Abstract B021: In vivo activation of neural gene signatures in fusion-positive rhabdomyosarcoma

Abstract Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer caused by a chromosomal translocation. The most common translocations occur between PAX3 or PAX7 and FOXO1, which generates a chimeric transcription factor with gain-of-function activities. These fusion proteins contain the PAX3/7 DNA-binding domains (paired and homeobox) and the FOXO1 transactivation domain. For over thirty years, these fusions have been established as the defining genetic drivers of this disease; however, they remain challenging to target pharmacologically. As such, improvements to the standard of care, which include surgery, radiation, and general chemotherapy, have focused on optimizing chemotherapy dosage and are rarely curative. Therapeutic development has, in part, been hindered by unclear in vivo regulatory mechanisms of PAX3/7::FOXO1 across cellular contexts and tumorigenic stages. Previously, our groups showed that PAX3::FOXO1, the most common fusion, is a pioneer transcription factor that reshapes chromatin architecture and can transform zebrafish cells to recapitulate human tumors. Recently, we aimed to understand how PAX3::FOXO1 initially interfaces with and modulates chromatin in vivo. We developed a zebrafish mRNA injection model and expressed human PAX3::FOXO1 during early vertebrate development. PAX3::FOXO1 utilized its homeobox domain and partial homeobox motif recognition for initial genomic binding, a new binding mode that varies from in vitro models. Moreover, PAX3::FOXO1 bound and activated inaccessible chromatin in vivo, two hallmarks of pioneer transcription factor activity. Chromatin and transcriptional profiling revealed that PAX3::FOXO1 binding resulted in activation of rhabdomyosarcoma and neural developmental gene signatures. Despite the common association of rhabdomyosarcoma with incomplete myogenesis, neural pathways and populations are present in primary patient tumors. The neural transcription factor HES3 is one example that we previously characterized as a PAX3::FOXO1 cooperating gene. The neural population is also enriched during chemotherapy treatment in patient-derived xenografts. We are continuing to utilize our zebrafish modeling and parallel approaches to understand the functional role and regulation of these neural pathways. Currently, we are investigating how HES3 modulates this activation, given that it developmentally promotes neural stem renewal, is an established target of PAX3::FOXO1, and correlates with worse patient outcomes. Neural signatures are seen in both PAX3::FOXO1 and PAX7::FOXO1 tumors, therefore we are leveraging in vivo comparative analyses between these fusions to assess if differences in neural activation can explain their divergent clinical outcomes. Altogether, this work highlights neural pathway activation as a core activity of PAX3::FOXO1 and suggests that these programs could play a critical role across multiple stages of tumorigenesis. Our long-term goal is to delineate how PAX3/7::FOXO1 cooperate with neural factors across cellular contexts to identify novel therapeutic vulnerabilities. Citation Format: Jack Kucinski, Alexi Tallan, Matthew Kent, Andrew Vontell, Katherine Silvius, Cenny Taslim, Benjamin Stanton, Genevieve Kendall. In vivo activation of neural gene signatures in fusion-positive rhabdomyosarcoma [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 B021.