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EWS-FLI1 and HOXD13 control tumor cell plasticity in Ewing sarcoma.

Authors
  • Apfelbaum, April A1
  • Wu, Feinan2
  • Hawkins, Allegra G3
  • Magnuson, Brian4
  • Jimenez, Jennifer A5
  • Taylor, Sean D6
  • Wrenn, Emma D7
  • Waltner, Olivia8
  • Pfaltzgraff, Elise R4
  • Song, Jane Y9
  • Hall, Cody1
  • Wellik, Deneen M10
  • Ljungman, Mats11
  • Furlan, Scott N12
  • Ryan, Russell J H1
  • Sarthy, Jay F12
  • Lawlor, Elizabeth R7
  • 1 University of Michigan-Ann Arbor, Ann Arbor, MI, United States. , (United States)
  • 2 Fred Hutchinson Cancer Research Center, United States. , (United States)
  • 3 Alex's Lemonade Stand Foundation, Philadelphia, PA, United States. , (United States)
  • 4 University of Michigan-Ann Arbor, Ann Arbor, Michigan, United States. , (United States)
  • 5 University of Michigan Medical School, Ann Arbor, Michigan, United States. , (United States)
  • 6 Seattle Children's Research Institute, United States. , (United States)
  • 7 Seattle Children's Research Institute, Seattle, WA, United States. , (United States)
  • 8 Fred Hutchinson Cancer Research Center, Seattle, United States. , (United States)
  • 9 Genentech Foundation, United States. , (United States)
  • 10 University of Wisconsin-Madison, United States. , (United States)
  • 11 University of Michigan-Ann Arbor, Ann Arbor, Mi, United States. , (United States)
  • 12 Fred Hutchinson Cancer Research Center, Seattle, WA, United States. , (United States)
Type
Published Article
Journal
Clinical Cancer Research
Publisher
American Association for Cancer Research
Publication Date
Jun 02, 2022
Identifiers
DOI: 10.1158/1078-0432.CCR-22-0384
PMID: 35653119
Source
Medline
Language
English
License
Unknown

Abstract

Propagation of Ewing sarcoma (EwS) 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 EwS 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-seq 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 geneset 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 EwS cells along a mesenchymal axis. EwS 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.

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