Mechanisms of transcriptional control by EWS-FLI1

The EWS-FLI1 chimeric transcription factor characteristic of Ewings sarcoma family tumors (ESFT) constitutes the prototype of an aberrant ETS transcription factor causally involved in oncogenesis. However, the mechanisms of transcriptional regulation leading to ETS driven tumorigenesis are poorly understood. In previous projects we have successfully established and tested a research plan that aims at leading from genomics data production, via bioinformatic generation of specific hypotheses to functional elucidation of EWS-FLI1 associated transcription network modules. Starting from deciphering and functionally annotating the EWS-FLI1 transcriptional signature in ESFT we characterized the promoter architecture of induced and repressed target genes of EWS-FLI1. While the promoter regions of EWS-FLI1 activated genes, which mainly annotate to proliferation-associated functions, were enriched in ETS-binding motifs, these motifs were underrepresented in EWS-FLI1 repressed genes, which annotated predominantly to differentiation. This suggested that EWS-FLI1 induced genes are enriched in direct targets of EWS-FLI1. In-silico analysis of time-resolved expression data revealed co-enrichment of ETS, E2F and NFY binding motifs in early EWS-FLI1 activated target genes, while EWS-FLI1 repressed genes showed enrichment of recognition motifs for forkhead box (FOX) and NFKB proteins. Genome-wide chromatin immunoprecipitation analysis followed by high through-put sequencing (ChIP-seq) confirmed the enrichment of EWS-FLI1 and E2F3/4 in EWS-FLI1 induced genes and their co-localization on about 50% of E2F target genes. Reporter gene assays for several target genes functionally confirmed co-regulation by EWS-FLI1 and E2F. These results successfully demonstrated the feasibility of all steps of our approach to uncover transcription factor networks around EWS-FLI1. Following our research plan, we propose to test hypotheses on major candidate effectors of EWS-FLI1 mediated transcriptional regulation, NFY, FOX and NFKB family members that were identified in our previous studies. Specifically, we will test the following two hypotheses: 1. EWS-FLI1 cooperates synergistically with E2F and/or NFY on proliferation associated target genes. 2. EWS-FLI1 mediates gene repression via inhibition of FOX and NFKB activation These two hypotheses will be functionally addressed using ectopic expression, RNA interference (RNAi) and reporter gene technologies combined with mutation analysis, and structurally studied using chromatin immunoprecipitation and protein co-immunoprecipitation. We will investigate the influence of recombinant DNA and drug-induced modulation of these putative EWS-FLI1 co-regulatory (NFY) or downstream (FOX and NFKB) factors on the EWS-FLI1 transcriptional signature, in-vitro ESFT cell line growth/differentiation, and in-vivo tumour development. This study will be the first to elucidate mechanisms of transcription factor cooperation in ETS-driven oncogenesis. The expected results may guide future therapeutic strategies to interfere with EWS-FLI1 function in ESFT and possibly other cancers in which aberrantly expressed ETS transcription factors play an essential role.

Ewing sarcoma is a malignant bone tumor in children and adolescents. It grows as a result of a gene rearrangement that leads to the expression of a DNA binding fusion protein, the ETS transcription factor EWSFLI1. In this project, we describe two new mechanisms of aberrant gene regulation driven by EWSFLI1. We demonstrated that binding of EWSFLI1 close to cell cycle regulatory genes leads to gene activation, while binding at far distance from differentiation genes is associated mainly with gene repression. For the activation of growth promoting genes, we define d a novel regulatory module, in which EWSFLI1 binding leads to the exchange of a repressive transcription factor of the E2F family (E2F4) for an activating E2F factor, E2F3, on the gene promoter. We demonstrated that this module is not specific to Ewing sarcoma but also present in prostate cancer, in which a related ETS transcription factor, ERG, is activated by gene rearrangement. In contrast, EWSFLI1 suppressed gene promoters were found specifically enriched in binding sites for members of the forkheadbox(FOX) transcription factor family. We identified FOXO1 as the key factor, whose expression is affected by EWSFLI1 in two ways: First, EWSFLI1 modulates FOXO1 RNA transcription, and second, it prohibits nuclear entry of FOXO1 by activation of two protein modifying enzymes, CDK2 and PI3K. In order to evaluate the therapeutic potential of our finding, we tested methyl selenenic acid as an exemplary FOXO1 reactivating substance in vitro and in a xenograft mouse model. We were able to demonstrate that pharmacological reactivatio n of FOXO1 results in Ewing sarcoma cell death leading to significantly delayed tumorgrowth. Thus, the concluded project did not only result in important new findings about novel oncogenic mechanisms, which extend beyond Ewing sarcoma, but it also provided proof of principle for innovative routes to novel future therapeutic strategies.