Surveillance of RAS-RAF dynamics in vivo: Tracking activity conformations and drug-induced interactions.

Abstract Live-cell recordings of protein conformations and interactions yield novel mechanistic insights into physiologic and pathologic protein functions. We have developed an adaptable and genetically encoded bioluminescence-based biosensor platform to precisely measure signaling dynamics of full-length RAS GTPases and RAF kinases upon physiologic pathway activation, post-translational modifications, mutagenesis, and drug binding. We have recorded the impact of both patient mutations and bioactive small-molecule binding on kinase conformations and binary interactions of GTP-loaded H, N, KRAS with kinases such as RAF. Systematic profiling of αC-helix-OUT BRAF inhibitors (BRAFi) vemurafenib, dabrafenib, encorafenib, and PLX8394 using the BRAF reporter reporter platform (displaying 10 different patient mutations) revealed differences in drug specificities and efficacies. Unexpectedly, the αC-helix-OUT BRAFi engagement with the catalytic pocket of mutated BRAF stabilized an inactive and intermediate full-length kinase conformation, which provoked the enhancement of binary RAS:RAF interactions. We showed that the interference with RAS interactions and nanoclustering has the potential to antagonize the sequential formation of drug-induced tetrameric RAS:RAF complexes. Recently, we have bioengineered a reporter toolbox for direct in vivo recordings of RAS activity conformations. First, we demonstrated that the wild-type RAS reporter—expressed at levels far below endogenous RAS proteins—responds to physiologic pathway activation. Second, we showed that hot-spot RAS mutations display a similar effect on the intramolecular dynamics. The systematic implementation, combination, and advancement of the GTPase and kinase reporter platform have the potential to be used to identify novel means for regulating or perturbing RAS-RAF-ERK signaling in vivo. Citation Format: Ruth Röck, Johanna Mayrhofer, Omar Torres-Quesada, Florian Enzler, Jakob Troppmair, Eduard Stefan. Surveillance of RAS-RAF dynamics in vivo: Tracking activity conformations and drug-induced interactions [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B45.