Abstact

Tri-complex inhibitors (TCIs) are molecular glues that bind the active, guanosine triphosphate (GTP)-bound state of RAS and recruit cyclophilin A (CYPA) to form a synthetic complex that blocks oncogenic signaling. Although these agents have shown clinical activity in RAS mutant cancers, resistance mechanisms remain poorly defined. Here, we analyzed paired baseline and end-of-treatment samples from 40 patients treated with the RAS inhibitor daraxonrasib and identified recurrent alterations in 18 cases. Structural and functional analyses revealed that acquired mutations confer resistance by disrupting interactions essential for daraxonrasib binding to RAS, including RAS Y64 mutations, or by enhancing the RAS-RAF interaction, thereby favoring native RAS-RAF signaling, including RAS Y71 or kinase-dead/hypoactive BRAF mutations. We then identified a TCI that targets RAS Y64 mutants and combination therapies to target resistance driven by kinase-dead BRAF. These findings uncover convergent resistance mechanisms that undermine the molecular glue function and offer a mechanistic blueprint for enhancing therapeutic efficacy in RAS-driven malignancies.