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，高度侵袭性的，无法治愈的鳞状细胞癌亚型，由NUT基因重排定义，称为中线癌（NMC）。所附建议的目的是1)确定NMC的发病机制和2)有效的治疗方法。NMC的特征是一个简单的核型，包含一个涉及NUT基因与含溴结构域的BET家族成员的单一易位，最常见的是BRD3或BRD4。由此产生的BRD- NUT融合蛋白在这种癌症中是致病的，其作用是阻止NMC细胞分化并维持其永久增殖状态。一些证据使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”来阻止分化，例如组蛋白乙酰转移酶（HATs），远离分化所需的基因到有限的基因组区域，BRD的溴结构域“读取”并激活促生长，抗分化基因的转录。事实上，乙酰组蛋白结合溴结构域（BETi）或组蛋白去乙酰化酶的药物抑制剂解除了BRD-NUT诱导的分化阻断，导致NMC分化和增殖受阻

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