Novel BRD4–NUT fusion isoforms increase the pathogenic complexity in NUT midline carcinoma

Abstract

Nuclear protein in testis (NUT)-midline carcinoma (NMC) is a rare, aggressive disease typically presenting with a single t(15;19) translocation that results in the generation of a bromodomain-containing protein 4 (BRD4)-NUT fusion. PER-624 is a cell line generated from an NMC patient with an unusually complex karyotype that gave no initial indication of the involvement of the NUT locus. Analysis of PER-624 next-generation transcriptome sequencing (RNA-Seq) using the algorithm FusionFinder identified a novel transcript in which Exon 15 of BRD4 was fused to Exon 2 of NUT, therefore differing from all published NMC fusion transcripts. The three additional exons contained in the PER-624 fusion encode a series of polyproline repeats, with one predicted to form a helix. In the NMC cell line PER-403, we identified the 'standard' NMC fusion and two novel isoforms. Knockdown by small interfering RNA in either cell line resulted in decreased proliferation, increased cell size and expression of cytokeratins consistent with epithelial differentiation. These data demonstrate that the novel BRD4-NUT fusion in PER-624 encodes a functional protein that is central to the oncogenic mechanism in these cells. Genomic PCR indicated that in both PER-624 and PER-403, the translocation 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. Finally, ectopic expression of wild-type NUT, a protein normally restricted to the testis, could be demonstrated in PER-403, indicating additional pathways for aberrant cell signaling in NMC. This study contributes to our understanding of the genetic diversity of NMC, an important step towards finding therapeutic targets for a disease that is refractory to current treatments.

Association of chromosome 19 to lung cancer genotypes and phenotypes

“…BRD4-NUT is the first fusion oncogene mechanism presenting a poorly differentiated carcinoma with high aggression [54]. Wild-type BRD4 was shown to inhibit G1 to S progression and fusion augments the inhibition of progression to S phase compared with wild-type BRD4, leading to lung cancer cell proliferation [55].…”

NSD3–NUT Fusion Oncoprotein in NUT Midline Carcinoma: Implications for a Novel Oncogenic Mechanism

“…In addition, induction of ET domain expression also negatively affected the proliferation rate of TC-797 cells, whereas the growth of heterologous, non-NMC cells, U2OS or 293T, was unaffected (Figure 3F) The findings indicate that expression of wild type NSD3 protein and the ET domain of BRD4-NUT are required for the blockade of differentiation in BRD4-NUT+ NMC. The specific requirement of the ET domain for the oncogenic function of BRD4-NUT is evidenced by its conservation in all characterized BRD-NUT fusions (1, 2, 12, 18, 19), including uncommon splice variants(16, 18), and by the lack of growth inhibition induced by ET domain expression in non-BRD-NUT-expressing cell lines (Figure 3F). …”

Functional Genomic Screening Reveals Splicing of the EWS-FLI1 Fusion Transcript as a Vulnerability in Ewing Sarcoma

“…This study also has implications for other tumor types dependent upon the expression of a fusion gene for cell survival. For example, as for the EWSR1 exon 8 containing EWS-FLI1 pre-mRNA, there is evidence that the first NUT exon must be spliced out to generate the in-frame BRD4–NUT fusion observed in nuclear protein in testis (NUT)-midline carcinomas harboring t(15;19) translocations (Thompson-Wicking et al, 2013). The application of a functional genomic approach akin to that used in this study has the potential to identify proteins required for specific steps in the maturation of other fusion transcripts and thereby provide new strategies for the treatment of fusion driven tumors.…”