Abstract 1976: Pathogenic germline KEAP1 variants drive constitutive NRF2 hyperactivation and enhanced oncogenic fitness in non small cell lung cancer.

Abstract KEAP1 is a key tumor suppressor, which is mutated in 15-20% of non-small cell lung cancers (NSCLC), ranking as the third most frequently altered gene. Somatic loss-of-function mutations disrupt the Cullin 3-based E3 ubiquitin ligase complex, preventing NRF2 degradation and enabling its nuclear translocation. This drives metabolic reprogramming, ferroptosis resistance, and adaptation to oxidative stress, which are hallmarks of aggressive disease and poor survival. While somatic KEAP1 alterations are established drivers of adverse prognosis, the role of germline KEAP1 variants in cancer predisposition remains largely unknown. Using MSK-IMPACT paired tumor-normal sequencing, we analyzed 49.000 patients across 65 cancer types for rare germline variants predicted to be deleterious by VEP, REVEL, and AlphaMissense. We identified 35 carriers of pathogenic KEAP1 candidates (12 truncating, 23 missense). In tumors from these carriers, we observed recurrent loss of the wild-type allele in NSCLC, and enrichment of somatic co-mutations in STK11.In this study we selected five novel KEAP1 germline missense mutations (1438GC, 1244GA, 779GT, 1524CG, 1649TA) to investigate their pathogenic impact on NRF2 signaling and tumor cell fitness in NSCLC. KEAP1 variant and wild-type (WT) plasmids were generated by site-directed mutagenesis and introduced via lentiviral transduction into NSCLC cell lines (A549, H460) using CMV-driven expression constructs. A549 and H460 harbor STK11 and KEAP1 co-mutations and were transduced with the NRF2-mediated ARE luciferase reporter plasmid. All five selected germline KEAP1 variants exhibited significant loss-of-function, failing to suppress NRF2 activity and resulting in 5-10-fold elevated ARE-luciferase reporter signal compared to WT KEAP1 (P0.001) across all time points. Western blotting confirmed disruption of KEAP1-NRF2 binding, with marked nuclear NRF2 accumulation and cytoplasmic depletion - phenocopying known somatic pathogenic mutants (959GT). Functionally, variant-expressing NSCLC cells demonstrated 2-3-fold increases in colony number and size (P0.01), enhanced proliferation rates and increased expression of ferroptosis protective proteins (AKR1C2, AKR1B10), directly linking germline KEAP1 dysfunction to oncogenic gain-of-function via NRF2 hyperactivation. This study provides the first functional validation of germline KEAP1 variants as pathogenic drivers of inherited NRF2 hyperactivation in NSCLC, with loss-of-function severity equivalent to somatic mutations. These findings establish germline KEAP1 testing as a critical tool for hereditary cancer risk assessment, enabling personalized therapeutic targeting strategies in high-risk families. This work has transformative implications for precision oncology and improved long-term outcomes in NSCLC. Citation Format: Laura Eibler, Miika M. Mehine, Rebecca Caeser, Esther Redin, Michael F. Berger, Chaitanya Bandlamudi, Charles M. Rudin. Pathogenic germline KEAP1 variants drive constitutive NRF2 hyperactivation and enhanced oncogenic fitness in non small cell lung cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1976.