Identifying Agents which Restored BRM expression

识别恢复 BRM 表达的药物

• 批准号: 7780209
• 负责人: DAVID N REISMAN
• 金额: $ 30.4万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-03 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7780209
• 关键词: Acetylation; Affect; Agonist; Binding Sites; Biological Assay; Cancer cell line; Catalytic Domain; Cell Cycle Arrest; Cell Cycle Regulation; Cell Line; Cells; Chemicals; Clinical; Complex; Control Animal; Data; Development; Dexamethasone; Gene Expression; Gene Silencing; Genes; Genetic Polymorphism; Genomics; Glucocorticoid Receptor; Goals; Grant; Growth; HDAC3 gene; Histones; Human; In Vitro; Incidence; Knockout Mice; Knowledge; Lead; Link; Luc Gene; Luciferases; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Michigan; Mouse Mammary Tumor Virus; Mutate; Odds Ratio; Output; Pathway interactions; Patients; Pharmaceutical Preparations; Play; Primary Neoplasm; Process; Promoter Regions; Property; Proteins; Reagent; Recruitment Activity; Reporter; Research; Retinoblastoma Protein; Retinoids; Role; Running; Screening procedure; Series; Site; Specificity; Testing; Therapeutic; Tissues; Tumor Suppressor Proteins; Universities; Work; base; cancer cell; cancer risk; cell growth; design; drug discovery; high throughput screening; in vitro Assay; inhibitor/antagonist; knock-down; novel; promoter; public health relevance; research study; response; restoration; senescence; transcription factor; tumor; tumor progression

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.

描述（由申请人提供）：BRM是主基因调节复合体SWI/SNF的亚基。该复合体控制多种基因的表达，并在生长控制、分化和发育中发挥关键作用。由于这些作用，BRM 表达在多种人类癌症中经常被靶向和破坏也就不足为奇了。当 BRM 在这些癌症中被沉默时，它并没有发生突变或改变，而是在表观遗传上被沉默。因此，在临床上有可能在缺乏 BRM 表达的癌症中恢复 BRM 表达。 BRM 在缺乏 BRM 表达的癌细胞系中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。为了进一步推进 BRM 作为靶向治疗，我们必须了解癌细胞中 BRM 表达如何被沉默。部分答案在于位于 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癌细胞系和原发性肿瘤中 BRM 表达的丧失密切相关，并且还与肺癌风险密切相关，优势比为 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 MEF2 结合位点即使不相同，也是高度相似的。为此，我们发现 MEF2D 或 HDAC3 的敲低会诱导 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。看来这些 BRM 多态性位点的功能是吸引 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，我们开发了一种测定方法来识别可以恢复 BRM 表达的化合物。这笔资助的主要重点是调整该检测方法，使其可用于高通量筛选：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大约 150,000 种化合物，以鉴定那些能够恢复 BRM 表达的化合物。然后，我们将通过一系列辅助筛选重新筛选这些潜在的点击，以识别真正的点击。然后我们将确定哪些化合物通过靶向 MEF2D 或 HDAC3 来激活 BRM。拟议的研究将进一步确定 BRM 如何被抑制、BRM 启动子多态性在 BRM 抑制中发挥的作用，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为将 BRM 恢复为可行的临床靶向治疗提供必要的先决知识。公共健康相关性：本研究的重点是开发高通量测定法，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发挥着重要作用，因此需要此类药物。