EWS–FLI1 Can Either Create or Inactivate Enhancers in Ewing Sarcoma

Ewing sarcoma is driven by translocations that fuse EWS to FLI1 or other ETS transcription factors. EWS–FLI1 has aberrant transcription factor activity, but how it orchestrates oncogenic gene expression programs is not well understood. Riggi, Knoechel, Gillespie, and colleagues characterized genome-wide EWS–FLI1 binding sites in Ewing sarcoma cell lines and primary tumors and noted enrichment for markers of cis-regulatory enhancer elements. Knockdown of EWS–FLI1 increased histone H3 lysine 27 acetylation, a marker of enhancer activity, at some EWS–FLI1 binding sites, whereas others showed equally strong decreases in this mark. These divergent effects were determined by the DNA sequence of each site: enhancers activated by EWS–FLI1 were significantly enriched for GGAA repeats, whereas most enhancers repressed by EWS–FLI1 contained canonical ETS transcription factor binding motifs. Analysis of evolutionary conservation and the chromatin landscape of other cell types did not indicate that GGAA repeats act as regulatory elements outside of Ewing sarcoma, suggesting that EWS–FLI1 creates de novo enhancers at these loci. Accordingly, expression of EWS–FLI1 in mesenchymal stem cells led to chromatin opening and creation of enhancers. In contrast, enhancers repressed by EWS–FLI1 are highly conserved and active in other mesenchymal cell types. Thus, the displacement of wild-type ETS proteins from these mesenchymal lineage enhancers by EWS–FLI1 may provide a potential explanation for how EWS–FLI1 disrupts mesenchymal differentiation. A strong correlation was observed between EWS–FLI1-bound enhancer activity and proximal gene expression, and several activated genes encode potential drug targets. One such kinase, VRK1, was strongly expressed in all Ewing sarcomas analyzed, and VRK1 knockdown significantly reduced Ewing sarcoma cell proliferation and survival in vitro and in vivo. Together, these results provide mechanistic insights into how EWS–FLI1 reprograms regulatory elements to alter gene expression and point to potential therapeutic targets for Ewing sarcoma.

Riggi N, Knoechel B, Gillespie SM, Rheinbay E, Boulay G, Suvà ML, et al. EWS-FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma. Cancer Cell 2014;26:668–81.

• ©2014 American Association for Cancer Research.