ATP-Dependent Chromatin Remodeling in Human Malignancy

描述(申请人提供)：最近对人类恶性肿瘤中驱动突变的筛查已反复鉴定哺乳动物SWI/SNF样BAF复合体的亚单位为肿瘤抑制因子。生化研究表明，已鉴定的BAF250a、Brg(BAF190)、BAF155、BAF60b、BAF53a和BAF47(HSNF5)亚基专用于这些复合体，而不是作为单个蛋白质或其他复合体的一部分。此外，我们最近发现SS18(在滑膜肉瘤中突变)、Bcl7和Bcl11似乎是BAF复合体的专用、稳定的亚基。这些研究表明，SWI/SNF样BAF复合体可能是人类癌症中最常见的突变染色质调节因子之一。BaF复合体调节染色质结构，由大约14个亚基组成，这些亚基是由编码亚基的基因家族的产物组合而成的。它们在癌症中频繁突变的机制尚不清楚。我们发现，致癌亚基的条件性缺失或缺失会导致有丝分裂和后期桥形成的停滞，这强烈地意味着在M期未能破坏DNA。重要的是，拓扑异构酶IIa(Topo IIa)与BAF复合体和Brg结合，在Topo IIa与染色质的结合中起着至关重要的作用。此外，在体外，需要纯化的BAF复合体才能使纯化的Topo IIa达到最佳浓缩效果。这些观察表明，Topo IIa的十年消亡失败有助于人类癌症的发生，这是这一应用的中心假说。这一假说得到了其他基因的高频率突变或携带BAF亚单位明显起始突变的肿瘤的非整倍体的支持。为了研究这些致癌的BAF突变，SS18到SSX的易位特别有用，因为它产生了SS18 BAF亚单位与位于X染色体上的SSX基因家族成员的立体型框内融合。这种精确的易位将78aa的SSX连接到SS18上，几乎可以肯定这是滑膜肉瘤(SS)的驱动事件，滑膜肉瘤约占肉瘤的8%。值得注意的是，一个等位基因的易位导致BAF复合体的部分溶解。我们将从确定SS18-SSX融合是否导致BAF复合体功能丧失或获得开始我们的研究。我们将确定这种易位的BAF复合体结合在基因组上的后果，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少量蛋白质。我们将研究Topo IIa对BAF复合体的依赖是否具有潜在的致癌作用。最后，我们将试图理解激活的α-连环蛋白和PI3K突变显然是如何与BAF亚单位的功能丧失突变一起导致癌症的。在我们的工作结束时，我们希望能够深入了解BAF复合体亚单位突变的转化机制，这些亚单位正在成为人类癌症的主要贡献者。

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.