Mutant RAS genes drive cancer more frequently than any other oncogene. Oncogenic RAS proteins transform cells only when associated with cellular membranes. Membrane association is mediated by post-translational modifications, including farnesylation, aaX proteolysis, carboxyl methylation, and palmitoylation. For more than two decades my laboratory has focused on the post-translational modification and membrane targeting of RAS and related small GTPases. We have made paradigm-shifting contributions to the field including the discovery that RAS traffics upon and signals from endomembranes as well as the plasma membrane (PM). These observations established the field of compartmentalized signaling of RAS. Early attempts to treat cancer with farnesyltransferase inhibitors (FTIs) failed in the clinic not because membrane association is dispensable for RAS function but rather because FTIs did not block membrane association. We have since sought more effective means of limiting membrane association of RAS. In recent work we have focused on KRAS and NRAS, the isoforms most often mutant in tumors. We have established phosphorylation of KRAS4B as a means of modulating membrane association and function, characterized the differential membrane trafficking of KRAS4A and KRAS4B, the two splice variants of the KRAS locus, developed quantitative assays for KRAS4B membrane association that were applied to genome-wide RNAi and CRISPR screens, and discovered differential effects of the two splice variants on tumor metabolism. Perhaps most remarkable is our recent discovery that hexokinase 1 (HK1), the enzyme that catalyzes the first committed step in glycolysis, is an effector of KRAS that is specific to the KRAS4A splice variant by virtue of its unique subcellular trafficking (in press in Nature). We have also discovered that NRAS is uniquely sensitive to inhibition of isoprenylcysteine carboxylmethytransferase (ICMT), the CaaX modifying enzyme we first identified. Over the seven years of funding that we seek through the R35 mechanism we propose to build on these discoveries. The overarching scientific question to be addressed is whether the differential modification and membrane trafficking of RAS proteins can reveal new therapeutic vulnerabilities. Specifically, we will a) characterize HK1 as an effector of KRAS4A and explore more broadly the differential effects on tumor metabolism driven by the two splice variants of the KRAS locus, b) pursue hits from a recent, innovative screen that revealed previously unappreciated genes, including several druggable protein kinases, that are required for efficient membrane association of KRAS4B, and c) determine if ICMT inhibition is viable approach to treating NRAS-driven melanoma. Our approach will be innovative, multidisciplinary, and collaborative. We have recruited experts to serve as collaborators in kinase biochemistry, super-resolution microscopy, structural biology, genome regulation, metabolomics, cancer genomics, ...