Project Abstract ASPSCR1::TFE3 (AT3) is a fusion oncoprotein transcription factor expressed from the junction site of a t(X;17) chromosomal translocation that defines alveolar soft part sarcomas, as well as the largest subset of Xp11- rearranged pediatric renal cell carcinomas. Both are related deadly cancers with poor prognoses that generally develop in young patients. Expression of AT3 in mouse models has proven its ability to drive both sarcomagenesis and renal cell carcinomagenesis, each independent of other genetic perturbations. We identified that VCP interacts with AT3 in cell lines and human and mouse tumor tissues. The interaction was also identified at the detailed level of chromatin distribution by heavily overlapping ChIP-seq for each protein, AT3 and VCP, which co-localize to promoters and enhancers across the genome, driving enhancer loop formation. VCP normally functions as a segregase or unfoldase, removing substrate proteins from membranes, complexes, or even chromatin. Typically, its inhibition or protein-level depletion leads to metabolic stress from the loss of its cytoplasmic role in governing quality control of protein folding. However, in AT3 expressing cells, VCP depletion mimics AT3 depletion itself. VCP depletion not only impacts the transcription of target genes, but also the 3D architecture of chromatin, where enhancer loops with AT3:VCP are lost upon VCP depletion. Inhibition of VCP enzymatic activity with a specific small molecular inhibitor also impacts AT3 transcriptional targets, but the correlation between VCP inhibition and depletion is limited, which suggests that there are impacts of VCP structural components that are distinct from its enzymatic activity. Preliminary biochemistry and structural analyses have demonstrated that AT3 interacts specifically with VCP hexamers that retain enzymatic activity. This all leads to an overall model for AT3-mediated transcriptional reprogramming depending on VCP as a co- factor. Yet, the precise mechanism of VCP regulating transcription at AT3 target loci remains a critical gap in knowledge. This proposal will determine the basis of whether VCP enables AT3 transcriptional impacts through its segregase enzymatic function and as a structural scaffold for higher order chromatin organization. Upon completion, this proposal will establish the role of VCP as an obligate co-factor of AT3 and its potential as a novel therapeutic target for the treatment of alveolar soft part sarcoma.