To identify potential therapeutic targets for overcoming resistance to KRAS G12D inhibitors in colorectal cancer using quantitative proteomic strategies.

MRTX1133-resistant KRAS G12D-mutant colorectal cancer cell lines were established via dose escalation and verified. Quantitative proteomic analysis was conducted using data-independent acquisition (DIA). Differentially expressed proteins were identified with the Limma package under the thresholds of adj. P < 0.05 and |logFC| > 1. Gene set enrichment analysis (GSEA) was conducted based on the Reactome database. Functional enrichment was analyzed using GO and KEGG databases. Protein-protein interaction (PPI) networks were constructed and analyzed using the STRING database.

MRTX1133-resistant cell lines were constructed. A total of 7124 proteins were quantified, 88 of which were significantly differentially expressed, including NF-κB inhibitor-interacting Ras-like 1 (NKIRAS1), IQ motif-containing protein B1 (IQCB1), keratin 17 (KRT17), and serine protease 2 (PRSS2). Enrichment analysis found that hypoxia, cytoskeleton organization, and amino acid metabolism pathways were significantly dysregulated. In addition, the hypoxia-related F-actin monooxygenase MICAL1 might contribute to resistance to KRAS G12D inhibitors by activating the WNT/β-catenin signaling pathway.

Resistance to the KRAS G12D inhibitor MRTX1133 is closely related to the dysregulation of hypoxia, cytoskeleton, and amino acid metabolism pathways. NKIRAS1, IQCB1, KRT17, PRSS2, and MICAL1 may serve as novel candidate targets for reversing drug resistance. In particular, MICAL1 may promote resistance to KRAS G12D inhibitors by activating the WNT/β-catenin pathway.