Epigenetic and clinical impact of SMARCB1 loss in cancer

SMARCB1(hSNF5/INI-1)基因编码INI-1蛋白，INI-1是SWI/SNF染色质的组成部分 重塑综合体。虽然已知SWI/SNF复合体在转录中发挥重要作用 监管，它是如何做到这一点的，在很大程度上是未知的。横纹肌样瘤是一种罕见的高度恶性的肉瘤。 表现出SMARCB1突变和/或缺失的婴儿期。我们最近报道了全球基因 RT的表达模式，并提供证据表明RT在非常早期的祖细胞中产生 SMARCB1缺失直接导致显著抑制SMARCB1基因表达的关键发育窗口 细胞周期蛋白依赖性激酶抑制的分化和缺失。我们提供更多的证据证明这些 这种变化在很大程度上是由组蛋白甲基化的变化所介导的。在这个提案中，我们测试了我们的假设 早期祖细胞中SMARCB1的丢失导致整体不能释放介导的多梳基团 H3K27me3抑制二价基因，导致发育停滞和异常增殖。 目的1：在hESC中敲除SMARCB1，并测量细胞增殖的变化。 与三种RT细胞系进行分化、基因表达和DNA甲基化的比较。我们将利用 RNA干扰在两个hESC中敲除SMARCB1并测量增殖的变化，EZH2 表达、分化以及RNA和DNA甲基化的变化。这些功能将与那些功能进行比较 在三个RT细胞系中。这些研究中的全球RNA和DNA甲基化分析将与组蛋白进行比较 全局CHIP-SEQ检测到的RT中的修改不在本提案范围内执行。 目的2：研究EZH2的药理抑制对细胞的直接和间接影响 SMARCB1基因缺陷细胞在体内外的增殖。EZH2代表了最重要的 多梳状蛋白在人类中存在，并已被证明在大量肿瘤中上调。它是 因此，这是一个有吸引力的治疗靶点。我们已经与葛兰素史克公司达成协议， 为临床前开发做好准备的有前景的EZH2抑制剂的研究。我们将在体外进行细胞毒性试验 对三个RT细胞系的检测，以及对由相同细胞系创建的异种移植的体内研究。这两个 在我们有能力进行早期临床试验之前，需要进行拟议的体外和体内研究。 相关性：鉴于总存活率为23%，RT迫切需要新的治疗方案。如果我们的 假设是正确的，它可能会为RTS和许多其他肿瘤带来新的治疗靶点 组蛋白修饰或EZH2过表达。此外，它还将推出一种独特而重要的模式 表观遗传学对早期胚胎分化和肿瘤发展的控制。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学相互作用的潜在机制 机械装置。这将使表观遗传调控的研究能够更广泛地在不同的 患者群体、发育阶段和疾病。

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 develop 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 for 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.