KEAP1 Mutations Research Articles

Genetic alterations of Keap1 confers chemotherapeutic resistance through functional activation of Nrf2 and Notch pathway in head and neck squamous cell carcinoma

Keap1 mutations regulate Nrf2 activity and lead to chemoresistance in cancers. Yet the underlying molecular mechanisms of chemoresistance are poorly explored. By focusing and genotyping head and neck squamous cell carcinoma (HNSCC) that had available pathologic and clinical data, we provide evidence that Keap1 displays frequent alterations (17%) in HNSCC. Functional loss of Keap1 results in significant activation of Nrf2 and promotes cancer cell growth, proliferation, and elevated cancer stem cell (CSCs) self-renewal efficiency and resistance to oxidative stress. Furthermore, decreased Keap1 activity in these cells increased nuclear accumulation of Nrf2 and activation of the Notch pathway, causing enhanced transcriptional alterations of antioxidants, xenobiotic metabolism enzymes, and resistance to chemotherapeutic treatment. Limiting the Nrf2 activity by either Keap1 complementation or by Nrf2 silencing increased the sensitivity to chemotherapy in Keap1-mutated cells and repressed the CSC self-renewal activity. Our findings suggest that Keap1 mutations define a distinct disease phenotype and the Keap1-Nrf2 pathway is one of the leading molecular mechanisms for clinical chemotherapeutic resistance. Targeting this pathway may provide a potential and attractive personalized treatment strategy for overcoming chemotherapeutic resistance conferred by Keap1 mutations.

STK11 and KEAP1 mutations in non-small cell lung cancer patients: Descriptive analysis and prognostic value among Hispanics (STRIKE registry-CLICaP)

BackgroundMutations in STK11 (STK11Mut) and, frequently co-occurring, KEAP1 mutations (KEAP1Mut) are associated with poor survival in metastatic Non-small Cell Lung Cancer (mNSCLC) patients treated with immunotherapy. However, there are limited data regarding the prognostic or predictive significance of these genomic alterations among Hispanics. MethodsThis retrospective study analyzed a cohort of Hispanic patients (N = 103) diagnosed with mNSCLC from the US and seven Latin American countries (LATAM) treated with immune checkpoint inhibitors (ICI) alone or in combination as first-line (Cohort A). All cases were treated in routine care between January 2016 and December 2021. The main objectives were to determine the association of mutations in STK11 or KEAP1 in these patients’ tumors with overall (OS) and progression-free survival (PFS), presence of KRAS mutations, tumor mutational burden (TMB), and other relevant clinical variables. To compare outcomes with a STK11Wt/KEAP1Wt population, historical data from a cohort of Hispanic patients (N = 101) treated with first-line ICI was used, matching both groups by country of origin, gender, and Programed Death-ligand 1 (PD-L1) expression level (Cohort B). ResultsMost tumors had mutations only in STK11 or KEAP1 (45.6%) without KRAS co-mutation or any other genomic alteration. Besides, 35%, 8.7%, 6.8%, and 3.9% were KRASMut + STK11Mut, KRASMut + STK11Mut + KEAP1Mut, STK11Mut + KEAP1Mut, and KRASMut + KEAP1Mut, respectively. Based on KRAS status, STK11 alterations were associated with significantly lower PD-L1 expression among those with KRASWt (p = 0.023), whereas KEAP1 mutations were predominantly associated with lower PD-L1 expression among KRASMut cases (p = 0.047). Tumors with KRASMut + KEAP1Mut had significantly higher median TMB when compared to other tumors (p = 0.040). For Cohort A, median PFS was 4.9 months (95%CI 4.3–5.4), slightly longer in those with KEAP1mut 6.1 months versus STK11Mut 4.7 months (p = 0.38). In the same cohort, PD-L1 expression and TMB did not influence PFS. OS was significantly longer among patients with tumors with PD-L1 ≥ 50% (30.9 months), and different from those with PD-L1 1–49% (22.0 months), and PD-L1 < 1% (12.0 months) (p = 0.0001). When we compared the cohorts A and B, OS was significantly shorter for patients carrying STK1 [STK11Mut 14.2 months versus STK11Wt 27.0 months (p = 0.0001)] or KEAP1 [KEAP1Mut 12.0 months versus KEAP1Wt 24.4 months (p = 0.005)] mutations. PD-L1 expression significantly affected OS independently of the presence of mutations in STK11, KEAP1, or KRAS. TMB-H favored better OS. ConclusionsThis is the first large Hispanic cohort to study the impact of STK11 and KEAP1 mutations in NSCLC patient treated with ICI. Our data suggest that mutations in the above-mentioned genes are associated with PD-L1 expression levels and poor OS.

Abstract 2181: Genomic correlates of Metastasis in KRAS mutant lung adenocarcinoma

Abstract Background: Lung adenocarcinoma (LUAD) is a disease classified by molecular markers. In KRAS-mutant LUAD, STK11 and KEAP1 mutations are associated with decreased overall survival (OS), but predictors of metastasis have been poorly defined. In this study, we identify clinical and genomic predictors of metastatic KRAS-mutant LUAD. Methods: Patients with KRAS-mutant LUAD profiled by targeted next generation sequencing (OncoPanel) were included. Stage, histology, recurrence-free and overall survival were assessed. Clinical and genomic features between metastatic vs non-metastatic samples were compared. KRAS-mutant LUAD samples profiled using MSK-IMPACT in the AACR GENIE database were used to validate our findings. Continuous variables were compared using the Mann-Whitney U test and categorical variables were compared using the Fisher’s Exact test. Survival analysis was performed using the Cox Proportional Hazards model. WExT was used to identify co-occurring and mutually exclusive genetic alterations. Benjamini-Hochberg was used to correct for multiple comparisons. P-values &amp;lt; 0.05 and q-values &amp;lt; 0.10 were considered significant. Results: In the OncoPanel cohort (metastatic n=290; non-metastatic n=324), tumor mutational burden (TMB) (p = .001) and KEAP1 mutations (q = 0.05) were enriched in metastatic samples, while NFKBIA amplifications (q = 0.07) were enriched in non-metastatic samples. KEAP1/STK11 mutations significantly co-occurred (q &amp;lt; 1e-8). Compared to double wild-type samples: KEAP1/STK11 co-mutations were significantly enriched in metastatic samples (n = 72, p = 0.0002, OR 3.4); KEAP1-mutant samples trended towards enrichment in metastatic samples, (n = 21,p = 0.07, OR 2.47); STK11 mutations did not associate with stage (n = 53, p = 0.88, OR = 0.94). In multivariable survival analysis, metastasis (p &amp;lt; 0.005), KEAP1 mutation (p=0.01), and STK11 mutation (p=0.02) were associated with worse OS. In the MSK-IMPACT validation cohort (metastatic site n=417, primary site n = 781), KEAP1 was the only gene enriched in metastatic samples (q &amp;lt; 0.001) at q &amp;lt; 0.05. Compared to double wild type samples: KEAP/STK11 co-mutations (n=138, p &amp;lt; 0.0001, OR 2.1) and KEAP1 mutations (n=59, p = 0.04, OR 1.77) were enriched in metastatic samples; STK11-mutations did not associate with metastasis (n = 190, p = 0.34, OR 0.83). Other predictors of metastasis included Fraction Genome Altered (FGA) (p &amp;lt; 1e-5), TMB (p &amp;lt; 1e-5), and CDKN2A/B deletions (q &amp;lt; 0.003). Conclusion: While both KEAP1 and STK11 mutations are associated with decreased OS in KRAS-mutant LUAD, we find in two independent cohorts that only KEAP1 mutations and KEAP1/STK11 co-mutations, but not STK11 mutations, are associated with metastasis. We also found that FGA, TMB, CDKN2A/B deletions are strongly associated with metastasis. Further research is necessary to understand the influence of KEAP1 mutations, independent of and in-conjunction with STK11 mutations, on metastasis. Citation Format: Daniel Boiarsky, Christine A. Lydon, Emily Chambers, Pasi A. Janne, Mark M. Awad, Eliezer M. Van Allen, David Barbie, Natalie I. Vokes. Genomic correlates of Metastasis in KRAS mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2181.

Abstract 2174: Sirpiglenastat (DRP-104), a novel broad acting glutamine antagonist, has therapeutic potential in targeting KEAP1-mutant lung cancer

Abstract Treating KRAS mutant lung adenocarcinoma (LUAD) remains a major challenge for clinical oncology. Approximately 20% of KRASmutant LUAD tumors carry loss-of-function mutations in KEAP1, a negative regulator of NRF2, which is the master transcriptional regulator of the endogenous antioxidant response. Emerging clinical studies demonstrate that KEAP1-mutant tumors are refractory to standard-of-care chemotherapy, immunotherapy, radiation and more recently KRASG12C inhibitors. Using a genetically engineered mouse model of KRAS-driven LUAD we demonstrated that loss of Keap1 hyper-activates Nrf2 and accelerates KRAS-driven LUAD. We observe that the ability of KEAP1 mutant 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 through the pharmacological inhibition of glutaminase and other glutamine metabolizing enzymes. Using sirpiglenastat (DRP-104), a novel broad acting glutamine antagonist which inhibits all glutamine-consuming reactions, we demonstrate robust anti-tumor efficacy in multiple mouse and human pre-clinical KEAP1-mutant NSCLC models. Furthermore, we have characterized the metabolic mechanisms underlying DRP-104 sensitivity. Our studies demonstrate that DRP-104 has a distinct and broader mechanism of action compared to glutaminase selective inhibition, suggesting broader inhibition of glutamine metabolism may be a more effective therapeutic approach against KEAP1-mutant tumors. We have recently demonstrated that KEAP1-mutant tumors evade anti-tumor immune responses and do not respond to immune checkpoint blockade, which is also supported by clinical observations. We are currently assessing whether DRP-104 is able to promote anti-tumor immune responses as a single agent or in combination with immune checkpoint blockade in KEAP1-mutant tumors. Our studies will provide a rationale for sub-stratification of KEAP1-mutant patients with hyperactivation of the NRF2 pathway, which is a genetic subset of NSCLC with great clinical need, as treatment candidates for sirpiglenastat. A first-in-human clinical trial of sirpiglenastat (DRP-104) is currently ongoing and will further explore this hypothesis in a LUAD cohort (NCT04471415). Citation Format: Sarah LeBoeuf, Ray Pillai, Shih Ming Huang, Triantafyllia Karakousi, Warren Wu, Volkan Sayin, Robert Wild, Thales Papagiannakopoulos. Sirpiglenastat (DRP-104), a novel broad acting glutamine antagonist, has therapeutic potential in targeting KEAP1-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2174.

Abstract 5742: The landscape of genetic alteration in Chinese lung adenosquamous carcinoma patients

Abstract Background: Lung adenosquamous carcinoma (LASC) is a mixed histologic component tumor type that contains both squamous cell carcinoma and adenocarcinoma, with each component accounting for at least 10% of tumors. The incidence of LASC is very low, only comprising 0.4-4% of all lung cancers. LASC yields a more aggressive clinical course, and its prognosis is generally worse than that of adenocarcinoma and squamous cell carcinoma of the lung. Due to the rarity of LASC, currently, there is no standard treatment. Also, to date, limited genomic data have been performed. In this study, we analyzed the genomic alterations and clinical signatures of LASC. Materials and Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor tissues and matched blood samples were collected for a targeted, next-generation sequencing (NGS) assay at OrigiMed (Shanghai, China), a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Genomic alterations including single base substitution, indel, copy number variations, and gene fusion and rearrangement were assessed. Results: A total of 124 LASC patients were enrolled in our study, including 77 (62.1%) males and 47 (37.9%) females, with a median age of 61 years old (range 27-82). Forty-three (43, 34.68%) patients had a smoking history and half of patients possessed a Stage III/IV disease. The most commonly altered genes were TP53 (66.9%), EGFR (54.8%), CDKN2A (21%), TERT (21%), and LRP1B (18.5%). Surprisingly, 91.1% and 52.4% patients harbored at least one druggable alteration based on the solid cancer and NSCLC NCCN guides, respectively. The most frequent druggable mutation, EGFR, is comprised of 35 (51.47%) single mutations and 33 (48.53%) complexed mutations, with most containing a 19 exon deletion and EGFR amplification (12%). The median tumor mutational burden (TMB) of patients with LASC was 5.15 muts/Mb (range, 0-69.3 muts/Mb), and 33 (26.6%) patients were TMB-H (TMB ≥ 10 Muts/Mb). Patients with an ARID2, BRCA1, or KEAP1 mutation had a significantly higher value of TMB than wild type (median TMB 16.1 vs. 5 muts/Mb, 29.7 vs. 5 muts/Mb, 14.6 vs. 4.85 muts/Mb, respectively, P &amp;lt; 0.05). Patients with HRR or DDR mutations also displayed a higher TMB value than for wild type. We additionally determined that mutation signature 1 (the clock-like signature), signature 2 (the APOBEC signature), and signature 3 (the BRCA mutational signature) exhibited a higher mutational load for the entire cohort. All patients with signature 2 or 3 displayed a HLA-B heterozygous subtype. Conclusions: Our study revealed genomic profiling for the largest sample size of Chinese LASC patients to date. Profiling obtained from this study demonstrates that LASC patients have the same chance for targeted therapy and immune checkpoint inhibitors as compared to lung adenocarcinoma and lung squamous cell carcinoma, respectively. Citation Format: Hongbiao Wang, Yingcheng Lin, Jun Liu, Yuyin Cai, Xiaofang Qi, Lujia Huang. The landscape of genetic alteration in Chinese lung adenosquamous carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5742.

Abstract 449: Machine learning models identify histological features that can predict KEAP1 mutations in lung adenocarcinoma

Abstract Background: In lung cancer, the KEAP1/NRF2 pathway modulates an anti-tumor response by regulating cellular metabolism and inflammatory processes. Approximately 19% of lung adenocarcinoma (LUAD) tumors have a mutation in KEAP1. Clinically, KEAP1-mutated LUAD has poor prognosis, and there is need for rapid and accurate patient genotyping to inform treatment decisions. Here, we describe machine learning (ML) models that can predict KEAP1 mutation status from tissue histology. Methods: ML models, pre-trained to identify and quantify areas of tissue (cancer epithelium, cancer stroma, and necrosis), counts of cancer, fibroblast, and immune cells (lymphocytes, macrophages, plasma cells) in non-small cell lung cancer (NSCLC), were applied to 208 hematoxylin and eosin (H&amp;E)-stained whole slide images (WSI) of LUAD from The Cancer Genome Atlas (TCGA) without further training. Genomic analyses indicated that 17% (N=35) of these cases are KEAP1MUT. Human Interpretable Features (HIFs), based on histology predictions, are automatically extracted from the model and provide a quantitative description of the tumor microenvironment of each WSI. Associations between HIFs and KEAP1MUT were determined by univariate analysis followed by false discovery rate (FDR) correction. Hierarchical clustering using cross correlation and combining p-values for HIF groups using an Empirical Brown’s method identified correlations between HIFs. Confounding factor influence was accounted for after positive associations were identified. Independent validation of associations between KEAP1MUT and HIFs was performed using TCGA transcriptomic data to correlate specific mutations with mRNA expression of relevant markers. Results: ML-models generated 4,443 HIFs from the TCGA LUAD WSI, which were reduced to 2,684 HIFs after removal of outlier HIFs, exclusion of HIFs that are degenerate, have missing features, or are of an absolute value. KEAP1MUT was significantly associated with 193 HIFs in univariate analyses (p&amp;lt;0.05 after FDR correction), and four groups of HIFs after taking correlations into account (p&amp;lt;0.05 after group-wise FDR correction; 211-264 HIFs per group). 161 HIFs were identified by both methods. Further assessment of the KEAP1MUT -associated HIFs showed that these mutations were correlated with a reduction in macrophages in the tumor microenvironment (TME). This was supported by analysis of transcriptomic data from KEAP1MUT (N=35) and KEAP1WT (N=164) samples, which showed significantly reduced expression of the macrophage marker CD14 (p&amp;lt;0.001) in KEAP1MUT tissue samples. Conclusions: ML model quantification of TME histological features can generate HIFs that correlate with the KEAP1MUT status of a LUAD biopsy. These results exemplify how ML-powered digital pathology could predict molecular markers directly from standard H&amp;E biopsy slides. Citation Format: Robert Egger, Martin Ieong, Murray Resnick, Amaro Taylor-Weiner, Victoria Mountain, Ilan Wapinski, Michael Montalto, Andrew Beck, Josie Hayes, Benjamin Glass. Machine learning models identify histological features that can predict KEAP1 mutations in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 449.

Genomic correlates of acquired resistance to PD-(L)1 blockade in patients with advanced non-small cell lung cancer (NSCLC).

9021 Background: Despite improvements in survival with immune checkpoint inhibition (ICI), the majority of patients develop acquired resistance to ICI after an initial benefit. However, the mechanisms underlying acquired resistance to ICI in NSCLC are largely unknown. Methods: Patients with advanced NSCLC treated with ICI at the Dana-Farber Cancer Institute (DFCI), and whose tumors underwent genomic profiling before and after ICI, with no intervening therapies, were included. Mutations, tumor mutational burden (TMB), copy number variations (CNVs), and PD-L1 tumor proportion score (TPS) were compared between pre- and post-ICI samples. Acquired resistance was defined as the development of disease progression after an initial objective response, or stable disease ≥3 months with PD-(L)1 blockade. Results: Among 1763 patients with advanced NSCLC who received ICI, 45 had matched pre- and post-ICI tissue samples available for genomic profiling. Putative mechanisms of resistance were identified in 55% of cases (N = 25). Five patients (20%) acquired an STK11 mutation, one patient (4%) acquired a KEAP1 mutation, and another patient (4%) developed concurrent KEAP1 and SMARCA4 mutations. A patient (4%) with KRAS G12C-mutant NSCLC developed concurrent STK11 and KEAP1 mutations at resistance. In 3 cases (12%) with pre-existing STK11 or KEAP1 mutations prior to ICI administration, we identified acquired copy losses of STK11 and KEAP1, respectively, resulting in bi-allelic inactivation of these genes. Acquired beta-2-microglobulin ( B2M) mutations were detected in 3 patients (12%), one of whom developed concurrent B2M copy loss, indicating bi-allelic inactivation. Eight additional patients (32%) developed B2M gene deletions. Other acquired alterations that have been implicated in ICI resistance included CDKN2A/B loss (N = 10, 40%), including 5 with bi-allelic deletion, acquired PTEN deletions (N = 5, 20%), and MDM2 amplification (N = 2, 8%). When we examined alterations in immune checkpoint genes, we identified acquired CD274 (PD-L1) and PDCD1LG2 (PD-L2) loss in 8% of cases (N = 2), and bi-allelic deletion in one case (4%). Intervening ICI did not affect TMB (median TMB: 8.7 [pre-ICI] vs 9.1 [post-ICI] mut/Mb, P = 0.6), PD-L1 expression (median PD-L1 TPS: 3% [pre-ICI] vs 5.0% [post-ICI] mut/Mb, P = 0.5), or aneuploidy levels (as fraction of genome altered [FGA]) (median FGA: 18.4% [pre-ICI] vs 21.1% [post-ICI], P = 0.2), indicating that acquired gene level CNVs were not a reflection of increased cancer aneuploidy. In a control cohort of 30 patients with pre- and post-chemotherapy matched samples which underwent genomic profiling, no acquired mutations in STK11, KEAP1, SMARCA4, or B2M were detected. Conclusions: Mechanisms of acquired resistance to PD-(L)1 blockade are heterogenous, and new therapeutic strategies are required to delay and overcome ICI resistance in patients with NSCLC.

Three-year outcomes and correlative analyses in patients with non–small cell lung cancer (NSCLC) and a very high PD-L1 tumor proportion score (TPS) ≥ 90% treated with first-line pembrolizumab.

9043 Background: Although 1st-line PD-1 monotherapy has improved survival in advanced NSCLC with a PD-L1 TPS ≥50%, responses occur in ̃45% of patients (pts). We previously showed that among pts treated with 1st-line pembrolizumab, clinical outcomes were significantly improved in those with a PD-L1 TPS of ≥90% compared to a TPS of 50-89%. Here, we report the 3-year survival analysis to 1st-line pembrolizumab in pts with a PD-L1 TPS ≥90% vs 50-89%, and characterize genomic and immunophenotypic differences between these PD-L1 expression groups. Methods: Pts with stage IV EGFR/ALK wild-type NSCLC and PD-L1 TPS ≥50% who received 1st-line pembrolizumab at the Dana-Farber Cancer Institute (DFCI) and Memorial Sloan Kettering Cancer Center (MSKCC), with a minimum follow-up of 3 years were included. Comprehensive tumor genomic profiling and multiplexed immunofluorescence (mIF) were performed to examine genomic and immunophenotypic correlates of very high PD-L1 expression on separate cohorts of NSCLC at the DFCI. Results: Among 396 pts, median age was 69, 53.3% were women, 90.1% had a history of tobacco use, 83.6% had a ECOG performance status of 0-1, and 28.8% had a KRAS mutation. At a median follow-up of 42.6 months, median progression-free (mPFS) and overall survival (mOS) in the entire cohort were 5.1 months, and 19.0 months, respectively. When compared to pts with a PD-L1 TPS of 50-89% (N = 252), those with PD-L1 TPS ≥90% (N = 144) had a significantly longer mPFS (6.0 vs 4.5 months, HR 0.67, p &lt; 0.001), and longer mOS (30.2 vs 16.9 months, HR 0.66, p &lt; 0.01). Kaplan-Meier estimates of the 3-year PFS and OS were 24.9% and 47.0% in the PD-L1 TPS ≥90% groups, and 9.4% and 27.7% in the PD-L1 TPS 50-89% group, respectively. A PD-L1 TPS ≥90% was confirmed to be an independent predictor of improved PFS (HR 0.68, p &lt; 0.01) and OS (HR 0.67, p &lt; 0.01) in multivariable analysis. Tumor genomic profiling from a separate cohort of 500 NSCLC samples revealed that mutations in STK11, KEAP1, FBXW7, and CTNNB1 were significantly more frequent in tumors with a PD-L1 TPS of 50-89% compared to those with a PD-L1 TPS ≥90% (q &lt; 0.05). mIF on 91 NSCLCs identified significantly higher CD8+PD1+ T cells and PD-L1+ immune cells in tumors with PD-L1 TPS ≥90% vs 50-89% (p &lt; 0.05). Conclusions: Pembrolizumab monotherapy continues to demonstrate a meaningful long-term survival benefit in pts with advanced NSCLC and a PD-L1 TPS ≥90%. NSCLCs with very high PD-L1 TPS may have a more favorable genomic and immunophenotypic profile. These findings have implications for treatment selection and clinical trial interpretation and design.

Genomic landscape of SMARCA4-deficient lung tumors by clinical RNA sequencing.

3118 Background: SMARCA4-deficient lung cancer is an undifferentiated lung cancer subtype associated with poor prognosis and morphological features that make them challenging to distinguish from sarcoma. These tumors are known to be resistant to standard of care surgery, radiation, and chemotherapy. Recent case reports suggest that these tumors might be resistant to immunotherapy. An assessment of the genomic and transcriptomic features of SMARCA4-deficient thoracic tumors may identify potential novel targets and treatment strategies for this new WHO lung cancer classification. Methods: We retrospectively analyzed de-identified NGS data from 8,484 thoracic formalin-fixed, paraffin-embedded tumor biopsies from lung cancer patients sequenced using the Tempus|xT solid tumor assay (DNA-seq of 595-648 genes at 500x coverage; whole-exome capture RNA-seq). Tumor-normal match sequencing was performed for all tumors, enabling the detection of incidental germline alterations across 46 genes. SMARCA4-deficiency was defined as tumors with a pathogenic or likely pathogenic SMARCA4 single nucleotide variant, insertion/deletion, or copy number alteration. Statistical significance was determined using Fisher's exact test and Wilcoxon rank-sum tests. Results: SMARCA4-deficiency was detected in 370 (4.4%) tumors, of which over 80% were stage III or IV. SMARCA4-deficient tumors included more male patients (63% vs 49%, p &lt; 0.001) and younger age at diagnosis (median 64 vs 68 years, p &lt; 0.001). There were more patients with high tumor mutational burden (TMB-H, ≥10 mutations per megabase) (34% vs 15%, p &lt; 0.001), and fewer patients with positive PD-L1 immunohistochemical staining (44% vs 54%, p = 0.009) compared to SMARCA4 wild-type tumors. Microsatellite instability status occurred at similar low frequencies across SMARCA4-deficient vs wild-type tumors (0.8% vs 0.5%, p = 0.5). SMARCA4-deficient tumors showed enrichment for somatic mutations in TP53 (71% vs 47%, q &lt; 0.001), STK11 (22% vs 6.8%, q &lt; 0.001), KEAP1 (15% vs 4.2%, q &lt; 0.001), and CDKN2A (15% vs 5.9%, q &lt; 0.001) compared to wild-type. Tumor normal-match sequencing identified incidental germline mutations in MUTYH (2.2%), ATM (1.1%), ATP7B (0.5%), and MSH6 (0.5%) for SMARCA4-deficient tumors. RNA-sequencing analysis confirmed reduced transcriptional expression of SMARCA4 (p &lt; 0.001), CD274 (PD-L1; p &lt; 0.001), TNFRSF18 (p &lt; 0.001), and TNFRSF4 (p = 0.035) in deficient tumors vs wild-type. Furthermore, SMARCA4-deficient tumors revealed reduced infiltration of CD4+ T cells (19% vs 22%, p &lt; 0.001). Conclusions: This study reveals the unique genomic and transcriptional characteristics of SMARCA4-deficient lung tumors. Further studies are needed to assess the impact of immunotherapies and targeted therapies among this patient population.

Machine learning modeling of real-world primary resistance and hyperprogression in immune checkpoint inhibitor (ICI) treated advanced non-small cell lung cancer (aNSCLC) patients.

e21127 Background: Many ICI-eligible patients display primary resistance (10-res), with some experiencing hyperprogressive disease (HPD). Machine learning (ML) on real-world data (RWD) can improve our clinical and mechanistic understanding related to ICI response. Methods: XGBoost models were built on ConcertAI’s oncology clinicogenomic database, comprising structured EMR, curated records from unstructured documents, and NGS reports, to predict 10-res and HPD in aNSCLC patients treated with ICI or chemotherapy (CT) within their 1st three lines between January 2015 and June 2021. HPD was defined as either tumor progression, treatment (Tx) discontinuation, or death within 49 days from Tx start (index date). 10-res was defined as any of these events after 49 days but within 180 days from Tx start. Models were trained on a clinical (N = 7647, ICI = 3265, 10-res = 3909, HPD = 2076), and a clinicogenomic (CG) (2304, 1271, 1084, 596) cohort. A 60:20:20 split was used for training, validation, and testing. Baseline period was defined as 180 days prior to the index date. Prognostic and predictive factors were identified from Shapley Additive Explanation (SHAP) values. Results: The clinical model was superior at predicting HPD; the CG model was better at predicting 10-res (AUROC = 0.67 for both). Total metastases (≥ 2) and lower levels of hemoglobin (HGB), and lymphocytes, higher ALP were risk factors for both HPD and 10-res. Elevated WBC and bilirubin, higher heart rate, lower body temperature, and history of anti-inflammatory medications were risk factors for HPD. Non-ICI-based regimens, low TMB, lower PD-L1 expression, and mutations in EGFR or KEAP1 were risk factors for 10-res. Mutation in KRAS was protective of 10-res. In addition to PD-L1 and TMB, the models identified prior lung radiation, smokers, higher baseline comorbidities, and positive MMR status as predictive of lower risk of 10-res from ICI but not in CT. STK11 mutation was predictive of higher risk of 10-res from ICI vs. CT. Non-adenocarcinoma histology was predictive of higher risk of HPD from ICI vs. CT. Conclusions: ML on RWD generated evidence to support both established and emerging prognostic and predictive markers for ICI response.[Table: see text]

A pancancer analysis of impact of MDM2/MDM4 on immune checkpoint blockade (ICB).

2630 Background: MDM2/MDM4 are implicated in hyperprogression after immune checkpoint blockade (ICB). Our preclinical studies showed reduced T-cell killing of MDM2-amplified tumor cells that was overcome by an MDM2 antagonist or gene knockdown, and we observed additional tumor killing by T-cells with MDM2 inhibition plus anti-PD1. We hypothesized that MDM2/4 gene amplification/overexpression correlates with resistance to ICB and investigated the association of MDM2/4 alterations to overall survival (OS) following ICB across multiple solid tumors. Methods: Solid tumors tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (NGS) were analyzed. MDM2/4 amplification (amp) was tested by NGS and determined as either amp4 (cutoff of &gt;=4.0 copies) or amp6 (&gt;=6.0) or amp8 (&gt;=8.0). Real-world OS was obtained from insurance claims data and calculated from treatment start or tissue collection to last contact. Kaplan-Meier estimates were calculated for molecularly defined groups. X2/Fisher-Exact were used and significance determined as P-value adjusted for multiple comparisons (q&lt;0.05). Results: In a large cohort of TP53-wild type solid tumors, 2785 had MDM2 amp4 (262 were ICB-treated), 2108 had MDM2 amp6 (ICB: 192), 1721 had MDM2 amp8 (ICB: 149); 1040 tumors had MDM4 amp4 (ICB: 59), 293 had MDM4 amp6 (ICB: 8) and 217 had amp8 (ICB: 4). NSCLC, GBM, breast, bladder cancer and liposarcoma had the highest MDM2 amp and breast cancer, GBM, uterine, NSCLC and ovarian cancers had the highest MDM4 amp. When all tumors were considered, MDM2 amp4, 6 and 8 significantly associated with shortened OS (amp4: HR 1.31; amp6: HR 1.31; amp8: 1.29, all p&lt;0.0001); similar results were seen with MDM4 (amp4: HR 1.14, p=0.004; amp6: HR 1.51, p&lt;0.00001 and amp8: HR 1.6, p&lt;0.00001). Of note, MDM4 amp4 but not MDM2 amp4 was associated with significantly worse post-ICB survival (HR: 1.55, p=0.009).When comparing molecular differences between MDM2 amp4 and MDM4 amp4, significantly higher PDL1 expression was associated with MDM2 amp (Ab clone 28.8: 58% vs. 28%; clone 22c3: 48% vs.25%, q&lt;0.05); while higher mutation rates of ARID1A, PIK3CA, PTEN, KRAS and CTNNB1 were associated with MDM4 amp (all q&lt;0.05). When investigating NSCLC, MDM4 amp4 was associated with decreased post-ICB survival in NSCLC (HR: 2.59, p=0.001) but not MDM2; when comparing the molecular alterations, MDM2 amp was associated with significantly higher PDL1 (22c3) (54% vs. 27%), EGFR mutation (36% vs. 14%) but less prevalent KRAS (15% vs. 50%), STK11 (11% vs. 41%), KEAP1 (2.5% vs. 27%) and BRAF (2% vs. 16%) mutations. The association with poor prognosis of MDM2 amp4/6 (HR: 1.2 and 1.3, p&lt;0.05) and MDM4 amp4/6 (HR: 1.3 and 1.6, p&lt;0.05) was seen in breast cancer, but not in NSCLC, GBM, bladder or uterine cancers. Conclusions: MDM2 and MDM4 amplification are negative prognostic factors in TP53-WT breast cancer while MDM4 amp is associated with reduced survival in ICB-treated NSCLC.

Clinicopathologic characteristics and outcomes for patients with KRAS G12D-mutant non-small cell lung cancer (NSCLC).

e21024 Background: Co-occurring mutations in KRAS-mutant NSCLC are associated with discrete biological properties and modulate therapeutic susceptibilities. As G12D-specific inhibitors are expected to enter the clinic, we sought to define the clinicopathologic characteristics and outcomes of patients (pts) with KRAS G12D-mutant NSCLC. Methods: This was a retrospective single-institution study. Pts with NSCLC and KRAS G12D mutations detected by the Massachusetts General Hospital SNaPshot next-generation sequencing assay were identified. Clinical and pathologic characteristics, including co-mutation status and survival outcomes, were collected by chart review. Results: Among pts who underwent SNaPshot testing between May 2014 and August 2021, 665 had cancers that were positive for KRAS G12D, of whom 107 (16.1%) had NSCLC. Most common histology was adenocarcinoma (93, 86.9%). PD-L1 level was &lt; 1% in 24 (22.4%), 1-49% in 19 (17.8%), &gt; 50% in 13 (12.2%), and not assessed in 51 (47.7%). Co-occurring mutations were frequent: 17 NSCLCs had co-occurring STK11 mutations (15.9%), 36 (33.6%) TP53 mutations, and 11 (10.3%) SMARCA4 mutations, of which 2 were truncating. Genetic alterations were detected in a variety of other genes including in the RTK/RAS/MAPK (20, 18.7%), PI3K/AKT/mTOR (12, 11.2%), cell-cycle (17, 15.9%), and WNT (11, 10.3%) pathways. Among 58 tumors with KEAP1 mutation testing, 10 (17.2%) were positive. 75 (70.1%) pts had metastatic disease, including 51 pts who had metastatic disease at initial diagnosis. CNS metastases were present in 27 (36.0%) pts, including 14 pts with CNS metastases at initial diagnosis. Among the 57 pts with metastatic disease who received first-line therapy, 28 (49.1%) received platinum-based chemotherapy, 17 (29.8%) immunotherapy alone, and 12 (21.1%) platinum chemotherapy plus immunotherapy. Median PFS in pts treated with first-line therapy was 4.0 months (95% CI 2.1– 5.7) and median OS 14.3 months (95% CI 8.1 – 27.4). When stratified by co-mutation status, pts with STK11 mutations had significantly worse outcomes than STK11-wild-type (median PFS: 1.2 months vs 4.8 months, p = 0.0165; median OS: 4.3 months vs 21.6 months, p = 0.0015), whereas TP53 exerted no influence. Conclusions: Co-occurring mutations were common in pts with KRAS G12D-mutant NSCLC. Outcomes were poorer for KRAS G12D/ STK11 pts, whereas co-occurring TP53 did not impact survival. Clinical trials examining G12D inhibitors should stratify by STK11.

Mutational Activation of the NRF2 Pathway Upregulates Kynureninase Resulting in Tumor Immunosuppression and Poor Outcome in Lung Adenocarcinoma.

Simple SummaryActivation of the Nuclear factor-erythroid factor 2-related factor 2 (NRF2) pathway through gain-of-function mutations or loss-of-function of its suppressor Kelch-like ECH-associated protein 1 (KEAP1) is frequent in lung cancer. NRF2 activation has also been reported to alter the tumor microenvironment. Proteomic profiles of 47 lung adenocarcinoma (LUAD) cell lines (11 KEAP1 mutant and 36 KEAP1 wild-type) revealed the tryptophan-kynurenine enzyme kynureninase (KYNU) as a top overexpressed protein associated with activated NRF2. Mechanistic studies demonstrated that NRF2 is a regulator of enzymatically functional KYNU in LUAD. Analysis of multiple independent gene expression datasets of human lung cancer and a LUAD tumor microarray demonstrated that elevated tumor KYNU expression was associated with immunosuppression, including potent induction of T-regulatory cells, increased levels of PD1 and PD-L1, and poorer overall survival. Our findings indicate a novel mechanism of NRF2 tumoral immunosuppression through upregulation of KYNU.Activation of the NRF2 pathway through gain-of-function mutations or loss-of-function of its suppressor KEAP1 is a frequent finding in lung cancer. NRF2 activation has been reported to alter the tumor microenvironment. Here, we demonstrated that NRF2 alters tryptophan metabolism through the kynurenine pathway that is associated with a tumor-promoting, immune suppressed microenvironment. Specifically, proteomic profiles of 47 lung adenocarcinoma (LUAD) cell lines (11 KEAP1 mutant and 36 KEAP1 wild-type) revealed the tryptophan-kynurenine enzyme kynureninase (KYNU) as a top overexpressed protein associated with activated NRF2. The siRNA-mediated knockdown of NFE2L2, the gene encoding for NRF2, or activation of the NRF2 pathway through siRNA-mediated knockdown of KEAP1 or via chemical induction with the NRF2-activator CDDO-Me confirmed that NRF2 is a regulator of KYNU expression in LUAD. Metabolomic analyses confirmed KYNU to be enzymatically functional. Analysis of multiple independent gene expression datasets of LUAD, as well as a LUAD tumor microarray demonstrated that elevated KYNU was associated with immunosuppression, including potent induction of T-regulatory cells, increased levels of PD1 and PD-L1, and resulted in poorer survival. Our findings indicate a novel mechanism of NRF2 tumoral immunosuppression through upregulation of KYNU.

Survival Outcomes and Treatment Patterns in Patients With NFE2L2 and/or KEAP1 Mutation-Positive Advanced Squamous Cell NSCLC Using a Real-World Clinico-Genomic Database

BackgroundNFE2L2 and/or KEAP1 mutations are associated with worse prognosis in all non-small cell lung cancer (NSCLC). We determined real-world survival outcomes and treatment patterns among patients with advanced squamous cell NSCLC by NFE2L2 and KEAP1 mutation status. Patients and MethodsA retrospective study (January 2011-December 2018) was conducted using a de-identified US-based clinico-genomic database. Adult patients with advanced squamous cell NSCLC with ≥ 2 in-network visits and comprehensive genomic profiling during the study period were included. Outcomes included real-world progression free survival (rwPFS) by line of therapy and overall survival (OS). The real-world effectiveness of anti-PD-1/PD-L1 first-line therapy was also evaluated in patients with a NFE2L2 and/or KEAP1 mutation. ResultsOf 703 patients included (median age: 70.0 years), 31.6% had a NFE2L2 and/or KEAP1 mutation. The most common first- and second-line treatments regardless of mutation status were platinum-based chemotherapies and anti-PD-1/PD-L1 therapies. The most common third-line treatment was anti-PD-1/PD-L1 therapy in patients with a NFE2L2 and/or KEAP1 mutation and single-agent chemotherapy in patients with wild-type disease. Patients with a NFE2L2 and/or KEAP1 mutation versus wild-type disease had significantly shorter rwPFS (4.54 vs. 6.25 months; P = .003) following first- but not second- or third-line therapy and shorter median OS (13.59 vs. 17.37 months; P = .4105). No survival differences were observed in patients with a NFE2L2 and/or KEAP1 mutation receiving first-line anti-PD-1/PD-L1 therapies versus other therapies. ConclusionsPatients with advanced squamous cell NSCLC with a NFE2L2 and/or KEAP1 mutation have poor real-world survival, highlighting the need for a genotype-directed therapeutic strategy in this population.

EMT, Stemness, and Drug Resistance in Biological Context: A 3D Tumor Tissue/In Silico Platform for Analysis of Combinatorial Treatment in NSCLC with Aggressive KRAS-Biomarker Signatures.

Simple SummaryThe phenotypic transition of tumor cells from epithelial to mesenchymal characteristics is called EMT and is widely discussed in the scientific community as a game changer in drug resistance and metastasis formation. However, clinical studies could not prove the efficacy of EMT-interfering treatments, and in clinical routine, EMT is not investigated to assess invasion. To fill this gap between bench and bedside, we use in this study a lung tumor tissue model with a preserved basement membrane for investigation of EMT functions with respect to invasion across this membrane and drug resistance. Our results suggest EMT is more a marker of drug resistance than a maker. Invasion is enhanced by EMT but more dependent on intrinsic factors, and EMT is not detected in the center of invasive tumor nodules. An in silico signaling network model is used to integrate these in vitro results and to reveal determinants for drug response.Epithelial-to-mesenchymal transition (EMT) is discussed to be centrally involved in invasion, stemness, and drug resistance. Experimental models to evaluate this process in its biological complexity are limited. To shed light on EMT impact and test drug response more reliably, we use a lung tumor test system based on a decellularized intestinal matrix showing more in vivo-like proliferation levels and enhanced expression of clinical markers and carcinogenesis-related genes. In our models, we found evidence for a correlation of EMT with drug resistance in primary and secondary resistant cells harboring KRASG12C or EGFR mutations, which was simulated in silico based on an optimized signaling network topology. Notably, drug resistance did not correlate with EMT status in KRAS-mutated patient-derived xenograft (PDX) cell lines, and drug efficacy was not affected by EMT induction via TGF-β. To investigate further determinants of drug response, we tested several drugs in combination with a KRASG12C inhibitor in KRASG12C mutant HCC44 models, which, besides EMT, display mutations in P53, LKB1, KEAP1, and high c-MYC expression. We identified an aurora-kinase A (AURKA) inhibitor as the most promising candidate. In our network, AURKA is a centrally linked hub to EMT, proliferation, apoptosis, LKB1, and c-MYC. This exemplifies our systemic analysis approach for clinical translation of biomarker signatures.

KEAP1-Mutant NSCLC: The Catastrophic Failure of a Cell-Protecting Hub

Mutations in the KEAP1-NRF2 pathway are common in NSCLC, albeit with a prevalence of KEAP1 mutations in lung adenocarcinoma and an equal representation of KEAP1 and NFE2L2 (the gene encoding for NRF2) alterations in lung squamous cell carcinoma. The KEAP1-NRF2 axis is a crucial modulator of cellular homeostasis, enabling cells to tolerate oxidative and metabolic stresses, and xenobiotics. The complex cytoprotective response orchestrated by NRF2-mediated gene transcription embraces detoxification mechanisms, ferroptosis protection, and metabolic reprogramming. Given that the KEAP1-NRF2 pathway controls core cellular functions, it is not surprising that a number of clinical studies connected KEAP1 mutations to increased resistance to chemotherapy, radiotherapy, and targeted agents. More recently, an immune-cold tumor microenvironment was described as a typical feature of KEAP1-mutant lung adenocarcinoma. Consistently, a reduced efficacy of immunotherapy was reported in the KEAP1-mutant background. Nevertheless, the connection between KEAP1 and immune resistance seems more complex and dependent on coexisting genomic alterations. Given the clinical implications of deregulated KEAP1-NRF2 pathway in lung cancer, the development of pathway-directed anticancer treatments should be considered a priority in the domain of thoracic oncology.

Identifying metabolic reprogramming phenotypes with glycolysis-lipid metabolism discoordination and intercellular communication for lung adenocarcinoma metastasis

Tumor metastasis imposes metabolic requirements for escaping from primary tissues, producing vulnerability in treatment. This study aimed to explore the metabolic reprogramming relevant to lung adenocarcinoma (LUAD) metastasis and decode the underlying intercellular alterations. Using the gene expression profiles of 394 LUAD samples derived from The Cancer Genome Atlas (TCGA), we identified 11 metastasis-related metabolic genes involved in glycolysis and lipid metabolism, and defined three metabolic reprogramming phenotypes (MP-I, -II, and -III) using unsupervised clustering. MP-III with the highest glycolytic and lowest lipid metabolic levels exhibited the highest metastatic potency and poorest survival in TCGA and six independent cohorts totaling 1,235 samples. Genomic analyses showed that mutations in TP53 and KEAP1, and deletions in SETD2 and PBRM1 might drive metabolic reprogramming in MP-III. Single-cell RNA-sequencing data from LUAD validated a metabolic evolutionary trajectory from normal to MP-II and MP-III, through MP-I. The further intercellular communications revealed that MP-III interacted uniquely with endothelial cells and fibroblasts in the ANGPTL pathway, and had stronger interactions with endothelial cells in the VEGF pathway. Herein, glycolysis-lipid dysregulation patterns suggested metabolic reprogramming phenotypes relevant to metastasis. Further insights into the oncogenic drivers and microenvironmental interactions would facilitate the treatment of LUAD metastasis in the future.

Coffee ingredients, hydroquinone, pyrocatechol, and 4-ethylcatechol exhibit anti-inflammatory activity through inhibiting NF-κB and activating Nrf2

• ・Pyrocatechol is the main coffee ingredient exhibiting anti-inflammatory activity. • ・Hydroquinone and 4-ethylcatechol in coffee exhibit anti-inflammatory activity. • ・Pyrocatechol, hydroquinone and 4-ethylcatechol inhibit NF-κB activation. • ・Pyrocatechol, hydroquinone and 4-ethylcatechol activate Nrf2. Pyrocatechol is a coffee ingredient that exhibits anti-inflammatory activity. We herein identified hydroquinone and 4-ethylcatechol as novel coffee ingredients inhibiting LPS-induced inflammatory responses. Similar to pyrocatechol, hydroquinone and 4-ethylcatechol significantly inhibited LPS-induced nitric oxide (NO) production and CCL2 secretion by suppressing the mRNA expression of iNOS and CCL2 in murine macrophage-like RAW264.7 cells. These ingredients also inhibited the LPS-induced activation of NF-κB, a pivotal transcription factor in inflammation, by preventing its nuclear localization, and induced the expression of Nrf2, a transcription factor negatively regulating LPS-induced inflammation. By utilizing Keap1 −/− MEFs reconstituted with Keap1 mutants in which major cysteine residues were substituted, we found that Cys151 in Keap1 was a critical sensor for pyrocatechol, hydroquinone, and 4-ethylcatechol to induce Nrf2 expression. However, the amounts of hydroquinone and 4-ethylcatechol in the coffee decoction were markedly lower than that of pyrocatechol, suggesting that pyrocatechol is the main coffee ingredient exhibiting anti-inflammatory activity.

Clinical efficacy of atezolizumab plus bevacizumab and chemotherapy in KRAS-mutated non-small cell lung cancer with STK11, KEAP1, or TP53 comutations: subgroup results from the phase III IMpower150 trial

BackgroundThe efficacy of atezolizumab (A) and/or bevacizumab (B) with carboplatin/paclitaxel (CP) chemotherapy was explored in the phase III, randomized IMpower150 study in patients with non-squamous non-small cell lung cancer (NSCLC)...

Present and Emerging Biomarkers in Immunotherapy for Metastatic Non-Small Cell Lung Cancer: A Review.

Targeting the immune system, especially the PDL-1/PD-1 axis, has significantly improved the outcomes of metastatic lung cancer patients. However, only a portion of patients will benefit significantly from PD(L)1 therapeutics alone or in combination with either chemotherapy or anti-CTLA4 antibody. It is therefore important to study predictive biomarkers to help select the patients who will experience the most benefit from immunotherapy. In this paper, the current status of PDL-1 expression on tumour cells, the smoking status of patients, tumour mutational burden, gut microbiome and STK11 and KEAP1 mutations in the tumour as predictive biomarkers for PD(L)-1-based immunotherapy are summarized.