Project Summary/Abstract Gene fusions between the transcriptional repressor Capicua (CIC) and several possible 3’ partner genes, including DUX4 and NUTM1, drive the progression of aggressive sarcomas with dismal prognoses and few treatment options. While CIC-fusions are increasingly recognized as a unique clinical entity, to date CIC-DUX4 is the only CIC fusion protein for which any mechanistic research has been published. Even for the limited available work, the suggested therapies are likely to be limited by a lack of specificity. A failure to understand the biology underpinning CIC-fused oncoproteins and their signaling pathways hampers the development of smarter and more effective treatment strategies. Due to the lack of existing research on CIC-fused oncoproteins, this proposal intends to leverage prior knowledge about the function of individual partner genes to inform hypotheses about how CIC-fusions function. Wild type CIC is known to be relocalized from the nucleus to the cytoplasm by FGF- or c-Src-mediated posttranslational mechanisms, and the WT CIC protein residues thought to be responsible for this shuttling are typically conserved in CIC-DUX4 proteins. Thus, Aim 1 of this proposal tests the hypothesis that FGF- or c-Src signaling can mediate CIC-DUX4 subcellular relocalization. This will be achieved using a novel EGFP-tagged CIC-DUX4 construct allowing for dynamic microscopic analysis of CIC-DUX4 localization, as well as using patient-derived cell lines for in vivo modeling. If this hypothesis is correct, then forced relocalization could be a novel therapeutic strategy. To move beyond the study of CIC-DUX4, Aim 2 will leverage structure-function studies to define the mechanistic underpinnings of the means by which CIC-NUTM1 drives tumorigenesis. CIC-NUTM1 is thought to recruit p300 via the NUTM1 moiety to activate CIC target genes, yet preliminary data suggests this is not the complete story. Thus, Aim 2 will test the hypothesis that NUTM1 possesses some yet unidentified p300-independent functional domain which contributes to the activity of the CIC-NUTM1 fusion. These experiments will leverage recently cloned synthetic, patient-based CIC-NUTM1 fusion plasmids that allow for the first modeling of CIC-NUTM1 tumors. Uncovering the mechanisms through which CIC-NUTM1 drives tumors will reveal targetable vulnerabilities that can shape future therapies. Together, the aims will provide foundations for improving treatments and outcomes in CIC-rearranged tumors. This proposal will be carried out in a world-class environment at UCSF, which specializes in translational cancer research. The fellowship will provide experimental training in advanced microscopy and mouse techniques as well as professional development opportunities in areas such as scientific writing and mentorship which will prepare the fellow for a career as an independent researcher.