Abstract 2599: Biological consequences and mechanisms of neural signatures in fusion-positive rhabdomyosarcoma

Abstract The chimeric transcription factor PAX3::FOXO1 is a core dependency and monogenic driver of fusion-positive rhabdomyosarcoma, an aggressive pediatric soft-tissue sarcoma. PAX3::FOXO1 consists of the PAX3 DNA binding domains (paired and homeobox) and the FOXO1 transactivation domain. Despite the identification of the fusion-oncogene over thirty years ago, there are no targeted therapies for children suffering from this disease. The current therapeutic regimen consists of surgery, radiation, and general chemotherapy, which has been largely unsuccessful in improving patient outcomes. This lack of targeted therapies is partly due to unclear in vivo regulatory mechanisms of PAX3::FOXO1 across cellular contexts and tumorigenic stages. Previously, we found that PAX3::FOXO1 has pioneer-like activity and is transforming in zebrafish models, which generate tumors that recapitulate the human disease. To understand initial PAX3::FOXO1 activities, we developed a zebrafish mRNA injection model that broadly expresses the human fusion oncoprotein during early vertebrate development. We find in vivo evidence to characterize PAX3::FOXO1 as a pioneer transcription factor and demonstrate its ability to bind to inaccessible chromatin. We identified partial homeobox motif recognition as a new mode for PAX3::FOXO1 pioneering activity that diverges from in vitro models. PAX3::FOXO1 binding sites and motif selection preference correlated with DNA methylation. Together, these findings highlight that context, specifically underlying DNA methylation, can impact how pioneer factors interact with chromatin. PAX3::FOXO1 subsequently induced chromatin accessibility and activated enhancers upon binding. This was observed at sites where PAX3::FOXO1 was bound to its full composite motif, showing that motif preferences drive different functional roles. This neomorphic chromatin landscape supports the transcriptional activation of known rhabdomyosarcoma gene signatures and neural-related pathways. Even though rhabdomyosarcoma is characterized by incomplete skeletal muscle differentiation, neural signatures specifically exist within this aggressive subtype and are further enriched upon chemotherapeutic treatment in patient-derived xenografts. One critical target of PAX3::FOXO1 in our zebrafish model is the neural transcription factor Her3, zebrafish ortholog to HES3. PAX3::FOXO1 mRNA injection into her3/HES3 knockout zebrafish embryos suppresses neural transcriptional programs. Altogether, this work highlights neural pathway activation as an initial activity of PAX3::FOXO1 and that these programs could play a critical role across multiple stages of tumorigenesis. Our long-term goal is to further elucidate and delineate how PAX3::FOXO1 cooperates with neural factors across cellular contexts to identify and assess novel therapeutic vulnerabilities to improve the lives of patients. Citation Format: Jack Kucinski, Alexi Tallan, Matthew Kent, Cenny Taslim, Meng Wang, Matthew V. Cannon, Katherine M. Silvius, Benjamin Z. Stanton, Genevieve C. Kendall. Biological consequences and mechanisms of neural signatures in fusion-positive rhabdomyosarcoma [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 2599.