Protomer Selectivity of RAF Inhibitors Within the RAS/RAF Signalosome

Abstract RAF dimer inhibitors offer therapeutic potential in RAF- and RAS-driven cancers. The utility of such drugs is predicated on their capacity to occupy both RAF protomers in the RAS-RAF signaling complex. Here we describe a method to conditionally quantify drug-target occupancy at selected RAF protomers within an active RAS-RAF signalosome in cells. RAF target engagement can be measured in the presence or absence of any mutant KRAS allele, enabling the high affinity state of RAF dimer inhibitors to be quantified in the cellular milieu. The intracellular selectivity of clinical-stage drugs for individual protomers within BRAF, CRAF, and ARAF heterodimers in complex with mutant KRAS-GTP revealed that ARAF protomer-engagement, but not engagement of BRAF or CRAF is commensurate with inhibition of MAPK signaling in various mutant RAS cell lines. Our results support a fundamental role for ARAF in mutant RAS signaling and highlight the avoidance of ARAF protomers for a cohort of RAF inhibitors undergoing clinical evaluation.