KEAP1 Mutations Research Articles

Abstract 1137: Deep targeted and whole exome sequencing identifies driver mutations in the airway epithelium of individuals at high risk for lung cancer

Abstract Insights into tumor mutational burden of the airway epithelium may play important role in stratifying individuals at different levels of risk for lung cancer. Comparison of the genetic profiles of normal airway epithelial cells to tumor mutations can yield valuable information regarding shared mutations as well as presence of neoantigens. We hypothesized that cytologically normal bronchial epithelium of patients with lung cancer and those at high risk for lung cancer harbor driver mutations that can be identified by deep targeted sequencing of selected genes implicated in cancer and whole exome sequencing. We performed next generation sequence-based CAPP-Seq (CAncer Personalized Profiling by deep Sequencing) and whole exome sequencing (WES) of DNA from the bronchial epithelial cells from the bronchial brushings of individuals at-risk (low and high) for lung cancer and patients with lung cancer. Bronchial brushings were collected from consented individuals at the time of bronchoscopy at Vanderbilt University Medical Center and the Nashville Veterans Affairs Medical Center. We used Tammemagi risk model to calculate the risk of developing lung cancer. Lung tumor tissues from matched individuals were also sequenced. Using CAPPSeq, a total of 129 mutations were identified in bronchial brushings. Among individuals without cancer, driver mutations included TP53, PIK3CA, NOTCH1, KRAS, MET, CUL3, NF1, KEAP1 and EGFR mutations in 5 out of 13 high risk and 2 out of 7 low risk individuals. Among individuals with cancer, 13 out of 22 brushings harbored driver mutations in TP53, PIK3CA, ARID1A, PTEN, NOTCH1, KRAS, MET, NFE2L2, CUL3, NF1, and KEAP1 of which mutations in TP53 and PTEN were identified in both tumor and bronchial brushings. At least one mutation was called in 18 brushings of individuals with lung cancer. Mutations detected in both individuals with and without cancer were TP53, PIK3CA, NOTCH1, KRAS, MET, CUL3, and NF1. Using WES on the DNA from the same bronchial brushings (low risk-9; high risk-8 and lung cancer-17), we identified 90 mutations that were also identified by CAPPSeq. Most of the mutations identified by CAPPSeq with >5% variant allelic frequencies (VAF) were also identified by WES. However, WES was not sensitive enough to identify mutations with less than 0.5% VAF. Out of 9 mutations in TP53 identified in the brushings by CAPPSeq, 4 were identified by WES. Mutation in KMTD2, a histone methylation enzyme and believed to be a tumor suppressor, was identified by WES in the brushings and the tumor tissues of patients with lung cancer as well as in one high risk brushings. We identified mutations in cancer driver genes in the bronchial brushings of individuals with and without lung cancer. This field of cancerization effect may provide a tool for risk assessment for lung cancer. This research was funded by U01CA196405 to PPM. Citation Format: SM Jamshedur Rahman, Shih-kai Chu, Shilin Zhao, Yong Zou, Angela Hui, Thomas Stricker, Maximilian Diehn, Pierre P. Massion. Deep targeted and whole exome sequencing identifies driver mutations in the airway epithelium of individuals at high risk for lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1137.

Abstract 1095: Comprehensive pan-cancer analyses of RAS genomic diversity

Abstract The RAS family of small GTPases represents the most commonly activated oncogenes and mutations in KRAS, NRAS and HRAS are found in approximately one third of all human cancers. To better understand the prevalence of somatic RAS mutations and the compendium of genes that have RAS-dependent co-mutation frequencies in an unbiased pan-cancer manner, we analyzed targeted next-generation sequence data from the AACR Project GENIE Registry (GENIE version 6.1-public). Our analyses utilized 644,757 mutations from 64,217 tumors in 97 cancer types. A Bayesian hierarchical model was implemented to estimate cancer-specific prevalence of RAS and non-RAS somatic mutations. To evaluate RAS co-mutations, we compared the observed co-mutation frequencies of RAS and non-RAS somatic mutations to posterior predictive distributions of their expected co-mutation frequencies under independence. The prevalence of RAS codon 12, 13 and 61 mutations varied among cancer types, ranging from 77.7% in pancreatic cancer-PAC, 44.8% in colorectal cancer-CRC, 31.2% in non-small cell lung cancer-NSCLC, 1.1% in breast cancer, and 0.5% in mesotheliomas. A cancer type-specific distribution was observed, such that KRAS was frequently mutated in >25% of the tumors, NRAS was frequently mutated in melanoma, hematologic malignancies and thyroid cancer, while HRAS was overall less frequently mutated. KRAS codon 12 mutations predominated in pancreatic cancers (69.8%), GI cancers (30.9-48.5%), NSCLC (26.3%), endometrial cancer (14.7%) and germ cell tumors (11.6%). KRAS G12C mutations were predominantly seen in NSCLC (12.4%, 95% CI: 11.8-13.1%) with lower frequencies in GI cancers (3.2% in CRC). RAS mutations co-occurred with mutations in STK11, ATM, KEAP1, RBM10 and EPHA5 in NSCLC, TERT in melanoma and PIK3CA in CRC. Patterns of mutual exclusivity emerged between RAS mutations and BRAF in melanoma, thyroid and NSCLC, EGFR and RB1 in NSCLC, GNA11 and GNAQ in melanoma, FGFR3 in bladder cancer, MEN1 in PAC, RNF43 in CRC, BAP1 in melanoma and BAP1 and TERT2 in hepatobiliary carcinoma. A cancer type-specific co-occurrence pattern was noted for RAS and TP53 genomic alterations, with a significant enrichment in TP53 mutations in RAS-mutant PAC but a significant under-representation of TP53 mutations in RAS-mutant endometrial cancer, NSCLC, ovarian cancer and CRC. Analysis of codon specific RAS variants revealed additional co-mutation patterns, including an enrichment in NTRK3 mutations in KRAS G12C-mutant NSCLC and mutual exclusivity between KRAS G12C mutations and EGFR and BRAF in NSCLC. The tissue-distinct differential enrichment in RAS mutations and co-mutation patterns may be reflective of selection of oncogenic RAS signaling under the tumor type-specific modifying effect of other driver mutations. The RAS genetic architecture points to tissue-specific therapeutic vulnerabilities that may be translated to targeted treatment strategies. Citation Format: Robert Scharpf, Gregory Riely, Mark Awad, Michele Lenoue-Newton, Biagio Ricciuti, Julia Rudolph, Leon Raskin, Andrew Park, Jocelyn Lee, Christine Lovly, Valsamo Anagnostou. Comprehensive pan-cancer analyses of RAS genomic diversity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1095.

Abstract 1800A: STK11 and KEAP1 co-mutation cooperatively promotes rapid tumor growth in lung adenocarcinoma

Abstract Background: Genomic profiling and targeted therapies have significantly improved outcomes for oncogene-addicted subgroups of lung adenocarcinoma (LUAD) patients (e.g., EGFR mutations or rearrangements of ALK, ROS1 and RET). However, we continue to lack effective targeted therapies for some of the common mutationally defined subsets, including those associated with poor outcome in patients with lung adenocarcinoma. A recent study from our group has shown that concomitant loss-of-function mutations in STK11/LKB1 and KEAP1 (LK) defines a cohort of LUAD patients with shortest overall survivals (Shen et al., 2019). Patients whose tumors harbor LK mutations across all stages of disease show a median overall survival of 11.8 months, compared to KEAP1 only (34.5 months), STK11 only (34.2 months), or neither (40.2 months). The biology and therapeutic vulnerabilities of LUAD subtype with concurrent loss of LKB1 and KEAP1 (either with or without KRAS mutation) is particularly understudied. Methods: We analyzed genomic characteristics of LK lung adenocarcinomas from patients (n=292) at Memorial Sloan Kettering. We performed CRISPR/Cas9 mediated knockout of LKB1, KEAP1, or both to develop matched isogenic clones for three adenocarcinoma cell lines to investigate phenotypic and molecular changes imparted by each single mutation and the co-mutation. We investigated growth in genetically manipulated isogenic groups of LUAD human cell lines (n=8).To investigate the gene expression changes and therapeutic vulnerabilities in samples with concurrent loss of LKB1/KEAP1 we performed bulk RNA-seq and druggable genome CRISPR screening respectively across derivatives of two of these lines, NCI-H358 and NCI-H292. Results: Study of variant allele frequency (VAF) on patient samples with mutations in both STK11and KEAP1 demonstrated a high correlation between VAF of the two genes suggesting clonal expansion of LK tumor cells without defining a preferred order in mutational timing. We show that loss of both genes increases cell proliferation and invasion in vitro and faster/enhanced tumor growth in vivo in all models independent of their carrier mutation status. RNA-sequencing of our isogenic cell lines showed that LK tumors have a distinct gene expression signature different from either of the single mutants with high expression of transcripts involved in cell division and cell survival. Conclusions: Our data suggests that co-mutation of STK11 and KEAP1 cooperate to enhance cell proliferation and tumor growth in human models of lung adenocarcinoma. This is in agreement with clinical observations demonstrating aggressiveness of this subpopulation. Furthermore, identified hits from the CRISPR gene dependency screening have clear translational potential and pave the way for the development of rational therapeutic strategies to improve treatment of LKB1/KEAP1-deficient tumors. Citation Format: Corrin A. Wohlhieter, Allison L. Richards, John T. Poirier, Fathema Uddin, Christopher Hulton, Elisa De Stanchina, Thales Papagiannakopoulos, Triparna Sen, Charles M. Rudin. STK11 and KEAP1 co-mutation cooperatively promotes rapid tumor growth in lung adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1800A.

Abstract 2569: Uncovering metabolic bottlenecks in KEAP1 mutant lung cancer

Abstract During tumorigenesis cancer cells continuously encountermetabolic bottlenecks as a result of accelerated growth, overall increased metabolic demand and increased oxidative stress due to the formation of reactive oxygen species. We use a combination of genetically-engineered mouse models, an accelerated CRISPR/Cas9-based experimentalplatformand biochemical approaches to identify metabolic liabilities that can be exploited using novel targeted therapies.Lung cancer, the leading cause of cancer-related deaths worldwide,is the most common cancer type to acquire mutually exclusive gain-of-function mutations in the anti-oxidant transcription factor NRF2or loss-of-function mutations in its negative regulator KEAP1. Loss of Keap1activates Nrf2, increases antioxidant production and dramatically accelerates KRAS-driven lung cancer. Our data are in line with mounting evidence demonstrating that antioxidants promote cancer progression. Our studies focus on elucidating the mechanisms underlying novel metabolic dependencies in KEAP1mutant tumors and explore the therapeutic potential of targeting metabolism in highly relevant pre-clinical mouse and human lung cancer models. We observe that the ability of KEAP1mutant tumors to divert their metabolism towards antioxidant production comes with a cost, generating multiple metabolic vulnerabilities, including a dependency on glutamine metabolism that can be therapeutically exploited using DRP-104, a novel broad acting glutamine antagonist which inhibits all glutamine-consuming reactions. In addition, we have extended our findings to demonstrate that cancers with high antioxidant capacity exhibit a general dependency on exogenous non-essential amino acids (NEAAs) that is driven by the Nrf2-dependent secretion of glutamate through system xc-(XCT), which limits intracellular glutamate pools that are required for NEAA synthesis. This dependency can be therapeutically targeted by dietary restriction or enzymatic depletion of individual NEAAs. Importantly, limiting endogenous glutamate levels by glutaminase inhibition can sensitize tumors without alterations in Keap1/Nrf2 pathway to dietary restriction of NEAAs. Our findings identify a metabolic strategy to therapeutically target cancers with genetic or pharmacologic activation of the Nrf2 antioxidant response pathway by restricting exogenous sources of NEAAs. Citation Format: Sarah E. LeBoeuf, Warren Wu, Triantafyllia Karakousi, Robert Wild, Thales Papagiannakopoulos. Uncovering metabolic bottlenecks in KEAP1 mutant lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2569.

Choosing the best first-line therapy: NSCLC with no actionable oncogenic driver.

Combination platinum-based therapy has been the standard of care for the treatment of advanced non-small-cell lung cancer (NSCLC). Immunotherapy has emerged and demonstrated to show benefit in the treatment of patients with advanced NSCLC. In this review, we discuss the pivotal trials that led to the US FDA approval of specific immunotherapy regimens in particular patient populations. We discuss the optimal use of immunotherapy as monotherapy based on the KEYNOTE-024, KEYNOTE-042 and IMpower110 trials, chemo-immunotherapy based on KEYNOTE-189, KEYNOTE-407, IMpower150 and IMpower130 trials, and as doublet immunotherapy based on CheckMate-227. We also discuss the role and limitations of PD-L1 expression and tumor mutational burden as predictive biomarkers in response to single-agent immunotherapy and combination chemoimmunotherapy. Furthermore, we discuss emerging resistance markers such as STK11 and KEAP1 mutations in immunotherapy response and briefly discuss the role of immunotherapy in elderly patients and in patients with actionable mutations.

Differences in molecular epidemiology of lung cancer among ethnicities (Asian vs. Caucasian).

BackgroundDifferences in carcinogenesis and therapeutic efficacy according to ethnicity have been reported for lung cancer, and understanding differences in genetic mutation profiles among ethnicities is important for interpreting the results of clinical trials, preventing carcinogenesis, and individualizing treatment. However, no studies have focused on differences in mutation profiles among different ethnicities using large-scale genomic analysis data with detailed information on smoking history, the main cause of lung cancer.MethodsTo clarify the differences in genetic mutation profiles between Caucasian and Japanese subjects, we compared data from The Cancer Genome Atlas, which mainly included Caucasians, with results from the Japan Molecular Epidemiology for lung cancer study, which is an epidemiological study only involving Japanese subjects. We divided the participants into four groups according to smoking status and performed comparative analysis by tissue type (lung adenocarcinoma and squamous cell lung cancer).ResultsIn patients with lung adenocarcinoma, the frequency of EGFR mutations was lower in Caucasian subjects than in Japanese subjects (14.6% vs. 51.1%), whereas the frequencies of mutations in other genes, namely KRAS (32.9% vs. 9.3%), TP53 (45.2% vs. 20.7%), BRAF (9.6% vs. 1.3%), PIK3CA (5.9% vs. 2.6%), KEAP1 (17.8% vs. 0.5%), NF1 (10.9% vs. 0.5%), STK11 (17.8% vs. 0.7%), RBM10 (8.7% vs. 0.1%), and MET (7.8% vs. 0.1%), were higher in Caucasian subjects. Among patients with squamous cell carcinoma, TP53 (81.2% vs. 49.1%), PIK3CA (14.5% vs. 6.8%), KEAP1 (12.7% vs. 0.9%), and NFE2L2 mutations (15.8% vs. 13.6%) were more common in Caucasian subjects.ConclusionsEthnicity is an important and complex characteristic that must be recognized and considered, even in the era of precision medicine. We should collaborate to share data for different ethnicities and incorporate them into clinical practice and the design of global clinical studies. Carefully designed molecular epidemiological studies focusing on ethnic differences are warranted.

Loss-of-function mutations in KEAP1 drive lung cancer progression via KEAP1/NRF2 pathway activation

Background and purposeTargeted therapy and immunotherapy have led to dramatic change in the treatment of lung cancer, however, the overall 5-year survival rate of lung cancer patients is still suboptimal. It is important to exploit new potential of molecularly targeted therapies. High-frequency somatic mutations in KEAP1/NRF2 (27.9%) have been identified in lung squamous cell carcinoma. In this research, we explored the role of KEAP1 somatic mutations in the development of LSCC and whether a nuclear factor erythroid 2-related factor 2(NRF2) inhibitor be potential to target lung cancer carrying KEAP1/NRF2 mutations.MethodsLung cancer cell lines A549 and H460 with loss-of-function mutations in KEAP1 stably transfected with wild-type (WT) KEAP1 or somatic mutations in KEAP1 were used to investigate the functions of somatic mutations in KEAP1. Flow cytometry, plate clone formation experiments, and scratch tests were used to examine reactive oxygen species, proliferation, and migration of these cell lines.ResultsThe expression of NRF2 and its target genes increased, and tumor cell proliferation, migration, and tumor growth were accelerated in A549 and H460 cells stably transfected with KEAP1 mutants compared to control cells with a loss-of-function KEAP1 mutation and stably transfected with WT KEAP1 in both in vitro and in vivo studies. The proliferation of A549 cell line trasfected with the R320Q KEAP1 mutant was inhibited more apparent than that of the A549 cell line trasfected with WT KEAP1 after treatment with NRF2 inhibitor ML385.ConclusionSomatic mutations of KEAP1 identified from patients with LSCC likely promote tumorigenesis mediated by activation of the KEAP1/NRF2 antioxidant stress response pathway. NRF2 inhibition with ML385 could inhibit the proliferation of tumor cells with KEAP1 mutation.27fX3rkGbRpRrWDtKhtxqBVideo abstract

Bioinformatic and computational analysis for predominant mutations of the Nrf2/Keap1 complex in pediatric leukemia

The levels of reactive oxygen species (ROS) are tightly controlled and regulated by Nuclear Factor Erythroid-2-Like 2 (Nrf2) transcription factor, which is the main regulator of antioxidant responses and its suppressor protein Kelch-like ECH-associated protein 1 (Keap1). Our previous study has identified six novel changes in Nrf2/Keap1 pathway in pediatric ALL, which were described for the first time. These changes in the pathway are likely to alter the evolutionary process of amino acids and cause structural changes in the final products of genes. In this study, we aimed to compare the pathogenicity of eight determined mutations reported in our previous study by utilizing different programs with different algorithms and molecular dynamics simulation. Since it is too difficult to handle each existing mutation in a wet laboratory, in silico methods may give suggestion to choose the important mutations for further analysis and to establish the appropriate patient population and conduct wet laboratory studies. For this purpose, four different algorithms were used to evaluate the effects of single amino acid mutation. In addition, root-mean-square deviation, root-mean-square fluctuation and free-energy landscape analyses were performed to observe stability, flexibility and energetically favorable conformations, respectively, for each amino acid mutation. As a result, our study emphasizes the importance of Keap1 mutations in pediatric ALL Nrf2/Keap1 pathway, a total of eight mutations, two of which were shown for the first time in our study. Especially the mutations in the Keap1 Broad-Complex, Tramtrack and Bric-à-brac domain are worthy of attention. Communicated by Ramaswamy H. Sarma.

Keap1 mutation renders lung adenocarcinomas dependent on Slc33a1.

Approximately 20-30% of human lung adenocarcinomas (LUAD) harbor loss-of-function (LOF) mutations in Kelch-like ECH Associated-Protein 1 (KEAP1), which lead to hyperactivation of the nuclear factor, erythroid 2-like 2 (NRF2) antioxidant pathway and correlate with poor prognosis1-3. We previously showed that Keap1 mutation accelerates KRAS-driven LUAD and produces a marked dependency on glutaminolysis4. To extend the investigation of genetic dependencies in the context of Keap1 mutation, we performed a druggable genome CRISPR-Cas9 screen in Keap1-mutant cells. This analysis uncovered a profound Keap1 mutant-specific dependency on solute carrier family 33 member 1 (Slc33a1), an endomembrane-associated protein with roles in autophagy regulation5, as well as a series of functionally-related genes implicated in the unfolded protein response. Targeted genetic and biochemical experiments using mouse and human Keap1-mutant tumor lines, as well as preclinical genetically-engineered mouse models (GEMMs) of LUAD, validate Slc33a1 as a robust Keap1-mutant-specific dependency. Furthermore, unbiased genome-wide CRISPR screening identified additional genes related to Slc33a1 dependency. Overall, our study provides a strong rationale for stratification of patients harboring KEAP1-mutant or NRF2-hyperactivated tumors as likely responders to targeted SLC33A1 inhibition and underscores the value of integrating functional genetic approaches with GEMMs to identify and validate genotype-specific therapeutic targets.

The association between STK11/LKB1 and/or KEAP1 mutations and response to PD-1/PD-L1 inhibitors in patients with advanced non-small cell lung cancer (NSCLC).

e21538 Background: Advanced non-small cell lung cancer (NSCLC) patients with tumors harboring STK11/LKB1 or KEAP1 mutations have inferior treatment outcomes when treated with PD-1/PD-L1 blockade, regardless of KRAS status, PD-L1 score, or TMB score (Skoulidis et al, Cancer Discovery 2018, ASCO abstract 102, 2019). Methods: Retrospective clinical databases from Indiana University and Karmanos Cancer Institute were queried to identify patients from 2008-2019 with advanced NSCLC treated with PD-1/PD-L1 monotherapy (group A) or concurrent chemotherapy + PD-1/PD-L1 inhibitors (group B) whose tumors harbored STK11/LKB1 and/or KEAP1 mutations. Patients were characterized by best response, progression of disease (PD) or non-PD (complete response (CR) + partial response (PR) + stable disease (SD)). Patient and disease characteristics, response, and duration of response (> vs. < 6 months) were recorded. Results: A total of 77 patients met the above criteria (55 in group A, 22 in group B). 52 of 55 patients in group A received prior chemo. Patient and disease characteristics and outcomes are summarized in the table. Conclusions: STK11/LKB1 and/or KEAP1 mutations are independent of PD-L1/TMB status, although most patients had PD-L1 of <1%. Nearly ½ of patients with STK11/LKB1 and/or KEAP1 mutations achieve at least SD with PD-1/PD-L1 inhibitor monotherapy and more than 50% of those with non-PD maintain that response status for > 6 mos. The frequency and duration of non-PD in this population is higher in patients receiving chemotherapy + PD-1/PD-L1 concomitantly. [Table: see text]

Association of a STK11/KEAP1-mutation gene expression signature in lung adenocarcinoma with immune desertion in squamous cell carcinomas and mediation by NFE2L2 deregulation.

3082 Background: KEAP1 and STK11 mutations are associated with resistance to immune checkpoint inhibition (ICI) in non-small cell lung cancer (NSCLC). Mechanisms are currently unknown. Methods: We examined mutation, methylation, copy number and gene expression data from the cancer genome atlas (TCGA) lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LSCC), head and neck squamous cell carcinoma (HNSCC) and cervical carcinoma (CESC) data sets as well as public single cell gene expression data from a HNSCC cohort. Pathway annotations were performed using gene set enrichment analysis. A previously published cohort of NSCLC patients treated with ICI was analyzed for the predictive value of NFE2L2 mutations on PFS. Results: Annotation of STK11 and KEAP1 mutant LUAD revealed identical gene set enrichment for mitochondrial metabolism and downregulation of the STING-pathway, immune checkpoints, and interferon signaling. A STK11/KEAP1-mutation derived gene expression signature was established in LUAD and found to be driven by NFE2L2-regulated genes. This gene expression signature was independently predictive of immune desertion in LSCC, CESC and HNSCC and associated with STING-pathway downregulation in single cell sequencing analyses in HNSCC. KEAP1 and STK11 mutations were less frequent in LSCC, CESC and HNSCC but NFE2L2 mutations were identified in 15, 6 and 5%, respectively. NFE2L2 mutant SCC exhibited upregulation of the 15-gene- signature as well as immune desertion. In NSCLC, NFE2L2 mutations were associated with significantly worse PFS with ICI. Conclusions: Alterations of KEAP1, STK11, NFE2L2 and other related genes are linked to NFE2L2 target gene upregulation and immune desertion in LUAD, CESC, LSCC and HNSCC alike. The NFE2L2 pathway should be investigated clinically as a putative negative predictive biomarker for ICI and a potential therapeutic target.

Racial diversity of genomic alterations in lung adenocarcinomas.

9543 Background: Lung cancer is the leading cause of cancer related death in the United States in all racial groups. However, the overall death rate from lung cancer is different among white, black and Asian. Although differences in socioeconomic status and treatments received might be the contributing factors, the biology, particularly genomic alteration of cancer might also have an important impact. To date, the differences of genomic alterations among all racial patients are poorly understood. Methods: The American Association for Cancer Research (AACR) Project Genomics Evidence Neoplasia Information Exchange (GENIE, version 7.0) database was analyzed. A total of 8908 patients with lung adenocarcinoma (LUAD) were identified. Among them, 5652 patients who had comprehensive sequencing results (gene panel ≥ 275) were selected for further analysis. Patients with unknown race, undefined and Pacific Island (only 3 patients) were excluded. Finally, 5360 patients were included in the study. The prevalence and distribution of genomic alteration cross all racial groups were analyzed. Results: Overall 5951 samples from 5360 patients were collected (85.7% white, 7.9% Asian, 4.7% black, 0.2% Native American and 1.4% other). Most patients have only one sample. The median mutation counts in white, black, Asian, Native American and others are 7, 6, 5, 5, 7 respectively (Kruskal–Wallis test, P< 0.001). Asian have significantly higher rates of insertion and deletions than other races (14.8% of Asian versus 9.8% of white, 10.7% of black, 10.4% of Native American, 11.1% of other; Pearson’s chi-square test, < 0.001). TP53, EGFR, KRAS and STK11 are the most frequent alterations in white, black and other. EGFR, TP53, KRAS and APC are the most frequent alterations in Asian. STK11 mutations are rare in both Asian and Native American. Native Americans have more alterations of LRP1B, ARID2 and ATM, although the patients’ number remains small. ATM and KEAP1 mutations are also common in white and black. EGFR is the highest discrepancy gene in racial distribution. 78.9% Asian, 47.7% black, 36.4% of native American had EGFR alteration in comparison to 29.6% in white (Fisher's exact test, P < 0.001). KRAS is the second highest discrepancy gene in racial distribution. 11.6% Asian, 26.3% black, 27.3% of Native American had KRAS alteration in comparison to 36.1% in white (Fisher's exact test, P< 0.001). Conclusions: Our study demonstrated the potential diversity of genomic alterations across all racial groups with potential impact on therapeutic decisions.

Impact of SWI/SNF complex mutations in patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors: Immuno-oncology biomarker study in LC-SCRUM-Japan (LC-SCRUM-IBIS).

9530 Background: The SWI/SNF chromatin remodeling complex is reported to be involved in sensitivity and resistance to immune checkpoint inhibitor (ICI). However, their role in non-small cell lung cancer (NSCLC) remains unclear. We examined the relationship between SWI/SNF complex mutations and clinical outcomes of ICI in patients with NSCLC. Methods: Of 1017 lung cancer patients enrolled in LC-SCRUM-IBIS, 350 patients were analyzable for whole-exome sequencing (WES). WES data were used to analyze the presence of mutations in 29 major subunits of the SWI/SNF complexes. ARID1A and SMARCA4 mutations were also evaluated in a targeted NGS panel (Oncomine comprehensive assay, OCA). PD-L1 expression by 22C3, tissue tumor mutational burden (tTMB) by WES, STK11 and KEAP1 mutations by WES or OCA were also assessed. Durable clinical benefit (DCB) including CR, PR and SD > 6 mos to ICI, progression-free survival (PFS) and overall survival (OS) were compared in status of each of SWI/SNF complex mutations and other factors. Results: At least one mutation in any subunits of the SWI/SNF complex was present in 28% of NSCLC patients. The most common mutated subcomplexes were SMARCA4 (12%), BAF (7%: ARID1A, 4%), non-canonical BAF (3%), PBAF (3%), and SMARCA2 (2%). Of 101 NSCLC patients treated with PD-1/PD-L1 inhibitors, SMARCA4 mutations tended to be associated with lower DCB (16 vs 31%) and shorter median PFS (1.9 vs 3.6 m) and OS (7.4 vs 18.1m). Patients with ARID1A mutations tended to have better clinical outcomes (DCB, 40 vs 28%) compared to those without mutations. No significant associations were found between PD-L1 expression and SMARCA4 or ARID1A mutations. Patients with STK11/KEAP1 mutations had lower rate of PD-L1 expression (TPS > 50%) (18% vs 48%, P = 0.03) and worse clinical outcomes (DCB, 6 vs 33%) compared to those without mutations. There was no significant association between a tTMB status and clinical outcome. Conclusions: SMARCA4 and ARID1A mutations appear to affect clinical outcomes of ICI in NSCLC patients. These findings indicate that SWI/SNF complex mutations may serve as a predictive biomarker for ICI in NSCLC patients.

Phase II study of TAK228 in patients with advanced non-small cell lung cancer (NSCLC) harboring NFE2L2 and KEAP1 mutations.

9607 Background: Despite past efforts, no targeted therapies exist for squamous cell lung cancer (LUSC) pts. We identified a heretofore untargeted oncogene (NFE2L2)/tumor suppressor (KEAP1) pair, each mutated in ~15% of LUSCs. NFE2L2 encodes NRF2, a transcription factor involved in the oxidative stress response and targeted for degradation by KEAP1. NFE2L2 mutations (mut) occur only in an exon 2 hotspot (Neh2 domain), which is the binding site for KEAP1. Mutations in this region disrupt KEAP1 binding, leading to NRF2 nuclear translocation and increased mTOR signaling via RagD. We report translational studies and results from a phase 2 trial of the oral TORC1/2 inhibitor TAK228 in biomarker-selected pts. Methods: Cell line and xenograft experiments were performed using LK-2 LUSC (NFE2L2 E79K mut), A549 ADCL (KRAS G12S + KEAP1 loss), and SK-MES-1 LUSC cells (NFE2L2/KEAP1 WT) treated with TAK-228, everolimus, rapamycin, or deforolimus. Pts with stage IV LUSC harboring NFE2L2 or KEAP1 mut and ADCL harboring KRAS + KEAP1 co-mut were treated on an NCI CTEP phase 2 study of TAK228 3mg po qd (NCT02417701). Primary endpoint: ORR. Secondary endpoint: PFS. The study used a Simon 2-stage design for each cohort with H0 = 5% (N≥1/5 responses), HA = 40% (N≥2/10 responses). Results: TAK228 exhibited differential anti-tumor activity over TORC1 rapalogs in LK-2 and A549 cells. TAK228 alone was cytotoxic at sub-[μM] (IC50 68nM) in LK-2 cells; all other rapalogs had IC50s > 10μM. This was associated with marked decrease in TORC1/2 & MAPK signaling (decreased pS6, pAKT, pERK). Anti-tumor response was seen in LK-2 and A549 xenografts treated with TAK228. No anti-tumor/growth inhibitory responses were seen with any other rapalog. N = 21 evaluable pts have been treated (10 NFE2L2, 6 KEAP1, 5 KRAS+NFE2L2/KEAP1). Median age = 70; median prior lines tx = 2, smokers = 100%, median pack yrs = 39. Most common AEs included hyperglycemia (72%), fatigue (32%), diarrhea (32%), decreased appetite (32%). In NFE2L2 mut LUSC pts, ORR = 20% (2/10 confirmed PR), DCR = 100% with median PFS = 8.9mos (95%CI 7-NR). In KEAP1 mut LUSC pts, ORR = 17% (1/6 confirmed PR), DCR = 67% with PFS range = 1.8-8.6 mos. In KRAS + NFE2L2/KEAP1 mut ADCL pts, ORR = 0% and DCR = 0%. Conclusions: TAK228 is tolerable with differential activity in NFE2L2 (primary endpoint met) and KEAP1 mutant LUSC. A randomized phase 2 trial of TAK228 + docetaxel vs. SoC chemotherapy in advanced LUSC pts with NFE2L2/KEAP1 mut is in development (LungMAP S1900D) as is an NCI CTEP phase 1/1b trial of TAK228 + CB-839 in advanced NSCLC patients with NFE2L2/KEAP1 mut (NCI #10327). Clinical trial information: NCT02417701 .

A mid-chemoradiation dynamic risk model integrating tumor features and ctDNA analysis for lung cancer outcome prediction.

9046 Background: Circulating tumor DNA (ctDNA) molecular residual disease after curative intent therapy predicts disease progression in localized lung cancer. We hypothesized that integrating pre-CRT features and ctDNA levels during chemoradiation therapy (CRT) can predict patient outcomes earlier to enable response-adapted therapy. Methods: We identified pre-CRT features prognostic of disease progression after CRT for Stage II-III non-small cell lung cancer (NSCLC) in a historical “pre-CRT” training cohort of 109 patients. In addition, we applied CAPP-Seq ctDNA analysis pre-CRT and a median of 21 days into CRT (mid-CRT) to a “ctDNA” training cohort of 42 patients treated at MD Anderson and an independent validation cohort of 21 patients treated at Stanford. Prognostic pre-CRT features and mid-CRT ctDNA concentration were integrated using a Bayesian proportional hazards approach to generate a Continuous Individualized Risk Index (Kurtz et al. Cell 2019) for NSCLC (CIRI-NSCLC) to predict freedom from progression (FFP). Results: Adenocarcinoma histology (HR 2.6, P = 0.0005) and KEAP1 mutation (HR 2.7, P = 0.002) but not stage (P = 0.16), age (P = 0.60), or gender (P = 0.98) were significantly associated with FFP in the pre-CRT training cohort. Mid-CRT ctDNA concentration as a continuous variable was significantly associated with FFP in the ctDNA training cohort (HR 1.6, P = 0.04), and applying an optimal threshold identified in the training cohort (3.2 hGE/ml) significantly stratified FFP in the independent ctDNA validation cohort (HR 4.8, P = 0.02). CIRI-NSCLC enabled individualized real-time updating of the probability of FFP as model features became available over the course of CRT. CIRI-NSCLC outperformed individual model features in the independent validation cohort when compared by C-statistic (CIRI-NSCLC: 0.85; mid-CRT ctDNA: 0.76; histology: 0.66; KEAP1: 0.60). Across the whole cohort, patients with a greater than 66% risk of progression predicted by CIRI-NSCLC (n = 10) had an FFP of 10.0% at 12 months while patients with a less than 33% risk of progression predicted by CIRI-NSCLC (n = 22) had an FFP of 79.7% at 12 months (HR 15.0, P < 0.001). Conclusions: Our results suggest that CIRI-NSCLC can identify patients at very high and low risk of progression. Prospective evaluation will be necessary to test the potential utility of adapting treatment based on CIRI-NSCLC.

An integrative investigation on significant mutations and their down-stream pathways in lung squamous cell carcinoma reveals CUL3/KEAP1/NRF2 relevant subtypes

BackgroundMolecular mechanism of lung squamous cell carcinoma (LUSC) remains poorly understood, hampering effective targeted therapies or precision diagnosis about LUSC. We devised an integrative framework to investigate on the molecular patterns of LUSC by systematically mining the genomic, transcriptional and clinical information.MethodsWe utilized the genomics and transcriptomics data for the LUSC cohorts in The Cancer Genome Atlas.. Both kinds of omics data for 33 types of cancers were downloaded from The NCI’s Genomic Data Commons (GDC) (https://gdc.cancer.gov/about-data/publications/pancanatlas). The genomics data were processed in mutation annotation format (maf), and the transcriptomics data were determined by RNA-seq method. Mutation significance was estimated by MutSigCV. Prognosis analysis was based on the cox proportional hazards regression (Coxph) model.ResultsSignificant somatic mutated genes (SMGs) like NFE2L2, RASA1 and COL11A1 and their potential down-stream pathways were recognized. Furthermore, two LUSC-specific and prognosis-meaningful subtypes were identified. Interestingly, the good prognosis subtype was enriched with mutations in CUL3/KEAP1/NRF2 pathway and with markedly suppressed expressions of multiple down-stream pathways like epithelial mesenchymal transition. The subtypes were verified by the other two cohorts. Additionally, primarily regulated down-stream elements of different SMGs were also estimated. NFE2L2, KEAP1 and RASA1 mutations showed remarkable effects on the subtype-determinant gene expressions, especially for the inflammatory relevant genes.ConclusionsThis study supplies valuable references on potential down-stream processes of SMGs and an alternative way to classify LUSC.

A phase II randomized study of telaglenastat, a glutaminase (GLS) inhibitor, versus placebo, in combination with pembrolizumab (Pembro) and chemotherapy as first-line treatment for KEAP1/NRF2-mutated non-squamous metastatic non-small cell lung cancer (mNSCLC).

TPS9627 Background: Mutational activation of the KEAP1/NRF2 pathway occurs in >20% of NSCLC patients (pts). KEAP1/NRF2 activation protects tumor cells from diverse forms of oxidative stress and promotes tumor growth and survival. In pts w/ advanced NSCLC, mutation of the KEAP1/NRF2 pathway is associated w/ dramatically reduced survival and poor outcomes following standard-of-care therapy. These tumors have increased dependence on GLS-mediated conversion of glutamine to glutamate due to upregulation of NRF2 target genes involved in glutamine metabolism. Telaglenastat (CB-839), an investigational, first-in-class, potent, oral GLS inhibitor, has demonstrated preclinical activity in KEAP1/NRF2-mutated NSCLC cell lines and xenograft models. This study will evaluate the safety and efficacy of telaglenastat + standard-of-care pembro and chemotherapy as 1L therapy for KEAP1/NRF2-mutated non-squamous mNSCLC (NCT04265534). Methods: This phase II, randomized, multicenter, double-blind study will enroll ~120 pts with histologically or cytologically documented stage IV non-squamous NSCLC w/ KEAP1 or NRF2 mutation, no prior systemic therapy for mNSCLC, measurable disease (RECIST v1.1), ECOG PS 0-1, and no EGFR, ALK, ROS, or other actionable mutation w/ available approved therapy in 1L setting. KEAP1 or NRF2 mutations will be determined by next generation sequencing (NGS), and study-provided liquid biopsy NGS will be available. Pts will be randomized 1:1 to receive telaglenastat (800 mg BID PO) or placebo, in combination with pembro, carboplatin, and pemetrexed at standard doses on day 1 of each 21-day cycle. Pts will be stratified by STK11/LKB1 mutational status and M stage of cancer (M1a-b vs M1c). The study will include an initial safety run-in period (n=12; 1 cycle). Co-primary endpoints are safety and investigator-assessed progression-free survival (RECIST v1.1). Secondary endpoints include overall response rate, duration of response, overall survival, and efficacy analysis in the subgroup of pts w/ biochemical confirmation of KEAP1/NRF2 pathway activation. Findings of this novel NGS biomarker-selected study will inform the efficacy and safety profile of telaglenastat + standard-of-care chemoimmunotherapy for 1L treatment of KEAP1/NRF2-mutated, non-squamous mNSCLC. Clinical trial information: NCT04265534 .

Adaptation of the master antioxidant response connects metabolism, lifespan and feather development pathways in birds

Birds (Aves) display high metabolic rates and oxygen consumption relative to mammals, increasing reactive oxygen species (ROS) formation. Although excess ROS reduces lifespan by causing extensive cellular dysfunction and damage, birds are remarkably long-lived. We address this paradox by identifying the constitutive activation of the NRF2 master antioxidant response in Neoaves (~95% of bird species), providing an adaptive mechanism capable of counterbalancing high ROS levels. We demonstrate that a KEAP1 mutation in the Neoavian ancestor disrupted the repression of NRF2 by KEAP1, leading to constitutive NRF2 activity and decreased oxidative stress in wild Neoaves tissues and cells. Our evidence suggests this ancient mutation induced a compensatory program in NRF2-target genes with functions beyond redox regulation—including feather development—while enabling significant metabolic rate increases that avoid trade-offs with lifespan. The strategy of NRF2 activation sought by intense clinical investigation therefore appears to have also unlocked a massively successful evolutionary trajectory.

Whole-exome mutational landscape of metastasis in patient-derived hepatocellular carcinoma cells

In order to explore the genomic basis for liver cancer metastasis, whole-exome sequencing (WES) was performed on patient-derived hepatocellular carcinoma (HCC) cell lines with differential metastatic potentials and analyzed their clonal evolution relationships. An evolutionary tree based on genomic single nucleotide polymorphism (SNP) was constructed in MegaX software. The WES data showed that the average percentage of heterogeneous mutations in each HCC cell lines was 16.55% (range, 15.38%–18.17%). C: G > T: A and T: A > C: G somatic transitions were the two most frequent substitutions. In these metastatic HCC cell lines, non-silent gene mutations were found in 21.88% of known driver genes and 10 classical signaling pathways. The protein interaction network was constructed by STRING, and hub genes were found in the shared trunk mutation genes and the heterogeneous branch mutations respectively. In cBioPortal database, some of the selected hub genes were found to be associated with poor overall survival (OS) of HCC patients. Among the mutated HCC driver genes, a novel KEAP1 mutation with a homozygous frameshift truncation at the c-terminal Nrf2 binding region was detected and verified in MHCC97-H and HCC97LM3 cells. In conclusion, WES data demonstrate that HCC cell lines from tumor biopsy specimens of the same patient have obtained different metastatic potentials through repeated selection in rodents in vivo, and they do indeed have a genetic relationship at the genomic level.

Less immune cell infiltration and worse prognosis after immunotherapy for patients with lung adenocarcinoma who harbored STK11 mutation

The STK11 mutation defined a special subtype for patients with lung adenocarcinoma. The cBioPortal data platform was applied to analyze STK11 mutation frequency and the relationship between STK11 mutation and immune prognostic markers. The TIMER database was used to analyze the relationship between STK11 mutation and immune cell infiltration. The survival difference for lung adenocarcinoma patients harbored STK11 mutation who received immunotherapy also used the cBioPortal database. The results showed that STK11 mutation co-occurrence more KRAS and KEAP1 mutation and fewer TP53 and EGFR mutation (all, P < 0.05); the patients harbored STK11 mutation had a lower expression of PDL1 (P = 0.002), higher TMB score (P = 0.002), higher proportion of males and smoking history; the patients harbored STK11 mutation had fewer immune cell infiltration including B cell (P < 0.01), CD8+ T cell (P < 0.001), CD4+ T cell (P < 0.001), Macrophage (P < 0.001), Neutrophil (P < 0.001) and Dendritic cell (P < 0.001). Importantly, we found the patients harbored STK11 mutation who received immune checkpoint inhibitors have worse overall survival (OS) with median survival only 6 months. In conclusion, our study demonstrated that STK11 mutation defined a special subtype for lung adenocarcinoma patients with different co-occurrence gene mutation, lower PDL1 expression, fewer immune cell infiltration and worse OS benefit from immune checkpoint inhibitors.