ATP-Dependent Chromatin Remodeling in Human Malignancy

描述（由申请人提供）：最近对人类恶性肿瘤中驱动突变的筛选已经反复鉴定出哺乳动物SWI/SNF样BAF复合物的亚基作为肿瘤抑制因子。生物化学研究表明，所鉴定的亚基：BAF 250 a、Brg（BAF 190）、BAF 155、BAF 60 b、BAF 53 a和BAF 47（hSNF 5）专用于这些复合物，而不是作为单独的蛋白质或作为其他复合物的一部分被发现。此外，我们最近发现，SS 18（滑膜肉瘤突变），Bcl 7和Bcl 11似乎是专用的，稳定的BAF复合物亚基。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突变染色质调节因子之一。BAF复合物调节染色质结构，由约14个亚基组成，这些亚基由编码亚基的基因家族的产物组合组装而成。其在癌症中频繁突变的机制尚不清楚。我们已经发现，致癌亚基的条件性缺失或耗竭导致有丝分裂停滞和后期桥形成，强烈暗示在M期DNA解链失败。重要的是，拓扑异构酶IIa（Topo IIa），其在M期分解链式DNA，与BAF复合物缔合，并且Brg对于Topo IIa结合染色质是必需的。此外，纯化的BAF复合物需要通过纯化的Topo IIa在体外进行最佳脱链。这些观察结果表明，Topo IIa的去连环化失败有助于人类癌症的发生，这是本申请的中心假设。这一假设得到了其他基因中的高频率并发突变或BAF亚基中携带明显起始突变的肿瘤中的非整倍体的支持。为了研究这些致癌BAF突变，SS 18易位到SSX是特别有用的，因为它产生SS 18 BAF亚基与位于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SSX的78 aa连接到SS 18上，并且几乎可以肯定是滑膜肉瘤（SS）中的驱动事件，其占肉瘤的约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。我们将开始我们的研究，确定是否SS 18-SSX融合导致BAF复合物的功能的损失或增益。我们将定义BAF复合物结合基因组的这种易位的后果，以确定靶基因是丢失还是获得。然后，我们将确定为什么野生型SS 18等位基因被抑制，并导致形成很少的蛋白质相比，易位的等位基因。我们将研究潜在的致癌作用拓扑IIa的依赖BAF复合物。最后，我们将试图了解α-连环蛋白和PI 3 K中的激活突变如何与BAF亚基中的功能丧失突变协同作用导致癌症。在我们的工作结束时，我们期望已经深入了解BAF复合物亚基突变的转化机制，BAF复合物亚基突变正在成为人类癌症的主要贡献者。 公共卫生相关性：最近的研究发现，许多人类肿瘤，包括常见的脑、肺、乳腺、卵巢和肾肿瘤，在一种复合物的亚单位内存在突变，该复合物使用ATP提供的能量来控制细胞核中DNA的包装和解包装。这些BAF复合物揭示了在发育的特定时间和特定组织中表达的基因。目前，对这些复合物在人体细胞中的功能只有初步的了解。此外，还不知道突变如何影响复合物的正常功能以及它们如何导致细胞恶性化。我们的研究将揭示肿瘤形成的分子机制，并为控制特定癌症开辟新的治疗策略。

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.