Abstract B015: PAX3::FOXO1 establishes dosage-dependent transcriptional states in rhabdomyosarcoma

Abstract We have investigated how expression levels, or dosage, of the oncogenic fusion transcription factor PAX3::FOXO1 alters cell state in a human myoblast model of fusion-positive rhabdomyosarcoma. Standard of care treatments for fusion-positive rhabdomyosarcoma have not changed in decades, and there are currently no targeted chemotherapies available. The PAX3::FOXO1 translocation is likely the first alteration that occurs in fusion-positive rhabdomyosarcoma. We have demonstrated that PAX3::FOXO1 has pioneering function, meaning it can associate with heterochromatin and contribute to increasing chromatin accessibility at its binding sites. Using human myoblasts transduced with a doxycycline-inducible PAX3::FOXO1 construct, we find that PAX3::FOXO1 protein expression level or dosage modulates oncogenicity and proliferation, with maximal oncogenicity at intermediate dosage and growth suppression at high dosage. To investigate the underlying gene regulatory mechanisms of these dosage-dependent observations, we examined PAX3::FOXO1’s chromatin occupancy. PAX3::FOXO1 is most enriched within euchromatin at intermediate dosage, and we observed with ChIP-seq that this dosage maximizes PAX3::FOXO1 genomic site occupancy. Intriguingly, PAX3::FOXO1’s presence in insoluble heterochromatin is maximal at the highest dosages. PAX3::FOXO1 occupies a subset of its sites regardless of dosage, while binding to other sites appears dependent on dosage. Different motifs are enriched at dosage-dependent sites, indicating either differential PAX3::FOXO1 chromatin recognition and/or cooperativity with other factors based on dosage. Corresponding to this variable DNA binding site usage, we find that PAX3::FOXO1 dosage modulates myogenic, epithelial-mesenchymal transition, S phase, and mitotic gene expression programs, among others. We have confirmed that PAX3::FOXO1 dosage perturbs cell cycle progression at high levels in our model. Additionally, we find that prolonged PAX3::FOXO1 expression increases heterogeneity of single cell gene expression. Cell subpopulations corresponding to published RMS single cell studies are present after prolonged PAX3::FOXO1 expression, with their proportions varying with dosage. One of these, a myogenic subpopulation, may expand following PAX3::FOXO1 withdrawal. Finally, we have begun to investigate how the transcriptional heterogeneity that produces these subpopulations may be propagated in fusion-positive rhabdomyosarcoma during the process of chromatin replication and maturation. PAX3::FOXO1 increases chromatin accessibility immediately behind the replication fork in a dosage-dependent manner, suggesting it may be able to influence epigenetic memory through this process. Together, our data indicate that PAX3::FOXO1 dosage modulates regulation of key pathways for fusion-positive rhabdomyosarcoma and modifies the transcriptional heterogeneity of single cell states. Citation Format: Rachel A. Hoffman, Meng Wang, Benjamin D. Sunkel, Thanh Hung. Nguyen, Jorge Lopez-Nava, Bishwanath Chatterjee, Wenyue Sun, Frederic G. Barr, Benjamin Z. Stanton. PAX3::FOXO1 establishes dosage-dependent transcriptional states in 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 B015.