Abstract 2612: Elucidation of PAX3::FOXO1 initiation mechanisms in human induced pluripotent stem cell models
Authors: Bradley T. Stevens, Yang Zhang, Jamaica F. Siwak, Shondra M. Pruett-Miller, Brian J. Abraham, Mark E. Hatley
Journal: Cancer Research (2025)

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Alveolar RMS (ARMS), an aggressive subtype of RMS, is driven by either t(2;13)(q35;q14) or t(1;13)(p36;q14) resulting in the PAX3-FOXO1 (P3F) or PAX7-FOXO1 (P7F) fusion oncoproteins. Despite rigorous scientific advances and clinical trials, the survival rate for high-risk ARMS has remained at 20% over the last three decades. A deeper understanding of the basic biology driving P3F mediated tumorigenesis is needed to discover novel therapeutic targets. The P3F oncofusion protein is the key oncogenic driver and a potential candidate for therapeutic intervention. Current in vivo model systems fail to faithfully recapitulate the human disease in terms of timing and location and are complicated by the formation of other tumor types. A deeper understanding of the cells of origin and mechanisms of P3F tumor initiation are essential to enhancing models and preclinical efforts. 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 blocked endothelial maturation and instead reprogrammed cells to skeletal muscle-like cells that form ARMS tumors in mice. Generation of iPSC-derived ARMS (iARMS) occurs over 15 days and provides a unique model to determine the mechanism of P3F mediated transformation from non-myogenic origins. To elucidate this mechanism, I utilized CUT&amp;RUN for P3F and H3K27ac to assess P3F occupancy and enhancer landscape, ATAC-seq to assess chromatin accessibility, and RNA-seq to assess gene expression changes at timepoints throughout the transformation event. These data define the fundamental genomic changes required for transformation and uncovers the key determinants of ARMS identity. Removal of P3F utilizing a guide RNA targeting the P3F breakpoint allowed iARMS cells to form endothelial-like tubes in vitro, indicating RMS cells retain endothelial potential after oncogene withdrawal. Finally, to explore alternative cells of origin and endogenous regulation of P3F during development, we created t(2;13) iPSCs (tiPSCs). In proof of principle experiments tiPSCs formed teratomas with discrete regions of ARMS in immunocompromised mice and differentiation of tiPSCs into muscle organoids revealed an abrogation in normal developmental trajectory upon P3F induction. Our novel cellular models provide a unique platform to uncover how P3F establishes and maintains ARMS cell state, dissect the specific dependencies required to maintain the ARMS cell state, and uncover how P3F is endogenously regulated during development. Understanding the fundamental mechanism of P3F-driven tumorigenesis from translocation regulation to transformation provides greater resolution into the key disease determinants to generate faithful model systems to focus pre-clinical efforts. Citation Format: Bradley T. Stevens, Yang Zhang, Jamaica F. Siwak, Shondra M. Pruett-Miller, Brian J. Abraham, Mark E. Hatley. Elucidation of PAX3::FOXO1 initiation mechanisms in human induced pluripotent stem cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2612.