Bromodomain-containing Protein 4 Expression Research Articles

Abstract 7578: BRD4 protein degradation for p53 mutant diffuse intrinsic pontine glioma (DIPG) via various delivery approaches for ARV-825 by using camel milk exosomes

Abstract DIPGs (diffuse intrinsic pontine gliomas) are a particularly aggressive and malignant type of pediatric brain tumor. Given the tumor's location, surgery has a restricted role, and it is insensitive to most chemotherapies. Although fractionated radiation is the standard of therapy for DIPG, the cancer invariably worsens in 6-12 months. TP53 tumor suppressor protein mutations are found in 70-80% of DIPG tumors. These TP53 mutations have been linked to radiation resistance in DIPG patients. Our goal was to find new pharmacological compounds that would improve the radiation sensitivity of p53 mutant DIPG cell lines and to determine their molecular mechanism of action. Numerous bromodomain-containing protein 4 (BRD4) inhibitors have entered clinical trials in recent years, yielding promising outcomes in tumor treatment since BRD4 expression in DIPG is substantially higher than in neighboring normal brain tissue. So consequently, BRD4 is a target for DIPG treatment. ARV-825 is an emerging PROTAC (Proteolysis Targeting Chimera) compound which can cause rapid and persistent BRD4 protein degradation. However, delivering ARV-825 to pons is a major challenge owing to poor oral bioavailability of PROTACS. In this study, we used camel milk derived exosomes (CME) for delivery of ARV-825 to pons by both nasal and oral routes. To begin with, goat milk exosomes were also considered but CME was chosen because of its innate cytotoxicity against DIPG cell lines. CME were isolated using ultracentrifugation and nanoparticle tracking analysis and revealed a size distribution of 112.5 ± 2.8 nm, zeta potential of -26.38 ± 0.12 mV. CME (1*1011) were cytotoxic to both SF8628 (with a H3.3 K27M mutation) and SF188 (grade IV pediatric glioblastoma with wild type H3.3) cell lines and had 51% and 44% cell viability respectively. ARV-825 showed IC50 of 546.58 ± 22.14 nM and 608.94 ± 30.04 nM in SF8628 cells and SF188 cells respectively. ARV-825’s IC50 was lowered when ARV-825 loaded CME were used. ARV-825 was loaded using sonication and entrapment efficiency was found to be 42.96 ± 2.14 % by HPLC analysis. CME were labelled with Vybrant DiO (CME-DIO) and instilled into each nostril of Sprague Dawley rats daily. Brain sections were viewed with fluorescent microscope and CME-DIO were seen in olfactory region after 3 days and in the pons region after 5 days demonstrating the ability of CME to reach the target site. In-vitro permeability studies using MDCK cells showed approximately 9µg/mL (60%), 15µg/mL (80%) and 16µg/mL (100%) of ARV-825 permeation in 2, 4 and 24 hours respectively when loaded into CME. The results of the present study suggest the possibility of using CME as a viable delivery strategy for ARV-825 against DIPG. Further in vitro and in vivo studies are currently undergoing to validate the results and understand kinetics and the molecular mechanisms of ARV-825 loaded CME. Citation Format: Aakash Nathani, Mounika Aare, Li Sun, Yan Li, Mandip Singh. BRD4 protein degradation for p53 mutant diffuse intrinsic pontine glioma (DIPG) via various delivery approaches for ARV-825 by using camel milk exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7578.

Gallic Acid Alleviates Psoriasis Keratinization and Inflammation by Regulating BRD4 Expression.

Psoriasis is a chronic non-contagious autoimmune disease. Gallic acid is a natural compound with potential health benefits, including antioxidant, anticancer, antiviral and antibacterial properties. Nevertheless, the influence of gallic acid on psoriasis has not been fully determined. This investigation aimed to discover the effect of gallic acid on psoriasis. Thirty-one pairs of psoriatic skin tissues and healthy adult human skin tissues were collected. Human keratinocytes (HaCaT cells) were transfected with interleukin 17A (IL-17A) to create the psoriatic keratinocyte model. The content of bromodomain-containing protein 4 (BRD4) microRNA was assessed using qRT-PCR testing. The content of BRD4 was detected by Western blotting. Cell migration was evaluated by conducting a wound healing assay. Cell proliferation was determined using an EdU assay. Apoptosis was detected by the TUNEL assay. The contents of interferon gamma (IFN-γ), IL-6, IL-8 and IL-17 were detected by ELISA. BRD4 was up-regulated in psoriatic skin tissues and in the IL-17A group compared to the healthy adult human skin tissues and the control group. Silencing BRD4 inhibited cell migration, proliferation and inflammatory response but induced apoptosis in IL-17A-treated HaCaT cells. Conversely, BRD4 over-expression promoted cell migration, proliferation and inflammatory response but suppressed apoptosis in IL-17A-treated HaCaT cells. Gallic acid repressed cell migration, proliferation and inflammatory response but indu-ced apoptosis in HaCaT cells transfected with IL-17A by down-regulating BRD4. Gallic acid represses cell migration, proliferation and inflammatory response but induces apoptosis in IL-17A-transfected HaCaT cells by down-regulating BRD4.

BRD4 facilitates osteogenic differentiation of human bone marrow mesenchymal stem cells through WNT4/NF-κB pathway

BackgroundHuman bone marrow mesenchymal stem cells (hBMSCs) are a major source of osteoblast precursor cells and are directly involved in osteoporosis (OP) progression. Bromodomain-containing protein 4 (BRD4) is an important regulator for osteogenic differentiation. Therefore, its role and mechanism in osteogenic differentiation process deserve further investigation.MethodshBMSCs osteogenic differentiation was evaluated by flow cytometry, alkaline phosphatase assay and alizarin red staining. Western blot was used to test osteogenic differentiation-related proteins, BRD4 protein, WNT family members-4 (WNT4)/NF-κB-related proteins, and glycolysis-related proteins. Metabolomics techniques were used to detect metabolite changes and metabolic pathways. BRD4 and WNT4 mRNA levels were determined using quantitative real-time PCR. Dual-luciferase reporter assay and chromatin immunoprecipitation assay were performed to detect BRD4 and WNT4 interaction. Glycolysis ability was assessed by testing glucose uptake, lactic acid production, and ATP levels.ResultsAfter successful induction of osteogenic differentiation, the expression of BRD4 was increased significantly. BRD4 knockdown inhibited hBMSCs osteogenic differentiation. Metabolomics analysis showed that BRD4 expression was related to glucose metabolism in osteogenic differentiation. Moreover, BRD4 could directly bind to the promoter of the WNT4 gene. Further experiments confirmed that recombinant WNT4 reversed the inhibition effect of BRD4 knockdown on glycolysis, and NF-κB inhibitors (Bardoxolone Methyl) overturned the suppressive effect of BRD4 knockdown on hBMSCs osteogenic differentiation.ConclusionBRD4 promoted hBMSCs osteogenic differentiation by inhibiting NF-κB pathway via enhancing WNT4 expression.

Fucoxanthin inhibits cardiac fibroblast transdifferentiation by alleviating oxidative stress through downregulation of BRD4.

Myocardial fibrosis can lead to ischemic damage of the myocardium, which can be life-threatening in severe cases. Cardiac fibroblast (CF) transdifferentiation is an important process in myocardial fibrosis. Fucoxanthin (FX) plays a key role in ameliorating myocardial fibrosis; however, its mechanism of action is not fully understood. This study investigated the role of FX in the angiotensin II (Ang II)-induced transdifferentiation of CFs and its potential mechanisms of action. We found that FX inhibited Ang II-induced transdifferentiation of CFs. Simultaneously, FX downregulated bromodomain-containing protein 4 (BRD4) expression in CFs and increased nuclear expression of nuclear factorerythroid 2-related factor 2 (Nrf2). FX reverses AngII-induced inhibition of the Keap1/Nrf2/HO-1 pathway and elevates the level of reactive oxygen species (ROS). FX failed to reverse Ang II-induced changes in fibrosis-associated proteins and ROS levels after Nrf2 silencing. BRD4 silencing reversed the inhibitory effect of Ang II on the Keap1/Nrf2/HO-1 antioxidant signalling pathway. In conclusion, we demonstrated that FX inhibited Ang II-induced transdifferentiation of CFs and that this effect may be related to the activation of the Keap1/Nrf2/HO-1 pathway by reducing BRD4 expression and, ultimately, oxidative stress.

Dopamine Transporter Knockout Rats Display Epigenetic Alterations in Response to Cocaine Exposure.

(1) Background: There is an urgent need for effective treatments for cocaine use disorder (CUD), and new pharmacological approaches targeting epigenetic mechanisms appear to be promising options for the treatment of this disease. Dopamine Transporter (DAT) transgenic rats recently have been proposed as a new animal model for studying susceptibility to CUD. (2) Methods: DAT transgenic rats were treated chronically with cocaine (10 mg/kg) for 8 days, and the expression of epigenetic modulators, Lysine Demethylase 6B (KDM6B) and Bromodomain-containing protein 4 (BRD4), was examined in the prefrontal cortex (PFC). (3) Results: We show that only full knockout (KO) of DAT impacts basal levels of KDM6B in females. Additionally, cocaine altered the expression of both epigenetic markers in a sex- and genotype-dependent manner. In response to chronic cocaine, KDM6B expression was decreased in male rats with partial DAT mutation (HET), while no changes were observed in wild-type (WT) or KO rats. Indeed, while HET male rats have reduced KDM6B and BRD4 expression, HET female rats showed increased KDM6B and BRD4 expression levels, highlighting the impact of sex on epigenetic mechanisms in response to cocaine. Finally, both male and female KO rats showed increased expression of BRD4, but only KO females exhibited significantly increased KDM6B expression in response to cocaine. Additionally, the magnitude of these effects was bigger in females when compared to males for both epigenetic enzymes. (4) Conclusions: This preliminary study provides additional support that targeting KDM6B and/or BRD4 may potentially be therapeutic in treating addiction-related behaviors in a sex-dependent manner.

Downregulation of BRD4 attenuates high glucose-induced damage of trophoblast cells by inhibiting activation of AKT/mTOR pathway.

It was elucidated that bromodomain-containing protein 4 (BRD4) has involvement with diabetic complication. However, the role and molecular mechanism of BRD4 in gestational diabetes mellitus (GDM) are still unclear. In this study, the mRNA and protein contents of BRD4 in placenta tissues of GDM patients and high glucose (HG)-induced HTR8/SVneo cells were detected by qRT-PCR and western blot assay. CCK-8, EdU staining, flow cytometry as well as western blot were applied for the appraisement of cell viability and apoptosis. Wound healing assay and transwell assay were conducted for the assessment of cell migration and invasion. Oxidative stress and inflammatory factors were detected. Additionally, the contents of AKT/mTOR pathway-related proteins were estimated applying western blot. It was discovered that BRD4 expression was ascended in tissues and HG-induced HTR8/SVneo cells. BRD4 downregulation cut down the contents of p-AKT and p-mTOR but had no effects on the total protein levels of AKT or mTOR in HG-induced HTR8/SVneo cells. BRD4 depletion promoted cell viability, enhanced proliferative capability, and reduced cell apoptotic level. Moreover, BRD4 depletion facilitated cell migrative and invasive capabilities, and repressed the oxidative stress as well as inflammatory damage in HG-induced HTR8/SVneo cells. The activation of Akt reversed the protective impacts of BRD4 depletion on HG-induced HTR8/SVneo cells. To sum up, BRD4 silencing may alleviate HG-induced HTR8/SVneo cell damage through the inhibition of the AKT/mTOR pathway.

Sevoflurane attenuates myocardial ischemia/reperfusion injury by up-regulating microRNA-99a and down-regulating BRD4.

It has been explored that sevoflurane (Sevo) is cardioprotective in myocardial ischemia/reperfusion injury (MI/RI) and mediates microRNA (miRNA) expression that control various physiological systems. Enlightened by that, the work was programmed to decode the mechanism of Sevo and miR-99a with the participation of bromodomain-containing protein 4 (BRD4). MI/RImodel was established on mice. MI/RI modeled mice were exposed to Sevo or injected with miR-99a or BRD4-related vectors to identify their functions in cardiac function, pathological injury, cardiomyocyte apoptosis, inflammation, and oxidative stress in MI/RI mice. MiR-99a and BRD4 expression in myocardial tissues were tested, and their relation was further validated. MiR-99a was down-regulated, and BRD4 was up-regulated in MI/RI mice. Sevo up-regulated miR-99a to inhibit BRD4 expression in myocardial tissues of MI/RI mice. Sevo improved cardiac function, relieved myocardial injury, repressed cardiomyocyte apoptosis, and alleviated inflammation and oxidative stress in mice with MI/RI. MiR-99a restoration further enhanced the positive effects of Sevo on mice with MI/RI. Overexpression of BRD4 reversed up-regulation of miR-99a-induced attenuation of MI/RI in mice. The work delineated that Sevo up-regulates miR-99a to attenuate MI/RI by inhibiting BRD4.

Early-immediate gene Egr1 is associated with TGFβ1 regulation of epigenetic reader Bromodomain-containing protein 4 via the canonical Smad3 signaling in hepatic stellate cells in vitro and in vivo.

Upon chronic damage to the liver, multiple cytokines stimulate hepatic stellate cells (HSCs), causing the alterations of gene expression profiles and thus leading to HSC activation, a key step in liver fibrogenesis. Activated HSCs are the dominant contributors to liver fibrosis. Bromodomain containing protein 4 (BrD4), an important epigenetic reader, was demonstrated to concentrate on hundreds of enhancers associated with genes involved in multiple profibrotic pathways, thereby directing HSC activation and the fibrotic responses. The present studies were designed to examine the effect of transforming growth factor beta-1 (TGFβ1), the most potent pro-fibrotic cytokine, on BrD4 expression in HSCs and, if so, elucidated the underlying mechanisms in vitro and in vivo. The experiments employed the heterogeneous TGFβ1 knockout (TGFβ1+/- ) mice, gene knockdown in vivo, and a model of thioacetamide (TAA)-induced liver injury. The results revealed that TGFβ1 enhanced BrD4 expression in HSCs, which was mediated, at least, by Smad3 signaling and early-immediate gene Egr1 (early growth response-1). TGFβ1-induced Smad3 signaling increased Egr1 expression and promoted Egr1 binding to BrD4 promoter at a site around -111 bp, promoting BrD4 expression. Egr1 knockdown reduced BrD4 expression in HSCs in a mouse model of TAA-induced liver injury and lessened liver fibrosis. Double fluorescence staining demonstrated a strong increase in BrD4 expression in activated HSCs in fibrotic areas of the human livers, paralleling the upregulation of p-Smad3 and Egr1. This research suggested novel molecular events underlying the roles of the master pro-fibrotic cytokine TGFβ1 in HSC activation and liver fibrogenesis.

Inhibition of BRD4 decreases fibrous scarring after ischemic stroke in rats by inhibiting the phosphorylation of Smad2/3

AimsFibrous scarring may play a much more important role in preventing secondary expansion of tissue damage and hindering repair and regeneration than glial scarring after central nervous system (CNS) injury. However, relatively little is known about how fibrous scars form and how fibrous scar formation is regulated after CNS injury. Bromodomain-containing protein 4 (BRD4) is involved in fibrosis in many tissues, and transforming growth factor-β1 (TGF-β1)/Smad2/3 signaling is one of the critical pathways of fibrosis. However, it is unclear whether and how BRD4 affects fibrous scar formation after ischemicbraininjury. In the present study, whether BRD4 can regulate the formation of fibrous scars after ischemic stroke via TGF-β1/Smad2/3 signaling was assessed. Materials and methodsPrimary meningeal fibroblasts isolated from neonatal SD rats were treated with TGF-β1, SB431542 (a TGF-β1 receptor inhibitor) and JQ1 (a small-molecule BET inhibitor that can also inhibit BRD4). BRD4 was knocked down in adult Sprague–Dawley (SD) rats by using adenovirus before middle cerebral artery occlusion/reperfusion (MCAO/R) injury. The proliferation and migration of meningeal fibroblasts in vitro were evaluated with the Cell Counting Kit-8 (CCK-8) assay and scratch test, respectively. Neurological function was assessed with Longa scores, modified Bederson Scores and modified neurological severity scores (mNSSs). The infarct volume was assessed with TTC staining. The protein expression of synaptophysin (SY), BRD4, Smad2/3, p-Smad2/3, α-smooth muscle actin (α-SMA), collagen-1 (COL1) and fibronectin (FN) in vivo and in vitro was examined with immunocytochemistry, immunofluorescence, and Western blotting. Key findingsBRD4 expression was upregulated in a TGF-β1-induced meningeal fibroblast fibrosis model and was downregulated by the TGF-β1 receptor inhibitor SB431542 in vitro. JQ1, a small-molecule BET inhibitor, inhibited BRD4 and decreased TGF-β1-induced meningeal fibroblast proliferation, migration and activation. Furthermore, MCAO/R injury induced fibrosis and upregulated BRD4 expression in the cerebral infarct center. BRD4 knockdown by adenovirus inhibited fibrous scarring, promoted synaptic survival, decreased the infarct volume, and improved neurological function after MCAO/R injury. Moreover, inhibition of BRD4, either by JQ1 in vitro or adenovirus in vivo, decreased the phosphorylation of Smad2/3. ConclusionsThis study is the first to indicate that inhibition of BRD4 delays fibrous scarring after ischemic stroke through mechanisms involving the phosphorylation of Smad2/3.

Abstract 421: PLX-3618, a potent, selective monovalent BRD4 degrader demonstrates activity in models of prostate cancer

Abstract Prostate cancer is the second most common cause of cancer related deaths in men in the United States. Pathogenesis is driven by the androgen receptor (AR), which has led to front-line treatment modalities that are based on androgen deprivation therapy (ADT). About 10-20% of all prostate cancers evolve to resist ADT and are classified as castration-resistant prostate cancer (CRPC) indicating the continued need for new treatment options. Bromodomain-containing protein 4 (BRD4) is an acetylated-chromatin associating protein that is involved in transcriptional elongation, mRNA splicing, epigenetic bookmarking, and super-enhancer activity. The BRD4 protein has been shown to both bind and colocalize with AR at androgen response elements (AREs) on chromatin. Furthermore, elevated BRD4 expression is prognostic of increased prostate specific antigen (PSA) levels following radical prostatectomy and is correlated with higher Gleason scores and poor overall survival. The intertwined activity of BRD4 with multiple essential driver mechanisms of prostate cancer suggests it may be a key target for developing novel therapeutics. Using our ultra-high throughput cell-based screening platform, which directly measures degradation of pathogenic proteins upon exposure to diverse chemical libraries, we identified a series of novel monovalent BRD4 degraders that was optimized to produce our lead compound PLX-3618. A cancer cell panel screen for antiproliferative effects of PLX-3618 indicated enhanced sensitivity in subsets of prostate cancer lines. PLX-3618 elicited selective, rapid, and deep degradation of BRD4 protein in prostate cancer cell models, without degrading the closely related BRD2 and BRD3 proteins. Addition of either proteosome or neddylation inhibitors blocked BRD4 degradation indicating a ubiquitin-proteosome system mediated clearance mechanism. Degradation of BRD4 led to sustained multimodal inhibition of the AR pathway and disruption of key oncogene enhancer networks. When compared to pan-BET inhibitors, treatment of select prostate cancer cell lines with PLX-3618 resulted in increased levels of the tumor suppressors p53 and p21, an aberrant DNA damage response, and substantially amplified apoptosis. Finally, in in vivo preclinical models of prostate cancer, PLX-3618 showed far superior efficacy over a pan-BET inhibitor. Taken together, the selective degradation of BRD4 via the potent monovalent degrader PLX-3618 represents a novel strategy in treating prostate cancer. Citation Format: Kenneth Steadman, Gregory S. Parker, Geoffray Leriche, Sarah Fish, Julia Toth, Mary E. Spalding, Elizabeth Daniele, Aleksandar Jamborcic, Xiaoming Li, E Adam Kallel, Farhana Barmare, Kenneth Chng, Erika Green, Michael Hocker, Elliot Imler, Yi Zhang, Peggy A. Thompson, Simon Bailey. PLX-3618, a potent, selective monovalent BRD4 degrader demonstrates activity in models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 421.

Design, synthesis, and anticancer evaluation of ammosamide B with pyrroloquinoline derivatives as novel BRD4 inhibitors

Bromodomain-containing protein 4 (BRD4), which is a member of the bromodomain and extra-terminal domain (BET) family, plays an important role in the regulation of gene expression as the “reader” of epigenetic regulation. BRD4 has become a promising target to treat cancer, because the up-regulation of BRD4 expression is closely associated with the occurrence and development of various cancers. At present, several BRD4 inhibitors are in clinical trials for cancer therapy, but no BRD4 inhibitors are on the market. Here, we designed and synthesized a series of compounds bearing pyrrolo[4,3,2-de]quinolin-2(1H)-one scaffold through structural modification of natural products ammosamide B, which is a natural pyrroloquinoline derivative reported for its potential antitumor activity. All target compounds were evaluated for their BRD4 BD1 inhibition activities via the protein thermal shift assays or AlphaSceen assay. The representative compound 49 showed potent activity (IC50 = 120 nM). The co-crystal of compound 49 with BRD4 BD1 was solved to study the structure activity relationship, which showed that 49 could combine with the acetyl lysine binding site and formed a hydrogen bond with the conserved residue Asn140. The results demonstrate that compound 49 is worthy of further investigation as a promising BRD4 inhibitor.

Pathological stage-associated non-coding RNA long intergenic non-protein coding RNA 1234 (LINC01234) participation in cell cycle regulation in adrenocortical carcinoma through bromodomain-containing protein 4 (BRD4) expression mediation via sponging microRNA (miR)-140-3p

ABSTRACT Many researches indicated that long non-coding RNAs (lncRNAs) were involved in the malignant progression of tumors, including Adrenocortical Carcinoma (ACC). However, as for most lncRNAs, their biological behaviors and molecular mechanism remain unclear in ACC. In the present research, weighted gene co-expression network analysis (WGCNA) was used to identify pathologically relevant gene, including lncRNAs. By comparing their expressions in GSE61359 tumors and normal controls, long intergenic non-protein coding RNA 1234 (LINC01234) was selected to investigate the clinical significance, biological function, and mechanism in ACC. Data mining revealed that LINC01234 expression was significantly up-regulated in ACC patients, and a shorter survival time presents in patients with higher LINC01234 expression compared to that in patients with lower LINC01234 expression. Further, LINC01234 silencing resulted in cells growth arrest in vitro and in vivo. Mechanism studies suggested that LINC01234 silencing induced cell cycle arrest, and bromodomain-containing protein 4 (BRD4) overexpression could restore this phenomenon. Further research showed that LINC01234 could mediate BRD4 expression through competitively sequestering microRNA (miR)-140-3p, as evidenced by the positive correlation of LINC01234 with BRD4 and inverse correlation with miR-140-3p expression. Luciferase activity assay also verified the targeting relationship between LINC01234, BRD4 and miR-140-3p. And up-regulated LINC01234 in ACC cells significantly reversed the degradation of BRD4 by miR-140-3p. Collectively, we deduce that LINC01234 functions as a ceRNA to regulate BRD4 expression by sponging miR-140-3p in ACC progress. Our findings have the potential to provide a new target for the diagnosis and treatment of ACC.

BRD4‐MK2 signaling: target for Crohn’s Disease‐associated fibrosis

IntroductionFibrosis is the major complication of Crohn’s disease (CD). No targeted therapies are currently available to revert CD‐associated fibrosis. While inflammatory mechanisms in CD have been extensively investigated, understanding the pathogenesis of fibrosis is relatively limited. Mesenchymal cells are the major effectors in profibrotic processes in CD. A dramatic increase in α‐SMA expressing subsets of mesenchymal cells, also known as myofibroblasts (MF), is among the main hallmarks of profibrotic changes in CD. Mitogen‐activated protein kinase‐activated protein kinase 2 (MK2) is involved in the increase of α‐SMAexpression by MFs. Along with an increase in MK2 activity in CD we also observed an increase in the bromodomain‐containing Protein 4 (BRD4), an important epigenetic reader. BRD4 is critical for the expression of enhancer‐mediated profibrotic genes and, thus, is a potential novel regulator of MK2. However, the presence of mechanistic link between BRD4 and MK2 activity with respect to profibrotic activation of MFs in CD is unknown. Herein, we tested hypothesis that BRD4 mediated increase in MK2 activity is critical to the profibrotic reprogramming of MFs in CD and this pathway has potential for novel anti‐fibrotic therapy.MethodsHuman normal (N)‐ and CD‐derived MFs were analyzed using RNAseq, cytokine/chemokine multiplex array, FACS, and immunofluorescent microscopy. The T cell transfer murine colitis model relevant to CD was also used in this study.ResultsAn Increase in BRD4 and MK2 expression was observed in primary human CD‐ when compared to N‐MFs and were associated with the expression of the profibrotic genes including Col1α2, α‐SMA, TNC, Fn1, and TGF‐β1. Inhibition of MK2 activity in CD‐MFs with the MK2 specific inhibitor PF‐3644022 and murine MK2 KO MFs showed significantly reduced basal and TGF‐β1‐induced profibrotic gene expression. Use of the BRD4 specific inhibitor ZL0454 significantly reduced MK2 mRNA expression in CD‐MFs Additionally, ex vivo treatment of the CD tissues with ZL0454 also abrogated MK2 expression. Therapeutic use of the BRD4 inhibitor ZL0454 (10 mg/kg, daily for 10 days) in T cell transfer murine colitis significantly decreased MK2 expression and downstream profibrotic responses as measured by collagen deposition and expression of fibrosis‐related genes.ConclusionsOur data suggests that BRD4 positively regulates MK2 expression in CD. Further, BRD4‐MK2 signaling is critical to profibrotic activation of mesenchymal cells in CD and is a potential therapeutic target to reverse CD‐associated fibrosis.

Post-Translational Modifications of BRD4: Therapeutic Targets for Tumor.

Bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extraterminal (BET) family, is considered to be a major driver of cancer cell growth and a new target for cancer therapy. Over 30 targeted inhibitors currently in preclinical and clinical trials have significant inhibitory effects on various tumors, including acute myelogenous leukemia (AML), diffuse large B cell lymphoma, prostate cancer, breast cancer and so on. However, resistance frequently occurs, revealing the limitations of BET inhibitor (BETi) therapy and the complexity of the BRD4 expression mechanism and action pathway. Current studies believe that when the internal and external environmental conditions of cells change, tumor cells can directly modify proteins by posttranslational modifications (PTMs) without changing the original DNA sequence to change their functions, and epigenetic modifications can also be activated to form new heritable phenotypes in response to various environmental stresses. In fact, research is constantly being supplemented with regards to that the regulatory role of BRD4 in tumors is closely related to PTMs. At present, the PTMs of BRD4 mainly include ubiquitination and phosphorylation; the former mainly regulates the stability of the BRD4 protein and mediates BETi resistance, while the latter is related to the biological functions of BRD4, such as transcriptional regulation, cofactor recruitment, chromatin binding and so on. At the same time, other PTMs, such as hydroxylation, acetylation and methylation, also play various roles in BRD4 regulation. The diversity, complexity and reversibility of posttranslational modifications affect the structure, stability and biological function of the BRD4 protein and participate in the occurrence and development of tumors by regulating the expression of tumor-related genes and even become the core and undeniable mechanism. Therefore, targeting BRD4-related modification sites or enzymes may be an effective strategy for cancer prevention and treatment. This review summarizes the role of different BRD4 modification types, elucidates the pathogenesis in the corresponding cancers, provides a theoretical reference for identifying new targets and effective combination therapy strategies, and discusses the opportunities, barriers, and limitations of PTM-based therapies for future cancer treatment.

Privileged Scaffolds Targeting Bromodomain-containing Protein 4.

In recent years, bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra terminal domain (BET) family, has been one of the most widely studied targets. BRD4 is a transcriptional regulation factor, which regulates cell transcription, marks mammalian biological mitosis, regulates cell cycle, and plays an important role in the biological process of cancer occurrence and development. It has been demonstrated that the imbalance or dysfunction of BRD4 expression leads to various types of cancers, including testicular gene nuclear protein melanoma, acute myeloid leukemia, colon cancer, breast cancer, liver cancer, and midline cancer. Therefore, inhibition of BRD4 has become a valuable approach in the treatment of these cancers. To date, there are numerous BRD4 inhibitors in preclinical development, some of which have entered human clinical trials. In this review, current progress in the development of privileged scaffolds designed as BRD4 inhibitors will be discussed by focusing on structure-activity relationship, selectivity, and mechanisms of action.

PLEK2 promotes the proliferation and migration of non-small cell lung cancer cells in a BRD4-dependent manner.

It has been reported that Pleckstrin 2 (PLEK2) acts as an oncogene in non-small cell lung cancer (NSCLC). Bromodomain containing protein 4 (BRD4), an important transcriptional regulator of tumorigenesis, has been shown to play a key role in NSCLC. However, whether BRD4 regulates the transcription of PLEK2 and further promotes the proliferation and migration of NSCLC remains unknown. In this study, we performed western blotting, real-time quantitative polymerase chain reaction, immunofluorescence, cell scratch wound assay and chromatin immunoprecipitation. According to these results, we found that PLEK2 plays a tumor‑promoting role in NSCLC via the PI3K/AKT signaling pathway. Moreover, BRD4 expression is significantly upregulated in NSCLC cell lines and suppression of BRD4 expression by siBRD4 and JQ-1 inhibits NSCLC cell lines proliferation and migration. Prominently, we first confirmed that BRD4 binds to the promoter region of the PLEK2 gene, which explains the mechanism by which BRD4 regulates the transcription of PLEK2 gene from the perspective of epigenetics. The present study suggested that PLEK2 promotes the proliferation and migration of NSCLC in a BRD4-dependent manner and provided key insights into the potential avenues for preventing and treating NSCLC.

Discovery of indol-6-yl-pyrrolo[2,3-c]pyridin-7-one derivatives as bromodomain-containing protein 4 (BRD4) inhibitors for the treatment of kidney fibrosis

Uncovering new therapeutics for kidney fibrosis hold promise for chronic kidney disease (CKD). Considerable studies confirmed that BRD4 inhibition ameliorated kidney injury and fibrosis. In the study, we synthesized a series of indol-6-yl-pyrrolo[2,3-c]pyridin-7-one derivatives and biologically evaluated against BRD4 for structure-activity relationship (SAR). Notably, compound 3r (ZLD2218) exhibited the most potent inhibitory activity against BRD4, with the IC50 value of 107 nM, which was comparative to 92 nM of positive control JQ-1. Importantly, at the dose of 15 and 30 mg/kg/d for consecutive 8 days, ZLD2218 alleviated kidney injury and fibrosis in unilateral ureteral obstruction (UUO) mice, with the 30 mg/kg/d being competitive to 100 mg/kd/d of JQ1. Mechanically, ZLD2218 inhibited BRD4 expression and further suppressed fibrotic signaling in the kidneys of UUO mice and TGF-β1-stimulated TCMK-1 cells. Furthermore, ZLD2218 at the dose of 30 mg/kg/d for 8 days to C57BL/6J mice did not affect liver, kidney function and organ pathological changes. Collectively, compound 3r (ZLD2218) might be a promising lead compound of BRD4 inhibitor for the treatment of kidney fibrosis.

BRD4 Inhibition Attenuates Inflammatory Pain by Ameliorating NLRP3 Inflammasome-Induced Pyroptosis.

Chronic pain, such as persistent inflammatory pain, remains a public health problem that has no effective treatment at present. Bromodomain-containing protein 4 (BRD4) inhibition, induced by JQ1 injection or BRD4 knockdown, has been used to attenuate inflammatory pain; However, it remains elusive whether BRD4 aggravates inflammatory pain by regulating inflammasome. Western blot and immunofluorescence staining showed that BRD4 expression increased after administration of complete Freund’s adjuvant (CFA) and reached its peak on day 3. Immunofluorescence staining showed that BRD4 was mainly colocalized with NeuN-positive neurons in the spinal cord, which was accompanied by upregulation of inflammasome component proteins, such as NLRP3, gasdermin D, and caspase-1. JQ1 was intrathecally injected into mice 1 h before CFA administration, and the mechanical and thermal hyperalgesia levels were measured on days 1, 3, and 7 after CFA administration. CFA-induced inflammatory pain, paw inflammation, and swelling were attenuated by pre-treatment with JQ1. To our knowledge, this study was the first to prove that NLRP3 inflammasome-induced neuronal pyroptosis participates in inflammatory pain. BRD4 inhibition decreased the expression of pyroptosis-related proteins by inhibiting the activation of NF-κB signaling pathway, both in vivo and in vitro. Taken together, BRD4 inhibition exerted analgesic and anti-inflammatory effects against inflammatory pain by inhibiting NF-κB and inflammasome activation, which protected neural cells from pyroptosis.

In Vivo Delivery of siRNAs Targeting EGFR and BRD4 Expression by Peptide-Modified Redox Responsive PEG-PEI Nanoparticles for the Treatment of Triple-Negative Breast Cancer.

The study aims to investigate the in vivo distribution, antitumor effect, and safety of cell membrane-penetrating peptide-modified disulfide bond copolymer nanoparticles loaded with small-interfering RNA (siRNA) targeting epidermal growth factor receptor (EGFR) and bromodomain-containing protein 4 (BRD4) in triple-negative breast cancer (TNBC). Polyethylene glycol disulfide bond-linked polyethylenimine (PEG-SS-PEI) was modified with peptides GALA and CREKA and used as vectors to prepare siRNA nanoparticles. The GALA- and CREKA-modified PEG-SS-PEI nanoparticles (GC-NPs) were prepared by mixing siEGFR and siBRD4 (1:1) with GALA-PEG-SS-PEI and CREKA-PEG-SS-PEI (1:1) in an aqueous solution at an N/P ratio of 30:1. Nanoparticles loaded with scrambled siRNA were prepared with the same method. The gene silencing effect on EGFR and BRD4 in vitro was evaluated by Western blotting analysis. TNBC xenograft models were established by subcutaneous injection of MDA-MB-231 cells into female nude mice. At 1, 3, 6, 12, and 24 h after administration of five formulations of Cy5-siRNA (133 μg/10 g) via the tail vein, the mice were observed and imaged for a biodistribution study using an in vivo imaging system. In the pharmacodynamics experiment, tumor-bearing mice were treated with respective siRNA preparations at a dose of 133 μg/10 g for 18 days, and the body weight and tumor size were recorded every other day. The protein expression levels of EGFR, p-EGFR, PI3K, p-PI3K, Akt, p-Akt, BRD4, and c-Myc were determined using Western blotting analysis. Hematological and serum biochemical parameters, organ indices, and HE staining results for the heart, liver, spleen, lung, and kidney were analyzed to evaluate the safety of the nanoparticles. GC-NPs loaded with siEGFR and siBRD4 significantly inhibited the expression of EGFR and BRD4 in vitro. The strongest fluorescence signals were observed in the GC-NP group, especially in tumors, indicating the excellent tumor-targeted delivery of GC-NPs we constructed. Tumor growth was significantly inhibited in the GC-NP-treated group, and the expression of EGFR, p-EGFR, PI3K, p-PI3K, Akt, p-Akt, BRD4, and c-Myc in the tumors decreased by 71%, 68%, 61%, 68%, 48%, 58%, 59%, and 74% compared to the control group, respectively. There was no significant change in hematological parameters, biochemical indices, or tissue morphology in GC-NP-treated mice. SiRNA cotargeting EGFR and BRD4 delivered by GALA- and CREKA-modified PEG-SS-PEI had favorable antitumor effects in vivo toward TNBC with tumor-targeting efficacy and good biocompatibility.

Epigenetic regulator BRD4 is involved in cadmium-induced acute kidney injury via contributing to lysosomal dysfunction, autophagy blockade and oxidative stress

Cadmium (Cd) is a known nephrotoxic heavy metal and proximal tubules are the major target of Cd-induced acute kidney injury (AKI). We previously demonstrated that lysosomal dysfunction and dysregulated autophagy contribute to Cd-induced AKI. Recent studies have revealed that bromodomain-containing protein 4 (BRD4) is a transcriptional repressor of autophagy and lysosomal function. Hence, in vivo and in vitro studies were performed to clarify the role of BRD4 in Cd-induced AKI. Firstly, Cd has no effect on BRD4 expression levels, but increases H4K16 acetylation. Resultantly, Cd promotes the recruitment of BRD4 to lysosomal gene promoter regions to make it as a transcriptional regulator. Pharmacological and genetic inhibition of BRD4 alleviates Cd-inhibited lysosomal gene transcript levels and lysosomal function, leading to the alleviation of Cd-induced autophagy inhibition. Moreover, inhibition of BRD4 relieves Cd-induced oxidative stress and concurrent cytotoxicity, which is counteracted by the inhibition of autophagy via Atg5 knockdown, indicating that alleviation of oxidative stress by BRD4 inhibition is ascribed to its restoration of autophagic flux. Collectively, these results demonstrate that BRD4 acts as a transcriptional repressor to mediate lysosomal dysfunction, autophagy blockade and oxidative stress during Cd exposure, which may be a potential therapeutic target for Cd-induced AKI.