Reporter-based screening identifies RAS-RAF mutations as drivers of resistance to active-state RAS inhibition in colorectal cancer

This work identifies and categorizes mechanisms of resistance to the broad-spectrum active-state RAS inhibition in colorectal cancer cell lines. It shows that KRAS-mutant colorectal cancer cell lines are universally sensitive to broad-spectrum active-state RAS inhibition, which inhibits the RAS-RAF-MEK-ERK axis, halts proliferation and in some cases induces apoptosis. However, long-term dose escalation with a broad-spectrum active-state RAS inhibitor revealed multiple patterns of ERK reporter reactivation in emerging resistant cell populations that correlated with phosphorylation states of compartment-specific ERK targets. Cells sorted for high, low, or cytoplasmic ERK 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. The study serves as a proof-of-concept for reporter-assisted cell sorting to disentangle resistance mechanisms by observing pathway reactivation in real time. Our findings endorse reporter-assisted screening together with single-cell analyses as a powerful approach for dissecting the complex landscape of therapy resistance.