PROJECT SUMMARY/ABSTRACT Small GTPases regulate diverse cellular functions and their aberrant activation plays a key role in disease. Perhaps most significant is their association with cancer, a disease where KRAS is mutated in ~1/3 of patients. With this in mind, the mechanism by which cancer hotspot mutations activate KRAS is a central concept in cancer biology. Under physiologic conditions, KRAS cycles between an active (GTP-bound) and an inactive (GDP-bound) conformation. Its slow intrinsic GTP hydrolysis is catalyzed by GTPase-activating proteins (GAPs). Common mutations in KRAS prevent the stabilization of the hydrolysis transition-state leading to oncoproteins that are thought to be deficient in hydrolysis, insensitive to GAPs, and constitutively active in cancer cells (i.e., `locked' in their active or GTP-bound, state). Emerging therapies are challenging the conventional model of KRAS oncoprotein activation. Perhaps the strongest evidence is provided by inhibitors selectively targeting KRAS G12C, the most common KRAS mutation in lung cancer. G12C inhibitors bind only to the GDP-bound (or hydrolyzed) conformation and trap the oncoprotein in an inactive state by preventing the exchange of GDP for GTP. To be effective, these inactive state selective drugs require intact GTP hydrolysis by mutant KRAS. In a similar fashion, inhibition of nucleotide-exchange (as achieved by targeting factors upstream of KRAS) has been reported to suppress mutant KRAS activation and/or tumor growth. This and other emerging therapeutic effects presented in the preliminary data of this application could not be possible if mutant KRAS GTPases were `locked' in their active state. Our proof-of-principle experiments suggest the presence of cellular proteins that enhance the GTPase activity of mutant KRAS and that KRAS oncoproteins are broadly susceptible to inactive state selective inhibition. We now propose (i) to isolate enhancers of mutant KRAS GTPase activity in cancer cells, (ii) to determine the tertiary structure of common KRAS mutants in complex with their enhancer and (iii) to characterize the effects of novel inactive state selective drugs that suppress common KRAS mutants found in cancer. This work will explain the mechanistic basis responsible for the physiologic inactivation of mutant KRAS and refine the conceptual model explaining how mutations activate KRAS in cancer. The proposed study will pave the way for key advances in cancer biology with a large potential for therapeutic and translational impact in patients.