Dynamic remodeling of the KRAS interactome by direct RAS inhibitors with distinct mechanism of action reveals convergent responses

Abstract

KRAS is mutated in 30% of all human cancers. Small molecule inhibitors targeting KRAS with distinct mechanisms-of-action and variable specificities have entered the clinic, but their full effect on the RAS signaling network remains unclear. Here, we described the impact of RAS inhibition on the KRAS interactome using Proximity-dependent Biotin Identification. Two inhibitors were used: panRAS-ON, which forms a ternary complex between cyclophilin and RAS in the GTP-bound state; panKRAS-off, which binds to the KRAS switch II pocket. RAS inhibitors were found to significantly alter 16.5% of proteins in proximity to KRAS. Despite their distinct modality, the KRAS inhibitors induced highly correlated changes in the KRAS interactomes. Among KRAS-proximal proteins, Afadin was highly affected by RAS inhibition. AFDN is a key regulator of cell motility, invasion, and metastasis. Analysis of AFDN phosphorylation revealed that AKT only partially modulates p-Ser1718, while KRAS inhibition sufficiently abolished EGF-mediated AFDN phosphorylation. Silencing AFDN in a KRAS-driven non-small cell lung cancer model abolished chemotaxis and disrupted directional migration in an EGF-driven wound healing model. We demonstrated that KRAS is a central node in regulating growth factor-induced cell migration, and that KRAS inhibition plays a broader role than MAPK-mediated cell proliferation and survival.