Abstract A020: Insights into human alveolar rhabdomyosarcoma initiation using a human t(2;13) induced pluripotent stem cell model

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) and PAX7-FOXO1 (P7F) fusion oncoproteins, respectively. 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. 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 cell(s) of origin and mechanisms of P3F tumor initiation are essential to enhancing models and preclinical efforts. To explore alternative cells of origin and endogenous regulation of P3F during development, we generated iPSCs harboring the canonical t(2;13) P3F translocation that we term t(2;13)-iPSCs (tiPSCs). tiPSCs injected into immunocompromised mice formed teratomas with discrete regions of FP-RMS that express the PAX3::FOXO1 oncofusion protein. Furthermore, directed differentiation of tiPSCs into muscle cells resulted in P3F expression and an abrogation in normal developmental trajectory. These cells injected into immunocompromised mice resulted in tumors with ARMS characteristics. Utilizing tiPSCs to explore developmental arrest we employed organoid assays that directly model human somitogenesis. Wild-type iPSCs undergo normal anterior-posterior patterning and somite formation; however, tiPSCs form somite-like condensations but fail to undergo anterior-posterior patterning upon P3F expression. Our novel cellular model provides a unique platform to 1) uncover how P3F establishes and maintains ARMS cell state(s), 2) dissect the specific dependencies required to maintain the ARMS cell state(s), and 3) uncover how P3F is endogenously regulated during human development. Understanding the fundamental mechanism of P3F-driven tumorigenesis from PAX3::FOXO1 oncofusion gene regulation to cellular transformation provides greater resolution into the key disease determinants to generate faithful model systems to focus pre-clinical efforts. Citation Format: Bradley T. Stevens, Jamaica F. Siwak, Yang Zhang, Shondra M. Pruett-Miller, Brian J. Abraham, Mark E. Hatley. Insights into human alveolar rhabdomyosarcoma initiation using a human t(2;13) induced pluripotent stem cell model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr A020.