BRD4

Gene Summary

Gene:BRD4; bromodomain containing 4
Aliases: CAP, MCAP, HUNK1, HUNKI
Location:19p13.12
Summary:The protein encoded by this gene is homologous to the murine protein MCAP, which associates with chromosomes during mitosis, and to the human RING3 protein, a serine/threonine kinase. Each of these proteins contains two bromodomains, a conserved sequence motif which may be involved in chromatin targeting. This gene has been implicated as the chromosome 19 target of translocation t(15;19)(q13;p13.1), which defines an upper respiratory tract carcinoma in young people. Two alternatively spliced transcript variants have been described. [provided by RefSeq, Jul 2008]
Databases:VEGA, OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:bromodomain-containing protein 4
Source:NCBIAccessed: 11 March, 2017

Ontology:

What does this gene/protein do?
Show (17)

Cancer Overview

Research Indicators

Publications Per Year (1992-2017)
Graph generated 11 March 2017 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

Tag cloud generated 11 March, 2017 using data from PubMed, MeSH and CancerIndex

Specific Cancers (4)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: BRD4 (cancer-related)

Andrieu G, Belkina AC, Denis GV
Clinical trials for BET inhibitors run ahead of the science.
Drug Discov Today Technol. 2016; 19:45-50 [PubMed] Article available free on PMC after 21/07/2017 Related Publications
Several cancer clinical trials for small molecule inhibitors of BET bromodomain proteins have been initiated. There is enthusiasm for the anti-proliferative effect of inhibiting BRD4, one of the targets of these inhibitors, which is thought to cooperate with MYC, a long-desired target for cancer therapeutics. However, no current inhibitor is selective for BRD4 among the three somatic BET proteins, which include BRD2 and BRD3; their respective functions are partially overlapping and none are functionally redundant with BRD4. Each BET protein controls distinct transcriptional pathways that are important for functions beyond cancer cell proliferation, including insulin production, cytokine gene transcription, T cell differentiation, adipogenesis and most seriously, active repression of dangerous latent viruses like HIV. BET inhibitors have been shown to reactivate HIV in human cells. Failure to appreciate that at concentrations used, no available BET inhibitor is member-selective, or to develop a sound biological basis to understand the diverse functions of BET proteins before undertaking for these clinical trials is reckless and likely to lead to adverse events. More mechanistic information from new basic science studies should enable proper focus on the most relevant cancers and define the expected side effect profiles.

Wang L, Wu X, Huang P, et al.
JQ1, a small molecule inhibitor of BRD4, suppresses cell growth and invasion in oral squamous cell carcinoma.
Oncol Rep. 2016; 36(4):1989-96 [PubMed] Related Publications
The present study aimed to evaluate whether bromodomain 4 (BRD4) is expressed in Cal27 cells and to assess the effect of JQ1 on cell proliferation, apoptosis, invasion and BRD4, C-Myc and Twist expression in Cal27 cells. Immunofluorescence staining was used to determine whether BRD4 was expressed in Cal27 cells. Cell viability and proliferation were evaluated using CCK-8 assay. Flow cytometry was used to determine the apoptosis and cell cycle distribution. The cell invasion was evaluated using Transwell plate. The expression levels of BRD4, C-Myc and Twist were determined by quantitative RT-PCR (qRT-PCR) and western blotting. BRD4 was highly expressed in Cal27 cells. JQ1 inhibited cell proliferation, induced cell apoptosis, induced cell cycle arrest, and inhibited cell invasion. Gene and protein expression levels of BRD4, C-Myc and Twist were downregulated in cells treated with JQ1. JQ1 inhibited Cal27 cell growth and invasion, and downregulated expression of several oncogenes. JQ1 may be a new drug for oral squamous cell carcinoma treatment.

Klijanienko J, Le Tourneau C, Rodriguez J, et al.
Cytological features of NUT midline carcinoma arising in sino-nasal tract and parotid gland: Report of two new cases and review of the literature.
Diagn Cytopathol. 2016; 44(9):753-6 [PubMed] Related Publications
Nuclear Protein in Testis (NUT) Midline Carcinoma (NMC) represents a recently described, uncommon, high-grade and extremely lethal malignancy mainly occurring in children and young adults. Such tumors are genetically characterized by chromosomal rearrangements of the NUT gene. Cytological description of NUT carcinoma is limited and only seven cases were reported up to date. We show here another two cases studied cytologically with molecular and immunohistochemical confirmation. In both cases smears were hypercellular and composed of isolated or clustered small to medium-sized in size with roundish and oval shape cells. Nuclei were either regular or roundish containing dusty chromatin and prominent nucleoli. Mitotic figures were prominent. Cytoplasm was scant, strongly basophilic. Cell debris, necrosis, and apoptosis were also prominent. One of the cases was studied by FISH and the second case was studied by RT-PCR and BRD4-NUT translocation was found in both cases. Moreover, the clinical evolution was aggressive in both cases with rapid fatal clinical outcome. NUT carcinomas are an underdiagnosed entity which should be taken into consideration when poorly differentiated carcinomas was diagnosed in children or young adults. Cytology material may be successfully used for morphological and molecular diagnosis. Diagn. Cytopathol. 2016;44:753-756. © 2016 Wiley Periodicals, Inc.

Bhagwat AS, Roe JS, Mok BY, et al.
BET Bromodomain Inhibition Releases the Mediator Complex from Select cis-Regulatory Elements.
Cell Rep. 2016; 15(3):519-30 [PubMed] Article available free on PMC after 21/07/2017 Related Publications
The bromodomain and extraterminal (BET) protein BRD4 can physically interact with the Mediator complex, but the relevance of this association to the therapeutic effects of BET inhibitors in cancer is unclear. Here, we show that BET inhibition causes a rapid release of Mediator from a subset of cis-regulatory elements in the genome of acute myeloid leukemia (AML) cells. These sites of Mediator eviction were highly correlated with transcriptional suppression of neighboring genes, which are enriched for targets of the transcription factor MYB and for functions related to leukemogenesis. A shRNA screen of Mediator in AML cells identified the MED12, MED13, MED23, and MED24 subunits as performing a similar regulatory function to BRD4 in this context, including a shared role in sustaining a block in myeloid maturation. These findings suggest that the interaction between BRD4 and Mediator has functional importance for gene-specific transcriptional activation and for AML maintenance.

Chang H, Liu Y, Xue M, et al.
Synergistic action of master transcription factors controls epithelial-to-mesenchymal transition.
Nucleic Acids Res. 2016; 44(6):2514-27 [PubMed] Article available free on PMC after 21/07/2017 Related Publications
Epithelial-to-mesenchymal transition (EMT) is a complex multistep process in which phenotype switches are mediated by a network of transcription factors (TFs). Systematic characterization of all dynamic TFs controlling EMT state transitions, especially for the intermediate partial-EMT state, represents a highly relevant yet largely unexplored task. Here, we performed a computational analysis that integrated time-course EMT transcriptomic data with public cistromic data and identified three synergistic master TFs (ETS2, HNF4A and JUNB) that regulate the transition through the partial-EMT state. Overexpression of these regulators predicted a poor clinical outcome, and their elimination readily abolished TGF-β-induced EMT. Importantly, these factors utilized a clique motif, physically interact and their cumulative binding generally characterized EMT-associated genes. Furthermore, analyses of H3K27ac ChIP-seq data revealed that ETS2, HNF4A and JUNB are associated with super-enhancers and the administration of BRD4 inhibitor readily abolished TGF-β-induced EMT. These findings have implications for systematic discovery of master EMT regulators and super-enhancers as novel targets for controlling metastasis.

Mio C, Lavarone E, Conzatti K, et al.
MCM5 as a target of BET inhibitors in thyroid cancer cells.
Endocr Relat Cancer. 2016; 23(4):335-47 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Anaplastic thyroid carcinoma (ATC) is an extremely aggressive thyroid cancer subtype, refractory to the current medical treatment. Among various epigenetic anticancer drugs, bromodomain and extra-terminal inhibitors (BETis) are considered to be an appealing novel class of compounds. BETi target the bromodomain and extra-terminal of BET proteins that act as regulators of gene transcription, interacting with histone acetyl groups. The goal of this study is to delineate which pathway underlies the biological effects derived from BET inhibition, in order to find new potential therapeutic targets in ATC. We investigated the effects of BET inhibition on two human anaplastic thyroid cancer-derived cell lines (FRO and SW1736). The treatment with two BETis, JQ1 and I-BET762, decreased cell viability, reduced cell cycle S-phase, and determined cell death. In order to find BETi effectors, FRO and SW1736 were subjected to a global transcriptome analysis after JQ1 treatment. A significant portion of deregulated genes belongs to cell cycle regulators. Among them, MCM5 was decreased at both mRNA and protein levels in both tested cell lines. Chromatin immunoprecipitation (ChIP) experiments indicate that MCM5 is directly bound by the BET protein BRD4. MCM5 silencing reduced cell proliferation, thus underlining its involvement in the block of proliferation induced by BETis. Furthermore, MCM5 immunohistochemical evaluation in human thyroid tumor tissues demonstrated its overexpression in several papillary thyroid carcinomas and in all ATCs. MCM5 was also overexpressed in a murine model of ATC, and JQ1 treatment reduced Mcm5 mRNA expression in two murine ATC cell lines. Thus, MCM5 could represent a new target in the therapeutic approach against ATC.

Zhang Z, Ma P, Jing Y, et al.
BET Bromodomain Inhibition as a Therapeutic Strategy in Ovarian Cancer by Downregulating FoxM1.
Theranostics. 2016; 6(2):219-30 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Ovarian cancer is responsible for the highest mortality among all gynecologic malignancies, and novel therapies are urgently needed to improve patient outcome. Here we performed an integrative genomic analysis and identified the bromodomain and extraterminal domain (BET) protein BRD4 as a potential therapeutic target in ovarian cancer. Suppression of BRD4 using small-molecule BET inhibitors JQ1 and I-BET151, or dual kinase-bromodomain inhibitor volasertib, led to robust and broad antitumor effects across all subclasses of ovarian cancer. In contrast to many other cancers which are susceptible to BET inhibition due to downregulation of super-enhancer-dependent MYC transcript, we discovered that JQ1-sensitive ovarian cancer cells exhibited marked disruption of Forkhead box protein M1 (FoxM1) pathway, a key driver of ovarian carcinoma. These in vitro findings were further supported by in vivo efficacies of JQ1 targeting both cell line-based and patient-derived xenograft models. Our data establish a new treatment strategy against ovarian cancer by employing epigenetic vulnerabilities, and provide a mechanistic rationale for the clinical investigation of BET bromodomain inhibitors in this deadly disease.

Liu X, Rice SJ, Jamis-Dow CA, et al.
A patient with complex multiple genomic ALK alterations.
Thorax. 2016; 71(4):383-5 [PubMed] Related Publications

Albrecht BK, Gehling VS, Hewitt MC, et al.
Identification of a Benzoisoxazoloazepine Inhibitor (CPI-0610) of the Bromodomain and Extra-Terminal (BET) Family as a Candidate for Human Clinical Trials.
J Med Chem. 2016; 59(4):1330-9 [PubMed] Related Publications
In recent years, inhibition of the interaction between the bromodomain and extra-terminal domain (BET) family of chromatin adaptors and acetyl-lysine residues on chromatin has emerged as a promising approach to regulate the expression of important disease-relevant genes, including MYC, BCL-2, and NF-κB. Here we describe the identification and characterization of a potent and selective benzoisoxazoloazepine BET bromodomain inhibitor that attenuates BET-dependent gene expression in vivo, demonstrates antitumor efficacy in an MV-4-11 mouse xenograft model, and is currently undergoing human clinical trials for hematological malignancies (CPI-0610).

Lin CY, Erkek S, Tong Y, et al.
Active medulloblastoma enhancers reveal subgroup-specific cellular origins.
Nature. 2016; 530(7588):57-62 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Here, using H3K27ac and BRD4 chromatin immunoprecipitation followed by sequencing (ChIP-seq) coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-seq, that is responsible for subgroup divergence, and implicates candidate cells of origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins.

Marcotte R, Sayad A, Brown KR, et al.
Functional Genomic Landscape of Human Breast Cancer Drivers, Vulnerabilities, and Resistance.
Cell. 2016; 164(1-2):293-309 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Large-scale genomic studies have identified multiple somatic aberrations in breast cancer, including copy number alterations and point mutations. Still, identifying causal variants and emergent vulnerabilities that arise as a consequence of genetic alterations remain major challenges. We performed whole-genome small hairpin RNA (shRNA) "dropout screens" on 77 breast cancer cell lines. Using a hierarchical linear regression algorithm to score our screen results and integrate them with accompanying detailed genetic and proteomic information, we identify vulnerabilities in breast cancer, including candidate "drivers," and reveal general functional genomic properties of cancer cells. Comparisons of gene essentiality with drug sensitivity data suggest potential resistance mechanisms, effects of existing anti-cancer drugs, and opportunities for combination therapy. Finally, we demonstrate the utility of this large dataset by identifying BRD4 as a potential target in luminal breast cancer and PIK3CA mutations as a resistance determinant for BET-inhibitors.

McCleland ML, Mesh K, Lorenzana E, et al.
CCAT1 is an enhancer-templated RNA that predicts BET sensitivity in colorectal cancer.
J Clin Invest. 2016; 126(2):639-52 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Colon tumors arise in a stepwise fashion from either discrete genetic perturbations or epigenetic dysregulation. To uncover the key epigenetic regulators that drive colon cancer growth, we used a CRISPR loss-of-function screen and identified a number of essential genes, including the bromodomain and extraterminal (BET) protein BRD4. We found that BRD4 is critical for colon cancer proliferation, and its knockdown led to differentiation effects in vivo. JQ1, a BET inhibitor, preferentially reduced growth in a subset of epigenetically dysregulated colon cancers characterized by the CpG island methylator phenotype (CIMP). Integrated transcriptomic and genomic analyses defined a distinct superenhancer in CIMP+ colon cancers that regulates cMYC transcription. We found that the long noncoding RNA colon cancer-associated transcript 1 (CCAT1) is transcribed from this superenhancer and is exquisitely sensitive to BET inhibition. Concordantly, cMYC transcription and cell growth were tightly correlated with the presence of CCAT1 RNA in a variety of tumor types. Taken together, we propose that CCAT1 is a clinically tractable biomarker for identifying patients who are likely to benefit from BET inhibitors.

Shu S, Lin CY, He HH, et al.
Response and resistance to BET bromodomain inhibitors in triple-negative breast cancer.
Nature. 2016; 529(7586):413-7 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. BET bromodomain inhibitors, which have shown efficacy in several models of cancer, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyl-lysine recognition modules, leading to inhibition of oncogenic transcriptional programs. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance.

Delcuratolo M, Fertey J, Schneider M, et al.
Papillomavirus-Associated Tumor Formation Critically Depends on c-Fos Expression Induced by Viral Protein E2 and Bromodomain Protein Brd4.
PLoS Pathog. 2016; 12(1):e1005366 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
We investigated the mechanism of how the papillomavirus E2 transcription factor can activate promoters through activator protein (AP)1 binding sites. Using an unbiased approach with an inducible cell line expressing the viral transcription factor E2 and transcriptome analysis, we found that E2 induces the expression of the two AP1 components c-Fos and FosB in a Brd4-dependent manner. In vitro RNA interference confirmed that c-Fos is one of the AP1 members driving the expression of viral oncogenes E6/E7. Mutation analysis and in vivo RNA interference identified an essential role for c-Fos/AP1 and also for the bromodomain protein Brd4 for papillomavirus-induced tumorigenesis. Lastly, chromatin immunoprecipitation analysis demonstrated that E2 binds together with Brd4 to a canonical E2 binding site (E2BS) in the promoter of c-Fos, thus activating c-Fos expression. Thus, we identified a novel way how E2 activates the viral oncogene promoter and show that E2 may act as a viral oncogene by direct activation of c-Fos involved in skin tumorigenesis.

Doran MG, Carnazza KE, Steckler JM, et al.
Applying ⁸⁹Zr-Transferrin To Study the Pharmacology of Inhibitors to BET Bromodomain Containing Proteins.
Mol Pharm. 2016; 13(2):683-8 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Chromatin modifying proteins are attractive drug targets in oncology, given the fundamental reliance of cancer on altered transcriptional activity. Multiple transcription factors can be impacted downstream of primary target inhibition, thus making it challenging to understand the driving mechanism of action of pharmacologic inhibition of chromatin modifying proteins. This in turn makes it difficult to identify biomarkers predictive of response and pharmacodynamic tools to optimize drug dosing. In this report, we show that (89)Zr-transferrin, an imaging tool we developed to measure MYC activity in cancer, can be used to identify cancer models that respond to broad spectrum inhibitors of transcription primarily due to MYC inhibition. As a proof of concept, we studied inhibitors of BET bromodomain containing proteins, as they can impart antitumor effects in a MYC dependent or independent fashion. In vitro, we show that transferrin receptor biology is inhibited in multiple MYC positive models of prostate cancer and double hit lymphoma when MYC biology is impacted. Moreover, we show that bromodomain inhibition in one lymphoma model results in transferrin receptor expression changes large enough to be quantified with (89)Zr-transferrin and positron emission tomography (PET) in vivo. Collectively, these data further underscore the diagnostic utility of the relationship between MYC and transferrin in oncology, and provide the rationale to incorporate transferrin-based PET into early clinical trials with bromodomain inhibitors for the treatment of solid tumors.

Gao X, Wu X, Zhang X, et al.
Inhibition of BRD4 suppresses tumor growth and enhances iodine uptake in thyroid cancer.
Biochem Biophys Res Commun. 2016; 469(3):679-85 [PubMed] Related Publications
Thyroid cancer is a common malignancy of the endocrine system. Although radioiodine (131)I treatment on differentiated thyroid cancer is widely used, many patients still fail to benefit from (131)I therapy. Therefore, exploration of novel targeted therapies to suppress tumor growth and improve radioiodine uptake remains necessary. Bromodomain-containing protein 4 (BRD4) is an important member of the bromodomain and extra terminal domain family that influences transcription of downstream genes by binding to acetylated histones. In the present study, we found that BRD4 was up-regulated in thyroid cancer tissues and cell lines. Inhibition of BRD4 in thyroid cancer cells by JQ1 resulted in cell cycle arrest at G0/G1 phase and enhanced (131)I uptake in vitro and suppressed tumor growth in vivo. Moreover, JQ1 treatment suppressed C-MYC but enhanced NIS expression. We further demonstrated that BRD4 was enriched in the promoter region of C-MYC, which could be markedly blocked by JQ1 treatment. In conclusion, our findings revealed that the aberrant expression of BRD4 in thyroid cancer is possibly involved in tumor progression, and JQ1 is potentially an effective chemotherapeutic agent against human thyroid cancer.

Sun H, McGuire MF, Zhang S, Brown RE
NUT Midline Carcinoma: Morphoproteomic Characterization with Genomic and Therapeutic Correlates.
Ann Clin Lab Sci. 2015; 45(6):692-701 [PubMed] Related Publications
NUT midline carcinoma is a rare entity arising primarily in the midline of teenagers and young adults. Genomically, it is associated with a translocation involving a nuclear protein in testis (NUT) gene with other genes, most commonly, the BRD4 gene. The resultant is a partial or near total block in differentiation of tumor cells into mature squamous elements. Such tumors are resistant to conventional therapy with a reported mean survival at less than 1 year. In this study, we investigated two cases with genomic confirmation as NUT midline carcinoma by morphoproteomic analysis using immunohistochemical antibodies. Our results showed overexpression, largely in the undifferentiated cells of the tumors of: 1) Stemness marker, SRY (sex determining region Y)-box 2 (Sox2); 2) Constitutive activation of the mTORC2 pathway with expression of total insulin-like growth factor-1 receptor (IGF-1R[Tyr1165/1166]), and nuclear p-mTOR (Ser 2448) and p-Akt (Ser 473); and 3) c-Myc, silent mating type information regulation 2 homolog 1 (Sirt1) and histone methyltransferase enhancer of Zeste, Drosophila, homolog 2 (EZH2) as molecular impediments to differentiation. These data were analyzed through the use of QIAGEN's Ingenuity(®) Pathway Analysis (IPA(®), QIAGEN Redwood City, www.qiagen.com/ingenuity). The results established the interconnection of these pathways and molecules, and identified several pharmacogenomic agents--melatonin, metformin, vorinostat, curcumin, and sulforaphane--that have the potential to remove the block in differentiation and lead to the establishment of a more benign form of NUT midline carcinoma.

Shen C, Ipsaro JJ, Shi J, et al.
NSD3-Short Is an Adaptor Protein that Couples BRD4 to the CHD8 Chromatin Remodeler.
Mol Cell. 2015; 60(6):847-59 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
The bromodomain and extraterminal (BET) protein BRD4 is a therapeutic target in acute myeloid leukemia (AML). Here, we demonstrate that the AML maintenance function of BRD4 requires its interaction with NSD3, which belongs to a subfamily of H3K36 methyltransferases. Unexpectedly, AML cells were found to only require a short isoform of NSD3 that lacks the methyltransferase domain. We show that NSD3-short is an adaptor protein that sustains leukemia by linking BRD4 to the CHD8 chromatin remodeler, by using a PWWP chromatin reader module, and by employing an acidic transactivation domain. Genetic targeting of NSD3 or CHD8 mimics the phenotypic and transcriptional effects of BRD4 inhibition. Furthermore, BRD4, NSD3, and CHD8 colocalize across the AML genome, and each is released from super-enhancer regions upon chemical inhibition of BET bromodomains. These findings suggest that BET inhibitors exert therapeutic effects in leukemia by evicting BRD4-NSD3-CHD8 complexes from chromatin to suppress transcription.

Hensel T, Giorgi C, Schmidt O, et al.
Targeting the EWS-ETS transcriptional program by BET bromodomain inhibition in Ewing sarcoma.
Oncotarget. 2016; 7(2):1451-63 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Ewing sarcomas (ES) are highly malignant bone or soft tissue tumors. Genetically, ES are defined by balanced chromosomal EWS/ETS translocations, which give rise to chimeric proteins (EWS-ETS) that generate an oncogenic transcriptional program associated with altered epigenetic marks throughout the genome. By use of an inhibitor (JQ1) blocking BET bromodomain binding proteins (BRDs) we strikingly observed a strong down-regulation of the predominant EWS-ETS protein EWS-FLI1 in a dose dependent manner. This was further enhanced by co-treatment with an inhibitor of the PI3K pathway. Microarray analysis further revealed JQ1 treatment to block a typical ES associated expression program. The effect on this expression program was mimicked by RNA interference with BRD3 or BRD4 expression, indicating that the EWS-FLI1 mediated expression profile is at least in part mediated via such epigenetic readers. Consequently, contact dependent and independent proliferation of different ES lines was strongly inhibited. Mechanistically, treatment of ES resulted in a partial arrest of the cell cycle as well as induction of apoptosis. Tumor development was suppressed dose dependently in a xeno-transplant model in immune deficient mice, overall indicating that ES may be susceptible to treatment with epigenetic inhibitors blocking BET bromodomain activity and the associated pathognomonic EWS-ETS transcriptional program.

Zhang L, Tong Y, Zhang X, et al.
Arsenic sulfide combined with JQ1, chemotherapy agents, or celecoxib inhibit gastric and colon cancer cell growth.
Drug Des Devel Ther. 2015; 9:5851-62 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
BACKGROUND: Arsenic compounds have modest cytotoxic activity in solid tumors. We investigated if arsenic sulfide (As4S4) in combination with other distinct agents could enhance its cytotoxic activity.
METHODS: We used gastric and colon cancer cell lines to study the synergistic effect of As4S4 in combination with BRD4 inhibitor JQ1, or with chemotherapy drug cisplatin and irinotecan or with COX2 inhibitor celecoxib. We investigated the mechanism of the cytotoxic effect of these novel combinations.
RESULTS: We found that when As4S4 was combined with JQ1, cisplatin, irinotecan or celecoxib, its cytotoxic activity was dramatically enhanced in both gastric and colon cancer cell lines. As4S4 and JQ1 inhibited BRD4 and c-Myc while activating p53 expression synergistically. As4S4 inhibited COX2 and cyclin D1 expression. When As4S4 was combined with chemotherapy drug cisplatin or COX2 inhibitor celecoxib, its inhibition of COX2, BCL2, and p38 expression was enhanced. As4S4 and cisplatin synergistically stimulated p53, phosphor-p38 (p-p38), and increased cleaved caspase 3 (c-caspase 3).
CONCLUSION: As4S4 in combination with JQ1, cisplatin, irinotecan or celecoxib showed enhanced cytotoxic effect on gastric and colon cancer cells, indicating the potential application of these novel drug combinations as part of treatment strategy that warrants further investigation. As4S4 and JQ1 demonstrate synergistic activation of p53 and inhibition of c-Myc. As4S4 and cisplatin and celecoxib activated multiple apoptosis pathways.

Jing Y, Zhang Z, Ma P, et al.
Concomitant BET and MAPK blockade for effective treatment of ovarian cancer.
Oncotarget. 2016; 7(3):2545-54 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Ovarian cancer is the most lethal gynecologic malignancy, and it is imperative to develop new treatments to ameliorate patient survival. Using an anti-cancer drug library containing 180 small molecule inhibitors, we performed a high-content image-based screen and found that BET and MEK inhibitors are among the candidates which were able to effectively inhibit ovarian cancer cell growth. However, BET inhibition alone was largely cytostatic, possibly due to feedback activation of the MAPK pathway. Consequently, the combination of MEK and BET inhibitors suppressed both cell proliferation and survival, and was more efficacious than single agent. Mechanistically, BET and MEK inhibitors exerted synergistic effects on apoptosis regulators including BIM and BAD. Our findings support concomitant BET and MAPK blockade as an effective therapeutic strategy in ovarian cancer.

Li GQ, Guo WZ, Zhang Y, et al.
Suppression of BRD4 inhibits human hepatocellular carcinoma by repressing MYC and enhancing BIM expression.
Oncotarget. 2016; 7(3):2462-74 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Bromodomain 4 (BRD4) is an epigenetic regulator that, when inhibited, has anti-cancer effects. In this study, we investigated whether BRD4 could be a target for treatment of human hepatocellular carcinoma (HCC). We show that BRD4 is over-expressed in HCC tissues. Suppression of BRD4, either by siRNA or using JQ1, a pharmaceutical BRD4 inhibitor, reduced cell growth and induced apoptosis in HCC cell lines while also slowing HCC xenograft tumor growth in mice. JQ1 treatment induced G1 cell cycle arrest by repressing MYC expression, which led to the up-regulation of CDKN1B (P27). JQ1 also de-repressed expression of the pro-apoptotic BCL2L11 (BIM). Moreover, siRNA knockdown of BIM attenuated JQ1-triggered apoptosis in HCC cells, suggesting an essential role for BIM in mediating JQ1 anti-HCC activity.

Choi SK, Hong SH, Kim HS, et al.
JQ1, an inhibitor of the epigenetic reader BRD4, suppresses the bidirectional MYC-AP4 axis via multiple mechanisms.
Oncol Rep. 2016; 35(2):1186-94 [PubMed] Related Publications
Bromodomain and extra-terminal domain (BET) family proteins are representative epigenetic modulators that read acetylated lysine residues and transfer cellular signals. Recently, the BET protein inhibitor JQ1 was developed and has been extensively studied in many cancer cell types. We demonstrated that JQ1 effectively suppressed the MYC-AP4 axis and induced antitumorigenic effects by targeting a bidirectional positive loop between MYC and AP4 which was first proposed in the present study. MYC and AP4 are the direct targets of BRD4, as demonstrated by chromatin immunoprecipitation (ChIP) assay and BRD4 loss-of-function experiments. Although inhibition of the MYC/MAC dimer suppressed AP4, the efficacy of suppression was not as effective as BRD4 inhibition. Notably, AP4 loss-of-function studies demonstrated that AP4 is a major critical target of JQ1 and that MYC is a novel downstream target of AP4, as demonstrated by AP4 binding to the MYC promoter. Taken together, our results suggest that the epigenetic reader BRD4 is a key mediator of the activated MYC-AP4 axis, which supports the possibility that targeting BET protein is a novel therapeutic strategy for MYC-AP4 axis-activated cancers.

Kobayashi Y
[Epigenetic therapy for hematologic malignancies].
Rinsho Ketsueki. 2015; 56(10):2015-23 [PubMed] Related Publications
Cumulative evidence suggests that at least some hematologic malignancies are derived from alterations of epigenetic machinery. Next generation sequencing has revealed recurrent mutations of genes related to DNA methylation and histone modification in myelodysplastic syndromes (MDS), acute myeloid leukemia, malignant lymphoma, and multiple myeloma. Both these pathways are targetable and specific inhibitors of their related proteins are currently in development. Among these novel therapies, hypomethylating agents have been approved for MDS, and recently, histone deacetylase inhibitors became available for T-cell lymphoma and multiple myeloma. Agents currently undergoing clinical trials include inhibitors of IDH2 targeting DNA methylation, and EZH2, Dot1L, and BRD4 inhibitors designed to target either writers or readers of post-translational modifications (PTMs) of histones. In a phase I setting, where the maximum tolerated dose has not been reached, efficacy was reported with these agents. Furthermore, Dot1bL and IDH2 inhibitors have been shown to induce differentiation of leukemic blasts in patients with MLL gene rearrangements and IDH2 mutations, respectively, thus providing functional evidence supporting the use of inhibitors of epigenetic mechanisms as a means of differentiation therapy for hematologic malignancies.

Zhou J, Li W, Guo J, et al.
Downregulation of miR-329 promotes cell invasion by regulating BRD4 and predicts poor prognosis in hepatocellular carcinoma.
Tumour Biol. 2016; 37(3):3561-9 [PubMed] Related Publications
Increasing evidence indicates that abnormal microRNA (miRNA) expression is related to hepatocellular carcinoma (HCC) development. Our study aimed to elucidate the essential role of miR-329 in HCC progression. Real-time PCR was used to analyze miR-329 and bromodomain containing 4 (BRD4) expression in HCC samples (n = 135). Cell Counting Kit-8 (CCK-8) and flow cytometric analysis were used to investigate cell proliferation and apoptosis. The transwell assay was used to examine the cell invasive ability. The regulation mechanism was confirmed by luciferase reporter and western blot assays. Kaplan-Meier analysis was used to detect the function of miR-329 on the prognosis of HCC patients. miR-329 was decreased in HCC samples and was related to tumor development. Furthermore, miR-329 significantly regulated cell invasion by targeting BRD4 but had no effect on cell proliferation and apoptosis. Moreover, downregulation of miR-329 predicted poor prognosis of HCC patients. miR-329 could control cell invasion via regulating BRD4 expression and may be a prognostic marker in HCC.

Pelish HE, Liau BB, Nitulescu II, et al.
Mediator kinase inhibition further activates super-enhancer-associated genes in AML.
Nature. 2015; 526(7572):273-6 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling transcription factors and oncogenes. BRD4 and CDK7 are positive regulators of SE-mediated transcription. By contrast, negative regulators of SE-associated genes have not been well described. Here we show that the Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We report that the natural product cortistatin A (CA) selectively inhibits Mediator kinases, has anti-leukaemic activity in vitro and in vivo, and disproportionately induces upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the transcription factors CEBPA, IRF8, IRF1 and ETV6 (refs 6-8). The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has anti-leukaemic activity. Individually increasing or decreasing the expression of these transcription factors suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to the dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types, and can be pharmacologically targeted as a therapeutic approach to AML.

Yao W, Yue P, Khuri FR, Sun SY
The BET bromodomain inhibitor, JQ1, facilitates c-FLIP degradation and enhances TRAIL-induced apoptosis independent of BRD4 and c-Myc inhibition.
Oncotarget. 2015; 6(33):34669-79 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Inhibition of BET bromodomains (BRDs) has emerged as a promising cancer therapeutic strategy. Accordingly, inhibitors of BRDs such as JQ1 have been actively developed and some have reached clinical testing. However, the mechanisms by which this group of inhibitors exerts their anticancer activity, including induction of apoptosis, have not been fully elucidated. This report reveals a previously uncovered activity of JQ1 in inducing c-FLIP degradation and enhancing TRAIL-induced apoptosis. JQ1 potently decreased c-FLIP (both long and short forms) levels in multiple cancer cell lines without apparently increasing the expression of DR5 and DR4. Consequently, JQ1, when combined with TRAIL, synergistically induced apoptosis; this enhanced apoptosis-inducing activity could be abolished by enforced expression of ectopic FLIPL or FLIPS. Hence it appears that JQ1 decreases c-FLIP levels, resulting in enhancement of TRAIL-induced apoptosis. Inhibition of proteasome with MG132 prevented JQ1-induced c-FLIP reduction. Moreover, JQ1 decreased c-FLIP stability. Therefore, JQ1 apparently decreases c-FLIP levels through facilitating its proteasomal degradation. Genetic inhibition of either BRD4 or c-Myc by knocking down their expression failed to mimic JQ1 in decreasing c-FLIP and enhancing TRAIL-induced apoptosis, suggesting that JQ1 induces c-FLIP degradation and enhances TRAIL-induced apoptosis independent of BRD4 or c-Myc inhibition. In summary, our findings in this study highlights a novel biological function of JQ1 in modulating apoptosis and warrant further study of the potential treatment of cancer with the JQ1 and TRAIL combination.

Raza A, Cao H, Conrad R, et al.
Nuclear protein in testis midline carcinoma with unusual elevation of α-fetoprotein and synaptophysin positivity: a case report and review of the literature.
Expert Rev Anticancer Ther. 2015; 15(10):1199-213 [PubMed] Related Publications
Nuclear protein in testis (NUT) midline carcinoma (NMC) is a rare cancer that displays a characteristic chromosomal rearrangement of BRD4-NUT t(15;19)(q14;q13.1). Despite occasional dramatic responses to radiation and chemotherapy, NMC usually behaves aggressively and becomes rapidly progressive. Immunohistochemical staining is usually limited to p63, cytokeratins, and monoclonal NUT antibody. Here, we report a NMC case in a 36-year-old man with elevated serum α-fetoprotein (AFP), synaptophysin positivity, and a 9.0 cm mass involving the right lung and mediastinum. Tumor cells demonstrated BRD4-NUT fusion on fluorescence in situ hybridization. To our knowledge, only one other case with elevated serum AFP and one case with synaptophysin positivity have been described. This diagnosis will undoubtedly grow more common as informed physicians become more aware of the disease and begin testing for NMC. Further study is needed to establish the prevalence of NMC and to elucidate the significance of elevated AFP and synaptophysin positivity in this rare tumor.

Ambrosini G, Sawle AD, Musi E, Schwartz GK
BRD4-targeted therapy induces Myc-independent cytotoxicity in Gnaq/11-mutatant uveal melanoma cells.
Oncotarget. 2015; 6(32):33397-409 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Uveal melanoma (UM) is an aggressive intraocular malignancy with limited therapeutic options. Both primary and metastatic UM are characterized by oncogenic mutations in the G-protein alpha subunit q and 11. Furthermore, nearly 40% of UM has amplification of the chromosomal arm 8q and monosomy of chromosome 3, with consequent anomalies of MYC copy number. Chromatin regulators have become attractive targets for cancer therapy. In particular, the bromodomain and extra-terminal (BET) inhibitor JQ1 has shown selective inhibition of c-Myc expression with antiproliferative activity in hematopoietic and solid tumors. Here we provide evidence that JQ1 had cytotoxic activity in UM cell lines carrying Gnaq/11 mutations, while in cells without the mutations had little effects. Using microarray analysis, we identified a large subset of genes modulated by JQ1 involved in the regulation of cell cycle, apoptosis and DNA repair. Further analysis of selected genes determined that the concomitant silencing of Bcl-xL and Rad51 represented the minimal requirement to mimic the apoptotic effects of JQ1 in the mutant cells, independently of c-Myc. In addition, administration of JQ1 to mouse xenograft models of Gnaq-mutant UM resulted in significant inhibition of tumor growth.Collectively, our results define BRD4 targeting as a novel therapeutic intervention against UM with Gnaq/Gna11 mutations.

Borbely G, Haldosen LA, Dahlman-Wright K, Zhao C
Induction of USP17 by combining BET and HDAC inhibitors in breast cancer cells.
Oncotarget. 2015; 6(32):33623-35 [PubMed] Article available free on PMC after 01/04/2017 Related Publications
Members of the bromodomain and extra-C terminal (BET) domain protein family and the histone deacetylase (HDAC) enzyme family regulate the expression of important oncogenes and tumor suppressor genes. Here we show that the BET inhibitor JQ1 inhibits proliferation and induces apoptosis of both triple negative and estrogen receptor positive breast cancer cells. Consistent with the critical role of histone acetylation in the regulation of gene expression, treatment with JQ1 or the HDAC inhibitor mocetinostat was associated with global changes in gene expression resulting in suppression of genes involved in cell-cycle regulation. Combining JQ1 with mocetinostat, further decreased cell viability. This synergistic effect was associated with increased suppression of genes essential for cell-cycle progression. Furthermore, we detected dramatic increase in the expression of several members of the ubiquitin-specific protease 17 (USP17) family of deubiquitinating enzymes in response to the combination treatment. Increased expression of USP17 enzymes were able to attenuate the Ras/MAPK pathway causing decrease in cell viability, while, siRNA mediated depletion of USP17 significantly decreased cytotoxicity after the combination treatment. In conclusion, our study demonstrates that co-treatment with BET inhibitors and HDAC inhibitors reduces breast cancer cell viability through induction of USP17.

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