Bromodomain Inhibitors Research Articles

Structure-Based Design of CBP/EP300 Degraders: When Cooperativity Overcomes Affinity.

We present the development of dCE-2, a structurally novel PROTAC targeting the CREB-binding protein (CBP) and E1A-associated protein (EP300)-two homologous multidomain enzymes crucial for enhancer-mediated transcription. The design of dCE-2 was based on the crystal structure of an in-house bromodomain (BRD) inhibitor featuring a 3-methyl-cinnoline acetyl-lysine mimic acetyl-lysine mimic discovered by high-throughput fragment docking. Our study shows that, despite its modest binding affinity to CBP/EP300-BRD, dCE-2's remarkable protein degradation activity stems from its good cooperativity, which we demonstrate by the characterization of its ternary complex formation both in vitro and in cellulo. Molecular dynamics simulations indicate that in aqueous solvents, this active degrader populates both folded and extended conformations, which are likely to promote cell permeability and ternary complex formation, respectively.

CDK9 inhibition constrains multiple oncogenic transcriptional and epigenetic pathways in prostate cancer

BackgroundCyclin-dependent kinase 9 (CDK9) stimulates oncogenic transcriptional pathways in cancer and CDK9 inhibitors have emerged as promising therapeutic candidates.MethodsThe activity of an orally bioavailable CDK9 inhibitor, CDKI-73, was evaluated in prostate cancer cell lines, a xenograft mouse model, and patient-derived tumor explants and organoids. Expression of CDK9 was evaluated in clinical specimens by mining public datasets and immunohistochemistry. Effects of CDKI-73 on prostate cancer cells were determined by cell-based assays, molecular profiling and transcriptomic/epigenomic approaches.ResultsCDKI-73 inhibited proliferation and enhanced cell death in diverse in vitro and in vivo models of androgen receptor (AR)-driven and AR-independent models. Mechanistically, CDKI-73-mediated inhibition of RNA polymerase II serine 2 phosphorylation resulted in reduced expression of BCL-2 anti-apoptotic factors and transcriptional defects. Transcriptomic and epigenomic approaches revealed that CDKI-73 suppressed signaling pathways regulated by AR, MYC, and BRD4, key drivers of dysregulated transcription in prostate cancer, and reprogrammed cancer-associated super-enhancers. These latter findings prompted the evaluation of CDKI-73 with the BRD4 inhibitor AZD5153, a combination that was synergistic in patient-derived organoids and in vivo.ConclusionOur work demonstrates that CDK9 inhibition disrupts multiple oncogenic pathways and positions CDKI-73 as a promising therapeutic agent for prostate cancer, particularly aggressive, therapy-resistant subtypes.

Identification of novel bromodomain inhibitors of Trypanosoma cruzi bromodomain factor 2 (TcBDF2) using a fluorescence polarization-based high-throughput assay.

Bromodomains are structural folds present in all eukaryotic cells that bind to other proteins recognizing acetylated lysines. Most proteins with bromodomains are part of nuclear complexes that interact with acetylated histone residues and regulate DNA replication, transcription, and repair through chromatin structure remodeling. Bromodomain inhibitors are small molecules that bind to the hydrophobic pocket of bromodomains, interfering with the interaction with acetylated histones. Using a fluorescent probe, we have developed an assay to select inhibitors of the bromodomain factor 2 of Trypanosoma cruzi (TcBDF2) using fluorescence polarization. Initially, a library of 28,251 compounds was screened in an endpoint assay. The top 350-ranked compounds were further analyzed in a dose-response assay. From this analysis, seven compounds were obtained that had not been previously characterized as bromodomain inhibitors. Although these compounds did not exhibit significant trypanocidal activity, all showed bona fide interaction with TcBDF2 with dissociation constants between 1 and 3 µM validating these assays to search for bromodomain inhibitors.

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

BackgroundOsteosarcoma (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.MethodsWe 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.ResultsIn 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.ConclusionsThis 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.

AB043. BRD4 promotes immune escape of glioma cells by upregulating PD-L1 expression.

Glioblastoma multiforme (GBM) poses significant challenges in treatment due to its aggressive nature and immune escape mechanisms. Despite recent advances in immune checkpoint blockade therapies, GBM prognosis remains poor. The role of bromodomain and extraterminal domain protein 4 (BRD4) in GBM, especially its interaction with immune checkpoints, is not well understood. Bioinformatic gene expression and survival analysis for BRD4 was utilized in The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases. Clone formation assay, Transwell, Cell Counting Kit-8 (CCK8), and wound healing assay were utilized to validate BRD4's promotion of glioma cell proliferation, invasion, and migration. Chromatin immunoprecipitation (ChIP) assay was conducted to confirm BRD4 binding to the programmed death ligand 1 (PD-L1) promoter. A co-culture model was utilized with activated cluster of differentiation 8 (CD8)+ T cells and glioma cells. GL261 cells with BRD4 short hairpin RNA (shRNA) and/or PD-L1 cDNA were intracranially injected into mice to investigate tumor growth and survival time. Tumor tissue characteristics were analyzed using hematoxylin-eosin (H&E) and immunohistochemistry (IHC) staining and immune cell infiltration were assessed by flow cytometry. Bioinformatics analyses reveal elevated BRD4 expression in high-grade gliomas, correlating with poor patient survival. In vitro studies confirm that BRD4 promotes proliferation, invasion, and migration in GBM cells. BRD4 is a regulator of PD-L1 at the transcriptional level, implying its involvement in GBM's immune escape mechanisms. Co-culture experiments with CD8+ T cells demonstrate that BRD4 inhibition enhances tumor cell apoptosis. In vivo studies indicate that BRD4 knockout reduces immunosuppression, improves prognosis. Simultaneous manipulation of BRD4 and PD-L1 levels provides insights into their intertwined roles in shaping the immune landscape of GBM. BRD4 has the capability to regulate the growth of glioblastoma and enhance immune suppression by promoting PD-L1 expression. Targeting BRD4 represents a promising direction for future research and treatment.

Resistance of estrogen receptor function to BET bromodomain inhibition is mediated by transcriptional coactivator cooperativity.

The Bromodomain and Extra-Terminal Domain (BET) family of proteins are critical chromatin readers that bind to acetylated histones through their bromodomains to activate transcription. Here, we reveal that bromodomain inhibition fails to repress oncogenic targets of estrogen receptor due to an intrinsic transcriptional mechanism. While bromodomains are necessary for the transcription of many genes, BRD4 binds to estrogen receptor binding sites and activates transcription of critical oncogenes independently of its bromodomains. BRD4 associates with the Mediator complex and disruption of Mediator complex reduces BRD4's enhancer occupancy. Profiling changes in the post-initiation RNA polymerase II (Pol II)-associated factors revealed that BET proteins regulate interactions between Pol II and elongation factors SPT5, SPT6, and PAF1 complex, which associate with BET proteins independently of their bromodomains and mediate their transcription elongation effect. Our findings highlight the importance of bromodomain-independent functions and interactions of BET proteins in the development of future therapeutic strategies.

Unlocking the therapeutic potential of selective CDK7 and BRD4 Inhibition against multiple myeloma cell growth.

Multiple myeloma (MM) is a plasma cell malignancy considered incurable despite the recent therapeutic advances. Effective targeted therapies are therefore needed. Our previous studies proved that inhibiting CDK7 impairs the cell cycle and metabolic programs by disrupting E2F1 and MYC transcriptional activities, making it an appealing therapeutic target for MM. Given that CDK7 and BRD4 operate in two distinct regulatory axes in MM, we hypothesized that targeting these two complementary pathways simultaneously would lead to a deeper and more durable response. Indeed, combination therapy had superior activity against MM cell growth and viability, and induced apoptosis to a greater extent than single-agent therapy in both cell lines and patient cells. This synergistic activity was also observed in Waldenström's Macroglobulinemia (WM) cells and with other inhibitors of E2F1 activity. Dual inhibition effectively impaired the MYC and E2F transcriptional programs and MM tumor growth and progression in xenograft animal models, providing evidence for combination therapy's potential as a therapeutic strategy in MM and WM.

CRISPR-Cas9-mediated deletion enhancer of MECOM play a tumor suppressor role in ovarian cancer.

MDS1 and EVI1 complex locus (MECOM), a transcription factor encoding several variants, has been implicated in progression of ovarian cancer. The function of regulatory regions in regulating MECOM expression in ovarian cancer is not fully understood. In this study, MECOM expression was evaluated in ovarian cancer cell lines treated with bromodomain and extraterminal (BET) inhibitor JQ-1. Oncogenic phenotypes were assayed using assays of CCK-8, colony formation, wound-healing and transwell. Oncogenic phenotypes were estimated in stable sgRNA-transfected OVCAR3 cell lines. Xenograft mouse model was assayed via subcutaneous injection of enhancer-deleted OVCAR3 cell lines. The results displayed that expression of MECOM is downregulated in cell lines treated with JQ-1. Data from published ChIP-sequencing (H3K27Ac) in 3 ovarian cancer cell lines displayed a potential enhancer around the first exon. mRNA and protein expression were downregulated in OVCAR3 cells after deletion of the MECOM enhancer. Similarly, oncogenic phenotypes both in cells and in the xenograft mouse model were significantly attenuated. This study demonstrates that JQ-1 can inhibit the expression of MECOM and tumorigenesis. Deletion of the enhancer activity of MECOM has an indispensable role in inhibiting ovarian cancer progress, which sheds light on a promising opportunity for ovarian cancer treatment through the application of this non-coding DNA deletion.

Expanding the landscape of oncogenic drivers and treatment options in acral and mucosal melanomas by targeted genomic profiling.

Despite advancements in treating cutaneous melanoma, patients with acral and mucosal (A/M) melanomas still have limited therapeutic options and poor prognoses. We analyzed 156 melanomas (101 cutaneous, 28 acral, and 27 mucosal) using the Foundation One cancer-gene specific clinical testing platform and identified new, potentially targetable genomic alterations (GAs) in specific anatomic sites of A/M melanomas. Using novel pre-clinical models of A/M melanoma, we demonstrate that several GAs and corresponding oncogenic pathways associated with cutaneous melanomas are similarly targetable in A/M melanomas. Other alterations, including MYC and CRKL amplifications, were unique to A/M melanomas and susceptible to indirect targeting using the BRD4 inhibitor JQ1 or Src/ABL inhibitor dasatinib, respectively. We further identified new, actionable A/M-specific alterations, including an inactivating NF2 fusion in a mucosal melanoma responsive to dasatinib in vivo. Our study highlights new molecular differences between cutaneous and A/M melanomas, and across different anatomic sites within A/M, which may change clinical testing and treatment paradigms for these rare melanomas.

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-1β. 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-1β 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.

Targeting BRD4 mitigates hepatocellular lipotoxicity by suppressing the NLRP3 inflammasome activation and GSDMD-mediated hepatocyte pyroptosis

Nod-like receptor family pyrin-containing protein 3 (NLRP3) inflammasome plays a pathologic role in metabolic dysfunction-associated steatohepatitis (MASH), but the molecular mechanism regulating the NLRP3 inflammasome activation in hepatocellular lipotoxicity remains largely unknown. Bromodomain-containing protein 4 (BRD4) has emerged as a key epigenetic reader of acetylated lysine residues in enhancer regions that control the transcription of key genes. The aim of this study is to investigate if and how BRD4 regulated the NLRP3 inflammasome activation and pyroptosis in MASH. Using the AML12 and primary mouse hepatocytes stimulated by palmitic acid (PA) as an in vitro model of hepatocellular lipotoxicity, we found that targeting BRD4 by genetic knockdown or a selective BRD4 inhibitor MS417 protected against hepatosteatosis; and this protective effect was attributed to inhibiting the activation of NLRP3 inflammasome and reducing the expression of Caspase-1, gasdermin D (GSDMD), interleukin (IL)-1β and IL-6. Moreover, BRD4 inhibition limited the voltage-dependent anion channel-1 (VDAC1) expression and oligomerization in PA-treated AML12 hepatocytes, thereby suppressing the NLRP3 inflammasome activation. Additionally, the expression of BRD4 enhanced in MASH livers of humans. Mechanistically, BRD4 was upregulated during hepatocellular lipotoxicity that in turn modulated the active epigenetic mark H3K27ac at the promoter regions of the Vdac and Gsdmd genes, thereby enhancing the expression of VDAC and GSDMD. Altogether, our data provide novel insights into epigenetic mechanisms underlying BRD4 activating the NLRP3 inflammasome and promoting GSDMD-mediated pyroptosis in hepatocellular lipotoxicity. Thus, BRD4 might serve as a novel therapeutic target for the treatment of MASH.Graphic abstract

Bromodomain-containing 4 is a positive regulator of the inflammatory cytokine response in the gut.

Bromodomain-containing protein 4 (BRD4), one of the components of the bromodomain and extraterminal domain (BET) family, is a transcriptional and epigenetic regulator of cellular proliferation and cytokine production. In this study, we assessed whether BRD4 regulates the cytokine response in inflammatory bowel diseases (IBD). BRD4 expression was analyzed in intestinal mucosal samples of patients with ulcerative colitis (UC), patients with Crohn's disease (CD), normal controls (CTRs), and mice with chemically-induced colitis by real-time PCR, Western blotting, and confocal microscopy. Cytokine production was evaluated in lamina propria mononuclear cells (LPMCs) of IBD patients and mucosal tissues of colitic mice treated with BRD4 inhibitors. Finally, we evaluated the effect of JQ1, an inhibitor of the BRD4 signaling pathway, on the course of murine colitis. BRD4 RNA and protein expression was up-regulated in the inflamed mucosa of patients with UC and patients with CD as compared to the uninvolved areas of the same patients and CTRs, and in the inflamed colon of colitic mice. Knockdown of BRD4 with a specific antisense oligonucleotide in IBD LPMCs led to reduced expression of TNF-α, IL-6, IFN-γ, and IL-17A. Administration of JQ1 to colitic mice inhibited the inflammatory cytokine response and attenuated the ongoing colitis. This is the first study showing the up-regulation of BRD4 in IBD and suggesting the role of such a protein in the positive control of the inflammatory cytokine response in the gut.

BET Inhibitor JQ1 Selectively Reduce Tregs by Upregulating STAT3 and Suppressing PD-1 Expression in Patients with Multiple Myeloma.

Multiple myeloma (MM) stands as a prevalent hematological malignancy, primarily incurable, originating from plasma cell clones. MM's progression encompasses genetic abnormalities and disruptions in the bone marrow microenvironment, leading to tumor proliferation, immune dysfunction, and compromised treatment outcomes. Emerging evidence highlights the critical role of regulatory T cells (Tregs) in MM progression, suggesting that targeting Tregs could enhance immune functionality and treatment efficacy. In this study, a notable increase in Treg proportions within MM patients' bone marrow (BM) compared to healthy individuals is observed. Additionally, it is found that the bromodomain and extraterminal domain (BET) inhibitor JQ1 selectively diminishes Treg percentages in MM patients' BM and reduces TGF-β1-induced Tregs. This reduction occurs via inhibiting cell viability and promoting apoptosis. RNA sequencing further indicates that JQ1's inhibitory impact on Tregs likely involves upregulating STAT3 and suppressing PD-1 expression. Collectively, these findings suggest JQ1's potential to modulate Tregs, bolstering the immune response in MM and introducing a promising avenue for MM immunotherapy.

BET Bromodomain Inhibition Potentiates Ocular Melanoma Therapy by Inducing Cell Cycle Arrest.

Ocular melanoma is a common primary malignant ocular tumor in adults with limited effective treatments. Epigenetic regulation plays an important role in tumor development. The switching/sucrose nonfermentation (SWI/SNF) chromatin remodeling complex and bromodomain and extraterminal domain family proteins are epigenetic regulators involved in several cancers. We aimed to screen a candidate small molecule inhibitor targeting these regulators and investigate its effect and mechanism in ocular melanoma. We observed phenotypes caused by knockdown of the corresponding gene and synergistic effects with BRD inhibitor treatment and SWI/SNF complex knockdown. The effect of JQ-1 on ocular melanoma cell cycle and apoptosis was analyzed with flow cytometry. Via RNA sequencing, we also explored the mechanism of BRD4. The best tumor inhibitory effect was observed for the BRD4 inhibitor (JQ-1), although there were no statistically obvious changes in the shBRD4 and shBRD9 groups. Interestingly, the inhibitory effect of JQ-1 was decrease in the shBRD4 group. JQ-1 inhibits the growth of melanoma in various cell lines and in tumor-bearing mice. We found 17 of these 28 common differentially expressed genes were downregulated after MEL270 and MEL290 cells treated with JQ-1. Four of these 17 genes, TP53I11, SH2D5, SEMA5A, and MDGA1, were positively correlated with BRD4. In TCGA database, low expression of TP53I11, SH2D5, SEMA5A, and MDGA1 improved the overall survival rate of patients. Furthermore, the disease-free survival rate was increased in the groups with low expression of TP53I11, SH2D5, and SEMA5A. JQ-1 may act downstream of BRD4 and suppress ocular melanoma growth by inducing G1 cell cycle arrest.

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

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. 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, β-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. In vitro, D-gal treatment induced noticeable aging-related changes such as inducing cell apoptosis and cell cycle arrest, increasing β-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 β-galactosidase (SA-β-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. 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.

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.

Structure- and Property-Based Optimization of Efficient Pan-Bromodomain and Extra Terminal Inhibitors to Identify Oral and Intravenous Candidate I-BET787.

The bromodomain and extra terminal (BET) family of bromodomain-containing proteins are important epigenetic regulators that elicit their effect through binding histone tail N-acetyl lysine (KAc) post-translational modifications. Recognition of such markers has been implicated in a range of oncology and immune diseases and, as such, small-molecule inhibition of the BET family bromodomain-KAc protein-protein interaction has received significant interest as a therapeutic strategy, with several potential medicines under clinical evaluation. This work describes the structure- and property-based optimization of a ligand and lipophilic efficient pan-BET bromodomain inhibitor series to deliver candidate I-BET787 (70) that demonstrates efficacy in a mouse model of inflammation and suitable properties for both oral and intravenous (IV) administration. This focused two-phase explore-exploit medicinal chemistry effort delivered the candidate molecule in 3 months with less than 100 final compounds synthesized.

Utilizing epigenetic regulators to improve HSC-based lentiviral gene therapy

AbstractThe curative benefits of autologous and allogeneic transplantation of hematopoietic stem cells (HSCs) have been proven in various diseases. However, the low number of true HSCs that can be collected from patients and the subsequent in vitro maintenance and expansion of true HSCs for genetic correction remains challenging. Addressing this issue, we here focused on optimizing culture conditions to improve ex vivo expansion of true HSCs for gene therapy purposes. In particular, we explored the use of epigenetic regulators to enhance the effectiveness of HSC-based lentiviral (LV) gene therapy. The histone deacetylase inhibitor quisinostat and bromodomain inhibitor CPI203 each promoted ex vivo expansion of functional HSCs, as validated by xenotransplantation assays and single-cell RNA sequencing analysis. We confirmed the stealth effect of LV transduction on the loss of HSC numbers in commonly used culture protocols, whereas the addition of quisinostat or CPI203 improved the expansion of HSCs in transduction protocols. Notably, we demonstrated that the addition of quisinostat improved the LV transduction efficiency of HSCs and early progenitors. Our suggested culture conditions highlight the potential therapeutic effects of epigenetic regulators in HSC biology and their clinical applications to advance HSC-based gene correction.

BET-directed PROTACs in triple negative breast cancer cell lines MDA-MB-231 and MDA-MB-436

PurposeThis study aims to find whether the proliferation and migration of triple negative breast cancer (TNBC) cell lines can be reduced by treatment with bromodomain and extra-terminal domain (BET) inhibitor JQ1 and BET protein targeting chimeras (PROTACs) ARV-771 and MZ1.MethodsCytotoxicity tests, scratch migration assays and western blot proteome profiler arrays for protein expression of cancer-related proteins were used to evaluate the impact of a BET-inhibitor and two BET-directed PROTACs on cell viability, migration and on protein expression.ResultsJQ1 and the PROTACs MZ1 and ARV-771 significantly inhibited the growth and migration of the KRAS G13D-mutated MDA-MB-231 cells. In this cell line, the PROTACs suppressed the residual expression of ERBB2/HER2, 3 and 4 that are essential for the proliferation of breast cancer cells and this cell line proved sensitive to HER2 inhibitors. In contrast, the effects of the PROTACs on the protein expression of MDA-MB-436 cells mostly affected cytokines and their cognate receptors.ConclusionThe degradation of BET-protein by PROTACs demonstrated significant anti-proliferative effects. The KRAS-mutated MDA-MB-231 cells belong to the low-HER2 expressing tumors that have a poorer prognosis compared to HER2-null patients. Since first oral PROTACs against tumor hormone receptors are in clinical trials, this mode of tumor therapy is expected to become an important therapeutic strategy in the future treatment of TNBC.

SYT4 binds to SNAP25 to facilitate exosomal secretion and prostate cancer enzalutamide resistance.

Prostate carcinoma represents a predominant malignancy affecting the male population, with androgen deprivation therapy (ADT) serving as a critical therapeutic modality for advanced disease states, but it often leads to the development of resistance. Enzalutamide (Enz), a second-generation antiandrogen drug, initially offers substantial therapeutic benefit, but its efficacy wanes as drug resistance ensues. In this study, we found that synaptotagmin 4 (SYT4) is an upregulated gene in enzalutamide-resistant (EnzR) cell lines. The downregulation of SYT4, in combination with enzalutamide therapy, substantially enhances the antiproliferative effect on resistant prostate cancer cells beyond the capacity of enzalutamide monotherapy. SYT4 promotes vesicle efflux by binding to the synaptosome-associated protein 25 (SNAP25), thereby contributing to cell resistance against enzalutamide. The elevated expression of SYT4 is mediated by bromodomain-containing protein 4 (BRD4), and BRD4 inhibition effectively suppressed the expression of SYT4. Treatment with a therapeutic dose of enzalutamide combined with ASO-1, an antisense oligonucleotide drug targeting SYT4, shows promising results in reversing the resistance of prostate cancer to enzalutamide.