Origin of Ewing sarcoma by embryonic reprogramming of neural crest to mesoderm

Ewing sarcoma is a malignant small round blue cell tumor of bones and soft tissues, predominantly affecting children, adolescents and young adults. Ewing sarcoma is caused by chromosomal translocations that generate aberrant chimeric transcription factors, most frequently EWSR1::FLI1. Ewing sarcomas exhibit features of both neural and mesenchymal cells. The cell of origin of Ewing sarcoma and the mechanisms of EWSR1::FLI1-driven cell transformation are the subjects of long-standing debates, largely due to the absence of a representative animal model. By developing a robust Ewing sarcoma model in zebrafish, we provide evidence for a neural crest origin of this cancer. We show that neural crest-derived cells (NCCs) uniquely tolerate expression of the human EWSR1::FLI1 oncofusion protein in vivo and that targeted expression of the oncofusion in NCCs is sufficient to generate Ewing sarcoma tumors. In the trunk, EWSR1::FLI1 reprogrammed NCCs to a mesoderm-like state, including upregulation of the early mesoderm specifier tbxta (Brachyury/T) and mesenchymal genes pdgfra, twist1a, and prrx1a. Mesodermal reprogramming of trunk neural crest was evident by the ability of EWSR1::FLI1-expressing cells to induce ectopic fins throughout the body. Single-nucleus analysis of RNA expression and chromatin accessibility, combined with genome-wide profiling of EWSR1::FLI1 binding, revealed that EWSR1::FLI1 targets single GGAA-containing ETS sites in mesodermal developmental enhancers for neural crest to mesoderm reprogramming during cancer initiation, also binding to GGAA repeats in established tumors. These findings show that EWSR1::FLI1 hijacks normal ETS-dependent developmental pathways in embryonic NCCs to drive initiation of Ewing sarcoma. Overall design: Dissected trunks of -28.5Sox10:BFP>dsRed -28.5Sox10:dsRed>eGFP-2A-EF1 zebrafish at 7dpf were dissociated and cells were identified by Fluorescence-activated cell sorting (FACS). Dissected tissue from a wild type fish at 3mpf, a -28.5Sox10:dsRed>eGFP-2A-EF1 trunk tumor at 3mpf, and a EWSFLI mosaic model head tumor at 9mpf were dissociated and processed with no sorting. All samples were subjected to nuclei isolation and processed for snATACseq and/or snRNAseq with 10x Genomics.