Presence Of EWS-FLI1 Research Articles

Targeting CD99 Compromises the Oncogenic Effects of the Chimera EWS-FLI1 by Inducing Reexpression of Zyxin and Inhibition of GLI1 Activity.

Ewing sarcoma, a highly aggressive pediatric tumor, is driven by EWS-FLI1, an oncogenic transcription factor that remodels the tumor genetic landscape. Epigenetic mechanisms play a pivotal role in Ewing sarcoma pathogenesis, and the therapeutic value of compounds targeting epigenetic pathways is being identified in preclinical models. Here, we showed that modulation of CD99, a cell surface molecule highly expressed in Ewing sarcoma cells, may alter transcriptional dysregulation in Ewing sarcoma through control of the zyxin-GLI1 axis. Zyxin is transcriptionally repressed, but GLI1 expression is maintained by EWS-FLI1. We demonstrated that targeting CD99 with antibodies, including the human diabody C7, or genetically inhibiting CD99 is sufficient to increase zyxin expression and induce its dynamic nuclear accumulation. Nuclear zyxin functionally affects GLI1, inhibiting targets such as NKX2-2, cyclin D1, and PTCH1 and upregulating GAS1, a tumor suppressor protein negatively regulated by SHH/GLI1 signaling. We used a battery of functional assays to demonstrate (i) the relationship between CD99/zyxin and tumor cell growth/migration and (ii) how CD99 deprivation from the Ewing sarcoma cell surface is sufficient to specifically affect the expression of some crucial EWS-FLI1 targets, both in vitro and in vivo, even in the presence of EWS-FLI1. This article reveals that the CD99/zyxin/GLI1 axis is promising therapeutic target for reducing Ewing sarcoma malignancy.

Abstract B52: Ewing sarcoma: A case study of clonal aneuploidy and DNA damage repair in pediatric cancer

Abstract Many tumor types harbor specific chromosome gains or losses. In Ewing sarcoma (ES) chromosome 8 gain is extremely common, indicating that this trisomy drives tumorigenesis. However, in primary cells, whole chromosome gains and losses are universally detrimental. We hypothesize that gain of chromosome 8 suppresses cellular stresses associated with oncogenic transformation in ES. In this study, we induced expression of EWS-FLI1 fusion oncogene, which drives ES, in various primary human mesenchymal progenitor and fibroblast cells. We showed that expression of EWS-FLI1 results in a strong cell cycle-dependent replication stress in euploid mesenchymal progenitor cells and fibroblast cells. As a consequence, proliferation is impaired when EWS-FLI1 is expressed in these cells. However, primary fibroblast cells carrying an extra copy of chromosome 8 (trisomy 8) exhibit much less replication stress, and the impairment of proliferation was also not seen in these cells. The levels of replication-associated DNA damage are lower in the trisomy 8 cells than in the euploid cells in the presence of EWS-FLI1. Then, we applied weighted co-expression network analysis (WGCNA) to RNA sequencing data obtained from ES patient tumor tissues in combination of mouse synteny analysis. We identified multiple potential genes and regions on chromosome 8, which are beneficial to ES oncogenesis when gain of extra copies. Above all, we demonstrate that gain of chromosome 8 in Ewing sarcoma facilitates the primary mesenchymal-lineage cells to overcome certain oncogenic stresses and reduces DNA damage associated with tumorigenic processes, which is achieved by cooperative upregulation of multiple gene expression on chromosome 8. Citation Format: Xiaofeng A. Su, Duanduan Ma, James V. Parsons, John M. Replogle, James F. Amatruda, Charles A. Whittaker, Kimberly Stegmaier, Angelika Amon. Ewing sarcoma: A case study of clonal aneuploidy and DNA damage repair in pediatric cancer [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B52.

Abstract 2882: Mithramycin impedes EWS-FLI1 driven transcription by preventing target promoter clearance

Abstract Background: Ewing sarcoma (ES) is a primary bone tumor of adolescence with poor prognosis. ES is characterized by the presence of EWS-FLI1 which acts as an oncogenic transcription factor to promote and maintain a tumorigenic phenotype. All ES cells express EWS-FLI1, however, other actionable mutations are exceedingly rare. We previously identified mithramycin (MMA) as a potent and specific EWS-FLI1 inhibitor. MMA effectively decreases ES tumor cell viability and tumor volume in vivo by reversing the EWS-FLI1 transcriptional signature. Structural and biochemical data suggest that sub-micromolar MMA increases the stability of the DNA binding domain of EWS-FLI1 at GGAA repeats. These data suggest that MMA may prevent EWS-FLI1 from successfully promoting Pol II transcriptional elongation. Methods: We used ChIP-qPCR to detect EWS-FLI1 binding at the promoter of well characterized targets. We used a Pol II processivity assay to measure the efficiency of transcriptional elongation at EWS-FLI1 targets. We use RT-qPCR, western blotting, and RNAseq to confirm that MMA sensitizes ES cells to elongation inhibitors. Finally, we use GROseq to identify the changes to Pol II dynamics at EWS-FLI1 targets following MMA treatment. Results: Our ChIP-qPCR data demonstrate an increased fraction of EWS-FLI1 DNA binding at the promoters of target genes with GGAA microsatellite regions. MMA significantly inhibits the processivity of Pol II at EWS-FLI1 targets when added both into a run-on reaction or following ES cell pretreatment. Combining MMA with elongation inhibitors induces strong synergy as measured by Bliss-Independence, renders multiple ES cell lines unviable by inhibiting the effects of EWS-FLI1 at the mRNA and protein level, and is effective in vivo. MMA biases GROseq reads toward the 5’ end of genes, a result consistent with a promoter trapping mechanism. We are currently correlating GROseq data to global EWS-FLI1 DNA binding using ChIPseq. Conclusions: We characterized a mechanism of MMA-induced EWS-FLI1 inhibition that involves increased stabilization of the fusion protein at target promoter regions. This mechanism is supported by changes to EWS-FLI1 DNA-binding, EWS-FLI1 target transcriptional processivity, and a reversal of the EWS-FLI1 transcriptional signature by combining MMA with downstream elongation inhibition. We hope that this work will inform future endeavors to develop a targeted therapy for ES that inhibits the main driver of disease, EWS-FLI1. Citation Format: Guillermo Flores, Susan Kitchen-Goosen, Brandon Oswald, Elissa Boguslawski, Marie Adams, Ian Beddows, Zachary Madaj, Patrick Grohar. Mithramycin impedes EWS-FLI1 driven transcription by preventing target promoter clearance [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 2882.

Abstract 1049: Targeting YAP/TAZ pathway inhibits Ewing sarcoma metastasis

Abstract The term epithelial to mesenchymal transition (EMT) and its reverse (MET) describe the dynamic and reversible metamorphosis of cells from a highly organized to a loose migratory phenotype through cytoskeletal reorganization causing cellular plasticity. EMT/MET are inherent to normal embryonal development and wound healing, but cancer cells hijack the underlying mechanisms to enable oscillations between proliferation, invasion and migration that cause metastasis, the major killer of cancer patients. Recently, fluctuations of the oncoprotein EWS-FLI1 were identified to drive EMT/MET in Ewing sarcoma. We previously demonstrated that, in presence of EWS-FLI1, transcriptional co-activators MRTFB and TAZ are largely blocked from associating with TEAD and its target genes keeping tumor cells in a poorly migratory, highly proliferative state. In contrast, under EWS-FLI-low conditions, MRTFB and TAZ associate with YAP/TEAD complexes on chromatin resulting in cytoskeletal target gene activation and phenotypic transition to a highly migratory and low proliferative state. We therefore hypothesized that pharmacologic inhibition of YAP/TAZ/TEAD protein interaction should prevent Ewing sarcoma cells from EMT and consequently interfere with their metastatic potential. Verteporfin is a small molecule safely used in the treatment of age-related macular degeneration. Independent of its photosensitizing activity exploited in ophthalmology, it is a YAP/TAZ pathway -blocking compound. In vitro treatment of EWS-FLI1-low Ewing sarcoma cells with verteporfin resulted in decreased YAP/TAZ/TEAD complex formation in the nanomolar range, reversal of the de-repression of a EWS-FLI1 controlled EMT transcriptional signature, and inhibition of tumor cell migration in a Boyden chamber assay. Upon orthotopic implantation of TC71 Ewing sarcoma cells into the tibial crest of SCID beige mice, intra-peritoneal treatment of mice with 25mg verteporfin/kg/day before and after amputation of the affected limb led to a drastic decrease of lung metastases without affecting primary tumor growth and without obvious general toxicity. Therefore, YAP/TAZ pathway blockade holds promise as a potential metastasis-preventive strategy in the treatment of Ewing sarcoma patients with primary localized disease. Citation Format: Lisa Bierbaumer, Anna M. Katschnig, Branka Radic-Sarikas, Jeffrey R. Petro, Karin Mühlbacher, Dave N. Aryee, Anna R. Pötsch, Sandra Högler, Lukas Kenner, Aykut Uren, Heinrich Kovar. Targeting YAP/TAZ pathway inhibits Ewing sarcoma metastasis [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 1049.

Abstract 4459: EWS-FLI1 orchestrates beta-catenin regulation by a mechanism involving post-translational KDM1A stabilization

Abstract Beta-catenin (CTNNB) fulfills different functions depending on its subcellular localization. At the membrane it is involved in homotypic cell adhesion. In the nucleus it acts as a transcriptional co-activator, while cytoplasmic CTNNB sequesters and stabilizes the transcriptional co-activator TAZ and prohibits its nuclear translocation. The most extensively characterized CTNNB function is that of a nuclear effector of canonical Wnt signaling, binding and activating TCF/LEF family transcription factors and leading to upregulation of master regulators of epithelial-to-mesenchymal transition. In Ewing sarcoma (EwS), the presence of the driver fusion oncogene EWS-FLI1 renders canonical Wnt signaling unresponsive to Wnts. The exact mechanism of EWS-FLI1 mediated suppression of this signaling pathway is still elusive, but may involve downregulation of the beta-catenin partner transcription factor TCF7L2. In the absence of Wnt/CTNNB/TCF signaling we still find nuclear localization of CTNNB in EwS cells, and demonstrate sensitivity of the tumor cells to the CTNNB targeting cell-permeable oxazole compound iCRT3, suggesting an essential TCF independent role of beta-catenin for EwS survival. The regulation of CTNNB stability is complex and involves phosphorylation and the activity of a multi-protein cytoplasmic destruction complex containing Axin, APC , GSK3B and other proteins. Here, we identify involvement of lysine specific demethylase KDM1A (LSD1) in EWS-FLI1 dependent nuclear CTNNB stabilization. We describe a mechanism, by which EWS-FLI1 directly activates expression of the ubiquitin specific protease USP1 leading to KDM1A post-translational stabilization and ultimately CTNNB nuclear accumulation. Consequently, knockdown of EWS-FLI1 resulted in modulation of USP1, depletion of KDM1A protein, activation of the LSD1 target and negative Wnt signaling regulator DKK1, and cytoplasmic sequestration of CTNNB. Strikingly, in presence of EWS-FLI1, genetic or pharmacologic inhibition of either USP1 or LSD1, or forced DKK1 expression, led to CTNNB degradation. As we find upregulation of TAZ upon EWS-FLI1 knockdown and loss of CTNNB upon modulation of TAZ by siRNA, we speculate that TAZ may be involved in CTNNB cytoplasmic stabilization in absence of EWS-FLI1, while LSD1 is required for nuclear stabilization in presence of the fusion oncogene. These findings indicate complex CTNNB regulation in EwS, orchestrated by EWS-FLI1, and suggest USP1, KDM1A and CTNNB as potential therapeutic targets in EwS. Citation Format: Jozef Ban, Dave N. Aryee, Anna M. Katschnig, Raphaela Schwentner, Maximilian O. Kauer, Heinrich Kovar. EWS-FLI1 orchestrates beta-catenin regulation by a mechanism involving post-translational KDM1A stabilization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4459.

Abstract A39: Alternative splicing in Ewing sarcoma may be driven by phase separation of spliceosome proteins

Abstract Alternative splicing has been implicated as an oncogenic process and provides both a categorization of cancer as well as an opportunity for more effective targeted treatments. Spliceosomal network interactions, including proteins that recognize splice enhancer and silencer regions, are critical for the regulation of alternative splicing, leading to oncogenic protein isoforms. Many proteins that participate in altering splicing have regions of low complexity amino acids, such as serine-arginine (SR) repeats. These SR repeats cause these proteins to have unique biophysical properties leading to phase separation and putatively formation of speckles in the nucleoplasm. EWS-FLI1 has low complexity regions and multiple validated direct interactions with spliceosome components. In addition, our work shows that EWS-FLI1 has novel direct binding to mRNA. We hypothesize that EWS-FLI1, the Ewing sarcoma (ES) oncoprotein, modulates post-transcriptional gene regulation through both novel protein interactions (trans-acting factors) and by directly binding to transcribed mRNA (cis-elements). Our initial results led us to build a novel EWS-FLI1 protein interaction model that identifies both direct and indirect binding throughout the spliceosome. Specific protein interactions of EWS-FLI1 include a newly discovered direct binding partner, DDX5, which is part of the U1 subunit. In addition, direct interactions with both U2 and U5 were identified. We have validated a small molecule probe, YK-4-279, as an enantio-specific inhibitor of EWS-FLI1 that directly blocks its binding to both RHA and p68 (DDX5). Reduction of EWS-FLI1 and YK-4-279 treatment alters exon skipping and intron inclusion events identified from RNA-seq. We then identified splicing events that were similarly reverted by both EWS-FLI1 reduction and YK-4-279, including CLK1, PPFIBP1, and CASP3. In addition, YK-4-279 reverts alternative splicing changes seen in the presence of EWS-FLI1, which was not an effect of altering RNA pol II activity. Our recent unpublished work explores the biophysical interactions of EWS-FLI1 in nucleoplasm. Inhibition of EWS-FLI1 protein interactions by YK-4-279 appear to alter nuclear speckle formation and may, through this mechanism, lead to altered spliceosomal activity. To establish that EWS-FLI1 alternative splicing changes the function of a protein through expression of a different isoform, we selected TERT as an enzyme with an isoform shift in the presence of EWS-FLI1 . When EWS-FLI1 is reduced, a γ-isoform of hTERT is synthesized that lacks exon 11. The γ-isoform of hTERT is reported by others to have increased hTERT activity and we confirm this in cell-based assays following EWS-FLI1 reduction. The cellular and oncogenic implications of this enzymatic activity change is under investigation. To confirm the relevance of splicing modulated by EWS-FLI1 occurring in human tumors, we determined splicing patterns from 75 ES patients and matched splicing patterns for 10 genes in cell line models. Exon-specific expression loci in this comparison were statistically significant for their overlap. In addition, overall gene expression levels did not stratify for overall survival, however, principal component isoform specific analyses did segregate survivors. In metastatic patients, this was significant (p=0.05) while in localized patients it approached significance (p=0.1). This work illuminates potentially critical oncogenic splicing shifts as well as provide a potential stratification mechanism for ES patients. A splicing map specific to ES will define and inform potential functional aspects of alternative isoforms. The biophysical role of EWS-FLI1 in creating or maintaining speckles is an ongoing investigation. Assessment of aberrant splicing driven by EWS-FLI1 may inform oncogenesis and reciprocally, EWS-FLI1 activities may inform splicing mechanisms. Citation Format: Jeffrey Toretsky, Garrett Graham, Uta Dirksen, Hayriye Verda Erkizan, Elizabeth Lawlor, Aykut Uren, Saravana Selvanathan. Alternative splicing in Ewing sarcoma may be driven by phase separation of spliceosome proteins. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A39.

Abstract 1407: The influence of EWS-FLI1 on the Rho/Actin/MRTF circuit in Ewing sarcoma

Abstract Ewing sarcoma (ES) is the second most common bone tumor in children and adolescents. The disease is characterized by the expression of the aberrant transcription factor EWS-FLI1, which is a fusion gene product originating from the chromosomal translocation t(11;22)(q24;q12). Despite good treatment outcomes for patients with localized ES, about 25% of patients already harbor metastatic disease upon presentation and are prevalently faced with poor overall survival. The metastatic cascade is a complex process. Remodeling of the actin cytoskeleton is the first step for a cell in order to acquire a migratory phenotype. The major regulator of the actin cytoskeleton is the Rho family of GTPases, which upon G-protein coupled receptor (GPCR) signaling gets activated and promotes the polymerization of monomeric G-actin into stress fibers. Concurrently G-actin releases the myocardin-related transcription factor family (MRTF) of transcriptional coactivators. MRTFs translocate into the nucleus where they, together with the transcription factor serum response factor (SRF), bind to serum response elements (SRE) in the promoter of genes involved in cell adhesion, migration and motility. However, the ternary complex factor (TCF) family, a class of transcriptional coactivators activated through Ras signaling, can compete with MRTFs for SRF complexing and binds to ets-motifs on SRE. The ets-transcription factor EWS-FLI1 has been shown to substitute for TCFs, potentially interfering with SRF target gene expression. We therefore hypothesize that EWS-FLI1 interferes with GPCR downstream signaling by competition with MRTFs. By gene expression profiling, we show that several putative MRTF target genes are repressed in the presence of EWS-FLI1 in ES cell lines and are strongly induced upon EWS-FLI1 knockdown. Using the MRTFA target gene transgelin (TAGLN) as an example, we show that knockdown of MRTFA can only reduce its serum inducibility in the absence but not in the presence of EWS-FLI1 supporting our hypothesis of EWS-FLI1 perturbing GPCR signaling in ES. With this study we want to elucidate the role of EWS-FLI1 in the regulation of early steps of the metastatic process in ES. Supported by the Liddy Shriver Sarcoma Initiative. Citation Format: Anna M. Katschnig, Raphaela Schwentner, Stephan Niedan, Maximilian O. Kauer, Elizabeth R. Lawlor, Dave N.T. Aryee, Heinrich Kovar. The influence of EWS-FLI1 on the Rho/Actin/MRTF circuit in Ewing sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1407. doi:10.1158/1538-7445.AM2014-1407

Suppression of FOXO1 is responsible for a growth regulatory repressive transcriptional sub-signature of EWS-FLI1 in Ewing sarcoma.

The Ewing sarcoma (ES) EWS-FLI1 chimeric oncoprotein is a prototypic aberrant ETS transcription factor with activating and repressive regulatory functions. We report that EWS-FLI1-repressed promoters are enriched in forkhead box (FOX) recognition motifs, and identify FOXO1 as a EWS-FLI1-suppressed regulator orchestrating a major subset of EWS-FLI1-repressed genes. In addition to FOXO1 regulation by direct promoter binding of EWS-FLI1, its subcellular localization and activity is regulated by cyclin-dependent kinase 2- and AKT-mediated phosphorylation downstream of EWS-FLI1. Restoration of nuclear FOXO1 expression in ES cells impaired proliferation and significantly reduced clonogenicity. Gene-expression profiling revealed a significant overlap between EWS-FLI1-repressed and FOXO1-activated genes. As a proof of principle for a potential therapeutic application of our findings, the treatment of ES cell lines with methylseleninic acid (MSA) reactivated endogenous FOXO1 in the presence of EWS-FLI1 in a dose- and time-dependent manner and induced massive cell death dependent on FOXO1. In an orthotopic xenograft mouse model, MSA increased FOXO1 expression in the tumor paralleled by a significant decrease in ES tumor growth. FOXO1 reactivation by small molecules may therefore serve as a promising strategy for a future ES-specific therapy.

Abstract 2646: Ewing sarcoma-derived iPS cell lines are resistant to chemotherapy but exhibit EWS-FLI1-independent, increased rapamycin sensitivity.

Abstract Ewing sarcoma (EWS) is a highly invasive, undifferentiated tumor of unknown histogenic origin and is the second most common type of primary bone malignancy in children and young adolescents. In approximately 85% of patients, tumor development is associated with the expression of the EWS-FLI1 fusion protein generated by the t(11;22)(q24;q12) chromosomal translocation. Although EWS tumors initially respond to aggressive combination cytotoxic chemotherapy, many tumors recur and metastasize. Disease relapse is thought to result from the outgrowth of a subpopulation of chemotherapy-resistant cancer stem cells (CSCs). Putative EWS CSCs have been identified in EWS-related cell lines and human xenografts as chemotherapy-resistant stem-like subpopulations expressing high ALDH. However, CSC isolation has proven to be challenging due to the low frequency of CSCs in vivo, the absence of specific cell surface markers for sorting, and the harsh conditions required to isolate CSCs from tumor tissues. Methods have been developed to induce the reprogramming of somatic tissues into a stem-like physiologic state similar to that of embryonic stem (ES) cells. Such strategies have been utilized to successfully generate induced pluripotent stem cells (iPSCs) from malignant tissues and have revealed significant similarities in the molecular and phenotypic characteristics of in vitro-derived iPSCs and in vivo-isolated CSCs. Thus, iPSCs have begun to serve as a prospective model for the CSC. We have generated iPSCs from the CHLA10 drug-resistant metastatic EWS cell line. These EWS iPSCs express characteristic ES cell surface markers, exhibit comparable DNA methylation patterns to previously established ES and iPS cell lines, and possess the capacity to differentiate into embryonic tissues in vivo. EWS iPSCs maintain expression of the EWS-FLI1 mutant transcript, show elevated ALDH expression, and exhibit increased drug resistance to vincristine, etoposide, and actinomycin D compared to the parent EWS cell line. Notably, EWS iPSCs are more sensitive to rapamycin than parent cells despite the persistent expression of the oncogenic EWS-FLI1 transcript. These results demonstrate that EWS iPSCs and EWS CSCs share numerous molecular and phenotypic characteristics and suggest that the mTOR signaling network participates in the regulation of iPSC maintenance and viability despite the presence of EWS-FLI1. This work highlights EWS iPSC as a potential model for the EWS CSC and provides a molecular foundation for the use of rapamycin, alone or in combination with other signal transduction pathway inhibitors, in targeting CSCs in Ewing sarcoma. Supported by Department of Defense Grant W81XWH-07-1-0580. RJA is supported by the endowed King Fahd Chair in Pediatric Oncology. Citation Format: Joseph B. Moore, David W. Lee, Michael Barbato, Timothy Triche, Daniel Wai, Robert J. Arceci. Ewing sarcoma-derived iPS cell lines are resistant to chemotherapy but exhibit EWS-FLI1-independent, increased rapamycin sensitivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2646. doi:10.1158/1538-7445.AM2013-2646

Abstract 2961: Reactivation of EWS-FLI1 suppressed FOXO1 expression as a novel therapeutic strategy for Ewing's sarcoma

Abstract The EWS-FLI1 chimeric protein, characterizing Ewing's sarcoma (ESFT), is prototypic for an aberrant oncogenic ETS transcription factor. The mechanisms of transcriptional regulation leading to ETS-driven tumorigenesis are poorly understood. In-silico analysis of time-resolved expression data revealed enrichment of recognition motifs for forkhead box (FOX) proteins in EWS-FLI1 repressed promoters. Several FOX genes were found to be bound by EWS-FLI1 in ChIP-seq and transcriptionally affected by EWS-FLI1 knockdown. We therefore hypothesized that EWS-FLI1 exerts an important part of its repressive activity via inhibiting FOX proteins. Upon silencing of EWS-FLI1, both FOXO1 and FOXO3 proteins were strongly induced in ESFT cells consistent with this hypothesis, but only FOXO1 translocated to the nucleus. However, in the presence of EWS-FLI1, FOXO1 is excluded from the nucleus as a consequence of phosphorylation. Nuclear translocation was restored by either inhibition of CDK2, augmented by chemical inhibition of PI3K, or by mutation of CDK2 or AKT phosphorylation sites. Furthermore, ChIP experiments revealed that EWS-FLI1 directly binds the promoter of FOXO1 in vitro suggesting a multi-layered regulation of FOXO1 expression by transcriptional repression and post-translational modification. Functional restoration of nuclear FOXO1 expression in ESFT cells resulted in impaired proliferation and significantly reduced soft agar colony formation ability. A significant overlap between EWS-FLI1 repressed and FOXO1 activated genes was observed. Treatment of ESFT cell lines with Methyl-Seleninic-Acid (MSA) led to re-activation of endogenous FOXO1 in the presence of EWS-FLI1 in a dose- and time-dependent manner, and induced massive cell death, which we found to be at least partially FOXO1-dependent. Taken together, these data confirm our hypothesis that a repressive sub-signature of EWS-FLI1 regulated genes is due to suppression of FOXO1. FOXO1 re-activation by small molecules may constitute a novel therapeutic strategy in the treatment of ESFT. This study was supported by grant 22328-B09 from the Austrian Science Fund FWF. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2961. doi:1538-7445.AM2012-2961

Abstract 5338: A repressive EWS-FLI1 subsignature is the result of FOXO1 suppression and nuclear exclusion

Abstract The EWS-FLI1 chimeric protein, characteristic of Ewing′s sarcoma (ESFT), constitutes the prototype of an aberrant oncogenic ETS transcription factor. The mechanisms of transcriptional regulation leading to ETS-driven tumorigenesis are poorly understood. In-silico analysis of time-resolved expression data revealed enrichment of ETS binding motifs in EWS-FLI1 activated target promoters, while EWS-FLI1 repressed promoters showed most prominently enrichment of recognition motifs for forkhead box (FOX) proteins. Several FOX genes were found to be bound by EWS-FLI1 in ChIP-seq and transcriptionally affected by EWS-FLI1 knockdown. We therefore hypothesized that EWS-FLI1 exerts an important part of its repressive activity via inhibition of FOX proteins. Upon silencing of EWS-FLI1, both FOXO1 and FOXO3 proteins were strongly induced in ESFT cells consistent with this hypothesis, but only FOXO1 translocated to the nucleus. However, in the presence of EWS-FLI1, ectopic FOXO1 was found excluded from the nucleus as consequence of phosphorylation. Nuclear translocation was restored by either inhibition of CDK2, a directly EWS-FLI1 activated kinase, augmented by chemical inhibition of PI3K, or by mutation of CDK2 phosphorylation sites. These results suggest an important role for CDK2 in FOXO1 repression by EWS-FLI1. To test the contribution of FOXO1 to the EWS-FLI1 repressive signature on the ESFT transcriptome, the overlap between genes activated by EWS-FLI1 knockdown and genes activated by ectopically expressed, nuclear directed FOXO1 was determined and compared to the effects of EWS-FLI1 knockdown under concomitant FOXO1 silencing. A significant overlap between FOXO1 activated and EWS-FLI1 repressed genes was identified (P<10e-20, hypergeometric test). Taken together, these data confirm our hypothesis that a repressive subsignature of EWS-FLI1 regulated genes is due to suppression of FOXO1. Functional restoration of nuclear FOXO1 expression in ESFT cells resulted in impaired proliferation and significantly reduced soft agar colony formation ability suggesting that FOXO1 plays an important role in ESFT oncogenesis. This study was supported by grant 22328-B09 from the Austrian Science Fund FWF. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5338. doi:10.1158/1538-7445.AM2011-5338

Neural differentiation arrest in embryonal carcinoma cells with forced expression of EWS-FLI1

Ewing's sarcoma/primitive neuroectodermal tumor (EWS/PNET) has a characteristic chimeric oncogene EWS-FLI1, which results from chromosomal translocation t (11; 22), that is believed to initiate tumorigenesis of EWS/PNET. However, the specific details of EWS/PNET oncogenesis and exact role of EWS-FLI1 remain largely unknown. In this study we explored the role of EWS-FLI1 in tumor differentiation using an embryonal carcinoma cell line P19 as a model, with forced expression of EWS-FLI1 in these cells. EWS-FLI1 has been reported to promote neural differentiation in fibroblasts, mesenchymal stem cells and rhabdomyosarcoma cells. We show forced expression of EWS-FLI1 causes absence of retinoic acid-induced neural morphology, and decreases expression of neural-specific proteins MAPT and NCAM. Critical transcriptional factors for neural differentiation and stem cells are also altered in the presence of EWS-FLI1, including decreases in levels of Oct-3 and Pax-6, and an increase in the level of Id2, which is a target of EWS-FLI1. Increased proliferation and decreased apoptotic rates are also observed in P19 cells with forced expression of EWS-FLI1. Our results raise the possibility that arrest of neural differentiation by forced expression of EWS-FLI1 as observed in this study may result from dysregulation of the cell cycle and cell proliferation. Taken together, our results demonstrate that the modulation of neural differentiation in P19 cells which have a stem cell-like pluripotency in vitro can provide a novel model system to study the neural differentiation effects of EWS-FLI1 tumorigenesis of EWS/PNET.