KEAP1 Mutations Research Articles

Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer

The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these is NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain, and we demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable transcriptional regulator that supports NRF2-dependent lung cancers.

Simultaneous K-ras activation and Keap1 deletion cause atrophy of pancreatic parenchyma.

The Kelch-like ECH-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) system has a wide variety of effects in addition to the oxidative stress response, such as growth promotion and chemoresistance of cancer cells. Nrf2 is constitutively activated in most cancer cells. However, the activation of Nrf2 together with oncogenic mutations does not always result in cancer promotion. K-rasLSL-G12D/+:: p53LSL-R172H/+:: Pdx-1-Cre (KPC) mice are an established model of pancreatic cancer that specifically express mutants of both K-ras and p53 in the pancreas by using Pdx-1-Cre. We here generated Pdx-1-Cre::K-rasLSL-G12D/+:: Keap1fl/fl (KC::Keap1) and KPC:: Keap1fl/fl (KPC::Keap1) mice in which Nrf2 is constitutively activated by Keap1 deletion. KC::Keap1 and KPC::Keap1 mice started to die or showed obvious weakness at approximately around 40 days after birth. Histological examination revealed that KC::Keap1 and KPC::Keap1 mice did not develop pancreatic cancer but, instead, progressive atrophy of the pancreatic parenchyma. In these mice, amylase-positive acinar cells as well as insulin- and glucagon-positive islet cells were decreased and surrounded by fibrotic tissues. KC::Keap1 and KPC::Keap1 mice presented lower body weight and glucose levels than C::Keap1 mice, presumably resulting from pancreatic exocrine insufficiency. Histological changes were not obvious in C::Keap1 and PC::Keap1 mice. The presence of the p53 mutation did not affect the phenotypes in KC::Keap1 mice. Heterologous or homologous Nrf2 deletion ( Nrf2+/- or Nrf2-/-) rescued the pancreatic phenotypes, weight loss, and hypoglycemia in KC::Keap1 mice, suggesting that Nrf2 is a major downstream target of Keap1. In conclusion, simultaneous K-ras activation and Keap1 deletion caused progressive atrophy of the pancreatic parenchyma in mice. NEW & NOTEWORTHY Aberrant activation of the Kelch-like ECH-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) system usually promotes carcinogenesis, and we assumed that simultaneous activation of K-ras and Nrf2 might promote pancreatic carcinogenesis. Conditional expression of mutant K-ras and Keap1 deletion did not result in pancreatic cancer development. Instead, these mice developed progressive loss of pancreatic parenchyma, accompanied by body weight loss and hypoglycemia, presumably because of pancreatic exocrine insufficiency. Nrf2 activation by Keap1 deletion concomitant with K-ras activation cause pancreatic atrophy.

Abstract 1034: Role of KEAP1/NRF2 and TP53 mutations in lung squamous cell carcinoma development and radiation resistance

Abstract Although lung squamous cell carcinomas (LSCC) comprise a large fraction of non-small cell lung cancers (NSCLCs), their pathogenesis and cell of origin remain incompletely understood and biomarkers that predict therapeutic responses are lacking. Here we describe novel, clinically relevant murine LSCC models driven by inactivation of Trp53 with or without Keap1, both of which are frequently mutated in human LSCCs. Homozygous inactivation of Keap1 or Trp53 promoted airway basal stem cell (ABSC) self-renewal both in vitro and in in vivo, suggesting that Trp53 or Keap1 mutations lead to expansion of mutant stem cell clones. Deletion of Trp53 with or without Keap1 in ABSCs, but not more differentiated tracheal cells, produced tumors recapitulating histologic and molecular features of human LSCCs. However, deletion of Trp53 with or without Keap1 in type II pneumocytes (ATIIs) or bronchioalveolar stem cells (BASCs) produced tumors with the features of adenocarcinoma, indicating that ABSCs represent the likely cell of origin for LSCC in this model. Deletion of Keap1 promoted tumor growth, metastasis and resistance to oxidative stress. N-acetylcysteine (NAC) treatment enhanced tumorsphere formation and metastasis in Keap1WT LSCCs, but not in Keap1-/- LSCCs, suggesting that NRF2-ROS pathway activation is the main mediator of Keap1 loss. Finally, Keap1 deletion induced radioresistance in vitro and in vivo in both LSCCs and lung adenocarcinomas (LUADs). Congruous with these findings, KEAP1/NRF2 mutation status strongly predicted risk of local recurrence in NSCLC patients treated with RT and these mutations could be non-invasively identified in circulating tumor DNA. These data suggest that Trp53 and Keap1 mutations in ABSCs play important roles in LSCC initiation and progression and identify KEAP1/NRF2 mutations as predictive biomarkers that could be used for personalization of therapeutic strategies for NSCLCs, and likely other cancers in which they are recurrently mutated. Citation Format: Youngtae Jeong, Ngoc Hoang, Henning Stehr, Alexander Lovejoy, Andrew Gentles, Aadel Chaudhuri, Billy Loo, Ash Alizadeh, Maximilian Diehn. Role of KEAP1/NRF2 and TP53 mutations in lung squamous cell carcinoma development and radiation resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1034. doi:10.1158/1538-7445.AM2017-1034

Abstract 593: Cross-platform integrative analysis of NSCLC reveals association between PD-L1 expression patterns and tumor genetic profile

Abstract Background: Immunotherapy with PD-1 inhibitors is associated with increased survival in NSCLC. While PD-L1 expression enriches for clinical benefit, additional predictive biomarkers are critically needed to further define patient subsets associated with specific response/resistance profiles and to develop optimal combination strategies. We hypothesized that integration of pathology features derived from H&E and PD-L1 immunohistochemistry with genetic/genomic data from NSCLC could identify phenotypic/genotypic associations that highlight specific immunosuppressive mechanisms. Towards that end, we present data from an initial pilot analysis. Methods: Twenty-nine NSCLC (23 adenocarcinoma; 6 squamous cell carcinoma) were included. H&E-stained sections were scored for tumor grade (1-3), relative proportion of tumor stroma (stroma score 1-3), percent necrosis and intensity of chronic inflammatory infiltrate (1-3+). PD-L1 immunohistochemistry was performed using the DAKO 28-8 antibody. PD-L1 expression was scored in tumor cells by modified H-score and by the predominant pattern observed - diffuse, heterogeneous, tumor-stroma interface or negative, and in immune cells using a semi-quantitative intensity scale (1-3+). DNA and RNA extracts from FFPE tissue were subjected to whole exome sequencing and RNAseq from which mutation load, oncogene/tumor suppressor genotypes and inflammation signatures were derived. Results: The highest PD-L1 H-scores were associated with diffuse expression patterns and were observed in high grade tumors (Grade 3). Deleterious mutations in TP53, STK11, KEAP1, KRAS, EGFR and MET occurred at expected frequencies. These mutations occurred across multiple PD-L1 expression patterns except for STK11 which was restricted to PD-L1 negative tumors (p=0.077, Fisher exact test). STK11-mutant tumors also displayed a lower PD-L1+ inflammation score (p=0.046, Fisher exact test). Trends toward an increase in mutation load with increasing levels of chronic inflammation on H&E stain as well as with PD-L1+ chronic immune infiltrates were noted. Inflammation signatures derived from RNAseq showed an association between diffuse PD-L1 expression by IHC and the highest levels of inflammation mRNA signatures comprising T-cells (CD8, Tregs), B-cells, macrophages and MDSCs. Conclusions: The potential association between tumor grade, PD-L1 expression, intensity of the immune infiltrate and mutation load raise the possibility that tumor morphology predicts mutation load and associated immune response. The finding that STK11 mutation is restricted to PD-L1 negative tumors suggests an immunosuppressive mechanism is invoked in this setting. This dataset is currently being expanded to establish the significance of these findings. Citation Format: Robin Edwards, Patrik Vitazka, Peter Szabo, Han Chang, John Cogswell, Hao Tang, Kaushal Desai, Darren Locke, Qiuyan Wu, Joseph Szustakowski, Steven Bernstein, Michele Cleary. Cross-platform integrative analysis of NSCLC reveals association between PD-L1 expression patterns and tumor genetic profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 593. doi:10.1158/1538-7445.AM2017-593

Abstract 1434: Mutational landscape in the normal-appearing airway cancerization field of early-stage non-small cell lung cancer

Abstract Lung cancer, of which non-small cell lung cancer (NSCLC) is the most common form, is the second most prevalent cancer in the U.S. and the leading cause of cancer mortality. Field cancerization phenomenon establishes that normal appearing tissues surrounding a tumor will exhibit a field effect – particularly in smokers – with the tissues proximal to the tumor showing more, and even sharing, alterations with the tumor. Previous studies in NSCLC provide evidence for both complex progression trajectories, yet, precise mechanisms remain unknown. To investigate the field cancerization phenomenon, we conducted a genome-wide survey of acquired DNA alterations in normal-appearing tissues including small airways adjacent to NSCLC, mainstem bronchi (large airways), nasal epithelium, distant normal lung and blood, along with multiple samples from their surgically resected paired tumors, for a total of 500 samples from 48 patients with early-stage NSCLC (11 squamous cell carcinomas and 37 adenocarcinomas). Since we expect low mutation burdens and mutant cell fractions in pathologically normal tissues, we assessed somatic point mutations via deep targeted sequencing of 409 genes and paired these data with our recent study of acquired copy number alterations (CNAs) inferred from high-density whole-genome SNP microarrays, thus offering a deep and wide survey of somatic DNA alterations. After aggregating results from multiple mutation callers, we observed somatic mutations (exonic, splicing and UTRs) in 257 samples of which 65 (in 35 patients) were from normal-appearing field (non-tumor, non-blood) samples. Tumor and field samples in smokers showed a higher mutation burden and large proportion of C:G>A:T changes. We also observed concordance of mutations in the airway and corresponding tumor profiles. Further, a statistically significant field effect was established with mutational burden (measured by the variant allele frequency) increasing with proximity to the tumor. Among the 35 NSCLC cases with field mutations, the small adjacent (to tumor) airways in six patients exhibited mutations in lung cancer drivers such as KRAS, STK11, TP53 and KEAP1. We also identified mutations in the large airway (TP53, SETD2, CDKN2A), distant normal lung parenchyma (RB1, RET) and nasal epithelium (AKT1). We then correlated point mutation and CNA mutation profiles. Of eight cases showing a nonsynonymous or stopgain variants in established lung cancer drivers in the airways, four exhibited putative “two-hit” progression models, e.g. both KEAP1 and STK11 mutations with 19p loss, overlapping TP53 mutation/17p loss, or KRAS mutation/12p gain. Our findings in normal-appearing tissues of the respiratory epithelium offer insights into the earliest mutational events in their progression to NSCLC and, possibly, in tumor relapse and that may represent suitable targets for early detection and chemoprevention. Citation Format: Smruthy Sivakumar, Yasminka Jakubek, Wenhua Lang, Tina McDowell, Melinda M. Garcia, Chi-Wan Chow, Zachary Weber, Carmen Behrens, Neda Kalhor, Cesar Moran, Randa El-Zein, Gareth Davies, Junya Fujimoto, Reza Mehran, Stephen G. Swisher, Jing Wang, Avrum E. Spira, Jerry Fowler, F Anthony San Lucas, Ignacio I. Wistuba, Erik Ehli, Paul Scheet, Humam Kadara. Mutational landscape in the normal-appearing airway cancerization field of early-stage non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1434. doi:10.1158/1538-7445.AM2017-1434

Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma with fully annotated clinical follow-up

Lung adenocarcinomas (LUADs) lead to the majority of deaths attributable to lung cancer. We performed whole-exome sequencing (WES) and immune profiling analyses of a unique set of clinically annotated early-stage LUADs to better understand the pathogenesis of this disease and identify clinically relevant molecular markers. We performed WES of 108 paired stage I-III LUADs and normal lung tissues using the Illumina HiSeq 2000 platform. Ten immune markers (PD-L1, PD-1, CD3, CD4, CD8, CD45ro, CD57, CD68, FOXP3 and Granzyme B) were profiled by imaging-based immunohistochemistry (IHC) in a subset of LUADs (n = 92). Associations among mutations, immune markers and clinicopathological variables were analyzed using ANOVA and Fisher's exact test. Cox proportional hazards regression models were used for multivariate analysis of clinical outcome. LUADs in this cohort exhibited an average of 243 coding mutations. We identified 28 genes with significant enrichment for mutation. SETD2-mutated LUADs exhibited relatively poor recurrence- free survival (RFS) and mutations in STK11 and ATM were associated with poor RFS among KRAS-mutant tumors. EGFR, KEAP1 and PIK3CA mutations were predictive of poor response to adjuvant therapy. Immune marker analysis revealed that LUADs in smokers and with relatively high mutation burdens exhibited increased levels of immune markers. Analysis of immunophenotypes revealed that LUADs with STK11 mutations exhibited relatively low levels of infiltrating CD4+/CD8+ T-cells indicative of a muted immune response. Tumoral PD-L1 was significantly elevated in TP53 mutant LUADs whereas PIK3CA mutant LUADs exhibited markedly down-regulated PD-L1 expression. LUADs with TP53 or KEAP1 mutations displayed relatively increased CD57 and Granzyme B levels indicative of augmented natural killer (NK) cell infiltration. Our study highlights molecular and immune phenotypes that warrant further analysis for their roles in clinical outcomes and personalized immune-based therapy of LUAD.

Mutation profiles in early-stage lung squamous cell carcinoma with clinical follow-up and correlation with markers of immune function

Lung squamous cell carcinoma (LUSC) accounts for 20–30% of non-small cell lung cancers (NSCLCs). There are limited treatment strategies for LUSC in part due to our inadequate understanding of the molecular underpinnings of the disease. We performed whole-exome sequencing (WES) and comprehensive immune profiling of a unique set of clinically annotated early-stage LUSCs to increase our understanding of the pathobiology of this malignancy. Matched pairs of surgically resected stage I-III LUSCs and normal lung tissues (n = 108) were analyzed by WES. Immunohistochemistry and image analysis-based profiling of 10 immune markers were done on a subset of LUSCs (n = 91). Associations among mutations, immune markers and clinicopathological variables were statistically examined using analysis of variance and Fisher’s exact test. Cox proportional hazards regression models were used for statistical analysis of clinical outcome. This early-stage LUSC cohort displayed an average of 209 exonic mutations per tumor. Fourteen genes exhibited significant enrichment for somatic mutation: TP53, MLL2, PIK3CA, NFE2L2, CDH8, KEAP1, PTEN, ADCY8, PTPRT, CALCR, GRM8, FBXW7, RB1 and CDKN2A. Among mutated genes associated with poor recurrence-free survival, MLL2 mutations predicted poor prognosis in both TP53 mutant and wild-type LUSCs. We also found that in treated patients, FBXW7 and KEAP1 mutations were associated with poor response to adjuvant therapy, particularly in TP53-mutant tumors. Analysis of mutations with immune markers revealed that ADCY8 and PIK3CA mutations were associated with markedly decreased tumoral PD-L1 expression, LUSCs with PIK3CA mutations exhibited elevated CD45ro levels and CDKN2A-mutant tumors displayed an up-regulated immune response. Our findings pinpoint mutated genes that may impact clinical outcome as well as personalized strategies for targeted immunotherapies in early-stage LUSC.

Abstract B04: NRF2 regulates serine biosynthesis in pancreatic ductal adenocarcinoma

Abstract Tumors have high energetic and anabolic needs for rapid growth and proliferation, and the serine biosynthetic pathway has recently been identified as an important source of metabolic intermediates for these processes. We recently demonstrated that a substantial fraction of NSCLC cell lines displayed significant serine/glycine biosynthetic activity due to KEAP1 and NRF2 mutations (DeNicola et al. Nature Genetics 2015). We demonstrated that NRF2 regulates glucose flux to serine by controlling transcription of serine biosynthetic genes through the transcription factor ATF4. Importantly, elevated expression of these genes conferred poor prognosis in human NSCLC. Thus, a substantial fraction of human NSCLC activates an NRF2-dependent transcriptional network that regulates serine and glycine metabolism and is linked to clinical aggressiveness. Pancreatic ductal adenocarcinoma (PDAC) also demonstrates high NRF2 activity due to mutations in KRAS and high MAPK signaling (DeNicola et al. Nature 2011). We demonstrate that serine biosynthesis enzyme expression is elevated in a mouse model of PDAC and that NRF2 regulates PHGDH expression in human PDAC cell lines. Furthermore, PDAC cell lines are resistant to serine starvation. PHGDH knockdown impairs the growth of tumor organoid orthotopic transplants, and the role of PHGDH in tumor maintenance is currently being investigated. These results suggest that the serine biosynthesis pathway is regulated by NRF2 in multiple cancers and may be an important therapeutic target. Citation Format: Gina M. DeNicola, Pei-Hsuan Chen, Edouard Mullarky, Christine Chio, John M. Asara, John D. Minna, David A. Tuveson, Ralph J. DeBerardinis, Lewis C. Cantley.{Authors}. NRF2 regulates serine biosynthesis in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B04.

Alkylating Agent-Induced NRF2 Blocks Endoplasmic Reticulum Stress-Mediated Apoptosis via Control of Glutathione Pools and Protein Thiol Homeostasis.

Alkylating agents are a commonly used cytotoxic class of anticancer drugs. Understanding the mechanisms whereby cells respond to these drugs is key to identify means to improve therapy while reducing toxicity. By integrating genome-wide gene expression profiling, protein analysis, and functional cell validation, we herein demonstrated a direct relationship between NRF2 and Endoplasmic Reticulum (ER) stress pathways in response to alkylating agents, which is coordinated by the availability of glutathione (GSH) pools. GSH is essential for both drug detoxification and protein thiol homeostasis within the ER, thus inhibiting ER stress induction and promoting survival, an effect independent of its antioxidant role. NRF2 accumulation induced by alkylating agents resulted in increased GSH synthesis via GCLC/GCLM enzyme, and interfering with this NRF2 response by either NRF2 knockdown or GCLC/GCLM inhibition with buthionine sulfoximine caused accumulation of damaged proteins within the ER, leading to PERK-dependent apoptosis. Conversely, upregulation of NRF2, through KEAP1 depletion or NRF2-myc overexpression, or increasing GSH levels with N-acetylcysteine or glutathione-ethyl-ester, decreased ER stress and abrogated alkylating agents-induced cell death. Based on these results, we identified a subset of lung and head-and-neck carcinomas with mutations in either KEAP1 or NRF2/NFE2L2 genes that correlate with NRF2 target overexpression and poor survival. In KEAP1-mutant cancer cells, NRF2 knockdown and GSH depletion increased cell sensitivity via ER stress induction in a mechanism specific to alkylating drugs. Overall, we show that the NRF2-GSH influence on ER homeostasis implicates defects in NRF2-GSH or ER stress machineries as affecting alkylating therapy toxicity. Mol Cancer Ther; 15(12); 3000-14. ©2016 AACR.

Abstract 122: Comparative analysis of small cell lung cancer and other pulmonary neuroendocrine tumors

Abstract Small cell lung cancer (SCLC) is accounting for 15% of all lung cancer cases and belongs to the family of pulmonary neuroendocrine tumors. This tumor entity additionally includes large-cell neuroendocrine carcinomas (LCNEC) and pulmonary carcinoids (PCA), which occur in 3% and 2% of all lung cancer patients, respectively. Whereas lung carcinoids are clinically benign, SCLC and LCNEC are characterized as high-grade malignant tumors, which are associated with heavy smoking and a poor 5-year survival rate of less than 5%. We aimed to characterize the genomic alterations in neuroendocrine lung tumors and performed whole genome, whole exome and transcriptome sequencing on up to 110 SCLC cases, over 40 pulmonary carcinoids and over 50 LCNEC tumors. SCLC and LCNEC tumors reveal high mutation rates with an average of 9.5 and 8.6 mutations per megabase, respectively. Our sequencing studies have shown that SCLC tumors are characterized by bi-allelic inactivation of TP53 and RB1 in almost 100% of the cases. Recurrent significant genomic alterations affected among others TP73 (13%) and NOTCH family genes (25%). Further studies in a pre-clinical mouse model confirmed NOTCH as tumor suppressor and as a regulator of neuroendocrine differentiation in SCLC (George et al., 2015). LCNEC tumors showed TP53 and RB1 alterations in up to 20% of the cases. Additionally, LCNECs were found with STK11 and KEAP1 mutations, which occurred mutually exclusive to RB1 alterations (P<0.05). In light of these distinct mutational characteristics, we analyzed the transcriptome sequencing data of neuroendocrine lung tumors in comparison to lung adenocarcinomas (LUAD) and lung squamous cell carcinomas (LUSQ). An unsupervised clustering approach led to the identification of five expression clusters: LUAD, LUSQ and PCA formed distinct transcriptional classes; the majority of SCLC and LCNEC tumors clustered into two subgroups. While a few LCNEC tumors clustered with LUAD, LUSQ and PCA, RB1-mutated LCNEC tumors predominantly clustered with SCLC samples (P <0.05). KEAP1 and STK11 mutated LCNECs did not segregate with a particular histological tumor type. Neuroendocrine lung tumors confirmed high expression levels of neuroendocrine markers. A detailed comparison of LCNEC and SCLC tumors revealed that the majority of the SCLC and a few LCNEC tumors were found in a transcriptional group that showed high expression of the neuroendocrine lineage transcription factor ASCL1, gastrin releasing peptide (GRP) and DLL3, which was less pronounced in transcriptional subsets formed by the majority of LCNEC tumors. Patient tumors that clustered to the transcriptional class marked by higher ASCL1 expression were identified to have a worse overall survival (P<0.05). In summary, these findings point to transcriptional differences in SCLC and LCNEC tumors and emphasize the precise analysis of these histological tumor types with respect to their molecular biology and therapeutic treatment option. Citation Format: Julie George, Lynnette Fernandez-Cuesta, Vonn Walter, Neil Hayes, Roman Thomas. Comparative analysis of small cell lung cancer and other pulmonary neuroendocrine tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 122.

Abstract 142: Mutation and immune profiles in early-stage lung squamous cell carcinoma

Abstract PURPOSE: Lung squamous cell carcinoma (LUSC) accounts for 20-30% of non-small cell lung cancers (NSCLCs). There are limited treatment strategies for LUSC in part due to our inadequate understanding of the molecular underpinnings of the disease. We sought to perform whole-exome sequencing (WES), comprehensive immune profiling and clinicopathological analysis of early-stage LUSCs to increase our understanding of the pathobiology of this malignancy. METHODS: Matched pairs of surgically resected stage I-III LUSCs and normal lung tissues (n = 108) were analyzed by WES. Immunohistochemistry and image analysis-based profiling of 10 immune markers was done on a subset of LUSCs (n = 91). Associations among mutations, immune markers and clinicopathological variables were statistically examined using ANOVA and Fisher tests. Cox proportional hazards regression models were used for statistical analysis of clinical outcome. RESULTS: This early-stage LUSC cohort displayed an average of 209 exonic mutations per tumor. Fourteen genes exhibited significant enrichment for mutation: TP53, MLL2, PIK3CA, NFE2L2, CDH8, KEAP1, PTEN, ADCY8, PTPRT, CALCR, GRM8, FBXW7, RB1 and CDKN2A. Among mutated genes associated with poor recurrence-free survival, MLL2 mutations predicted poor prognosis in both TP53 mutant and wild type LUSCs. We also found that in treated patients, FBXW7 and KEAP1 mutations were associated with poor response to adjuvant therapy, particularly in TP53-mutant tumors. Analysis of mutations with immune markers revealed that ADCY8 and PIK3CA mutations were associated with markedly decreased tumoral PD-L1 expression, LUSCs with PIK3CA mutations exhibited elevated CD45ro levels and CDKN2A-mutant tumors displayed an up-regulated immune response. CONCLUSION: Our findings pinpoint mutated genes that may impact clinical outcome as well as personalized strategies for targeted immunotherapies in early-stage LUSC. Citation Format: Murim Choi, Humam Kadara, Jiexin Zhang, Edwin Parra Cuentas, Jaime Rodriguez Canales, Stephen G. Gaffney, Zi-Ming Zhao, Carmen Behrens, Junya Fujimoto, Chi-Wan Chow, Neda Kalhor, Cesar Moran, David Rimm, Stephen Swisher, Don L. Gibbons, John V. Heymach, Edward Kaftan, Jeffrey Townsend, Thomas J. Lynch, Joseph Schlessinger, J. Jack Lee, Richard Lifton, Roy S. Herbst, Ignacio I. Wistuba. Mutation and immune profiles in early-stage lung squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 142.

Abstract 89: Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma

Abstract PURPOSE: Lung adenocarcinomas (LUADs) lead to the preponderance of deaths attributable to lung cancer. We performed whole-exome sequencing (WES), comprehensive immune profiling and clinicopathological analysis of LUADs to better understand the molecular pathogenesis of this disease and identify clinically relevant molecular markers. METHODS: We performed WES of 108 paired surgically resected stage I-III LUADs and normal lung tissues using the Illumina HiSeq 2000 platform. Additionally, ten immune related markers (PD-L1, PD-1, CD3, CD4, CD8, CD45ro, CD57, CD68, FOXP3 and Granzyme B) were profiled by imaging-based immunohistochemistry in a subset of LUADs (n = 92). Associations among mutations, immune markers and clinicopathological variables were analyzed using ANOVA and Fishers Exact tests. Cox proportional hazards regression models were employed for multivariate analysis of clinical outcome. RESULTS: LUADs in this cohort exhibited an average of 243 coding mutations per tumor. We identified 28 genes with significant enrichment for mutation. SETD2-mutant LUADS exhibited relatively poor recurrence-free survival (RFS) and mutations in STK11 and ATM were associated with poor RFS in KRAS-mutant tumors. EGFR, KEAP1 and PIK3CA mutations were predictive of poor response to adjuvant therapy. Immune marker analysis demonstrated that PD-L1 expression was increased in smoker compared to non-smoker LUADs and, along with other immune markers, was positively correlated with somatic mutation burden. Moreover, immune marker levels including PD-L1 were elevated in TP53-mutant LUADs. In contrast, STK11 and U2AF1 mutant tumors exhibited a suppressed immune response and LUADs with PIK3CA mutations exhibited markedly decreased tumoral PD-L1 expression. CONCLUSION: Our study highlights mutations that may impact clinical outcome and personalized strategies for immune-based therapy of early-stage LUAD patients. Citation Format: Humam Kadara, Murim Choi, Jiexin Zhang, Edwin Parra Cuentas, Jaime Rodriguez Canales, Stephen Gaffney, Zi-Ming Zhao, Carmen Behrens, Junya Fujimoto, Chi-Wan Chow, Neda Kalhor, Cesar Moran, David Rimm, Stephen G. Swisher, Don L. Gibbons, John V. Heymach, Edward Kaftan, Jeffrey Townsend, Thomas J. Lynch, Joseph Schlessinger, J. Jack Lee, Richard Lifton, Ignacio I. Wistuba, Roy S. Herbst. Whole-exome sequencing and immune profiling of early-stage lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 89.

Abstract LB-311: Elevated kynureninase protein expression is associated with KEAP1 mutant non-small cell lung cancer and is predictive of poor overall survival

Abstract Purpose: To identify signatures related to tryptophan metabolism independent of indoleamine 2,3-dioxygenase (IDO) that are predictive of tumor behavior. Introduction: Recent evidence has pointed to the importance of tryptophan catabolites on immune suppression and promotion of tumor growth. IDO1 is a rate-limiting enzyme in tryptophan catabolism currently targeted in clinical trials. Tryptophan catabolism can occur in IDO-negative tumors and resulting metabolites may have immunosuppressive and potential autocrine functions. Our late breaking finding is that we have uncovered signatures related to tryptophan metabolism independent of IDO that impact clinical outcome in NSCLC. Experimental Procedures: We initially carried out integrated genomic and proteomic profiling of 114 cancer cell lines. Small interfering RNA transfection experiments were used to evaluate the effect of KYNU knockdown on cell viability. TCGA data was analyzed to determine clinical relevance. Results: We identified kynureninase (KYNU), an enzyme involved in tryptophan metabolism that mediates formation of anthranilate and 3-hydroxyanthranilate, as uniquely overexpressed at the RNA and protein levels in NSCLC compared to other cancer types. These findings were not conserved for IDO or tryptophan 2,3-dioxygenase (TDO2), rate-limiting enzymes in tryptophan metabolism. Knockdown of KYNU reduced cell viability of NSCLC cell lines tested. Upregulation of KYNU was highly associated with KEAP1 mutational status and positively correlated with numerous Nrf-2 regulated-genes at both the protein and mRNA level. These findings were confirmed using TCGA data. KYNU mRNA levels predicted worse outcomes in stage II and III NSCLC adenocarcinoma and squamous cell carcinoma in TCGA cohorts independently of other covariates (P = : 0.008). Conclusion: We have uncovered a unique signature in NSCLC reflective of aberrant tryptophan metabolism mediated by elevated KYNU but not IDO/TDO2. KYNU expression is highly associated with KEAP1 mutational status. High KYNU mRNA levels was predictive of worse clinical outcomes in NSCLC. Citation Format: Johannes F. Fahrmann, Jennifer Dennison, Eunice Murage, Hong Wang, Edwin J. Ostrin, Ayumu Taguchi, Samir Hanash. Elevated kynureninase protein expression is associated with KEAP1 mutant non-small cell lung cancer and is predictive of poor overall survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-311.

Methylseleninic acid induces NAD(P)H:quinone oxidoreductase-1 expression through activation of NF-E2-related factor 2 in Chang liver cells.

Selenium has been reported to induce the expression of some cytoprotective enzymes, which may account for its chemoprotective and chemopreventive effects. However, it remains largely unresolved whether these effects are exerted by selenium itself or mediated by its metabolite(s). In the present study, methylseleninic acid (MSeA), a monomethylated selenium, induced the expression of NAD(P)H:quinone oxidoreductase-1 (NQO-1) in human Chang liver cells. Expression of NQO-1 and other antioxidant/stress response genes is primarily regulated by the transcription factor NF-E2-related factor2 (Nrf2). Exposure of human Chang liver cells to MSeA (3 μM) increased nuclear translocation of Nrf2 and binding to antioxidant response elements. Silencing Nrf2 markedly reduced the MSeA-induced NQO-1 expression. In comparison with embryonic fibroblasts from Nrf2 wild-type mice, those from Nrf2 knockout mice failed to induce NQO-1 expression when treated with MSeA. Moreover, MSeA treatment enhanced ubiquitination of Keap1, but repressed Nrf2 ubiquitination. Pretreatment of cells with dithiothreitol abrogated the MSeA-induced NQO-1 expression, suggesting that MSeA causes Keap1 thiol modification. MSeA-induced NQO-1 upregulation was attenuated in cells harbouring the mutant Keap1 in which the cysteine 151 residue was replaced by serine. Oral administration of MSeA (1 mg/kg) by gavage to mice induced hepatic NQO-1 expression. Similar to MSeA, methylselenol generated from selenomethionine by methioninase activity induced NQO-1 expression. In conclusion, MSeA, the immediate precursor of methylselenol, upregulates the expression of NQO-1 via the Keap1-Nrf2 signaling. The above findings suggest that biological activities of selenium are dependent on the nature of the metabolites as well as the type of ingested selenium formulations.

NAD(P)H:Quinone Oxidoreductase-1 Expression Sensitizes Malignant Melanoma Cells to the HSP90 Inhibitor 17-AAG

The KEAP1-NRF2 pathway regulates cellular redox homeostasis by transcriptional induction of genes associated with antioxidant synthesis and detoxification in response to oxidative stress. Previously, we reported that KEAP1 mutation elicits constitutive NRF2 activation and resistance to cisplatin (CDDP) and dacarbazine (DTIC) in human melanomas. The present study was conducted to clarify whether an HSP90 inhibitor, 17-AAG, efficiently eliminates melanoma with KEAP1 mutation, as the NRF2 target gene, NQO1, is a key enzyme in 17-AAG bioactivation. In melanoma and non-small cell lung carcinoma cell lines with or without KEAP1 mutations, NQO1 expression and 17-AAG sensitivity are inversely correlated. NQO1 is highly expressed in normal melanocytes and in several melanoma cell lines despite the presence of wild-type KEAP1, and the NQO1 expression is dependent on NRF2 activation. Because either CDDP or DTIC produces reactive oxygen species that activate NRF2, we determined whether these agents would sensitize NQO1-low melanoma cells to 17-AAG. Synergistic cytotoxicity of the 17-AAG and CDDP combination was detected in four out of five NQO1-low cell lines, but not in the cell line with KEAP1 mutation. These data indicate that 17-AAG could be a potential chemotherapeutic agent for melanoma with KEAP1 mutation or NQO1 expression.

Application of Mass Spectrometry Profiling to Establish Brusatol as an Inhibitor of Global Protein Synthesis

The KEAP1/Nrf2 pathway senses and responds to changes in intracellular oxidative stress. Mutations that result in constitutive activation of Nrf2 are present in several human tumors, especially non-small cell lung cancer. Therefore, compounds that inhibit Nrf2 activity might be beneficial in treating patients whose tumors show activation of this pathway. Recent reports suggest that the natural product brusatol can potently and selectively inhibit Nrf2 activity, resulting in cell cytotoxicity, and can be effectively combined with chemotherapeutic agents. Here, we analyzed the effects of brusatol on the cellular proteome in the KEAP1 mutant non-small cell lung cancer cell line A549. Brusatol was found to rapidly and potently decrease the expression of the majority of detected proteins, including Nrf2. The most dramatically decreased proteins are those that display a short half-life, like Nrf2. This effect was confirmed by restricting the analysis to newly synthesized proteins using a labeled methionine analogue. Moreover, brusatol increased the expression of multiple components of the ribosome, suggesting that it regulates the function of this macromolecular complex. Finally, we show that brusatol induces its potent cellular cytotoxicity effects on multiple cancer cell lines in a manner independent of KEAP1/Nrf2 activity and with a profile similar to the protein translation inhibitor silvestrol. In conclusion, our data show that the activity of brusatol is not restricted to Nrf2 but, rather, functions as a global protein synthesis inhibitor.

Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response.

The Keap1-Nrf2 system plays a central role in cytoprotection against electrophilic/oxidative stresses. Although Cys151, Cys273, and Cys288 of Keap1 are major sensor cysteine residues for detecting these stresses, it has not been technically feasible to evaluate the functionality of Cys273 or Cys288, since Keap1 mutants that harbor substitutions in these residues and maintain the ability to repress Nrf2 accumulation do not exist. To overcome this problem, we systematically introduced amino acid substitutions into Cys273/Cys288 and finally identified Cys273Trp and Cys288Glu mutations that do not affect Keap1's ability to repress Nrf2 accumulation. Utilizing these Keap1 mutants, we generated stable murine embryonic fibroblast (MEF) cell lines and knock-in mouse lines. Our analyses with the MEFs and peritoneal macrophages from the knock-in mice revealed that three major cysteine residues, Cys151, Cys273, and Cys288, individually and/or redundantly act as sensors. Based on the functional necessity of these three cysteine residues, we categorized chemical inducers of Nrf2 into four classes. Class I and II utilizes Cys151 and Cys288, respectively, while class III requires all three residues (Cys151/Cys273/Cys288), while class IV inducers function independently of all three of these cysteine residues. This study thus demonstrates that Keap1 utilizes multiple cysteine residues specifically and/or collaboratively as sensors for the detection of a wide range of environmental stresses.

Cancer Cell Growth Is Differentially Affected by Constitutive Activation of NRF2 by KEAP1 Deletion and Pharmacological Activation of NRF2 by the Synthetic Triterpenoid, RTA 405.

Synthetic triterpenoids are antioxidant inflammation modulators (AIMs) that exhibit broad anticancer activity. AIMs bind to KEAP1 and inhibit its ability to promote NRF2 degradation. As a result, NRF2 increases transcription of genes that restore redox balance and reduce inflammation. AIMs inhibit tumor growth and metastasis by increasing NRF2 activity in the tumor microenvironment and by modulating the activity of oncogenic signaling pathways, including NF-κB, in tumor cells. Accumulating evidence suggests that KEAP1 loss or mutation—which results in high levels of sustained NRF2 activity—may promote cancer growth and increase chemoresistance. Loss of KEAP1 also increases the levels of other oncogenic proteins, including IKKβ and BCL2. The apparent survival advantage provided to some tumor cells by loss of functional KEAP1 raises the question of whether pharmacological inhibition of KEAP1 could promote tumor growth. To address this issue, we characterized the basal levels of KEAP1 and NRF2 in a panel of human tumor cell lines and profiled the activity of an AIM, RTA 405. We found that in tumor cell lines with low or mutant KEAP1, and in Keap1 -/- murine embryonic fibroblasts, multiple KEAP1 targets including NRF2, IKKβ, and BCL2 were elevated. Keap1 -/- murine embryonic fibroblasts also had higher rates of proliferation and colony formation than their wild-type counterparts. In cells with functional KEAP1, RTA 405 increased NRF2 levels, but not IKKβ or BCL2 levels, and did not increase cell proliferation or survival. Moreover, RTA 405 inhibited growth at similar concentrations in cells with different basal NRF2 activity levels and in cells with wild-type or mutant KRAS. Finally, pre-treatment with RTA 405 did not protect tumor cells from doxorubicin- or cisplatin-mediated growth inhibition. Collectively, these data demonstrate that pharmacological activation of NRF2 by AIMs is distinct from genetic activation and does not provide a growth or survival advantage to tumor cells.

An Integrated Molecular Analysis of Lung Adenocarcinomas Identifies Potential Therapeutic Targets among TTF1-Negative Tumors, Including DNA Repair Proteins and Nrf2.

Thyroid transcription factor-1 (TTF1) immunohistochemistry (IHC) is used clinically to differentiate primary lung adenocarcinomas (LUAD) from squamous lung cancers and metastatic adenocarcinomas from other primary sites. However, a subset of LUAD (15%-20%) does not express TTF1, and TTF1-negative patients have worse clinical outcomes. As there are no established targeted agents with activity in TTF1-negative LUAD, we performed an integrated molecular analysis to identify potential therapeutic targets. Using two clinical LUAD cohorts (274 tumors), one from our institution (PROSPECT) and The Cancer Genome Atlas, we interrogated proteomic profiles (by reverse phase protein array, RPPA), gene expression, and mutational data. Drug response data from 74 cell lines were used to validate potential therapeutic agents. Strong correlations were observed between TTF1 IHC and TTF1 measurements by RPPA (Rho = 0.57, P < 0.001) and gene expression (NKX2-1, Rho = 0.61, P < 0.001). Established driver mutations (e.g., BRAF and EGFR) were associated with high TTF1 expression. In contrast, TTF1-negative LUAD had a higher frequency of inactivating KEAP1 mutations (P = 0.001). Proteomic profiling identified increased expression of DNA repair proteins (e.g., Chk1 and the DNA repair score) and suppressed PI3k/mTOR signaling among TTF1-negative tumors, with differences in total proteins confirmed at the mRNA level. Cell line analysis showed drugs targeting DNA repair to be more active in TTF1-low cell lines. Combined genomic and proteomic analyses demonstrated infrequent alteration of validated lung cancer targets (including the absence of BRAF mutations in TTF1-negative LUAD), but identified novel potential targets for TTF1-negative LUAD, including KEAP1/Nrf2 and DNA repair pathways.

Toll-Like Receptor Signaling Induces Nrf2 Pathway Activation through p62-Triggered Keap1 Degradation.

Toll-like receptors (TLRs) induce inflammation and tissue repair through multiple signaling pathways. The Nrf2 pathway plays a key role in defending against the tissue damage incurred by microbial infection or inflammation-associated diseases. The critical event that mediates TLR-induced Nrf2 activation is still poorly understood. In this study, we found that lipopolysaccharide (LPS) and other Toll-like receptor (TLR) agonists activate Nrf2 signaling and the activation is due to the reduction of Keap1, the key Nrf2 inhibitor. TLR signaling-induced Keap1 reduction promoted Nrf2 translocation from the cytoplasm to the nucleus, where it activated transcription of its target genes. TLR agonists modulated Keap1 at the protein posttranslation level through autophagy. TLR signaling increased the expression of autophagy protein p62 and LC3-II and induced their association with Keap1 in the autophagosome-like structures. We also characterized the interaction between p62 and Keap1 and found that p62 is indispensable for TLR-mediated Keap1 reduction: TLR signaling had no effect on Keap1 if cells lacked p62 or if cells expressed a mutant Keap1 that could not interact with p62. Our study indicates that p62-mediated Keap1 degradation through autophagy represents a critical linkage for TLR signaling regulation of the major defense network, the Nrf2 signaling pathway.