Regulation and function of HOX genes in Ewing sarcoma pathogenesis

项目总结 劫持正常发育程序是肿瘤发生和表观遗传的常见机制 解除对发育转录程序的管制是大多数(如果不是全部)儿科疾病发生的核心 癌症。尤文肉瘤，侵袭性骨和软组织肿瘤，主要发生在青春期， 仍然与高死亡率有关，需要新的治疗方法。尤因 肉瘤的特征是存在致病的驱动融合癌基因，最常见的是 EWS/FLI1，其细胞来源可能为间充质干细胞/祖细胞(MSC)。EWS/FLI1启动 由于基因改变导致转录调控的广泛中断而导致肿瘤的发生 招募染色质重构体作为基因增强子和启动子的靶标。我们已经报告了后部 Hoxd基因，特别是Hoxd13，在尤因肉瘤和后遗症中异常过度表达 HOXD基因有助于维持肿瘤的形成表型。我们的初步研究表明 EWS/FLI1可以细胞上下文依赖的方式诱导Hoxd13的表达，并且这种激活是 由发育促进剂的异常激活所介导，否则这些促进剂只在非常离散的情况下活跃 时空发育窗口。此外，染色质免疫沉淀(CHIP)研究 在EWS/FLI1+细胞中，Hoxd13启动子优先富含MLL- 依赖于激活组蛋白修饰H3K4me3，并与MLL和Menin蛋白结合。值得注意的是， 尤文肉瘤细胞对MLL：Menin相互作用的新型小分子抑制剂的作用 在MLL融合阳性白血病的临床前发展中，导致显著的致瘤性丧失和 伴随的后遗症HOXD基因表达缺失。因此，这些研究表明，就像MLL-融合一样 阳性白血病，尤文肉瘤致癌表型的维持严重依赖于 MLL：发育中HOX基因的依赖于MENIN的激活。在这个提案中，我们将检验这一假设 Hoxd13在尤文肉瘤中发挥专有协作性癌基因及其对其依赖性 持续表达呈现一种以前未被识别的肿瘤特异性易感性，可 在治疗上被剥削。在Aim 1中，将使用创新的Bru-seq技术和功能验证研究来 确定促进肿瘤发生的Hoxd13下游转录靶点。在《目标2》中，我们将使用 靶向芯片分析和染色质构象研究以确定EWS/FLI1如何导致表观遗传学 Hoxd13的激活。在目标3中，我们将测试MI-503的治疗潜力，MI-503是一种小分子抑制物 MLL：Menin Protein：蛋白质相互作用，体内肿瘤模型。这些研究将共同阐明 Hoxd13在尤文肉瘤发病机制中的作用及一类新的表观遗传学的可能性 尤文肉瘤靶向治疗的改良剂。

PROJECT SUMMARY Hijacking of normal developmental programs is a common mechanism of tumorigenesis and epigenetic deregulation of developmental transcription programs is central to the genesis of most, if not all, pediatric cancers. Ewing sarcomas, aggressive bone and soft tissue tumors that predominately arise in adolescence, continue to be associated with high rates of mortality and novel approaches to therapy are needed. Ewing sarcomas are characterized by the presence of pathognomonic driver fusion oncogenes, most commonly EWS/FLI1, and their likely cellular origin is mesenchymal stem/progenitor cells (MSC). EWS/FLI1 initiates tumorigenesis by inducing widespread disruption of transcriptional regulation as a consequence of altered recruitment of chromatin remodelers to target gene enhancers and promoters. We have reported that posterior HOXD genes, in particular HOXD13, are aberrantly over-expressed by Ewing sarcoma and that posterior HOXD genes contribute to maintenance of the tumorigenic phenotype. Our preliminary studies suggest that EWS/FLI1 can induce expression of HOXD13, in a cell context dependent fashion, and that this activation is mediated by aberrant activation of developmental enhancers that are otherwise active in only very discrete spatiotemporal developmental windows. In addition, chromatin immunoprecipitation (ChIP) studies have demonstrated that in EWS/FLI1+ cells the HOXD13 promoter is preferentially enriched with the MLL- dependent activating histone modification H3K4me3 and bound by both MLL and menin proteins. Significantly, exposure of Ewing sarcoma cells to novel small molecule inhibitors of MLL:menin interaction, that are currently in preclinical development for MLL-fusion positive leukemias, leads to a dramatic loss tumorigenicity and concomitant loss posterior HOXD gene expression. Thus, these studies demonstrate that that, like MLL-fusion positive leukemias, maintenance of the oncogenic phenotype of Ewing sarcoma is critically dependent on MLL:menin-dependent activation of developmental HOX genes. In this proposal, we will test the hypothesis that HOXD13 functions an obligate cooperative oncogene in Ewing sarcoma and that dependency on its continued expression presents a previously unrecognized tumor-specific vulnerability that can be therapeutically exploited. In Aim 1 will use innovative Bru-seq technologies and functional validation studies to define the downstream transcriptional targets of HOXD13 that promote tumorigenicity. In Aim 2 we will use targeted ChIP assays and chromatin conformation studies to determine how EWS/FLI1 leads to epigenetic activation of HOXD13. In Aim 3 we will test the therapeutic potential of MI-503, a small molecule inhibitor of MLL:menin protein:protein interaction, in in vivo tumor models. These studies will together elucidate the contribution of HOXD13 to Ewing sarcoma pathogenesis and test the potential of a new class of epigenetic modifying agents for Ewing sarcoma-targeted therapies.