Abstract 5205: EWS-FLI1 activates HOXD13 expression through enhancer reprogramming

Abstract

Abstract Ewing sarcomas (ES) are bone tumors of mesenchymal stem cell (MSC) origin. ES is defined by EWS-FLI1 fusions, which initiate tumorigenesis via enhancer reprogramming. Posterior homeobox D (HOXD) genes, in particular HOXD13, are over-expressed by ES, where they function as obligate, cooperative oncogenes. These genes play crucial roles in limb development and spatiotemporal control of HOXD13 expression depends on long-range enhancers that flank the HOXD locus. We hypothesize that HOXD13 is aberrantly activated in the ES cell of origin as a result of EWS-FLI1-dependent hijacking of developmental enhancers. We interrogated ChIP-seq and RNA-seq data to identify EWS-FLI1 binding sites and HOXD13 activation states in ES cells. These analyses revealed an EWS-FLI1 binding site within the developmentally conserved posterior HOXD enhancer region, which we will refer to as Desert Enhancer 268 (DE268). These sites were also marked with the active enhancer marks, H3K27Ac and H3K4me1. EWS-FLI1 knockdown was associated with loss of EWS-FLI1 binding, loss of H3K27Ac and H3K4me1, and loss of HOXD13 expression. Findings were independently validated by ChIP-qPCR and RT-qPCR. Through BruUV-seq we also identified that DE268 has eRNA expression that is lost upon EWS-FLI1 knockdown. Thus, HOXD13 expression in ES cells is maintained by EWS-FLI1-mediated enhancer activation. To address whether hijacking of HOXD13 enhancers is an initiating event in tumorigenesis, we evaluated enhancer profiles in MSCs ± EWS-FLI1. Whereas DE268 was not active in parent MSC, EWS-FLI1 binding and enhancer activation were evident in EWS-FLI1+ MSC. However, although EWS-FLI1 reproducibly binds and activates DE268, expression of HOXD13 is limited to discrete conditions. Together, these results suggest that EWS-FLI1 is necessary for enhancer activation, but that the fusion is alone is insufficient for induction of HOXD13 expression. Ongoing studies will utilize CRISPRi technologies to determine the contribution of DE268 on HOXD13 gene expression. Citation Format: April Ann Apfelbaum, Laurie Svoboda, Brian Magnuson, Mats Ljungman, Elizabeth Lawlor. EWS-FLI1 activates HOXD13 expression through enhancer reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5205.