PROJECT SUMMARY/ABSTRACT Fusion-positive alveolar rhabdomyosarcoma (FP-RMS) remains a poorly understood but highly fatal cancer of childhood. The pathognomonic fusion oncoprotein of ARMS is PAX3-FOXO1 (i.e., PAX3::FOXO1, P3F), a transcription factor fusion driving ARMS progression through dysregulation of gene expression and chromatin state. Major obstacles to the development of therapies for FP-RMS and other oncogenic fusion proteins include the high degree of conformational plasticity of the fusion protein drivers and the lack of knowledge about protein partners or suitable mechanisms for inhibiting or clearing the fusions. To solve these gaps, we need a comprehensive, coordinated approach to discover and develop chemical agents that clarify the relevance of various molecular mechanisms capable of inhibiting PAX3-FOXO1-mediated tumorigenesis. We assembled a team of chemical biologists, medicinal chemists and RMS-focused physician-scientists whose unique and complementary expertise in high-throughput screening, chemoproteomics, quantitative molecular interaction analyses, transcriptional biology, medicinal chemistry, pharmacology and RMS biology will be applied to the discovery and development of small molecules that impact PAX3-FOXO1 function in FP-RMS. A key goal of the Center is to explore multiple mechanisms of action for targeting PAX3-FOXO1 and efforts will emphasize 1) identifying direct small-molecule binders of PAX3-FOXO1 or nearest neighbor proteins that may be pursued as direct functional modulators or silent binders for degrader development, 2) development of PAX3-FOXO1 degraders, including heterobifunctional proteolysis targeting chimeras (PROTACs) and molecular glues that degrade a target by hijacking the ubiquitin-proteasome system, and 3) indirect targeting of PAX3-FOXO1 by targeting transcriptional network collaborators that modulate the function of the fusion or that control transcription of the fusion. We propose three complementary Projects supported by four Research Groups, including the High-Throughput Screening Group (HTS), the Molecular & Cellular Mechanism Group (MCM), the Medicinal Chemistry Group (Med Chem), and the Pharmacology Group (Pharm). The Specific Aims involve all four Research Groups and include: 1) Ligand Discovery for PAX3-FOXO1 (Project 1); 2. Discovery and Development of Targeted Protein Degraders for PAX3-FOXO1 (Project 2); and 3. Targeting the PAX3-FOXO1-associated core regulatory transcriptional complex (Project 3). While the aims to explore multiple pharmacologic modes of perturbing PAX3-FOXO1 activities are ambitious, our combined expertise and experience in balancing foraging and focusing in drug discovery or development will enable our goal of accelerating the identification of therapies for PAX3-FOXO1-positive FP-RMS. Our unique strengths will also inform the TFCC Network, resulting in knowledge generalizable to targeting of fusion oncoproteins in childhood cancer and accelerating advances in clinical care.

PROJECT NARRATIVE Fusion-positive alveolar rhabdomyosarcoma (FP-RMS or ARMS) is one the most fatal cancers of childhood. While the disease is still poorly understood, a fusion protein called PAX3-FOXO1 (i.e., PAX3::FOXO1, P3F) is a critical driver of ARMS and dysregulates the expression of many disease genes. The PAX3-FOXO1 fusion protein was discovered three decades ago, yet it has not been successfully targeted for treating FP-RMS. Our comprehensive, coordinated discovery approach will identify small molecules that impact PAX3-FOXO1 activity, exploring multiple mechanisms of action and accelerating the development of agents to treat this cancer of unmet need.