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bioRxiv · 10.1101/2025.11.14.688534

Remodeling of KRAS interactome induced by clinically relevant RAS inhibitors reveals convergent responses and a KRAS-mediated regulation of directional cell migration

Yang, Y.·Cho, C. Y.·Brock, A.·Quackenbush, D.·Chen, K.·Lo, F.·Benes, C. H.·Haling, J. R.
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Abstract

KRAS is commonly mutated in lung, colorectal, and pancreatic cancers. Small molecule inhibitors targeting KRAS with distinct mechanisms-of-action and variable specificities have entered the clinic, but a comprehensive view of their effect on the RAS signaling network has not been reported. Here, we describe the impact of RAS inhibition on the KRAS protein interactome using Proximity-dependent Biotinylation Identification. Two inhibitors were used: panRAS-ON, which forms a ternary complex between cyclophilin and RAS in the GTP bound state; and panKRAS-off, which binds specifically to the KRAS switch II pocket. RAS inhibitors were found to significantly alter 16.5% of proteins in proximity to KRAS. Despite their distinct mechanisms-of-action, the KRAS inhibitors induced highly correlated changes in the KRAS interactomes. Among proteins in close proximity to KRAS, Afadin was found to be highly regulated by RAS inhibition. AFDN has been characterized as a key regulator of cell motility, invasion, and metastasis. Analysis of AFDN phosphorylation revealed that AKT only partially modulates p-Ser1718, while inhibition of KRAS is sufficient to abolish EGF-mediated AFDN phosphorylation. Knockdown of AFDN in a KRAS-driven non-small cell lung cancer model abolished chemotaxis in a transwell migration assay and disrupted directional movement in an EGF-driven wound healing model. These results suggest 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.

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Yang, Y., Cho, C. Y., Brock, A., Quackenbush, D., Chen, K., Lo, F., Benes, C. H., Haling, J. R.. 2025-11-16. Remodeling of KRAS interactome induced by clinically relevant RAS inhibitors reveals convergent responses and a KRAS-mediated regulation of directional cell migration. https://doi.org/10.1101/2025.11.14.688534

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