Identifying Agents which Restored BRM expression

描述(申请人提供)：BRM是主基因调控复合体SWI/SNF的一个亚基。这种复合体控制着多种基因的表达，并在生长控制、分化和发育中发挥关键作用。由于这些作用，BRM的表达在各种人类癌症中经常被靶向和干扰也就不足为奇了。当BRM在这些癌症中沉默时，它不会发生突变或改变，而是在表观遗传学上沉默。因此，在缺乏BRM表达的癌症中恢复BRM表达在临床上是可能的。BRM在缺乏BRM表达的癌细胞株中的重新表达会导致细胞周期停滞和衰老；这一观察表明恢复BRM表达的潜在临床益处。为了进一步推动BRM作为一种靶向治疗，我们必须了解BRM在癌细胞中的表达是如何沉默的。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BRM在癌细胞系和原发肿瘤中的表达缺失密切相关，也与肺癌风险密切相关，优势比为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MEF2D或HDAC3的敲除可以诱导BRM的表达。此外，MEF2转录因子将HDAC招募到启动子区域以沉默基因。似乎这些BRM多态位点的功能是吸引MEF2D，然后MEF2D招募HDAC3，导致BRM基因的沉默。在这个建议中，我们将通过目标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方法来鉴定能够恢复BRM表达的化合物。这笔赠款的主要重点是调整这种检测方法，使其能够用于高通量筛查：AIM1。然后，我们将从密歇根高通量筛选设施和密歇根大学化学基因组学中心筛选约150,000种化合物，以鉴定那些恢复BRM表达的化合物。然后，我们将用一系列次级屏幕重新筛选这些潜在的热门歌曲，以确定真正的热门歌曲。然后我们将确定这些化合物中的哪一种通过靶向MEF2D或HDAC3来激活BRM。这项拟议的研究将进一步确定BRM是如何被抑制的，BRM启动子的多态在抑制BRM中所起的作用，以及HDAC和MEF2D是否通过这些多态来抑制BRM。我们期望，这项工作将提供必要的先决条件知识，以发展BRM的修复作为一种可行的临床靶向治疗。公共卫生相关性：这项研究的重点是开发一种高通过化验来寻找重新激活BRM的药物。由于BRM在细胞周期控制和其他抗癌过程中发挥着重要作用，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.