NFE2L2 Mutations Research Articles

Clinical factors associated with comprehensive genomic variation profiling of cervical cancer.

62 Background: To better understand the mechanism contributing to cervical carcinogenesis, we perform a comprehensive analysis of genomic alterations in cervical cancer (CC). Methods: We conducted whole-exome sequencing of 43 CCs and established strict integrated workflow of genomic analysis. Correlation analysis was performed to measure the relationships between gene mutations and clinical characteristics of CC patients. Results: Genomic analysis revealed 28 genes frequently mutated in CC including BRD4 (4/43), AXL (4/43), MED12 (4/43), which are undetermined genomic features in CC, and identified recurrent genetic mutations in PIK3CA (16/43), KMT2D (11/43), KMT2C (6/43), FBXW7 (5/43), FAT1 (5/43), ERBB2 (4/43), EP300 (3/43) and NFE2L2 (3/43). Intriguingly, CC patients harbored high frequent mutations in BRCA2 (7/43), but not in BRCA1. The relationship between BRCA2 mutations and clinical factors is yet to be determined. The identified mutations predominately resulted in the dysregulation of the PI3K/AKT/mTOR pathway. Interestingly, we found that AXL mutations were positively correlated with FIGO stage ( P = 0.028), regional lymph node metastasis ( P = 0.011) and distant metastasis ( P = 0.022). KMT2C mutations were positively correlated with FIGO stage ( P = 0.004) and distant metastasis ( P = 0.009). SF3B1 mutations were positively correlated with regional lymph node metastasis ( P = 0.027) and distant metastasis ( P< 0.001). NFE2L2, ERBB2, RAC1, MED12, MET, BAP1, PTPRD and HNF1Amutations were positively correlated with distant metastasis ( P = 0.045, P = 0.004, P = 0.022, P = 0.001, P< 0.001, P< 0.001, P = 0.014, P< 0.001, respectively). POLD1 mutations were positively associated with regional lymph node metastasis ( P = 0.029). However, NOTCH1 mutations were negatively associated with regional lymph node metastasis ( P = 0.047). STK11 mutations were negatively associated with FIGO stage ( P = 0.013). Conclusions: Our results highlight the application of deep sequencing for understanding the molecular mechanisms and uncovering potential diagnostic and therapeutic targets of CC.

2034P - Genetic landscape of KEAP1 and NFE2L2 mutated cancers from the AACR GENIE database

BackgroundNFE2L2 encodes Nrf2, which is activated at times of cellular stress to neutralize reactive oxygen species. KEAP1 acts as a negative regulator of Nrf2. Nrf2 has been implicated in tumorigenesis of many human cancers via the mTOR pathway. The prevalence and genetic landscape of NFE2L2 and KEAP1 mutants remains poorly characterized. MethodsWe extracted data from AACR GENIE 5.0 database, including only subjects with complete demographic, mutational, and CNA data (n=54,237). We quantified the prevalence of NFE2L2 and KEAP1 mutations, determined location frequency by gene subdomain, copy number alterations, and most frequent co-mutated genes. ResultsWe identified 720 NFE2L2 mutations and 1422 KEAP1 mutations. NFE2L2-mutated samples showed a difference in age (61.6 vs 55.9 years, p<.01) but had no gender difference (48.9% vs 49.1% female, p=0.8). NFE2L2 was most commonly mutated in head and neck (6.0%), anal (5.1%), and endometrial (4.5%) cancer samples. 391 (54.3%) mutations were seen in the Neh2 binding domain. TP53(51%), KMT2D(29.4%), and PIK3CA(26.4%) were most commonly co-mutated. KEAP1-mutated samples showed no difference in age (58.3 vs 59.0 years, p=0.75) nor gender (40.2% vs 48.4% female, p=0.31). KEAP1 mutations were most common in non-small cell lung (9.4%), unknown primary (6.9%), and small cell lung (4.0%) cancers. 696 (48.9%) mutations were seen in the KELCH binding domain. TP53 (53.8%), STK11(35.0%), and KRAS (30.9%) were the most common co-mutations. Based on prevalence identified in this study and publicly available epidemiological data, we estimate an annual incidence of nearly 60,000 cancers with NFE2L2 or KEAP1 mutations in the United States alone. ConclusionsNFE2L2 and KEAP1 mutations in their respective binding domains were found in a wide variety of cancer types. Based on estimated incidence of these mutations, mTOR inhibitors may play an important role in treating a signficant number of cancers. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

ERBB3 and IGF1R Signaling Are Required for Nrf2-Dependent Growth in KEAP1-Mutant Lung Cancer.

Mutations in KEAP1 and NFE2L2 (encoding the protein Nrf2) are prevalent in both adeno and squamous subtypes of non-small cell lung cancer, as well as additional tumor indications. The consequence of these mutations is stabilized Nrf2 and chronic induction of a battery of Nrf2 target genes. We show that knockdown of Nrf2 caused modest growth inhibition of cells growing in two-dimension, which was more pronounced in cell lines expressing mutant KEAP1. In contrast, Nrf2 knockdown caused almost complete regression of established KEAP1-mutant tumors in mice, with little effect on wild-type (WT) KEAP1 tumors. The strong dependency on Nrf2 could be recapitulated in certain anchorage-independent growth environments and was not prevented by excess extracellular glutathione. A CRISPR screen was used to investigate the mechanism(s) underlying this dependence. We identified alternative pathways critical for Nrf2-dependent growth in KEAP1-mutant cell lines, including the redox proteins thioredoxin and peroxiredoxin, as well as the growth factor receptors IGF1R and ERBB3. IGF1R inhibition was effective in KEAP1-mutant cells compared with WT, especially under conditions of anchorage-independent growth. These results point to addiction of KEAP1-mutant tumor cells to Nrf2 and suggest that inhibition of Nrf2 or discrete druggable Nrf2 target genes such as IGF1R could be an effective therapeutic strategy for disabling these tumors. SIGNIFICANCE: This study identifies pathways activated by Nrf2 that are important for the proliferation and tumorigenicity of KEAP1-mutant non-small cell lung cancer.

Abstract 1: A small molecule inhibits Nrf2 expression and activity in cancer cells

Abstract Activation of the Nrf2-Keap1-ARE pathway is a master defense mechanism protecting against oxidative and electrophilic stress. Nrf2 was traditionally considered cancer-preventing, as Nrf2 deficiency enhances susceptibility to carcinogens in a variety of mouse models. However, increasing evidence suggests a tumor-promoting role for Nrf2 in established tumors: gain of function mutations in NFE2L2, which activates the Nrf2 pathway, or loss of function mutations in its negative regulator, Keap1, have been found most frequently in lung cancer. Constitutive activation of the Nrf2 pathway is associated with chemoresistance and poor survival. Nrf2 inhibitors, therefore, become potential novel drugs for cancer treatment and needed tool compounds to study the Nrf2 pathway in cancer. To identify Nrf2 inhibitors, we conducted a high throughput screen with a diverse set of small molecules (~6500 compounds). 890 compounds were identified as hits, and a funnel strategy was applied to filter through the hits and select lead compounds. Although other known Nrf2 inhibitors were identified, a potentially novel compound, MSU225, was chosen as a lead for further evaluation. MSU225 downregulated tBHQ (tert-butyl hydroquinone)-induced Nrf2 transcriptional activity in a luciferase assay without inducing cell death. When A549 human lung cancer cells, in which Keap1 is mutated inducing constitutive activation of Nrf2, were treated with 5 µM MSU225, the mRNA expression of HMOX1, NQO1, GST1A1, GCLC, GCLM, and UGT1A6, all downstream targets of Nrf2, were decreased. Moreover, MSU225 significantly (p&amp;lt;0.05) decreased the protein level of Nrf2 in A549 cells. The effect of MSU225 on Nrf2 protein levels was blocked by the proteasome inhibitor MG132, suggesting MSU225 enhances Nrf2 degradation through the proteasome system. In addition, MSU225 inhibited the growth of human lung cancer cells (A549, H460, A427) in soft agar. Cells addicted to Nrf2 were more susceptible to MSU225 for suppression of cell proliferation than cells not addicted to Nrf2. MSU225 also sensitized A549 cells to carboplatin treatment. Our results suggest that MSU225 is a novel Nrf2 inhibitor, and additional in vivo studies will be done to determine if it can be used to treat experimental lung cancer. Citation Format: Di Zhang, Corbin Livingston, Thomas Dexheimer, Edmund Ellsworth, Aaron Odom, Karen Liby. A small molecule inhibits Nrf2 expression and activity in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1.

Abstract 693: Deep learning analysis of circulating tumor DNA identified a pan-tumor molecular subtype with enhanced response to durvalumab (anti-PDL1)

Abstract Tumor mutation burden (TMB) has emerged as a promising predictive biomarker for anti-PD1/L1; however, whether somatic mutations in specific genes can sensitize to immunotherapy is poorly understood. We used deep learning to identify molecular subtypes from mutations detectable in circulating tumor DNA (ctDNA) and associated these with outcomes with anti-PDL1 (durvalumab) and durvalumab + anti-CTLA4 (tremelimumab) therapy. Patient blood samples from 4 nonrandomized phase 2 trials evaluating durvalumab (NCT01693562, 1L+ solid tumors; NCT02087423, 3L+ NSCLC) or durvalumab+ tremelimumab (NCT02000947, 2L+ NSCLC; NCT02261220, 2L+ solid tumors) were sequenced with the Guardant 360 assay of 73 genes. CtDNA mutations were examined pre-treatment in a discovery cohort of 818 patients composed of &amp;gt;15 tumor types and validated in a cohort of 94 patients composed of &amp;gt;5 tumor types. Deep learning neuron network was computed using Tensorflow to identify 4 unique molecular subgroups. Kaplan-Meier analysis was calculated and objective response rates (ORR) were assessed with RECISIST v1.1. The subgroups consisted of: DS1 (N = 215/818, 26%) enriched in RB1 mutations (17%) and low TMB, DS2 (N = 237/818, 29%) enriched in ARID1A (33%), NFE2L2 (8%), and cKIT (13%) mutations, DS3 (N = 251/818, 31%) enriched in EGFR, ERBB2, BRAF and STK11 mutations, and DS4 (N = 115/818, 14%) enriched in TERT, FGFR3, and PIK3CA mutations. In the discovery cohort, the DS2 subgroup had most improved outcomes (mOS=24.6 months, mPFS=3.7 months, ORR=27%) compared to the other 3 subgroups. DS1 subgroup had the worst outcomes (mOS=6.3 months, mPFS=1.6 months, ORR=10%), while the biomarker evaluable population (BEP, n=818) had ORR=17%. In the validation cohort, DS2 also had the most improved outcomes (mOS=not reached, mPFS=13.2 months, and ORR=64%), with the BEP (n=94) having an ORR=37%. PD-L1 was not correlated with any specific molecular subgroup, while an IFNg mRNA signature trended with correlation with subgroup DS2. Deep learning analysis of ctDNA identified a molecular subgroup with enhanced response to anti-PDL1 treatment in multiple tumor types. Citation Format: Song Wu, Sriram Sridhar, Han Si, Michael Kuziora, Judson Englert, Shaad E. Abdullah, Philip Dennis, Feng Xiao, Guozhi Gao, Koustubh Ranade, Brandon W. Higgs. Deep learning analysis of circulating tumor DNA identified a pan-tumor molecular subtype with enhanced response to durvalumab (anti-PDL1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 693.

Nrf2 Activation Promotes Lung Cancer Metastasis by Inhibiting the Degradation of Bach1

Approximately 30% of human lung cancers acquire mutations in either Keap1 or Nfe2l2, resulting in the stabilization of Nrf2, the Nfe2l2 gene product, which controls oxidative homeostasis. Here, we show that heme triggers the degradation of Bach1, a pro-metastatic transcription factor, by promoting its interaction with the ubiquitin ligase Fbxo22. Nrf2 accumulation in lung cancers causes the stabilization of Bach1 by inducing Ho1, the enzyme catabolizing heme. In mouse models of lung cancers, loss of Keap1 or Fbxo22 induces metastasis in a Bach1-dependent manner. Pharmacological inhibition of Ho1 suppresses metastasis in a Fbxo22-dependent manner. Human metastatic lung cancer display high levels of Ho1 and Bach1. Bach1 transcriptional signature is associated with poor survival and metastasis in lung cancer patients. We propose that Nrf2 activates a metastatic program by inhibiting the heme- and Fbxo22-mediated degradation of Bach1, and that Ho1 inhibitors represent an effective therapeutic strategy to prevent lung cancer metastasis.

Targeting NFE2L2/KEAP1 mutations in advanced NSCLC with the TORC1/2 inhibitor TAK-228.

9085 Background: Despite efforts over the past decade, no targeted therapy options exist for SQCLC pts. We have identified a heretofore untargeted oncogene (NFE2L2)/tumor suppressor (KEAP1) pair, each mutated in ~20% of patients with SQCLC. NFE2L2 encodes Nrf2, a transcription factor involved in the oxidative stress response which is targeted for degradation by Keap1. NFE2L2 mutations occur exclusively in an exon 2 hotspot that encodes the Neh2 domain (aa.1-86), which is the binding site for Keap1. Mutations in this region disrupt Keap1 binding, leading to Nrf2 nuclear translocation and increased mTOR signaling through RagD (Shibata Cancer Res 2010). We report translational studies and results from an ongoing phase 2 trial of the oral TORC1/2 inhibitor TAK-228. Methods: Cytotoxicity, signaling, and xenograft experiments were performed using LK-2 SQCLC cells (NFE2L2 E79K mut) treated with TAK-228, everolimus, rapamycin, or deforolimus. Pts with stage IV SQCLC harboring NFE2L2 or KEAP1 mutations and NSCLC harboring KRAS + NFE2L2 or KEAP1 co-mutations are treated on an NCI CTEP phase 2 study of TAK228 3mg po qd (continuous 28 day cycles; NCT02417701). Primary endpoint: ORR. Secondary endpoints: PFS. The study utilizes a Simon 2-stage design for each cohort with H0 = 5% (N≥1/5 responses), HA = 40% (N≥2/10 responses). Results: TAK-228 exhibited increased anti-tumor activity over TORC1 rapalogs in LK-2 cells. TAK-228 alone was cytotoxic at sub-[μM] (IC50 68nM); all other rapalogs exhibited IC50s &gt; 10μM. This was associated with marked decrease in pS6, pAKT, and pERK. Tumor response (-55%) was seen in an LK-2 xenograft treated with TAK228. No anti-tumor/growth inhibitory response was seen with any other rapalog. N = 15 evaluable pts have been treated on study (7 NFE2L2, 3 KEAP1, 5 KRAS + NFE2L2 or KEAP1). 2 related-SAEs (G3 hyperglycemia, G3 confusion) were seen; there were no unexpected AEs. Within SQCLC NFE2L2 pts ORR = 29% (2/7 PR) and DCR = 100%. Prolonged duration of response is present, with PFS = 10.5mo, 11.4mo (18.2mo tx beyond PD), 6mo (8mo tx beyond PD), 5.5mo+, 4.5mo, and 1.5mo+. Within SQCLC KEAP1 pts DCR = 66% (2SD, 1PD) with PFS = 7.4mo+, 3mo+, 1mo. Data for the KRAS + NFE2L2/KEAP1 arm will be presented. Conclusions: TAK228 is tolerable with evidence of pre-clinical activity, radiographic responses, and prolonged DOR in biomarker-selected NSCLC pts. Accrual is ongoing. Clinical trial information: NCT02417701.

Impact of KEAP1/NFE2L2/CUL3 mutations on duration of response to EGFR tyrosine kinase inhibitors in EGFR mutated non-small cell lung cancer

ObjectivesFor patients with Epidermal Growth Factor Receptor (EGFR)-mutated non-small cell lung cancer (NSCLC), frontline EGFR-tyrosine kinase inhibitor (TKI) therapy compared to chemotherapy improves outcomes. However, resistance to these agents uniformly develops. Recently, mutations in the KEAP1-NFE2L2 pathway have been implicated as a potential mechanism of acquired EGFR TKI resistance. Materials and methodsWe examined all patients with metastatic NSCLC with mutations in both EGFR and KEAP1/NFE2L2/CUL3 identified on next generation sequencing from 2015 - 2018. These patients were compared to a NSCLC control cohort with mutations in EGFR and wild type in KEAP1/NFE2L2/CUL3 matched on the basis of sex, smoking status, age and race. Time to treatment failure on EGFR TKI therapy and overall survival were examined. ResultsAmong 228 EGFR mutant NSCLCs, 17 (7%) also carried mutations in KEAP1, NFE2L2, or CUL3. The most common co-mutation in both the KEAP1/NFE2L2/CUL3 mutant and wild-type cohort was TP53. Patients with KEAP1/NFE2L2/CUL3 mutations had a shorter median time to treatment failure on EGFR TKI (4.7 months) compared with the wild-type matched cohort (13.0 months), p= 0.0014. There was no difference in overall survival. ConclusionFor NSCLC patients with mutations in EGFR, co-mutations in KEAP1/NFE2L2/CUL3 are associated with significantly decreased time to treatment failure. Our results suggest that these mutations represent a mechanism of intrinsic resistance to TKI treatment.

NFE2L2 synergizes with beta‐catenin gene mutations to induce HCC in patients and mice

Nrf/Keap signaling and Wnt/b‐catenin signaling are important in liver pathophysiology. Mutations in NFE2L2 (encoding for Nrf2) and CTNNB1 (encoding for beta‐catenin) can co‐occur in a subset of HCC patients. However, the precise relevance of these co‐occurrences remains obscure. In the current study, we explored the relevance of any cross‐talk between Nrf2 and Wnt signaling using the sleeping beauty transposon/transposase and hydrodynamic tail vein injection (HDTVI) to co‐express relevant mutant forms of CTNNB1 and NFE2L2 in murine livers. TCGA data showed that among the 377 HCC patients, there were 99 patients with CTNNB1 mutation, 15 patients with NFE2L2 mutation and 18 with mutations in KEAP1. 12 patients of the 377 cases had concomitant mutations in CTNNB1 and NFE2L2 or KEAP1, representing around 3% of all and 12% of CTNNB1 mutated cases. We next co‐expressed beta‐catenin‐T41A and NFE2L2‐WT or NFE2LE‐G3A1 or NFE2L2‐T80K in mouse livers, either individually or together. The NFE2L2‐WT:beta‐catenin‐T41A injected mice showed normal gross morphology and histology by H&amp;E test at 14‐weeks post injection. At this time, NFE2L2‐T80K:beta‐catenin‐T41A and NFE2L2‐G31A:beta‐catenin‐T41A groups develop HCC. HCC developed faster in the NFE2L2‐G31A:beta‐catenin‐T41A group versus the NFE2L2‐T80K:beta‐catenin‐T41A group. Several downstream target genes of Nrf2 were up‐regulated significantly in HCCs occurring in these models, including NAD(P)H dehydrogenase [quinone]1 (NQO1) and peroxiredoxin 1 (PRDX1), suggesting the mutations in NFE2L2 to be GOF. There was also an increase in activation of AKT, mTOR and ERK. Likewise, beta‐catenin targets such as GS were increased remarkably. Thus, we have a generated a highly relevant mouse model of HCC representing a subset of human HCC which is characterized by activation of both Nrf2 and Wnt signaling and hence will lend itself well for investigating biology and precision therapies.Support or Funding Information1R01DK62277, 1R01DK100287,1R01CA204586 and Endowed Chair for Experimental Pathology (S.P.M.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The NRF2 transcriptional target NQO1 has low mRNA levels in TP53-mutated endometrial carcinomas.

BackgroundNRF2 is a major transcription factor regulating the expression of antioxidative/detoxifying enzymes, involved in oncogenic processes and drug resistance. We aimed to identify molecular alterations associated with NRF2 activation in endometrial carcinoma (EC).MethodsNinety patients treated (2012–2017) for localized/locally advanced EC were included in this study. Formalin-fixed paraffin-embedded tissue samples were processed for immunohistochemical (NRF2 and Mismatch Repair proteins) analyses. Next generation sequencing (NGS) of a panel of genes including POLE, TP53, NFE2L2, KEAP1 and CUL3 was performed using Ampliseq panels on Ion Torrent PGM (ThermoFisher). NRF2 activity was assessed by NQO1, GCLC, and AKR1C3 mRNA expressions, using TaqMan assays and quantitative RT-PCR.ResultsTumors were classified as POLE exonuclease domain mutated (N = 3, 3%), MMR-deficient (MSI-like) (N = 28, 31%), TP53 mutated (Copy-number high-like) (N = 22, 24%), and other tumors (Copy-number low-like) (N = 32, 36%). NRF2 nuclear immunostaining did not correlate with NRF2 target genes expression. The 3 tumors with highest NRF2 target genes expression harbored oncogenic KEAP1 or NFE2L2 mutations. Low NQO1 mRNA and protein levels were observed in the TP53 mutated subgroup compared to others tumors (p < .05) and in silico analyses of The Cancer Genome Atlas data further indicated that NQO1 mRNA levels were lower in serous compared to endometrioid copy-number high EC.ConclusionIn contrast with previous reports based on immunohistochemistry, our study indicates that NRF2 activation is a rare event in EC, associated with NFE2L2 or KEAP1 mutations. The subset of aggressive EC with low NQO1 mRNA level might represent a specific subgroup, which could be sensitive to combination therapies targeting oxidative stress.

2O - Somatic mutations in BRCA2, NFE2L2, ARID1A and NOTCH1 sensitize to anti-PDL1 therapy in multiple tumor types

2O - Somatic mutations in BRCA2, NFE2L2, ARID1A and NOTCH1 sensitize to anti-PDL1 therapy in multiple tumor types

PD-1 and PD-L1 Related Comprehensive Analysis of Lung Squamous Cell Carcinoma

Lung squamous cell carcinoma (LUSC) is a subtype of non-small cell cancer associated with poor clinical prognosis and lacks targeted agents. PD-1 and PD-L1 play a critical role in cancer immune. However, genomic alterations associated with PD-1 and PD-L1 expression in LUSC have not been comprehensively characterized. To characterize genomic landscape, we integrally analyzed related genomic and clinical prognostic value associated with PD-1 and PD-L1 in Cancer Genome Atlas (TCGA) and GEO data. Here, we showed that PD-1 and PD-L1 expression were found to demonstrate the existence of heterogeneity in tumor samples. The degrees of PD-1 and PD-L1 expression did not show prognostic significance in patients with LUSC in TCGA. Higher tumor mutation burden (TMB) showed significantly increased expression of PD-1. NFE2L2 mutations led to higher PD-L1 expression in LUSC. The expression of PD-1 and PD-L1 were correlated with tumor microenvironment cells and immune-related molecules such as CTLA4, ICOS, FOXP3, HAVCR2, CD3G, CD8A, LAG3, and PDCD1LG2. The over-expression of PD-1 and PD-L led to genomic alterations of cell adhesion molecules cams; natural killer cell mediated cytotoxicity, antigen processing and presentation, and toll-like receptor signaling pathway. Moreover, survival analysis of PD-1 related module genes shows that ITIH3, EDEM1, EPS15, SETDB2, EHD1, and ANK26 are potent predictors of prognosis in patients with LUSC. Our results indicated that PD-1 and PD-L1 alterations were involved in crucial steps of tumor immune process, and present new therapeutic opportunities for manipulation of PD-1 and PD-L1 to achieve optimal immune responses in LUSC. Funding Statement: This project was supported by National major research program on Natural Science of China (91542164). Declaration of Interests: The authors declare that they have no competing financial interests.

P2.13-44 Targeting NFE2L2 Mutations in Advanced Squamous Cell Lung Cancers with the TORC1/2 Inhibitor TAK-228

Despite research efforts over the past decade, no targeted therapy options exist for patients with SQCLC. Analyses by TCGA and others (Paik Cancer Disc 2015) have identified a heretofore untargeted, frequently mutated oncogene (NFE2L2)/tumor suppressor (KEAP1) pair, each mutated in ∼20% of patients with SQCLC. NFE2L2 encodes Nrf2, a transcription factor involved in the oxidative stress response which is targeted for degradation by Keap1. NFE2L2 mutations occur exclusively in an exon 2 hotspot that encodes the Neh2 domain (∼aa.1-86), which is the binding site for Keap1. Mutations in this region disrupt Keap1 binding, leading to Nrf2 nuclear translocation and increased mTOR signaling through regulation of RagD (Shibata Cancer Res 2010). We now report translational studies and preliminary results from a phase 2 trial of the oral TORC1/2 inhibitor TAK-228 in SQCLC patients with these mutations. Cytotoxicity, signaling, and xenograft experiments were performed using LK-2 SQCLC cells harboring an NFE2L2 E79K mutation treated with TAK-228, everolimus, rapamycin, or deforolimus with requisite vehicle controls. Patients with stage IV SQCLC harboring NFE2L2 mutations were treated on an NCI CTEP- single-institution phase 2 study of TAK228 3mg po qd (continuous, 28 day cycles; NCT02417701). Primary endpoint: ORR. Secondary endpoints: PFS/OS. The study utilizes a Simon 2-stage design with H0=5% (N≥1/5 responses), HA=40% (N≥2/10 responses). TAK-228 exhibited significantly increased anti-tumor activity over TORC1 rapalogs in LK-2 cells. TAK-228 alone was cytotoxic at sub-[μM] (IC50 68nM); all other rapalogs exhibited IC50s >10μM. This was associated with an 80% decrease in downstream S6 phosphorylation. Tumor response (-55% shrinkage) was also seen in an LK-2 xenograft treated with TAK228. No anti-tumor/growth inhibitory response was seen with any other rapalog tested. N=4 patients have been treated on study (exon 2 del, D29H, F37V, W24C). 3 are evaluable for response. 2 related-SAEs (G3 hyperglycemia, G3 confusion) were seen; no other G3 AEs reported. ORR=67% (2 PR, 1 SD, 0 PD). Prolonged disease response is present, with DOR= 12mo, 10mo (ongoing), 8mo (ongoing). TAK228 is tolerable with evidence of pre-clinical and clinical activity in this biomarker-selected population. The trial has met its first-stage endpoint and has expanded to N=10 patients. The trial has also expanded to included patients with KRAS mutant lung cancers harboring co-alterations in NFE2L2 or KEAP1.

1348PD - Genomic landscape and its correlation with TMB, CD8 TILs and PD-L1 expression in Chinese lung squamous cell carcinoma

Background: To comprehensively depict the genomic landscape of Chinese lung squamous cell carcinoma (LSCC) and investigate its correlation with tumor mutation burden (TMB), CD8 tumor infiltrating lymphocytes (TILs) density and PD-L1 expression. Methods: Whole-exome sequencing were performed on tumor tissue collected from 189 patients with surgically resected LSCC. TMB was defined as total number of nonsynonymous single nucleotide and indel variants. High TMB was defined as greater than 75th percentile. CD8+ TILs and PD-L1 expression were assessed by immunohistochemistry. The cut-off point was 5% for high/low CD8+ TIL or PD-L1 +/- expression. Results: We found recurrent mutations (>5%) in 8 genes, including TP53, KMT2C, NFE2L2, KEAP1, CDKN2A, PTEN and FBXW7. FGFR1 and PIK3CA amplifications were found in 19% and 11% of samples. 24.9% of patients had high TMB. Baseline clinical variables were similar except for smoking status. Interestingly, FGFR1, PIK3CA or SOX2 amplification was independently associated with higher TMB (P = 0.020, P = 0.017, P = 0.029; respectively). Patients with copy number variations had significantly higher TMB than those without (P = 0.009). Positive PD-L1 and CD8+ TILs expression were identified in 24.3% and 78.8% of all cases. NFE2L2 mutation and PIK3CA amplification were associated with significantly higher PD-L1 expression (P = 0.003, P = 0.014; respectively). TP53 mutations were associated with higher CD8+ TILs expression (P = 0.008), but FGFR1 amplification was correlated with lower CD8+ TILs expression (P = 0.042). Of note, there is no association between TMB and PD-L1 expression (r = 0.052, P = 0.476), or CD8+ TILs expression (r = 0.026, P = 0.718). None of TMB, PD-L1 and CD8+ TIL expression could individually predict overall survival (OS). However, combination of TMB and PD-L1 could stratify total populations into two groups with distinct prognosis. Patients with negative PD-L1 expression and high TMB had the worst prognosis (P = 0.008). Conclusions: This was the most large-scale study to comprehensively portray genomic landscape of Chinese LSCC and provides several meaningful and referential findings for the future design of clinical trials in LSCC, especially immunotherapy. Legal entity responsible for the study: Caicun Zhou. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

The mutational landscape of recurrent versus nonrecurrent human papillomavirus-related oropharyngeal cancer.

Human papillomavirus-related (HPV-related) oropharyngeal squamous cell carcinomas (OPSCCs) have an excellent response rate to platinum-based chemoradiotherapy. Genomic differences between primary HPV-related OPSCCs that do or do not recur are unknown. Furthermore, it is unclear if HPV-related OPSCCs that recur share a genomic landscape with HPV-negative head and neck cancers (HNCs). We utilized whole exome sequencing to analyze somatic nucleotide (SNVs) and copy number variants (CNVs) among a unique set of 51 primary HPV-related OPSCCs, including 35 that did not recur and 16 that recurred. We evaluated 12 metachronous recurrent OPSCCs (7 with paired primary OPSCCs) and 33 primary HPV-unrelated oral cavity and OPSCCs. KMT2D was the most frequently mutated gene among primary HPV-related OPSCCs (n = 51; 14%) and among metachronous recurrent OPSCCs (n = 12; 42%). Primary HPV-related OPSCCs that recurred shared a genomic landscape with primary HPV-related OPSCCs that did not recur. However, TSC2, BRIP1, NBN, and NFE2L2 mutations occurred in primary OPSCCs that recurred but not in those that did not recur. Moreover, primary HPV-related OPSCCs that recur harbor features of HPV-unrelated HNCs, notably including MAPK, JAK/STAT, and differentiation signaling pathway aberrations. Metachronous recurrent OPSCCs shared a genomic landscape with HPV-unrelated HNCs, including a high frequency of TP53, CASP8, FAT1, HLA-A, AJUBA, and NSD1 genomic alterations. Overall, primary HPV-related OPSCCs that recur share a genomic landscape with nonrecurrent OPSCCs. Metachronous recurrent OPSCCs share genomic features with HPV-negative HNCs. These data aim to guide future deescalation endeavors and functional experiments. This study is supported by the American Cancer Society (RSG TBG-123653), funding support for RAH (T32DC00018, Research Training in Otolaryngology, University of Washington), funds to EM from Seattle Translational Tumor Research (Fred Hutchinson Cancer Research Center), and center funds from the Fred Hutchinson Cancer Research Center to EM. UD is supported by the Department of Veterans Affairs, Biomedical Laboratory Research and Development (BLR&D), grant IO1-oo23456, and funds from the Pittsburgh Foundation and PNC Foundation.

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.

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&amp;lt;.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&amp;lt;.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 620: Associations of genomic alteration, tumor mutational burden with PD-L1 expression and response to immune checkpoint inhibitors in Chinese lung patients

Abstract Background: For advanced or metastatic lung cancer patients (pts) in the absence of actionable genetic alterations, whose disease progressed after chemotherapy, the effective treatment options are limited. Recently, PD-1/PD-L1 inhibitors have demonstrated improved survival in advanced lung cancer. However, sufficient evidence is still lacking in Chinese lung cancer pts. Methods: Twenty lung cancer pts including 12 adenocarcinomas and 5 squamous cell carcinomas who received anti-PD-1/PD-L1 treatments as first- to fourth-line therapy were retrospectively reviewed. The majority of the pts were stage IV (17 pts). PD-L1 expression was analyzed by immunohistochemical staining. Genomic alterations were detected by using a next-generation sequencing (NGS) assay targeting 450 cancer genes. Tumor mutational burden (TMB) and microsatellite instability (MSI) status were acquired by NGS algorithm. Results: The median age of the 20 pts was 57 years (range: 36-69) and male vs. female was 3. The median time from date of diagnosis to the date of the first anti-PD-1/PD-L1 treatment was 9.5 months (range: 1-69 months). The overall response rate was 89%, including 8 partial response (PR), 9 stable disease (SD) and 2 progressive disease (PD), and the median PFS (mPFS) was not reached (NR) (range: 0.5-NR). Seven pts had one or more targetable oncogenic mutations including EGFR L858R (2) and amplification, PIK3CA (1), BRAF, ERBB2, PTEN, and ROS1 fusion. The median TMB value of the 20 pts was 11 muts/Mb (range: 2.4-38 muts/Mb). 45% of the pts had TMB greater than 20 muts/Mb, and 60% had TMB greater than 10 muts/Mb. Among the 9 pts who had the TMB greater than 20 muts/Mb, 5 pts achieved PR, 4 achieved SD, mPFS was not reached. Also, 5 pts including 2 PR and 3 SD were identified as MSI-high. In the PD-L1 positive subgroup (N=11), 5 pts achieved PR, 5 achieved SD and 1 had a PD. Furthermore, we found 5 pts with NFE2L2 mutations including 2 adenocarcinomas and 3 squamous cell carcinomas, associated with high TMB values (&amp;gt;20 muts/Mb) and PD-L1 positivity; all 5 pts had response to anti-PD-1/PD-L1 treatment, which include 3 PR and 2 SD with mPFS not reached (range: 6-NR). Conclusion: Our preliminary results based on a small number of pts showed a significant association between high TMB values and response of immune checkpoint inhibitors in Chinese lung cancer pts, and a strong correlation between high TMB values and high PD-L1 expression. We also reported that NFE2L2 mutation was tightly associated with high TMB value (P=0.011) and PD-L1 positivity (P=0.038), which may serve as a new predictive biomarker for advanced or metastatic lung cancer pts receiving anti-PD-1/PD-L1 treatments. Further larger cohort study is warranted. Citation Format: Jing Hu, Bixun Li, Bing Zou, Senming Wang, Ye Qiu, Maolin Yan, Zhiming Zeng, Han Yang, Yan Guan, Lei Zhang, Wei Chen, Yuan Zhang, Lei Mei, Xiaowei Dong, Ming Yao, Kai Wang. Associations of genomic alteration, tumor mutational burden with PD-L1 expression and response to immune checkpoint inhibitors in Chinese lung patients [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 620.

Targeting NFE2L2 mutations in advanced squamous cell lung cancers with the TORC1/2 inhibitor TAK228.

9098Background: Despite efforts over the past decade, no targeted treatments exist for pts w/ SQCLC. Analyses by TCGA and others (Paik Cancer Disc 2015) have identified a heretofore untargeted, fre...

Comparative genomic analysis of esophageal squamous cell carcinoma between Asian and Caucasian patient populations

Esophageal squamous cell carcinoma is a major histological type of esophageal cancer, with distinct incidence and survival patterns among races. Although previous studies have characterized somatic mutations in this disease, a rigorous comparison between different patient populations has not been conducted. Here we sequence the samples of 316 Chinese patients, combine them with those from The Cancer Genome Atlas, and perform a comparative analysis between Asian and Caucasian patients. We find that mutated CSMD3 is associated with better prognosis in Asian patients. Applying a robust computational strategy that adjusts for both technical and biological confounding factors, we find that TP53, EP300, and NFE2L2 show higher mutational frequencies in Asian patients. Moreover, NFE2L2 mutations correlate with the allele status of a nearby high-Fst SNP, suggesting their potential interaction. Our study provides insights into the molecular basis underlying the striking racial disparities of this disease, and represents a general computational framework for such a cross-population comparison.