Epigenetic and clinical impact of SMARCB1 loss in cancer

描述（由申请人提供）：SMARCB 1（hSNF 5/INI-1）基因编码INI-1蛋白，是SWI/SNF染色质重塑复合物的一种组分。虽然已知SWI/SNF复合物在转录调控中发挥重要作用，但其如何发挥作用在很大程度上是未知的。 未知横纹肌样瘤（RT）是一种罕见的婴儿高度恶性肉瘤，表现出SMARCB 1的突变和/或缺失。我们最近报道了RT的全球基因表达模式，并提供了证据表明，RT出现在非常早期的祖细胞在一个关键的发育窗口期间，SMARCB 1的损失直接导致显着的抑制分化和细胞周期蛋白依赖性激酶抑制的损失。我们提供了额外的证据表明，这些变化主要是由组蛋白甲基化的变化介导的。在这个提议中，我们测试了我们的假设，即早期祖细胞中SMARCB 1的缺失导致在二价基因上释放polycomb组介导的抑制性H3 K27 me 3的整体失败，导致发育停滞和异常增殖。目标1：在hESC中敲低SMARCB 1，并测量与三种RT细胞系相比细胞增殖、分化、基因表达和DNA甲基化的变化。我们将利用RNA干扰技术在两个hESC中敲低SMARCB 1，并测量增殖，EZH 2表达，分化以及RNA和DNA甲基化变化的变化。这些将与三种RT细胞系中的那些特征进行比较。这些研究中的全局RNA和DNA甲基化分析将与在本提案之外通过全局ChIP-SEQ检测的RT中的组蛋白修饰进行比较。目标二：研究EZH 2的药理学抑制对SMARCB 1缺陷细胞体外和体内细胞增殖的直接和间接影响。EZH 2代表人类中最重要的多梳组蛋白，并且已显示在大量肿瘤中上调。因此，它是一个有吸引力的治疗靶点。我们已经与葛兰素史克达成协议，研究一种有前途的EZH 2抑制剂，该抑制剂已准备好进行临床前开发。我们将进行体外细胞毒性试验 在三种RT细胞系上，以及在从相同细胞系产生的异种移植物上的体内研究。在我们能够开展早期临床试验之前，需要进行体外和体内研究。相关性：总生存率为23%，新的治疗选择是 如果我们的假设是正确的，它可能会导致新的治疗靶点RT和许多其他肿瘤，显示组蛋白修饰或EZH 2过表达。此外，它将介绍一个独特的和重要的模型，早期胚胎分化和肿瘤发展的表观遗传控制。从长远来看，这些研究将有助于确定组蛋白修饰与其他表观遗传机制相互作用的潜在机制。这将使研究表观遗传调控更广泛地在不同的患者群体，发育阶段和疾病。 公共卫生相关性：该项目使用人类肿瘤（横纹肌样肿瘤）作为模型来研究SMARCB 1基因的丢失，该基因是SWI/SNF染色质重塑复合物的关键成员。这与胚胎干细胞分化的最早阶段所涉及的机制以及癌症的发展有关。治疗干预可能有助于治疗儿童横纹肌样瘤以及大量其他显示异常SWI/SNF功能的肿瘤。

DESCRIPTION (provided by applicant): The SMARCB1 (hSNF5/INI-1) gene encodes the INI-1 protein, a component of the SWI/SNF chromatin remodeling complex. While the SWI/SNF complex is known to play an important role in transcriptional regulation, how it does so is largely unknown. Rhabdoid Tumors (RT) are rare, highly malignant sarcomas of infancy that demonstrate mutation and/or deletion of SMARCB1. We recently reported the global gene expression pattern of RT, and provided evidence that RTs arise within very early progenitor cells during a critical developmental window during which loss of SMARCB1 directly results in striking repression of differentiation and loss of cyclin dependent kinase inhibition. We provide additional evidence that these changes are largely mediated by changes in histone methylation. In this proposal we test our hypothesis that loss of SMARCB1 in early progenitor cells results in a global failure to release the polycomb group mediated repressive H3K27me3 on bivalent genes, resulting in arrested development and abnormal proliferation. Aim 1: To knock-down SMARCB1 within hESC and to measure changes in cell proliferation, differentiation, gene expression and DNA methylation compared with three RT cell lines. We will utilize RNA interference to knock-down SMARCB1 in two hESC and measure changes in proliferation, EZH2 expression, differentiation, and RNA and DNA methylation changes. These will be compared to those features in three RT cell lines. Global RNA and DNA methylation analysis in these studies will be compared with histone modifications in RT detected by global ChIP-SEQ being performed outside of this proposal. Aim 2: To investigate the direct and indirect impact of pharmacologic inhibition of EZH2 on cell proliferation in SMARCB1-deficient cells in vitro and in vivo. EZH2 represents the most important polycomb group protein in humans, and has been shown to be upregulated in a large number of tumors. It is therefore an attractive therapeutic target. We have established an agreement with GlaxoSmithKline for the study of a promising EZH2 inhibitor that is ready for preclinical development. We will perform in vitro cytotoxic assays on the three RT cell lines, and in vivo studies on xenografts created from the same cell lines. Both the in vitro and in vivo studies that are proposed are required prior to our ability to develo early clinical trials. Relevance: With an overall survival rate of 23%, new therapeutic options are urgently needed for RT. If our hypothesis is correct, it may result in novel therapeutic targets fo RTs and the many other tumors that show histone modification or EZH2 overexpression. In addition, it will introduce a unique and important model of epigenetic control of both early embryonic differentiation and tumor development. Long-term, these studies will help to identify the underlying mechanisms by which histone modifications interact with other epigenetic mechanisms. This would then enable the investigation of epigenetic regulation more broadly in different patient populations, developmental stages, and diseases. PUBLIC HEALTH RELEVANCE: This project uses a human tumor, rhabdoid tumor, as a model for investigating the loss of the SMARCB1 gene, a key member of the SWI/SNF chromatin remodeling complex. This has relevance to the mechanisms involved in the very earliest stages of differentiation of embryonic stem cells, and to the development of cancer. Therapeutic intervention may contribute to the treatment of children with rhabdoid tumor as well as a large number of other tumors that show aberrant SWI/SNF function.