title: 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.
authors.affiliation: Division of Children's Leukaemia and Cancer Research, Telethon Institute for Child Health Research, University of Western Australia Centre for Child Health Research, Perth, Australia.
authors.forename: K
authors.initials: K
authors.lastname: Thompson-Wicking
authors.forename: R W
authors.initials: RW
authors.lastname: Francis
authors.forename: A
authors.initials: A
authors.lastname: Stirnweiss
authors.forename: E
authors.initials: E
authors.lastname: Ferrari
authors.forename: M D
authors.initials: MD
authors.lastname: Welch
authors.forename: E
authors.initials: E
authors.lastname: Baker
authors.forename: A R
authors.initials: AR
authors.lastname: Murch
authors.forename: A M
authors.initials: AM
authors.lastname: Gout
authors.forename: K W
authors.initials: KW
authors.lastname: Carter
authors.forename: A K
authors.initials: AK
authors.lastname: Charles
authors.forename: M B
authors.initials: MB
authors.lastname: Phillips
authors.forename: U R
authors.initials: UR
authors.lastname: Kees
authors.forename: A H
authors.initials: AH
authors.lastname: Beesley
mesh_terms: D000293:Adolescent; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D001483:Base Sequence; D002277:Carcinoma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D002454:Cell Differentiation; D045744:Cell Line, Tumor / Q000378:metabolism / Q000648:ultrastructure; D048429:Cell Size; D002648:Child; D002884:Chromosomes, Human, Pair 15 / Q000235:genetics* / Q000648:ultrastructure; D002888:Chromosomes, Human, Pair 19 / Q000235:genetics* / Q000648:ultrastructure; D019008:Drug Resistance, Neoplasm; D005091:Exons / Q000235:genetics; D017809:Fatal Outcome; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D015514:Oncogene Proteins, Fusion / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D020033:Protein Isoforms / Q000235:genetics / Q000502:physiology; D017433:Protein Structure, Secondary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013953:Thymus Neoplasms / Q000235:genetics* / Q000473:pathology; D014178:Translocation, Genetic; D055815:Young Adult
chemical_list: C471990:BRD4-NUT fusion oncogene protein, human; D009687:Nuclear Proteins; D015514:Oncogene Proteins, Fusion; D020033:Protein Isoforms; D034741:RNA, Small Interfering