Dual RAF inhibition outperforms RAF-MEK combinations for suppressing ERK signaling in KRAS mutant cells.

Complex interactions among RAS, RAF, KSR, and MEK isoforms, together with their interplay with clinically used kinase inhibitors, hinder accurate predictions of drug efficacy and the choice of optimal inhibitor combinations. Here, we combine structure-and-rule-based computational modeling with experiments to systematically study how KSR1 abundance modulates responses to RAF and MEK inhibitors (RAFi and MEKi) in PSN1 mutant KRASG12R/WT pancreatic cancer and MCF7 breast cancer WT KRAS cells. KSR1 knockdown did not substantially affect ppERK responses to Type I½ RAF inhibitor (Encorafenib) in both cell types, whereas ppERK sensitivity slightly decreased for Type II RAFi (TAK-632) in MCF7 cells, aligning with simulations. The efficacy of MEKi (Cobimetinib) slightly increased in MCF7 cells following KSR1 knockdown but slightly decreased in PSN1 cells where higher MEKi concentrations were required to suppress ERK signaling, as predicted by the model. Our computational models predict, and experiments validate that in RAS-mutant cells, two conformation-specific RAF inhibitors used in combination suppress the ERK pathway more effectively than a combination of MEK and RAF inhibitors irrespective of KSR1 levels.