Identifying Agents which Restored BRM expression

描述（由申请人提供）：BRM是主基因调控复合体SWI/SNF的一个亚基。该复合物控制着多种基因的表达，在生长控制、分化和发育中起着关键作用。由于这些作用，在各种人类癌症中，BRM的表达经常被靶向和破坏就不足为奇了。当BRM在这些癌症中沉默时，它不会发生突变或改变，而是在表观遗传上沉默。因此，在临床上恢复缺乏BRM表达的肿瘤的表达是可能的。BRM在缺乏表达的癌细胞中重新表达导致细胞周期停滞和衰老；这一观察结果表明恢复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在癌细胞中的表达是如何被沉默的。部分答案在于BRM基因启动子区域的两个多态位点。这两个多态位点的存在与肿瘤细胞系和原发肿瘤中BRM表达的缺失密切相关，也与肺癌风险密切相关，比值比为2.2。因此，了解这些多态位点的功能对于确定BRM在癌症中是如何沉默的至关重要。此外，对这些位点的分析表明，它们与MEF2结合位点即使不完全相同，也非常相似。为此，我们发现敲低MEF2D或HDAC3可诱导BRM表达。此外，MEF2转录因子将hdac招募到启动子区域以沉默基因。这些BRM多态位点的功能似乎是吸引MEF2D，然后MEF2D招募HDAC3，导致BRM基因沉默。在本提案中，我们将通过Aim 2中的一系列实验来验证这一假设。作为开发BRM激活药物的第一步，我们已经开发了一种检测方法来鉴定可以恢复BRM表达的化合物。这笔拨款的主要重点是调整这种检测方法，使其可以用于高通量筛选：Aim1。然后，我们将筛选来自密歇根高通量筛选设施和密歇根大学化学基因组学中心的化合物，大约150,000个化合物，以确定那些恢复BRM表达的化合物。然后，我们将通过一系列的二次筛选来重新筛选这些潜在的命中目标，以确定真正的命中目标。然后，我们将确定哪些化合物通过靶向MEF2D或HDAC3激活BRM。该研究将进一步确定BRM是如何被抑制的，BRM启动子多态性在BRM抑制中的作用，以及HDAC和MEF2D是否通过这些多态性抑制BRM。我们期望这项工作将提供必要的前提知识，以发展BRM的恢复作为一种可行的临床靶向治疗。

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.