Abstract IA06: Myogenic and neural transcriptional circuitry determines PAX3::FOXO1-driven initiation of rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Alveolar RMS (ARMS) has a poor prognosis and is driven by either t(2;13)(q35;q14) or t(1;13)(p36;q14) translocations resulting in the PAX3::FOXO1 (P3F) and PAX7::FOXO1 (P7F) fusion oncoproteins, respectively. Current in vivo model systems fail to faithfully recapitulate the human disease and are complicated by the formation of other tumor types. A deeper understanding of P3F-mediated tumorigenesis is needed to discover novel therapeutic targets. Previously, our lab generated an ARMS model system derived from TP53-null human induced pluripotent stem cells (iPSCs), in which forced P3F expression during endothelial cell directed differentiation reprogrammed cells to skeletal muscle-like cells that form ARMS tumors in mice. Given the human iPSC-derived ARMS (iARMS) model does not derive from skeletal muscle progenitors, it offers unique insights into the principal components necessary to establish ARMS cell fate. Analysis of cells along the course of cell reprogramming provides understandings into the order of operations necessary for P3F-mediated transformation. To elucidate the P3F-mediated tumor initiation mechanism, we utilized CUT&RUN for P3F binding and H3K27ac to assess P3F occupancy and enhancer landscape and RNA-seq to assess gene expression changes at timepoints throughout the transformation event. These data revealed that ARMS cell fate commitment occurred within two days of P3F expression that was reinforced throughout the time course. P3F established a novel enhancer landscape resulting in the early expression of one myogenic and several neural transcription factors. Single cell multi-ome profiling along the time course confirmed early fate commitment and revealed heterogeneous expression of the core TFs with both myogenic and neural transcription factors not necessarily expressed in the same cells. We generated knock-out iPSCs of each TF to define their necessity for the P3F-mediated ARMS transformation. We then refined the iARMS system allowing temporal control over P3F expression with a doxycycline-inducible, degron-fused P3F-FKBP12F36V (ddP3F cells). Doxycycline removal and P3F degradation 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 over 20 passages and retained the ability to form foci. Transcriptional analyses revealed ARMS cell states remained stable upon P3F loss. These data suggest that P3F is essential for ARMS cell fate initiation but not maintenance. This human iPSC-derived model revealed insights into the mechanism of P3F-mediated establishment of ARMS cell state, provides a platform to dissect the specific dependencies required for ARMS cell state(s), and uncovered P3F-independent maintenance of ARMS cell fate. Prior to embarking on resource-intense efforts to therapeutically target the P3F oncofusion, a rigorous investigation of the roles of P3F in FP-RMS induction, maintenance, and recurrence is needed.