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.