Evaluating the effects of the Pan-KRas inhibitor RMC-6236 in KRas-mutant colorectal cancer cells

Background: KRas mutations occur in ~40-45% of colorectal cancers (CRC) and are highly heterogeneous, with distinct codon variants. This diversity makes KRas a challenging therapeutic target in CRC. RMC-6236 is a novel, non-covalent pan-KRas inhibitor that selectively targets active, GTP-bound KRas across multiple KRas variants. This study evaluates its effects on viability, apoptosis, and cell-cycle progression in KRas-mutant CRC cells. Methods: SW480 cells were treated with increasing RMC-6236 concentrations (0, 10, 25 and 50 nM). Cell viability was assessed using Trypan Blue exclusion and XTT assays. DNA fragmentation and Giemsa staining evaluated morphological changes and apoptosis. Flow cytometry was used to assess cell-cycle distribution (PI staining) and apoptosis (Annexin V/PI). All experiments were performed in triplicate. Expression levels of apoptotic markers (procaspase-3 and cleaved caspase-3) were assessed using western blotting. Statistical significance was determined using one-way ANOVA (p < 0.05). Results: Trypan Blue and XTT assays demonstrated a significant, dose-dependent reduction in cell viability in RMC-6236-treated SW480 cells (p < 0.001). DNA fragmentation increased (p = 0.03), and Giemsa staining showed characteristic apoptotic morphology. Flow cytometry demonstrated dose-dependent G0/G1 cell-cycle arrest, increased sub-G0 accumulation, and reduced S and G2/M phase populations. Annexin V/PI analysis confirmed a dose-dependent increase in apoptotic cells. Western blotting showed increased cleaved caspase-3 and decreased procaspase-3, consistent with activation of apoptotic pathways. Conclusion: RMC-6236 induces cytotoxicity, apoptosis, and cell-cycle disruption in KRas-mutant CRC cells, supporting its further evaluation as a therapeutic approach capable of addressing KRas mutation heterogeneity. The impact of the inhibitor on downstream KRas oncogenic pathways, including PI3K and MAPK signaling will also be investigated in future studies.