Project Summary Tumor Initiating Cells (TICs) are a subpopulation of tumor cells defined by their ability to self-renew and regenerate the heterogeneous tumor. TICs have altered metabolic and drug efflux properties, allowing them to persist as a sanctuary population of resistant cells during treatment. As such, TICs mediate tumor recurrence and metastasis, which are key contributors to mortality. To date, however, there are no therapies targeting the TIC population in solid tumors. My data show that the formation and maintenance of TICs in Ras-driven colorectal and lung tumor cell lines are critically dependent on the molecular scaffold Kinase Suppressor of Ras 1 (KSR1). KSR1 coordinates signaling through the Raf-MEK-ERK cascade downstream of oncogenic Ras. KSR1 is necessary for Ras-driven tumor formation, but dispensable for normal cell growth. Moreover, ksr1-/- mice are phenotypically normal but resistant to cancer formation. These characteristics highlight the value of KSR1 as a potential therapeutic target. Our data further show that KSR1 KO can prevent MEK inhibitor trametinib-mediated expansion of the TIC population and restore sensitivity to trametinib in both NSCLC and in CRC where resistance currently limits clinical utility of this drug. Further characterization using KSR1 transgenes with defined mutations in downstream effector-binding regions has identified KSR1 interaction with both ERK and AMPK to be implicated in maintenance of the TIC population, in vitro clonogenicity, and resistance to trametinib. These data suggest the hypothesis that KSR1-dependent ERK and AMPK activation in Ras-driven cancers plays a selective role in the formation of tumor-initiating cells and the drug resistance conveyed by this subpopulation. I will test this hypothesis by 1) defining the proximal KSR1-dependent signaling pathways that control TICs and trametinib resistance; and 2) assessing the ability of targeted KSR1 degradation to impair tumor-initiating capacity in a preclinical tumor organoid model. Completion of these aims will expand mechanistic understanding of Ras-driven TIC formation and maintenance and may reveal a novel therapeutic approach to treat TIC-mediated resistance, recurrence, and metastasis.