American Association for Cancer Research
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Supplementary Table from EWS::FLI1 and HOXD13 Control Tumor Cell Plasticity in Ewing Sarcoma

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posted on 2023-04-01, 00:06 authored by April A. Apfelbaum, Feinan Wu, Allegra G. Hawkins, Brian Magnuson, Jennifer A. Jiménez, Sean D. Taylor, Emma D. Wrenn, Olivia Waltner, Elise R. Pfaltzgraff, Jane Y. Song, Cody Hall, Deneen M. Wellik, Mats Ljungman, Scott N. Furlan, Russell J.H. Ryan, Jay F. Sarthy, Elizabeth R. Lawlor
Supplementary Table from EWS::FLI1 and HOXD13 Control Tumor Cell Plasticity in Ewing Sarcoma


National Cancer Institute (NCI)

United States Department of Health and Human Services

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National Institutes of Health (NIH)

1 million 4 Anna Foundation

Alex's Lemonade Stand Foundation for Childhood Cancer (ALSF)

University of Michigan (UM)



Propagation of Ewing sarcoma requires precise regulation of EWS::FLI1 transcriptional activity. Determining the mechanisms of fusion regulation will advance our understanding of tumor progression. Here we investigated whether HOXD13, a developmental transcription factor that promotes Ewing sarcoma metastatic phenotypes, influences EWS::FLI1 transcriptional activity. 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. 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 widespread 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 activated EWS::FLI1-repressed genes, leading to adoption of mesenchymal 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. 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.See related commentary by Weiss and Bailey, p. 4360

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