Abstract

KRAS G12C-targeted therapies have transformed the treatment of KRAS G12C-mutant lung adenocarcinoma. However, acquired resistance to these therapies, whose underlying molecular mechanisms are not fully understood, presents a major obstacle to achieving long-term therapeutic success. The purpose of this study was to elucidate the mechanisms of acquired resistance to KRAS G12C inhibitors and identify potential regulators of resistance in lung adenocarcinoma. Two lung adenocarcinoma cell lines (H23 and H2122) were exposed to escalating doses of two novel KRAS G12C inhibitors, fulzerasib and garsorasib, to generate resistant variants. Transcriptomic profiling was conducted to identify genes consistently upregulated in resistant cells. CRISPR/Cas9-mediated knockout (MXD4-KO) and siRNA-mediated knockdown of MXD4 were performed to assess its role in drug resistance. Mechanistic investigations employed inhibitors of ferroptosis, apoptosis, and necrosis, along with assays measuring lipid peroxidation and malondialdehyde levels. Further analysis included ferroptosis-related gene expression profiling, lipidomic profiling, ChIP-Seq, ChIP-qPCR, and dual-luciferase reporter assays. The findings were validated in patient-derived organoids (PDOs) and nude mouse models. Transcriptomic profiling identified 11 genes consistently upregulated in resistant cells, with MXD4 emerging as a key resistance regulator. CRISPR/Cas9-mediated knockout of MXD4 restored sensitivity to fulzerasib, garsorasib, sotorasib, and adagrasib, an effect fully reversed upon MXD4 re-overexpression. Similarly, siRNA-mediated knockdown of MXD4 in resistant cells restored drug sensitivity. Mechanistic studies revealed that MXD4 specifically suppresses ferroptosis, rather than apoptotic or necrotic pathways, by repressing ACSL4 expression and blocking its catalytic synthesis of phosphatidylethanolamine-polyunsaturated fatty acids (PE-PUFAs).