Pathogenesis and Treatment of NUT-Midline Carcinoma

描述（由申请人提供）：该项目的重点是一种罕见的，高度侵袭性的，不可治愈的鳞状细胞癌亚型，由NUT基因重排遗传定义，称为中线癌（NMC）。所附建议的目标是1）确定NMC的发病机制和2）有效治疗。 NMC的特征在于具有涉及NUT基因与含溴结构域的BET家族成员（最常见的是BRD 3或BRD 4）的单个易位的简单核型。所得的BRD-NUT融合蛋白在这种癌症中是致病的，其作用是阻断分化并将NMC细胞维持在永久增殖状态。几个证据已经引导我们制定了一个模型，其中BRD-NUT通过隔离转录所需的染色质“写入者”（如组蛋白乙酰转移酶（HAT））来阻断分化，使其远离分化所需的基因，以限制基因组区域，在该区域中BRD的溴结构域“读取”并激活促生长、抗分化基因的转录。实际上，乙酰基-组蛋白结合溴结构域（BETi）或组蛋白脱乙酰基酶的药物抑制剂解除了BRD-NUT诱导的对分化的阻断，导致NMC的分化和增殖停滞 细胞在体外和体内。这两种方法都已用于治疗患者。 为了支持上述模型，最近的研究结果表明，BRD 4-NUT对常见癌症基因MYC的转录上调是必需的，并且可以取代BRD 4-NUT阻断分化的功能。 第一个目的是鉴定BRD 4-NUT导致NMC细胞中MYC依赖性分化阻断的分子机制，这将通过分离和测序与BRD 4-NUT相关的基因，并与在促进NMC细胞分化的条件下这些基因表达中发生的变化相关联来实现。在这些研究中鉴定的BRD 4-NUT的基因靶标将与从全基因组siRNA敲低获得的发现相关，这将鉴定其表达是阻断NMC细胞中的分化所需的特定基因。在基于途径分析确定关键靶基因的列表并进行优先排序后，将测试它们的 对MYC蛋白和RNA水平的影响，以鉴定上调MYC水平所需的BRD 4-NUT靶基因。此外，将通过质谱法鉴定含BRD 4-NUT的蛋白质复合物，以鉴定与BRD 4-NUT协同上调MYC的蛋白质。 第二个目的是探索性目的，即鉴定与BRD 4-NUT合作阻断NMC细胞分化和维持NMC细胞生长的NMC细胞突变，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。该目的的目的还在于鉴定有助于BRD 4-NUT致癌活性的突变蛋白，其可以在NMC的治疗中被小分子抑制剂靶向。PHS 398/2590（Rev.09/04）

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