MXD4 enhances resistance to KRAS G12C-targeted therapy in lung adenocarcinoma by suppressing ACSL4-mediated ferroptosis

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). ACSL4 knockout or overexpression abolished MXD4’s ability to promote ferroptosis suppression and resistance to KRAS G12C inhibitors. ChIP-Seq, ChIP-qPCR, and dual-luciferase reporter assays confirmed that MXD4 directly binds to and represses the ACSL4 promoter. These findings were validated in PDOs and nude mouse models, where MXD4 knockout restored therapeutic sensitivity, while ACSL4 knockout blocked MXD4’s resistance-promoting effects. This study uncovers a novel resistance mechanism in which MXD4 transcriptionally silences ACSL4 to suppress ferroptosis, enabling cancer cells to evade KRAS G12C inhibitors. Targeting the MXD4-ACSL4 axis represents a promising strategy to overcome therapeutic resistance in KRAS G12C-mutant lung cancer, potentially improving long-term treatment outcomes.