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// Laurie K. Svoboda 1 , Natashay Bailey 1 , Raelene A. Van Noord 2 , Melanie A. Krook 1 , Ashley Harris 1 , Cassondra Cramer 1 , Brooke Jasman 1 , Rajiv M. Patel 3, 4 , Dafydd Thomas 3 , Dmitry Borkin 3 , Tomasz Cierpicki 3 , Jolanta Grembecka 3 , Elizabeth R. Lawlor 1, 3 1 Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, MI 48109, USA 2 Department of Surgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA 3 Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA 4 Department of Dermatology, University of Michigan Medical School, Ann Arbor, MI 48109, USA Correspondence to: Elizabeth R. Lawlor, email: elawlor@med.umich.edu Keywords: Ewing sarcoma, trithorax, MLL, menin, HOX Received: August 15, 2016 Accepted: November 14, 2016 Published: November 18, 2016 ABSTRACT Developmental transcription programs are epigenetically regulated by the competing actions of polycomb and trithorax (TrxG) protein complexes, which repress and activate genes, respectively. Ewing sarcoma is a developmental tumor that is associated with widespread de-regulation of developmental transcription programs, including HOX programs. Posterior HOXD genes are abnormally over-expressed by Ewing sarcoma and HOXD13 , in particular, contributes to the tumorigenic phenotype. In MLL1 fusion-driven leukemia, aberrant activation of HOXA genes is epigenetically mediated by the TrxG complex and HOXA gene expression and leukemogenesis are critically dependent on the protein-protein interaction between the TrxG proteins MLL1 and menin. Based on these data, we investigated whether posterior HOXD gene activation and Ewing sarcoma tumorigenicity are similarly mediated by and dependent on MLL1 and/or menin. Our findings demonstrate that Ewing sarcomas express high levels of both MLL1 and menin and that continued expression of both proteins is required for maintenance of tumorigenicity. In addition, exposure of Ewing sarcoma cells to MI-503, an inhibitor of the MLL1-menin protein-protein interaction developed for MLL1-fusion driven leukemia, leads to loss of tumorigenicity and down-regulated expression of the posterior HOXD gene cluster. Together these data demonstrate an essential role for MLL1 and menin in mediating tumor maintenance and posterior HOXD gene activation in Ewing sarcoma. A critical dependency of these tumors on the MLL1-menin interaction presents a potentially novel therapeutic target.

The polycomb proteins BMI-1 and EZH2 are highly overexpressed by Ewing sarcoma (ES), a tumor of stem cell origin that is driven by EWS-ETS fusion oncogenes, most commonly EWS-FLI1. In the current study we analyzed expression of transcription programs that are controlled by polycomb proteins during embryonic development to determine if they are abnormal in ES. Our results show that polycomb target gene expression in ES deviates from normal tissues and stem cells and that, as expected, most targets are relatively repressed. However, we also discovered a paradoxical up regulation of numerous polycomb targets and these were highly enriched for homeobox (HOX) genes. Comparison of HOX profiles between malignant and non-malignant tissues revealed a distinctive HOX profile in ES, which was characterized by overexpression of posterior HOXD genes. In addition, ectopic expression of EWS-FLI1 during stem cell differentiation led to aberrant up regulation of posterior HOXD genes. Mechanistically, this up regulation was associated with altered epigenetic regulation. Specifically, ES and EWS-FLI1+ stem cells displayed a relative loss of polycomb-dependent H3K27me3 and gain of trithorax-dependent H3K4me3 at the promoters of posterior HOXD genes and also at the HOXD11.12 polycomb response element. In addition, a striking correlation was evident between HOXD13 and other genes whose regulation is coordinately regulated during embryonic development by distal enhancer elements. Together, these studies demonstrate that epigenetic regulation of polycomb target genes, in particular HOXD genes, is altered in ES and that these changes are mediated downstream of EWS-FLI1.

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.

Background: Ewing sarcoma (ES) is the second most common malignant bone tumor in pediatric patients. While intensification of chemotherapy has improved the outlook for patients with localized disease, little progress has been made for those with metastatic or relapsed disease. Prognosis for these patients remains dismal with fewer than 20% surviving. Moving forward, we need to develop molecularly targeted therapy for patients with high-risk disease. The mechanisms underlying metastatic relapse in ES remain unknown but gene encoding the chemokine receptor CXCR4 has been shown to be upregulated in metastatic ES. The CXCL12-CXCR4 axis contributes to tumor metastasis in other sarcomas but its function in ES is not yet known. Objective: In the current study we are investigating whether CXCR4 is a mediator of ES cell metastasis and may be useful as a biomarker of high-risk disease. Results: Using qRT-PCR and flow cytometry we have shown that expression of CXCR4 transcript and protein varies significantly across ES cell lines. Importantly, the frequency of CXCR4+ cells and CXCR4 expression levels were found to be highly dynamic and were altered under different experimental conditions. Specifically, both serum deprivation and hypoxia induced upregulation of CXCR4 in TC32, TC71, and A673 ES cells. CXCR4+ cell frequency was found to be consistently highest in TC32 where the frequency ranged from 35% in ambient conditions to 62% in serum deprivation. When sorted into CXCR4+ and CXCR4- populations TC32 populations returned to their baseline state of 40% CXCR4+ cells within 3 weeks indicating that CXCR4+ cells can change into CXCR4- cells and vice versa. Interestingly, CXCR4 transcript was identified to be increased in slowly proliferating (PKH-dye-retaining) TC71 and A673 cells compared to more rapidly dividing counterparts. These data implicate CXCR4 as a potential marker of putative cancer stem cells. Finally, initial immunohistochemical studies have demonstrated highly variable patterns of CXCR4 expression in primary ES tumor samples. Summary: ES cells express CXCR4 both in primary tumors and in cultured cell lines. However, the expression is both variable and highly dynamic. Specifically, we have found that ES cells can switch back and forth between CXCR4- and CXCR4+ states in response to environmental cues including serum deprivation and hypoxia. Ongoing work is now testing the hypothesis that CXCR4+ ES cells are more migratory than CXCR4- cells and may thus be a source of metastasis. To further test this hypothesis we are using correlative studies of clinically annotated tumor microarrays to determine if a high frequency of CXCR4+ cells at diagnosis is associated with an increased risk of metastatic relapse. Citation Format: Melanie A. Krook, Lauren A. Nicholls, Natashay J. Bailey, Amy Han, Chris Scannell, Dafydd Thomas, Elizabeth R. Lawlor. Elucidating the role of CXCR4 in Ewing sarcoma. [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 5032. doi:10.1158/1538-7445.AM2013-5032