Multimodal single-cell analyses reveal distinct fusion-regulated transcriptional programs in Ewing sarcoma

Ewing sarcoma (EwS) is a fusion-driven malignancy, peaking in adolescence. Although EwS tumors are driven uniquely by EWS::FLI1 and related fusions, patient outcomes vary greatly. If and how tumor plasticity of EWS::FLI1-regulated transcriptional signatures contribute to disease progression is not known. We utilized a single-cell co-assay of RNA and ATAC sequencing to identify gene regulatory networks in EwS. By comprehensively characterizing regulatory elements across cell lines, we identified multiple unique modules of gene regulation. Differential usage and prevalence of these modules was evident across cell lines, associated with distinct epigenetic and transcriptomic signatures, and in specific cases, modifiable by exogenous TGF-β. Evaluating these modules in transcriptomic data from patients confirmed their enrichment across and within EwS primary tumors. We conclude that multiple gene regulatory networks co-exist but are differentially abundant within EwS tumors providing insight into how heterogenous transcriptional networks may contribute to clinical outcomes.