Mutant KRAS drives human cancers from several sites, including pancreatic ductal adenocarcinoma (PDAC) and low-grade serous ovarian cancer (LGSOC). Despite the prevalence of RAS mutations in different cancers, effective RAS-targeted treatment remains a challenge. KRAS monomers form homodimers and nanoclusters in the cell membrane to optimize signaling and to transform cells efficiently. Dimerization of KRAS is required for RAS-driven transformation and cancer growth. Agents that disrupt mutant RAS dimers and clusters can block oncogenic activity. Recent evidence indicates that inhibition of RAS signaling induces autophagy and enhances the response to anti-autophagic therapy. Despite four decades of effort, development of effective strategies for the treatment for mutant KRAS-driven cancers remains a work in progress. Our laboratory has discovered a novel endogenous physiological RAS inhibitor designated DIRAS3, a 26 KDa GTPase sharing 50-60% homology with classical RAS family members, but with a distinctive 34 amino acid N-terminal extension that reverses RAS function. Like RAS, DIRAS3 is prenylated at the C-terminal CAAX site, binds GTP with high affinity, exhibits weak GTPase activity, and requires membrane association for its biological function. DIRAS3 is downregulated in a number of cancers including PDAC and LGSOC, and re-expression of DIRAS3 blocks cancer cell proliferation, inhibits motility, and, importantly, induces autophagy by multiple mechanisms. Recently we have found that DIRAS3 and a DIRAS3-derived stapled peptide from its α5 domain interact directly with mutant KRAS, reducing KRAS dimerization and nanoclustering, and inhibiting KRAS signaling. Both intact DIRAS3 and DIRAS3-derived stapled peptide induce autophagy and potentiate the pro-apoptotic activity of autophagy inhibitors in PDAC and LGSOC cells. In this proposal, we will study the effect of DIRAS3 on KRAS-dependent cell growth, migration and effector signaling in MEF cells and genetically engineered mouse model with mutant KRAS, as well as in KRAS-driven PDAC and LGSOC, better defining the mechanism by which DIRAS3 inhibits KRAS (Aim 1). We will investigate the mechanisms by which DIRAS3 induces autophagy in KRAS-driven PDAC and LGSOC (Aim 2). Finally, we will test the ability of DIRAS3 or a DIRAS3-derived stapled peptide in combination with autophagy inhibitors (CQ/DC661) to enhance apoptosis and growth inhibition in PDAC and LGSOC (Aim3). These studies will not only lay the groundwork for exploring new therapeutic strategies targeting KRAS-mutant cancers, but also contribute to a fundamental understanding of the mechanisms by which DIRAS3, as a tumor suppressor, inhibits mutant KRAS activity and induces autophagy.