Resistant cell panel-based discovery of multi-target combinations and mechanisms to overcome KRAS inhibitor resistance

Background: KRAS mutations are common oncogenic drivers in solid tumors, and KRAS-targeted inhibitors have revolutionized treatment. However, inherent and acquired resistance limits long-term clinical benefit, highlighting the need for effective combination strategies and their mechanisms. Methods: 1) Generation of KRAS inhibitor-resistant cell lines: Over 10 resistant cell lines were established by long-term exposure of KRAS-mutant cancer cells to clinically approved KRAS inhibitors and novel agents (e.g., KRAS molecular glues, PI3K/RAS breakers). 2) Resistance mechanism exploration: Multi-omics bioinformatics analyses (transcriptomics, proteomics) were performed to dissect molecular drivers of resistance and predict potential synergistic combination partners. 3) High-throughput combination screening: More than 100 drug combinations (including KRAS inhibitors, targeted agents against bypass pathways, and novel scaffolds) were tested using a customized resistant cell panel to identify regimens that overcome resistance. 4) Mechanistic validation: Key signaling pathways (e.g., MAPK, PI3K-AKT, STAT3) were interrogated via Western blot, and phospho-protein arrays to confirm the functional relevance of predicted resistance mechanisms and combination efficacy. Results: Bioinformatics analyses of resistant cell lines revealed diverse resistance mechanisms, including upregulation of bypass signaling cascades, KRAS isoform switching, and adaptive metabolic rewiring. High-throughput screening identified several promising drug combinations that restored sensitivity to KRAS inhibitors in resistant cells, with combinations of KRAS inhibitors plus PI3K/RAS breakers or pathway-specific inhibitors showing the most potent synergistic effects. Mechanistic validation confirmed that these combinations effectively abrogated aberrantly activated resistance-related signaling pathways, reversing the resistant phenotype. Additionally, the customized cell panel enabled rapid ranking of KRAS inhibitor monotherapy and combination efficacy across distinct KRAS mutation subtypes. Conclusions: Our study establishes a robust platform of KRAS inhibitor-resistant cell lines and a high-throughput screening system for identifying resistance-overcoming combinations. The identified synergistic regimens and their validated mechanisms provide critical preclinical evidence to guide the development of next-generation KRAS-targeted combination therapies, addressing the unmet clinical need of overcoming resistance in KRAS-mutant cancers.