Abstract 6997: Impact of KRASG12C oxidation state on non-small-cell lung cancer response to targeted therapeutics

Abstract RAS proteins are critical signaling nodes that regulate cellular growth, with activating mutations in KRAS found in approximately 25% of human cancers. These mutations lead to chronic KRAS activation and sustained oncogenic signaling. Among >130 cancer-associated point mutations in KRAS, 99% cluster at hotspots (G12, G13, and Q61), with G12C mutations predominant in non-small cell lung cancer (NSCLC, ∼80%). The G12C mutation introduces a reactive cysteine, enabling covalent drug discovery strategies. Therapeutics such as AMG510 (Sotorasib) target KRASG12C by locking it in an inactive state; however, resistance arises in ∼60% of treated patients. To address this challenge, we aim to investigate how oxidation of KRAS and at Cys12 inKRASG12C influences its ability to bind covalent inhibitors, potentially offering insights into resistance mechanisms and novel therapeutic strategies. To explore the effects of oxidative modifications on KRASG12C function, we have started by engineering a recombinant KRASG12C protein that lacks other cysteines (denoted KRASG12C-CL) and introduced a Trp substitution (Y137W) to enable fluorescent kinetic analyses (KRASG12C-CLW; CL stands for cysteine-light). Recombinant protein samples were exposed to oxidants (H2O2) in the presence or absence of GDP, subsequently desalted using Bio-Spin Chromatography columns, and then analyzed by mass spectrometry to quantify the kinetics of oxidation to sulfinic acid state (-SO2H) under these conditions. Further fluorescent kinetic assays of reduced and oxidized KRASG12C with AMG510 and other KRAS inhibitors showed complex profiles distinguishing inhibitor binding to the reduced and oxidized KRASG12C. Additionally, we conducted targeted analyses of endogenous KRASG12C oxidation in Calu-1 non-small cell lung cancer cells. Calu-1 cells treated with 1 mM H2O2for 5 minutes demonstrated rapid oxidation of KRASG12C. Data will be presented showing the impact of KRASG12C oxidation on the therapeutic efficacy of clinical and experimental inhibitors. These approaches aim to better evaluate the impact of KRASG12C oxidation state on inhibitor engagement and therapeutic outcomes, addressing mechanisms of resistance and optimizing treatment strategies. Citation Format: Yismeilin R. Feliz Mosquea, Maria Luiza Carneiro Buchele, Derek Parsonage, Jingyun Lee, Surbhi Mundra, Sharon L. Campbell, Leslie B. Poole, Cristina M. Furdui. Impact of KRASG12C oxidation state on non-small-cell lung cancer response to targeted therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6997.