Abstract 490: The Trithorax proteins menin and MLL promote Ewing sarcoma tumorigenicity

Abstract

Abstract 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