EWS::FLI1 and HOXD13 Control Tumor Cell Plasticity in Ewing Sarcoma

Purpose: Propagation of Ewing sarcoma requires precise regula-tion of EWS::FLI1 transcriptional activity. Determining the mechan-isms of fusion regulation will advance our understanding of tumor progression. Here we investigated whether HOXD13, a developmen-tal transcription factor that promotes Ewing sarcoma metastatic phenotypes, influences EWS::FLI1 transcriptional activity. Experimental Design: Existing tumor and cell line datasets were used to define EWS::FLI1 binding sites and transcriptional targets. Chromatin immunoprecipitation and CRISPR interference were employed to identify enhancers. CUT&RUN and RNA sequencing defined binding sites and transcriptional targets of HOXD13. Transcriptional states were investigated using bulk and single -cell transcriptomic data from cell lines, patient-derived xenografts, and patient tumors. Mesenchymal phenotypes were assessed by gene set enrichment, flow cytometry, and migration assays.Results: We found that EWS::FLI1 creates a de novo GGAA microsatellite enhancer in a developmentally conserved regulatory region of the HOXD locus. Knockdown of HOXD13 led to wide-spread changes in expression of developmental gene programs and EWS::FLI1 targets. HOXD13 binding was enriched at established EWS::FLI1 binding sites where it influenced expression of EWS:: FLI1-activated genes. More strikingly, HOXD13 bound and acti-vated EWS::FLI1-repressed genes, leading to adoption of mesen-chymal and migratory cell states that are normally suppressed by the fusion. Single-cell analysis confirmed that direct transcriptional antagonism between HOXD13-mediated gene activation and EWS::FLI1-dependent gene repression defines the state of Ewing sarcoma cells along a mesenchymal axis.Conclusions: Ewing sarcoma tumors are comprised of tumor cells that exist along a mesenchymal transcriptional continuum. The identity of cells along this continuum is, in large part, determined by the competing activities of EWS::FLI1 and HOXD13.