Abstract 114: Unraveling the origin of Ewing sarcoma using zebrafish transgenic models

Abstract Ewing sarcoma (ES) is a pediatric cancer of bones and soft tissues with enigmatic origin. Ewing sarcoma cells are characterized by the presence of a driver fusion oncogene, most frequently EWSR1-FLI1. Previous reports have suggested that bone-marrow mesenchyme or/and neural crest cells may serve as potential cells of origin for Ewing sarcoma. These hypotheses have never been tested in vivo, largely due to the absence of a representative mouse model as the EWSR1-FLI1 fusion causes severe developmental toxicity in most cell types. To address these questions, we developed a stable zebrafish transgenic model enabling tissue-specific expression of the human EWSR1-FLI1 oncofusion in neural crest cells (Vasileva et al., 2024). Using this model, we demonstrated that expression of human EWSR1-FLI1 oncofusion in neural crest cells can lead to their transformation and the development of tumors in vivo. Single-cell analysis of tumor initiation shows that EWSR1::FLI1 reprograms neural crest-derived cells to a mesoderm-like state, strikingly resulting in ectopic fins formation throughout the body. EWSR1-FLI1 hindered the normal differentiation of neural crest cells into glial and neuronal lineages. Instead, EWSR1-FLI1 strongly reprogrammed neural crest cells by hijacking developmental enhancers and upregulating the expression of mesodermal regulators, including the key mesodermal regulator tbxta (Brachyury or T) transcription factor. tbxta/TBXT expression was maintained in subset of zebrafish and human Ewing sarcomas. Our model provides a mechanism by which a neural crest cell lineage can be transformed to Ewing sarcoma, a malignancy with predominant mesenchymal features. Taken together, these findings show how a single mutation profoundly alters embryonic cell fate decisions reprogramming neural crest cells to a mesoderm-like state to initiate a devastating childhood cancer. Citation Format: Elena Vasileva, Claire Arata, Gage Crump, James Amatruda. Unraveling the origin of Ewing sarcoma using zebrafish transgenic 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 114.