Berger, Alice HRiley, Amanda2024-04-262024-04-262024-04-262024Riley_washington_0250E_26512.pdfhttp://hdl.handle.net/1773/51387Thesis (Ph.D.)--University of Washington, 2024Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer is the most diagnosed type of lung cancer, and lung adenocarcinoma is the most prevalent subtype. Approximately 50% of lung adenocarcinoma tumors harbor druggable mutations in genes such as EGFR and ALK, and targeted therapies are highly effective at reducing tumor burden. Indeed, targeted therapies have revolutionized cancer treatment and are becoming standard of care over cytotoxic chemotherapy; however, many mutations are not clinically actionable. Up to 15% of lung adenocarcinoma tumors are driven by mutation or amplification of the RAS-family gene RIT1, and RIT1 mutations do not co-occur with other canonical driver mutations. There is a growing understanding that the protein abundance of RIT1 is essential for its function. Therefore, inhibiting positive regulators of RIT1 abundance could be a tractable means of reducing tumor burden and abrogating the growth of tumors driven by RIT1 mutations and amplifications. Development of a RIT1-specific inhibitor is unlikely to succeed due to the structure of RIT1 as a GTPase. In 2013, groundbreaking work on KRAS resulted in the development of the first mutant-specific inhibitors, which represents a major advance in this field and for patient care. Such an approach for RIT1, however, would be quite difficult due to the resources required and our lack of knowledge pertaining to RIT1 biology and oncogenic mechanisms. Because of this, innovative approaches are needed to understand RIT1 genetic dependencies and uncover druggable targets. The Berger Lab developed a CRISPR screening approach to discover genes required for RIT1-driven cellular transformation. From this work, we found that RIT1-mutant cells are uniquely dependent on genes associated with the Spindle Assembly Checkpoint (SAC), including Aurora kinases A and B. RIT1-mutant cells are more sensitive than KRAS-mutant cells to alisertib (an Aurora kinase A inhibitor) and barasertib (an Aurora kinase B inhibitor). Expression of mutant RIT1 weakens the SAC, prompting cells to prematurely exit mitosis and accumulate mitotic abnormalities. In addition to the SAC vulnerability, we identified the deubiquitinase USP9X as a top essential gene in RIT1-mutant cells. This was particularly intriguing given that previous work has suggested that the protein abundance of RIT1 is important for its function. Indeed, although RIT1 shows high sequence homology to KRAS, RIT1 does not appear to be regulated in a similar manner (i.e. at the level of GAP resistance). Instead, RIT1 appears to be regulated at the level of protein abundance. Here, I explore the hypothesis that RIT1 is a substrate of USP9X and found that USP9X binds to and deubiquitinates RIT1. I find that USP9X depletion decreases RIT1 protein abundance and stability, and loss of USP9X abrogates RIT1-driven cell growth and proliferation. These findings increase our understanding of RIT1 biology and oncogenic mechanisms and nominate USP9X as a therapeutic target for the treatment of RIT1-driven diseases.application/pdfen-USCC BY-NC-NDdeubiquitinaselung cancerRASRIT1USP9XCellular biologyOncologyMolecular biologyMolecular and cellular biologyThesis