The ETS gene family is frequently involved in chromosome translocations that cause human cancer, including prostate cancer, leukemia, and sarcoma. However, the mechanisms by which oncogenic ETS proteins, which are DNA-binding transcription factors, target genes necessary for tumorigenesis is not well understood. Ewing's sarcoma serves as a paradigm for the entire class of ETS-associated tumors because nearly all cases harbor recurrent chromosomal translocations involving ETS genes. The most common translocation in Ewing's sarcoma encodes the EWS/FLI oncogenic transcription factor. We used whole genome localization (ChIP-chip) to identify target genes that are directly bound by EWS/FLI. Analysis of the promoters of these genes demonstrated a significant over-representation of highly repetitive GGAA-containing elements (microsatellites). In a parallel approach, we found that EWS/FLI uses GGAA microsatellites to regulate the expression of some of its target genes including NR0B1, a gene required for Ewing's sarcoma oncogenesis. The microsatellite in the NR0B1 promoter bound EWS/FLI in vitro and in vivo and was both necessary and sufficient to confer EWS/FLI regulation to a reporter gene. Genome wide computational studies demonstrated that GGAA microsatellites were enriched close to EWS/FLI-up-regulated genes but not down-regulated genes. Mechanistic studies demonstrated that the ability of EWS/FLI to bind DNA and modulate gene expression through these repetitive elements depended on the number of consecutive GGAA motifs. These findings illustrate an unprecedented route to specificity for ETS proteins and use of microsatellites in tumorigenesis.

Ewing sarcoma provides an important model for transcription-factor mediated oncogenic transformation because of its reliance on the ETS-type fusion oncoprotein EWS/FLI. EWS/FLI functions as a transcriptional activator and transcriptional activation is required for its oncogenic activity. Here we demonstrate that a previously less-well characterized transcriptional repressive function of the EWS/FLI fusion is also required for the transformed phenotype of Ewing sarcoma. Through comparison of EWS/FLI transcriptional profiling and genome-wide localization data, we define the complement of EWS/FLI direct downregulated target genes. We demonstrate that LOX is a previously undescribed EWS/FLI-repressed target that inhibits the transformed phenotype of Ewing sarcoma cells. Mechanistic studies demonstrate that the NuRD co-repressor complex interacts with EWS/FLI, and that its associated histone deacetylase and LSD1 activities contribute to the repressive function. Taken together, these data reveal a previously unknown molecular function for EWS/FLI, demonstrate a more highly coordinated oncogenic transcriptional hierarchy mediated by EWS/FLI than previously suspected, and implicate a new paradigm for therapeutic intervention aimed at controlling NuRD activity in Ewing sarcoma tumors.

Purpose Ewing sarcoma is a pediatric bone tumor which absolutely relies on the transcriptional activity of the EWS/ETS family of fusion oncoproteins. While the most common fusion, EWS/FLI, utilizes lysine-specific demethylase 1 (LSD1) to repress critical tumor suppressors, small molecule blockade of LSD1 has not yet been thoroughly explored as a therapeutic approach for Ewing sarcoma. We therefore evaluated the translational potential of potent and specific LSD1 inhibition with HCI2509 on the transcriptional program of both EWS/FLI and EWS/ERG as well as the downstream oncogenic phenotypes driven by EWS/ETS fusions in both in vitro and in vivo models of Ewing sarcoma. Experimental Design RNA-seq was used to compare the transcriptional profiles of EWS/FLI, EWS/ERG, and treatment with HCI-2509 in both EWS/FLI and EWS/ERG containing cell lines. We then evaluated morphological phenotypes of treated cells with immunofluorescence. The induction of apoptosis was evaluated using caspase 3/7 activation and TUNEL staining. Colony forming assays were used to test oncogenic transformation and xenograft studies with patient-derived cell lines were used to evaluate the effects of HCI-2509 on tumorigenesis. Results HCI2509 caused a dramatic reversal of both the up- and down-regulated transcriptional profiles of EWS/FLI and EWS/ERG accompanied by the induction of apoptosis, and disruption of morphological and oncogenic phenotypes modulated by EWS/FLI. Importantly, HCI2509 displayed single-agent efficacy in multiple xenograft models. Conclusions These data support epigenetic modulation with HCI2509 as a therapeutic strategy for Ewing sarcoma, and highlight a critical dual role for LSD1 in the oncogenic transcriptional activity of EWS/ETS proteins.

Ewing’s sarcoma is a highly aggressive bone and soft tissue tumor of children and young adults. At the molecular genetic level Ewing’s sarcoma is characterized by a balanced reciprocal translocation, t(11;22)(q24;q12), which encodes an oncogenic fusion protein and transcription factor EWS/FLI. This tumor-specific chimeric fusion retains the amino terminus of EWS, a member of the TET (TLS/EWS/TAF15) family of RNA-binding proteins, and the carboxy terminus of FLI, a member of the ETS family of transcription factors. In addition to EWS/FLI, variant translocation fusions belonging to the TET/ETS family have been identified in Ewing’s sarcoma. These studies solidified the importance of TET/ETS fusions in the pathogenesis of Ewing’s sarcoma and have since been used as diagnostic markers for the disease. EWS fusions with non-ETS transcription factor family members have been described in sarcomas that are clearly distinct from Ewing’s sarcoma. However, in recent years there have been reports of rare fusions in “Ewing’s-like tumors” that harbor the amino-terminus of EWS fused to the carboxy-terminal DNA or chromatin-interacting domains contributed by non-ETS proteins. This review aims to summarize the growing list of fusion oncogenes that characterize Ewing’s sarcoma and Ewing’s-like tumors and highlights important questions that need to be answered to further support the existing concept that Ewing’s sarcoma is strictly a “TET/ETS” fusion-driven malignancy. Understanding the molecular mechanisms of action of the various different fusion oncogenes will provide better insights into the biology underlying this rare but important solid tumor.