Bromodomain Inhibitors Research Articles

Identification of novel transcription factors regulated by H3K27 acetylation in myogenic differentiation of porcine skeletal muscle satellite cells.

H3K27 acetylation (H3K27ac) is crucial in muscle development as it regulates gene expression. Dysregulation of H3K27ac level has been linked to muscle-related diseases such as Duchenne muscular dystrophy, yet the mechanisms through which H3K27ac influences myogenic differentiation are not fully understood. Here, we utilized the SGC-CBP30 drug, a CBP/p300 bromodomain inhibitor, to reduce H3K27ac level and investigated its effect on myogenic differentiation of porcine skeletal muscle satellite cells. The results demonstrated an increased H3K27ac level during normal muscle satellite cell differentiation. We found that the addition of SGC-CBP30 resulted in a reduced level of H3K27ac based on ATAC-seq and CUT&Tag data. Our analysis revealed that a cluster characterized by reduced levels of H3K27ac and increased levels of H3K27me3 was enriched with motifs corresponding to Bach2, MafK, and Fosl2 transcription factors. Furthermore, knockdown of Bach2, MafK, and Fosl2 produced a similar suppression effect on myogenic differentiation. Taken together, our study contributes to a better understanding of how H3K27ac influences myogenic differentiation.

Lipid metabolism-related gene signature predicts prognosis and unveils novel anti-tumor drugs in specific type of diffuse large B cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma which possess highly aggressive and heterogeneous. Despite advances in understanding heterogeneity and development of novel targeted agents, the prognosis of DLBCL patients remains unsatisfied. Lipids are crucial components of biological membranes and signal transduction while accumulating evidence has supported the vital roles of abnormal lipid metabolism in tumorigenesis. Furthermore, some related pathways could serve as prognostic biomarkers and potential therapeutic targets. However, the clinical significance of abnormal lipid metabolism reprogramming in DLBCL has not been investigated. In the current study, we developed a prognostic risk model for DLBCL based on the abnormal expressed lipid metabolism genes and moreover based on our risk model we classified patients with DLBCL into novel subtypes and identified potential drugs for DLBCL patients with certain lipid metabolism profiles. We utilized univariate Cox regression analysis to identify the prognosis-related lipid metabolism genes, and then performed LASSO Cox regression to identify prognostic related lipid metabolism related genes. Multivariate cox regression was used to establish the prognostic model. Patients were divided in to high and low risk groups based on the median risk score. Immune cell infiltration and GSEA were used to identify the pathways between high and low risk groups. Oncopredict algorithm was utilized to identify potential drug for high-risk patients. In vitro cell apoptosis and viability analysis were employed to verify the specific tumor inhibition effects of AZD5153. Nineteen survival related lipid metabolism genes TMEM176B, LAYN, RAB6B, MMP9, ATAD3B, SLC2A11, CD3E, SLIT2, SLC2A13, SLC43A3, CD6, SIRPG, NEK6, LCP2, CTTN, CXCL2, SNX22, BCL6 and FABP4 were identified and subjected to build the prognostic model which was further verified in four external microarray cohorts and one RNA seq cohorts. Tumor immune microenvironment analysis and GSEA results showed that the activation of MYC targets genes rather than immunosuppression contribute to the poor survival outcome of patients in the high-risk group. AZD5153, a novel bivalent BET bromodomain inhibitor which could inhibit the transcription of MYC and E2F exhibited specific antitumor function for cells with high-risk score. Our results provide the first lipid metabolism-based gene signature for predicting the survival of patients with DLBCL. Furthermore, by determining novel subtypes with our lipid metabolism prognostic model we illustrated that drugs that compromising MYC target genes rather than immune checkpoint inhibitors may be beneficial to DLBCL patients with certain lipid metabolism profiles.

Enhanced cellular death in liver and breast cancer cells by dual BET/BRPF1 inhibitors.

The acetylpyrrole scaffold is an acetylated lysine mimic that has been previously explored to develop bromodomain inhibitors. When tested on the hepatoma cell line Huh7 and the breast cancer cell line MDA-MB-231, a few compounds in our acetylpyrrole-thiazole library induced peculiar morphological changes, progressively causing cell death at increasing concentrations. Their evaluation on a panel of human bromodomains revealed concurrent inhibition of BRPF1 and BET bromodomains. To dissect the observed cellular effects, the acetylpyrrole derivatives were compared to JQ1 and GSK6853, chemical probes for the bromodomains of BET and BRPF1, respectively. The appearance of neurite-like extrusions, accompanied by βIII-tubulin overexpression, is caused by BET inhibition, with limited effect on cellular viability. Conversely, interference with BRPF1 induces cellular death but not phenotypic alterations. Combined treatment with JQ1 and GSK6853 showed additivity in reducing cellular viability, comparably to the acetylpyrrole-thiazole-based BET/BRPF1 inhibitors. In addition, we determined the crystallographic structures of the BRD4 and BRPF1 bromodomains in complex with the acetylpyrrole-thiazole compounds. The binding modes in the two bromodomains show similar interactions for the acetylpyrrole and different orientations of the moiety that point to the rim of the acetyl-lysine pocket.

Targeting BRD4-HK2 reverses the meta-inflammatory shift in perivascular adipose tissue and rescues cardiometabolic vascular dysfunction

Abstract Background/Introduction Reducing vascular inflammation is crucial to halt and reverse cardiometabolic damage. BRD4, an epigenetic pan-inflammatory activator whose inhibition has shown promising results in cancer, may play an important role in this context. However, its role in cardiometabolic disease remains unclear. Purpose To investigate BRD4-related transcriptional programmes and therapeutic modulation in translational models of cardiometabolic disease. Methods We dissected small arteries (100-300 μM) from visceral fat biopsies in healthy volunteers (n=16) and patients with obesity and hypertension (n=16), a highly prevalent cardiometabolic phenotype. We assessed the ex vivo effects of BRD4 inhibition on vascular function by pressure myography in the presence or absence of perivascular adipose tissue (PVAT), at baseline and after incubation with the BRD4 inhibitor RVX-208 or with anti-inflammatory/anti-metabolic drugs. We used a cardiometabolic mouse model (high-fat diet+L-NAME supplementation) that mirrored our human phenotype and orally administered RVX-208 (150 mg/kg) or vehicle for 5 days (n=6 per group) to confirm the in vivo effect of chronic BRD4 inhibition. Finally, we investigated the molecular pathways involved in the perivascular cross-talk in an in vitro model of human adipocytes (hADSCs) and endothelial cells (HAECs) using gene overexpression/silencing strategies. We assessed ROS and nitric oxide levels (confocal microscopy), protein and gene expressions (Western blot; qPCR), transcriptional (custom qPCR array; chromatin immunoprecipitation (ChIP)) and metabolic changes (metabolomics, lipidomics, mitochondrial swelling). Results Vascular inflammation, remodeling and function were altered in cardiometabolic patients/mice. RVX-208 attenuated ex vivo vascular dysfunction to a greater extent than anti-IL-1β, anti-IL-6 receptor and anti-TNF-α. In vessels with intact PVAT we observed a stronger effect, due to the restoration of healthy PVAT phenotype (Figure 1). In PVAT, Hexokinase-2 (Hk2) - a glycolytic enzyme implicated in mitochondrial dysfunction and inflammation - was the top downregulated gene by RVX-208 treatment; ChIP confirmed increased binding of BRD4 to the Hk2 promoter. In line, metabolomics and lipidomics assays revealed a PVAT glycolytic shift with increased fatty acid storage in disease states, which was reversed by BRD4 inhibition. The in vitro study showed that: i) healthy adipocytes overexpressing HK2 shift to an inflammatory phenotype; ii) their secretome induces an inflammatory phenotype in HAECs; iii) ex vivo PVAT-selective inhibition of HK2 ameliorates vascular dysfunction (Figure 2). Conclusions We identified BRD4-HK2 interaction at the PVAT level as a novel mediator of cardiometabolic damage. Its targeting rescues vascular dysfunction by reversing the PVAT meta-inflammatory shift. Epigenetic modulators of meta-inflammation may represent a promising strategy in patients with obesity and hypertension.

Effects of BET Inhibition on lung micro-environmental changes in obesity-related pulmonary Arterial Hypertension (PAH)

Abstract Background Obesity is emerging as a pivotal risk factor for pulmonary arterial hypertension (PAH) but the underlying molecular mechanisms are poorly explored. The lung microenvironment, namely the cell-cell communication among fibroblasts, immune and endothelial cells, may significantly impact on vascular structure and function thus promoting microvascular disease in this setting. Obesity-induced chromatin modifications lead to maladaptive transcriptional programs altering cell function in different organs. Apabetalone (APA), a selective inhibitor of bromodomain and extra-terminal containing protein family (BET) proteins, prevents bromodomain-containing protein 4 (BRD4) interactions with chromatin thus modulating gene expression. Purpose To determine whether a new epi-drug, namely APA, affects the lung microenvironment in obesity-related PAH. Methods Mice were fed a normal diet (ND, control) or high-fat-diet (HFD) and L-NAME for 15 weeks to induce obesity-related PAH (obPAH). Echocardiography was performed. Pathways regulating obPAH were identified by single-cell nuclei RNA sequencing in lung specimens. Primary endothelial cells (ECs) and Fibroblasts (mFs) were freshly isolated from obPAH lungs and treated with APA for 48 hours. Cells and lung samples were used for molecular biology and histology. Cellular migration was investigated by in vitro gap closure. Results Obese mice displayed PAH as shown by increased pulmonary artery (PA) pressure, PA resistance, right ventricular wall thickness and reduced PA acceleration time. Of note, chronic APA treatment prevented PAH-related features in obese mice. To determine the cell types affected by APA, we performed scRNAseq. We found that fibroblast and endothelial cells had the highest enrichment of genes whose expression significantly correlated with echo-determined PAH features. ECs and mFs from obese mice displayed an altered phenotype whereas treatment with APA rescued obesity-driven ECs and mFs dysfunction. In obPAH ECs, we found increased expression of NF-kB p65-related inflammatory genes (IL-1β, IL-6, TNF-alpha) as well as enhanced p53 signaling. Moreover, obPAH-ECs expressed high levels of hypoxia-inducible factor (HIF1-α) and NADPH-oxidase-NOX4 with subsequent increase in ROS generation. Furthermore, APA treatment protected against cellular inflammation, senescence and oxidative stress. To evaluate the effect of APA and the paracrine response of fibroblasts, we performed a scratch assay on control mFs exposed to TGF-β or conditioned media collected from obPAH-ECs with and without APA. In both experimental settings, APA affected gap closure, suggesting that the treatment influences fibroblast migration through both a direct effect and paracrine mechanisms. Conclusions APA is able to reset the lung micro-envirnment in obesity thus reducing inflammation and senescence. BET inhibitors could represent potential therapeutic approaches in this setting.

The BET inhibitor Apabetalone protects against heart failure with preserved ejection fraction by suppressing myocardial inflammation

Abstract Background Posttranslational histone modifications, play a major role in cardiac hypertrophy and dysfunction. Apabetalone (APA), a selective inhibitor of bromodomain and extraterminal containing protein family (BET) proteins, prevents bromodomain-containing protein 4 (BRD4) interactions with chromatin thus modulating transcriptional programs in different organs. Purpose We sought to investigate whether APA could be beneficial in cardiometabolic heart failure with preserved ejection fraction (cHFpEF). Methods Mice were subjected to high fat diet feeding and L-NAME treatment for 15 weeks to induce cHFpEF. Histology, mouse echocardiography (Vevo3100) and Treadmill exhaustion test were performed. Unbiased gene expression profiling by PCR array and proteomics analysis (Olink) was employed in left ventricular (LV) myocardial specimens from HFpEF mice and control animals. Cultured cardiomyocytes (CMs) treated with palmitic acid (PA) were used as an in vitro model of metabolic stress. cHFpEF mice were chronically treated with the BET inhibitor APA (150mg/kg/day) or vehicle (DMSO). In order to translate our findings to the human setting, passive stiffness of skinned CMs collected from cHFpEF patients was assessed before and after APA treatment. Results HFpEF mice displayed LV hypertrophy, diastolic dysfunction, myocardial fibrosis, lung congestion and impaired exercise tolerance as compared to controls. Interestingly, diastolic dysfunction - assessed by E/A ratio and isovolumic relaxation time (IVRT) - and lung congestion were significantly reduced in HFpEF mice treated with APA, while exercise tolerance was improved. Transcriptomic analysis in PA-treated CMs and LV mouse specimens from cHFpEF mice showed a profound deregulation of genes controlling inflammation, namely IL-6, TNF-alpha, and IL-1beta. ChIP assays showed BRD4 occupancy on the promoter of several inflammatory genes, including IL6. Treatment with APA suppressed most of inflammatory genes, with a pronounced effect on IL6 expression. Moreover, APA reduced circulating levels of several inflammatory chemokines. The beneficial effects of APA were explained by modulation of IL-6/CaMKII/STAT3 pathway both in cHFpEF hearts and PA-treated CMs. In skinned CMs from cHFpEF patients, both APA and IL6 blockade were able to attenuate passive stiffness. Conclusions Our findings set the stage for preclinical studies and exploratory clinical trials testing APA in patients with cHFpEF.

Functional inversion of circadian regulator REV-ERBα leads to tumorigenic gene reprogramming

Profound functional switch of key regulatory factors may play a major role in homeostasis and disease. Dysregulation of circadian rhythm (CR) is strongly implicated in cancer with mechanisms poorly understood. We report here that the function of REV-ERBα, a major CR regulator of the orphan nuclear receptor subfamily, is dramatically altered in tumors in both its genome binding and functional mode. Loss of CR is linked to a functional inversion of REV-ERBα from a repressor in control of CR and metabolic gene programs in normal tissues to a strong activator in different cancers. Through changing its association from NCoR/HDAC3 corepressor complex to BRD4/p300 coactivators, REV-ERBα directly activates thousands of genes including tumorigenic programs such as MAPK and PI3K-Akt signaling. Functioning as a master transcriptional activator, REV-ERBα partners with pioneer factor FOXA1 and directly stimulates a large number of signaling genes, including multiple growth factors, receptor tyrosine kinases, RASs, AKTs, and MAPKs. Moreover, elevated REV-ERBα reprograms FOXA1 to bind new targets through a BRD4-mediated increase in local chromatin accessibility. Pharmacological targeting with SR8278 diminishes the function of both REV-ERBα and FOXA1 and synergizes with BRD4 inhibitor in effective suppression of tumorigenic programs and tumor growth. Thus, our study revealed a functional inversion by a CR regulator in driving gene reprogramming as an unexpected paradigm of tumorigenesis mechanism and demonstrated a high effectiveness of therapeutic targeting such switch.

Triple Combination of Entinostat, a Bromodomain Inhibitor, and Cisplatin Is a Promising Treatment Option for Bladder Cancer.

Cisplatin is part of the first-line treatment of advanced urothelial carcinoma. Cisplatin resistance is a major problem but may be overcome by combination treatments such as targeting epigenetic aberrances. Here, we investigated the effect of the class I HDACi entinostat and bromodomain inhibitors (BETis) on the potency of cisplatin in two pairs of sensitive and cisplatin-resistant bladder cancer cell lines. Cisplatin-resistant J82cisR and T24 LTT were 3.8- and 24-fold more resistant to cisplatin compared to the native cell lines J82 and T24. In addition, a hybrid compound (compound 20) comprising structural features of an HDACi and a BETi was investigated. We found complete (J82cisR) or partial (T24 LTT) reversal of chemoresistance upon combination of entinostat, JQ1, and cisplatin. The same was found for the BETis JQ35 and OTX015, both in clinical trials, and for compound 20. The combinations were highly synergistic (Chou Talalay analysis) and increased caspase-mediated apoptosis accompanied by enhanced expression of p21, Bim, and FOXO1. Notably, the combinations were at least 4-fold less toxic in non-cancer cell lines HBLAK and HEK293. The triple combination of entinostat, a BETi, and cisplatin is highly synergistic, reverses cisplatin resistance, and may thus serve as a novel therapeutic approach for bladder cancer.

Discovery of CZL-046 with an (S)-3-Fluoropyrrolidin-2-one Scaffold as a p300 Bromodomain Inhibitor for the Treatment of Multiple Myeloma.

E1A binding protein (p300) is a promising therapeutic target for the treatment of cancer. Herein, we report the discovery of a series of novel inhibitors with an (S)-3-fluoropyrrolidin-2-one scaffold targeting p300 bromodomain. The best compound 29 (CZL-046) shows potent inhibitory activity of p300 bromodomain (IC50 = 3.3 nM) and antiproliferative activity in the multiple myeloma (MM) cell line (OPM-2 IC50 = 51.5 nM). 29 suppressed the mRNA levels of c-Myc and IRF4 and downregulated the expression of c-Myc and H3K27Ac. Compared to the lead compound 5, 29 exhibits significantly improved in vitro and in vivo metabolic properties. Oral administration of 29 with 30 mg/kg achieved a TGI value of 44% in the OPM-2 xenograft model, accompanied by good tolerability. The cocrystal structure of CREB binding protein bromodomain with 29 provides an insight into the precise binding mode. The results demonstrate that 29 is a promising p300 bromodomain inhibitor for the treatment of MM.

Bromodomain containing 4 inhibition combats gastric precancerous lesions via modulating macrophage polarization

ObjectiveGastric precancerous lesions (GPL), characterized by intestinal metaplasia and dysplasia, marks a pivotal juncture in the transformation from gastritis to gastric cancer. Research on GPL could offer fresh perspectives on preventing cancer occurrence. MethodsThis study employed 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) to establish GPL rat models and knocked BRD4 down in vivo to assess its impact on the lesions and macrophage morphology. Following that, the impacts of BRD4 knockdown on the malignant phenotypes of human gastric epithelial GES-1 cells were determined. Moreover, conditioned medium from macrophage was gathered and used for GES-1 cell culture. The involvement of macrophage polarization in the BRD4 regulatory mechanism in GES-1 cells was assessed. ResultsThis study elucidated that MNNG induced an increase level of BRD4 in the rat models. BRD4 knockdown reduced lesions based on pathological sections and immunohistochemistry to detect proliferative antigens. Western blotting and immunofluorescence showed that BRD4 knockdown suppressed epithelial-mesenchymal transition and macrophage M2 polarization. In in vitro experiments, BRD4 knockdown inhibited the malignant phenotype of GES-1 cells and the differentiation of THP-1 cells into M2 macrophages, respectively. The conditioned medium from M2 macrophages with BRD4 knockdown was co-incubated with GES-1 cells, which attenuated the malignant phenotypes compared with the medium from M2 macrophages. ConclusionThrough in vivo and in vitro experiments, BRD4 upregulation was found to already occur during GPL, affecting macrophage polarization and epithelial cell cancerization. This finding provides an experimental basis for strategies targeting BRD4 inhibition at this critical stage.

Enhancing therapeutic efficacy in homologous recombination-proficient pancreatic cancer via the combination of PARP1-PROTAC and a BRD4 inhibitor

Currently, poly (ADP-ribose) polymerase inhibitors (PARPi) have been approved by the U.S. Food and Drug Administration for BRCA-mutated pancreatic cancer therapy. However, limited indications hinder their further application. Bromodomain-containing protein 4 (BRD4) repression can block the homologous recombination (HR) repair pathway and has the potential to enhance the response to PARPi in HR-proficient pancreatic cancer therapy. In addition, proteolysis targeting chimeras (PROTACs) can hijack E3 ligase within the cell to ubiquitinate degradation-targeted proteins effectively and quickly, thus enhancing the therapeutic effect on tumors. In the present study, the LB23 compound (with the ability to induce PARP1 degradation) was employed in combination with the BRD4 inhibitor, JQ1, and their synergistic effect in HR-proficient pancreatic cancer was confirmed via various methods. Moreover, compared with the JQ1 and PARPi olaparib combination, it was revealed that PARP1-PROTAC and JQ1 had more notable synergistic effects. Further research into the synergistic mechanism demonstrated that combination therapy enhanced DNA damage and suppressed DNA repair by inducing cell cycle arrest and cell apoptosis. The present study therefore provides the experimental data for this type of combination therapy, which is expected to be an innovative approach for the treatment of HR-proficient pancreatic cancer.

Chidamide Combined with (+) -JQ-1 to Kill MLL-Rearrangement Acute Myeloid Leukemia Cells by Disrupting the DNA Damage Response Pathway

To investigate the mechanism of DNA damage and repair in MLL -rearranged acute myeloid leukemia（ MLL-r AML）cells by the combination of Chidamide and the BRD4 inhibitor(+)-JQ-1. MLL-r AML cell lines Molm-13, MV4-11 and non- MLL-r AML cell line Kasumi were divided into control group（contr）, Chidamide group（chida）, (+)-JQ-1 group and Combination group（combi）, respectively. Cell viability of Molm-13 was measured by CCK-8 to determine optimal the concentrations of Chidamide and(+)-JQ-1. The cell cycle was detected by flow cytometry, and apoptosis-related factors Bcl-2, Bax and caspase-3 were detected by Western blot. DNA damage marker γH2AX was detected by immunofluorescence. The protein expressions of DNA damage factor γH2AX, DNA damage checkpoint kinases p-ATR, p-CHK1, p-ATM, p-CHK2 and DNA damage repair factors Rad51 and 53BP1 were detected by Western blot. The expression of DNA damage repair factors Rad51 and 53BP1 mRNA was detected by qRT-PCR. Under the treatment of Chidamide (300 nmol/L) and (+)-JQ-1 (400 nmol/L), the proportion of G1 phase cells in MLL-r AML cell lines Molm-13 and MV4-11 was increased in combination group compared with control group. In non- MLL-r AML cell line Kasumi, compared with control group, the proportion of G1 phase cells in combination group was increased (P < 0.05). In Molm-13 and MV4-11 cell lines, compared with control group, the expression level of DNA damage marker γH2AX in combination group was increased (P < 0.05). The expression levels of DNA damage checkpoint and damage repair factors p-ATR, p-CHK1, p-ATM, p-CHK2, Rad51, 53BP1 were decreased (P < 0.05). In Kasumi cell line, compared with control group, there was no significant change in the expression of some of the above factors in combination group (P >0.05), but the expression trend of some factors was opposite. In MLL-r AML cell lines Molm-13 and MV4-11, compared with control group, the expression levels of Bax and caspase-3 protein were increased in combination group, while the expression levels of Bcl-2 protein were decreased (P < 0.05). In non- MLL-r AML cell line Kasumi, there was no significant change in apoptotic factor protein expression in combination group compared with control group (P >0.05). Chidamide combined with (+)-JQ-1 can inhibit the proliferation of MLL-r AML cells, inhibit the initiation of protective self-repair of these leukemia cells by inhibiting the DNA damage response pathway, and ultimately increase the apoptosis of these cells, but non- MLL-r AML cells have no similar results.

Rational Design of HER2-Targeted Combination Therapies to Reverse Drug Resistance in Fibroblast-Protected HER2+ Breast Cancer Cells.

Fibroblasts, an abundant cell type in the breast tumor microenvironment, interact with cancer cells and orchestrate tumor progression and drug resistance. However, the mechanisms by which fibroblast-derived factors impact drug sensitivity remain poorly understood. Here, we develop rational combination therapies that are informed by proteomic profiling to overcome fibroblast-mediated therapeutic resistance in HER2+ breast cancer cells. Drug sensitivity to the HER2 kinase inhibitor lapatinib was characterized under conditions of monoculture and exposure to breast fibroblast-conditioned medium. Protein expression was measured using reverse phase protein arrays. Candidate targets for combination therapy were identified using differential expression and multivariate regression modeling. Follow-up experiments were performed to evaluate the effects of HER2 kinase combination therapies in fibroblast-protected cancer cell lines and fibroblasts. Compared to monoculture, fibroblast-conditioned medium increased the expression of plasminogen activator inhibitor-1 (PAI1) and cell cycle regulator polo like kinase 1 (PLK1) in lapatinib-treated breast cancer cells. Combination therapy of lapatinib with inhibitors targeting either PAI1 or PLK1, eliminated fibroblast-protected cancer cells, under both conditions of direct coculture with fibroblasts and protection by fibroblast-conditioned medium. Analysis of publicly available, clinical transcriptomic datasets revealed that HER2-targeted therapy fails to suppress PLK1 expression in stroma-rich HER2+ breast tumors and that high PAI1 gene expression associates with high stroma density. Furthermore, we showed that an epigenetics-directed approach using a bromodomain and extraterminal inhibitor to globally target fibroblast-induced proteomic adaptions in cancer cells, also restored lapatinib sensitivity. Our data-driven framework of proteomic profiling in breast cancer cells identified the proteolytic degradation regulator PAI1 and the cell cycle regulator PLK1 as predictors of fibroblast-mediated treatment resistance. Combination therapies targeting HER2 kinase and these fibroblast-induced signaling adaptations eliminates fibroblast-protected HER2+ breast cancer cells. The online version contains supplementary material available at 10.1007/s12195-024-00823-0.

121 (PB109): TT125-802, a highly selective bromodomain inhibitor of CBP/p300, in patients with advanced solid tumors: an update on the phase I study

121 (PB109): TT125-802, a highly selective bromodomain inhibitor of CBP/p300, in patients with advanced solid tumors: an update on the phase I study

Platelet and Erythrocyte Membranes Coassembled Biomimetic Nanoparticles for Heart Failure Treatment.

Cardiac fibrosis is a prevalent pathological process observed in the progression of numerous cardiovascular diseases and is associated with an increased risk of sudden cardiac death. Although the BRD4 inhibitor JQ1 has powerful antifibrosis properties, its clinical application is extremely limited due to its side effects. There remains an unmet need for effective, safe, and low-cost treatments. Here, we present a multifunctional biomimetic nanoparticle drug delivery system (PM&EM nanoparticles) assembled by platelet membranes and erythrocyte membranes for targeted JQ1 delivery in treating cardiac fibrosis. The platelet membrane endows PM&EM nanoparticles with the ability to target cardiac myofibroblasts and collagen, while the participation of the erythrocyte membrane enhances the long-term circulation ability of the formulated nanoparticles. In addition, PM&EM nanoparticles can deliver sufficient JQ1 with controllable release, achieving excellent antifibrosis effects. Based on these advantages, it is demonstrated in both pressures overloaded induced mouse cardiac fibrosis model and MI-induced mouse cardiac fibrosis that injection of the fusion membrane biomimetic nanodrug carrier system effectively reduced fibroblast activation, collagen secretion, and improved cardiac fibrosis. Moreover, it significantly mitigated the toxic and side effects of long-term JQ1 treatment on the liver, kidney, and intestinal tract. Mechanically, bioinformatics prediction and experimental validation revealed that PM&EM/JQ1 NPs reduced liver and kidney damage via alleviated oxidative stress and mitigated cardiac fibrosis via the activation of oxidative phosphorylation activation. These results highlight the potential value of integrating native platelet and erythrocyte membranes as a multifunctional biomimetic drug delivery system for treating cardiac fibrosis and preventing drug side effects.

Integrator complex subunit 12 knockout overcomes a transcriptional block to HIV latency reversal.

The latent HIV reservoir is a major barrier to HIV cure. Combining latency reversal agents (LRAs) with differing mechanisms of action such as AZD5582, a non-canonical NF-kB activator, and I-BET151, a bromodomain inhibitor is appealing towards inducing HIV-1 reactivation. However, even this LRA combination needs improvement as it is inefficient at activating proviruses in cells from people living with HIV (PLWH). We performed a CRISPR screen in conjunction with AZD5582 & I-BET151 and identified a member of the Integrator complex as a target to improve this LRA combination, specifically Integrator complex subunit 12 (INTS12). Integrator functions as a genome-wide attenuator of transcription that acts on elongation through its RNA cleavage and phosphatase modules. Knockout of INTS12 improved latency reactivation at the transcriptional level and is more specific to the HIV-1 provirus than AZD5582 & I-BET151 treatment alone. We found that INTS12 is present on chromatin at the promoter of HIV and therefore its effect on HIV may be direct. Additionally, we observed more RNAPII in the gene body of HIV only with the combination of INTS12 knockout with AZD5582 & I-BET151, indicating that INTS12 induces a transcriptional elongation block to viral reactivation. Moreover, knockout of INTS12 increased HIV-1 reactivation in CD4 T cells from virally suppressed PLWH ex vivo. We also detected viral RNA in the supernatant from CD4 T cells of all three virally suppressed PLWH tested upon INTS12 knockout suggesting that INTS12 prevents full-length HIV RNA production in primary T cells.

Discovery of electrophilic degraders that exploit SNAr chemistry.

Targeted covalent inhibition (TCI) and targeted protein degradation (TPD) have proven effective in pharmacologically addressing formerly 'undruggable' targets. Integration of both methodologies has resulted in the development of electrophilic degraders where recruitment of a suitable E3 ubiquitin ligase is achieved through formation of a covalent bond with a cysteine nucleophile. Expanding the scope of electrophilic degraders requires the development of electrophiles with tempered reactivity that enable selective ligase recruitment and reduce cross-reactivity with other cellular nucleophiles. In this study, we report the use of chemical moieties that enable nucleophilic aromatic substitution (SNAr) reactions in the rational design of electrophilic protein degraders. Appending an SNAr covalent warhead to several preexisting small molecule inhibitors transformed them into degraders, obviating the need for a defined E3 ligase recruiter. The SNAr covalent warhead is versatile; it can recruit various E3 ligases, including DDB1 and CUL4 associated factor 11 (DCAF11), DDB1 and CUL4 associated factor 16 (DCAF16), and possibly others. The incorporation of an SNAr covalent warhead into the BRD4 inhibitor led to the discovery of degraders with low picomolar degradation potency. Furthermore, we demonstrate the broad applicability of this approach through rational functional switching from kinase inhibitors into potent degraders.

GDF3 promotes adipose tissue macrophage-mediated inflammation via altered chromatin accessibility during aging.

Age-related susceptibility to sepsis and endotoxemia is poorly defined, although hyperactivation of the immune system and the expansion of the visceral adipose as an immunological reservoir are underlying features. Macrophages from older organisms exhibit substantial changes, including chronic NLRP3 inflammasome activation, genomic remodeling and a dysfunctional, amplified inflammatory response upon new exposure to pathogen. However, the mechanisms by which old macrophages maintain their inflammatory phenotype during endotoxemia remains elusive. We previously identified Gdf3 , a TGFβ superfamily cytokine, as a top-regulated gene by age and the NLRP3 inflammasome in adipose tissue macrophages (ATMs). Here, we demonstrate that endotoxemia increases inflammatory (CD11c + ) ATMs in a Gdf3- dependent manner in old mice. Lifelong systemic or myeloid-specific deletion of Gdf3 leads to reduced endotoxemia- induced inflammation, with decreased CD11c + ATMs and inflammatory cytokines, and protection from hypothermia. Moreover, acute blockade of Gdf3 using JQ1, a BRD4 inhibitor, phenocopies old mice with lifelong Gdf3- deficiency. We show that GDF3 promotes the inflammatory phenotype in ATMs by phosphorylating SMAD2/3. Mechanistically, the differential chromatin landscape of ATMs from old mice with or without myeloid-driven Gdf3 indicates that GDF3- SMAD2/3 signaling axis shifts the chromatin accessibility of ATMs towards an inflammatory state during aging. Furthermore, pharmaceutical inhibition of SMAD3 with a specific inhibitor of SMAD3 (SIS3) mimics Gdf3 deletion. SIS3 reduces endotoxemia-mediated inflammation with fewer CD11c + ATMs and less severe hypothermia in old, but not young mice, as well as reduced mortality. In human adipose tissue, age positively correlates with GDF3 level, while inflammation correlates with pSMAD2/3 level. Overall, these results highlight the importance of GDF3-SMAD2/3 axis in driving inflammation in older organisms and identify this signaling axis as a promising therapeutic target for mitigating endotoxemia-related inflammation in the aged.

Targeting CBP and p300: Emerging Anticancer Agents.

CBP and p300 are versatile transcriptional co-activators that play essential roles in regulating a wide range of signaling pathways, including Wnt/β-catenin, p53, and HIF-1α. These co-activators influence various cellular processes such as proliferation, differentiation, apoptosis, and response to hypoxia, making them pivotal in normal physiology and disease progression. The Wnt/β-catenin signaling pathway, in particular, is crucial for cellular proliferation, differentiation, tissue homeostasis, and embryogenesis. Aberrant activation of this pathway is often associated with several types of cancer, such as colorectal tumor, prostate cancer, pancreatic and hepatocellular carcinomas. In recent years, significant efforts have been directed toward identifying and developing small molecules as novel anticancer agents capable of specifically inhibiting the interaction between β-catenin and the transcriptional co-activators CBP and p300, which are required for Wnt target gene expression and are consequently involved in the regulation of tumor cell proliferation, migration, and invasion. This review summarizes the most significant and original research articles published from 2010 to date, found by means of a PubMed search, highlighting recent advancements in developing both specific and non-specific inhibitors of CBP/β-catenin and p300/β-catenin interactions. For a more comprehensive view, we have also explored the therapeutic potential of CBP/p300 bromodomain and histone acetyltransferase inhibitors in disrupting the transcriptional activation of genes involved in various signaling pathways related to cancer progression. By focusing on these therapeutic strategies, this review aims to offer a detailed overview of recent approaches in cancer treatment that selectively target CBP and p300, with particular emphasis on their roles in Wnt/β-catenin-driven oncogenesis.

Exploration of the tunability of BRD4 degradation by DCAF16 trans-labelling covalent glues

Chemically induced proximity modalities such as targeted protein degradation (TPD) hold promise for expanding the number of proteins that can be manipulated pharmacologically. However, current TPD strategies are often limited to proteins with preexisting ligands. Molecular glues (e.g. glutarimide ligands for CUL4CRBN), offer the potential to target undruggable proteins. Yet, their rational design is largely unattainable due to the unpredictability of the ‘gain-of-function’ nature of the glue interaction upon chemical modification of ligands. We recently reported a covalent trans-labelling glue mechanism which we named ‘Template-assisted covalent modification’, where an electrophile decorated BRD4 inhibitor was effectively delivered to a cysteine residue on DCAF16 due to an electrophile-induced BRD4-DCAF16 interaction. Herein, we report our efforts to evaluate how various electrophilic modifications to the BRD4 binder, JQ1, affect DCAF16 recruitment and subsequent BRD4 degradation efficiency. We discovered a moderate correlation between the electrophile-induced BRD4-DCAF16 ternary complex formation and BRD4 degradation. Moreover, we show that a more solvent-exposed warhead presentation optimally recruits DCAF16 and promotes BRD4 degradation. The diversity of covalent attachments in this class of BRD4 degraders suggests a high tolerance and tunability for the BRD4-DCAF16 interaction. This offers a new avenue for rational glue design by introducing covalent warheads to known binders.