Stephan Niedan

Hypoxia Modulates EWS-FLI1 Transcriptional Signature and Enhances the Malignant Properties of Ewing's Sarcoma Cells In vitro

Hypoxia is an important condition in the tumor cell microenvironment and approximately 1% to 1.5% of the genome is transcriptionally responsive to hypoxia with hypoxia-inducible factor-1 (HIF-1) as a major mediator of transcriptional activation. Tumor hypoxia is associated with a more aggressive phenotype of many cancers in adults, but data on pediatric tumors are scarce. Because, by immunohistochemistry, HIF-1alpha expression was readily detectable in 18 of 28 primary Ewings sarcoma family tumors (ESFT), a group of highly malignant bone-associated tumors in children and young adults, we studied the effect of hypoxia on ESFT cell lines in vitro. Intriguingly, we found that EWS-FLI1 protein expression, which characterizes ESFT, is upregulated by hypoxia in a HIF-1alpha-dependent manner. Hypoxia modulated the EWS-FLI1 transcriptional signature relative to normoxic conditions. Both synergistic as well as antagonistic transcriptional effects of EWS-FLI1 and of hypoxia were observed. Consistent with alterations in the expression of metastasis-related genes, hypoxia stimulated the invasiveness and soft agar colony formation of ESFT cells in vitro. Our data represent the first transcriptome analysis of hypoxic ESFT cells and identify hypoxia as an important microenvironmental factor modulating EWS-FLI1 expression and target gene activity with far-reaching consequences for the malignant properties of ESFT.

Suppression of deacetylase SIRT1 mediates tumor suppressive NOTCH response and offers a novel treatment option in metastatic Ewing sarcoma

The developmental receptor NOTCH plays an important role in various human cancers as a consequence of oncogenic mutations. Here we describe a novel mechanism of NOTCH-induced tumor suppression involving modulation of the deacetylase SIRT1, providing a rationale for the use of SIRT1 inhibitors to treat cancers where this mechanism is inactivated because of SIRT1 overexpression. In Ewing sarcoma cells, NOTCH signaling is abrogated by the driver oncogene EWS-FLI1. Restoration of NOTCH signaling caused growth arrest due to activation of the NOTCH effector HEY1, directly suppressing SIRT1 and thereby activating p53. This mechanism of tumor suppression was validated in Ewing sarcoma cells, B-cell tumors, and human keratinocytes where NOTCH dysregulation has been implicated pathogenically. Notably, the SIRT1/2 inhibitor Tenovin-6 killed Ewing sarcoma cells in vitro and prohibited tumor growth and spread in an established xenograft model in zebrafish. Using immunohistochemistry to analyze primary tissue specimens, we found that high SIRT1 expression was associated with Ewing sarcoma metastasis and poor prognosis. Our findings suggest a mechanistic rationale for the use of SIRT1 inhibitors being developed to treat metastatic disease in patients with Ewing sarcoma.

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.

Variability in functional p53 reactivation by PRIMA-1(Met)/APR-246 in Ewing sarcoma.

Background:Though p53 mutations are rare in ES, there is a strong indication that p53 mutant tumours form a particularly bad prognostic group. As such, novel treatment strategies are warranted that would specifically target and eradicate tumour cells containing mutant p53 in this subset of ES patients.Methods:PRIMA-1Met, also known as APR-246, is a small organic molecule that has been shown to restore tumour-suppressor function primarily to mutant p53 and also to induce cell death in various cancer types. In this study, we interrogated the ability of APR-246 to induce apoptosis and inhibit tumour growth in ES cells with different p53 mutations.Results:APR-246 variably induced apoptosis, associated with Noxa, Puma or p21WAF1 upregulation, in both mutant and wild-type p53 harbouring cells. The apoptosis-inducing capability of APR-246 was markedly reduced in ES cell lines transfected with p53 siRNA. Three ES cell lines established from the same patient at different stages of the disease and two cell lines of different patients with identical p53 mutations all exhibited different sensitivities to APR-246, indicating cellular context dependency. Comparative transcriptome analysis on the three cell lines established from the same patient identified differential expression levels of several TP53 and apoptosis-associated genes such as APOL6, PENK, PCDH7 and MST4 in the APR-246-sensitive cell line relative to the less APR-246-sensitive cell lines.Conclusion:This is the first study reporting the biological response of Ewing sarcoma cells to APR-246 exposure and shows gross variability in responses. Our study also proposes candidate genes whose expression might be associated with ES cells’ sensitivity to APR-246. With APR-246 currently in early-phase clinical trials, our findings call for caution in considering it as a potential adjuvant to conventional ES-specific chemotherapeutics.

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.

Factors Affecting EWS-FLI1 Activity in Ewing's Sarcoma

Ewings 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.

Abstract 444: Hypoxia modulates EWS-FLI1 transcriptional signature and enhances invasiveness and anchorage-independent growth of Ewing's sarcoma cells in-vitro

Hypoxia is an important factor in the tumor cell microenvironment and approximately 1-1.5% of the genome is transcriptionally responsive to hypoxia. Hypoxia-inducible factor-1 (HIF-1) is the major mediator of transcriptional activation under hypoxia. Tumor hypoxia is associated with a more aggressive phenotype of many cancers in adults, but data on pediatric tumors are scarce. By immunohistochemistry, HIF-1α expression was readily detectable in 18/28 primary Ewing′s sarcoma family tumors (ESFT), a group of highly malignant bone-associated tumors in children and young adults, hence we studied the effect of hypoxia on ESFT cell lines in-vitro. Intriguingly, we found that EWS-FLI1 protein expression, which characterizes ESFT, is transiently up-regulated by hypoxia in a HIF-1α-dependent manner. Hypoxia modulated the EWS-FLI1 transcriptional signature relative to normoxic conditions. Both synergistic as well as antagonistic transcriptional effects of EWS-FLI1 and of hypoxia were observed. Consistent with alterations in the expression of metastasis related genes, hypoxia stimulated the invasiveness and soft-agar colony formation of ESFT cells in-vitro. Our data represents the first transcriptome analysis of hypoxic ESFT cells and identifies hypoxia as an important microenvironmental factor modulating EWS-FLI1 expression and target gene activity that may impact the malignant properties of ESFT. This study was funded by: “European Embryonal Tumor Pipeline” 6 th framework program of the European Commission (STREP “E.E.T. Pipeline” contract LSHC-CT-2006-037260) and grant 12675 from the Austrian National Bank Jubilaeumsfonds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 444.

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

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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

Abstract 1436: Investigating the NAD metabolome in Ewing sarcoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ewing Sarcoma (ES) is the second most common bone cancer in children and adolescents with a high metastatic potential. Tumor development is driven by the specific t(11;22)(q24;q12) chromosomal translocation resulting in generation of the chimeric transcription factor EWS-FLI1. Recently, ES has been reported to be exquisitely sensitive to inhibitors of poly(ADP-ribose) polymerase 1 (PARP1). This enzyme uses NAD+ as substrate and was demonstrated to regulate EWS-FLI1 in a feed-back mechanism. Another major mammalian NAD+ consumer is the deacetylase SIRT1 which we observed to be highly expressed in ES metastases, validated with immunohistochemistry of 250 primary tumors and 30 metastases. PARP1 and SIRT1 play pivotal roles in coupling cellular metabolism to transcriptional gene regulation as well as to stress response. Both of them regulate pro-apoptotic transcription factors that are suppressed in ES including p53, FOXO1 and 3, and NFκB. It has been demonstrated that PARP1 activity is at least partially dependent on acetylation which is counteracted by SIRT1 activity. Severe cell stress such as DNA damage leads to massive activation of PARP1, thus resulting in depletion of the cellular NAD+ pool and finally cell death. Usually, NAD+ is regenerated from nicotinamide via NAMPT or from the reduction of pyruvate via LDHA (Warburg effect), but can also be synthesized de novo from tryptophan. Interestingly, the knockdown of EWS-FLI1 in ES cells comes along with increased TDO and suppressed KMO expression, both being critical enzymes in tryptophan metabolism and NAD+ de novo synthesis. As a consequence, cellular tryptophan consumption drastically increases, which might indicate a regulatory function of EWS-FLI1 in maintaining the balance of the cellular NAD+ metabolism. In addition, we observed a striking sensitivity of ES cells to interferon gamma treatment at very low doses (5U/ml) resulting in the induction of the tryptophan metabolizing enzyme IDO and the production of the immune-suppressive metabolite kynurenine. We are currently studying how these changes in tryptophan metabolism affect cellular NAD+ pools and SIRT1 and PARP1 activities in ES. This study serves for a better understanding of EWS-FLI1 and of the microenvironment in the post-translational control of SIRT1 and PARP1 regulated gene transcription, and the consequences for PARP1 and/or SIRT1 directed therapies of ES. Supported by the Austrian Science fund, grant I1225-B19. Citation Format: Cornelia N. Mutz, Jozef Ban, Stephan Niedan, Maximilian O. Kauer, Dave N.T. Aryee, Dietmar Fuchs, Andreas Heitger, Heinrich Kovar. Investigating the NAD metabolome 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 1436. doi:10.1158/1538-7445.AM2014-1436

Abstract PR10: Inhibition of deacetylase SIRT1 offers a novel treatment option in metastatic Ewing sarcoma

Metastasis is the major cause of disease-related death in Ewing sarcoma. Patients, who present with clinically overt disseminated disease at diagnosis and those who relapse early with distant metastases have a poor outcome despite multi-modal high-dose chemotherapy. Therefore, new treatment options are highly warranted. Ewing sarcoma pathogenesis is driven by the chimeric ETS oncogene EWS-FLI1. We here describe regulation of sirtuin SIRT1 by EWS-FLI1 and its role in metastasis. SIRT1 belongs to a family of NAD+-dependent group III deacetylases that target histone and non-histone proteins in response to metabolic stress resulting in widespread gene expression changes through epigenetic and direct transcriptional mechanisms. Among SIRT1 target proteins is the tumor suppressor p53. SIRT1 overexpression prevents p53 acetylation, which is otherwise required for the induction of its transcriptional activity. Also, SIRT1 is involved in negative regulation of several signaling pathways including NOTCH that we had previously described as being inactivated in Ewing sarcoma by the EWS-FLI1 fusion oncogene. Studying the molecular basis of EWS-FLI1 mediated functional p53 perturbation in Ewing sarcoma, we here describe a feed-back regulatory loop in which the NOTCH effector HEY1, which is epigenetically repressed by SIRT1, in turn suppresses SIRT1 expression leading to histone H4K16 and H3K56 acetylation and to p53 activation, and consequently to Ewing sarcoma cell death in vitro. By immunohistochemistry, the study of almost 400 Ewing sarcoma patient samples revealed high SIRT1 expression in about one third of primary tumors and a highly significant association of SIRT1 positivity with Ewing sarcoma metastases. The analysis of 18 paired primary tumor/metastasis samples indicated that SIRT1 was more frequently observed in lung metastases (88%) than in bone marrow metastases (55%), and was already detectable in the corresponding primary tumors in more than 60% of cases. In vitro treatment of Ewing sarcoma cell lines with a pharmacological SIRT1/2 inhibitor resulted in cell death with IC50 values between 0.8 and 3.5 μmol that depended on SIRT1 expression level and p53 status. The greatest sensitivity was obtained with cells that express high SIRT1 and are wildtype for p53. Consistent with this in vitro result, SIRT1 inhibition prohibited tumor cell migration and proliferation in a zebrafish yolk sac xenotransplantation model of a SIRT1 positive, wildtype p53 Ewing sarcoma cell line, but not of a mutant p53 cell line with low SIRT1 expression. Together, these results suggest that SIRT1 expression, which is enabled by EWS-FLI1 mediated suppression of the NOTCH signaling pathway resulting in p53 inactivation, may be therapeutically targeted to fight metastasis using pharmacological sirtuin inhibitors. Supported by EU-FP7 grant 259348 (“ASSET”) and by the Austrian Research Fund FWF, ERA-NET grant I1225-B19 (“PROVABES”). This abstract is also presented as Poster A38. Citation Format: Jozef Ban, Argyrou Fourtouna, Isidro Machado, Stephan Niedan, Ewa Snaar-Jagalska, Dave NT Aryee, Maximilian Kauer, Marco Alberghini, Adrienne Flanagan, Katia Scotlandi, Sandra J. Strauss, Elisabeth R. Lawlor, Antonio Llombart-Bosch, Heinrich Kovar. Inhibition of deacetylase SIRT1 offers a novel treatment option in metastatic Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR10.