BRD4 Research Articles

Abstract LB542: Overexpression of the short isoform of Brd4 in ovarian carcinoma leads to highly aggressive tumor phenotype

Abstract High-grade serous ovarian carcinoma (HGSOC) is one of the deadliest types of cancer in the US. Occurrence of sporadic HGSOC is associated with high genomic instability, which causes amplification and deletion of several different genes. BRD4 is one of the most frequently amplified genes in ovarian carcinoma patients. BRD4 amplification leads to overexpression of BRD4 proteins including long and short BRD4 isoforms (BRD4-L and BRD4-S, respectively), and is associated with poor patient prognosis. Recently, it has been shown that the short isoform BRD4-S contributes to oncogenic properties of several tumor types; however, little is known about its role in ovarian cancer. Therefore, in order to interrogate the function of different BRD4 isoforms in HGSOC, we generated ovarian cancer cell lines stably overexpressing BRD4 isoforms (BRD4-L and BRD-S). Our data revealed that overexpression of BRD4-S in ovarian carcinoma cell lines induces a highly aggressive tumor phenotype in vitro and in vivo. Ovcar4-BRD4-S cells showed significantly higher cell proliferation rate and increased anchorage-independent growth when compared with parental cell lines. In addition, Ovcar4-BRD4-S presents a high level of DNA damage reflected by increased pH2AX foci. We also observed a disruption of cell cycle in Ovcar4-BRD4-S cells associated with a higher number of cells arrested in G2/M phase and increased population of polyploid cells. In vivo experiments demonstrated that BRD4-S expressing tumors grow significantly faster than parental tumors. BRD4-S overexpressing tumors are also very resistant to DNA-damage agents including standard of care platinum drugs. Finally, our preliminary drug screening demonstrated that Ovcar4-BRD4-S cells are highly sensitive to Wee1 inhibitor MK-1775. Furthermore, the combination of MK-1775 with paclitaxel synergistically increased the antitumor effect. In summary, our studies indicate that amplification of the BRD4 gene in HGSOC patients might lead to an increase in splicing of BRD4-S transcript, which in consequence promotes highly aggressive and platinum-resistant tumors. Precision therapies targeting BRD4 amplified tumors, such as Wee1 inhibitors in combination with paclitaxel hold great promise to improve ovarian cancer treatment and reduce chemotherapy resistance, which is a critical problem for eradication of this aggressive tumor. Citation Format: Ana Luiza Drumond-Bock, Luyao Wang, Lin Wang, Magdalena Cybula, Maria Rostworowska, Magdalena Bieniasz. Overexpression of the short isoform of Brd4 in ovarian carcinoma leads to highly aggressive tumor phenotype [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 LB542.

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.

Identification of differentially expressed long noncoding RNAs in the ovarian tissue of Shal and Sangsari ewes using RNA-seq.

BackgroundThe ovary has an important role in reproductive function. Animal reproduction is dominated by numerous coding genes and noncoding elements. Although long noncoding RNAs (LncRNAs) are important in biological activity, little is known about their role in the ovary and fertility.MethodsThree adult Shal ewes and three adult Sangsari ewes were used in this investigation. LncRNAs in ovarian tissue from two breeds were identified using bioinformatics analyses, and then target genes of LncRNAs were discovered. Target genes were annotated using the DAVID database, and their interactions were examined using the STRING database and Cytoscape software. The expression levels of seven LncRNAs with their target genes were assessed by real‐time PCR to confirm the RNA‐seq.ResultsAmong all the identified LncRNAs, 124 LncRNAs were detected with different expression levels between the two breeds (FDR < 0.05). According to the DAVID database, target genes were discovered to be engaged in one biological process, one cellular component, and 21 KEGG pathways (FDR < 0.05). The PES1, RPS9, EF‐1, Plectin, SURF6, CYC1, PRKACA MAPK1, ITGB2 and BRD2 genes were some of the most crucial target genes (hub genes) in the ovary.ConclusionThese results could pave the way for future efforts to address sheep prolificacy barriers.

A direct high-throughput protein quantification strategy facilitates discovery and characterization of a celastrol-derived BRD4 degrader

We describe a generalizable time-resolved Förster resonance energy transfer (TR-FRET)-based platform to profile the cellular action of heterobifunctional degraders (or proteolysis-targeting chimeras [PROTACs]) that is capable of both accurately quantifying protein levels in whole-cell lysates in less than 1h and measuring small-molecule target engagement to endogenous proteins, here specifically for human bromodomain-containing protein 4 (BRD4). The detection mix consists of a single primary antibody targeting the protein of interest, a luminescent donor-labeled anti-species nanobody, and a fluorescent acceptor ligand. Importantly, our strategy can readily be applied to other targets of interest and will greatly facilitate the cell-based profiling of small-molecule inhibitors and PROTACs in a high-throughput format with unmodified cell lines. We furthermore validate our platform in the characterization of celastrol, a p-quinone methide-containing pentacyclic triterpenoid, as a broad cysteine-targeting E3 ubiquitin ligase warhead for potent and efficient targeted protein degradation.

Long Noncoding RNA MALAT1 Promotes Laryngocarcinoma Development by Targeting miR-708-5p/BRD4 Axis to Regulate YAP1-Mediated Epithelial-Mesenchymal Transition.

Objective The objective of this study was to investigate whether long noncoding RNA Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) contributes to laryngocarcinoma development via regulating the Yes-associated protein 1- (YAP1-) mediated epithelial-mesenchymal transition (EMT) and the underlying mechanism. Methods The effects of MALAT1 suppression and BET inhibitor JQ1 on the malignant phenotypes and cancer stem cell- (CSC-) like properties of laryngocarcinoma cells as well as the expression of bromodomain-containing protein 4 (BRD4), YAP1, and EMT markers were investigated. Moreover, the relationships between MALAT1 and miR-708-5p as well as between miR-708-5p and BRD4 were explored. Furthermore, whether MALAT1 regulated the malignant phenotypes of laryngocarcinoma cells via sponging miR-708-5p to target BRD4 was revealed by both in vitro and in vivo experiments. Results MALAT1 suppression inhibited the malignant phenotypes of laryngocarcinoma cells, such as decreased proliferation, promoted apoptosis, suppressed migration, and inhibited the CSC properties. Suppression of MALAT1 increased miR-708-5p expression and decreased the expression of BRD4 and YAP1 and inhibited EMT. Moreover, there were target relationships between MALAT1 and miR-708-5p as well as between miR-708-5p and BRD4. miR-708-5p overexpression and MALAT1 suppression had synergistic inhibitory effects on the malignant phenotypes of laryngocarcinoma cells and the expression of BRD4, YAP1, and EMT. Furthermore, in vivo experiments confirmed that MALAT1/miR-708-5p regulated tumorigenicity by regulating BRD4 and YAP1-mediated EMT. Conclusions Our results indicate that suppression of MALAT1 may inhibit laryngocarcinoma development by sponging miR-708-5p/BRD4 to regulate YAP1-mediated EMT. Targeting MALAT1/miR-708-5p/BRD4 axis may provide a promising therapeutic strategy for laryngocarcinoma.

CircKCNQ5 controls proliferation, migration, invasion, apoptosis, and glycolysis of multiple myeloma cells by modulating miR-335-5p/BRD4 axis.

Circular RNAs (circRNAs) are key players in tumorigenesis progression. However, the role and molecular mechanisms of circKCNQ5 in multiple myeloma (MM) progression remain unclear. The quantitative real-time polymerase chain reaction was used for examining circKCNQ5, miR-335-5p, and Bromodomain-containing protein 4 (BRD4) levels. The proliferation ability of MM cells was determined by Cell Counting Kit-8 and colony-forming assays. The migration and invasion were analyzed by transwell assay. Flow cytometry was used to assess cell apoptosis. The lactate production, glucose consumption, and ATP/ADP ratios were determined by commercialized kits. The protein levels were quantified by western blot analysis. The interactions between circKCNQ5 and miR-335-5p, along with miR-335-5p and BRD4 were analyzed by dual-luciferase reporter and RNA immunoprecipitation assays. The overexpression of circKCNQ5 was confirmed in MM tissues and cells. Importantly, knockdown of circKCNQ5 suppressed proliferation, migration, invasion, and glycolysis while it increased apoptosis of MM cells in vitro. Interestingly, the downregulation of miR-335-5p was able to rescue the circKCNQ5 inhibition-induced effects on MM cells. MiR-335-5p interacted with circKCNQ5, and was able to target BRD4 in MM cells. MiR-335-5p upregulation inhibited malignant phenotypes of MM cells depending on BRD4. CircKCNQ5 was found to stimulate MM progression through competitively sponging to miR-335-5p.

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.

Upregulation of BRD7 protects podocytes against high glucose-induced apoptosis by enhancing Nrf2 in a GSK-3β-dependent manner

Bromodomain-containing protein 7 (BRD7) is linked to a variety of pathophysiological conditions. However, it is still unclear whether BRD7 is connected with diabetic nephropathy. This research explored the relevance of BRD7 in diabetic nephropathy using high glucose (HG)-stimulated podocytes in vitro. BRD7 expression in podocytes was decreased after HG stimulation. Podocytes with forced BRD7 expression were protected from HG-induced apoptosis, oxidative stress and inflammation. Further data revealed that forced expression of BRD7 led to enhanced nuclear factor erythroid-2-related factor 2 (Nrf2) activation in HG-stimulated podocytes, associated with the upregulation of glycogen synthase kinase-3β (GSK-3β) phosphorylation. Reactivation of GSK-3β diminished BRD7-elicited Nrf2 activation. In addition, restraining of Nrf2 diminished the BRD7 overexpression-induced beneficial effects on HG-induced podocyte damage. Taken together, these data document that BRD7 defends against HG-induced podocyte damage by enhancing Nrf2 via regulation of GSK-3β. Our work indicates that the BRD7/GSK-3β/Nrf2 axis may play a key role in mediating podocyte injury in diabetic nephropathy.

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.

Structural and Biophysical Characterization of Plasmodium falciparum Bromodomain Protein 1

Epigenetic regulatory mechanisms are important for the growth and survival of eukaryotic organisms. Post‐translational modifications found on histone proteins in the nucleosome function as a dynamic cell signaling mechanism that controls many essential functions such as cell growth, DNA replication, and gene expression. Bromodomains are conserved protein interaction modules that specifically recognize acetylated lysine residues on histones. Among five Plasmodium species, P. falciparum is the major cause of malaria cases worldwide. According to a WHO report in 2019 there were 409,000 deaths reported worldwide and the most affected age group was of children under 5 years of age. Interestingly, the P. falciparum genome encodes eight bromodomain‐containing proteins, and bromodomain protein 1 (PfBDP1) has been shown to play an important role in malaria pathogenesis. The domain architecture of PfBDP1 includes a single C‐terminal bromodomain and several ankyrin repeats. PfBDP1 is thought to act by tethering a transcriptional activator complex to acetylated histone H3 to control genes required for parasite invasion. Additionally, PfBDP1 forms a complex with the bromodomain protein 2 (PfBDP2) to carry out its cellular functions. We hypothesized that the bromodomain is crucial for the function of PfBDP1 via recognition of acetylated lysine modifications. We carried out several histone binding assays to identify novel histone interactions of PfBDP1 bromodomain. We also utilized a combination of structural biology and biophysical approaches to identify amino acid residues critical for ligand coordination. The rise of drug‐resistance towards P. falciparum has created an urgent need to outline the molecular mechanisms driving pathogenesis in hopes of uncovering new potential drug targets. Importantly, the results from this study may improve the development of new therapeutics to treat malaria.

Design and Synthesis of Bi‐aryl Methylated Lactam Derivatives to Inhibit the BRD7 Bromodomain Function in Prostate Cancer

Targeting bromodomain‐containing proteins has emerged as a promising therapeutic strategy and multiple clinical studies indicate that bromodomain (BD) inhibitors can act as anticancer and anti‐inflammatory agents. Bromodomain‐containing protein 7 (BRD7) is a subunit of the SWI/SNF chromatin remodeling complex, which is involved in the fine‐tuning of gene expression through changes in the organization of DNA. It regulates the progression of different types of cancers such as nasopharyngeal carcinoma and hormone‐dependent breast and ovarian cancers. BRD7 is also implicated in progression of prostate cancer, another hormone‐dependent malignancy; however, the role of BRD7 in this type of cancer is undefined. (Figure 1) We have characterized BRD7 as an oncogenic driver across a variety of prostate cancer cell lines, implicating BRD7 inhibitors as a potential therapeutic strategy to combat therapy resistant cancers.To develop inhibitors of BRD7 we are developing small molecules that bind selectively to the bromodomain of BRD7. The main challenge when developing BRD7 BD inhibitors is the high similarity to the BRD9 BD, its closest homolog. Several dual inhibitors of BRD7 and BRD9 have been developed, all of them being more selective for the BRD9 BD over the BRD7 BD. Using the X‐ray crystal structure of both BDs bound to a BRD9‐selective inhibitor, we identified an accessible hydrophobic region in the binding pocket of BRD7 that is not present in BRD9, which we hypothesized could be exploited to develop BRD7‐selective inhibitors. Based on this structural difference in the BRD7 binding pocket, we designed a library of compounds that share a bi‐aryl methylated lactam core. (Figure 2)From in silicomolecular docking simulations and in vitro affinity screenings, we have identified several ligands selective for BRD7. These compounds also display potent anti‐proliferative effects in prostate cancer cell lines, providing further evidence for proposing BRD7 as a therapeutic target in prostate cancer. Our on‐going work focuses on optimizing the potency and solubility properties of BRD7 inhibitors, and characterizing their selectivity using cell‐based models.

Reading the message: Connections between the epigenetic landscape bromodomain activity

Bromodomains are conserved epigenetic reader domains known to recognize acetylated lysine residues on the N‐terminal histone tails protruding from the nucleosome. The 61 human bromodomains are found in 41 different proteins, and have been classified into eight different subfamilies based on sequence conservation. These subfamilies often share unique sequence variations that drive differences in histone ligand preference for specific patterns of post‐translational modifications on core and variant histone proteins. The BET bromodomains, which are an attractive drug target in multiple disease states, have been the focus of many studies to elucidate the molecular mechanism of histone recognition. However, much less is known about non‐BET bromodomain‐containing proteins. In this study, we examined the ligand specificity the family IV ATAD2B bromodomain and compared it to its closely related paralog in ATAD2. We show that the ATAD2B bromodomain recognizes mono‐ and di‐acetyllysine modifications on histones H4 and H2A. A structure‐function approach using X‐ray crystallography, isothermal titration calorimetry experiments, and site‐directed mutagenesis coupled to ligand binding assays identified key residues in the acetyllysine binding pocket that dictate the molecular recognition process. Furthermore, our results demonstrate how cross‐talk between multiple modifications alters the binding activity of the ATAD2B bromodomain. The structure of the ATAD2B bromodomain in complex with a small molecule ATAD2 bromodomain inhibitor revealed that critical contacts required for coordination are conserved between the ATAD2/B bromodomains, and many of these residues play a dual role in acetyllysine recognition. We further characterized an alternative splice variant of ATAD2B that results in a loss of bromodomain function. Our results outline the structural and functional features of the ATAD2B bromodomain, and identify novel mechanisms regulating the interaction of the ATAD2B protein with chromatin.

Discovery of 4-Hydroxyquinazoline Derivatives as Small Molecular BET/PARP1 Inhibitors That Induce Defective Homologous Recombination and Lead to Synthetic Lethality for Triple-Negative Breast Cancer Therapy.

The effective potency and resistance of poly(ADP-ribose) polymerase (PARP) inhibitors limit their application. Here, we exploit a new paradigm that mimics the effects of breast cancer susceptibility genes (BRCA) mutations to trigger the possibility of synthetic lethality, based on the previous discovery of a potential synthetic lethality effect between bromodomain-containing protein 4 (BRD4) and PARP1. Consequently, the present study describes compound BP44 with high selectivity for BRD4 and PARP1. Fortunately, BP44 inhibits the homologous recombination in triple-negative breast cancer (TNBC) and triggers synthetic lethality, thus leading to cell cycle arrest and DNA damage. In conclusion, we optimized the BRD4-PARP1 inhibitor based on previous studies, and we expect it to become a candidate drug for the treatment of TNBC in the future. This strategy aims to expand the use of PARPi in BRCA-competent TNBC, making an innovative approach to address unmet oncology needs.

MUS81 Inhibition Enhances the Anticancer Efficacy of Talazoparib by Impairing ATR/CHK1 Signaling Pathway in Gastric Cancer.

MUS81 is a critical endonuclease involved in heterodimer formation with Eme1/Mms4 and an important DNA damage repair regulatory molecule. Our previous study suggested that MUS81 was overexpressed and its high expression was positively correlated with gastric cancer metastasis. However, the therapeutic potential of targeting MUS81 in gastric cancer requires further exploration. Therefore, in this study, the Cancer Genome Atlas (TCGA) data were analyzed and showed that MUS81 is a key regulator of cell cycle distribution and DNA damage repair in gastric cancer. In vitro and in vivo, MUS81 knockdown significantly enhanced the anticancer effect of the PARP inhibitor talazoparib. Mechanistically, MUS81 inhibition impaired the activation of the ATR/CHK1 cell cycle signaling pathway and promoted gastric cancer cells with talazoparib-induced DNA damage to continue mitosis. Moreover, addition of the bromodomain-containing protein 4 inhibitor AZD5153 increased the anticancer effect of talazoparib via MUS81 inhibition in gastric cancer cells, and this combination effect was largely impaired when MUS81 was knocked down. In conclusion, these data suggested that MUS81 regulated ATR/CHK1 activation, a key signaling pathway in the G2M checkpoint, and targeting MUS81 enhanced the antitumor efficacy of talazoparib. Therefore, AZD5153 combined with talazoparib may represent a promising therapeutic strategy for patients with MUS81 proficient gastric cancer.

Bromodomain-containing protein 4 activates cardiotrophin-like cytokine factor 1, an unfavorable prognostic biomarker, and promotes glioblastoma in vitro

BackgroundGlioblastoma (GBM) is one of the most common and malignant brain tumors. Cardiotrophin-like cytokine factor 1 (CLCF1) is a member of the IL-6 superfamily. However, the clinical significance, potential role, and molecular mechanism of CLCF1 in GBM remain obscure. Here, the expression and prognostic significance of CLCF1 was investigated in GBM.MethodsThe Cancer Genome Atlas (TCGA) GBM and Chinese Glioma Genome Atlas (CGGA) datasets were downloaded and analyzed by using Gene Expression Profiling Interactive Analysis (GEPIA). Next, 3 shRNAs targeting CLCF1 were designed, and silencing efficiency was examined with real-time polymerase chain reaction (PCR). Cell Counting Kit 8 (CCK-8), flow cytometry, transwell, and wound healing assays were used to study the function of CLCF1 in glioma cells.ResultsWe found increased expression of CLCF1 as an unfavorable prognostic marker in GBM. Functionally, down-regulation of CLCF1 significantly reduced cell proliferation, induced cell apoptosis and cell cycle G2 phase arrest, and weakened the migration and invasion of GBM cells. Downstream pathway analysis was conducted, and potential targets in cytokine receptors, extracellular matrix (ECM) receptors, apoptosis, and the cell cycle were uncovered. Finally, transcriptional regulators were analyzed, and bromodomain-containing protein 4 (BRD4) was found to activate CLCF1 in GBM.ConclusionsCLCF1, transcriptionally activated by BRD4, promotes glioma and serves as an unfavorable marker in GBM.

Alternatively mechanistic insights into acetylation in p53-mediated transcriptional regulation of cancer cell-intrinsic PD-1

Since the recent discovery of cancer cell-intrinsic programmed cell death protein-1 (PD-1), the mechanisms that manipulate PD-1 functions in tumor development beyond its immune checkpoint roles have become attractive research topics in oncology. Our previous study validated that PD-1 exists in lung cancer cells and is directly transactivated by p53 in a DNA-binding domain (DBD) acetylation-dependent manner. Here, we report that the carboxyl-terminal domain (CTD) of p53 likewise participates in PD-1 transcriptional regulation in cancer cells under different regulatory mechanisms. By mutating the lysine residues within the CTD to mimic either acetylation-deficient or fully acetylated status, we proved that acetylated CTD dramatically impeded p53-mediated transactivation of PD-1. Furthermore, we identified bromodomain-containing protein 4 (BRD4) as a transcriptional coactivator of p53 that facilitates p53-mediated PD-1 transcription. Mechanistically, BRD4 specifically bound to the unacetylated CTD of p53, while CTD acetylation almost completely destroyed the BRD4-p53 interaction and thus led to compromised PD-1 expression. Collectively, this study unveils an alternative mechanism of p53 acetylation-directed PD-1 transcriptional regulation, which would broaden our current understanding of the molecular regulatory network of cancer cell-intrinsic PD-1.

Calcium-Calmodulin-Involved Heat Shock Response of Neoporphyra haitanensis

Increasing global temperatures have seriously affected the sustainable development of Neoporphyra haitanensis cultivation. Although several pathways are reported to be involved in the response of N. haitanensis to heat stress, it is unknown which ones are activated by signal transduction. Previously, we detected a large influx of calcium ions (Ca2+) in N. haitanensis under heat stress. In this study, we further investigated the specificity of Ca2+ signaling and how is it transduced. Transmission electron microscopy and Fv/Fm analyses showed that the Ca2+ signal derived from extracellular Ca2+ formed at the early stage of the response to heat stress, and the signal was recognized and decoded by N. haitanensis calmodulin (NhCaM). In yeast two-hybrid assays, DnaJ, a voltage-dependent anion channel, and a bromodomain-containing protein interacted with PhCaM1 in vivo. The transcript levels of the genes encoding these proteins increased significantly in response to heat stress, but decreased upon inhibition of NhCaM1, indicating that these interacting factors were positively related to NhCaM1. Additionally, a comparative transcriptome analysis indicated that Ca2+ signal transduction is involved in phosphatidylinositol, photosystem processes, and energy metabolism in N. haitanensis under heat stress. Our results suggest that Ca2+-CaM plays important roles in signal transduction in response to heat stress in N. haitanensis.

BRD4 Targets the KEAP1-Nrf2-G6PD Axis and Suppresses Redox Metabolism in Small Cell Lung Cancer.

Accumulating evidence has witnessed the Kelch-like ECH-associated protein 1(KEAP1)- nuclear factor (erythroid-derived 2)-like 2 (Nrf2) axis is the main regulatory factor of cell resistance to endogenous and exogenous oxidative assaults. However, there are few studies addressing the upstream regulatory factors of KEAP1. Herein, bioinformatic analysis suggests bromodomain-containing protein 4 (BRD4) as a potential top transcriptional regulator of KEAP1 in lung cancer. Using molecular and pharmacological approaches, we then discovered that BRD4 can directly bind to the promoter of KEAP1 to activate its transcription and down-regulate the stability of Nrf2 which in turn transcriptionally suppresses glucose-6-phosphate dehydrogenase (G6PD) in small cell lung cancer (SCLC), a highly proliferative and aggressive disease with limited treatment options. In addition, BRD4 could associate with the Nrf2 protein in a non-KEAP1-dependent manner to inhibit Nrf2 activity. Furthermore, simultaneous application of JQ1 and ATRA or RRx-001 yielded synergistic inhibition both in vitro and in vivo. These data suggest metabolic reprogramming by JQ1 treatment improves cell resistance to oxidative stress and might be a resistance mechanism to bromodomain and extra-terminal domain (BET) inhibition therapy. Altogether, our findings provide novel insight into the transcriptional regulatory network of BRD4 and KEAP1 and transcriptional regulation of the pentose phosphate pathway in SCLC.

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.

NUT Carcinoma: Clinicopathologic Features, Molecular Genetics and Epigenetics.

Nuclear protein in testis (NUT) carcinoma is a rare, highly aggressive, poorly differentiated carcinoma occurring mostly in adolescents and young adults. This tumor usually arises from the midline structures of the thorax, head, and neck, and exhibits variable degrees of squamous differentiation. NUT carcinoma is defined by the presence of a NUTM1 (15q14) rearrangement with multiple other genes. In about 70-80% of the cases, NUTM1 is involved in a balanced translocation with the BRD4 gene (19p13.12), leading to a BRD4-NUTM1 fusion oncogene. Other variant rearrangements include BRD3-NUTM1 fusion (~15-20%) and NSD3-NUTM1 fusion (~6%), among others. The diagnosis of NUT carcinoma requires the detection of nuclear expression of the NUT protein by immunohistochemistry. Additional methods for diagnosis include the detection of a NUTM1 rearrangement by fluorescence in situ hybridization or by reverse transcriptase PCR. NUT carcinoma is usually underrecognized due to its rarity and lack of characteristic histological features. Therefore, the goal of this review is to provide relevant recent information regarding the clinicopathologic features of NUT carcinoma, the role of the multiple NUTM1 gene rearrangements in carcinogenesis, and the impact of understanding these underlying molecular mechanisms that may result in the development of possible novel targeted therapies.