Pilot Studies of PAX3-FOXO1 Fusions Proteins in Alveolar Rhabdomyosarcoma

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PROJECT SUMMARY The druggable human proteome is vast and yet only a small fraction of it is targeted by current FDA approved pharmaceuticals. This situation is understandable as the diseases targeted are relatively common and pose profound burdens in terms of lost years of human life, quality of life prior to mortality, and economic productivity. Yet there remains a significant ethical and economic need to relieve the burden from rare diseases. To meet this need, the NIH has released RFA-TR-22-030: Pilot Projects Investigating Understudied Proteins Associated with Rare Diseases, to which we respond with the current proposal. This pilot project aims to develop biochemical reagents and assays capable of defining the structure and molecular-scale function of a PAX3-FOXO1 fusion protein that is known to drive the phenotype of the rare disease alveolar rhabdomyosarcoma. Motivated by our interest in transcription factor transactivation domains, the PI's laboratory has contributed novel nuclear magnetic resonance spectroscopy techniques that empower high resolution investigation of transcription factor transactivation domains and their interactions with downstream coregulators, yielding a unique opportunity to address the unmet need for mechanistic studies of PAX3-FOXO1 fusion protein function that will enable downstream lead compound screening and eventual drug development. In this context, the first specific aim of this project is to generation recombinant PAX3-FOXO1 fusion protein constructs and completion of preliminary structure screening for both the fusion protein's DNA binding region and transactivation domain. Following screening for solubility and stability, selected constructs will be analyzed through our laboratory's innovative 13C direct-detect strategy that is uniquely well-suited to analysis of transcription factors and their disordered transactivation domains. Importantly, the region of FOXO1 included in this fusion is known to be regulated by lysine acetylation and we have recently developed biochemical and spectroscopic techniques that enable direct study of these post-translational modifications. The second specific aim is to establish in vitro functional studies of PAX3-FOXO1 fusion protein interactions with both DNA and protein coregulators. PAX3-FOXO1 interactions with DNA will be investigated through fluorescence-detected binding assays and targets of opportunity screened for co-crystallization. Protein-protein interactions mediated by the transactivation domain will be screened using a combination of sedimentation velocity analytical ultracentrifugation and nuclear magnetic resonance spectroscopy to identify interactions that may be druggable in future studies. Together, these aims will facilitate exploration of the relationship between the PAX3-FOXO1 fusion protein and the phenotype of the rare disease alveolar rhabdomyosarcoma. Ultimately, the in vitro system developed through this pilot study will be leveraged to define how the PAX3-FOXO1 fusion protein interacts with partners, advance understanding of the molecular basis for alveolar rhabdomyosarcoma, and set the stage for downstream high-throughput library screening for lead compounds capable of disrupting interactions mediated by the PAX3-FOXO1 transactivation domain.