BRD4 Expression Research Articles

IL-34 in hepatoblastoma cells potentially promote tumor progression via autocrine and paracrine mechanisms.

Hepatoblastoma is the most common pediatric liver tumor, but little research has been done on the role of macrophages in hepatoblastoma. The purpose of this study was to gain insight into potential roles for macrophages in hepatoblastoma. Paraffin‐embedded specimens from 56 patients who underwent surgical resection were examined with immunohistochemical staining for the macrophage‐specific markers, Iba1 and CD163. Significant differences were seen among histological subtypes. Significantly increased numbers of macrophages were detected in embryonal components compared to fetal components in the mixed epithelial type. In vitro studies using human monocyte‐derived macrophages and two hepatoblastoma cell lines (HepG2 and Huh6) were performed. Conditioned medium from these cell lines induced increased CD163 expression in macrophages. Direct co‐culture with macrophages induced tumor cell proliferation via induction of protumor cytokine secretion from macrophages. Direct co‐culture with macrophages also induced interleukin (IL)‐34 overexpression by Huh6 cells via Brd4 signaling. IL‐34 overexpression promoted tumor cell proliferation and chemoresistance. High IL‐34 and Brd4 expression was detected in embryonal components, which have potentially higher proliferation activity than fetal components. In conclusion, IL‐34 expression in embryonal components may induce macrophage chemotaxis in a paracrine manner, and tumor cell proliferation and chemoresistance in an autocrine manner. IL‐34 is a potential therapeutic target for hepatoblastoma.

Remote ischemic preconditioning protects against cerebral ischemia injury in rats by upregulating miR-204-5p and activating the PINK1/Parkin signaling pathway.

Remote ischemic preconditioning (RiPC) is the process where preconditioning ischemia protects the organs against the subsequent index ischemia. RiPC is a protective method for brain damage. This study is to explore the effect and mechanism of RiPC in cerebral ischemia injury in rats through regulation of miR-204-5p/BRD4 expression. Middle cerebral artery occlusion (MCAO) rat model and glucose deprivation (OGD) neuron model were established. The effect of RiPC on neurological deficits, cerebral infarct size, autophagy marker, inflammatory cytokines and apoptosis was evaluated. miR-204-5p expression was analyzed using RT-qPCR, and then downregulated using miR-204-5p antagomir to estimate its effect on MCAO rats. The downstream mechanism of miR-204-5p was explored. RiPC promoted autophagy, reduced cerebral infarct volume and neurological deficit score, and alleviated apoptosis and cerebral ischemia injury in rats, with no significant effects on healthy rat brains. RiPC up-regulated miR-204-5p expression in MCAO rats. miR-204-5p knockdown partially reversed the effect of RiPC. RiPC promoted autophagy in OGD cells, and attenuated inflammation and apoptosis. miR-204-5p targeted BRD4, which partially reversed the effect of miR-204-5p on OGD cells. RiPC activated the PINK1/Parkin pathway via the miR-204-5p/BRD4 axis. In conclusion, RiPC activated the PINK1/Parkin pathway and prevented cerebral ischemia injury by up-regulating miR-204-5p and inhibiting BRD4.

Design, synthesis and mechanism studies of novel dual PARP1/BRD4 inhibitors against pancreatic cancer

Inducing the deficiency of homologous recombination (HR) repair is an effective strategy to broaden the indication of PARP inhibitors in pancreatic cancer treatment. Repression of BRD4 has been reported to significantly elevate HR deficiency and sensitize cancer cells to PARP1/2 inhibitors. Inspired by the concept of synthetic lethality, we designed, synthetized and optimized a dual PARP1/BRD4 inhibitor III-7, with a completely new structure and high selectivity against both targets. III-7 repressed the expression and activity of PARP1 and BRD4 to synergistically inhibit the malignant growth of pancreatic cancer cells in vitro and in vivo. Based on the results of bioinformatic analysis, we found that Olaparib induced the acceleration of mitosis and recovery of DNA repair to cause the generation of drug resistance. III-7 reversed Olaparib-induced adaptive resistance and induced cell cycle arrest and DNA damage by perturbing PARP1 and BRD4-involved signaling pathways. We believe that the PARP1/BRD4 dual inhibitors are novel and promising antitumor agents, which provide an efficient strategy for pancreatic cancer treatment.

Propofol Inhibits Thyroid Cancer Cell Proliferation, Migration, and Invasion by Suppressing SHH and PI3K/AKT Signaling Pathways via the miR-141-3p/BRD4 Axis.

Objective This study explores the effect and mechanism of propofol for thyroid tumor. Methods Culture human normal thyroid cells Nthy-ori 3-1 and thyroid cancer cell line TPC-1. TPC-1 cells were divided into the propofol group (treated with propofol), miR-141-3p group (transfected with the miR-141-3p mimic), negative control group (transfected with miR-NC), miR-141-3p + pcDNA-BRD4 group (transfected with the miR-141-3p mimic and pcDNA-BRD4), miR-141-3p + pcDNA group (transfected with the miR-141-3p mimic and pcDNA), siBRD4 group (transfected with siBRD4), and si-control group (transfected with si-control). The detection of miR-141-3p and BRD4 expression in cells was done by RT-qPCR, and the dual-luciferase reporter gene method and western blotting were used to verify the targeting relationship between miR-141-3p and BRD4. MTT method was used to test cell proliferation, transwell method was used to test cell migration and invasion, and western blotting was used to test SHH, GLI1, p-PI3K, and p-AKT protein expression. Results Compared with Nthy-ori 3-1 cells, the expression of miR-141-3p in TPC-1 cells was markedly decreased. Propofol treatment and excessive expression of miR-141-3p could influence the phenotype of TPC-1 cells. BRD4 is one of the target genes of miR-141-3p, and its expression is negatively regulated by miR-141-3p. Overexpression of BRD4 can partially reverse the restraining effect of miR-141-3p on the TPC-1 cell phenotype. Both miR-141-3p and BRD4 can regulate the activity of SHH and PI3K/AKT signaling pathways. Conclusion Propofol can inhibit the activity of SHH and PI3K/AKT pathways by targeting downregulating BRD4 through miR-141-3p, thereby inhibiting the phenotype of TPC-1 cells.

Abstract P109: Targeting BRD4 in T cells with self-delivering RNAi PH-894 for immunotherapy

Abstract BRD4 is a member of the family of BET proteins that act as epigenetic readers and regulators of gene transcription. BRD4 is a known driver of tumor growth via enhancement of transcription of oncogenes such as Myc. More recent evidence also suggests a role of BRD4 in T cells. T cell differentiation and T cell exhaustion is epigenetically regulated and BRD4 has been associated with T cell differentiation. Available small molecule inhibitors of BET proteins do not target BRD4 specifically, but also inhibit other members of the BET protein family. We therefore investigated the effects of BRD4 silencing on T cells with the INTASYL™ compound PH-894, a self-delivering RNAi specifically targeting BRD4. INTASYL self-delivering RNAi molecules consist of an asymmetric duplex structure, a small duplex region (≤ 15 base pairs), a single-stranded phosphorothioate tail, and chemical modifications to confer stability, hydrophobicity, and efficient cellular uptake. Primary human T cells from healthy donors, either unstimulated or activated by OKT3 or anti-CD3/CD28 beads, were treated with PH-894. Additional studies were performed with CD8+ T cells magnetically sorted from primary human T cells. Silencing of BRD4 mRNA was assessed by RT-qPCR, and protein by flow cytometry. Phenotypic markers of T cell activation, differentiation and function were assessed by flow cytometry and cytokine secretion by cytometric bead array. Expanded and PH-894-treated CD8+ T cells were studied for IFNγ production upon coculture with allogeneic melanoma cell lines. Silencing of BRD4 mRNA and protein by PH-894 was concentration dependent and durable for up to 10 days in activated and naïve T cells. Silencing was specific for BRD4 with no effect on BRD2 or BRD3 expression. In activated T cells there was a suppression of IL-10 secretion and no effect on inflammatory cytokine levels. Markers of T cell activation were elevated on naïve PH-894-treated T cells with a shift to an increased CD8+/CD4+ ratio. In addition, BRD4 silencing in activated CD8+ T cells coincided with a decreased expression of transcription factors typically induced immediately upon TCR activation and are known drivers of T cell differentiation towards an effector phenotype. Moreover, CD8+ T cells expanded in the presence of PH-894 produced increased levels of IFNγ when cocultured with two allogeneic melanoma cell lines. These data translated in vivo with PH-894 treatment eliciting a dose associated inhibition of tumor growth in a CT26 murine colon carcinoma model. Ex vivo analysis of tumors found PH-894 efficacy was associated with decreased expression of BRD4 in tumor infiltrating CD3 T cells, and a reduction of Foxp3hi CD25hi CD4+ CD3+ Tregs, compared with control tumors. Specific silencing of BRD4 with PH-894 resulted in phenotypic changes on T cells associated with T cell activation and reduced immunosuppression. These data suggest that BRD4 not only plays a role on tumor cells but can also regulate T cell function and that PH-894 can reprogram T cells to provide enhanced immunotherapeutic activity. Citation Format: Melissa Maxwell, Daisuke Ujiie, Benjamin Cuiffo, Brianna Rivest, Dingxue Yan, James Cardia, Jeroen Melief, Rolf Kiessling, Simon P. Fricker. Targeting BRD4 in T cells with self-delivering RNAi PH-894 for immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P109.

BRD4 drives esophageal squamous cell carcinoma growth by promoting RCC2 expression.

The low survival rate of esophageal squamous cell carcinoma patients is primarily attributed to technical limitations and a lack of insight regarding the molecular mechanisms contributing to its progression. Alterations in epigenetic modulators are critical to cancer development and prognosis. BRD4, a chromatin reader protein, plays an essential role in regulating oncogene expression. Here, we investigated the contributing role of BRD4 and its related mechanisms in the context of ESCC tumor progression. Our observations showed that BRD4 transcript and protein expression levels are significantly increased in ESCC patient tissues. Genetic or pharmacological inhibition of BRD4 suppressed ESCC cell proliferation in vitro and in vivo. Proteomic and transcriptomic analyses were subsequently used to deduce the potential targets of BRD4. Mechanistic studies showed that RCC2 is a downstream target of BRD4. Inhibition of either BRD4 or RCC2 resulted in decreased ESCC cell proliferation. The BRD4-TP73 interaction facilitated the binding of BRD4 complex to the promoter region of RCC2, and subsequently modulated RCC2 transcription. Furthermore, targeting BRD4 with inhibitors significantly decreased tumor volume in ESCC PDX models, indicating that BRD4 expression may contribute to tumor progression. Collectively, these findings suggest that BRD4 inhibition could be a promising strategy to treat ESCC by downregulating RCC2.

Combined Targeting of Distinct c-Myc and JunB Transcriptional Programs Inducing Synergistic Anti-Myeloma Activity

INTRODUCTIONTranscription factors (TFs) are convergence points of signaling cascades that coordinate cell differentiation, proliferation and survival and are commonly deregulated in cancer, including multiple myeloma (MM). They contribute to the initiation of MM and promote tumor cell growth and drug resistance. Both cMyc, a merging point of the PI3K-, and JunB, a merging point of the MEK/MAPK-signaling pathway, play pivotal roles in MM. Exciting novel approaches to inhibit TFs like proteolysis-targeting-chimera (PROTAC) promise to lead to selective tumor cell death with little/no consequence for normal cells. However, redundancy phenomena of transcriptional programs are likely to challenge their efficacy. Here, we report our final results on combined targeting of distinct c-Myc & JunB transcriptional programs for MM therapy.METHODSMM cell lines and patient MM cells were analyzed. Following CRISPR-loss-of-function screens for cMyc & JunB across MM cell lines and correlation analyses in MM patient datasets, the functional relevance of BRD4/c-Myc- and MEK/JunB-induced TF programs was delineated using genomic and chemical approaches in 2D and 3D models of the bone marrow (BM) microenvironment. Specifically, effects of single or combined targeting of cMyc- and JunB-induced TF-programs were analyzed by flow cytometry, western blot, RNAseq, qPCR and luciferase assays. In vitro and ex vivo results were finally verified in a MM xenograft mouse model.RESULTSWhile CRISPR loss-of-function screens across various MM cell lines confirmed their growth dependency on cMyc and JunB, we did not observe correlative expression levels among these TFs, neither in the publicly available GSE6477 nor in the CoMMpass dataset. In contrast, a significant positive correlation was observed between Brd4 and cMyc, and MEK and JunB expression levels, respectively. The existence of two distinct Brd4/cMyc and MEK/JunB transcriptional programs in MM cells was subsequently supported by a lack of changes in cMyc mRNA/protein levels and resultant transcriptional activity upon JunB knockdown, and vice versa. Likewise, MZ-1, a novel PROTAC which targets Brd4, resulted in the inhibition of BMSC/IL-6- induced cMyc- but not JunB- upregulation. Conversely, neither the MEK inhibitor trametinib nor doxycycline-induced knockdown of BMSC/IL-6- induced JunB upregulation in TetshJunB/MM.1S cells reduced Brd4/c-Myc mRNA/protein levels. Importantly, the activity of MZ-1 and trametinib was predicted by Brd4 and JunB expression levels using mathematical models, respectively. Further, combination of MZ-1 with trametinib or JunB knockdown synergistically inhibited tumor cell proliferation, and induced cell death in a 2D and a dynamic 3D model of the MM-BM milieu. Finally, our in vitro and ex vivo results were confirmed in vivo, utilizing BMSC:TetshJunB/MM.1S vs. BMSC:TetshSCR/MM.1S-carrying NSG mice treated with MZ-1 with/without doxycycline or trametinib.CONCLUSIONIn summary, our data demonstrate for the first time the existence of non-overlapping cMyc and JunB-regulated TF programs providing a rationale for combined cMyc:JunB targeting treatment strategies in MM. DisclosuresVallet: Pfizer: Honoraria; MSD: Honoraria; Roche Pharmaceuticals: Consultancy. Podar: Celgene: Consultancy, Honoraria; Roche Pharmaceuticals: Research Funding; Janssen Pharmaceuticals: Consultancy, Honoraria; Amgen Inc.: Consultancy, Honoraria.

OAB-030: Combined targeting of distinct c-Myc and JunB transcriptional programs induces synergistic anti-myeloma activity

<h3>Background</h3> Transcription factors (TF) are convergence points of signaling cascades that coordinate cell differentiation, proliferation and survival and are commonly deregulated in cancer, including multiple myeloma (MM). They contribute to the initiation of MM, promote tumor cell growth and drug resistance. Both cMyc, a merging point of the PI3K-, and JunB, a merging point of the MEK/MAPK signaling pathway, play pivotal roles in MM pathogenesis. Exciting novel approaches to inhibit TFs like proteolysis-targeting-chimera (PROTAC) promise to lead to selective tumor cell death with little consequence for normal cells. However, redundancy phenomena of TF-programs are likely to challenge their efficacy. Here, we report our final results on the combined targeting of distinct cMyc and JunB transcriptional programs. <h3>Methods</h3> Following CRISPR-loss-of-function screens for cMyc and JunB across MM cell lines and correlation analyses in MM patient datasets, the functional relevance of Brd4/cMyc- and MEK/JunB-induced TF programs was delineated using genomic and pharmacological approaches in 2D and 3D models of the BM microenvironment. Specifically, effects of single or combined targeting of cMyc- and JunB-induced transcriptional programs were analyzed by FACS, WB, qPCR and luciferase assays. In vitro and ex vivo results were finally verified in a MM xenograft mouse model. <h3>Results</h3> While CRISPR-loss-of-function screens of MM cell lines confirmed the dependency on cMyc and JunB, we did not observe correlative expression levels among these TFs, neither in the publicly available GSE6477 nor in the CoMMpass dataset. In contrast, a significant positive correlation was observed between MEK and JunB, and Brd4 and cMyc expression levels, respectively. The existence of two distinct Brd4/cMyc and MEK/JunB transcriptional programs in MM cells was supported by stable cMyc levels and resultant transcriptional activity upon JunB knockdown, and vice versa. Likewise, MZ-1, a novel PROTAC which targets Brd4, resulted in inhibition of BMSC/IL-6-induced cMyc but not JunB-upregulation. Conversely, neither the MEK inhibitor trametinib nor doxycycline-induced knockdown of BMSC/IL-6-triggered JunB upregulation in TetshJunB/MM.1S cells reduced Brd4/cMyc mRNA/protein levels. Importantly, the activity of MZ-1 and trametinib was predicted by Brd4 and JunB expression levels using mathematical scoring models, respectively. Moreover, combinations of MZ-1 with trametinib or JunB knockdown synergistically inhibited tumor cell proliferation and induced cell death in a 2D and a dynamic 3D model of the MM-BM milieu. Finally, our results were verified in BMSC:TetshJunB/MM.1S vs BMSC:TetshSCR/MM.1S-carrying NSG mice treated with MZ-1 with/without doxycycline or trametinib. <h3>Conclusions</h3> In summary, our data demonstrate for the first time the existence of non-overlapping cMyc and JunB-regulated transcriptional programs providing the rationale for combined cMyc:JunB targeting strategies for MM therapy.

Synthesis, molecular structure, DFT studies, in silico docking and molecular dynamics simulations of 2,6 dimethoxychalcone derivatives as BRD4 inhibitors

Methoxyphenyl chalcone derivatives (MAHB1 and MAHB3) have been synthesized and characterised by NMR (1H&13C) spectra and single crystal X-ray diffraction techniques. The crystal structures revealed the crystallization of the both compounds in the monoclinic systems and P2(1)/c space groups. Supramolecular architecture unveiled the stability of compounds and build of crystal packings upon short contacts and weak interactions. Density functional theory (DFT) with B3LYP functional under 6–311++G(d,p) basis set have been adopted to calculate the geometry and investigate intramolecular charge transfer (ICT) using natural bond orbital analysis (NBO) as well as the frontier molecular orbitals (FMOs), hardness, softness, energy gap and other chemical reactivity parameters. Hirshfeld surface and energy frameworks were adopted to evaluate the different intermolecular interaction and energies based on the anisotropy of the topology. The inhibitory activity of the compounds against the first bromodomain of human BRD4 protein have been assessed by using in silico molecular docking and molecular dynamics simulations against 5H21 protein (homo sapiens), which revealed prominent interactions of both the ligands at the active site of BRD4 protein showing with a reasonable conformity as similar with the co-crystal ligand interactions. Furthermore, apo and holo states in the molecular dynamic simulations confirmed the stability and structural constancy of the protein-ligand complexes with minimal deviation based on RMSD and RMSF plots. Both the simulation results confirmed the hydrogen bonds of the compounds with the vital amino acid ASN140 which is necessary for the inhibition of BRD4 as well as exhibiting similar hydrophobic residues interactions. Furthermore, the newly synthesized compounds were screened against human leukemia MV4–11 cells based on MTT and apoptosis assays system. These compounds exhibited anti-proliferation activity and induced the apoptotic effect. This study indicated that the MAHB1 is a promising compound against leukaemia with IC50 value of 79.10 μM and exhibited better apoptogenic effect. Furthermore, the expression of BRD4 in MV4–11 cell line was measured by western blotting, while the protein thermal shift assay was conducted to determine the impact of MAHB1 compound against the thermal stability of BRD4.

Abstract 2106: Combined deacetylase and bromodomain inhibition downregulates ERCC2 and suppresses growth of metastatic colon cancer cells

Abstract DNA repair genes have potential clinical value in predicting cancer prognosis and treatment outcomes. Nucleotide excision repair (NER) proteins like ERCC2 play a critical role in maintaining genome integrity by recognizing and unwinding DNA at sites of damage1.Aberrant expression of ERCC2 alters NER capacity, influencing treatment outcomes2. The current investigation examined the expression of ERCC2 following epigenetic combination treatment in colorectal cancer (CRC) cells and preclinical models. Attention was drawn to ERCC2 based on three observations. First, from online databases, when ERCC2 was overexpressed in colon tumors the corresponding CRC patients had reduced overall survival3. Second, ERCC2 was the most highly downregulated gene when dietary histone deacetylase 3 (HDAC3) inhibitor sulforaphane (SFN) was combined withJQ1, an inhibitor of the bromodomain and extra terminal domain family, in human colon cancer cells and in colon polyps from the polyposis in rat colon (Pirc) model4. Third, as reported here for the first time, RNA-seq analyses of Pirc colon polyps from rats treated withJQ1 and the SFN analog 6-SFN5 identified Ercc2 as the most highly downregulated gene. RNA-seq data were corroborated by RT-qPCR and immunoblotting experiments. There is much interest in combination approaches that target epigenetic ‘readers, writers, and erasers' that are deregulated in cancer and other pathologies. The current work identified promising second-generation inhibitors with enhanced synergy and antitumor efficacy, especially in metastatic cells cultured in three-dimensions. Drug combinations decreased HDAC3, BRD4 and ERCC2 expression, while DNA damage and apoptosis markers were increased both in spheroids and in a mouse xenograft model. This investigation has potential clinical relevance for the identification of robust biomarkers that predict enhanced antitumor outcomes in CRC patients.1. Gillet LC, Schärer OD. Molecular mechanisms of mammalian global genome nucleotide excision repair. Chem Rev 2006;106:253-76.2. Shuck SC, Short EA, Turchi JJ. Eukaryotic nucleotide excision repair: from understanding mechanisms to influencing biology. Cell Res 2008;18:64-72.3. Liu J et al., The Differential Expression of Core Genes in Nucleotide Excision Repair Pathway Indicates Colorectal Carcinogenesis and Prognosis. Biomed Res Int 2018;2018:9651320.4. Rajendran P et al., Acetylation of CCAR2 establishes a BET/BRD9 acetyl switch in response to combined deacetylase and bromodomain inhibition. Cancer Res 2019;79:918-27.5. Rajendran Pet al., HDAC turnover, CtIP acetylation and dysregulated DNA damage signaling in colon cancer cell treated with sulforaphane and related dietary isothiocyanates. Epigenetics 2013;8:612-23. Citation Format: Sabeeta Kapoor, Trace Gustafson, Mutian Zhang, Ying-Shiuan Chen, Jia Li, Nhung Nguyen, Jorge Enrique Perez, Wan Mohaiza Dashwood, Praveen Rajendran, Roderick Dashwood. Combined deacetylase and bromodomain inhibition downregulates ERCC2 and suppresses growth of metastatic colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2106.

MUC1-C activates the PBAF chromatin remodeling complex in integrating redox balance with progression of human prostate cancer stem cells

The polybromo-associated PBAF (SWI/SNF) chromatin remodeling complex, which includes PBRM1, ARID2, and BRD7, regulates cell differentiation and genomic integrity. MUC1-C is an oncogenic protein that drives lineage plasticity in prostate cancer (PC) progression. The present work demonstrates that MUC1-C induces PBRM1, ARID2, and BRD7 expression by the previously unrecognized E2F1-mediated activation of their respective promoters. The functional significance of the MUC1-C→PBAF pathway is supported by demonstrating involvement of MUC1-C in associating with nuclear PBAF and driving the NRF2 antioxidant gene transcriptome in PC cells. Mechanistically, MUC1-C forms a complex with NRF2 and PBRM1 on the NRF2 target SLC7A11 gene that encodes the xCT cystine-glutamate antiporter, increases chromatin accessibility and induces SLC7A11/xCT expression. We also show that MUC1-C and PBRM1 are necessary for induction of other NRF2 target genes, including G6PD and PGD that regulate the pentose phosphate pathway. Our results further demonstrate that MUC1-C integrates activation of PBRM1 with the regulation of antioxidant genes, ROS levels, pluripotency factor expression and the cancer stem cell (CSC) state. These findings reveal a role for MUC1-C in regulating PBAF, redox balance and lineage plasticity of PC CSC progression. Our findings also uncover involvement of MUC1-C in integrating the PBAF and BAF pathways in cancer.

Inhibiting Brd4 alleviated PTSD-like behaviors and fear memory through regulating immediate early genes expression and neuroinflammation in rats.

Post-traumatic stress disorder (PTSD) is characterized by depression/anxiety and memory failure, primarily fear memory. According to the reports, neuroinflammation and synaptic plasticity can play a role in the neurophysiological mechanisms underlying PTSD. Bromodomain-containing protein 4 (Brd4) intriguingly affects regulating of inflammatory responses and learning and memory. This study aimed to explore the effect of inhibiting Brd4 on depression/anxiety-like behaviors, spatial and fear memory, and underlying mechanisms in a model of PTSD. Inescapable foot shocks (IFS) with a sound reminder in 6days were used to induce PTSD-like behaviors which were tested using contextual and cue fear tests, sucrose preference test, open-field test, elevated plus maze test, and Y-maze test. Meanwhile, the Brd4 inhibitor JQ1 was used as an intervention. The results found that IFS induced PTSD-like behaviors and indicated obvious Brd4 expression in microglia of the prefrontal cortex (PFC), hippocampus, and amygdala, pro-inflammatory cytokines over-expression, microglial activation, and nuclear factor-kappa B over-expression in PFC and hippocampus but not in amygdala. Meanwhile, the alterations of immediate early genes (IEGs) were found in PFC, hippocampus, and amygdala. Besides, dendritic spine density was reduced in PFC and hippocampus but was elevated in amygdala of rats with IFS. In addition, treatment with JQ1 significantly reduced freezing time in the contextual and cue fear test, reversed the behavioral impairment, decreased the elevated neuroinflammation, and normalized the alteration in IEGs and dendritic spine densities. The results suggested that Brd4 was involved in IFS-induced PTSD-like behaviors through regulating neuroinflammation, dynamics of IEGs, and synaptic plasticity.

BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL.MethodsExpression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP).ResultsBRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo.ConclusionsBRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

LEDGF/p75-mediated chemoresistance of mixed-lineage leukemia involves cell survival pathways and super enhancer activators.

MLL is an aggressive subtype of leukemia with a poor prognosis that mostly affects pediatric patients. MLL-rearranged fusion proteins (MLLr) induce aberrant target gene expression resulting in leukemogenesis. MLL and its fusions are tethered to chromatin by LEDGF/p75, a transcriptional co-activator that specifically recognizes H3K36me2/3. LEDGF/p75 is ubiquitously expressed and associated with regulation of gene expression, autoimmune responses, and HIV replication. LEDGF/p75 was proven to be essential for leukemogenesis in MLL. Apart from MLL, LEDGF/p75 has been linked to lung, breast, and prostate cancer. Intriguingly, LEDGF/p75 interacts with Med-1, which co-localizes with BRD4. Both are known as co-activators of super-enhancers. Here, we describe LEDGF/p75-dependent chemoresistance of MLLr cell lines. Investigation of the underlying mechanism revealed a role of LEDGF/p75 in the cell cycle and in survival pathways and showed that LEDGF/p75 protects against apoptosis during chemotherapy. Remarkably, LEDGF/p75 levels also affected expression of BRD4 and Med1. Altogether, our data suggest a role of LEDGF/p75 in cancer survival, stem cell renewal, and activation of nuclear super enhancers.

BRD4 inhibition sensitizes cervical cancer to radiotherapy by attenuating DNA repair.

Cisplatin-based chemoradiotherapy is the recommended treatment for local advanced cervical cancer, but radioresistance remains one of the most important and unresolved clinical problems. Investigations have revealed aberrant epigenetic modifications as one of the chief culprits for the development of radioresistance. Here, we attempt to identify a radiosensitizer from an epigenetic drug synergy screen and explore the underlying mechanism. We integrated epigenetic inhibitors and radiotherapy in cervical cancer cell lines to identify potential radiosensitizers. We further verified the sensitization effect of the drug and the function of its target gene both in vitro and in vivo. Finally, we validated the clinical significance of its target gene in clinical cervical cancer specimens. We identified JQ1, a BRD4 inhibitor, as a potent radiosensitizer. Functional assays demonstrated that repressing BRD4 activity led to significant radiosensitization and potentiation of DNA damage in cervical cancer cell lines. By using RNA-seq to determine JQ1-mediated changes in transcription, we identified RAD51AP1 as a major BRD4 target gene involved in radiosensitivity. A dual-luciferase reporter assay and ChIP-qPCR showed that BRD4 binds to the promoter region of RAD51AP1 and promotes its transcription, whereas this activity was attenuated by BRD4 inhibition. The in vivo experiments also suggested a synergy between BRD4 inhibition and radiotherapy. High BRD4 expression was found to be related to a worse prognosis and radiation resistance. BRD4 inhibition sensitizes cervical cancer to radiotherapy by inhibiting RAD51AP1 transcription. The combination of JQ1 with radiotherapy merits further evaluation as a therapeutic strategy for improving local control in cervical cancer.

Resistance to BET inhibitors in lung adenocarcinoma is mediated by casein kinase phosphorylation of BRD4

Targeting the epigenome to modulate gene expression programs driving cancer development has emerged as an exciting avenue for therapeutic intervention. Pharmacological inhibition of the bromodomain and extraterminal (BET) family of chromatin adapter proteins has proven effective in this regard, suppressing growth of diverse cancer types mainly through downregulation of the c-MYC oncogene, and its downstream transcriptional program. While initially effective, resistance to BET inhibitors (BETi) typically occurs through mechanisms that reactivate MYC expression. We have previously shown that lung adenocarcinoma (LAC) is inhibited by JQ1 through suppression of FOSL1, suggesting that the epigenetic landscape of tumor cells from different origins and differentiation states influences BETi response. Here, we assessed how these differences affect mechanisms of BETi resistance through the establishment of isogenic pairs of JQ1 sensitive and resistant LAC cell lines. We found that resistance to JQ1 in LAC occurs independent of FOSL1 while MYC levels remain unchanged between resistant cells and their JQ1-treated parental counterparts. Furthermore, while epithelial–mesenchymal transition (EMT) is observed upon resistance, TGF-β induced EMT did not confer resistance in JQ1 sensitive LAC lines, suggesting this is a consequence, rather than a driver of BETi resistance in our model systems. Importantly, siRNA knockdown demonstrated that JQ1 resistant cell lines are still dependent on BRD4 expression for survival and we found that phosphorylation of BRD4 is elevated in resistant LACs, identifying casein kinase 2 (CK2) as a candidate protein mediating this effect. Inhibition of CK2, as well as downstream transcriptional targets of phosphorylated BRD4—including AXL and activators of the PI3K pathway—synergize with JQ1 to inhibit BETi resistant LAC. Overall, this demonstrates that the mechanism of resistance to BETi varies depending on cancer type, with LAC cells developing JQ1 resistance independent of MYC regulation, and identifying CK2 phosphorylation of BRD4 as a potential target to overcome resistance in this cancer.

Co-targeting BET bromodomain BRD4 and RAC1 suppresses growth, stemness and tumorigenesis by disrupting the c-MYC-G9a-FTH1axis and downregulating HDAC1 in molecular subtypes of breast cancer.

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.

Effect of HIF-1α and BRD4 on autophagy level of breast cancer cell in hypoxic microenvironment

Objective: To investigate the expressions of HIF-1α, BRD4, Beclin1, LC3B and p62 in breast cancer tissues and their clinicopathological significance, and to study alterations of their expression in breast cancer cells under hypoxic microenvironment. Methods: Immunohistochemistry was used to detect HIF-1α, BRD4, Beclin1, LC3B and p62 protein expressions in 125 breast cancer tissues and 50 para-cancer normal breast tissues, and their correlation with clinicopathologic characteristics were analyzed. The expression of these proteins were also measured after 24 hours of hypoxia stimulation was detected in different breast cancer cell lines and normal breast epithelial cells. Results: The expression of HIF-1α, BRD4, Beclin1 and LC3B proteins in breast cancer tissues were significantly higher than in para-cancer normal breast tissues (P<0.05). There was a positive association between histologic grade, the expression of HIF-1α, BRD4, Beclin1 and LC3B (P<0.05). High expressions of HIF-1a and Beclin1 were often correlated with lymph node metastasis and lymphatic invasion (P<0.05). Increased HIF-1α, BRD4, Beclin1 and LC3B expression was associated with ER or PR negativity, but only HIF-1α was associated with HER2 positivity (P<0.05). HIF-1α, BRD4, Beclin1, and LC3B were positively correlated with each other in breast cancer tissues (P<0.01). After 24 hours of hypoxic stimulation, the expression of HIF-1α, BRD4, Beclin1 and LC3B was up-regulated in breast cancer cells. Conclusions: Hypoxia induces autophagy in breast cancer tissues. HIF-1α is positively correlated with BRD4, suggesting that BRD4 is involved in the regulation of autophagy by hypoxic microenvironment in breast cancer. High expression of HIF-1α, BRD4 and autophagy may play an important role in the development of breast cancer.

LncRNA TUG1 knockdown mitigates inflammatory injury induced by cigarette smoke extract in chronic obstructive pulmonary disease via miR-34c/BRD4 axis.

Chronic obstructive pulmonary disease (COPD) is a type of respiratory disease. The dysregulation of long non-coding RNA (lncRNA) taurine upregulated gene 1 (TUG1) has been reported in diverse diseases. This study aimed to explore the functions and mechanism of TUG1 in COPD. The levels of TUG1 and BRD4 were significantly up-regulated, and the level of miR-34c was apparently down-regulated in COPD patients or CSE-treated BEAS-2B cells. TUG1 was verified to sponge to miR-34c, and BRD4 was validated as a target of miR-34c. TUG1 depletion or miR-34c overexpression promoted cell proliferation but restrained apoptosis, inflammatory response in CSE-treated BEAS-2B cells. MiR-34c inhibitor mitigated the inhibitory effect on cell proliferation and the promotion effects on cell apoptosis, inflammatory response in CSE-treated BEAS-2B cells induced by TUG1 silencing. Mechanistically, TUG1 modulated BRD4 expression in CSE-treated BEAS-2B cells by sponging miR-34c. TUG1 modulated BRD4 to regulate cell proliferation, cell apoptosis and inflammatory response in CSE-treated BEAS-2B cells by sponging miR-34c.

PROTAC Bromodomain Inhibitor ARV-825 Displays Anti-Tumor Activity in Neuroblastoma by Repressing Expression of MYCN or c-Myc.

Neuroblastoma (NB) is one of the most common solid tumors in childhood. To date, targeting MYCN, a well-established driver gene in high-risk neuroblastoma, is still challenging. In recent years, inhibition of bromodomain and extra terminal (BET) proteins shows great potential in multiple of Myc-driven tumors. ARV-825 is a novel BET inhibitor using proteolysis-targeting chimera (PROTAC) technology which degrades target proteins by the proteasome. In this study, we investigated the effect of ARV-825 in neuroblastoma in vitro and in vivo. Our results showed that ARV-825 treatment robustly induced proliferative suppression, cell cycle arrest, and apoptosis in NB cells. Moreover, ARV-825 efficiently depleted BET protein expression, subsequently repressing the expression of MYCN or c-Myc. In the NB xenograft model, ARV-825 profoundly reduced tumor growth and led to the downregulation of BRD4 and MYCN expression in mice. Taken together, these findings provide evidence that PROTAC BET inhibitor is an efficient way to achieve MYCN/c-Myc manipulation, and ARV-825 can be used as a potential therapeutic strategy for the treatment of neuroblastoma.