Molecular mechanisms of transcriptional repression by the EWS-FLI1 oncogene in Ewing’s Sarcoma

Ewing-Sarkome sind die am zweithäufigsten auftretenden Knochen- und Weichteil-tumore bei Kindern. Sie sind klein-rund-blauzellige Tumore, die hohe Spiegel des Zelloberflächen-Glykoproteins CD99 exprimieren und sich durch die t(11;22) (q24;q12) Translokation auszeichnen. Diese führt zum EWS-FLI1 Fusionsgen, welches in 85% aller Tumore der Ewing-Sarkom Familie gefunden werden kann. Mittels der Affymetrix Gen Chip Technologie analysierte unser Labor die Gen-expression von sechs Ewing-Sarkom-Zelllinien und fand 73 überexprimierte Gene und 52 Gene mit verringerter Expression. Diese Daten legen nahe, dass EWS-FLI1 nicht nur als transkriptionaler Aktivator, sondern auch als Repressor funktioniert. Unter den reprimierten Genen fanden wir den Notch-Liganden JAG1, das Notch-Zielgen Hey1 und den Zellzyklus-Inhibitor p21. Die direkte Reprimierung von p21 durch die Bindung von EWS-FLI1 an putative ETS Motive wurde bereits publiziert. p21 arretiert den Zellzyklus in der G1-Phase nach der Aktivierung von p53. In den 10kb der p21-Promoter-Region sind mehrere p53 und Ets Bindungsmotive zu finden, aber nur an zwei Stellen beide innerhalb weniger Nukleotide. Wir beschreiben hier erstmalig eine Genreporteranalyse von 14 Genreporterkonstrukten, welche in überlappenden Fragmenten 10kb der p21-Promoter-Region abdecken. EWS-FLI1 Knock-down resultierte in erhöhter Expression von p21 in den beiden p53 und Ets enthaltenden Konstrukten, in einer p53wt Ewing-Sarkom-Zelllinie, nicht aber in einer Zelllinie mit mutiertem p53. Induktion von p53 und Knock-down von EWS-FLI1 erhöhte die Expression von p21 noch stärker, während ein Knock-down von p53 p21 verringerte. Diese Daten legen nahe, dass p53 in die Genregulation von p21 durch EWS-FLI1 involviert ist. EWS-FLI1 Knock-down resultierte in einer erhöhten Expression von JAG1 in Reporter Gen Assays mit zwei JAG1-Konstrukten, die 1.7kb bzw. 573bp der JAG1-Promoter-Region enthalten. Unsere Daten lassen vermuten, dass sich das regulatorische Element für die Repression von JAG1 in den 573bp befindet. EWS-FLI1 Knock-down führte zu erhöhter Expression von Hey1 in Reporter Gen Assays mit zwei Hey1-Konstrukten, die 3.9kb bzw. 1.7kb der Hey1-Promoter-Region beinhalten. Unsere Daten deuten darauf hin, dass für die Repression von Hey1 durch EWS-FLI1 die 3.9kb Sequenz notwendig ist. Wir konnten in dieser Arbeit zeigen, dass EWS-FLI1 als transkriptionaler Repressor, nicht nur p21, sondern auch die Notch-Signalübertragungsweg Komponenten JAG1 und Hey1 reprimiert.Ewing’s sarcoma is the second most common bone and soft tissue cancer in children with an occurrence of 1-3 cases in 1 million people. Ewing’s sarcoma are small round blue cell tumors that express high levels of the cell surface glycoprotein CD99 and carry the t(11;22) (q24;q12) translocation. This generates the EWS-FLI1 fusion gene which can be found in 85% of all Ewing’s sarcoma family tumors. Using Affymetrix gene chip technology, our lab had analyzed the gene expression in six Ewing’s sarcoma cell lines and found 73 overexpressed genes and 52 genes repressed by EWS-FLI1. These data indicate that EWS-FLI1 may act not only as transcriptional activator, but also as a transcriptional repressor. Among these repressed genes we found, the Notch ligand JAG1, the Notch target Hey1 and the cell cycle inhibitor p21. It has already been published, that p21 is directly repressed by EWS-FLI1 by binding to a putative Ets motif within the p21 promoter. p21 is a cyclin-dependent kinase inhibitor, and as a direct target of p53, it arrests the cell cycle in G1 after activation of p53. Several p53 and Ets binding motifs are distributed over 10kb of the p21 5’ flanking sequence, but remarkably there are two sites where both, p53 and Ets motifs were found in a cluster. We describe here for the first time a gene reporter analysis of 14 reporter gene constructs which contain overlapping parts covering 10kb of the p21 promoter region. shRNA mediated knock down of EWS-FLI1 resulted in the upregulation of p21 in those two constructs which contain both Ets and p53 binding sites in a p53wt Ewing’s sarcoma cell line, but not in a p53mut cell line. Induction of p53 via Etoposide treatment and knock down of EWS-FLI1 resulted in even higher induction of those two constructs in the p53wt cell line, whereas knock down of p53 reduced them. These data indicate strongly that p53 is involved in the gene expression regulation of p21 by EWS-FLI1. Reporter gene assays using two JAG1 constructs encoding for 1.7kb and 573bp of the JAG1 promoter region resulted in an induction for both constructs upon knock down of EWS-FLI1 in several cell lines. Our data indicate that the regulatory element responsible for the repression of JAG1 by EWS-FLI1 is within the 573bp. Knock down of EWS-FLI1 resulted in an induction of Hey1 in reporter gene assays using two constructs encoding for 3.9kb and 1.7kb of the Hey1 promoter region. Our data suggest that the 3.9kb sequence is necessary for full repression by EWS-FLI1. We could demonstrate here that EWS-FLI1 acts as a transcriptional repressor, not only for p21, but as well for the Notch pathway components JAG1 and Hey1

Mechanisms of target regulation by the chimeric oncogene EWS-FLl1 in Ewing`s sarcoma

Ewing Sarkome sind hoch aggressive pediatrische Tumore, die sich durch eine chromosomale Translokation auszeichnen, welche in einem ETS Transkriptionsfaktor resultiert. Wir führten Genom weite Expressionsanalysen durch um transkriptionell regulierte Zielgene von EWS-FLI1 zu identifizieren. Um herauszufinden welche davon direkt reguliert werden, wurden Chromatin Immunoprezipitationen gefolgt von Sequenzierung (ChIP-seq) durchgeführt. Bioinformatische Analyen zeigten dass EWS-FLI1 an 50% der E2F Zielgene bindet. Darüber hinaus sind einige E2F Faktoren ebenfalls direkt durch EWS-FLI1 reguliert. Da E2F Faktoren den Zellzyklus regulieren, könnte die Regulierung von E2F Zielgenen durch EWS-FLI1 ein Weg sein, auf dem der Zellzyklus im Ewing Sarkom dereguliert wird. Auf Grund dessen untersuchten wir die funktionelle Interaktion zwischen EWS-FLI1 und E2F. Zehn zufällig ausgewählte Zielgene deren Promotoren von EWS-FLI1 und E2F gebunden waren, unter anderem E2F3, RAD51 und ATAD2, wurden in detaillierten Promoteranalysen durch Luziferase Reporter Assays in Kombination mit Mutationsanalysen für die EWS-FLI1 und E2F DNA Bindungsstellen studiert. Alle Promotoren der untersuchten Zielgene zeigten nach Knock-down von EWS-FLI1 verminderte Aktivität. Durch ChIPs konnten wir die direkte Bindung von EWS-FLI1 and die oben genannten Promotoren zeigen. Weiters konnten wir durch eine Analyse der Promoter Belegung demonstrieren, dass durch den Knock-down von EWS-FLI1 ein aktivierender E2F Faktor, E2F3, durch einen reprimierenden, E2F4, ausgetauscht wird. Interessant ist, dass dieser Austausch nur bei intakter ETS Bindungsstelle funktioniert, nicht aber wenn EWS-FLI1 nicht mehr an den Promoter binden kann. Die Mutation der ETS Bindungsstelle resultierte in reduzierter E2F3 Bindung. Unsere Daten stehen mit einem Modell in Einklang, in welchem EWS-FLI1 aktiv E2F3 zu Promotoren von gemeinsamen Zielgene rekrutiert und dadurch den reprimierenden Faktor E2F4 verdrängt. Dabei ist die Bindung von EWS-FLI1 essentiell für diesen Vorgang. Besonders hervorzuheben ist, dass dieser Mechanismus auch in einer TMPRSS2-ERG expremierenden Prostatakarzinom Zelllinie, nicht aber in einer nicht ETS-Fusion exprimierenden Zelllinie, gezeigt werden konnte.Ewing sarcoma is a highly aggressive pediatric cancer characterized by a chromosomal translocation leading to the chimeric ETS transcription factor EWS-FLI1 in 85% of cases. We have performed gene expression analysis to identify genes regulated at the transcriptional level by EWS-FLI1. To elucidate which of these are direct targets of EWS-FLI1 chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed. Integrative bioinformatic analysis revealed that EWS-FLI1 directly binds to 50% of E2F target genes in Ewing Sarcoma. Furthermore, several E2F factors were also found to be directly regulated by EWS-FLI1. Since E2F family members regulate cell cycle progression, regulation of E2F target genes by EWS-FLI1 might be a major pathway of cell cycle deregulation in Ewing Sarcoma. Therefore, we studied the functional interaction of EWS-FLI1 with E2F. Ten arbitrarily chosen EWS-FLI1/E2F candidate targets including E2F3, RAD51, GEMIN4 and ATAD2 were chosen for an in-depth promoter activity analysis. We confirmed direct EWS-FLI1 promoter binding (ChIP-PCR) and observed decreased reporter activity for all ten studied promoters upon knockdown of EWS-FLI1 by RNAi. Furthermore, the study of promoter occupancy by different E2F family members revealed that silencing of EWS-FLI1 results in the exchange of activating E2F3 for repressive E2F4 on the promoters of their jointly regulated target genes. Importantly, testing E2F3 promoter occupancy on wildtype and ETS motif mutated promoter constructs in Ewing sarcoma cell lines revealed that binding of E2F3 to its target promoters is dependent on an intact ETS binding site. Mutation of the ETS motif resulted in decreased binding of E2F3. These data suggest a model in which EWS-FLI1 actively recruits an activating E2F factor thereby replacing a repressing E2F factor and in which EWS-FLI1 binding is essential for E2F binding. Strikingly, the functional E2F/ETS transcriptional module detected in Ewing Sarcoma for the chimeric ETS factor EWS-FLI1 was found in TMPRSS2-ERG expressing prostate cancer cells. Our findings therefore suggest that this mechanism might be also relevant to other ETS fusion driven cancers

Factors Affecting EWS-FLI1 Activity in Ewing's Sarcoma

Ewing's sarcoma family tumors (ESFT) are characterized by specific chromosomal translocations, which give rise to EWS-ETS chimeric proteins. These aberrant transcription factors are the main pathogenic drivers of ESFT. Elucidation of the factors influencing EWS-ETS expression and/or activity will guide the development of novel therapeutic agents against this fatal disease

Identifying the druggable interactome of EWS-FLI1 reveals MCL-1 dependent differential sensitivities of Ewing sarcoma cells to apoptosis inducers

Ewing sarcoma (EwS) is an aggressive pediatric bone cancer in need of more effective therapies than currently available. Most research into novel targeted therapeutic approaches is focused on the fusion oncogene EWSR1-FLI1, which is the genetic hallmark of this disease. In this study, a broad range of 3,325 experimental compounds, among them FDA approved drugs and natural products, were screened for their effect on EwS cell viability depending on EWS-FLI1 expression. In a network-based approach we integrated the results from drug perturbation screens and RNA sequencing, comparing EWS-FLI1-high (normal expression) with EWS-FLI1-low (knockdown) conditions, revealing novel interactions between compounds and EWS-FLI1 associated biological processes. The top candidate list of druggable EWS-FLI1 targets included genes involved in translation, histone modification, microtubule structure, topoisomerase activity as well as apoptosis regulation. We confirmed our in silico results using viability and apoptosis assays, underlining the applicability of our integrative and systemic approach. We identified differential sensitivities of Ewing sarcoma cells to BCL-2 family inhibitors dependent on the EWS-FLI1 regulome including altered MCL-1 expression and subcellular localization. This study facilitates the selection of effective targeted approaches for future combinatorial therapies of patients suffering from Ewing sarcoma.(VLID)471264

The First European Interdisciplinary Ewing Sarcoma Research Summit

The European Network for Cancer Research in Children and Adolescents (ENCCA) provides an interaction platform for stakeholders in research and care of children with cancer. Among ENCCA objectives is the establishment of biology-based prioritization mechanisms for the selection of innovative targets, drugs, and prognostic markers for validation in clinical trials. Specifically for sarcomas, there is a burning need for novel treatment options, since current chemotherapeutic treatment protocols have met their limits. This is most obvious for metastatic Ewing sarcoma (ES), where long term survival rates are still below 20%. Despite significant progress in our understanding of ES biology, clinical translation of promising laboratory results has not yet taken place due to fragmentation of research and lack of an institutionalized discussion forum. To fill this gap, ENCCA assembled 30 European expert scientists and five North American opinion leaders in December 2011 to exchange thoughts and discuss the state of the art in ES research and latest results from the bench, and to propose biological studies and novel promising therapeutics for the upcoming European EWING2008 and EWING2012 clinical trials

The role of miR-17-92 in the miRegulatory landscape of Ewing sarcoma

MicroRNAs serve to fine-tune gene expression and play an important regulatory role in tissue specific gene networks. The identification and validation of miRNA target genes in a tissue still poses a significant problem since the presence of a seed sequence in the 3'UTR of an mRNA and its expression modulation upon ectopic expression of the miRNA do not reliably predict regulation under physiological conditions. The chimeric oncoprotein EWS-FLI1 is the driving pathogenic force in Ewing sarcoma. MiR-17-92, one of the most potent oncogenic miRNAs, was recently reported to be among the top EWS-FLI1 activated miRNAs. Using a combination of AGO2 pull-down experiments by PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) and of RNAseq upon miRNA depletion by ectopic sponge expression, we aimed to identify the targetome of miR-17-92 in Ewing sarcoma. Intersecting both datasets we found an enrichment of PAR-CLIP hits for members of the miR-17-92 cluster in the 3'UTRs of genes up-regulated in response to mir-17-92 specific sponge expression. Strikingly, approximately a quarter of these genes annotate to the TGFB/BMP pathway, the majority mapping downstream of SMAD signaling. Testing for SMAD phosphorylation, we identify quiet but activatable TGFB signaling and cell autonomous activity of the BMP pathway resulting in the activation of the stemness regulatory transcriptional repressors ID1 and ID3. Taken together, our findings shed light on the complex miRegulatory landscape of Ewing Sarcoma pointing miR-17-92 as a key node connected to TGFB/BMP pathway.This study was supported in part by the Austrian Science Fund (FWF), [grants 24708-B21 and I1225-B19], and by the 7th framework program of the European Commission, [grant FP7-259348] (‘ASSET’).S

An integrated genomics approach unravels a role for micro-RNAs in EWS-FLI1 driven Ewings sarcoma pathogenesis

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Evidence for E2F/EWS-FLI1 oncoprotein synergism using systems biology [abstract]

No abstract available