Transcriptional Programs Define Intratumoral Heterogeneity of Ewing Sarcoma at Single-Cell Resolution

EWSR1-FLI1, the chimeric oncogene specific for Ewing sarcoma (EwS), induces a cascade of signaling events leading to cell transformation. However, it remains elusive how genetically homogeneous EwS cells can drive the heterogeneity of transcriptional programs. Here, we combine independent component analysis of single-cell RNA sequencing data from diverse cell types and model systems with time-resolved mapping of EWSR1-FLI1 binding sites and of open chromatin regions to characterize dynamic cellular processes associated with EWSR1-FLI1 activity. We thus define an exquisitely specific and direct enhancer-driven EWSR1-FLI1 program. In EwS tumors, cell proliferation and strong oxidative phosphorylation metabolism are associated with a well-defined range of EWSR1-FLI1 activity. In contrast, a subpopulation of cells from below and above the intermediary EWSR1-FLI1 activity is characterized by increased hypoxia. Overall, our study reveals sources of intratumoral heterogeneity within EwS tumors. [Display omitted] •Time-resolved single-cell RNA-seq of EWSR1-FLI1 induction in an Ewing sarcoma cell line•Highly specific transcriptomic signature of EWSR1-FLI1 chimeric oncogene•Sources of transcriptomic heterogeneity in Ewing tumors•Correlations between EWSR1-FLI1 activity and metabolic and other phenotypic states Aynaud et al. reveal a highly specific enhancer-driven transcriptional signature of EWSR1-FLI1 through independent component analysis of time-resolved single-cell RNA-seq analysis. Cell-to-cell variation of the intensity of this signature constitutes a major source of heterogeneity in Ewing tumors and is associated with proliferative, migratory, or metabolic states of the cells.