Abstract B013: Reversing the ferroptosis-resistant phenotype of <i>KEAP1</i> -mutant lung adenocarcinoma through glutaminase 1 inhibition enhances the efficacy of KRAS inhibitors

Abstract Background: KEAP1 is one of the most commonly mutated tumor suppressors in lung adenocarcinoma and is frequently co-altered with KRAS and STK11/LKB1 mutations. Mechanistically, KEAP1 mutations impair NRF2 degradation, resulting in the upregulation of antioxidant and ferroptosis response genes. Non-small cell lung cancer (NSCLC) tumors with KEAP1 mutations are resistant to various treatment modalities, including KRAS G12C inhibitors. KRAS inhibitors are changing the treatment landscape for several malignancies, including NSCLC, and understanding mechanisms of resistance to these agents is of utmost importance. Here, we examined the effects of the ferroptosis-resistant phenotype of KEAP1 mutation on sensitivity to KRAS inhibitors in KEAP1/KRAS-mutant lung adenocarcinoma. Methods: DepMap was used to assess the drug sensitivity profiles for sotorasib, MRTX1133, erastin, RSL3, ML162, and ML210 as well as RNAi and CRISPR screen data in human NSCLC cell lines. The Cancer Therapeutics Response Portal database was used to create a NSCLC ferroptosis resistance gene signature by identifying the genes with Pearson correlation z-score of &amp;gt;3 with resistance to both RSL3 and erastin. Kras MUT (K), Kras MUT/Keap1 KO (KK), Kras MUT/Lkb1 KO (KL), and Kras MUT/Lkb1 KO/Keap1 KO (KLK) tumor cells were created by knocking-out Keap1 and Stk11 from LKR13 KRAS G12C and KRAS G12D mutant murine cell lines. BODIPY™ 581/591 C11 was used to quantify lipid peroxidation by flow cytometry. Results: Using murine and human models, we found that KEAP1 deficiency promoted resistance to ferroptosis inducers (erastin, RSL3, ML162, ML210) as well as to KRAS G12C (sotorasib, adagrasib) and KRAS G12D (MRTX1133) inhibitors. MRTX1133 and adagrasib induced lipid peroxidation -a hallmark of ferroptosis- in LKR13 K G12D and G12C cell lines. RSL3 and erastin synergized with adagrasib and MRTX1133 in KEAP1-wt, but not KEAP1-mutant, LKR13 G12C and G12D models. Through RNAi and CRISPR screens conducted in a large panel of human KRAS-mutant NSCLC cell lines, we found that KEAP1 mutation and a higher NSCLC ferroptosis resistance signature also correlated with diminished sensitivity to genetic disruption of KRAS. Glutaminase 1 (GLS1) inhibition can deplete glutathione pools necessary for antioxidant response. While RSL3 alone could not effectively induce lipid peroxidation in the KEAP1-mutant LKR13 KLK G12D model, combining it with the GLS1 inhibitor CB-839 resulted in an increased lipid peroxidation in the KLK, but not KEAP1-wt, model and this approach increased the effectiveness of RSL3 treatment in KEAP1-mutant samples. Finally, CB-839 treatment enhanced the effectiveness of adagrasib and MRTX1133 in LKR13 KK and KLK G12C and G12D cell lines. Conclusions: KRAS inhibitors induce ferroptosis in KRAS-mutant lung adenocarcinoma cell lines. KEAP1 mutation and enhanced anti-ferroptosis response correlated with lack of response to KRAS inhibition. Our data indicates that GLS1 blockade could sensitize KEAP1-mutant lung adenocarcinoma to ferroptosis and KRAS inhibitors. Citation Format: Amirali Karimi, Yu Qian, David Molkentine, Ana Galan Cobo, Alvaro Guimaraes Paula, Büsra Ernhofer, David Peng, Monique Nilsson, John V. Heymach. Reversing the ferroptosis-resistant phenotype of KEAP1-mutant lung adenocarcinoma through glutaminase 1 inhibition enhances the efficacy of KRAS inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_1):Abstract nr B013.