NUT-midline carcinoma (NMC) is a fatal cancer affecting both children and adults, with an average survival time of only nine months. The hallmark is a translocation between the bromodomain gene BRD4 and the testis-specific gene NUT, but our understanding of other cellular features is limited. Although extremely rare, our laboratory has generated cell lines from three NMC patients, an important resource for the investigation of the biology of this disease and the search for improved therapies. Using next-generation transcriptome sequencing, and an in-house bioinformatic algorithm FusionFinder, we have identified novel BRD4-NUT translocation breakpoints in these lines which give rise to altered protein structures and which therefore have implications for the potential design of targeted therapies for NMC. Knockdown of expressed fusions resulted in decreased proliferation, increased cell size and expression of cytokeratins consistent with the restoration of epithelial differentiation, demonstrating that the novel fusions are central to the oncogenic mechanism in these cells.Genomic PCR indicated that in each NMC line the translocation event fuses an intron of BRD4 to a region upstream of the NUT coding sequence. Thus the generation of BRD4-NUT fusion transcripts through post-translocation RNA-splicing appears to be a common feature of these carcinomas that has not previously been appreciated, with the mechanism facilitating the expression of alternative isoforms of the fusion. Drug screening in these lines has begun to evaluate the relative cytotoxicity of existing chemotherapeutic agents in NMC, and has identified the CDK9 inhibitor flavopiridol as a promising novel therapy. This study contributes to our understanding of the genetic and biological diversity of NMC, an important step towards finding effective treatment strategies for an orphaned disease that is refractory to current therapy approaches.