SPECIFIC AIMS Oncogenic RAS mutations, which are among the most common molecular alterations in cancer, encode mutant proteins that dominantly drive aberrant growth. Unfortunately, structural and biochemical properties of the mutant Ras/GTPase activating protein (Ras/GAP) molecular switch pose formidable barriers to mechanism-based drug discovery and no targeted therapies have been approved for Ras-driven cancers to date. The Ras palmitoylation/depalmitoylation cycle regulates the subcellular trafficking of the N-Ras, H-Ras, and K-Ras4a isoforms, but not of K-Ras4b. Thus, developing potent and selective inhibitors of the Ras palmitoylation/depalmitoylation cycle has the potential to treat malignancies dependent on oncogenic N-Ras without interfering with K-Ras4b signaling in normal tissues. This ongoing project involves a cross-disciplinary collaboration between investigators with complementery expertise in biochemistry, chemical biology, hematologic cancer, Ras signaling, and preclinical therapeutics. During the current funding period we: (1) validated N-Ras palmitoylation as a promising therapeutic target for NRAS mutant cancers in a novel strain of NrasG12D,C181S “knock in” mice; (2) identified the ABHD17 family of serine hydrolase (SH) enzymes as N-Ras depalmitoylases; (3) showed that the Palmostatin class of SH inhibitors are too promiscuous for use as selective probes of ABHD17 function; (4) identified, in collaboration with Lundbeck, a structurally distinct class of selective ABHD17 inhibitors that reduce the growth of NRAS mutant acute myeloid leukemia (AML) cells and exhibit genotype-specific activity in isogenic models; and, (5) demonstrated that reduced growth of NRAS mutant AML cells treated with ABHD17 inhibigtors correlates with biochemical inhibition of Ras effector pathways. The experiments proposed in this renewal application will extend these exciting studies through the following specific aims: Aim 1. To investigate the roles of ABHD17s and additional SH enzymes in N-Ras depalmitoylation and to test the potent and bioavailable ABHD17 inhibitor ABD778 in preclinical models of AML; and, Aim 2. To identify palmitoyl acyl transferase (PAT) proteins that modify N-Ras and to validate them as targets for N-Ras mutant cancers. We anticipate that the studies described in this interdisciplinary project will determine fundamental mechanisms of N-Ras palmitoylation, ascertain the efficacy and mechanism of action of chemical inhibitors of ABHD17s alone and in combination with other targeted agents, and generate essential foundational knowledge for developing PAT inhibitors as anti-cancer drugs. The development of selective inhibitors of oncogenic N-Ras signaling would have significant therapeutic impact for a number of different aggressive adult and pediatric cancers.