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Supplementary Figures S1-S5. MDM4 modulation by HEY1 occurs downstream of TP53 (S1); Modulation of SIRT1 and TP53 induction upon ectopic expression of Flag-tagged HEY1 in the B-cell malignancy cell lines "697" and NALM6, and in primary keratinocytes (S2); The degree of SIRT1 modulation depends on HEY1 levels (S3); Flow-diagram of samples analysed on tissue micro arrays (TMA1, TMA2, TMA3), and FFPE sections used in this study (S4); Kaplan-Meier survival estimates for patients analysed for SIRT1 expression on TMA3 (S5).

Supplementary Tables S1-S4. Identification of HEY1 regulated genes downstream of EWS-FLI1 silencing in Ewing sarcoma (S1); Combined immunohistochemical staining results for samples from patients with metastases at diagnosis on TMA1 and 2 according to site of metastasis (S2); Clinical characteristics and SIRT1 staining results of primary tumors from 43 ES patients treated at St. Jude Children´s Hospital between 1979 and 1987 using vincristine, doxorubicin, and cyclophosphamide-based regimens, with subsequent addition of ifosfamide and etoposide (S3); Materials used in the study (S4).

Supplementary Figure Legends. Legend for Supplementary Figures S1-S5.

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. Cancer Res; 74(22); 6578–88. ©2014 AACR.

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.

Besides its important role in organismal development, NOTCH receptor signaling exerts tissue specific proliferative or antiproliferative functions. While the oncogenic role of NOTCH has been extensively investigated due to its constitutive activation in T-cell leukemias and several epithelial cancers, little is known about NOTCH mediated tumor suppression. We have previously reported that in Ewing's sarcoma, a pediatric bone tumor driven by the chimeric ETS oncogene EWS-FLI1, auto-stimulatory NOTCH signaling is suppressed and that reactivation results in p53 and consequently growth inhibitory p21 induction via activation of the NOTCH effector HEY1. We now demonstrate that HEY1-mediated p53 stimulation is accompanied by C-terminal p53 acetylation as a consequence of downregulation of nuclear deacetylase sirtuin 1 (SIRT1). We found that both EWS-FLI1 and HEY1 bind to the SIRT1 promoter with opposite transcriptional consequences. Thus, knockdown of EWS-FLI1 and ectopic HEY1 expression resulted in similar SIRT1 modulation and p53 acetylation which could be reversed by ectopically expressed SIRT1. Consistent with these results, treatment of Ewing's sarcoma cell lines with the sirtuin inhibitor Tenovin 6 resulted in massive cell death. Immunohistochemical analysis of more than 310 Ewing's sarcoma samples identified moderate (25%-50% positive nuclei) to strong (>50% positive nuclei) SIRT1 expression in 35% of cases. Additional 20% showed sporadic positivity (between 10-25% positive nuclei). The involvement of SIRT1 in tumor suppressive NOTCH signaling is not restricted to Ewing's sarcoma, but also relevant at least to B-cell malignancies and some normal tissues, since we found that in several B-cell leukemia and lymphoma cell lines and in keratinocytes HEY1 was able to lead to activating p53 acetylation in a SIRT1 suppression-dependent manner. Supported by grants from the Austrian Science Fund (P22328-B09) and the European Comission (EU-FP7 STREP 259348). 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 1190. doi:1538-7445.AM2012-1190

Notch can act as an oncogene or as a tumor suppressor and thus can either promote or inhibit tumor cell growth. To establish Notch status in Ewing's sarcoma family of tumors (ESFT), we investigated the Notch pathway by gene expression profiling meta-analysis or immunohistochemistry in samples obtained from 96 and 24 ESFT patients, respectively. We find that although Notch receptors were highly expressed, Notch did not appear to be active as evidenced by the absence of Notch receptors in cell nuclei. High expression of the Notch effector HES1 transcription factor, usually used as a surrogate marker for active Notch, was also restricted to outside of the nucleus in the majority of tumors, and analysis of HES1 gene targets indicated HES1 to be transcriptionally inactive. Neither forced activation nor pharmacological or genetic blocking of Notch affected HES1 expression in ESFT cells, indicating HES1 expression to be uncoupled from the Notch pathway. Additional functional studies in ESFT cell lines confirmed Notch to be switched off. Finally, unlike experiments in which HES1 expression was modulated, experimental activation of Notch in ESFT cell lines via several means blocked cell proliferation. These indicate that HES1 is uncoupled from Notch in ESFT, that EWS-FLI1-mediated inhibition of Notch contributes to ESFT aggressive cell growth, and supports a role for Notch in ESFT tumor suppression, at least partly through the Notch effector HEY1. 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 1936. doi:10.1158/1538-7445.AM2011-1936