KEAP1 Mutations Research Articles

Systematic Identification of Multi Omics-based Biomarkers in KEAP1 Mutated TCGA Lung Adenocarcinoma.

Mutations in KEAP1 and/or NRF2 genes have been identified across many cancers and the dysregulation of the NRF2 pathway due to these mutations leads to drug and radioresistance in several cancers. Identification of biomarkers associated with these mutations allows the researchers and clinicians to identify the personalized medicine and quicker diagnosis. In this current study, we carried out an integrated, multi-omics, multi-database analysis of exome, transcriptomics data's of KEAP1 mutated TCGA- Lung adenocarcinoma (LUAD) patients against non-mutated counterparts. Finally, we discovered the gene signature associated with KEAP1 mutations, prognostic genes which were highly correlated with the upregulation of the NRF2 pathway in the KEAP1 mutated LUAD patients. Our finding might be useful to identify the early diagnosis of KEAP1 mutated LUAD patients.

Molecular Mechanism of Cellular Oxidative Stress Sensing by Keap1

The Keap1-Nrf2 system plays a central role in the oxidative stress response, however the identity of the reactive oxygen sensor within Keap1 remains poorly understood. Here, we show that a Keap1 mutant lacking eleven cysteine residues retains the ability to target Nrf2 for degradation, but is unable to respond to cysteine-reactive Nrf2 inducers. Of the eleven mutated cysteine residues, we found that four are important for sensing hydrogen peroxide. Our analyses of multiple mutant mice lines, complemented by MEFs expressing a series of Keap1 mutants, revealed that Keap1 utilizes the cysteine residues redundantly to set up an elaborate fail-safe mechanism in which specific combinations of these four cysteine residues can form a disulfide bond to sense hydrogen peroxide. Importantly, this sensing mechanism is distinct from that used for electrophilic Nrf2 inducers, demonstrating that Keap1 is equipped with multiple cysteine-based sensors to detect various endogenous and exogenous stresses.

Clinicopathological, microenvironmental and genetic determinants of molecular subtypes in KEAP1/NRF2-mutant lung cancer.

Somatic KEAP1-NRF2 pathway alterations are frequently detected in both lung adenocarcinomas and squamous cell carcinomas. However, the biological characteristics and molecular subtypes of KEAP1/NRF2-mutant lung cancer remain largely undefined. Here, we performed a stepwise, integrative analytic and experimental interrogation of primary tumors and cancer cell lines harboring KEAP1 or NFE2L2 (encoding NRF2) gene mutations. First, we discovered that KEAP1/NRF2-mutant lung cancer presented APOBEC-mediated mutational signatures, impaired tumor angiogenesis, elevated hypoxic stress and deficient immune-cell infiltrates. Second, gene expression-based subtyping revealed three molecular subsets of KEAP1/NRF2-mutant lung adenocarcinomas and two molecular subsets of KEAP1/NRF2-mutant lung squamous cell carcinomas, each associated with distinguishing genetic, differentiation, immunological and clinicopathological properties. Third, single-sample prediction allowed for de novo identification of KEAP1/NRF2-active tumors within KEAP1/NRF2-wild-type samples. Our data demonstrate that KEAP1/NRF2-mutant lung cancer is a microenvironmentally distinct, biologically heterogeneous, and clinically underestimated disease. These new pathological and molecular insights may accelerate the development of efficacious therapeutic strategies against human malignancies featured by KEAP1-NRF2 pathway activation.

Four-octyl itaconate activates Keap1-Nrf2 signaling to protect neuronal cells from hydrogen peroxide

BackgroundFour-octyl itaconate (OI), the itaconate’s cell-permeable derivative, can activate Nrf2 signaling via alkylation of Keap1 at its cysteine residues. The current study tested the potential neuroprotective function of OI in hydrogen peroxide (H2O2)-treated neuronal cells.MethodsSH-SY5Y neuronal cells and epigenetically de-repressed (by TSA treatment) primary murine neurons were treated with OI and/or H2O2. Nrf2 pathway genes were examined by Western blotting assay and real-time quantitative PCR analysis. Neuronal cell death was tested by the LDH and trypan blue staining assays. Apoptosis was tested by TUNEL and Annexin V assays.ResultsIn SH-SY5Y neuronal cells and primary murine neurons, OI activated Nrf2 signaling, causing Keap1-Nrf2 disassociation, Nrf2 protein stabilization and nuclear translocation, as well as expression of Nrf2-regulated genes (HO1, NQO1 and GCLC) and ninjurin2 (Ninj2). Functional studies showed that OI attenuated H2O2-induced reactive oxygen species (ROS) production, lipid peroxidation and DNA damage as well as neuronal cell death and apoptosis. shRNA-mediated knockdown, or CRISPR/Cas9-induced knockout of Nrf2 almost abolished OI-induced neuroprotection against H2O2. Keap1 is the primary target of OI. Keap1 knockout by CRISPR/Cas9 method mimicked and abolished OI-induced actions in SH-SY5Y cells. Introduction of a Cys151S mutant Keap1 in SH-SY5Y cells reversed OI-induced Nrf2 activation and anti-H2O2 neuroprotection.ConclusionsOI activates Keap1-Nrf2 signaling to protect SH-SY5Y cells and epigenetically de-repressed primary neurons from H2O2 in vitro.

P62-Dependent Phase Separation of Patient-Derived KEAP1 Mutations and NRF2.

Cancer-derived loss-of-function mutations in the KEAP1 tumor suppressor gene stabilize the NRF2 transcription factor, resulting in a prosurvival gene expression program that alters cellular metabolism and neutralizes oxidative stress. In a recent genotype-phenotype study, we classified 40% of KEAP1 mutations as ANCHOR mutants. By immunoprecipitation, these mutants bind more NRF2 than wild-type KEAP1 and ubiquitylate NRF2, but they are incapable of promoting NRF2 degradation. BioID-based protein interaction studies confirmed increased abundance of NRF2 within the KEAP1 ANCHOR mutant complexes, with no other statistically significant changes to the complexes. Discrete molecular dynamic simulation modeling and limited proteolysis suggest that the ANCHOR mutations stabilize residues in KEAP1 that contact NRF2. The modeling supports an intramolecular salt bridge between the R470C ANCHOR mutation and E493; mutation of the E493 residue confirmed the model, resulting in the ANCHOR phenotype. In live cells, the KEAP1 R320Q and R470C ANCHOR mutants colocalize with NRF2, p62/SQSTM1, and polyubiquitin in structured spherical droplets that rapidly fuse and dissolve. Transmission electron microscopy coupled with confocal fluorescent imaging revealed membraneless phase-separated biomolecular condensates. We present a model wherein ANCHOR mutations form p62-dependent biomolecular condensates that may represent a transitional state between impaired proteasomal degradation and autophagy.

Articles of Significant Interest in This Issue

This article refers to:p62-Dependent Phase Separation of Patient-Derived KEAP1 Mutations and NRF2Transient Overexpression of Vascular Endothelial Growth Factor A in Adipose Tissue Promotes Energy Expenditure via Activation of the Sympathetic Nervous System

KEAP1 Mutation Is Correlated to Increased Rates of Local Failure after Radiation Therapy for Spine Metastases From Non-Small Cell Lung Cancer

KEAP1 Mutation Is Correlated to Increased Rates of Local Failure after Radiation Therapy for Spine Metastases From Non-Small Cell Lung Cancer

MA19.09 Concurrent Mutations in STK11 and KEAP1 is Associated with Resistance to PD-(L)1 Blockade in Patients with NSCLC Despite High TMB

MA19.09 Concurrent Mutations in STK11 and KEAP1 is Associated with Resistance to PD-(L)1 Blockade in Patients with NSCLC Despite High TMB

Importance of the Keap1-Nrf2 pathway in NSCLC: Is it a possible biomarker?

Worldwide, lung cancer remains the most common cause of cancer-related mortality, with non-small cell lung cancer (NSCLC) accounting for 85% of all diagnosed lung cancer cases. Chemotherapy is considered the standard of care for patients with advanced NSCLC; however, the tumors can develop mechanisms that inactivate these drugs. Comparative genomic analyses have revealed that disruptions in the kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid-2-related factor-2 (Nrf2) pathway are frequent in NSCLC, although Nrf2 mutations occur less frequently than Keap1 mutations. As the Keap1-Nrf2 pathway appears to be a primary regulator of key cellular processes that aid to resist the action of chemotherapy drugs, the clinical implementation of Nrf2 inhibitors in patients with advanced NSCLC may be a useful therapeutic approach for patients harboring KEAP1-NRF2 mutations. The aim of the present review was to highlight findings of how constitutive Nrf2 activation may be a specific biomarker for predicting patients most likely to benefit from classical chemotherapy drugs, overall improving patient survival rate.

Clinical and Pathological Characteristics of KEAP1- and NFE2L2-Mutated Non-Small Cell Lung Carcinoma (NSCLC).

Purpose:KEAP1 and NFE2L2 mutations are associated with impaired prognosis in a variety of cancers and with squamous cell carcinoma formation in non-small cell lung cancer (NSCLC). However, little is known about frequency, histology dependence, molecular and clinical presentation as well as response to systemic treatment in NSCLC.Experimental Design: Tumor tissue of 1,391 patients with NSCLC was analyzed using next-generation sequencing (NGS). Clinical and pathologic characteristics, survival, and treatment outcome of patients with KEAP1 or NFE2L2 mutations were assessed.Results:KEAP1 mutations occurred with a frequency of 11.3% (n = 157) and NFE2L2 mutations with a frequency of 3.5% (n = 49) in NSCLC patients. In the vast majority of patients, both mutations did not occur simultaneously. KEAP1 mutations were found mainly in adenocarcinoma (AD; 72%), while NFE2L2 mutations were more common in squamous cell carcinoma (LSCC; 59%). KEAP1 mutations were spread over the whole protein, whereas NFE2L2 mutations were clustered in specific hotspot regions. In over 80% of the patients both mutations co-occurred with other cancer-related mutations, among them also targetable aberrations like activating EGFR mutations or MET amplification. Both patient groups showed different patterns of metastases, stage distribution and performance state. No patient with KEAP1 mutation had a response on systemic treatment in first-, second-, or third-line setting. Of NFE2L2-mutated patients, none responded to second- or third-line therapy.Conclusions:KEAP1- and NFE2L2-mutated NSCLC patients represent a highly heterogeneous patient cohort. Both are associated with different histologies and usually are found together with other cancer-related, partly targetable, genetic aberrations. In addition, both markers seem to be predictive for chemotherapy resistance. Clin Cancer Res; 24(13); 3087-96. ©2018 AACR.

Highlights of This Issue

Actinic keratoses (AK) are very common premalignant skin lesions (presquamous cell carcinoma) in patients with chronic sun damage. Recently, physicians have been empirically combining two treatment approaches, topical 5-fluorouracil (5FU) and photodynamic therapy (PDT), but without any scientific basis. In a mechanistic clinical study using surface fluorimetry and skin biopsies, Maytin and colleagues show that sequential 5FU and PDT improves AK clearance by upregulating mitochondrial heme enzymes, increasing target photosensitizer (protoporphyrin IX) expression, and increasing proapoptotic p53. The results provide a scientific rationale for combining two FDA-approved treatments in an effective, practical approach that reduces cutaneous precancer burden.Besides therapeutically targetable mutations in signal transduction pathways, mutations related to the KEAP1/NRF2-pathway are involved in malignant transformation in various cancers, including lung cancer. Based on the NGS-analysis of a large cohort of NSCLC patients, Frank and colleagues show that mutations in KEAP1 and NRF2 occurred nearly mutually exclusive even though strongly linked functionally. They are also associated with different histologies, clinical characteristics, and co-occurring molecular aberrations. Thus, patients with KEAP1- or NRF2-mutations are a heterogenous group. This work argues against a driver function of these mutations but suggests a pivotal role in chemotherapy resistance.Around 50% of non-small cell lung cancer (NSCLC) patients receiving first-generation EGFR inhibitors develop resistance by T790M mutation. Osimertinib has proven effective in overcoming T790M mutation; however, resistance inevitably develops. Yang and colleagues surveyed 416 cancer-related genes in 93 NSCLC patients who progressed on osimertinib by next-generation sequencing (NGS). EGFR mutation on G796/C797, L792 or L718/G719 residues were identified in approximately 1/3 of the patients; further, resistance mechanisms were functionally studied in vitro. In EGFR nonmutated patients, MET, KRAS, and PIK3CA mutations were discovered. This study uncovers novel osimertinib-resistant mechanisms using cell-free DNA-based liquid biopsy and provides grounds for future therapeutic strategies.Dysregulated zinc transport is involved in many malignancies, including pancreatic cancer, and it promotes tumor growth. To further understand the mechanism of zinc transporter-mediated cancer progression, Liu and colleagues used genetic inactivation and rescue assays in human pancreatic cancer cells and mouse models to demonstrate that a zinc importer ZIP4 promotes pancreatic cancer migration and invasion through decreasing ZO-1 and claudin-1 via activation of ZEB1, leading to upregulation of their targets FAK and Paxillin. These findings suggest a novel signaling pathway initiated by ZIP4 controlling pancreatic cancer invasiveness and metastasis, and may be a new therapeutic target for this devastating disease.

Abstract 2408: LKB1 and KEAP1/NRF2 pathways cooperatively promote glutamine dependence and vulnerability to glutaminase inhibitors in KRAS-mutant lung adenocarcinoma

Abstract KRAS is the most commonly mutated oncogenic driver in non-small cell lung cancer (NSCLC) and other solid tumors. Recently we conducted an integrative analysis and found three major subgroups of KRAS-mutated cancer defined by co-occurring genomic events with distinct biology, molecular vulnerabilities, and therapeutic sensitivities. One of these genes, the serine/threonine kinase STK11 (LKB1), represents the second most commonly altered tumor suppressor in NSCLC and there are currently no treatment strategies tailored for LKB1-deficient NSCLC. KRAS-mutant/LKB1-deficient (KL) tumors are characterized by high co-occurrence of KEAP1 mutational inactivation. Inactivation of KEAP1 protects cells against REDOX stress via upregulation of NRF2 target genes, in part by production of glutathione. We evaluated the effects of blocking glutamine metabolism using an isogenic series of NSCLC cell lines harboring mutations in STK11 and KEAP1. Through sequential silencing or overexpression of LKB1, KEAP1, or NRF2 we demonstrated that glutaminase inhibitors (GLSi) can block cell proliferation while increasing energetic and REDOX stress specifically in LKB1 deficient cells with hyperactivation of the KEAP1/NRF2 pathway driven by KEAP1 mutations (KLK subtype). In KLK models, overexpression of LKB1 or KEAP1 partially reduced GLSi sensitivity, while siRNA-mediated down-regulation of NRF2 showed a similar effect. Furthermore, the combination of LKB1 add back coupled with down-regulation of NRF2 conferred even greater resistance to GLSi. To confirm the LKB1/KEAP1-driven response to GLSi, we performed in vivo experiments examining the response of subcutaneous xenografts of an A549 isogenic series; A549 (KLK), A549 LKB1 add back (KK) or A549 KEAP1 add back (KL); to GLS inhibition. These experiments demonstrated that GLSi impaired tumor growth in A549 (KLK) tumors, exhibiting significant statistical differences compared with the vehicle group from 18 days of treatment. Conversely, GLS inhibition did not significantly affect the growth of A549/LKB1 (KK) or A549/KEAP1 (KL) tumors. Collectively, our data indicate that in KLK tumors both pathways, LKB1 and KEAP1/NRF2, cooperatively drive a glutamine-addicted metabolic program, making KLK tumors selectively vulnerable to GLSi treatment. These findings have immediate clinical implications and support the future clinical testing of GLS inhibitors in KLK NSCLC. Citation Format: Ana Galan-Cobo, Piyada Sitthideatphaiboon, Xiao Qu, Jeffrey J. Kovacs, Alissa Poteete, Pan Tong, Sungnam Cho, Varsha V. Gandhi, Ferdinandos Skoulidis, Jing Wang, Timothy P. Heffernan, John V. Heymach. LKB1 and KEAP1/NRF2 pathways cooperatively promote glutamine dependence and vulnerability to glutaminase inhibitors in KRAS-mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2408.

Abstract 4609: High-grade TP53-mutated endometrial carcinomas have decreased NRF2 antioxidant activity

Abstract Background NRF2 is a major transcription factor regulating the expression of antioxidative/detoxifying enzymes. Its activation, due to mutations or activation of signaling pathways, has been associated with chemoresistance and poor prognosis in several tumors. NRF2 status remains unclear in endometrial carcinoma (EC). We aimed to identify molecular alterations associated with NRF2 activation in the four molecular subgroups of EC described by the Cancer Genome Atlas (TCGA), and explored its impact on EC prognosis. Methods Ninety patients treated in Cochin Hospital (2012 to 2017) for EC were included. Formalin-fixed paraffin-embedded tissue samples were processed for histopathological analysis and DNA and RNA extractions. Next generation sequencing (NGS) of NFE2L2 (encoding NRF2), KEAP1 and CUL3 genes and a panel of 15 genes significantly mutated in EC was performed using AmpliseqTM panels on Ion TorrentTM PGM (Thermo Fisher). The nuclear expression of mismatch repair (MMR) and NRF2 proteins were analyzed by immunochemistry (IHC). NRF2 activity was assessed by NQO1, GCLC, and AKR1C3 mRNA expressions, normalized to MRPL19 and TBP housekeeping genes, using TaqManTM assays and quantitative RT-PCR. Clinical event of interest was event-free survival (EFS) (progression, relapse, or death). Results Tumors were classified according to NGS and IHC data as POLE exonuclease domain mutated (N=3, 3%), MMR-deficient (dMMR) (N=28, 31%), TP53 mutated (N=23, 25%), and MMR-proficient tumors (N=32, 36%). NRF2 nuclear immunostaining was not correlated to NRF2 activity. The 3 tumors with highest NRF2 target genes expression harbored known NRF2 pathway activating mutations (NFE2L2 p.W24G, KEAP1 p.R336*, KEAP1 p.D422N). In addition, 2 dMMR tumors showed intermediate/high (1st quartile) NRF2 target genes expression and low allele ratio NFE2L2 mutations, suggesting NRF2 subclonal activation. No correlation was observed between NRF2 activity and PI3K or KRAS pathways mutations. The TP53 subgroup showed a strikingly lower NQO1 expression compared to dMMR or pMMR tumors (ANOVA p<.05). This observation was confirmed by in silico analyses of publicly available TCGA data. NQO1 low expression was significantly associated with poor EFS, independently of tumor stage (Cox p<.01). Conclusion In contrast with previous reports based on IHC, NRF2 activation is a rare event in EC, associated with NFE2L2 or KEAP1 mutations. NQO1 downregulation in the TP53-mutated subgroup might be explained by the interaction of NRF2 with TP53 missense mutants, which has been shown to impair its anti-oxidant transcriptional activity while enhancing proteasome expression (Walerych et al. Nat Cell Biol 2016). Very low levels of NQO1 expression identifies a subset of EC with a poor prognosis, which might be sensitive to specific combination therapies (Liu et al. Nat Commun. 2017). Citation Format: Guillaume Beinse, Pierre-Alexandre Just, Bastien Rance, Brigitte Izac, Franck Letourneur, Nathaniel Edward Saidu, Sandrine Chouzenoux, François Goldwasser, Eric Pasmant, Frederic Batteux, Bruno Borghese, Karen Leroy, Jérome Alexandre. High-grade TP53-mutated endometrial carcinomas have decreased NRF2 antioxidant activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4609.

Abstract 1447: Aberrant tryptophan catabolism marked by high kynureninase expression contributes to immunosuppression and poor outcome in lung adenocarcinoma

Abstract Low tryptophan levels promote tumor immune evasion by suppressing immune function. Elucidation of the immunomodulatory effects of altered tryptophan catabolism has relevance to cancer immunotherapy. Previous studies have largely focused on the role of the rate-limiting enzyme indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan depletion in the microenvironment. However, IDO1 levels are highly heterogeneous in different cancers, and numerous other downstream tryptophan catabolites have also been shown to have immunomodulatory activity. We surveyed the proteomes of 123 cancer cell lines for regulators of tryptophan metabolism which revealed overexpression of kynureninase (KYNU) in KEAP1 mutant lung adenocarcinomas. Functional analysis indicated that KYNU expression is regulated through nuclear factor, erythroid 2-like (Nrf2) activation. KYNU-high cell lines exhibited increased secretion of KYNU-derived anthranilate/3-hydroxyanthranilate. Ex-vivo studies using isolated PBMCs demonstrated that 3-hydroxyanthranilate reduced CD8+ T-cell viability. KYNU mRNA expression in three independent datasets of lung adenocarcinomas and additionally KYNU protein expression in lung adenocarcinoma tissue microarrays revealed that high KYNU expression predicts poor survival and disease recurrence. Our findings indicate that KYNU promotes immune suppression and is an independent prognostic marker for lung adenocarcinoma. Citation Format: Edwin J. Ostrin, Johannes F. Fahrmann, Ichidai Tanaka, Muge Celiktas, Clemente Aguilar, Mitzi Aguilar, Jennifer B. Dennison, Eunice Murage, Satyendra C. Tripathi, Oliver Delgado, Hong Wang, Jaime Rodriguez-Canales, Carmen Behrens, Ignacio I. Wistuba, Ayumu Taguchi, Samir M. Hanash. Aberrant tryptophan catabolism marked by high kynureninase expression contributes to immunosuppression and poor outcome in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1447.

A 5-hydroxyoxindole derivative attenuates LPS-induced inflammatory responses by activating the p38-Nrf2 signaling axis

5-Hydroxyoxindole is a urinary metabolite of indole that exhibits antioxidant activity. In the present study, we found that a 5-hydroxyoxindole derivative (5-HI) significantly inhibited LPS-induced inflammatory effects in the murine macrophage cell line, RAW264.7. 5-HI induced the expression of the transcription factor, Nrf2, which is typically ubiquitinated by Keap1, an adaptor component of the ubiquitin E3 ligase complex, resulting in its proteasomal degradation. By utilizing Keap1−/− MEFs reconstituted with Keap1 mutants harboring substitutions in their major cysteine residues, we clarified the importance of Cys151 in Keap1 as a sensor for 5-HI in the induction of Nrf2 expression. Furthermore, 5-HI induced the activation of the MKK3/6-p38 pathway, which is required for the transcriptional activation of Nrf2. The knockdown of Nrf2 enhanced the LPS-induced expression of inflammatory mediators, including iNOS, NO, and CCL2, and effectively repressed the inhibitory effects of 5-HI on their expression. Although 5-HI and antioxidant N-acetyl cysteine (NAC) both reduced LPS-induced ROS generation, the treatment with NAC did not affect the LPS-induced expression of inflammatory mediators, suggesting that the anti-inflammatory activity of 5-HI mediated by Nrf2 is independent of redox control. Furthermore, when injected into mice with 5-HI, the expression of Nrf2 was significantly increased, and the LPS-induced mRNA expression of CXCL1, CCL2, TNFα, and IL-6 were remarkably inhibited in the kidneys, liver, and lungs, and the production of these cytokines in serum was effectively reduced. Collectively, these results suggest that 5-HI has potential in the treatment of inflammatory diseases through the activation of Nrf2.

Resistance to gefitinib and cross-resistance to irreversible EGFR-TKIs mediated by disruption of the Keap1-Nrf2 pathway in human lung cancer cells.

The development of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) occurs by various mechanisms and appears to be almost inevitable, even in patients with lung cancer who initially respond well to EGFR-TKIs. Consequently, considerable efforts have been made to develop more effective EGFR-TKIs. Therefore, an understanding of the mechanisms behind TKI resistance is essential for improving EGFR-TKI therapeutic efficacy in non-small cell lung cancer (NSCLC) patients. In this study, we discovered that overexpression of antioxidant-responsive element (ARE)-containing Nrf2 target genes by increased transactivation of Nrf2 occurred because of an acquired Keap1 mutation in the gefitinib-resistant (GR) NSCLC cell line we established. These GR cells also acquired cross-resistance to the irreversible EGFR-TKIs, afatinib and osimertinib, and showed increased viability, invasiveness, proliferation, and tumorigenicity both in vitro and in vivo. These results were confirmed by the fact that inhibition of Nrf2 activity, either by treatment with brusatol or by inducing expression of exogenously introduced wild-type Keap1, suppressed tumor cell proliferation and tumorigenicity in vitro and in vivo. Our data suggest that disruption of the Keap1-Nrf2 pathway is one of the mechanisms by which EGFR-TKI resistance occurs, a fact that must be considered when treating patients with EGFR-TKI.-Park, S.-H., Kim, J. H., Ko, E., Kim, J.-Y., Park, M.-J., Kim, M. J., Seo, H., Li, S., Lee, J.-Y. Resistance to gefitinib and cross-resistance to irreversible EGFR-TKIs mediated by disruption of the Keap1-Nrf2 pathway in human lung cancer cells.

Oncogenic mutations in KEAP1 disturbing inhibitory Nrf2-Keap1 interaction: Activation of antioxidative pathway in papillary thyroid carcinoma.

Nuclear factor erythroid 2-like 2 (NFE2L2) encodes Nrf2, transcription factor of antioxidative genes. In the presence of reactive oxygen species, Keap1 (Kelch-ECH-associating protein-1) inhibitor complex undergoes conformational changes disrupting Keap1-Nrf2 binding and Nrf2 translocates into nucleus. We evaluated the presence of mutations in NFE2L2 and KEAP1 in papillary thyroid carcinomas (PTCs) and correlated them with clinical presentation. Coding regions of NFE2L2 and KEAP1 were sequenced in 131 patients with PTC. Clinical and histopathological features were analyzed. Immunohistochemical analysis of Nrf2 expression was performed in mutated carcinomas. Although no mutations were found in NFE2L2, missense mutations in KEAP1 were observed in 6 patients with PTC (4.6%). Immunohistochemistry showed increased Nrf2 expression in nuclei of all mutated carcinomas, which presented poor prognostic features in histopathology. We identified mutations in KEAP1 associated with Nrf2 overexpression in PTC. Mutations favored disruption of inhibitory interaction Nrf2-Keap1 to enable increased antioxidant Nrf2 activity, possibly with prognostic consequences.

P3.02-076 Glutaminase Inhibitor CB-839 Radiosensitizes KRAS-Mutant Lung Cancer Cells in a LKB1- and KEAP1/NRF2-Pathway Dependent Manner

KRAS mutant non-small cell lung cancers (NSCLC) bearing co-mutations in LKB1 (KL tumors) have a distinct biology, molecular vulnerabilities, and therapeutic sensitivities. KL tumors are characterized by upregulation of the NRF2/KEAP1 pathway which may serve as a compensatory mechanism to maintain redox homeostasis in the face of oxidative stress. The NRF2/KEAP1 pathway plays a role in regulating glutathione synthesis from glutamate. We hypothesized that glutaminase inhibition would reduce intracellular glutamate, reduce the ability to tolerate oxidative stress, and enhance radiotherapy sensitivity in KL tumors with KEAP1/NRF2 pathway upregulation. Expression of NRF2 and NRF2 related genes were analyzed by Western blotting in isogenic KRAS-mutant LKB1 deficient/proficient NSCLC cell lines. Intracellular reactive oxygen species (ROS) levels were monitored using 2’7’-dichlorofluorescein (DCFDA) and flow cytometry. Cell apoptosis analysis was determined using PE-conjugated annexin-V/7-AAD staining and carried out by flow cytometry. Cell growth was evaluated by Cell Titer Glo Assays. Ionizing radiation sensitivity was assessed by clonogenic cell survival assay (CSA). LKB1 loss increased ROS accumulation and resulted in up-regulation of NRF2 and NRF2 target genes in isogenic KRAS-mutant cell lines. KL cells were significantly more resistant to ROS than cells with KRAS mutation alone, as determined by the treatment with the ROS donor H2O2. NRF knockdown abrogated this resistance to in KL cells. KL cells also were more resistant to radiotherapy. Re-expression of LKB1 or NRF2 pathway suppression (via KEAP1 expression or NRF2 knockdown) enhanced radiotherapy sensitivity as measured by CSA with a dose enhancement ratio (DER) 1.6 and 1.2, respectively, at a surviving fraction of 0.5. The glutaminase inhibitor CB839 enhanced the radio-sensitivity of KL cells with NRF2 pathway activation (via KEAP1 mutation). Re-expression of LKB1 or NRF2 pathway suppression abrogated the CB839-induced radiosensitization. Our results suggest that upregulation of the NRF2/KEAP1 pathway in NSCLC bearing co-mutations in KRAS and LKB1 is critical to maintain redox homeostasis and GLS inhibition can sensitize KL tumors to radiotherapy in an LKB1- and KEAP1/NRF2-dependent manner.

139 - A Distinct Class of Antioxidant Response Elements is Consistently Activated in Tumors with Oncogenic NRF2 Mutations

The transcription factor NRF2 (encoded by the gene Nuclear Factor, Erythroid 2 Like 2 [NFE2L2]) is a master regulator of the transcriptional response to oxidative stress. In the presence of excess reactive oxygen species, NRF2 induces expression of antioxidant genes via its interaction with regulatory DNA sequences known as antioxidant response elements (AREs). However, oxidative stress is not the only context in which NRF2 can be activated. Somatic mutations that disrupt the interaction between NRF2 and KEAP1, an important inhibitor of NRF2, lead to NRF2 hyperactivation and promote tumorigenesis. The exact mechanisms underlying NRF2’s oncogenic properties remain unclear, but likely involve aberrant expression of select NRF2 target genes. We tested this possibility using an integrative genomics approach, combining our own NRF2-centric functional genomics data with thousands of tumor genome and transciptome profiles from The Cancer Genome Atlas (TCGA), to get a precise view of the direct NRF2 target genes dysregulated in tumors with NRF2 hyperactivating mutations. This approach revealed a core set of 32 direct NRF2 targets that are consistently upregulated in NRF2 hyperactivated tumors. This set of NRF2 “cancer target genes” includes canonical redox-related NRF2 targets (NQO1, TRX, GCLC, etc.), as well as several target genes that have not been previously linked to NRF2 activation; NRF2-driven upregulation of this gene set is largely independent of the organ system where the tumor developed. This critical subset of NRF2 targets is significantly enriched for genes functionally linked to oncogenesis, and several features differentiate these genes from other NRF2 targets. One of the key distinguishing features is that the NRF2 cancer target genes are regulated by high affinity, switch-like AREs that fall within genomic regions that possess a distinct, ubiquitously permissive chromatin signature. This finding implies that the NRF2 cancer target genes are highly responsive to oncogenic NRF2 in most tissues because the AREs at these loci lack the epigenetic constraints that restrict expression of most other NRF2 targets genes. Notably, the NRF2 cancer target gene set also serves as a reliable proxy for NRF2 activity. Identification of tumors with hyperactive NRF2 is difficult because there are many routes to its activation – mutations in NRF2, KEAP1, or CUL3; epigenetic silencing of KEAP1; etc. – so we used this 32 gene set to infer NRF2 activity based on RNA-seq data from thousands of tumors profiled by the TCGA project. This approach revealed that high NRF2 activity is associated with significant decreases in survival in at least 10 different cancers, including liver hepatocellular carcinoma, papillary kidney carcinoma, urothelial bladder cancer, and head and neck squamous cell carcinoma. Overall, the pervasive upregulation of these NRF2 cancer targets across multiple cancers, and their association with negative prognostic outcomes, suggest that these genes will be central to dissecting the functional implications of NRF2 hyperactivation in several cancer contexts.

Keap1 loss promotes Kras-driven lung cancer and results in dependence on glutaminolysis.

Treating KRAS-mutant lung adenocarcinoma (LUAD) remains a major challenge in cancer treatment given the difficulties associated with directly inhibiting the KRAS oncoprotein1. One approach to addressing this challenge is to define frequently co-occurring mutations with KRAS, which themselves may lead to therapeutic vulnerabilities in tumors. Approximately 20% of KRAS-mutant LUAD tumors carry loss-of-function (LOF) mutations in Kelch-like ECH-associated protein 1 (KEAP1)2-4, a negative regulator of nuclear factor erythroid 2-like 2 (NFE2L2; hereafter NRF2), which is the master transcriptional regulator of the endogenous antioxidant response5-10. The high frequency of mutations in KEAP1 suggests an important role for the oxidative stress response in lung tumorigenesis. Using a CRISPR/Cas9-based approach in a mouse model of Kras-driven LUAD we examined the effects of Keap1 loss in lung cancer progression. We show that loss of Keap1 hyper-activates Nrf2 and promotes Kras-driven LUAD. Combining CRISPR/Cas9-based genetic screening and metabolomic analyses, we show that Keap1/Nrf2-mutant cancers are dependent on increased glutaminolysis, and this property can be therapeutically exploited through the pharmacological inhibition of glutaminase. Finally, we provide a rationale for sub-stratification of human lung cancer patients with KRAS-KEAP1 or -NRF2-mutant tumors as likely to respond to glutaminase inhibition.