Role Of Bromodomain-containing Protein 4 Research Articles

BRD4 inhibitors broadly promote erastin-induced ferroptosis in different cell lines by targeting ROS and FSP1

Ferroptosis, an iron-dependent form of programmed cell death, is a promising strategy for cancer treatment. Bromodomain-containing protein 4 (BRD4) is an epigenetic reader and a promising target for cancer therapeutics. However, the role of BRD4 in ferroptosis is controversial and the value of the interaction between BRD4 inhibitors and ferroptosis inducers remains to be explored. Here, we found that BRD4 inhibition greatly enhanced erastin-induced ferroptosis in different types of cells, including HEK293T, HeLa, HepG2, RKO, and PC3 cell lines. Knocking down BRD4 in HEK293T and HeLa cells also promoted erastin-induced cell death. BRD4 inhibition by JQ-1 and I-BET-762 or BRD4 knockdown resulted in substantial accumulation of reactive oxygen species (ROS) in both HEK293T and HeLa cells. The effect of BRD4 inhibition on ferroptosis-associated genes varied in different cells. After using BRD4 inhibitors, the expression of FTH1, Nrf2, and GPX4 increased in HEK293T cells, while the levels of VDAC2, VDAC3, and FSP1 decreased. In HeLa cells, the expression of FTH1, VDAC2, VDAC3, Nrf2, GPX4, and FSP1 was reduced upon treatment with JQ-1 and I-BET-762. Consistently, the level of FSP1 was greatly reduced in HEK293T and HeLa cells with stable BRD4 knockdown compared to control cells. Furthermore, ChIP-sequencing data showed that BRD4 bound to the promoter of FSP1, but the BRD4 binding was greatly reduced upon JQ-1 treatment. Our results suggest that ROS accumulation and FSP1 downregulation are common mechanisms underlying increased ferroptosis with BRD4 inhibitors. Thus, BRD4 inhibitors might be more effective in combination with ferroptosis inducers, especially in FSP1-dependent cancer cells.

Abstract 15185: BRD4-Mediated Genome Folding Controls Fibroblast Cell State Transition

Introduction: Cardiac fibrosis is mediated by the activation of fibroblasts to myofibroblasts, a cell state transition which involves the coordination of expression of hundreds of genes. Previous studies have demonstrated that inhibition of bromodomain and extra-terminal domain (BET) proteins attenuates fibrosis. We recently discovered a novel role of bromodomain-containing protein 4 (BRD4), a BET family member, in maintaining genome folding by stabilizing the cohesin complex (necessary for genome-genome interactions). We hypothesize that BRD4-mediated genome folding is critical for maintaining the enhancer-promoter interactions at genes required for fibroblast activation. Methods: A cardiac fibroblast cell line was generated in which a degron epitope tag was appended biallelically to Brd4 to enable acute BRD4 degradation. Fibroblasts were activated via addition of TGFβ. Meox1 , a critical transcription factor mediating a broad fibrotic gene program, was used as a model locus to investigate chromatin looping. The proximity between the Meox1 enhancer and promoter, Meox1 expression, and protein occupancy at the locus were determined using DNA fluorescence in situ hybridization, RT-qPCR, and chromatin immunoprecipitation (ChIP)-qPCR, respectively. Results: The proximity between the Meox1 enhancer and promoter and Meox1 expression increased upon TGFβ-induced activation. BRD4 occupancy was enriched at the enhancer of Meox1 upon activation, and BRD4 depletion reduced Meox1 expression and the co-localization of the Meox1 enhancer and promoter. BRD4 physically interacts with the cohesin agonist NIPBL, and we found that co-depletion of BRD4 and a cohesin antagonist normalizes the Meox1 enhancer and promoter proximity and Meox1 expression levels. Ongoing studies include genome-wide occupancy studies and expanding our findings to in vivo models of cardiac fibrosis. Conclusions: Our studies provide a mechanistic understanding of the functional relevance of genome organization during fibroblast activation. These studies will also expand our knowledge on how genome folding regulates cell plasticity and inform therapeutic approaches for targeting pathologic fibrotic remodeling.

Bromodomain-containing protein 4 inhibitor JQ1 promotes melanoma cell apoptosis by regulating mitochondrial dynamics.

Although the role of bromodomain‐containing protein 4 (BRD4) in ovarian cancer, pancreatic cancer, lymphoma, and many other diseases is well known, its function in cutaneous melanoma is only partially understood. The results of the present study show that the BRD4 inhibitor JQ1 promotes the apoptosis of B16 melanoma cells by altering mitochondrial dynamics, thereby inducing mitochondrial dysfunction and increasing oxidative stress. We found that treatment of B16 cells with different concentrations of JQ1 (125 nmol/L or 250 nmol/L) significantly downregulated the expression of protein subunits involved in mitochondrial respiratory chain complexes I, III, IV, and V, increased reactive oxygen species, induced energy metabolism dysfunction, significantly enhanced apoptosis, and activated the mitochondrial apoptosis pathway. At the same time, JQ1 inhibited the activation of AMP‐activated protein kinase, a metabolic energy sensor. In addition, we found that the mRNA and protein levels of mitochondrial dynamin‐related protein 1 increased, whereas the levels of mitochondrial fusion protein 1 and optic atrophy protein 1 decreased. Mechanistically, we determined that JQ1 inhibited the expression of c‐Myc and altered mitochondrial dynamics, eventually leading to changes in the mitochondrial function, metabolism, and apoptosis of B16 melanoma cells.

A Bromodomain-Containing Protein 4 (BRD4) Inhibitor Suppresses Angiogenesis by Regulating AP-1 Expression.

Angiogenesis dysregulation contributes to inflammation, infections, immune disorders, and carcinogenesis. Bromodomain-containing protein 4 (BRD4) is an epigenetic reader that recognizes histone proteins and acts as a transcriptional regulator to trigger tumor growth and the inflammatory response. The pan-bromodomain and extra-terminal domain (BET) inhibitor, (+)-JQ1 (1), was reported to inhibit angiogenesis. However, owing to the non-selectivity action of (+)-JQ1 towards all BET family members, the role of BRD4 and that of its bromodomains (BD1 and BD2) in angiogenesis remains elusive. Herein, we identified a potent BRD4 inhibitor, ZL0513 (7), which exhibited significant anti-angiogenic effects in chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) models. This inhibitor also directly suppressed the viability and tube formation of human umbilical vascular endothelial cells (HUVECs). Moreover, ZL0513 (7) was found to inhibit the phosphorylation of c-jun and c-fos, important members of activating protein-1 (AP-1) transcription factor complexes that enhance angiogenesis. The findings on this novel BRD4 inhibitor indicate that, in addition to being a powerful pharmacological tool for further elucidating the roles and functions of BRD4 and its BD domains in angiogenesis, it may serve as a potential therapeutic strategy for targeting the vasculature in various angiogenesis-dysregulated human diseases.

Opposing Functions of BRD4 Isoforms in Breast Cancer

Bromodomain-containing protein 4 (BRD4) is a cancer therapeutic target in ongoing clinical trials disrupting primarily BRD4-regulated transcription programs. The role of BRD4 in cancer has been attributed mainly to the abundant long isoform (BRD4-L). Here we show, by isoform-specific knockdown and endogenous protein detection, along with transgene expression, the less abundant BRD4 short isoform (BRD4-S) is oncogenic while BRD4-L is tumor-suppressive in breast cancer cell proliferation and migration, as well as mammary tumor formation and metastasis. Through integrated RNA-seq, genome-wide ChIP-seq, and CUT&RUN association profiling, we identify the Engrailed-1 (EN1) homeobox transcription factor as a key BRD4-S coregulator, particularly in triple-negative breast cancer. BRD4-S and EN1 comodulate the extracellular matrix (ECM)-associated matrisome network, including type II cystatin gene cluster, mucin 5, and cathepsin loci, via enhancer regulation of cancer-associated genes and pathways. Our work highlights the importance of targeted therapies for the oncogenic, but not tumor-suppressive, activity of BRD4.

Genetic variants involved in bromodomain-containing protein 4 (BRD4) regulating pathway to predict outcomes in patients with metastatic colorectal cancer: Results from FIRE3 and MAVERICC trials.

232 Background: BRD4 plays an important role in transcription, DNA repair and drug resistance. High expression and polymorphisms of BRD4 regulating pathways were reported to be related to worse prognosis in colorectal cancer. Therefore, we hypothesized that genetic variants in BRD4 regulating pathway may predict first-line treatment outcome in mCRC pts. Methods: The impact on outcome of 22 SNPs in 7 genes involved in BRD4 regulating pathway (BRD4, SIPA1, MYC, 53BP1, H2AX, BATF, CD47) was analyzed through the OncoArray, a customized array manufactured by Illumina, on genomic DNA from blood samples of pts enrolled in 2 randomized trials. MAVERICC FOLFIRI/bevacizumab (bev) arm served as discovery cohort (N = 107), FIRE3 FOLFIRI/bev arm as validation (N = 107) and FOLFIRI/cetuximab (cet) arm as control (N = 129). Results: In the discovery cohort, right(R)-sided pts with BRD4 rs4808272 any G allele (N = 46) showed significantly shorter PFS (9.5 vs 18 m) compared to carriers of A/A (N = 21) in both uni- and multi-variable analysis ( p < .01); R-sided pts carrying any T allele of BATF rs7161377 (N = 50) showed longer PFS (12.3 vs 6.8 m) compared to carriers of C/C (N = 14) in univariate analysis ( p < .05) and had a strong trend in multivariable analysis ( p = .06). These findings were all validated in R-sided pts in FIRE3 bev arm (BRD4 rs4808272, PFS 9.8 vs 18.7 m; BATF rs7161377, PFS 15.1 vs 4.2 m) in uni- (both p < .01) and multi-variable ( p = .08 and p < .05 respectively) analysis. No significant association was observed in the control arm. Interestingly, pts carrying CD47 rs3206652 any C allele (N = 13) only showed significant longer PFS (9.0 vs 3.0 m, univariable p < .01 and multivariable p = .07) in the R-sided pts of FIRE3 cet cohort, but no association was observed in the bev-based treatment. Conclusions: Our study demonstrates for the first time that BRD4 and BATF polymorphisms may predict outcomes of bev-based treatment in R-sided mCRC pts; Meanwhile CD47 polymorphism could predict outcomes of cet-based treatment in R-sided mCRC pts. This finding supports a possible role of BRD4 regulating pathway in contributing to resistance to anti-VEGF/EGFR treatment.

Bromodomain-containing Protein 4 Activates Voltage-gated Sodium Channel 1.7 Transcription in Dorsal Root Ganglia Neurons to Mediate Thermal Hyperalgesia in Rats.

Bromodomain-containing protein 4 binds acetylated promoter histones and promotes transcription; however, the role of bromodomain-containing protein 4 in inflammatory hyperalgesia remains unclear. Male Sprague-Dawley rats received hind paw injections of complete Freund's adjuvant to induce hyperalgesia. The dorsal root ganglia were examined to detect changes in bromodomain-containing protein 4 expression and the activation of genes involved in the expression of voltage-gated sodium channel 1.7, which is a key pain-related ion channel. The intraplantar complete Freund's adjuvant injections resulted in thermal hyperalgesia (4.0 ± 1.5 s; n = 7). The immunohistochemistry and immunoblotting results demonstrated an increase in the bromodomain-containing protein 4-expressing dorsal root ganglia neurons (3.78 ± 0.38 fold; n = 7) and bromodomain-containing protein 4 protein levels (2.62 ± 0.39 fold; n = 6). After the complete Freund's adjuvant injection, histone H3 protein acetylation was enhanced in the voltage-gated sodium channel 1.7 promoter, and cyclin-dependent kinase 9 and phosphorylation of RNA polymerase II were recruited to this area. Furthermore, the voltage-gated sodium channel 1.7-mediated currents were enhanced in neurons of the complete Freund's adjuvant rats (55 ± 11 vs. 19 ± 9 pA/pF; n = 4 to 6 neurons). Using bromodomain-containing protein 4-targeted antisense small interfering RNA to the complete Freund's adjuvant-treated rats, the authors demonstrated a reduction in the expression of bromodomain-containing protein 4 (0.68 ± 0.16 fold; n = 7), a reduction in thermal hyperalgesia (7.5 ± 1.5 s; n = 7), and a reduction in the increased voltage-gated sodium channel 1.7 currents (21 ± 4 pA/pF; n = 4 to 6 neurons). Complete Freund's adjuvant triggers enhanced bromodomain-containing protein 4 expression, ultimately leading to the enhanced excitability of nociceptive neurons and thermal hyperalgesia. This effect is likely mediated by the enhanced expression of voltage-gated sodium channel 1.7.

Research Advances in the Role of Bromodomain-containing Protein 4 in Epithelial-mesenchymal Transition in Asthma.

Asthma is a chronic airway disease characterized by airway inflammation and airway remodeling. Chronic airway inflammation can be involved in airway remodeling in asthmatic patients by incuding epithelial-mesenchymal transition (EMT).Bromodomain-containing protein 4 (BRD4) is a key transcriptional regulator in mammals,and many evidences have shown that BRD4 plays a pivotal role in airway remodeling via nuclear factor-κB/RelA signaling pathway. This review summarizes the recent advances in the role of BRD4 in regulating EMT,with an attempt to elucidate the molecular mechanisms of asthma and inform the prevention and control of asthma.

BRD4 promotes p63 and GRHL3 expression downstream of FOXO in mammary epithelial cells.

Bromodomain-containing protein 4 (BRD4) is a member of the bromo- and extraterminal (BET) domain-containing family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelial-specific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXO transcription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression.

Genome-Wide Mapping Reveals BRD4 in Regulation of Tumor-Driver Genes in Cutaneous T-Cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) is a non-Hodgkin's lymphoma of skin homing malignant CD4+ T cells. Early treatment of patients with skin-directed therapies often yields successful short-term outcomes; however, survival of patients with late-stage CTCL is extremely poor, highlighting the need to identify novel therapies to inhibit key oncogenic processes. Although the underlying factors driving CTCL pathogenesis are poorly understood, recent findings from our lab highlight the critical role of epigenetic dysregulation in its development (Mishra et al., Blood, 122:1826, 2013). While many studies have been carried out on the role of epigenetic writers (histone acetyltransferases) and erasers (histone deacetylases) in CTCL gene regulation, little is known about the epigenetic readers (bromodomains; tandem PHD fingers; Pleckstrin homology domains) that coordinate transcriptional reprogramming. Previous studies have identified bromodomain-containing protein-4 (BRD4) as a super-enhancer transcriptional regulator that interacts with pairs of acetylated lysine residues on histone H3 to upregulate oncogenic transcription in multiple myeloma and diffuse large B-cell lymphoma (Loven, J. et al. Cell 153, 2013; Chapuy, B. et al. Cancer Cell 24, 2013). We analyzed BRD4 occupancy patterns in the CTCL genome and characterized downstream oncogenic pathways. Additionally, we investigated the effect of treatment with a BRD4 inhibitor, JQ1, on CTCL in vitro and in vivo. In order to gain insights into the role of BRD4 in CTCL, we generated a genome-wide map of BRD4 occupancy in CD4+ T-cells from CTCL patients and normal donors using "ChIP-seq", which combines chromatin immunoprecipitation (ChIP) with sequencing of BRD4-associated DNA. The analysis revealed an increased occupancy of BRD4 in patient's CD4+ T-cells compared to normal donor CD4+ T-cells at putative active regions, promoter active regions, distal active regions, and super-enhancers (Figure 1). The abnormal binding patterns in patients were reversed within 24 hours of treatment with JQ1 (Figure 1). Patient's cells treated with 1.0 μM JQ1 showed reduced cell viability (mean ± SEM of relative % viability in JQ1 vs control treated cells: 39.67 ± 1.838 vs 100.0 ± 2.335, n=4 each). Furthermore, we saw a similar effect of JQ1 in all the patient-derived CTCL cell lines tested (HH, Hut78, Hut102, SeAx, and MyLa) with EC50 concentrations ranging from 0.167 to 21.0 μM. Since both primary CD4+ T-cells from CTCL patients and CTCL cell lines displayed sensitivity to the cytotoxic effect of JQ1, we performed subsequent functional assays on Hut78 cell line. Propidium iodide/Annexin staining of JQ1-treated Hut78 cells (1.0 μM for 48 hours) revealed an increased frequency of cells in the G1 phase, indicating significant disruption of cell cycle progression upon BRD4 inhibition. Furthermore, considering that a large portion of BRD4 binding was detected in promoter and super-enhancer regions, we speculated that BRD4 regulates tumor driver genes. When examining BRD4 occupancy, we found that BRD4 binding at NOTCH1, and MYC was higher in patients' vs normal donors' CD4+ T-cells. Similar differential increase in BRD4 occupancy was also noted at the DNA-binding factor gene RBPJ, a known NOTCH1 activator and T-cell oncogenic co-factor. In order to determine whether BRD4 occupancy at these genes exerts transcriptional regulation, we examined expression levels of RBPJ, NOTCH1, and MYC in JQ1 treated cells. Semi-quantitative RT-PCR analysis on HuT78 cells treated with JQ1 showed significant reduction in RBPJ (mean ± SEM of relative expression in Control vs JQ1 treatment= 100.5 ± 07.086 vs. 35.39 ± 0.7832, n=3, P =0.0008), NOTCH1 (mean ± SEM of relative expression in Control vs JQ1 treatment= 100.0 ± 0.95 vs 0.61.77 ± 02.844, n=3, P =0.0002), and MYC (mean ± SEM of relative expression in Control vs JQ1 treatment= 100.1 ± 03.019 vs. 57.97 ± 01.997, n=3, P =0.0003). Additionally, in vivo treatment of CTCL mice with JQ1 (50mg/kg, 5 days/week for 4-weeks) halted as well as regressed progression of CTCL compared to placebo treated controls. Together, these findings demonstrate that BRD4 regulates the expression of the oncogenic drivers in CTCL, and that bromodomain inhibitors offer the opportunity to interrogate the mechanisms of BRD4-mediated T-cell oncogenesis in the CTCL mouse model and design new anti-cancer therapies in CTCL patients. [Display omitted] DisclosuresPorcu:Cell Medica: Research Funding; Celgene: Research Funding; Shape: Research Funding; Seattle Genetics: Research Funding; Infinity: Research Funding.