Identifying Agents which Restored BRM expression

描述（申请人提供）：BRM是主基因调节复合物SWI/SNF的亚基。该复合体控制多种基因的表达，并在生长控制、分化和发育中起关键作用。由于这些作用，BRM表达在各种人类癌症中经常被靶向和破坏也就不足为奇了。当BRM在这些癌症中沉默时，它不会突变或改变，而是表观遗传沉默。因此，在临床上有可能恢复缺乏其表达的癌症中的BRM表达。BRM在缺乏其表达的癌细胞系中的再表达导致细胞周期停滞和衰老;该观察结果表明恢复BRM表达的潜在临床益处。为了进一步推进BRM作为靶向治疗，我们必须了解BRM表达如何在癌细胞中沉默。部分答案在于BRM基因启动子区域的两个多态性位点。这两个多态性位点的存在与两种癌细胞系和原发性肿瘤中BRM表达的丧失密切相关，并且也与肺癌风险密切相关，比值比为2.2。因此，了解这些多态性位点如何发挥作用对于确定BRM如何在癌症中沉默至关重要。此外，对这些位点的分析表明，它们与MEF 2结合位点高度相似（如果不相同的话）。为此，我们发现MEF 2D或HDAC 3的敲低诱导BRM表达。此外，MEF 2转录因子将HDAC募集到启动子区域以沉默基因。似乎这些BRM多态性位点的功能是吸引MEF 2D，然后招募HDAC 3，导致BRM基因沉默。在本提案中，我们将通过目标2中的一系列实验来检验这一假设。作为开发BRM激活药物的第一步，我们已经开发了一种鉴定可以恢复BRM表达的化合物的测定法。这项资助的主要重点是调整这种检测方法，使其可用于高通量筛选：Aim 1。然后，我们将从密歇根州高通量筛选设施和密歇根大学化学基因组学中心筛选化合物，约15万种化合物，以确定那些恢复BRM表达的化合物。然后，我们将重新筛选这些潜在的命中与一系列的二级屏幕，以确定真正的命中。然后，我们将确定这些化合物中的哪一种通过靶向MEF 2D或HDAC 3激活BRM。这项研究将进一步确定BRM是如何被抑制的，BRM启动子多态性在抑制BRM中所起的作用，以及HDAC和MEF 2D是否通过这些多态性抑制BRM。我们希望这项工作将提供必要的先决条件知识，以开发BRM作为一种可行的临床靶向治疗的恢复。公共卫生相关性：本研究的重点是开发一种高穿透试验，以寻找重新激活BRM的药物。由于BRM在细胞周期控制和其他抗癌过程中起着重要作用，因此需要此类药物。

DESCRIPTION (provided by applicant): BRM is a subunit of the master gene-regulating complex SWI/SNF. This complex controls the expression of a wide variety of genes and plays a critical role in growth control, differentiation, and development. Because of these roles, it is not surprising that BRM expression is frequently targeted and disrupted in a variety of human cancers. When BRM is silenced in these cancers, it is not mutated or altered, but rather it is epigenetically silenced. Hence it is clinically possible to restore BRM expression in cancers that lack its expression. The re- expression of BRM in cancer cell lines devoid of its expression results in cell cycle arrest and senescence; this observation indicates the potential clinical benefit of restoring BRM expression. To further advance BRM as a targeted therapy, we must understand how BRM expression is silenced in cancer cells. Part of the answer lies with two polymorphic sites that lie in the promoter region of the BRM gene. The presence of these two polymorphic sites strongly correlates with the loss of BRM expression in both cancer cell lines and in primary tumors and is also strongly associated with lung cancer risk, with an odds ratio of 2.2. Hence, understanding how these polymorphic sites function is central to determining how BRM is silenced in cancer. Furthermore, analysis of the sites shows that they are highly similar if not identical to the MEF2 binding sites. To this end, we have found that knockdown of MEF2D or HDAC3 induces BRM expression. Moreover, MEF2 transcription factors recruit HDACs to the promoter region in order to silence genes. It appears that these BRM polymorphic sites function to attract MEF2D, which then recruits HDAC3, resulting in the silencing of the BRM gene. In this proposal, we will test this hypothesis with a series of experiments in Aim 2. As a first step in developing BRM- activating drugs, we have developed an assay to identify compounds that can restore BRM expression. The main focus of this grant is to adapt this assay so it can be used for high throughput screening: Aim1. We will then screen compounds from both the Michigan High Throughput Screening Facility and the University of Michigan Center for Chemical Genomics, approximately 150,000 compounds, to identify those that restore BRM expression. We will then rescreen these potential hits with series of secondary screens to identify true hits. We will then determine which of these compounds activate BRM by targeting either MEF2D or HDAC3. The proposed research will further determine how BRM is suppressed, the roles that BRM promoter polymorphisms play in the suppression of BRM, and if HDAC and MEF2D inhibit BRM via these polymorphisms. We expect that this work will give the prerequisite knowledge necessary to develop the restoration of BRM as a viable clinically targeted therapy. PUBLIC HEALTH RELEVANCE: This research is focused of the development of a High Through Assay to find drugs which reactivate BRM. Such drugs are needed because of the important roles that BRM plays in cell cycle control and other anticancer process.