Abstract A001: PAX3-FOXO1-induced transcriptional dysregulation in rhabdomyosarcoma

Abstract The survival rate for high-risk rhabdomyosarcoma (RMS) has not increased above 20% in the last thirty years. This insufficiency persists despite thorough molecular characterization of existing models and patient tumors, which has demonstrated that the most challenging RMS tumors harbor chromosomal translocations generating PAX3-FOXO1 (P3F) or PAX7-FOXO1 (P7F) oncofusion transcription factors. The presence of P3/7F largely explains the alveolar subtype of RMS. However, the molecular mechanisms by which P3/7F drives tumor onset and the extent to which they are required after establishment of RMS remain unclear. In recent work, we challenged the dogma that fusion-positive RMS arises from incompletely differentiated muscle cells by demonstrating that P3F-bearing endothelial precursors form RMS tumors ex vivo and in vivo. This observation proposes an explanation for why RMS is observed throughout the body including in sites devoid of skeletal muscle. Here, we describe a novel constructionist model of RMS in which the P3F protein is added to induced pluripotent stem cell-derived endothelial precursors. The inducible alveolar rhabdomyosarcoma (iARMS) system faithfully recapitulates tumor-relevant aspects of RMS biology, including histology, tumor-formation, and the upregulation of signature expression programs. P3F expression during endothelial-directed differentiation blocked endothelial maturation and reprogrammed cell identity toward skeletal muscle-like cells that form ARMS tumors in mice. The iARMS system reproducibly generates RMS over a 15-day timecourse, providing a unique model to examine gene-regulatory dynamics induced by P3F during RMS transformation. In addition to the expected upregulation of hallmark RMS factors, including MYOD1 and MYF6, we observed a cadre of additional transcription factor proteins that were induced and may contribute to transformation, as well as signature endothelial genes that became downregulated. P3F activated a set of enhancers that drove these expression patterns. P3F induced specific genetic dependencies in iARMS cells as observed in knockout screens. Our results define the fundamental cell identity changes underpinning P3F-induced transformation into FP-RMS and nominate collaborative transcription and non-transcription proteins that might be vulnerable to therapeutic attack. Citation Format: Bradley T. Stevens, Yang Zhang, Randolph K. Larsen, Grace E. Adkins, Jack D. Hopkins, Darden W. Kimbrough, Matthew R. Garcia, Mark E. Hatley, Brian J. Abraham. PAX3-FOXO1-induced transcriptional dysregulation in rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr A001.