PROJECT SUMMARY Fusion transcription factors (TFs) are an enticing class of cancer targets. When present in a malignancy, these chimeric proteins are often the primary oncogenic drivers, and genetic knockdown of the fusion TF often leads to cell death or differentiation. However, fusion TFs are an exceptionally difficult class of proteins to target with small molecule inhibitors. Chief among the challenges of targeting fusion TFs is the significant degree of intrinsic disorder and the lack of mechanistic characterization of individual fusions, limiting the ability to execute structure and/or function-driven approaches to developing inhibitors. Strategies that enable direct targeting of fusion TF function without the need for extensive functional characterization of the fusion TF are therefore highly valuable. In this proposal, I will develop generalizable and mechanistically unbiased approaches to discover inhibitors of the fusion TF PAX3-FOXO1 using small molecule microarray (SMM) screening strategies. Via the training plan detailed herein, I will acquire expertise in several chemical biology strategies that are critical to accomplishing this important objective. The proposed research will be further facilitated by an exceptional training environment that provides me with mentorship from scientific leaders in cancer biology and TF inhibitor development. Preliminary data indicates that PAX3-FOXO1 is ligandable; a small proof-of-concept screen produced two selective hit molecules, one of which was confirmed to directly bind PAX3-FOXO1 and inhibit its function in follow-up assays. Building on this preliminary data, I hypothesize molecules that directly target PAX3-FOXO1 or its binding partners can be identified via joint analysis of SMM screens against PAX3-FOXO1 in cell lysate and purified protein formats. In Aim 1, I will identify functionally active direct binders of PAX3-FOXO1 and identify binding sites for a series of the most potent molecules. Structural characterization of one of these sites and its interactions with binders will provide critical insights about small molecule recognition that will serve as the basis for future structure-based optimization efforts. In Aim 2, I will identify active molecules that bind to functionally vulnerable PAX3-FOXO1 interaction partners. Detailed target identification efforts using photo-affinity labeling approaches will identify the molecular target and binding site of a lead molecule, and target validation experiments will characterize the function of the target protein in PAX3-FOXO1-driven transcription. In Aim 3, I will develop a PAX3-FOXO1 proteolysis targeting chimaera (PROTAC) degrader by identifying high-affinity PAX3-FOXO1 binders and subjecting them to an efficient screening and optimization strategy to identify the combination of PAX3-FOXO1 binder, linker, and E3 ligase binder that leads to the most potent degradation. This PAX3-FOXO1 PROTAC, representing the first targeted degrader of any fus...