Pathogenesis and Treatment of NUT-Midline Carcinoma

描述(由申请人提供)：本项目的重点是一种罕见的、高度侵袭性和不可治愈的鳞状细胞癌亚型，从基因上由NUT基因重排定义，并被称为中线癌(NMC)。所附提案的目标是1)确定NMC的发病机制和2)有效的治疗。NMC的特点是具有单一的核型，涉及NUT基因与含有溴域的BET家族的成员，最常见的是BRD3或BRD4。由此产生的BRD-NUT融合蛋白是导致这种癌症的原因，它阻止分化并保持NMC细胞处于永久增殖状态。几项证据使我们建立了一个模型，BRD-NUT通过将转录所需的染色质“编写者”，如组蛋白乙酰转移酶(HATS)，从分化所需的基因隔离到有限的基因组区域，在那里BRD的溴域“读取”并激活促生长、反分化基因的转录，从而阻止分化。事实上，乙酰组蛋白结合溴域(Beti)或组蛋白去乙酰基酶的药物抑制剂解除了BRD-NUT对分化的阻断，导致NMC分化和增殖受阻。 细胞在体外和体内。这两种方法都已用于患者的治疗。为了支持上述模型，最近的研究结果表明，BRD4-NUT需要上调常见的癌症基因MYC的转录，并可以取代BRD4-NUT的功能来阻止分化。第一个目的是确定BRD4-NUT导致NMC细胞MYC依赖的分化抑制的分子机制，将通过分离和测序BRD4-NUT相关的基因，并与这些基因在促进NMC细胞分化的条件下发生的表达变化相关联来实现。在这些研究中确定的BRD4-NUT的基因靶点将与全基因组siRNA敲除获得的结果相关联，这将识别阻止NMC细胞分化所需的特定基因的表达。在根据通径分析确定了一系列关键目标基因并确定其优先顺序后，将对它们进行测试 对MYC蛋白和RNA水平的影响，以确定哪些BRD4-NUT靶基因需要上调MYC水平。此外，还将通过质谱学鉴定含有BRD4-NUT的蛋白质复合体，以确定与BRD4-NUT协同上调MYC的蛋白质。第二个目标是探索性目标，即识别与BRD4-NUT合作阻止NMC细胞分化和维持其生长的NMC细胞中的突变，将通过对大约10个NMC肿瘤的所有编码基因进行测序来实现。这一目的也是为了确定有助于BRD4-NUT致癌活性的突变蛋白，这些突变蛋白可以作为小分子抑制剂治疗NMC的靶点。PHS 398/2590(09/04版)页面续格式页面

DESCRIPTION (provided by applicant): The focus of this project is a rare, highly aggressive, and incurable subtype of squamous cell carcinoma, genetically defined by rearrangement of the NUT gene, and termed midline carcinoma (NMC). The goals of the enclosed proposal are 1) to determine the pathogenesis and 2) effective therapy of NMC. NMC is characterized by a simple karyotype harboring a single translocation involving the NUT gene with members of the bromodomain-containing BET family, most commonly BRD3 or BRD4. The resultant BRD- NUT fusion protein is causative in this cancer, acting to block differentiation and maintain NMC cells in a perpetually proliferative state. Several pieces of evidence have led us formulate a model whereby BRD-NUT blocks differentiation by sequestering chromatin "writers" necessary for transcription, such as histone acetyl- transferases (HATs), away from genes required for differentiation to limited genomic regions where the bromodomains of BRD "read" and activate transcription of pro-growth, anti-differentiative genes. Indeed, drug inhibitors of the acetyl-histone binding bromodomains (BETi) or of histone deacetylases unblock the BRD-NUT induced blockade on differentiation, resulting in differentiation and arrested proliferation of NMC cells in vitro and in vivo. Both approaches have been used therapeutically in patients. In support of the above model, recent findings indicate that the transcriptional upregulation of the common cancer gene, MYC, by BRD4-NUT is required and can replace BRD4-NUT's function to block differentiation. The first aim, to identify the molecular mechanisms by which BRD4-NUT leads to the MYC-dependent blockade of differentiation in NMC cells, will be accomplished by isolating and sequencing genes with which BRD4-NUT associates, and correlating with changes that occur in expression of those genes under conditions which promote differentiation in NMC cells. The gene targets of BRD4-NUT identified in these studies will be correlated with findings acquired from the genome-wide siRNA knockdown, which will identify specific genes whose expression is required for the blockade of differentiation in NMC cells. After a list of key target genes is identified and prioritized based on pathway analysis, they will be tested for their effects on MYC protein and RNA levels to identify which BRD4-NUT target genes are required to upregulate MYC levels. In addition, identification of BRD4-NUT-containing protein complexes by mass-spectrometry will be performed to identify proteins which collaborate with BRD4-NUT to upregulate MYC. The second aim, an exploratory aim, to identify mutations in NMC cells that collaborate with BRD4-NUT in the blockade of differentiation and maintenance of growth of NMC cells, will be accomplished by sequencing all coding genes of approximately ten NMC tumors. The purpose of this aim is also to identify mutant proteins which aid BRD4-NUT oncogenic activity that can be targeted by small molecule inhibitors in the treatment of NMC. PHS 398/2590 (Rev. 09/04) Page Continuation Format Page