Abstract LB278: KEAP1 loss-of-function modulates ROS and AKT-mTOR pathway leading to resistance to KRAS inhibitor

Abstract Resistance to KRAS G12C inhibitors remains a significant clinical challenge in the treatment of non-small cell lung cancer (NSCLC). Among KRAS-mutated NSCLC cases, which account for approximately 30% of all NSCLC, the G12C subtype is the most prevalent, representing about 40% of KRAS-positive mutations. Clinical evidence demonstrates that KEAP1 loss-of-function (LoF) mutations are associated with early disease progression and poor responses to KRAS G12C inhibitor monotherapy, underscoring the urgent need to decipher and counteract this resistance mechanism. This study aims to elucidate the molecular basis of KEAP1-mediated resistance to KRAS G12C inhibitors and explore potential therapeutic strategies to overcome it. We performed in vitro experiments using KRAS G12C-mutated NSCLC cell lines, both with and without KEAP1 LoF mutations, and in vivo mouse models bearing KEAP1 LoF xenografts. Experimental procedures included treating cells with KRAS G12C inhibitors, measuring ROS levels via flow cytometry, assessing GSH and GSSG levels with a colorimetric enzymatic assay, and analyzing AKT-mTOR pathway activity through western blotting. These analyses revealed that KEAP1 mutations suppress ROS accumulation, promote glutathione synthesis, and activate the AKT signaling pathway, thereby promoting resistance. Combinatorial therapeutic approaches targeting glutaminase (e.g., CB-839) and mTOR pathways (e.g., TAK-228) have shown potential for effectively inhibiting tumor cell growth in KEAP1-mutant contexts. Our findings demonstrate that KEAP1 mutations confer resistance to KRAS G12C inhibitors in NSCLC through a multifaceted mechanism involving oxidative stress regulation and compensatory pathway activation. This resistance extends beyond KRAS inhibitors, mirroring resistance mechanisms observed with other therapies, including epidermal growth factor receptor (EGFR) inhibitors, BRAF, MEK, and ALK inhibitors, as well as chemotherapeutic agents, radiotherapy, and anti-angiogenic therapies. These observations underscore the broader role of KEAP1 mutations in enhancing tumor adaptability and survival under diverse therapeutic pressures. In conclusion, our results suggest that combinatorial therapeutic approaches targeting glutaminase (e.g., CB-839) and mTOR pathways (e.g., TAK-228) hold promise for overcoming resistance in KEAP1-mutant tumors. These strategies may restore sensitivity to KRAS G12C inhibitors and improve clinical outcomes. Citation Format: Weijia Lu, Mei Ning, Zhui Chen. KEAP1 loss-of-function modulates ROS and AKT-mTOR pathway leading to resistance to KRAS inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB278.