Pathogenesis of Malignant Mesothelioma by the Human Polycomb Complex BAP1-ASXL

描述(由申请人提供)：恶性间皮瘤(MM)是一种与石棉接触有关的侵袭性、抗药性癌症。多发性骨髓瘤的遗传学基础历来集中于CDKN2A和NF2的体细胞突变，它们是影响发病和进展的关键改变。最近，BAP1泛素羧基末端水解酶首次以CH.3p21抑癌基因(Rauscher Lab)在1998年被认为是多发性骨髓瘤的主要致病基因。在MM和其他癌症高发的两个家系中发现了胚系BAP1突变，在MMS中发生了体细胞BAP1变化，这与肿瘤抑制基因(Testa Lab)的双等位基因失活一致。此外，体细胞BAP1突变在散发性MMS以及散发性和家族性葡萄膜黑色素瘤(UM)中都很常见。BAP1突变诱发MM和UM的遗传和生化机制以及BAP1如何与CDKN2A和NF2基因相互作用影响MM的病理、预后和治疗反应在很大程度上尚不清楚。然而，BAP1编码一种核定位的去泛素酶，它与ASXL1/2结合，ASXL1/2是一种专有的以组蛋白为靶标的多梳家庭伙伴蛋白，这一事实强烈表明，BAP1在基因转录的表观遗传调控中发挥了作用。此外，由于BAP1与ASXL1/2的结合是酶活性和肿瘤抑制所必需的，因此灭活BAP1抑癌活性的另一种方法可能是突变或沉默ASXL1/2基因。为了确定MM中BAP1、ASXL1/2、CDKN2A和NF2之间的生化机制和遗传相互作用，Rauscher和Testa实验室联手追求以下具体目标：1)使用体内直接遗传方法来确定杂合BAP1突变(+/mut)小鼠是否易患包括MM在内的各种自发肿瘤，并加速石棉诱导的MM。2)使用条件基因敲除小鼠来确定仅在间皮细胞中BAP1的躯体丢失是否能够驱动间皮细胞增生和/或坦率的MM，如果体细胞BAP1失活结合NF2和CDKN2a的缺失导致更快的发展和/或更具侵袭性的疾病表型，具有潜在的预后和新的治疗意义。3)确定人多发性骨髓瘤标本/细胞系中BAP1和ASXL1/2突变的谱，并确定这些突变如何影响BAP1与ASXL1/2和其他PR-DUB复合体蛋白的结合和功能，并确定由于MM细胞中BAP1和其他PR-DUB组分的改变(通过敲除和重组)而改变的转录组和细胞表型。4)测定间皮细胞和BAP1和/或ASXL1/2缺陷的MM细胞中含有野生型BAP1和ASXL1/2的天然PR-DUB大分子蛋白复合体的组成，以比较正常和肿瘤特异性亚基结构以及这些复合体在表观遗传失调中的作用。由两个具有互补专业知识的PI进行的拟议多管齐下的研究代表了一种全面的方法，以产生对MM发病机制的新见解，同时为治疗和预防提供新的靶点。

DESCRIPTION (provided by applicant): Malignant mesothelioma (MM) is an aggressive, treatment-resistant cancer linked to asbestos exposure. The genetic basis for MM has historically focused on somatic mutations of CDKN2A and NF2 as key alterations influencing initiation and progression. Very recently, the BAP1 ubiquitin carboxy-terminal hydrolase, first isolated as a Ch. 3p21 tumor suppressor in 1998 (Rauscher Lab), has been strongly implicated as a major player in MM based on genetic analyses. Germline BAP1 mutations were found in two families with a high incidence of MM and other cancers, and somatic BAP1 alterations occurred in MMs, consistent with biallelic inactivation of a tumor suppressor (Testa Lab). Moreover, somatic BAP1 mutations are common in sporadic MMs and in both sporadic and familial uveal melanoma (UM). The genetic and biochemical mechanisms by which BAP1 mutations predispose to MM and UM and how BAP1 interacts genetically with CDKN2A and NF2 to influence MM pathology, prognosis, and therapeutic response are largely unknown. However, the fact that BAP1 encodes a nuclear-localized de-ubiquitinase that binds to ASXL1/2, an obligate polycomb family partner protein that targets histones, strongly suggests a role in epigenetic regulation of gene transcription. Moreover, since binding of BAP1 to ASXL1/2 is required for enzyme activity and tumor suppression, an alternative way to inactivate BAP1 tumor suppressor activity may be to mutate or silence ASXL1/2 genes. To define both the biochemical mechanisms and the genetic interactions among BAP1, ASXL1/2, CDKN2A and NF2 in MM, the Rauscher and Testa Labs have joined forces to pursue the following Specific Aims: 1) Use a direct in vivo genetic approach to determine if heterozygous Bap1-mutant (+/mut) mice are predisposed to the development of various spontaneous tumors, including MM, and accelerate asbestos-induced MM. 2) Use conditional knockout mice to determine if somatic loss of Bap1 alone in the mesothelium is capable of driving mesothelial hyperplasia and/or frank MM, and if somatic Bap1 inactivation combined with loss of Nf2 and Cdkn2a results in more rapid development and/or a more aggressive disease phenotype, with potential prognostic and novel therapeutic implications. 3) Define the spectrum of BAP1 and ASXL1/2 mutations in human MM specimens/cell lines and determine how these mutations affect binding and function of BAP1 in complex with ASXL1/2 and other PR-DUB complex proteins, and define the transcriptome and cellular phenotype which are altered as a consequence of changes (via knockdown and reconstitution) in BAP1 and other PR-DUB components in MM cells. 4) Determine the composition of the native PR-DUB macromolecular protein complexes that contain wild- type BAP1 and ASXL1/2 in mesothelial cells and in BAP1- and/or ASXL1/2-deficient MM cells to compare normal- and tumor-specific subunit structure and the role of these complexes in epigenetic dysregulation. The proposed multipronged studies by two PIs with complementary expertise represent a comprehensive approach to yield novel insights into MM pathogenesis while providing new targets for therapy and prevention.