned plastic films (Figures 45 and 46), or in methylcellulose (Figure 47). Growth in soft agar was used to more precisely characterize the results of NRF2 knockdown. NRF2 knockdown completely abrogated colony formation in three KEAP1 mutant cell lines but had little effect in two wild-type KEAP1 NSCLC cell lines, H1048 and H441 (Figures 43 and 44). Since this pathway has been shown to mediate survival properties promoted by high NRF2 activity, the role of the glutathione pathway in the response to NRF2 knockdown was evaluated. The addition of reduced glutathione generally increased the ability of all tested cell lines to form colonies in soft agar but was unable to rescue the results of NRF2 knockdown (Figures 43 and 44). Similar negative results were seen with N-acetylcysteine (NAC, Figure 48). Exogenous glutathione was able to enter cells and reduce reactive oxygen species (ROS) levels as measured by dichlorofluorescein staining (Figure 49). Thus, the requirement for NRF2 activity is surprisingly independent of the glutathione synthesis pathway.

[0182] To further investigate the effect of the glutathione pathway in NRF2 response, the expression and activity of the xCT glutamate / cysteine antiporter, one of the rate-limiting steps in glutathione synthesis, were monitored. SLC7A11 expression was reduced after NRF2 knockdown (Figure 50), causing a decrease in cystine uptake (Figure 51) associated with reduced glutathione (Figure 52). NRF2 knockdown also caused a substantial increase in ROS levels (Figure 53). To determine whether inhibition of SLC7A11 expression and cystine uptake contributed to the decreased viability after NRF2 knockdown, xCT function was initiated using erastin, which inhibited cystine uptake (Figure 51) and increased oxidative stress (Figure 53). However, this was not sufficient to decrease the viability of the KEAP1 mutant cell line A549 (Figure 54) or most other KEAP1 mutant cell lines (Figure 55). The combination of erastin and NRF2 knockdown, however, resulted in a dramatic decrease in viability (Figure 54). Similarly, the glutathione synthase inhibitor buthionine sulfoximine (BSO) or the glutaminase inhibitor BPTES also did not show preferential toxicity for KEAP1 mutant cell lines (Figures 56 and 57). These results indicate that glutathione replenishment was not sufficient to rescue the lethality induced by NRF2 knockdown, and glutathione depletion was also not sufficient to kill KEAP1 mutant cell lines.

[0183] To understand which pathways were activated as a result of NRF2 activation or KEAP1 loss, CRISPR screens were performed using a library of genes that decreased after NRF2 knockdown in A549 cells and increased in a panel of KEAP1 mutant NSCLC cell lines. Since different outcomes were observed after NRF2 knockdown in 2D, 3D, and xenograft conditions, the screens were performed in all three environments to determine whether individual dependencies could be identified. At the 15-day time point for all three conditions, all three screens functioned similarly, and the gRNAs represented only a small number of genes showing significant dropout (Figures 58 - 60). NFE2L2 and its binding partner, MAFG, were among the most significant genes, indicating that the screen functioned as expected. Pentose phosphate pathway genes PGD, G6PD, and TKT, known NRF2 target genes, also showed strong dropout. Other strong hits in the screen were two growth factor receptor genes, IGF1R and ERBB3, and genes encoding three components of the redox signaling relay, PRDX1, TXN, and TXNRD1.

[0184] The expression of ErbB3 decreased after NRF2 knockdown in A549 cells (Figures 58 - 60). Treatment with YW57.88.5 in the tumor xenograft model indicated that ErbB3 is required for A549 proliferation (Figure 61).

[0185] The expression of IGF1R was greater in KEAP1 mutant NSCLC cells compared to KEAP1 wild-type NSCLC cell lines. To test the effect of IGF1R inhibition on KEAP1 mutant and KEAP1 wild-type cells, cell lines were treated with linsitinib, a potent and selective IGF1R small molecule inhibitor. Linsitinib showed little effect on proliferation when tested in three wild-type and three mutant KEAP1 NSCLC cell lines. However, this compound was highly effective at inhibiting colony growth of A549 cells in soft agar, with an IC 50It had. Also, when tested against a large panel of NSCLC cell lines, there appeared to be selective growth inhibition of this compound in soft agar in KEAP1 mutant cell lines. A similar selective effect on KEAP1 mutant cell lines when grown under anchorage-independent conditions was also seen with the independent IGF1R inhibitor NVP-AEW541 (Figure 62).

[0186] Therefore, growth factors that signal through IGF1R and ErbB3 are significant mediators of the growth of KEAP1 mutant cells.

[0187] Other embodiments The foregoing invention has been described in some detail by way of illustration and example for purposes of clear understanding, but the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific documents cited herein are hereby expressly incorporated by reference in their entirety.

Claims

1. 1. A method of diagnosing cancer in a subject, the method comprising: (a) determining the expression level of at least one gene selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in a sample obtained from the subject; (b) comparing the expression level of the at least one gene to a reference expression level of the at least one gene, wherein an increase in the expression level of the at least one gene in the sample relative to the reference expression level of the at least one gene identifies the subject as having cancer.

2. 1. A method for identifying a subject having a cancer that is an NRF2 dependent cancer, the method comprising: (a) determining the expression level of at least one gene selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL in a sample obtained from the subject; (b) comparing the expression level of the at least one gene to a reference expression level of the at least one gene; (c) determining whether the subject's cancer is an NRF2-dependent cancer, wherein an increase in the expression level of the at least one gene in the sample relative to the reference expression level of the at least one gene identifies the subject as having an NRF2-dependent cancer.

3. The method of claim 1 or 2, wherein the expression levels of at least two genes selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL are determined in a sample obtained from the subject.

4. 4. The method of claim 3, wherein the expression levels of at least three genes selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL are determined in a sample obtained from the subject.

5. 5. The method of claim 4, wherein the expression levels of at least four genes selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL are determined in a sample obtained from the subject.

6. 6. The method of claim 5, wherein the expression levels of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL are determined in a sample obtained from the subject.

7. 7. The method of any one of claims 1 to 6, wherein the expression level of one or more of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, or NQO1 is determined.

8. 7. The method of any one of claims 1 to 6, wherein the expression level of one or more of AKR1B10, AKR1C2, ME1, KYNU, CABYR, TRIM16L, AKR1C4, CYP4F11, RSPO3, AKR1B15, NR0B1, and AKR1C3 is determined.

9. (a) the expression levels of the at least two genes in the sample are the average of the expression levels of the at least two genes in the sample; (b) the reference expression level of the at least two genes is the average of the at least two genes of the reference; The method of any one of claims 3 to 6, wherein (c) the average of said at least two genes of said sample is compared with the average of said at least two genes of said reference.

10. The method of any one of claims 1 to 9, wherein said reference expression level is the average level of expression of said at least one gene in a population of subjects.

11. The method of claim 10 , wherein the population of subjects is a population of subjects sharing a common ethnicity.

12. The method of any one of claims 1 to 11, wherein said reference expression level is the average level of expression of said at least one gene in a population of subjects with cancer.

13. 13. The method of claim 12, wherein the reference expression level is the average level of expression of the at least one gene in a population of subjects with lung cancer.

14. 14. The method of claim 13, wherein the lung cancer is non-small cell lung cancer (NSCLC).

15. 15. The method of claim 14, wherein the NSCLC is squamous NSCLC.

16. The method according to any one of claims 1 to 15, wherein the expression level is an mRNA expression level.

17. 17. The method of claim 16, wherein the mRNA expression level is determined by PCR, RT-PCR, RNA-seq, gene expression profiling, serial analysis of gene expression, or microarray analysis.

18. The method of any one of claims 1 to 15, wherein the expression level is a protein expression level.

19. 20. The method of claim 18, wherein the protein expression level is determined by Western blot, immunohistochemistry, or mass spectrometry.

20. The method of any one of claims 1 to 19, further comprising determining the DNA sequence of NRF2.

21. 21. The method of claim 20, wherein the DNA sequence is determined by PCR, exome-seq, microarray analysis, or whole genome sequencing.

22. 1. A method for diagnosing cancer in a subject, the method comprising determining the DNA sequence of NRF2 in a sample obtained from the subject, wherein the presence of NRF2 DNA containing a deletion of all or part of exon 2 identifies the subject as having cancer.

23. 23. The method of claim 22, wherein the DNA sequence is determined by PCR, exome-seq, microarray analysis, or whole genome sequencing.

24. 1. A method for identifying a subject having cancer, the method comprising determining the mRNA expression level of NRF2 that contains a deletion of all or part of its exon 2 in a sample obtained from the subject, wherein the presence of NRF2 that contains a deletion of all or part of its exon 2 identifies the subject as having cancer.

25. 25. The method of claim 23 or 24, wherein the mRNA expression level is determined by PCR, RT-PCR, RNA-seq, gene expression profiling, serial analysis of gene expression, or microarray analysis.

26. 26. The method of claim 24 or 25, further comprising determining the DNA sequence of the NRF2.

27. 27. The method of claim 26, wherein the DNA sequence is determined by PCR, exome-seq, microarray analysis, or whole genome sequencing.

28. The method of any one of claims 22 to 27, wherein the NRF2 further comprises a deletion of all or part of exon 3 thereof.

29. A method for diagnosing cancer in a subject, the method comprising determining the protein expression level of NRF2 that includes a deletion of all or part of its Neh2 domain in a sample obtained from the subject, wherein the presence of NRF2 that includes a deletion of all or part of its Neh2 domain identifies the subject as having cancer.

30. 1. A method for identifying a subject having cancer, the method comprising determining the protein expression level of NRF2 that contains a deletion of all or part of its Neh2 domain in a sample obtained from the subject, wherein the presence of NRF2 that contains a deletion of all or part of its Neh2 domain identifies the subject as having cancer.

31. 31. The method of claim 29 or 30, wherein the NRF2 further comprises a deletion of all or part of its Neh4 domain.

32. The method of any one of claims 29 to 31, wherein the protein expression is determined by Western blot, immunohistochemistry, or mass spectrometry.

33. 33. The method of any one of claims 1 to 32, further comprising administering to the subject a therapeutically effective amount of an NRF2 pathway antagonist.

34. The method of any one of claims 1 to 33, further comprising administering to the subject a therapeutically effective amount of an anti-cancer agent.

35. 35. The method of any one of claims 1-34, wherein the method comprises administering an anti-cancer agent and an NRF2 pathway antagonist.

36. 36. The method of claim 34 or 35, wherein the anti-cancer agent and the NRF2 pathway antagonist are administered simultaneously.

37. 36. The method of claim 34 or 35, wherein the anti-cancer agent and the NRF2 pathway antagonist are administered sequentially.

38. 38. The method of any one of claims 34 to 37, wherein the anti-cancer agent is selected from the group consisting of anti-angiogenic agents, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, and immunotherapy.

39. 39. The method of claim 38, wherein the antiangiogenic agent is a VEGF antagonist.

40. 40. The method of any one of claims 33-39, wherein the NRF2 pathway antagonist is selected from the group consisting of a CREB antagonist, a CREB binding protein (CBP) antagonist, a Maf antagonist, an activating transcription factor 4 (ATF4) antagonist, a protein kinase C (PKC) antagonist, a Jun antagonist, a glucocorticoid receptor antagonist, a UbcM2 antagonist, a HACE1 antagonist, a c-Myc agonist, a SUMO agonist, a KEAP1 agonist, a CUL3 agonist, or a retinoic acid receptor alpha (RARα) agonist.

41. 1. A method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of an NRF2 pathway antagonist, wherein an expression level of at least one of the following genes, AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL, in a sample obtained from the subject has been determined to be increased relative to a reference expression level of the at least one gene.

42. 42. The method of claim 41, wherein the expression levels of at least two genes selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL are determined in a sample obtained from the subject.

43. 43. The method of claim 42, wherein the expression levels of at least three genes selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL are determined in a sample obtained from the subject.

44. 44. The method of claim 43, wherein the expression levels of at least four genes selected from the group consisting of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL are determined in a sample obtained from the subject.

45. 45. The method of claim 44, wherein the expression levels of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, ME1, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, CYP4F11, RSPO3, ABCC2, AKR1B15, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, NQO1, and FTL are determined in a sample obtained from the subject.

46. 46. ​​The method of any one of claims 41 to 45, wherein the expression level of one or more of AKR1B10, AKR1C2, SRXN1, OSGIN1, FECH, GCLM, TRIM16, KYNU, CABYR, SLC7A11, TRIM16L, AKR1C4, NR0B1, UGDH, TXNRD1, GSR, AKR1C3, TALDO1, PGD, TXN, or NQO1 is determined.

47. 46. ​​The method of any one of claims 41 to 45, wherein the expression level of one or more of AKR1B10, AKR1C2, ME1, KYNU, CABYR, TRIM16L, AKR1C4, CYP4F11, RSPO3, AKR1B15, NR0B1, and AKR1C3 is determined.

48. (a) the expression level of at least two genes in said sample is the average of the expression levels of said at least two genes in said sample; (b) the reference expression level of the at least two genes is the average of the at least two genes of the reference; 46. ​​The method of any one of claims 41 to 45, wherein (c) the average of said at least two genes of said sample is compared with the average of said at least two genes of said reference.

49. The method of any one of claims 41 to 48, wherein said reference expression level is the average level of expression of said at least one gene in a population of subjects.

50. 50. The method of claim 49, wherein the population of subjects is a population of subjects sharing a common ethnicity.

51. 51. The method of any one of claims 41 to 50, wherein said reference expression level is the average level of expression of said at least one gene in a population of subjects with cancer.

52. 52. The method of claim 51, wherein the lung cancer is non-small cell lung cancer (NSCLC).

53. 53. The method of claim 52, wherein the NSCLC is squamous NSCLC.

54. The method of any one of claims 41 to 53, wherein the expression level is an mRNA expression level.

55. 55. The method of claim 54, wherein the mRNA expression level is determined by PCR, RT-PCR, RNA-seq, gene expression profiling, serial analysis of gene expression, or microarray analysis.

56. 56. The method of claim 55, wherein the mRNA expression levels are determined by RNA-seq.

57. The method of any one of claims 41 to 53, wherein the expression level is a protein expression level.

58. 58. The method of claim 57, wherein the protein expression is determined by Western blot, immunohistochemistry, or mass spectrometry.

59. 59. The method of any one of claims 41 to 58, further comprising determining the DNA sequence of the NRF2.

60. 60. The method of claim 59, wherein the DNA sequence is determined by PCR, exome-seq, microarray analysis, or whole genome sequencing.

61. 1. A method of treating a subject having cancer, the method comprising: (a) determining in a sample obtained from the subject an expression level of NRF2 mRNA that contains a deletion of all or part of its exon 2, wherein the presence of NRF2 mRNA that contains a deletion of all or part of its exon 2 identifies the subject as having cancer; (b) administering to the subject a therapeutically effective amount of an NRF2 pathway antagonist.

62. 62. The method of claim 61, wherein the mRNA expression is determined by PCR, RT-PCR, RNA-seq, gene expression profiling, serial analysis of gene expression, or microarray analysis.

63. 63. The method of claim 62, wherein the mRNA expression is determined by RNA-Seq.

64. 64. The method of any one of claims 61 to 63, further comprising determining the DNA sequence of the NRF2.

65. 65. The method of claim 64, wherein the DNA sequence is determined by PCR, exome-seq, microarray analysis, or whole genome sequencing.

66. 1. A method of treating a subject having cancer, the method comprising: (a) determining in a sample obtained from the subject a DNA sequence of NRF2 that contains a deletion of all or part of its exon 2, wherein the presence of NRF2 DNA that contains a deletion of all or part of its exon 2 identifies the subject as having cancer; (b) administering to the subject a therapeutically effective amount of an NRF2 pathway antagonist.

67. 67. The method of claim 66, wherein the DNA sequence is determined by PCR, exome-seq, microarray analysis, or whole genome sequencing.

68. 68. The method of any one of claims 61 to 67, wherein the NRF2 further comprises a deletion of all or part of exon 3 thereof.

69. 1. A method of treating a subject having cancer, the method comprising: (a) determining in a sample obtained from the subject a protein expression level of NRF2 that contains a deletion of all or part of Neh2, wherein the presence of an NRF2 protein that contains a deletion of all or part of Neh2 identifies the subject as having cancer; (b) administering to the subject a therapeutically effective amount of an NRF2 pathway antagonist.

70. 70. The method of claim 69, wherein the NRF2 protein further comprises a deletion of all or a portion of its Neh4 domain.

71. 71. The method of claim 69 or 70, wherein the protein expression is determined by Western blot, immunohistochemistry, or mass spectrometry.

72. 72. The method of any one of claims 41 to 71, wherein the method comprises administering to the subject a therapeutically effective amount of an anti-cancer agent.

73. 73. The method of claim 72, wherein the anti-cancer agent and the NRF2 pathway antagonist are administered simultaneously.

74. 73. The method of claim 72, wherein the anti-cancer agent and the NRF2 pathway antagonist are administered sequentially.

75. 75. The method of any one of claims 72 to 74, wherein the anti-cancer agent is selected from the group consisting of anti-angiogenic agents, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, and immunotherapy.

76. 76. The method of claim 75, wherein the antiangiogenic agent is a VEGF antagonist.

77. 77. The method of any one of claims 41-76, wherein the NRF2 pathway antagonist is selected from the group consisting of a CREB antagonist, a CREB binding protein (CBP) antagonist, a Maf antagonist, an activating transcription factor 4 (ATF4) antagonist, a protein kinase C (PKC) antagonist, a Jun antagonist, a glucocorticoid receptor antagonist, a UbcM2 antagonist, a HACE1 antagonist, a c-Myc agonist, a SUMO agonist, a KEAP1 agonist, a CUL3 agonist, or a retinoic acid receptor alpha (RARα) agonist.

78. The method of any one of claims 1 to 77, wherein the sample obtained from the subject is a tumor sample.

79. The method of any one of claims 1 to 78, wherein the sample obtained from the subject is a biopsy sample.

80. The method of any one of claims 1 to 79, wherein the sample is obtained from a subject who has not previously been treated.

81. The method of any one of claims 1 to 80, wherein the subject has lung cancer or head and neck cancer.

82. 82. The method of claim 81, wherein the lung cancer is non-small cell lung cancer (NSCLC).

83. 83. The method of claim 82, wherein the NSCLC is squamous NSCLC.

84. 82. The method of claim 81, wherein the head and neck cancer is squamous cell head and neck cancer.

Images