Project Grant R01CA290740

CHEMICAL BIOLOGICAL ANALYSIS OF RAF KINASES - ABSTRACT THE SERINE/THREONINE RAF KINASES FUNCTION AS DOWNSTREAM MODULATORS OF RAS GTPASE-DRIVEN SIGNALING AND ARE KEY DRIVERS OF MANY ONCOGENIC SIGNALING NETWORKS. DESPITE EXTENSIVE EFFORTS TO CHARACTERIZE THE MOLECULAR FUNCTIONS OF RAF KINASES AND TO DEVELOP PHARMACOLOGICAL MODULATORS AS CANCER THERAPIES, MANY ASPECTS OF RAF KINASE REGULATION AND INTRACELLULAR FUNCTION ARE NOT WELL UNDERSTOOD. RECENT WORK SUGGESTS THAT THE INTERPLAY OF DIFFERENT RAF ISOFORMS (A/B/C-R RAF) CAN HAVE A PROFOUND EFFECT ON DOWNSTREAM SIGNALING AND MAY BE A MAJOR DETERMINANT IN THE ABILITY TO PHARMACOLOGICALLY TARGET CANCERS THAT ARE DRIVEN BY MUTANT RAS AND RAF. HERE, WE PROPOSE A SYSTEMATIC BIOCHEMICAL ANALYSIS OF RAF REGULATION, INTRACELLULAR FUNCTION, AND PHARMACOLOGY. LEVERAGING A SUITE OF MOLECULAR TOOLS FOR DISSECTING THE "DRUGGABILITY" OF RAF ISOFORMS, WE WILL DEFINE PATHWAYS THAT MAKE RAF KINASES SENSITIVE OR RESISTANT TO SPECIFIC CLASSES OF ATP-COMPETITIVE INHIBITORS AND CO-INHIBITION STRATEGIES. IN ADDITION, BY APPLYING A NEW RNA BARCODING METHODOLOGY THAT WE'VE DEVELOPED, WE WILL PERFORM DEEP MUTATIONAL SCANS OF THE STRUCTURE, FUNCTION, AND PHARMACOLOGY OF RAF ISOFORMS. FURTHERMORE, WE WILL SYSTEMATICALLY ANALYZE HOW UPSTREAM AND DOWNSTREAM MODULATORS OF RAF KINASE SIGNALING AFFECT THE ACTIVITY AND INTRACELLULAR INTERACTIONS OF RAF KINASES. TOGETHER, THESE STUDIES WILL PROVIDE A MORE COMPLETE PICTURE OF RAF KINASE REGULATION, INTRACELLULAR FUNCTION, AND MAY FACILITATE MORE EFFECTIVE THERAPEUTIC TARGETING OF RAS- AND RAF- DRIVEN CANCERS.