Rhabdomyosarcoma fusion oncoprotein initially pioneers a neural signature in vivo

Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer molecularly characterized by arrested myogenesis. The defining genetic driver, PAX3::FOXO1, encodes a chimeric gain-of-function transcription factor. An incomplete understanding of the in vivo chromatin regulatory mechanisms of PAX3::FOXO1 has hindered therapeutic development. Here, we establish a PAX3::FOXO1 zebrafish injection model and a semi-automated ChIP-seq normalization strategy to evaluate how PAX3::FOXO1 initially interfaces with and modulates chromatin in a developmental context. We find that PAX3::FOXO1 interacts with inaccessible chromatin through partial/homeobox motif recognition consistent with pioneering activity. However, PAX3::FOXO1-genome binding through a composite paired box/homeobox motif alters chromatin accessibility and redistributes H3K27ac to activate neural transcriptional programs. We uncover neural signatures that are highly representative of clinical rhabdomyosarcoma gene expression programs that are enriched following chemotherapy. Overall, we identify partial/homeobox motif recognition as a key mode for PAX3::FOXO1 pioneer function and identify neural signatures as a potentially critical PAX3::FOXO1 tumor initiation event.