Elucidation of PAX3::FOXO1 tumor initiation mechanisms in human induced pluripotent stem cell models

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Alveolar RMS (ARMS) is driven by either t(2;13)(q35;q14) or t(1;13)(p36;q14) resulting in the PAX3-FOXO1 (P3F) and PAX7-FOXO1 (P7F) fusion oncoproteins, respectively and has a poor prognosis. A deeper understanding of P3F mediated tumorigenesis is needed to discover novel targets. Current model systems fail to recapitulate the human disease in terms of timing, location, and the formation of other tumor types. Previously, our lab generated an ARMS model system derived from human induced pluripotent stem cells (iPSCs), in which forced P3F expression during endothelial directed differentiation blocked endothelial maturation instead reprogramming cells to skeletal muscle-like that form ARMS tumors in mice. Building off this model, we generated a doxycycline inducible iARMS model driven by degradable P3F-FKBP12 F36V (ddP3F cells) allowing for fine control over P3F expression. Degradation of P3F did not significantly reduce viability or proliferation but reduced the ability of ddP3F cells to undergo myogenic differentiation. P3F-negative ddP3F cells continued to proliferate for multiple passages and retained the ability to form foci. Transcriptional analyses revealed ARMS cell states remained stable upon P3F loss. Taken together, this data shows that P3F is important for ARMS fate initiation but not maintenance, indicating further examination of the initiation event was needed for insight into ARMS biology. To elucidate the P3F-mediated initiation mechanism, I turned to our previously established iARMS model. I utilized CUT&RUN for P3F and H3K27ac to assess P3F occupancy and enhancer landscape and RNA-seq to assess gene expression changes at timepoints throughout the transformation event. Analysis of this data revealed that ARMS fate commitment occurred within two days of P3F expression that was reinforced throughout the time course. Mechanistically, P3F established a novel enhancer landscape resulting in the early expression of one myogenic and a few neural transcription factors (core TFs). Single cell multiome profiling confirmed early fate commitment and revealed heterogeneous expression of the core TFs. Taken together, these data show that P3F initially establishes cell fate through enhancer landscape alterations with both myogenic and neural transcription factors but not necessarily in the same cells. Overall, our novel cellular models revealed insights into P3F independent maintenance of ARMS cell fate, uncovered a mechanism of P3F-mediated establishment of ARMS cell state, and provides a platform to dissect the specific dependencies required for ARMS cell state. Understanding the fundamental mechanism of ARMS tumorigenesis provides greater resolution into the key disease determinants to generate better model systems, focus pre-clinical efforts, and identify novel targets.