Abstract Secondary resistance limits the clinical effectiveness of mutation-specific RAS inhibitors in colorectal cancer. It is unknown whether broad-spectrum RAS inhibitors meet similar limitations. Here, we identify and categorize mechanisms of resistance to the broad-spectrum active-state RAS inhibitor RMC-7977 in colorectal cancer cell lines. We found that KRAS-mutant colorectal cancer cell lines are universally sensitive to RMC-7977, inhibiting the RAS-RAF-MEK-ERK axis, halting proliferation and in some cases inducing apoptosis. To monitor KRAS downstream effector pathway activity, we developed a compartment-specific dual-color ERK activity reporter system. RMC-7977 treatment reduced reporter activity. However, long-term dose escalation with RMC-7977 revealed multiple patterns of reporter reactivation in emerging resistant cell populations that correlated with phosphorylation states of compartment-specific ERK targets. Cells sorted for high, low, or cytoplasmic reporter activity exhibited distinct patterns of genomic mutations, phospho-protein, and transcriptional activities. Notably, all resistant subpopulations showed dynamic ERK regulation in the presence of the RAS inhibitor, unlike the parental sensitive cell lines. High levels of RAS downstream activities were observed in cells characterized by a KRAS Y71H resistance mutation. In contrast, RAS inhibitor-resistant populations with low, or cytoplasmic ERK reporter reactivation displayed different genetic alterations, among them RAF1 S257L and S259P mutations. Colorectal cancer cells resistant to RMC-7977 and harboring the RAF1 mutation specifically exhibited synergistic sensitivity to concurrent RAS and RAF inhibition. Our findings endorse reporter-assisted screening together with single-cell analyses as a powerful approach for dissecting the complex landscape of therapy resistance. The strategy offers opportunities to develop clinically relevant combinatorial treatments to counteract emergence of resistant cancer cells.

Reporter-based screening identifies RAS-RAF stabilizing mutations as drivers of resistance to broad-spectrum RAS inhibition in colorectal cancer