PROJECT SUMMARY/ABSTRACT Recent advances in targeted therapies have revolutionized lung cancer clinical practice. Lung adenocarcinomas harbor frequent mutations/amplifications/fusions in receptor tyrosine kinase (RTK) and RAS pathway oncogenes, many of which can be targeted by FDA-approved therapies. However, the majority of patients do not have targeted treatment options. Our previous work identified somatic RIT1 mutations in lung adenocarcinomas and discovered that RIT1 variants act as gain-of-function mutations to promote cellular transformation and drug resistance. RIT1 amplification and overexpression may play a similar pathogenic role. RIT1 mutations also are found in myeloid leukemias and in the germline of individuals with Noonan Syndrome. In all diseases, mutations in RIT1 are mutually exclusive with other RAS-pathway mutations, implicating RIT1 as a RAS-pathway driver gene. However, our recent preliminary data show that RIT1 and KRAS substantially differ in the downstream effectors needed to promote tumorigenesis. Further understanding the cellular consequences of RIT1 mutations will open up new strategies for treatment of RIT1-mutant cancers. In this proposal, we define the mechanism of action of RIT1 mutations in lung cancer and test the efficacy of two new treatment strategies. Building on our preliminary studies that constitute the first global profiling of RIT1 function, we now will: (1) Identify the mechanism of RIT1-YAP1 synergy in lung cancer, (2) Determine how a USP9X-RIT1 axis regulates the spindle assembly checkpoint and sensitivity to anti-mitotic therapies, and (3) Define the therapeutic potential of anti-YAP1/TEAD and anti-mitotic therapies in RIT1-mutant lung cancer. Ultimately, this work will advance our understanding of the role and mechanism of RIT1 mutations in cancer and contribute the rationale and pre-clinical data needed to translate these findings into new clinical trials. Our access to novel patient-derived and genetically-engineered mouse models, coupled with our expertise in both functional genomics and pre-clinical studies, make our laboratory uniquely well-suited to discover new therapeutic options and improve outcomes for patients with RIT1-mutant cancers.