Pilot Project Investigating the PAX3-FOXO1 Protein in the Rare Disease Rhabdomyosarcoma

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SUMMARY The rare childhood muscle cancer alveolar rhabdomyosarcoma (ARMS) is generally not survivable when metastatic. However, ARMS does usually respond clinically to chemotherapy initially. With respect to the cause of recurrences, clinicians observe that the translocation-mediated PAX3:FOXO1 chimeric protein present in most ARMS cases mediates treatment resistance, causing a 45% drop in 10-year survival – a clinical outcome not associated with the less common PAX7:FOXO1 protein. PAX3, PAX7 and FOXO1 are all individually key transcription factor proteins in normal muscle, brain and heart development – yet incompletely studied. The PAX3/7:FOXO1 fusion proteins are even less well characterized. We hypothesize that selective small molecule inhibitors of these chimeric transcription factor can be designed as probes to uncover these proteins' molecular, epigenetic, cellular and in vivo functions. Thus, our aim is to: (1) Develop small molecule inhibitors of PAX3-FOXO1 and PAX7:FOXO1 as tool compounds. Our approach is to establish PAX homeodomain and paired domain transcriptional reporter assays in a variety of cell model systems with different epigenetic contexts (skeletal muscle myoblasts, cardiomyocytes, RMS, control fibroblasts and neuronal cells); counter-screen for an unrelated transcriptional response element target (e.g., NFkB); perform surface plasmon resonance affinity testing of compound hits with recombinant wildtype and mutant PAX3:FOXO1 or PAX7:FOXO1 proteins; and perform ARMS cell compartment fractionation to define PROTAC-compatible nuclear E3 ligases towards generation of a PROTAC tool for PAX3/7-FOXO1. From these studies, we hope to facilitate the study of PAX3/7:FOXO1 function as transcription factors without necessarily disturbing complexes (via small molecule inhibitors), and to allow study of epigenetic protein function in real time without secondary effects (via eventual PROTAC tools).