Summary The RAS/RAF/MEK/ERK (RAS/MAPK) signaling pathway is an established driver of many cancers, commonly hyperactivated by genetic alterations in RAS or BRAF. RAF and MEK inhibitors (RAFi and MEKi) have been clinically successful in BRAF-mutant (BRAF-MUT) tumors, but their clinical efficacy in RAS-mutant (RAS-MUT) tumors has been so far modest, limited by toxicities. Based on the experience with BRAF-MUT tumors, we posit that clinical efficacy of RAS pathway-targeting inhibitors requires selective MAPK inhibition in RAS-MUT tumors over RAS-WT normal cells. To achieve such RAS-MUT tumor-selective potency of RAS pathway inhibitors, we need better understanding of how RAS activation affects target drug binding and MAPK inhibition in cells. The goal of this proposal is to accomplish a better understanding of how the cellular context affects tumor-selective therapeutic potency of RAFi and MEKi, with the goal of designing more effective therapeutic strategies for RAS- MUT cancers. Our preliminary findings suggest that RAS activation differentially affects RAFi binding to RAF paralogs, as well as their protein stability. We have further evidence that although RAFi suppress MAPK preferentially in RAS-MUT over RAS-WT normal cells, combinations of RAFi and MEKi result in similar MAPK inhibition in the two contexts, they are thus predicted to promote dose-limiting toxicities. Our hypothesis is that RAS activation affects protein expression levels and conformations of RAF paralogs and MEK resulting in differential binding to RAFi and MEKi, determining tumor-selective MAPK and cell growth inhibition by these drugs. This hypothesis will be tested in three Aims: Aim 1 will characterize differential RAFi binding to RAF paralogs in RAS-MUT and normal cells using in-cell binding assays and structural and biophysical approaches. Aim 2 will define effects of RAS activation on RAF paralog protein stability and expression. Aim 3 will characterize biochemically, biophysically and in vivo combinations of diverse RAFi and MEKi to determine whether the lack of tumor-selective suppression of MAPK signaling in RAS-MUT tumor over RAS-WT normal cells increases toxicity and limits the efficacy of combined RAFi+MEKi. The proposed research will develop experimental tools and concepts to delineate mechanisms determining differential potency of RAFi and MEKi in RAS-MUT over normal cellular contexts. Defining such mechanisms should enable the rational design of more effective RAS pathway-targeting drugs and drug combinations for the treatment of RAS-MUT cancers.