BRD-NUT oncoproteins: A family of closely related nuclear proteins that block epithelial differentiation and maintain the growth of carcinoma cells

Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
Oncogene (Impact Factor: 8.46). 05/2008; 27(15):2237-42. DOI: 10.1038/sj.onc.1210852
Source: PubMed


An unusual group of carcinomas, here termed nuclear protein in testis (NUT) midline carcinomas (NMC), are characterized by translocations that involve NUT, a novel gene on chromosome 15. In about 2/3rds of cases, NUT is fused to BRD4 on chromosome 19. Using a candidate gene approach, we identified two NMCs harboring novel rearrangements that result in the fusion of NUT to BRD3 on chromosome 9. The BRD3-NUT fusion gene encodes a protein composed of two tandem chromatin-binding bromodomains, an extra-terminal domain, a bipartite nuclear localization sequence, and almost the entirety of NUT that is highly homologous to BRD4-NUT. The function of NUT is unknown, but here we show that NUT contains nuclear localization and export sequences that promote nuclear-cytoplasmic shuttling via a leptomycin-sensitive pathway. In contrast, BRD3-NUT and BRD4-NUT are strictly nuclear, implying that the BRD moiety retains NUT in the nucleus via interactions with chromatin. Consistent with this idea, FRAP studies show that BRD4, BRD4-NUT and BRD3-NUT have significantly slower rates of lateral nuclear diffusion than that of NUT. To investigate the functional role of BRD-NUT fusion proteins in NMCs, we investigated the effects of siRNA-induced BRD3-NUT and BRD4-NUT withdrawal. Silencing of these proteins in NMC cell lines resulted in squamous differentiation and cell cycle arrest. Together, these data suggest that BRD-NUT fusion proteins contribute to carcinogenesis by associating with chromatin and interfering with epithelial differentiation.

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Available from: Jeffery L Kutok, Feb 12, 2014
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    • "The resulting cell lines were maintained in medium (above) supplemented with blasticidin (Invitrogen) and hygromycin (Sigma). pEGFPC1-BRD4-NUT was created as described (French et al. 2008) and transfected into 293T cells using Lipofectamine 2000 according to the manufacturer's instructions (Life Technologies). "
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    ABSTRACT: NUT midline carcinoma (NMC), a subtype of squamous cell cancer, is one of the most aggressive human solid malignancies known. NMC is driven by the creation of a translocation oncoprotein, BRD4-NUT, which blocks differentiation and drives growth of NMC cells. BRD4-NUT forms distinctive nuclear foci in patient tumors, which we found correlate with ∼100 unprecedented, hyperacetylated expanses of chromatin that reach up to 2 Mb in size. These "megadomains" appear to be the result of aberrant, feed-forward loops of acetylation and binding of acetylated histones that drive transcription of underlying DNA in NMC patient cells and naïve cells induced to express BRD4-NUT. Megadomain locations are typically cell lineage-specific; however, the cMYC and TP63 regions are targeted in all NMCs tested and play functional roles in tumor growth. Megadomains appear to originate from select pre-existing enhancers that progressively broaden but are ultimately delimited by topologically associating domain (TAD) boundaries. Therefore, our findings establish a basis for understanding the powerful role played by large-scale chromatin organization in normal and aberrant lineage-specific gene transcription. © 2015 Alekseyenko et al.; Published by Cold Spring Harbor Laboratory Press.
    Genes & Development 07/2015; 29(14):1507-1523. DOI:10.1101/gad.267583.115 · 10.80 Impact Factor
    • "Several recent studies establish that BRD4 is preferentially located at super-enhancer regions, which often reside upstream of important oncogenes such as c-myc, bcl-xL, and bcl-6, and play a key role in regulating their expressions (Chapuy et al., 2013; Loven et al., 2013). Owing to its pivotal role in modulating the expression of essential oncogenes, BRD4 has emerged as a promising therapeutic target in multiple cancer types, including midline carcinoma , acute myeloid leukemia (AML), multiple myeloma (MM), Burkitt's lymphoma (BL), and prostate cancer (Asangani et al., 2014; Delmore et al., 2011; French et al., 2008; Loven et al., 2013; Mertz et al., 2011; Wyce et al., 2013; Zuber et al., 2011). "
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    ABSTRACT: BRD4, a bromodomain and extraterminal domain (BET) family member, is an attractive target in multiple pathological settings, particularly cancer. While BRD4 inhibitors have shown some promise in MYC-driven malignancies such as Burkitt's lymphoma (BL), we show that BRD4 inhibitors lead to robust BRD4 protein accumulation, which may account for their limited suppression of MYC expression, modest antiproliferative activity, and lack of apoptotic induction. To address these limitations we designed ARV-825, a hetero-bifunctional PROTAC (Proteolysis Targeting Chimera) that recruits BRD4 to the E3 ubiquitin ligase cereblon, leading to fast, efficient, and prolonged degradation of BRD4 in all BL cell lines tested. Consequently, ARV-825 more effectively suppresses c-MYC levels and downstream signaling than small-molecule BRD4 inhibitors, resulting in more effective cell proliferation inhibition and apoptosis induction in BL. Our findings provide strong evidence that cereblon-based PROTACs provide a better and more efficient strategy in targeting BRD4 than traditional small-molecule inhibitors. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Chemistry & biology 06/2015; 22(6). DOI:10.1016/j.chembiol.2015.05.009 · 6.65 Impact Factor
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    • "In addition, bromodomain-containing proteins have been implicated in a wide range of diseases. Overexpression of numerous family members has been reported in a variety of cancers [2-6], and translocations of BRD3 or BRD4 with NUT and CREBBP with MLL, MOZ or MORF have been observed in NUT midline carcinoma [7] and acute myeloid and lymphoblastic leukaemia [8], respectively. Bromodomain-containing proteins have also been implicated in proinflammatory processes as well as in a number of neurological diseases [2,3]. "
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    ABSTRACT: Background Acetylation of lysine residues in histone tails plays an important role in the regulation of gene transcription. Bromdomains are the readers of acetylated histone marks, and, consequently, bromodomain-containing proteins have a variety of chromatin-related functions. Moreover, they are increasingly being recognised as important mediators of a wide range of diseases. The first potent and selective bromodomain inhibitors are beginning to be described, but the diverse or unknown functions of bromodomain-containing proteins present challenges to systematically demonstrating cellular efficacy and selectivity for these inhibitors. Here we assess the viability of fluorescence recovery after photobleaching (FRAP) assays as a target agnostic method for the direct visualisation of an on-target effect of bromodomain inhibitors in living cells. Results Mutation of a conserved asparagine crucial for binding to acetylated lysines in the bromodomains of BRD3, BRD4 and TRIM24 all resulted in reduction of FRAP recovery times, indicating loss of or significantly reduced binding to acetylated chromatin, as did the addition of known inhibitors. Significant differences between wild type and bromodomain mutants for ATAD2, BAZ2A, BRD1, BRD7, GCN5L2, SMARCA2 and ZMYND11 required the addition of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) to amplify the binding contribution of the bromodomain. Under these conditions, known inhibitors decreased FRAP recovery times back to mutant control levels. Mutation of the bromodomain did not alter FRAP recovery times for full-length CREBBP, even in the presence of SAHA, indicating that other domains are primarily responsible for anchoring CREBBP to chromatin. However, FRAP assays with multimerised CREBBP bromodomains resulted in a good assay to assess the efficacy of bromodomain inhibitors to this target. The bromodomain and extraterminal protein inhibitor PFI-1 was inactive against other bromodomain targets, demonstrating the specificity of the method. Conclusions Viable FRAP assays were established for 11 representative bromodomain-containing proteins that broadly cover the bromodomain phylogenetic tree. Addition of SAHA can overcome weak binding to chromatin, and the use of tandem bromodomain constructs can eliminate masking effects of other chromatin binding domains. Together, these results demonstrate that FRAP assays offer a potentially pan-bromodomain method for generating cell-based assays, allowing the testing of compounds with respect to cell permeability, on-target efficacy and selectivity.
    Epigenetics & Chromatin 07/2014; 7(1). DOI:10.1186/1756-8935-7-14 · 5.33 Impact Factor
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