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Ewing sarcoma is driven by the oncogenic fusion protein, EWS-FLI1, and arises via de-regulation of developmental transcriptional programs. Control of normal developmental transcription programs is governed by epigenetic regulation which, in the case of HOX programs, is dependent on coordinated and reciprocal actions of polycomb (PcG) and trithorax (TrxG) proteins. We recently reported that in Ewing sarcoma posterior HOX genes, in particular HOXD13, are abnormally over-expressed and that the promoters of these genes are marked with the TrxG-dependent activating histone modification, H3K4me3. H3K4me3 is deposited by the MLL methyltransferase, a key enzyme that is frequently mutated in leukemia, largely as a result of chromosomal translocations that induce the creation of oncogenic MLL fusion-proteins. In leukemia, MLL fusion-proteins cooperate with wild-type MLL and the scaffolding protein menin to induce malignant transformation via deregulation of HOXA genes. The purpose of this study was to test the hypothesis that MLL and menin contribute to tumorigenesis and to posterior HOX gene deregulation in Ewing sarcoma. Gene and protein expression were determined by microarray, qRT-PCR, western blot and immunohistochemistry. Loss of function was achieved by lentiviral shRNAs and by exposing cells to MI-503, a small molecule inhibitor of menin-MLL protein-protein interactions. Changes in gene and protein expression after MI-503 treatment were assessed by qRT-PCR and western blot. Chromatin immunoprecipitation (ChIP) was used to assess binding of MLL and menin at gene promoters. Our results confirm that MLL, menin and HOXD13 are all highly expressed by Ewing sarcoma tumors and cell lines relative to non-malignant tissues and stem cells, and loss of function studies implicate each as a tumor promoting oncogene. Specifically, knockdown of MLL, menin or HOXD13 resulted in reduced cell proliferation, increased death and/or reduced tumorigenic capacity, as determined by anchorage-independent growth in soft agar and subcutaneous tumor formation in vivo. At a molecular level, knockdown of MLL and menin led to reduced expression of HOXD13, implicating these proteins as key mediators of HOXD13 over-expression. Exposure of Ewing sarcoma cells to MI-503 inhibited proliferation and colony formation in soft agar, and resulted in reduced expression of HOXD13 as well as other posterior HOX genes. In contrast, MI-NC, a control compound with similar structure that lacks affinity for the menin-MLL interaction had no effect on cell growth, viability or HOX gene expression. Significantly, MI-503-treated cells also showed a marked reduction in EWS-FLI1 and wild-type EWSR1 expression, both of which are regulated by the EWSR1 promoter, and of EZH2, a well-established oncogene in Ewing sarcoma. ChIP studies of the EWSR1 and EZH2 promoters demonstrated reduced binding of MLL and menin in MI-503-treated cells. These data demonstrate a key role for MLL and menin in Ewing sarcoma pathogenesis and directly implicate the TrxG complex in regulation of EWS-FLI1, thus highlighting a novel opportunity for therapeutic intervention. Citation Format: Laurie K. Svoboda, Natashay Bailey, Melanie Krook, Raelene Van Noord, Ashley Harris, Rajiv M. Patel, Dafydd Thomas, Tomek Cierpicki, Jolanta Grembecka, Elizabeth R. Lawlor. Tumorigenicity of Ewing sarcoma is critically dependent on the trithorax proteins MLL and menin. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A20.

Normal development is governed by covalent modifications to chromatin that are mediated by the reciprocal actions of Polycomb group (PcG) and Trithorax (TrxG) proteins, which promote gene repression and activation, respectively. We recently reported that HOX genes, key developmental transcription factors that are regulated by PcG and TrxG during embryogenesis, are abnormally expressed in Ewing sarcoma (ES). In particular, we found that the posterior HOXD genes, including HOXD13, are markedly overexpressed, and that the promoters of these genes are aberrantly marked with the TrxG-dependent histone mark, H3K4me3. The TrxG proteins MLL and menin cooperate to drive HOX gene expression and promote tumorigenesis in leukemia. We therefore hypothesized that MLL and menin might also contribute to ES pathogenesis. Gene and protein expression were determined by microarray, q-RT-PCR and western blot of ES cell lines and tumors. Lentiviral shRNA constructs were used to knock down HOXD13, menin and MLL. Proliferation, survival, and tumorigenicity of genetically modified cells were assessed in vitro and in vivo. Inhibition of TrxG function was achieved by exposing cells to MI-503, a small molecule inhibitor of menin-MLL interactions. Our results confirm that menin, MLL and HOXD13 are all highly expressed by ES relative to non-malignant tissues and stem cells. Moreover, loss of function studies implicate each as a tumor promoting oncogene. Specifically, knockdown of HOXD13, menin or MLL inhibited cell proliferation and cell death was also induced by loss of either MLL or HOXD13. Colony formation in soft agar was likewise inhibited by MLL and HOXD13 knockdown and tumor formation in vivo was significantly delayed in ES cells with reduced expression of HOXD13. Importantly, knockdown of MLL led to reduced expression of HOXD13 further implicating MLL as a key mediator of HOXD13 over-expression in ES and supporting the hypothesis that, like MLL-fusion-dependent leukemias, HOX deregulation and tumorigenicity in ES are dependent on the TrxG complex. To further test this hypothesis we exposed ES cells to MI-503, an inhibitor of menin-MLL protein-protein interaction that disrupts the TrxG complex. Exposure of ES cells to MI-503 resulted in a dose-dependent inhibition of cell proliferation and marked inhibition of colony formation in soft agar. In contrast, MI-NC, a control compound with similar structure that lacks affinity for the menin-MLL interaction had no effect on ES cell growth or viability. Together these data for the first time demonstrate a key role for menin and MLL in ES pathogenesis and highlight a novel opportunity for therapeutic intervention in these aggressive tumors. Citation Format: Laurie K. Svoboda, Cassondra Cramer, Ashley Harris, Natashay Bailey, Tomek Cierpicki, Jolanta Grembecka, Elizabeth Lawlor. The Trithorax proteins menin and MLL promote Ewing sarcoma tumorigenicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 490. doi:10.1158/1538-7445.AM2015-490

Disordered control of developmentally regulated polycomb target genes leads to severe abnormalities in embryonic development. Pediatric cancers often arise as a consequence of disordered embryonic development and thus, we hypothesize that disruption of polycomb-regulated transcriptional programs may be a key driver of pediatric tumor initiation. Ewing sarcoma (ES) is a bone and soft tissue tumor that predominantly affects children and young adults. Significantly, established ES constitutively over-express EZH2 and BMI-1 and both of these polycomb proteins have been implicated in ES pathogenesis. ES is initiated by EWS-ETS fusion oncogenes that result from chromosomal translocation events. In the current study we used in vitro models of human neural crest-derived stem cells, ES cell lines and xenografts and established primary ES tumors to define the pattern of polycomb target gene expression in ES and determine whether EWS-FLI1 disrupts normal developmental regulation of polycomb target gene expression during stem cell differentiation. Neuromesenchymal stem cells (NMSC), candidate cells of ES origin, were generated from human embryonic stem cells and then transduced with an EWS-FLI1-EGFP (EF) or EGFP (control) lentivirus. Western blot analysis of transduced cells after six weeks in differentiation media demonstrated persistently high expression of BMI-1 and EZH2 in the EF transduced cells. Comparison of whole genome expression profiles of the cells before and after exposure to differentiation media for six to thirteen weeks revealed that while control cells activated a transcriptional program consistent with mesenchymal differentiation, the mesenchymal program was inhibited in EF cells and a neuronal transcriptional program was instead dominant. Significantly, although expression of polycomb target genes was altered in both conditions following exposure to differentiation media, the pattern of expression was notably divergent in EF cells. In particular, HOX gene expression was found to be reproducibly and globally altered in EF cells. Evaluation of primary ES tumors by gene expression microarrays confirmed global dysregulation of polycomb target gene expression in tumor cells and again revealed a marked variance in HOX gene profiles compared to both normal stem cells as well as normal terminally differentiated adult tissues. Although the HOX gene expression pattern in ES was found to be globally abnormal, HOXD13 was reproducibly highly elevated in all EWS-FLI1+ samples and this was confirmed by RT-PCR of ES cell lines. To determine if abnormal polycomb target gene expression is dependent on EWS-FLI1 in established ES we knocked down EWS-FLI1 in ES cell line xenografts using a doxycline-inducible shRNA. Knockdown of EWS-FLI1 led to marked inhibition of tumor growth and this was associated with downregulation of HOXD13. Studies to further delineate the mechanistic contribution of HOX gene dysregulation to ES pathogenesis are now underway. Together these studies demonstrate that disordered regulation of polycomb target genes is ubiquitous in ES and results from EWS-FLI1-mediated disruption of polycomb-modulated transcriptional programs during stem cell differentiation. In addition, these studies for the first time implicate altered expression of HOX genes in ES tumor initiation and progression. Citation Format: Ashley Harris, Natashay Bailey, Elizabeth R. Lawlor. The EWS-FLI1 oncogene disrupts normal developmental regulation of polycomb-modulated transcriptional programs. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A26.