ATP-Dependent Chromatin Remodeling in Human Malignancy

描述（由申请人提供）：最近对人类恶性肿瘤驱动突变的筛选已经反复发现哺乳动物SWI/ snf样BAF复合物亚基作为肿瘤抑制因子。生化研究表明，鉴定出的亚基：BAF250a、Brg （BAF190）、BAF155、BAF60b、BAF53a和BAF47 （hSNF5）是这些复合物专用的，而不是作为单个蛋白或其他复合物的一部分发现的。此外，我们最近发现SS18（在滑膜肉瘤中突变）、Bcl7和Bcl11似乎是BAF复合物专用的、稳定的亚基。这些研究表明SWI/ snf样BAF复合物可能是人类癌症中最常见的染色质突变调节因子之一。BAF复合物调节染色质结构，由大约14个亚基组成，这些亚基是由编码这些亚基的基因家族的产物组合而成。它们在癌症中频繁突变的机制尚不清楚。我们发现，致癌亚基的条件缺失或耗尽导致有丝分裂和后期桥形成的停滞，强烈暗示在M期DNA失decatenate的失败。重要的是，拓扑异构酶IIa （Topo IIa）在M期分解与BAF复合物和Brg相关的链状DNA，是Topo IIa结合染色质所必需的。此外，纯化的BAF配合物需要在体外通过纯化的Topo IIa实现最佳的十烷二烯化。这些观察结果表明，Topo IIa的十烷化失败有助于人类癌症的发生，这是该应用的中心假设。这一假设得到了其他基因的高频率同步突变或肿瘤的非整倍体的支持，这些肿瘤在BAF亚基中具有明显的起始突变。为了研究这些致癌的BAF突变，SS18易位到SSX是特别有用的，因为它产生了SS18 BAF亚基与位于X染色体上的SSX基因家族成员的刻板框架内融合。这种精确的易位将78aa的SSX附着在SS18上，几乎可以肯定是滑膜肉瘤（SS）的驱动事件，约占肉瘤的8%。值得注意的是，一个等位基因的易位导致BAF复合物的部分溶解。我们将通过确定SS18-SSX融合是否导致BAF复合物功能的丧失或获得来开始我们的研究。我们将定义BAF复合体在基因组上结合的这种易位的后果，以确定目标基因是丢失还是获得。然后，我们将确定为什么野生型SS18等位基因被抑制，并导致与易位等位基因相比形成很少的蛋白质。我们将研究Topo IIa依赖BAF复合物的潜在致癌作用。最后，我们将试图了解a- catenin和PI3K的激活突变如何明显地与BAF亚基的功能丧失突变合作，从而导致癌症。在我们的工作结束时，我们希望能够深入了解BAF复合物亚基突变的转化机制，这是人类癌症的主要原因。

DESCRIPTION (provided by applicant): Recent screens for driving mutations in human malignancy have repeatedly identified subunits of mammalian SWI/SNF-like BAF complexes as tumor suppressors. Biochemical studies indicate that the identified subunits: BAF250a, Brg (BAF190), BAF155, BAF60b, BAF53a, and BAF47 (hSNF5) are dedicated to these complexes and not found as individual proteins or as parts of other complexes. In addition, we have recently found that SS18 (mutated in Synovial Sarcoma), Bcl7 and Bcl11 appear to be dedicated, stable subunits of BAF complexes. These studies suggest that SWI/SNF-like BAF complexes might be one of the most commonly mutated chromatin regulators in human cancer. BAF complexes regulate chromatin structure and are composed of about 14 subunits that are combinatorially assembled from the products of gene families encoding the subunits. The mechanisms underlying their frequent mutation in cancer are unclear. We have found that conditional deletion or depletion of the oncogenic subunits leads to stalling in mitosis and anaphase bridge formation, strongly implicating a failure to decatenate DNA during M phase. Importantly, Topoisomerase IIa (Topo IIa), which resolves catenated DNA at M phase associates with BAF complexes and Brg is essential for chromatin binding by Topo IIa. Furthermore, purified BAF complexes are required for optimal decatenation by purified Topo IIa in vitro. These observations suggest that a failure of decatenation by Topo IIa contributes to the genesis of human cancers, which is the central hypothesis of this application. This hypothesis is supported by the high frequency of concurrent mutations in other genes or aneuploidy in tumors bearing apparent initiating mutations in BAF subunits. To study these oncogenic BAF mutations, the SS18 translocation to SSX is particularly useful since it produces a sterotypic in-frame fusion of the SS18 BAF subunit to a member of the SSX gene family located on the X chromosome. This precise translocation attaches 78aa of SSX to SS18 and is almost certainly the driving event in synovial sarcoma (SS), which account for about 8% of sarcomas. Remarkably, the translocation of one allele leads to partial dissolution of BAF complexes. We will begin our studies by determining whether the SS18-SSX fusion causes a loss or gain of function for BAF complexes. We will define the consequences of this translocation for BAF complex binding over the genome to determine if target genes are lost or gained. We will then determine why the wildtype SS18 allele is repressed and leads to the formation of little protein compared to the translocated allele. We will examine the potentially oncogenic role of Topo IIa's dependence upon BAF complexes. Finally, we will attempt to understand how activating mutations in a- catenin and PI3K apparently cooperate with loss-of-function mutations in BAF subunits to lead to cancer. At the conclusion of our work we expect to have gained insight into the mechanism of transformation by mutations of the subunits of BAF complexes, which are emerging as major contributors to human cancer. PUBLIC HEALTH RELEVANCE: Recent studies have found that many human tumors, including common tumors of the brain, lung, breast, ovary and kidney harbor mutations within the subunits of a complex that uses energy provided by ATP to control the packaging and unpackaging of DNA in the nucleus. These BAF complexes unveil genes for their expression at specific times in development and in specific tissues. At present there is only rudimentary knowledge of the function of these complexes in human cells. Furthermore, it is not known how the mutations affect the normal functions of the complexes and how they cause cells to become malignant. Our studies will shed light upon the molecular mechanisms involved in tumor formation and open new therapeutic strategies for control of specific cancers.