BRD4 expression and its regulatory interaction with miR-26a-3p, DLG5-AS1, and JMJD1C-AS1 lncRNAs in gastric cancer progression.

Gastric cancer remains a significant health challenge despite advancements in diagnosis and treatment. Early detection is critical to reducing mortality, necessitating the investigation of molecular mechanisms underlying gastric cancer progression. This study focuses on BRD4 expression and its correlation with miR-26a-3p, DLG5-AS1, and JMJD1C-AS1 lncRNAs in gastric cancer. Analysis of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets revealed significant upregulation of BRD4 in gastric cancer tissues compared to normal tissues, correlating negatively with miR-26a-3p and positively with DLG5-AS1 and JMJD1C-AS1 lncRNAs. Quantitative RT-PCR confirmed these findings in 25 gastric cancer tissue samples and 25 normal samples. BRD4's overexpression was associated with reduced survival rates and older patient age. MiR-26a-3p, a known tumor suppressor, showed decreased expression in gastric cancer tissues, with ROC analysis suggesting it, alongside BRD4, as a potential diagnostic biomarker. Additionally, bioinformatics predicted miR-26a-3p's interaction with BRD4 mRNA. Upregulated lncRNAs DLG5-AS1 and JMJD1C-AS1 likely act as competing endogenous RNAs, sponging miR-26a-3p, thus promoting BRD4 dysregulation. These lncRNAs have not been previously studied in gastric cancer. The findings propose a novel BRD4/lncRNA/miRNA regulatory axis in gastric cancer, highlighting the potential of BRD4, DLG5-AS1, and JMJD1C-AS1 as biomarkers for early diagnosis. Further studies with larger sample sizes and in vivo and in vitro experiments are needed to elucidate this regulatory mechanism's role in gastric cancer progression.

Impact of JQ1 treatment on seizures, hippocampal gene expression, and gliosis in a mouse model of temporal lobe epilepsy.

Epilepsy is a neurological disease characterised by recurrent seizures with complex aetiology. Temporal lobe epilepsy, the most common form in adults, can be acquired following brain insults including trauma, stroke, infection or sustained status epilepticus. The mechanisms that give rise to the formation and maintenance of hyperexcitable networks following acquired insults remain unknown, yet an extensive body of literature points towards persistent gene and epigenomic dysregulation as a potential mediator of this dysfunction. While much is known about the function of specific classes of epigenetic regulators (writers and erasers) in epilepsy, much less is known about the enzymes, which read the epigenome and modulate gene expression accordingly. Here, we explore the potential role for the epigenetic reader bromodomain and extra-terminal domain (BET) proteins in epilepsy. Using the intra-amygdala kainic acid model of temporal lobe epilepsy, we initially identified widespread dysregulation of important epigenetic regulators including EZH2 and REST as well as altered BRD4 expression in chronically epileptic mice. BRD4 activity was also notably affected by epilepsy-provoking insults as seen by elevated binding to and transcriptional regulation of the immediate early gene Fos. Despite influencing early aspects of epileptogenesis, blocking BET protein activity with JQ1 had no overt effects on epilepsy development in mice but did alter glial reactivity and influence gene expression patterns, promoting various neurotransmitter signalling mechanisms and inflammatory pathways in the hippocampus. Together, these results confirm that epigenetic reader activity is affected by epilepsy-provoking brain insults and that BET activity may exert cell-specific actions on inflammation in epilepsy.

SOX2 represses c-MYC transcription by altering the co-activator landscape of the c-MYC super-enhancer and promoter regions.

Our previous studies determined that elevating SOX2 in a wide range of tumor cells leads to a reversible state of tumor growth arrest. Efforts to understand how tumor cell growth is inhibited led to the discovery of a SOX2:MYC axis that is responsible for downregulating c-MYC (MYC) when SOX2 is elevated. Although we had determined that elevating SOX2 downregulates MYC transcription, the mechanism responsible was not determined. Given the challenges of targeting MYC clinically, we set out to identify how elevating SOX2 downregulates MYC transcription. In this study, we focused on the MYC promoter region and an upstream region of the MYC locus that contains a MYC super-enhancer encompassing five MYC enhancers and which is associated with several cancers. Here we report that BRD4 and p300 associate with each of the MYC enhancers in the upstream MYC super-enhancer as well as the MYC promoter region and that elevating SOX2 decreases the recruitment of BRD4 and p300 to these sites. Additionally, we determined that elevating SOX2 leads to increases in the association of SOX2 and H3K27me3 within the MYC super-enhancer and the promoter region of MYC. Importantly, we conclude that the increases in SOX2 within the MYC super-enhancer precipitate a cascade of events that culminates in the repression of MYC transcription. Together, our studies identify a novel molecular mechanism able to regulate MYC transcription in two distinctly different tumor types and provide new mechanistic insights into the molecular interrelationships between two master regulators, SOX2 and MYC, widely involved in multiple cancers.

Transcriptomic profiling of a late recurrent nuclear protein in testis carcinoma of the lung 14 years after the initial operation: a case report.

Background: Nuclear protein in testis (NUT) carcinoma (NC) of the lung is a rare cancer that occurs mainly in young adolescents and adults. NC is genetically characterized by NUTM1 rearrangements, which usually take the form of BRD4-NUT fusions. The prognosis for NC is dismal, and treatment with conventional chemotherapeutic regimens is ineffective. Case Description: We herein describe the case of a 53-year-old woman with recurrent NC of the lung 14 years after surgery for nasal cavity cancer. Chest computed tomography revealed a 5.5-cm tumor in the lower lobe of the left lung. We completely resected the recurrent lung NC via thoracotomy. Immunohistochemistry (IHC) of the lung and nasal cavity cancers showed diffuse strong expression of NUT. RNA-seq of the lung NC revealed NUTM1 rearrangement, with a fusion of BRD4 exon 10 to NUTM1 exon 4. This breakpoint has never been reported before. In addition, IHC revealed elevated expression of parathyroid hormone-like hormone in the lung NC but not in the nasal cavity NC, indicating that the lung and nasal cavity NCs were metachronous multiple primary cancers. Conclusions: We experienced a rare recurrence of lung NC 14 years after the initial surgery. The BRD4-NUT fusion consisted of a new breakpoint. Furthermore, the expression pattern of parathyroid hormone-like hormone (PTHLH) suggested that the NCs in the nasal cavity and lung may be metachronous multiple lung cancers. This extremely rare case highlighted the possibility of identifying less malignant NCs in patients with poorly differentiated tumors via fusion gene analysis and the need to develop more effective treatment strategies for this malignancy.

Phase separation of PML/RARα and BRD4 coassembled microspeckles governs transcriptional dysregulation in acute promyelocytic leukemia.

In acute promyelocytic leukemia (APL), the promyelocytic leukemia-retinoic acid receptor alpha (PML/RARα) fusion protein destroys PML nuclear bodies (NBs), leading to the formation of microspeckles. However, our understanding, largely learned from morphological observations, lacks insight into the mechanisms behind PML/RARα-mediated microspeckle formation and its role in APL leukemogenesis. This study presents evidence uncovering liquid-liquid phase separation (LLPS) as a key mechanism in the formation of PML/RARα-mediated microspeckles. This process is facilitated by the intrinsically disordered region containing a large portion of PML and a smaller segment of RARα. We demonstrate the coassembly of bromodomain-containing protein 4 (BRD4) within PML/RARα-mediated condensates, differing from wild-type PML-formed NBs. In the absence of PML/RARα, PML NBs and BRD4 puncta exist as two independent phases, but the presence of PML/RARα disrupts PML NBs and redistributes PML and BRD4 into a distinct phase, forming PML/RARα-assembled microspeckles. Genome-wide profiling reveals a PML/RARα-induced BRD4 redistribution across the genome, with preferential binding to super-enhancers and broad-promoters (SEBPs). Mechanistically, BRD4 is recruited by PML/RARα into nuclear condensates, facilitating BRD4 chromatin binding to exert transcriptional activation essential for APL survival. Perturbing LLPS through chemical inhibition (1, 6-hexanediol) significantly reduces chromatin co-occupancy of PML/RARα and BRD4, attenuating their target gene activation. Finally, a series of experimental validations in primary APL patient samples confirm that PML/RARα forms microspeckles through condensates, recruits BRD4 to coassemble condensates, and co-occupies SEBP regions. Our findings elucidate the biophysical, pathological, and transcriptional dynamics of PML/RARα-assembled microspeckles, underscoring the importance of BRD4 in mediating transcriptional activation that enables PML/RARα to initiate APL.

Targeting BRD4 to attenuate RANKL-induced osteoclast activation and bone erosion in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that can cause destruction of cartilage and bone's extracellular matrix. Bromodomain 4 (BRD4), as a transcriptional and epigenetic regulator, plays a key role in cancer and inflammatory diseases. While, the role of BRD4 in bone destruction in RA has not been extensively reported. Our study aimed to investigate the effect of BRD4 on the bone destruction in RA and, further, its mechanism in the pathogenesis of the disease. In this study, receiving approval from the Ethical Committee of the Affiliated Hospital of Qingdao University, we evaluated synovial tissues from patients with RA and OA for BRD4 expression through advanced techniques such as immunohistochemistry, quantitative real-time PCR (qRT-PCR), and Western blotting. We employed a collagen-induced arthritis (CIA) mouse model to assess the therapeutic efficacy of the BRD4 inhibitor JQ1 on disease progression and bone destruction, supported by detailed clinical scoring and histological examinations. Further, in vitro osteoclastogenesis assays using RAW264.7 macrophages, facilitated by TRAP staining and resorption pit assays, provided insights into the mechanistic effects of JQ1 on osteoclast function. Statistical analysis was rigorously conducted using SPSS, applying Kruskal-Wallis, one-way ANOVA, and Student's t-tests to validate the data. In our study, we found that BRD4 expression significantly increased in the synovial tissues of RA patients and the ankle joints of CIA mice, with JQ1, a BRD4 inhibitor, effectively reducing inflammation, arthritis severity (p < 0.05), and bone erosion. Treatment with JQ1 not only improved bone mass and structural integrity in CIA mice but also downregulated osteoclast-related gene expression and the RANKL/RANK signaling pathway, indicating a suppression of osteolysis. Furthermore, in vitro assays demonstrated that JQ1 markedly inhibited osteoclast differentiation and function, underscoring the pivotal role of BRD4 in osteoclastogenesis and its potential as a target for therapeutic intervention in RA-induced bone destruction. Our study concludes that targeting BRD4 with the inhibitor JQ1 significantly mitigates inflammation and bone destruction in rheumatoid arthritis, suggesting that inhibition of BRD4 may be a potential therapeutic strategy for the treatment of bone destruction in RA.

The BET PROTAC inhibitor GNE-987 displays anti-tumor effects by targeting super-enhancers regulated gene in osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is one of the most common primary malignant tumors of bone in children, which develops from osteoblasts and typically occurs during the rapid growth phase of the bone. Recently, Super-Enhancers(SEs)have been reported to play a crucial role in osteosarcoma growth and metastasis. Therefore, there is an urgent need to identify specific targeted inhibitors of SEs to assist clinical therapy. This study aimed to elucidate the role of BRD4 inhibitor GNE-987 targeting SEs in OS and preliminarily explore its mechanism. METHODS: We evaluated changes in osteosarcoma cells following treatment with a BRD4 inhibitor GNE-987. We assessed the anti-tumor effect of GNE-987 in vitro and in vivo by Western blot, CCK8, flow cytometry detection, clone formation, xenograft tumor size measurements, and Ki67 immunohistochemical staining, and combined ChIP-seq with RNA-seq techniques to find its anti-tumor mechanism. RESULTS: In this study, we found that extremely low concentrations of GNE-987(2-10 nM) significantly reduced the proliferation and survival of OS cells by degrading BRD4. In addition, we found that GNE-987 markedly induced cell cycle arrest and apoptosis in OS cells. Further study indicated that VHL was critical for GNE-987 to exert its antitumor effect in OS cells. Consistent with in vitro results, GNE-987 administration significantly reduced tumor size in xenograft models with minimal toxicity, and partially degraded the BRD4 protein. KRT80 was identified through analysis of the RNA-seq and ChIP-seq data. U2OS HiC analysis suggested a higher frequency of chromatin interactions near the KRT80 binding site. The enrichment of H3K27ac modification at KRT80 was significantly reduced after GNE-987 treatment. KRT80 was identified as playing an important role in OS occurrence and development. CONCLUSIONS: This research revealed that GNE-987 selectively degraded BRD4 and disrupted the transcriptional regulation of oncogenes in OS. GNE-987 has the potential to affect KRT80 against OS.

SHP2 mediates the ROS/JNK/NFAT4 signaling pathway in gastric cancer cells prompting lncRNA SNHG18 to drive gastric cancer growth and metastasis via CAR-T cells.

OBJECTIVE: In gastric cancer cells, the influence of CAR T cells can be produced in the process of inhibiting the progression of gastric cancer, and the role of tyrosine phosphatase SHP2 can be explored in this study, along with its molecular mechanisms. METHODS: The research utilized subcutaneous tumor models in nude mice to assess gastric cancer progression. Protein expression was detected using Western blotting, while Q-PCR examined the expression levels of lncRNA SNHG18 and miR-211-5p in MGC-803 cells. The relationship between miR-211-5p and lncRNA SNHG18 can be analyzed by dual luciferase reporter genes. The migratory ability of MGC-803 cells was determined through wound healing and transwell experiments, and cell proliferation was evaluated using a CCK-8 assay. RESULTS: SHP2 was found to inhibit the cytotoxic effects of CAR-T cells on MGC-803 cells, and it suppressed the expression of proteins related to the ROS/JNK/NFAT4 signaling pathway in MGC-803 cells and the miR-211-5p/BRD4 axis in CAR-T cells. In addition, the proliferation, invasion and migration of MGC-803 cells were promoted, and the expression of miR-211-5p could be inhibited specifically by ncRNA SNHG18, as shown below:SHP2 in gastric cancer cells mediates the ROS/JNK/NFAT4 signaling pathway and induces lncRNA SNHG18, which, through the miR-211-5p/BRD4 axis in CAR-T cells, promotes gastric cancer growth and metastasis.

A novel 7-phenoxy-benzimidazole derivative as a potent and orally available BRD4 inhibitor for the treatment of melanoma.

The bromodomain-containing protein 4 (BRD4), which is a key epigenetic regulator in cancer, has emerged as an attractive target for the treatment of melanoma. In this study, we investigate 7-phenoxy-benzimidazole derivative 12, which is a novel BRD4 inhibitor for the treatment of melanoma, by performing scaffold hopping on the previously reported benzimidazole derivative 1. Despite their good oral and intravenous exposure, the compounds obtained by modifying derivate 1 exhibit mutagenicity, which was confirmed by the positive Ames test results. Based on our hypothesis that the cause of the Ames test positivity is the metabolic intermediates generated from those chemical series, we implemented a scaffold hopping strategy to avoid the N-benzyl moiety by relocating the substituent groups to preserve the essential interaction. Based on this strategy, we successfully obtained compound 12; the Ames test results of this compound were negative. Notably, compound 12 not only exhibited a favorable pharmacokinetic (PK) profile but also significant tumor growth inhibition in a mouse melanoma xenograft model, indicating its potential as a therapeutic agent for the treatment of melanoma.

Repression of BRD4 mitigates NLRP3 inflammasome-mediated pyroptosis in Mycobacterium-infected macrophages by repressing endoplasmic reticulum stress.

Tuberculosis (TB) is the leading cause of human death worldwide due to Mycobacterium tuberculosis (Mtb) infection. Multiple lines of evidences have illuminated the emerging role of NLRP3 inflammasome-mediated pyroptosis in the clearance of pathogenic infection. In the current study, we sought to investigate the functional role and feasible potential mechanism of BRD4 in Mtb-infected macrophages. We observed that BRD4 was distinctly ascended in THP-1 macrophages upon Mtb infection. Functionally, intervention of BRD4 or pretreated with JQ1 obviously restricted Mtb-triggered cell pyroptosis, as evidenced by declination of protein level of the specific pyroptosis markers including Cleaved Caspase 1, gasdermin D (GSDMD-N) and Cleaved-IL-1ß. In the meanwhile, disruption of BRD4 or JQ1 application remarkably prohibited excessive inflammatory responses as characterized by reduce the production of the inflammatory factors such as IL-1ß and IL-18. Concomitantly, disruption of BRD4 or administrated with JQ1 manifestly repressed Mtb-aroused Nod-like receptor family pyrindomain-containing 3 (NLRP3) inflammasome activation, as witnessed by attenuation of protein levels of NLRP3, Pro-Caspase1 and apoptosis-associated speck-like protein (ASC). The above findings clearly demonstrated that suppression of BRD4 exerted great influence on regulating Mtb-elicited inflammatory response by coordinating NLRP3 inflammasome-mediated pyroptosis. More importantly, perturbation of BRD4 or JQ1 employment notably restrained endoplasmic reticulum (ER) stress triggered by Mtb-infection, as reflected by noticeably lessened the levels of GRP78, CHOP and ATF6. In terms of mechanism, ER stress agonist tunicamycin profoundly abrogated the favorable effects of BRD4 inhibition on Mtb-triggered pyroptosis, inflammation reaction and inflammasome activation. Collectively, these preceding outcomes strongly illuminated that inhibition of BRD4 targeted ER stress to retard NLRP3 inflammasome activation and subsequent cell pyroptosis and prevention of inflammatory response in Mtb-infected macrophages, highlighting that blocking BRD4 might serve as a promising candidate for protection against Mtb-triggered inflammatory injury.

[Research Progress on Pathogenesis and Treatment of NUT Carcinoma].

NUT carcinoma (nuclear protein in testis carcinoma) is a rare and highly invasive malignant tumor, which is most common in midline organs and lungs. The characteristic genetic change of NUT carcinoma is the rearrangement of NUT middle carcinoma family member 1 (NUTM1) gene. In this article, we will review the pathogenic mechanism of its most common fusion form, bromodomaincontaining protein 4 (BRD4)-NUTM1 fusion gene, and the progress in the research and development of targeting drugs.
.

The Megacomplex protects ER-alpha from degradation by Fulvestrant in epithelial ovarian cancer.

Ovarian cancer, a significant contributor to cancer-related mortality, exhibits limited responsiveness to hormonal therapies targeting the estrogen receptor (ERα). This study aimed to elucidate the mechanisms behind ERα resistance to the therapeutic drug Fulvestrant (ICI182780 or ICI). Notably, compared to the cytoplasmic version, nuclear ERα was minimally degraded by ICI, suggesting a mechanism for drug resistance via the protective confines of the nuclear substructures. Of these substructures, we identified a 1.3MDa Megacomplex comprising transcription factors ERα, FOXA1, and PITX1 using size exclusion chromatography (SEC) in the ovarian cancer cell line, PEO4. ChIP-seq revealed these factors colocalized at 6,775 genomic positions representing sites of Megacomplex formation. Megacomplex ERα exhibited increased resistance to degradation by ICI compared to cytoplasmic and nuclear ERα. A small molecule inhibitor of active chromatin and super-enhancers, JQ1, in combination with ICI significantly enhanced ERα degradation from Megacomplex as revealed by SEC and ChIP-seq. Interestingly, this combination degraded both the cytoplasmic as well as nuclear ERa. Pathway enrichment analysis showed parallel results for RNA-seq gene sets following Estradiol, ICI, or ICI plus JQ1 treatments as those defined by Megacomplex binding identified through ChIP-seq. Furthermore, similar pathway enrichments were confirmed in mass-spec analysis of the Megacomplex macromolecule fractions after modulation by Estradiol or ICI. These findings implicate Megacomplex in ERα-driven ovarian cancer chromatin regulation. This combined treatment strategy exhibited superior inhibition of cell proliferation and viability. Therefore, by uncovering ERα's resistance within the Megacomplex, the combined ICI plus JQ1 treatment elucidates a novel drug treatment vulnerability.

Progestogen-driven B7-H4 contributes to onco-fetal immune tolerance.

Immune tolerance mechanisms are shared in cancer and pregnancy. Through cross-analyzing single-cell RNA-sequencing data from multiple human cancer types and the maternal-fetal interface, we found B7-H4 (VTCN1) is an onco-fetal immune tolerance checkpoint. We showed that genetic deficiency of B7-H4 resulted in immune activation and fetal resorption in allogeneic pregnancy models. Analogously, B7-H4 contributed to MPA/DMBA-induced breast cancer progression, accompanied by CD8+ T cell exhaustion. Female hormone screening revealed that progesterone stimulated B7-H4 expression in placental and breast cancer cells. Mechanistically, progesterone receptor (PR) bound to a newly identified -58 kb enhancer, thereby mediating B7-H4 transcription via the PR-P300-BRD4 axis. PR antagonist or BRD4 degrader potentiated immunotherapy in a murine B7-H4+ breast cancer model. Thus, our work unravels a mechanistic and biological connection of a female sex hormone (progesterone) to onco-fetal immune tolerance via B7-H4 and suggests that the PR-P300-BRD4 axis is targetable for treating B7-H4+ cancer.

Elevating PLK1 overcomes BETi resistance in prostate cancer via triggering BRD4 phosphorylation-dependent degradation in mitosis.

Bromodomain-containing protein 4 (BRD4) has emerged as a promising therapeutic target in prostate cancer (PCa). Understanding the mechanisms of BRD4 stability could enhance the clinical response to BRD4-targeted therapy. In this study, we report that BRD4 protein levels are significantly decreased during mitosis in a PLK1-dependent manner. Mechanistically, we show that BRD4 is primarily phosphorylated at T1186 by the CDK1/cyclin B complex, recruiting PLK1 to phosphorylate BRD4 at S24/S1100, which are recognized by the APC/CCdh1 complex for proteasome pathway degradation. We find that PLK1 overexpression lowers SPOP mutation-stabilized BRD4, consequently rendering PCa cells re-sensitized to BRD4 inhibitors. Intriguingly, we report that sequential treatment of docetaxel and JQ1 resulted in significant inhibition of PCa. Collectively, the results support that PLK1-phosphorylated BRD4 triggers its degradation at M phase. Sequential treatment of docetaxel and JQ1 overcomes BRD4 accumulation-associated bromodomain and extra-terminal inhibitor (BETi) resistance, which may shed light on the development of strategies to treat PCa.

Research progress of multi-target HDAC inhibitors blocking the BRD4-LIFR-JAK1-STAT3 signaling pathway in the treatment of cancer.

Histone deacetylase inhibitors (HDACis) show beneficial effects on different hematological malignancy subtypes. However, their impacts on treating solid tumors are still limited due to diverse resistance mechanisms. Recent studies have found that the feedback activation of BRD4-LIFR-JAK1-STAT3 pathway after HDACi incubation is a vital mechanism inducing resistance of specific solid tumor cells to HDACis. This review summarizes the recent development of multi-target HDACis that can concurrently block BRD4-LIFR-JAK1-STAT3 pathway. Moreover, our findings hope to shed novel lights on developing novel multi-target HDACis with reduced BRD4-LIFR-JAK1-STAT3-mediated drug resistance in some tumors.

Bromodomain Protein Inhibition Protects ß-Cells from Cytokine-Induced Death and Dysfunction via Antagonism of NF-κB Pathway.

Cytokine-induced ß-cell apoptosis is a major pathogenic mechanism in type 1 diabetes (T1D). Despite significant advances in understanding its underlying mechanisms, few drugs have been translated to protect ß-cells in T1D. Epigenetic modulators such as bromodomain-containing BET (bromo- and extra-terminal) proteins are important regulators of immune responses. Pre-clinical studies have demonstrated a protective effect of BET inhibitors in an NOD (non-obese diabetes) mouse model of T1D. However, the effect of BET protein inhibition on ß-cell function in response to cytokines is unknown. Here, we demonstrate that I-BET, a BET protein inhibitor, protected ß-cells from cytokine-induced dysfunction and death. In vivo administration of I-BET to mice exposed to low-dose STZ (streptozotocin), a model of T1D, significantly reduced ß-cell apoptosis, suggesting a cytoprotective function. Mechanistically, I-BET treatment inhibited cytokine-induced NF-kB signaling and enhanced FOXO1-mediated anti-oxidant response in ß-cells. RNA-Seq analysis revealed that I-BET treatment also suppressed pathways involved in apoptosis while maintaining the expression of genes critical for ß-cell function, such as Pdx1 and Ins1. Taken together, this study demonstrates that I-BET is effective in protecting ß-cells from cytokine-induced dysfunction and apoptosis, and targeting BET proteins could have potential therapeutic value in preserving ß-cell functional mass in T1D.

BRD4 plays an antiaging role in the senescence of renal tubular epithelial cells.

Background: Age-related kidney failure is often induced by a decrease in the bioavailability of tubular epithelial cells in elderly chronic kidney disease (CKD) patients. BRD4, an epigenetic regulator and a member of the bromodomain and extraterminal (BET) protein family, acts as a super-enhancer (SE) organizing and regulating genes expression during embryogenesis and cancer development. But the physiological function of BRD4 in normal cells has been less studied. This study aimed to research certain biological roles of BRD4 in the process of normal cell aging and discuss the potential mechanisms. Methods: In this study, we investigated the biological functions of BRD4 proteins in the aging of renal tubular cells. At first, we used a D-galactose (D-gal) and BRD4 inhibitor (Abbv-075) to replicate kidney senescence in vivo. D-gal and Abbv-075 were then used to measure the aging-related changes, such as changes in cell cycle, ß-galactosidase activity, cell migration, and p16 protein expression in vitro. At last, we knocked down and over-expressed BRD4 to investigate the aging-related physiological phenomena in renal tubular cells. Results: In vitro, D-gal treatment induced noticeable aging-related changes such as inducing cell apoptosis and cell cycle arrest, increasing ß-galactosidase activity as well as up-regulating p16 protein expression in primary human tubular epithelial cells. In the aging mice model, D-gal significantly induced renal function impairment and attenuated BRD4 protein expression. At the same time, the BRD4 inhibitor (Abbv-075) was able to mimic D-gal-induced cell senescence. In vivo, Abbv-075 also decreased kidney function and up-regulated p21 protein expression. When we knocked down the expression of BRD4, the senescence-associated ß-galactosidase (SA-ß-gal) activity increased dramatically, cell migration was inhibited, and the proportion of cells in the G0/G1 phase increased. Additionally, the knockdown also promoted the expression of the senescence-related proteins p16. When the renal tubular cells were overexpressed with BRD4, cell aging-related indicators were reversed in the D-gal-induced cell aging model. Conclusions: BRD4 appears to have an active role in the aging of renal tubular cells in vivo and in vitro. The findings also suggest that BRD4 inhibitors have potential nephrotoxic effects for oncology treatment. BRD4 may be a potential therapeutic biomarker and drug target for aging-related kidney diseases, which warrants additional studies.

Ultrasound-Activated PROTAC Prodrugs Overcome Immunosuppression to Actuate Efficient Deep-Tissue Sono-Immunotherapy in Orthotopic Pancreatic Tumor Mouse Models.

The degradation of oncoproteins mediated by proteolysis-targeting chimera (PROTAC) has emerged as a potent strategy in cancer therapy. However, the clinical application of PROTACs is hampered by challenges such as poor water solubility and off-target adverse effects. Herein, we present an ultrasound (US)-activatable PROTAC prodrug termed NPCe6+PRO for actuating efficient sono-immunotherapy in a spatiotemporally controllable manner. Specifically, US irradiation, which exhibits deep-tissue penetration capability, results in Ce6-mediated generation of ROS, facilitating sonodynamic therapy (SDT) and inducing immunogenic cell death (ICD). Simultaneously, the generated ROS cleaves the thioketal (TK) linker through a ROS-responsive mechanism, realizing the on-demand activation of the PROTAC prodrug in deep tissues. This prodrug activation results in the degradation of the target protein BRD4, while simultaneously reversing the upregulation of PD-L1 expression associated with the SDT process. In the orthotopic mouse model of pancreatic tumors, NPCe6+PRO effectively suppressed tumor growth in conjunction with US stimulation.

A novel magnetic ligand-based assay for the electrochemical determination of BRD4.

The first magnetic ligand-based electrochemical assay aimed at the determination of BRD4 was developed and validated. BRD4 is an epigenetic regulator of great interest in oncology in relation to its overexpression observed in the pathogenesis of several cancer diseases. BRD4 also represents a major target for the development of innovative treatments aimed at protein inhibition or degradation. Despite the relevance of BRD4 both for diagnostics and therapeutic purposes, current methodologies for its determination are limited to commercial ELISA kits. We present a novel magnetic ligand-based assay for the electrochemical determination of BRD4. The developed assay is based on the use of a small synthetic fragment of the natural protein ligand for BRD4 as receptor, thus exploiting the intrinsic biological protein-protein recognition mechanism. In addition, the assay features the use of magnetic beads as immobilization platforms and peroxidase-conjugated monoclonal anti-BRD4 antibody for the generation of the electrochemical signal. The ligand-based assay shows outstanding performance in terms of rapidity, with results achievable in less than 20 min, no matrix effect when applied to human plasma or cell lysate samples, and excellent specificity. The proposed method exhibits a limit of detection of 2.66 nM and a response range tunable as a function of the amount of immobilized receptor. The developed ligand-based assay was successfully applied to the accurate determination of BRD4 in untreated cell lysates, as proven by the ELISA reference method. The good performance of the proposed bioassay for determination of BRD4 showed potential application of this strategy in convenient point-of-care testing.

Up-regulation of BRD4 contributes to gestational diabetes mellitus-induced cardiac hypertrophy in offspring by promoting mitochondria dysfunction in sex-independent manner.

Gestational diabetes mellitus (GDM) is associated with cardiovascular disease in postnatal life. The current study tested the hypothesis that GDM caused the cardiac hypertrophy in fetal (ED18.5), postnatal day 7 (PD7), postnatal day 21 (PD21) and postnatal day 90 (PD90) offspring by upregulation of BRD4 and mitochondrial dysfunction. Pregnant mice were divided into control and GDM groups. Hearts were isolated from ED18.5, PD7, PD21 and PD90. GDM increased the body weight (BW) and heart weight (HW) in ED18.5 and PD7, but not PD21 and PD90 offspring. However, HW/BW ratio was increased in all ages of GDM offspring compared to control group. Electron microscopy showed disorganized myofibrils, mitochondrial swelling, vacuolization, and cristae disorder in GDM offspring. GDM resulted in myocardial hypertrophy in offspring, which persisted from fetus to adult in a sex-independent manner. Echocardiography analysis revealed that GDM caused diastolic dysfunction, but had no effect on systolic function. Meanwhile, myocardial BRD4 was significantly upregulated in GDM offspring and BRD4 inhibition by JQ1 alleviated GDM-induced myocardial hypertrophy in offspring. Co-immunoprecipitation showed that BRD4 interacted with DRP1 and there was an increase of BRD4 and DRP1 interaction in GDM offspring. Furthermore, GDM caused the accumulation of damaged mitochondria in hearts from all ages of offspring, including mitochondrial fusion fission imbalance (upregulation of DRP1, and downregulation of MFN1, MFN2 and OPA1) and myocardial mitochondrial ROS accumulation, which was reversed by JQ1. These results suggested that the upregulation of BRD4 is involved in GDM-induced myocardial hypertrophy in the offspring through promoting mitochondrial damage in a gender-independent manner.