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Experimental evidence suggests that, in the inflamed gut of inflammatory bowel disease (IBD) patients, interleukin-34 (IL-34) triggers detrimental signaling pathways. Factors/mechanisms regulating IL-34 production in IBD remain poorly characterized. Bromodomain-containing 4 (BRD4), a transcriptional and epigenetic regulator, is over-expressed in IBD, and studies in cancer cells suggest that BRD4 might positively control IL-34 expression. This study aimed to assess whether, in IBD, BRD4 regulates IL-34 expression. In IBD, there was an up-regulation of both IL-34 and BRD4 compared to the controls, and the two proteins co-localized in both lamina propria mononuclear cells (LPMCs) and epithelial cells. Flow cytometry analysis of CD45+ LPMCs confirmed that the percentages of IL-34- and BRD4-co-expressing cells were significantly higher in IBD than in the controls and showed that more than 80% of the IL-34-positive CD45-LPMCs expressed BRD4. IL-34 and BRD4 were mainly expressed by T cells and macrophages. IL-34 expression was reduced in IBD LPMCs transfected with BRD4 antisense oligonucleotide and in the colons of mice with dextran sulfate sodium-induced colitis treated with JQ1, a pharmacological inhibitor of BRD4. These data indicate that BRD4 is a positive regulator of IL-34 in IBD, further supporting the pathogenic role of BRD4 in IBD-associated mucosal inflammation.

Uterine leiomyosarcoma (uLMS) is the most common type of uterine sarcoma, associated with poor prognosis, high rates of recurrence, and metastasis. Currently, the molecular mechanism of the origin and development of uLMS is limited. Bromodomain and extra-terminal (BET) proteins are involved in both physiological and pathological events. However, the role of BET proteins in the pathogenesis of uLMS is unknown. Here, we show for the first time that BET protein family members, BRD2, BRD3, and BRD4, are aberrantly overexpressed in uLMS tissues compared to the myometrium, with a significant change by histochemical scoring assessment. Furthermore, inhibiting BET proteins with their small, potent inhibitors (JQ1 and I-BET 762) significantly inhibited the uLMS proliferation dose-dependently via cell cycle arrest. Notably, RNA-sequencing analysis revealed that the inhibition of BET proteins with JQ1 and I-BET 762 altered several critical pathways, including the hedgehog pathway, EMT, and transcription factor-driven pathways in uLMS. In addition, the targeted inhibition of BET proteins altered several other epigenetic regulators, including DNA methylases, histone modification, and m 6 A regulators. The connections between BET proteins and crucial biological pathways provide a fundamental structure to better understand uterine diseases, particularly uLMS pathogenesis. Accordingly, targeting the vulnerable epigenome may provide an additional regulatory mechanism for uterine cancer treatment.

Bromodomain and extra-terminal (BET) proteins are relevant chromatin adaptors involved in the transcriptional control of thousands of genes. Two tandem N-terminal bromodomains are essential for chromatin attachment through acetyl-histone recognition. Recently, the BET proteins members BRD2 and BRD4 were found to interact with the SARS-CoV-2 envelope (E) protein, raising the question of whether the interaction constitutes a virus hijacking mechanism for transcription alteration in the host cell. To shed light on this question, we have compared the transcriptome of cells overexpressing E with that of cells treated with the BET inhibitor JQ1. Notably, E overexpression leads to a strong upregulation of natural immunity- and interferon response-related genes. However, BET inhibition results in the downregulation of most of these genes, indicating that these two conditions, far from causing a significant overlap of the altered transcriptomes, course with quite different outputs. Concerning the interaction of E protein with BET members, and differing from previous reports indicating that it occurs through BET bromodomains, we find that it relies on SEED and SEED-like domains, BET regions rich in Ser, Asp, and Glu residues. By taking advantage of this specific interaction, we have been able to direct selective degradation of E protein through a PROTAC system involving a dTAG-SEED fusion, highlighting the possible therapeutic use of this peptide for targeted degradation of a viral essential protein., (© 2024. The Author(s).)

Background: Injury to contractile organs such as the heart, vasculature, urinary bladder and gut can stimulate a pathological response that results in loss of normal contractility. PDGF and TGFβ are among the most well studied initiators of the injury response and have been shown to induce aberrant contraction in mechanically active cells of hollow organs including smooth muscle cells (SMC) and fibroblasts. However, the mechanisms driving contractile alterations downstream of PDGF and TGFβ in SMC and fibroblasts are incompletely understood, limiting therapeutic interventions., Methods: To identify potential molecular targets, we have leveraged the analysis of publicly available data, comparing transcriptomic changes in mechanically active cells stimulated with PDGF and TGFβ. Additional Analysis of publicly available data sets were performed on SMC and fibroblasts treated in the presence or absence of the MYC inhibitor JQ1. Validation of in silico findings were performed with qPCR, immunoblots, and collagen gel contraction assays measure the effect of JQ1 on cytoskeleton associated genes, proteins and contractility in mechanically active cells. Likelihood ratio test and FDR adjusted p-values were used to determine significant differentially expressed genes. Student ttest were used to calculate statistical significance of qPCR and contractility analyses., Results: Comparing PDGF and TGFβ stimulated SMC and fibroblasts identified a shared molecular profile regulated by MYC and members of the AP-1 transcription factor complex. Additional in silico analysis revealed a unique set of cytoskeleton-associated genes that were sensitive to MYC inhibition with JQ1. In vitro validation demonstrated JQ1 was also able to attenuate TGFβ and PDGF induced changes to the cytoskeleton and contraction of smooth muscle cells and fibroblasts in vitro., Conclusions: These findings identify MYC as a key driver of aberrant cytoskeletal and contractile changes in fibroblasts and SMC, and suggest that JQ1 could be used to restore normal contractile function in hollow organs., (© 2024. The Author(s).)

The BET family protein BRD4, which forms the CDK9-containing BRD4-PTEFb complex, is considered to be a master regulator of RNA polymerase II (Pol II) pause release. Because its tandem bromodomains interact with acetylated histone lysine residues, it has long been thought that BRD4 requires these bromodomains for its recruitment to chromatin and transcriptional regulatory function. Here, using rapid depletion and genetic complementation with domain deletion mutants, we demonstrate that BRD4 bromodomains are dispensable for Pol II pause release. A minimal, bromodomain-less C-terminal BRD4 fragment containing the PTEFb-interacting C-terminal motif (CTM) is instead both necessary and sufficient to mediate Pol II pause release in the absence of full-length BRD4. Although BRD4-PTEFb can associate with chromatin through acetyl recognition, our results indicate that a distinct, active BRD4-PTEFb population functions to regulate transcription independently of bromodomain-mediated chromatin association. These findings may enable more effective pharmaceutical modulation of BRD4-PTEFb activity., Competing Interests: Declaration of interests The authors declare no conflict of interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

An X-ray structure of a CLICK chemistry-based BET PROTAC bound to BRD2(BD2) inspired synthesis of JQ1 derived heterocyclic amides. This effort led to the discovery of potent BET inhibitors displaying overall improved profiles when compared to JQ1 and birabresib. A thiadiazole derived 1q (SJ1461) displayed excellent BRD4 and BRD2 affinity and high potency in the panel of acute leukaemia and medulloblastoma cell lines. A structure of 1q co-crystalised with BRD4-BD1 revealed polar interactions with the AZ/BC loops, in particular with Asn140 and Tyr139, rationalising the observed affinity improvements. In addition, exploration of pharmacokinetic properties of this class of compounds suggest that the heterocyclic amide moiety improves drug-like features. Our study led to the discovery of potent and orally bioavailable BET inhibitor 1q (SJ1461) as a promising candidate for further development., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ZR, MF, WCKP reports financial support was provided by ALSAC. VS reports financial support was provided by The Science Fund of The Republic of Serbia., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Combretastatin A4 (CA4) inhibits microtubule polymerization, and clinical trials of the prodrug, CA4 disodium phosphate (CA4DP), as an anti-cancer agent have been conducted. However, CA4DP has not been marketed to date because the margin between the effective dose and the cardiotoxic dose is insufficient. Meanwhile, bromodomain-containing protein 4 (BRD4) has been reported to be required for recovery from mitotic arrests induced by anti-microtubule drugs. BRD4 has also been reported to be involved in the progression of heart failure. Therefore, we hypothesized that the combined use of CA4DP with BRD4 inhibitors can enhance the antitumor effect and attenuate CA4DP-induced cardiotoxicity. In this study, the antitumor effect and cardiotoxicity caused by the co-administration of CA4DP with JQ1, a BRD4 inhibitor, were evaluated. CA4 or JQ1 alone reduced the viability of cultured canine mammary tumor cells (CHMp-13a). Viability was further reduced by co-administration, through the suppression of c-Myc. BRD4 positivity in CHMp-13a cytoplasm showed a significant increase when treated with CA4 alone, while the increase was not significant following co-administration. In CHMp-13a xenograft-transplanted mice, co-administration of CA4DP and JQ1 suppressed tumor growth significantly. In CA4DP-induced cardiac injury model rats, echocardiography showed a CA4DP-induced decrease in cardiac function and histopathology showed cardiomyocyte necrosis. Meanwhile, these cardiac changes tended to be milder following the co-administration of CA4DP and JQ1. These results suggest that CA4DP-JQ1 co-administration enhances the antitumor effect of CA4DP while attenuating its cardiotoxicity and therefore potentially open the doors to the development of a novel cancer chemotherapy with reduced cardiotoxicity risks., Competing Interests: Conflict of interest statement The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yoshiharu Tsuru is part of Primetech Corp., which is the vendor of the instruments used for echocardiography in this study., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Proteins of the bromodomain and exterminal domain (BET) family mediate critical host functions such as cell proliferation, transcriptional regulation, and the innate immune response, which makes them preferred targets for viruses. These multidomain proteins are best known as transcriptional effectors able to read acetylated histone and non-histone proteins through their tandem bromodomains. They also contain other short motif-binding domains such as the extraterminal domain, which recognizes transcriptional regulatory proteins. Here, we describe how different viruses have evolved to hijack or disrupt host BET protein function through direct interactions with BET family members to support their own propagation. The network of virus-BET interactions emerges as highly intricate, which may complicate the use of small-molecule BET inhibitors-currently in clinical development for the treatment of cancer and cardiovascular diseases-to treat viral infections.

Analysis of The Cancer Genome Atlas and other published data of head and neck squamous cell carcinoma (HNSCC) reveals somatic alterations of the Hippo-YAP pathway in approximately 50% of HNSCC. Better strategies to target the YAP1 transcriptional complex are sought. Here, we show that FAT1, an upstream inhibitor of YAP1, is mutated either by missense or by truncating mutation in 29% of HNSCC. Comprehensive proteomic and drug-screening studies across pan-cancer models confirm that FAT1-mutant HNSCC exhibits selective and higher sensitivity to BRD4 inhibition by JQ1. Epigenomic analysis reveals an active chromatin state in FAT1-mutant HNSCC cells that is driven by the YAP/TAZ transcriptional complex through recruitment of BRD4 to deposit active histone marks, thereby maintaining an oncogenic transcriptional state. This study reveals a detailed cooperative mechanism between YAP1 and BRD4 in HNSCC and suggests a specific therapeutic opportunity for the treatment of this subset of head and neck cancer patients., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lysine acetylation creates docking sites for epigenetic reader domains of BET bromodomain proteins that have emerged as principal regulators of linage specific gene transcription. The development of potent and highly selective inhibitors, that have been soon widely available, enabled mechanistic studies in a diversity of disease models leading to a rapid translation into the clinic. Initial studies on pan-BET inhibitors lead now to second generation inhibitors with improved domain selectivity, but also to highly potent bifunctional and dual inhibitors extending the toolbox for basic research on acetylation dependent transcription, BET associated diseases and further translational efforts targeting this interesting family of epigenetic reader domains., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships that may be considered as potential competing interests: Prof Stefan Knapp, PhD reports a relationship with Pfizer Global Research and Development that includes: paid expert testimony. Prof Stefan Knapp has no patents to disclose. He is editor for Current Research in Chemical Biology, (Copyright © 2022 Elsevier Ltd. All rights reserved.)

BET bromodomain BRD4 and RAC1 oncogenes are considered important therapeutic targets for cancer and play key roles in tumorigenesis, survival and metastasis. However, combined inhibition of BRD4-RAC1 signaling pathways in different molecular subtypes of breast cancer including luminal-A, HER-2 positive and triple-negative breast (TNBC) largely remains unknown. Here, we demonstrated a new co-targeting strategy by combined inhibition of BRD4-RAC1 oncogenic signaling in different molecular subtypes of breast cancer in a context-dependent manner. We show that combined treatment of JQ1 (inhibitor of BRD4) and NSC23766 (inhibitor of RAC1) suppresses cell growth, clonogenic potential, cell migration and mammary stem cells expansion and induces autophagy and cellular senescence in molecular subtypes of breast cancer cells. Mechanistically, JQ1/NSC23766 combined treatment disrupts MYC/G9a axis and subsequently enhances FTH1 to exert antitumor effects. Furthermore, combined treatment targets HDAC1/Ac-H3K9 axis, thus suggesting a role of this combination in histone modification and chromatin modeling. C-MYC depletion and co-treatment with vitamin-C sensitizes different molecular subtypes of breast cancer cells to JQ1/NSC23766 combination and further reduces cell growth, cell migration and mammosphere formation. Importantly, co-targeting RAC1-BRD4 suppresses breast tumor growth in vivo using xenograft mouse model. Clinically, RAC1 and BRD4 expression positively correlates in breast cancer patient's samples and show high expression patterns across different molecular subtypes of breast cancer. Both RAC1 and BRD4 proteins predict poor survival in breast cancer patients. Taken together, our results suggest that combined inhibition of BRD4-RAC1 pathways represents a novel and potential therapeutic approach in different molecular subtypes of breast cancer and highlights the importance of co-targeting RAC1-BRD4 signaling in breast tumorigenesis via disruption of C-MYC/G9a/FTH1 axis and down regulation of HDAC1., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Glioblastoma (GBM) is the deadliest brain malignancy without effective treatments. Here, we reported that epidermal growth factor receptor-targeted chimeric antigen receptor T cells (EGFR CAR-T) were effective in suppressing the growth of GBM cells in vitro and xenografts derived from GBM cell lines and patients in mice. However, mice soon acquired resistance to EGFR CAR-T cell treatment, limiting its potential use in the clinic. To find ways to improve the efficacy of EGFR CAR-T cells, we performed genomics and transcriptomics analysis for GBM cells incubated with EGFR CAR-T cells and found that a large cohort of genes, including immunosuppressive genes, as well as enhancers in vicinity are activated. BRD4, an epigenetic modulator functioning on both promoters and enhancers, was required for the activation of these immunosuppressive genes. Accordingly, inhibition of BRD4 by JQ1 blocked the activation of these immunosuppressive genes. Combination therapy with EGFR CAR-T cells and JQ1 suppressed the growth and metastasis of GBM cells and prolonged survival in mice. We demonstrated that transcriptional modulation by targeting epigenetic regulators could improve the efficacy of immunotherapy including CAR-T, providing a therapeutic avenue for treating GBM in the clinic., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Bromodomain 4 (BRD4), an important epigenetic regulator, is involved in many bone-related pathologies via promoting osteoclast formation. However, whether and how it participates in the process of osteoblast formation remain unclear. This study aimed to investigate the potential role of BRD4 in osteogenic differentiation of bone marrow stromal cells (BMSCs). Our experiments revealed that an inhibitor of BRD4, JQ1, attenuated osteogenic differentiation of BMSCs. The recombinant adenoviruses for AdBRD4 and AdsiBRD4 could infect BMSCs with high efficiency. Exogenous BRD4 expression potentiated differentiation, and silencing endogenous BRD4 expression decreased it. In addition, the Wnt/β-catenin signaling pathway is known to be important for osteogenic differentiation. Our results showed that AdBRD4 increased the expressions of Wnt3a and β-catenin while AdsiBRD4 decreased the expressions. What's more, the recombinant adenovirus for Adsiβ-catenin, which obviously decreased in β-catenin expression, inhibited BRD4-induced osteogenic differentiation. Conclusion: Our data indicates that the epigenetic reader BRD4 participates in the process of BMSC osteogenic differentiation via the Wnt/β-catenin signaling pathway. This finding may pave the way into further understanding the mechanism of BMSC osteogenic differentiation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Inaccurate fear memories can be maladaptive and potentially portrait a core symptomatic dimension of fear adaptive disorders such as post-traumatic stress disorder (PTSD), which is generally characterized by an intense and enduring memory for the traumatic events. Evidence exists in support of epigenetic regulation of fear behavior. Brd4, a member of the bromodomain and extra-terminal domain (BET) protein family, serves as a chromatin "reader" by binding to histones in acetylated lysine residues, and hence promotes transcriptional activities. However, less is known whether Brd4 participates in modulating cognitive activities especially memory formation and extinction. Here we provide evidence for a role of Brd4 in modulation of auditory fear memory. Auditory fear conditioning resulted in a biphasic Brd4 activation in the anterior cingulate cortex (ACC) and hippocampus of adult mice. Thus, Brd4 phosphorylation occurred 6 h and 3-14 days, respectively, after auditory fear conditioning. Systemic inhibition of Brd4 with a BET inhibitor, JQ1, impaired the extinction of remote (i.e., 14 days after conditioning) fear memory. Further, conditional Brd4 knockout in excitatory neurons of the forebrain impaired remote fear extinction as observed in the JQ1-treated mice. Herein, we identified that Brd4 is essential for extinction of remote fear in rodents. These results thus indicate that Brd4 potentially plays a role in the pathogenesis of PTSD., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Endotoxemia is a severe inflammation response induced by infection especially bacterial endotoxin translocation, which severely increases mortality in combination with acute colon injury. Bromodomain-containing protein 4 (BRD4) is an important Bromo and Extra-Terminal (BET) protein to participate in inflammatory responses. However, it is still unknown about the specific connection between BRD4 and inflammation-related pyroptosis in endotoxemia colon. Here, through evaluating the mucous morphology and the expression of tight junction proteins such as occludin and ZO1, we found the upregulation of BRD4 in damaged colon with poor tight junction in an endotoxemia mouse model induced by lipopolysaccharides (LPS). Firstly, the BRD4 inhibitor JQ1 was used to effectively protect colon tight junction in endotoxemia. As detected, high levels of pro-inflammation cytokines IL6, IL1β and IL18 in endotoxemia colon were reversed by JQ1 pretreatment. In addition, JQ1 injection reduced endotoxemia-induced elevation of the phosphorylated NF κB and NLRP3/ASC/caspase 1 inflammasome complex in colon injury. Furthermore, activated pyroptosis markers gasdermins in endotoxemia colon were also blocked by JQ1 pretreatment. Together, our data indicate that BRD4 plays a critical role in regulating pyroptosis-related colon injury induced by LPS, and JQ1 as a BRD4 inhibitors can effectively protect colon from endotoxemia-induced inflammation injury., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen, Zhong, Cao, Mao, Li, Yang, Li, Shi, Zhang, Deng, Zu and Liu.)

Retinoblastoma (RB) is the most common intraocular cancer in children, and chemotherapy has been the first-line treatment. However, due to the side effects of chemotherapy drugs, novel treatments must be developed. JQ1, a selective inhibitor of BRD4, suppresses cell growth in several cancers in which BRD4 is overexpressed. In the present study, BRD4 was overexpressed in retinoblastoma, and JQ1 effectively inhibited RB cell proliferation and colony formation by inducing cell cycle arrest and promoting apoptosis. Furthermore, the Myc-P21-CDK2 and Myc-cyclinD3/CDK6 pathways were activated in RB cells treated with JQ1, and an animal experiment suggested that JQ1 significantly inhibited tumour growth in vivo. In conclusion, JQ1 may be a potential drug treatment for retinoblastoma., (Copyright © 2020. Published by Elsevier Ltd.)

BRD4 is a member of the BET family of epigenetic regulators. Inhibition of BRD4 by the selective bromodomain inhibitor JQ1, alleviates thoracic aortic constriction-induced cardiac hypertrophy and heart failure. However, whether BRD4 inhibition by JQ1 has therapeutic effect on diabetic cardiomyopathy, a major cause of heart failure in patients with Type 2 diabetes, remains unknown. Here, we discover a novel link between BRD4 and PINK1/Parkin-mediated mitophagy during diabetic cardiomyopathy. Upregulation of BRD4 in diabetic mouse hearts inhibits PINK1/Parkin-mediated mitophagy, resulting in accumulation of damaged mitochondria and subsequent impairment of cardiac structure and function. BRD4 inhibition by JQ1 improves mitochondrial function, and repairs the cardiac structure and function of the diabetic heart. These effects depended on rewiring of the BRD4-driven transcription and repression of PINK1. Deletion of Pink1 suppresses mitophagy, exacerbates cardiomyopathy, and abrogates the therapeutic effect of JQ1 on diabetic cardiomyopathy. Our results illustrate a valid therapeutic strategy for treating diabetic cardiomyopathy by inhibition of BRD4., (Copyright © 2020. Published by Elsevier Ltd.)

Vincristine (VCR) is a well-known anticancer drug, and frequently causes painful neuropathy and impairs the quality of life of patients. However, the molecular mechanisms revealing VCR-induced neuropathy are still unclear, and effectively therapeutic strategy is still necessary. Bromodomain-containing protein 4 (BRD4) has long been implicated in many different pathological processes, in particular, the development of oxidative stress and inflammation. In the present study, we showed that BRD4 played a mechanistic role in VCR-induced peripheral neuropathy. Using the in vivo transfection of BRD4 siRNA, we found that BRD4 suppression markedly alleviated VCR-induced neuropathic pain. Macrophage infiltration in sciatic nerve was effectively inhibited in VCR-challenged mice with BRD4 knockdown, as evidenced by the markedly reduced expression of F4/80. In the VCR-induced sciatic nerve tissues, we found that the mRNA and protein expression levels of C-X3-C motif chemokine receptor 1 (CX3CR1) and C-C chemokine receptor type 2 (CCR2) were greatly elevated, which were, however, mitigated by siBRD4 injection. In addition, oxidative stress induced by VCR was markedly restrained in sciatic nerve from mice with BRD4 knockdown, which was closely associated with the improved activation of nuclear factor erythroid 2-related factor 2 (Nrf-2) signaling. The in vitro studies indicated that in H 2 O 2 -stimulated primary neurons, BRD4 silence markedly reduced reactive oxygen species (ROS) production and improved Nrf-2 activation, exhibiting anti-oxidant effects. Finally, BRD4 selective inhibitor JQ1 was subjected to mice challenged with VCR. The results confirmed that reducing BRD4 expression by JQ1 effectively ameliorated VCR-induced peripheral neuropathy also through repressing macrophage infiltration, inflammatory response and oxidative stress. Taken together, these findings demonstrated that BRD4 played a critical role in VCR-induced neuropathy, and developing novel and new therapies might be effective for the treatment of VCR-induced neuropathic pain., (Copyright © 2020. Published by Elsevier Inc.)

Effective spatio-temporal control of transcription and replication during S-phase is paramount to maintaining genomic integrity and cell survival. Dysregulation of these systems can lead to conflicts between the transcription and replication machinery, causing DNA damage and cell death. BRD4 allows efficient transcriptional elongation by stimulating phosphorylation of RNA polymerase II (RNAPII). We report that bromodomain and extra-terminal domain (BET) protein loss of function (LOF) causes RNAPII pausing on the chromatin and DNA damage affecting cells in S-phase. This persistent RNAPII-dependent pausing leads to an accumulation of RNA:DNA hybrids (R-loops) at sites of BRD4 occupancy, leading to transcription-replication conflicts (TRCs), DNA damage, and cell death. Finally, our data show that the BRD4 C-terminal domain, which interacts with P-TEFb, is required to prevent R-loop formation and DNA damage caused by BET protein LOF., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Programmed death-ligand 1 (PD-L1) expression on the surface of tumour cells can cause tumour immune evasion. Benefits of combining anti-PD-L1 therapy with nab-paclitaxel in patients with advanced triple-negative breast cancer (TNBC) have been reported. However, some patients cannot tolerate the immune-related adverse effects (irAEs) caused by antibody-based immunotherapy. BRD4 is a member of the bromodomain and extra-terminal domain (BET) family. BRD4 inhibition has shown antitumour effects in many tumours, but its role in TNBC has not been definitively concluded. In particular, the immune regulation of BRD4 in TNBC has been rarely studied. In this study, we used JQ1, a BET inhibitor, and small interfering RNAs (siRNAs) targeting BRD4 to explore the influence of BRD4 on PD-L1 expression in TNBC. The results indicated that BRD4 inhibition suppressed PD-L1 expression and the PD-L1 upregulation induced by interferon-γ (IFN-γ). In the in vivo experiments, we found that JQ1 not only reduced the PD-L1 expression level but also changed the proportions of T lymphocyte subsets in the spleens of tumour-bearing mice, which helped to relieve immunosuppression. Briefly, our study reveals that BRD4 regulates PD-L1 expression and may provide a potential method for blocking the programmed death 1 (PD-1)/PD-L1 immune checkpoint in TNBC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Treatment of medulloblastoma (MB) is challenging due to diverse genetic make-up, chemoresistance and inefficient drug transport across the blood brain barrier (BBB). Since hedgehog (Hh) signaling regulates cancer cell proliferation and tumorigenicity, Hh inhibitors have the potential to treat sonic Hh driven MB (SHH-MB), but their repeated use develops chemoresistance due to mutations in smoothened (SMO). Herein, we aimed to overcome these problems by modulating GLI transcription using JQ1, which is a small molecule BRD4 inhibitor. JQ1 inhibited HD-MB03 and DAOY cell proliferation, with the IC 50 of 402 and 4220 nM, respectively. JQ1 inhibited colony formation, but increased apoptosis in HD-MB03 and DAOY cells. Western blot analysis confirmed significant inhibition of GLI1 and c-MYC protein expression in DAOY and HD-MB03 cells, respectively. JQ1 was encapsulated into apolipoprotein (ApoE) mimetic peptide decorated nanoparticles (ApoE-NPs), with the mean particle size of 64 nm and drug loading of 10% (w/w). ApoE-NPs increased JQ1 concentration in the tumor by 5 and 8 folds at 6 and 24 h after systemic administration into orthotopic MB tumor bearing NSG mice compared to non-targeted JQ1 loaded NPs. Although there was also modest increase in JQ1 delivery to the liver, there was no hepatotoxicity as evidenced by H&E staining and little increase in serum ALT and AST after treatment with JQ1 loaded ApoE-NPs. There was also significant decrease in the orthotopic MB tumor burden after systemic administration of JQ1 loaded ApoE- NPs at the dose of 10 mg/kg every 3rd day for a total of 8 injections. In conclusion, JQ1 loaded NPs have the potential to treat Group 3 and SHH driven MB in mice., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Rett syndrome (RTT), mainly caused by mutations in methyl-CpG binding protein 2 (MeCP2), is one of the most prevalent intellectual disorders without effective therapies. Here, we used 2D and 3D human brain cultures to investigate MeCP2 function. We found that MeCP2 mutations cause severe abnormalities in human interneurons (INs). Surprisingly, treatment with a BET inhibitor, JQ1, rescued the molecular and functional phenotypes of MeCP2 mutant INs. We uncovered that abnormal increases in chromatin binding of BRD4 and enhancer-promoter interactions underlie the abnormal transcription in MeCP2 mutant INs, which were recovered to normal levels by JQ1. We revealed cell-type-specific transcriptome impairment in MeCP2 mutant region-specific human brain organoids that were rescued by JQ1. Finally, JQ1 ameliorated RTT-like phenotypes in mice. These data demonstrate that BRD4 dysregulation is a critical driver for RTT etiology and suggest that targeting BRD4 could be a potential therapeutic opportunity for RTT., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Hippo/YAP1 signaling is a major regulator of organ size, cancer stemness, and aggressive phenotype. Thus, targeting YAP1 may provide a novel therapeutic strategy for tumors with high YAP1 expression in esophageal cancer (EC). Chromatin immunoprecipitation (ChiP) and quantitative ChiP-PCR were used to determine the regulation of the chromatin remodeling protein bromodomain-containing protein 4 (BRD4) on YAP1. The role of the bromodomain and extraterminal motif (BET) inhibitor JQ1, an established BRD4 inhibitor, on inhibition of YAP1 in EC cells was dissected using western blot, immunofluorescence, qPCR, and transient transfection. The antitumor activities of BET inhibitor were further examined by variety of functional assays, cell proliferation (MTS), tumorsphere, and ALDH1+ labeling in vitro and in vivo. Here, we show that BRD4 regulates YAP1 expression and transcription. ChiP assays revealed that BRD4 directly occupies YAP1 promoter and that JQ1 robustly blocks BRD4 binding to the YAP1 promoter. Consequently, JQ1 strongly suppresses constitutive or induced YAP1 expression and transcription in EC cells and YAP1/Tead downstream transcriptional activity. Intriguingly, radiation-resistant cells that acquire strong cancer stem cell traits and an aggressive phenotype can be effectively suppressed by JQ1 as assessed by cell proliferation, tumorsphere formation, and reduction in the ALDH1+ cells. Moreover, effects of JQ1 are synergistically amplified by the addition of docetaxel in vitro and in vivo. Our results demonstrate that BRD4 is a critical regulator of Hippo/YAP1 signaling and that BRD4 inhibitor JQ1 represents a new class of inhibitor of Hippo/YAP1 signaling, primarily targeting YAP1 high and therapy-resistant cancer cells enriched with cancer stem cell properties., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Listeria monocytogenes (LM) is a facultative intracellular bacterium that causes septicemia-associated acute hepatic injury. However, the pathogenesis of this process is still unclear, and there is still a lack of effective therapeutic strategy for the treatment of LM-induced liver injury. In this study, we attempted to explore the effects of necroptosis on bacterial-septicemia-associated hepatic disease and to explore the contribution of JQ1, a selective BRD4 inhibitor, to the suppression of necroptosis and inhibition of LM-triggered hepatic injury. The results indicated that hepatic BRD4 was primarily stimulated by LM both in vitro and in vivo, along with significantly up-regulated expression of receptor-interacting protein kinase (RIPK)-1, RIPK3, and p-mixed lineage kinase-like (MLKL), showing the elevated necroptosis. However, JQ1 treatment and RIPK1 knockout were found to significantly alleviate LM-induced acute liver injury. Histological alterations and cell death in hepatic samples in LM-infected mice were also alleviated by JQ1 administration or RIPK1 deletion. However, JQ1-improved hepatic injury by LM was abrogated by RIPK1 over-expression, suggesting that the protective effects of JQ1 took place mainly in an RIPK1-dependent manner. In addition, LM-evoked inflammatory response in liver tissues were also alleviated by JQ1, which was similar to the findings observed in mice lacking RIPK1. The anti-inflammatory effects of JQ1 were diminished by RIPK1 over-expression in LM-infected mice. Finally, both in vivo and in vitro experiments suggested that JQ1 dramatically improved hepatic mitochondrial dysfunction in LM-injected mice, but this effect was abolished by RIPK1 over-expression. In conclusion, these results indicated that suppressing BRD4 by JQ1 could ameliorate LM-associated liver injury by suppressing necroptosis, inflammation, and mitochondrial dysfunction by inhibiting RIPK1., (Copyright © 2020. Published by Elsevier Masson SAS.)

Background: Pharmacologic inhibition of bromodomain and extra-terminal (BET) proteins is currently being explored as a new therapeutic approach in cancer. Some studies have also implicated BET proteins as regulators of cell identity and differentiation through their interactions with lineage-specific factors. However, the role of BET proteins has not yet been investigated in melanocyte differentiation. Melanocyte inducing transcription factor (MITF) is the master regulator of melanocyte differentiation, essential for pigmentation and melanocyte survival. In this study, we tested the hypothesis that BET proteins regulate melanocyte differentiation through interactions with MITF., Results: Here we show that chemical inhibition of BET proteins prevents differentiation of unpigmented melanoblasts into pigmented melanocytes and results in de-pigmentation of differentiated melanocytes. BET inhibition also slowed cell growth, without causing cell death, increasing the number of cells in G1. Transcriptional profiling revealed that BET inhibition resulted in decreased expression of pigment-specific genes, including many MITF targets. The expression of pigment-specific genes was also down-regulated in melanoma cells, but to a lesser extent. We found that RNAi depletion of the BET family members, bromodomain-containing protein 4 (BRD4) and bromodomain-containing protein 2 (BRD2) inhibited expression of two melanin synthesis enzymes, TYR and TYRP1. Both BRD4 and BRD2 were detected on melanocyte promoters surrounding MITF-binding sites, were associated with open chromatin structure, and promoted MITF binding to these sites. Furthermore, BRD4 and BRD2 physically interacted with MITF., Conclusion: These findings indicate a requirement for BET proteins in the regulation of pigmentation and melanocyte differentiation. We identified changes in pigmentation specific gene expression that occur upon BET inhibition in melanoblasts, melanocytes, and melanoma cells.

Bromodomain‑containing 4 (BRD4) is a histone modification reader and transcriptional regulator that has been reported to interact with acetylated lysine histone motifs transcription factors (TFs), transcription co‑activators and RNA polymerase II. The selective small molecule inhibitor JQ1, which binds competitively to bromodomains, has been reported to exhibit anti‑proliferative effects in various types of cancer. Previous studies on the mechanism of action of JQ1 mostly focused on a specific tumor type or disease; however, the general mechanism through which JQ1 affects various tumors remains to be determined. In the present study, chromatin immunoprecipitation sequencing data for BRD4 and its expression profiles in six cancer cell lines were integrated and analyzed systematically. The results indicated that BRD4 binds to enhancers with histone H3 acetylated at lysine 27 (H3K27Ac) and mediator complex subunit 1 in a cell type‑specific manner, as well as binds to promoter regions with the oncogenic TFs MYC and E2F1 in a cell type‑common manner. The cell type‑common sites across the six cell types investigated were found to be functionally important for tumorigenesis, whereas the cell type‑specific sites were functionally enriched with the cell identity, all of which were sensitive to JQ1 treatment. Furthermore, a core set of JQ1‑regulated BRD4 binding genes were obtained, which were significantly inhibited by JQ1 in various cancer cell lines and contributed to hallmarks of cancer. These results implied a common mechanism underlying the therapeutic effects of JQ1 and suggested its potential suitability as an anti‑cancer drug targeting BRD4‑mediated transcriptional regulation.

In gastrointestinal stromal tumours (GISTs), the function of bromodomain-containing 4 (BRD4) remains underexplored. BRD4 mRNA abundance was quantified in GISTs. In the current study, we investigated the role of BRD4 in GISTs. Our results show a significant enhancement in BRD4 mRNA and a shift from very low-risk/low-risk to high-risk levels as per NCCN specifications. Overexpression of BRD4 correlated with unfavourable genotype, nongastric location, enhanced risk and decreased disease-free survival, which were predicted independently. Knockout of BRD4 in vitro suppressed KIT expression, which led to inactivation of the KIT/PI3K/AKT/mTOR pathway, impeded migration and cell growth and made the resistant GIST cells sensitive to imatinib. The expression of KIT was repressed by a BRD4 inhibitor JQ1, which also induced myristoylated-AKT-suppressible caspases 3 and 9 activities, induced LC3-II, exhibited dose-dependent therapeutic synergy with imatinib and attenuated the activation of the PI3K/AKT/mTOR pathway. In comparison with their single therapy, the combination of JQ1/imatinib more efficiently suppressed the growth of xenografts and exhibited a reduction in KIT phosphorylation, a decrease in Ki-67 and in the levels of phosphorylated PI3K/AKT/mTOR and enhanced TUNEL staining. Thus, we characterized the biological, prognostic and therapeutic implications of overexpressed BRD4 in GIST and observed that JQ1 suppresses KIT transactivation and nullifies the activation of PI3K/AKT/mTOR, providing a potential strategy for treating imatinib-resistant GIST through dual blockade of KIT and BRD4., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Ischemic stroke is a major cause of death and disability worldwide. Hyperneuroinflammation significantly contributes to ischemic stroke. Bromodomain-containing protein 4 (BRD4) is a member of the Bromo and Extra-Terminal (BET) family, and promotes inflammatory response in various types of tissue and cells. Thereby, we examined the contribution of BRD4 after cerebral ischemic/reperfusion (I/R) injury in a mouse middle cerebral artery occlusion (MCAO) model. Here, we showed that BRD4 expression was correlated with glial activation and cerebral I/R injury after MCAO in mice. Intriguingly, we found that BRD4 inhibition using its selective inhibitor, JQ1, showed a protective role in cerebral I/R injury in mice. Suppressing BRD4 by JQ1 reduced the infarction volume, brain water contents and neurological deficit score of MCAO mice. In addition, MCAO-induced glial activation was also blunted by JQ1, as proved by the significantly reduced expression of glial fibrillary acidic protein (GFAP) and Iba-1. Consistently, JQ1 treatment decreased the expression of pro-inflammatory factors by blocking nuclear factor kappa B (NF-κB) signaling. Furthermore, inflammasome activation and pyroptosis found in MCAO mice were markedly attenuated by JQ1, which were through suppressing the expression of NLRP3 (nucleotide-binding domain, leucine-rich repeat containing protein 3), ASC (apoptosis-associated speck-like protein containing a CARD), Caspase-1 and GSDMD (gasdermin D). The protective effects of BRD4 inhibition on cerebral ischemia-induced brain injury were verified in astrocytes and microglial cells via the inhibition of inflammation and pyroptosis. In summary, blocking BRD4 expression might serve as a potential therapeutic strategy for stroke therapy., (Copyright © 2019. Published by Elsevier Inc.)

Brd4 protein is an important epigenetic regulator involved in the process of inflammatory cytokine production in many diseases. However, whether and how Brd4 participates in the process of wear-particle-induced inflammation remain unclear. This study aimed to investigate the potential role of Brd4 in titanium (Ti) particle-induced inflammatory cytokine production in mouse macrophage RAW264.7 cells. Our experiment detected Brd4 expressed in both normal synovium and periprosthetic osteolysis interface membrane, but the expression increased in the interface membrane as compared with that in normal synovium. Treatment with Ti particles significantly increased TNF-α, IL-6, and IL-1β production in RAW264.7 cells, which was inhibited by JQ1 or Brd4-siRNA. Ti particles enhanced the expression of Brd4, which was abrogated by JQ1. Ti particles enhanced NF-κB p65 and IKK phosphorylation and attenuated IκBα protein expression, which were abrogated by JQ1. Co-immunoprecipitation analysis indicated that Ti particles promoted the binding of Brd4 to acetylated NF-κB p65 (lysine-310), which was also abrogated in JQ1-treated RAW264.7 cells. In conclusion, Brd4 expression increases in interface membrane and Brd4 participates in the production of pro-inflammatory cytokines induced by Ti particles via promoting the activation of NF-κB signaling and binding to acetylated NF-κB p65 (lysine-310) in mouse macrophages.

Drug resistance substantially limits the curative capability of chemotherapy in head and neck cancers such as oral squamous cell carcinoma. Immunosuppression is considered a potential cause of drug resistance. A key discovery in the past decade is that chemotherapeutics can alter tumor cell immunogenicity via inducing release of damage-associated molecular patterns (DAMPs), including ecto-calreticulin (ecto-CALR), high mobility group box 1 (HMGB1) and ATP, causing tumor cells to die in a manner known as bona fide immunogenic apoptosis or immunogenic cell death (ICD). Intriguingly, JQ1 was found in this study to exhibit therapeutic potential in tongue squamous cell carcinoma (TSCC) by inducing ICD. JQ1 induced significant release of calreticulin (CALR), HMGB1 and ATP from Cal27 and SCC7 cells in vitro. Immature dendritic cells (Im-DCs) cocultured with JQ1-pretreated Cal27 cells exhibited significant upregulation of mature markers on their surface and an increase in the secretion of cytokines. In vivo experiments demonstrated that JQ1-pretreated dying SCC7 cells protected immunocompetent mice from rechallenge of SCC7 cells. Intravenous injection of JQ1 efficiently reduced tumor growth and increased tumor-infiltration of CD3 + /CD8 + T cells in C3H mice., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The pathophysiology of spinal cord injury (SCI) involves primary injury and secondary injury. For the irreversibility of primary injury, therapies of SCI mainly focus on secondary injury, whereas inflammation is considered to be a major target for secondary injury; however the regulation of inflammation in SCI is unclear and targeted therapies are still lacking. In this study, we found that the expression of BRD4 was correlated with pro-inflammatory cytokines after SCI in rats; in vitro study in microglia showed that BRD4 inhibition either by lentivirus or JQ1 may both suppress the MAPK and NF-κB signalling pathways, which are the two major signalling pathways involved in inflammatory response in microglia. BRD4 inhibition by JQ1 not only blocked microglial M1 polarization, but also repressed the level of pro-inflammatory cytokines in microglia in vitro and in vivo. Furthermore, BRD4 inhibition by JQ1 can improve functional recovery and structural disorder as well as reduce neuron loss in SCI rats. Overall, this study illustrates that microglial BRD4 level is increased after SCI and BRD4 inhibition is able to suppress M1 polarization and pro-inflammatory cytokine production in microglia which ultimately promotes functional recovery after SCI., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Although a vast amount of research underlines the roles of the HR HPV E6 and E7 oncogenes in HPV-induced carcinogenesis of cervical cancer, it remains unclear whether these oncogenes are also involved in the resistance of the cancer against chemotherapy. We examined the role of the HPV16 E6 oncogene in cisplatin resistance by analyzing its expression in newly established cisplatin-sensitive versus -resistant cervical cancer cell lines (CC7, CC10). Resistant variants were obtained by interval exposure treatment with 1-2 μM cisplatin for 8-9 months. Our results demonstrate that the expression level of HPV16 E6 directly correlates with the extent of cisplatin resistance in novel as well as established (SiHa) drug resistant cervical cancer cell lines. Overexpression of HPV16 E6 in cisplatin-naïve cells rendered these cells more resistant to cisplatin. Reducing E6 expression by JQ1 treatment reversed the drug resistant phenotype and strongly enhanced chemoresponse only in HPV-positive cisplatin-resistant variants and not in HPV-negative C33A cervical cancer cells. The level of E6 directly correlated with the extent of cisplatin sensitivity and was shown to be increased in newly established drug-resistant cell line variants, while reducing E6 expression using Brd4-inhibitors enhanced chemoresponse when co-delivered with cisplatin. Inhibition of Brd4 could represent a new therapeutic option by increasing treatment response in cervical cancer cells and might allow lower cisplatin dosages, thus reducing negative side effects., (© 2018 UICC.)

SOX9 is a key transcription factor during cell differentiation, sex determination, and tumorigenesis. However, the detailed mechanisms of its targeting strategy remain elusive. To investigate possibilities of targeting SOX9 with epigenetic drugs and the precise underlying mechanisms, two human cancer cell lines were chosen as model systems, which showed high SOX9 expression and anti-tumorigenic effects upon loss of SOX9. Histone acetylation-related screening of a small panel of epigenetic drugs revealed that the bromodomain reader inhibitor JQ1 dramatically downregulated SOX9 through multiple regulation steps, namely, transcription, BRD4-SOX9 protein-protein interaction, and further protein stability. These findings suggest that BRD4 inhibition is a novel therapeutic strategy for diseases characterized by SOX9 overexpression., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The bromodomain and extraterminal family members are epigenetic readers and transcriptional coactivators which are critically involved in various biological processes including tumorigenesis. BRD4 has been increasingly appreciated as a key oncogene and promising anticancer target. Here, we sought to characterize the expression of BRD4 and its tumorigenic roles as well as therapeutic targeting in HNSCC. Methods: Expression of BRD4 mRNA and protein was determined by bioinformatics interrogation of publically available databases, primary HNSCC samples and 4NQO-induced HNSCC animal model. The tumorigenic roles of BRD4 in HNSCC were evaluated by genetic and pharmacological approach in vitro and in vivo . Therapeutic efficiency of BRD4 targeting by JQ1 was assessed in three preclinical models including xenograft model, 4NQO-induced model and patients-derived xenograft model. Gene candidates responsible for therapeutic effects of JQ1 were identified by transcriptional profiling in HNSCC cells after JQ1 exposure. Results: Significant upregulation of BRD4 was found in primary HNSCC samples and 4NQO-induced HNSCC model. Its overexpression associated with aggressive clinicopathological features and inferior overall and disease-free survival. BRD4 depletion by genetic silencing or pharmacological inhibition impaired cell proliferation, migration and invasion and reduced tumor growth and metastasis in vivo . Transcriptional profiling of HNSCC cells following JQ1 exposure identified hundreds of genes which might mediated its antitumor effects and enriched in cancer-relevant pathways. A novel prognostic risk score derived from JQ1-regulated genes was developed to stratify patients into subgroups with favorable or inferior prognosis. Conclusions: Our findings reveal that BRD4 serves as a novel and critical mediator underlying tumorigenesis and a robust prognostic biomarker in HNSCC. Therapeutic targeting of BRD4 represents a potent and promising strategy against HNSCC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Despite advances in understanding the molecular pathogenesis of acute myeloid leukaemia (AML), overall survival rates remain low. The ability to predict treatment response based on individual cancer genomics using computational modeling will aid in the development of novel therapeutics and personalize care. Here, we used a combination of genomics, computational biology modeling (CBM), ex vivo chemosensitivity assay, and clinical data from 100 randomly selected patients in the Beat AML project to characterize AML sensitivity to a bromodomain (BRD) and extra-terminal (BET) inhibitor. Computational biology modeling was used to generate patient-specific protein network maps of activated and inactivated protein pathways translated from each genomic profile. Digital drug simulations of a BET inhibitor (JQ1) were conducted by quantitatively measuring drug effect using a composite AML disease inhibition score. 93% of predicted disease inhibition scores matched the associated ex vivo IC 50 value. Sensitivity and specificity of CBM predictions were 97.67%, and 64.29%, respectively. Genomic predictors of response were identified. Patient samples harbouring chromosomal aberrations del(7q) or -7, +8, or del(5q) and somatic mutations causing ERK pathway dysregulation, responded to JQ1 in both in silico and ex vivo assays. This study shows how a combination of genomics, computational modeling and chemosensitivity testing can identify network signatures associating with treatment response and can inform priority populations for future clinical trials of BET inhibitors., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: To investigate the therapeutic mechanism of the BRD4 inhibitor JQ1 in SACC-83 cells and explore strategies to enhance its therapeutic potential., Material and Methods: SACC-83 cells were used in the experiment. Immunohistochemistry was used to assess BRD4 expression in SACC tissues and corresponding adjacent non-tumor tissues. Cell viability and proliferation were evaluated using the Cell Counting Kit-8 assay. Flow cytometry was used to quantitate apoptosis. Levels of cleaved caspase-3, BRD4, c-Myc, pEGFR (γ-1173), and EGFR were determined by quantitative real-time PCR and Western blot. To study the role of EGFR in JQ1 resistance, we generated EGFR knockdown SACC-83 cells by siRNA transfection., Results: Our study revealed that BRD4 was overexpressed and could be a treatment target in SACC. The BRD4 inhibitor JQ1 markedly inhibited c-Myc expression in SACC-83 cells, which produced modest therapeutic effects. Nevertheless, the EGFR pathway was strongly activated following JQ1 treatment, which led to JQ1 resistance. Combined JQ1 and PI3K inhibitor treatment effectively increased the therapeutic potential by inhibiting the EGFR and c-Myc signaling pathways in SACC-83 cells. Moreover, EGFR knockdown sensitized SACC-83 cells to JQ1., Conclusion: These data demonstrate that EGFR and c-Myc signaling synergistically drive SACC progression. The JQ1 and PI3K inhibitor combination exhibited a strong synergistic effect by suppressing c-Myc and EGFR in SACC-83 cells, identifying a novel rational combinatorial treatment. Moreover, EGFR expression influences the sensitivity of SACC-83 cells to JQ1, which is useful for planning treatment., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Diabetic cardiomyopathy is one of the major complications of diabetes, and due to the increasing number of patients with diabetes it is a growing concern. Diabetes‑induced cardiomyopathy has a complex pathogenesis and histone deacetylase‑mediated epigenetic processes are of prominent importance. The olfactory bromodomain‑containing protein 4 (BRD4) is a protein that recognizes and binds acetylated lysine. It has been reported that the high expression of BRD4 is involved in the process of cardiac hypertrophy. The aim of the present study was to investigate the function of BRD4 in the process of high glucose (HG)‑induced cardiac hypertrophy, and to clarify whether epigenetic regulation involving BRD4 is an important mechanism. It was revealed that BRD4 expression levels were increased in H9C2 cells following 48 h of HG stimulation. This result was also observed in a diabetic rat model. Furthermore, HG stimulation resulted in the upregulation of the myocardial hypertrophy marker, atrial natriuretic peptide, the cytoskeletal protein α‑actin and fibrosis‑associated genes including transforming growth factor‑β, SMAD family member 3, connective tissue growth factor and collagen, type 1, α1. However, administration of the specific BRD4 inhibitor JQ1 (250 nM) for 48 h reversed this phenomenon. Furthermore, protein kinase B (AKT) phosphorylation was activated by HG stimulation and suppressed by JQ1. In conclusion, BRD4 serves an important role in the pathogenesis of HG‑induced cardiomyocyte hypertrophy through the AKT pathway.

Inhibiting BRD4 has emerged as a promising anticancer strategy, and inhibitors such as JQ1 can suppress cell growth in oral squamous cell carcinoma (OSCC). However, the mechanism through which JQ1 exerts its anticancer activity has not been reported. Moreover, JQ1 does not markedly inhibit proliferation and increase apoptosis in OSCC when used as a monotherapy. Herein, we explore the mechanism of JQ1 in OSCC and probe ways to increase its therapeutic potential. In this study, we used two cell lines, Cal27, and Scc25. We found that BRD4 was highly expressed in OSCC tissues when compared with adjacent non-tumor tissues, and JQ1 worked through the EGFR-mediated signaling pathway in tumor cells. Furthermore, we demonstrated that JQ1 induced an increased treatment effect in vitro and in vivo when combined with a PI3K inhibitor. Interestingly, subsequent mechanistic analyses indicated that further suppressing EGFR and BRD4 expression was instrumental to this functional synergism. Moreover, we found that upregulating EGFR expression by EGF stimulation protected cells treated with JQ1 from apoptosis, while knockdown of EGFR before addition of JQ1 successfully mimicked the combination treatment results. In summary, our findings revealed that JQ1 can act by inhibiting the EGFR-mediated signaling pathway, and EGFR expression influences the sensitivity of OSCC to JQ1. Regarding clinical use, this study demonstrates that BRD4 is a novel therapeutic target and EGFR can be used as a biomarker to identify the most appropriate anti-BRD4 treatment strategy in OSCC., (© 2018 Wiley Periodicals, Inc.)

Background: Glioblastoma (GBM) is the most common and most malignant primary brain cancer in adults. Despite multimodality treatment, the prognosis is still poor. Therefore, further work is urgently required to discover novel therapeutic strategies for GBM treatment., Methods: The synergistic effects of cotreatment with the histone deacetylase (HDAC) inhibitor panobinostat and bromodomain inhibitor JQ1 or OTX015 were validated using cell viability assays in GBM cell lines. Furthermore, the inhibitory mechanisms were investigated via an EdU proliferation assay, an apoptosis assay, qPCR, Western blot and RNAseq analyses., Results: We found that the cotreatment with panobinostat and JQ1 or OTX015 synergistically inhibited cell viability in GBM cells. The cotreatment with panobinostat and JQ1 or OTX015 markedly inhibited cell proliferation and induced apoptosis in GBM cells. Compared with treatment with each drug alone, the cotreatment with panobinostat and JQ1 induced more profound caspase 3/7 activation and cytotoxicity. Mechanistic investigation showed that combination of panobinostat with JQ1 or OTX015 results in stronger repression of GBM-associated oncogenic genes or pathways as well as higher induction of GBM-associated tumor-suppressive genes., Conclusion: Our study demonstrated that HDAC inhibitor and bromodomain inhibitor had synergistical efficacy against GBM cells. The cotreatment with HDAC inhibitor and bromodomain inhibitor warrants further attention in GBM therapy.

Although the significance of BRD4 in the epigenetic memory and cancer genesis has been intensively investigated, little is known about its function and potential roles during the generation and progression of gastric cancer. We report here that BRD4 increases the proliferation and represses the apoptosis of gastric cancer cells through activating c-MYC via transcriptional and epigenetic regulation mechanisms. Expression analyses in both small and large cohort of sample show that BRD4 is highly expressed in gastric cancer tissues/cells when compared with the adjacent non-tumor tissues/normal cells. We also find a positive correlation between the expression of BRD4 and c-MYC in patient samples. The repression of BRD4 by siRNAs leads to the down-regulation of c-MYC in gastric cancer cells. Chromatin immunoprecipitation-qPCR and luciferase assays show that BRD4 binds to and coordinately activates c-MYC promoter, indicating that c-MYC is transcriptional target of BRD4 and BRD4 regulates its basal expression. Further evidence show that the histone acetylation inhibitor reduces the binding of BRD4 as well as the histone activation level on c-MYC promoter, and leads to the down-regulation of c-MYC, suggesting that BRD4 regulates the expression of c-MYC through epigenetic mechanism. Functionally, the suppression of BRD4 leads to growth inhibition and apoptosis in gastric cancer cells. Force expression of c-MYC alongside with BRD4 repression rescue the anti-cancer effects caused by BRD4 repression. Collectively, our data not only uncovered the mechanism of BRD4 in regulating the proliferation of gastric cancer cells but also provides a new therapeutic strategy for this type of cancer., (© 2017 Wiley Periodicals, Inc.)

Background: Malignant Pleural Mesothelioma (MPM) is an asbestos-associated tumor with poor prognosis and few therapeutic options. JQ1, a selective antagonist of BRD4, modulates transcription of oncogenes, including MPM chemoresistance-associated c-Myc and Fra-1., Objective: We investigated if JQ1 could enhance the efficacy of cisplatin against MPM., Methods: The antiproliferative activity of cisplatin in combination with JQ1 was assessed on MPM cell lines representative of the cellular phenotypes of this tumor (epithelioid, sarcomatoid and biphasic), and on one cisplatin resistant sub-line. The combination schedule was optimized adopting a 3Dspheroid model. Drug combination effects were correlated with cell cycle distribution and senescence- associated β-galactosidase positive cells. The expression of c-Myc and Fra-1 proteins and some apoptosis markers was assessed by immunoblotting and RT-qPCR. DNA damage and repair were evaluated by means of alkaline comet assay., Results: JQ1 in combination with cisplatin elicited additive or synergistic (superadditive) antiproliferative effects on MPM cells, depending on the cell line. The combination showed tumor regression on the 3D-spheroid model. It induced increased apoptosis, along with decreased c-Myc and, sometimes, Fra-1 expression. JQ1 decreased cisplatin-induced DNA breaks in all MPM cells and increased senescence even in less proficient cells, thus enhancing the DNA Damage Response (DDR)., Conclusion: The superadditive effect is due to c-Myc repression. The consequent DDR enhancement triggers to apoptosis induction and/or permanent growth arrest (senescence), depending on the MPM cellular context, leading to tumor regression. Thus, the pharmacological modulation of BET activity could represent a promising tool for future MPM therapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

BET proteins commonly activate cellular gene expression, yet inhibiting their recruitment paradoxically reactivates latent HIV-1 transcription. Here we identify the short isoform of BET family member BRD4 (BRD4S) as a corepressor of HIV-1 transcription. We found that BRD4S was enriched in chromatin fractions of latently infected T cells, and it was more rapidly displaced from chromatin upon BET inhibition than the long isoform. BET inhibition induced marked nucleosome remodeling at the latent HIV-1 promoter, which was dependent on the activity of BRG1-associated factors (BAF), an SWI/SNF chromatin-remodeling complex with known repressive functions in HIV-1 transcription. BRD4S directly bound BRG1, a catalytic subunit of BAF, via its bromodomain and extraterminal (ET) domain, and this isoform was necessary for BRG1 recruitment to latent HIV-1 chromatin. Using chromatin immunoprecipitation sequencing (ChIP-seq) combined with assay for transposase-accessible chromatin coupled to high-throughput sequencing (ATAC-seq) data, we found that the latent HIV-1 promoter phenotypically resembles endogenous long terminal repeat (LTR) sequences, pointing to a select role of BRD4S-BRG1 complexes in genomic silencing of invasive retroelements., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Lytic infection by the Epstein-Barr virus (EBV) poses numerous health risks, such as infectious mononucleosis and lymphoproliferative disorder. Proteins in the bromodomain and extraterminal (BET) family regulate multiple stages of viral life cycles and provide promising intervention targets. Synthetic small molecules can bind to the bromodomains and disrupt function by preventing recognition of acetylated lysine substrates. We demonstrate that JQ1 and other BET inhibitors block two different steps in the sequential cascade of the EBV lytic cycle. BET inhibitors prevent expression of the viral immediate-early protein BZLF1. JQ1 alters transcription of genes controlled by the host protein BACH1, and BACH1 knockdown reduces BZLF1 expression. BET proteins also localize to the lytic origin of replication (OriLyt) genetic elements, and BET inhibitors prevent viral late gene expression. There JQ1 reduces BRD4 recruitment during reactivation to preclude replication initiation. This represents a rarely observed dual mode of action for drugs.

Aim: BET proteins have been shown to regulate gene expression including inflammatory genes., Methods: In order to investigate the role of the BET proteins in immunoglobulin production we treated the human B-cell line CLNH11.4 and primary human B cells and ozone-exposed mice with BET inhibitors (JQ1 or IBET151)., Results: Both proliferation and IgG production were reduced by JQ1 in a concentration-dependent manner. JQ1 significantly reduced immunoglobulin gene transcription. In vivo treatment of ozone-exposed mice with the BET inhibitor IBET151 similarly inhibited ozone-induced immunoglobulin production. JQ1 did not reduce the protein levels of Brd4 or Oct2 per se but reduced the ability of Brd4 and Oct2 to co-immunoprecipitate and of Oct2 to bind to immunoglobulin gene promoters., Conclusion: Our results indicate that BET proteins including Brd4 play a crucial role regulation B-cell-specific gene expression and immunoglobulin production.

Background/aims: Renal cell carcinoma (RCC) remains an intractable genitourinary malignancy. Resistance to chemotherapy or targeted therapies in RCC is presumably due to the complicated underlying molecular mechanisms and insufficient understanding. The aim of this research was to assess the expression and role of bromodomain-4 protein (BRD4) in RCC and evaluate the effects of BRD4 inhibitor JQ1 for RCC treatment., Methods: BRD4 expressionlevels were assessed by qRT-PCR and western blot in RCC tissues and cells. The effects of BRD4 knockdown or JQ1 on RCC cells were assessed by MTT assay and flow cytometry. The effects of in vivo treatment were evaluated through xenograft experiments., Results: BRD4 is significantly overexpressed in RCC, and is related to tumor stage and lymph node metastasis. Inhibition of BRD4 suppressed RCC cell proliferation, induced cell apoptosis in vitro and repressed tumor growth in vivo. Inhibition of BRD4 decreased BCL2 and C-MYC expression while increased BAX and cleaved caspase3 expression, and strikingly diminished the recruitment of BRD4 to BCL2 promoter., Conclusions: Our research reveals that BRD4 probably play a critical role in RCC progression, and is a new promising target for pharmacological treatment directed against this intractable disease., (© 2017 The Author(s)Published by S. Karger AG, Basel.)

Bromodomain and extraterminal domain protein inhibitors (BETi) hold great promise as a novel class of cancer therapeutics. Because acquired resistance typically limits durable responses to targeted therapies, it is important to understand mechanisms by which tumor cells adapt to BETi. Here, through pooled shRNA screening of colorectal cancer cells, we identified tripartite motif-containing protein 33 (TRIM33) as a factor promoting sensitivity to BETi. We demonstrate that loss of TRIM33 reprograms cancer cells to a more resistant state through at least two mechanisms. TRIM33 silencing attenuates down-regulation of MYC in response to BETi. Moreover, loss of TRIM33 enhances TGF-β receptor expression and signaling, and blocking TGF-β receptor activity potentiates the antiproliferative effect of BETi. These results describe a mechanism for BETi resistance and suggest that combining inhibition of TGF-β signaling with BET bromodomain inhibition may offer new therapeutic benefits.

Deregulation of the epigenome component affects multiple pathways in the cancer phenotype since the epigenome acts at the pinnacle of the hierarchy of gene expression. Pioneering work over the past decades has highlighted that targeting enzymes or proteins involved in the epigenetic regulation is a valuable approach to cancer therapy. Very recent results demonstrated that inhibiting the epigenetic reader BRD4 has notable efficacy in diverse cancer types. We investigated the potential of BRD4 as a therapeutic target in liver malignancy. BRD4 was overexpressed in three different large cohort of hepatocellular carcinoma (HCC) patients as well as in liver cancer cell lines. BRD4 inhibition by JQ1 induced anti-tumorigenic effects including cell cycle arrest, cellular senescence, reduced wound healing capacity and soft agar colony formation in liver cancer cell lines. Notably, BRD4 inhibition caused MYC-independent large-scale gene expression changes in liver cancer cells. Serial gene expression analyses with SK-Hep1 liver cancer cells treated with JQ1 to delineate the key player of BRD4 inhibition identified E2F2 as the first line of downstream direct target of BRD4. Further experiments including chromatin immunoprecipitation (ChIP) assay and loss of function study confirmed E2F2 as key player of BRD4 inhibition. Overexpressed E2F2 is a crucial center of cell cycle regulation and high expression of E2F2 is significantly associated with poor prognosis of HCC patients. Our findings reveal BRD4-E2F2-cell cycle regulation as a novel molecular circuit in liver cancer and provide a therapeutic strategy and innovative insights for liver cancer therapies., Competing Interests: The authors have declared that no competing interests exist.

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.

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)