Dual mechanism: Epigenetic inhibitor apabetalone reduces SARS-CoV-2 Delta and Omicron variant spike binding and attenuates SARS-CoV-2 RNA induced inflammation.

The SARS-CoV-2 virus initiates infection via interactions between the viral spike protein and the ACE2 receptors on host cells. Variants of concern have mutations in the spike protein that enhance ACE2 binding affinity, leading to increased virulence and transmission. Viral RNAs released after entry into host cells trigger interferon-I (IFN-I) mediated inflammatory responses for viral clearance and resolution of infection. However, overreactive host IFN-I responses and pro-inflammatory signals drive COVID-19 pathophysiology and disease severity during acute infection. These immune abnormalities also lead to the development of post-COVID syndrome if persistent. Novel therapeutics are urgently required to reduce short- and long-term pathologic consequences associated with SARS-CoV-2 infection. Apabetalone, an inhibitor of epigenetic regulators of the BET protein family, is a candidate for COVID-19 treatment via a dual mechanism of action. In vitro, apabetalone downregulates ACE2 gene expression to limit SARS-CoV-2 entry and propagation. In pre-clinical models and patients treated for cardiovascular disease, apabetalone inhibits expression of inflammatory mediators involved in the pathologic cytokine storm (CS) stimulated by various cytokines. Here we show apabetalone treatment of human lung epithelial cells reduces binding of viral spike protein regardless of mutations found in the highly contagious Delta variant and heavily mutated Omicron. Additionally, we demonstrate that apabetalone counters expression of pro-inflammatory factors with roles in CS and IFN-I signaling in lung cells stimulated with SARS-CoV-2 RNA. Our results support clinical evaluation of apabetalone to treat COVID-19 and post-COVID syndrome regardless of the SARS-CoV-2 variant.

Epigenetic BET reader inhibitor apabetalone (RVX-208) counters proinflammatory aortic gene expression in a diet induced obesity mouse model and in human endothelial cells.

BACKGROUND AND AIMS: Obese patients are at risk for type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). A lipid-rich diet promotes arterial changes by inducing hypertension, oxidative stress, and inflammation. Bromodomain and extraterminal (BET) proteins contribute to endothelial and immune cell activation in vitro and in atherosclerosis mouse models. We aim to determine if BET inhibition can reduce lipid-rich diet-induced vascular inflammation in mice. METHODS: Body weight, serum glucose and lipid levels were measured in mice fed a high-fat diet (HFD) or low-fat diet (LFD) for 6 weeks and at study termination. BET inhibitors apabetalone and JQ1 were co-administered with the HFD for additional 16 weeks. Aortic gene expression was analyzed post necropsy by PCR, Nanostring nCounter® Inflammation Panel and bioinformatics pathway analysis. Transcription changes and BRD4 chromatin occupancy were analyzed in primary human endothelial cells in response to TNFα and apabetalone. RESULTS: HFD induced weight gain, visceral obesity, high fasting blood glucose, glucose intolerance and insulin resistance compared to LFD controls. HFD upregulated the aortic expression of 47 genes involved in inflammation, innate immunity, cytoskeleton and complement pathways. Apabetalone and JQ1 treatment reduced HFD-induced aortic expression of proinflammatory genes. Congruently, bioinformatics predicted enhanced signaling by TNFα in the HFD versus LFD aorta, which was countered by BETi treatment. TNFα-stimulated human endothelial cells had increased expression of HFD-sensitive genes and higher BRD4 chromatin occupancy, which was countered by apabetalone treatment. CONCLUSIONS: HFD induces vascular inflammation in mice through TNFα signaling. Apabetalone treatment reduces this proinflammatory phenotype, providing mechanistic insight into how BET inhibitors may reduce CVD risk in obese patients.

The BET inhibitor apabetalone decreases neuroendothelial proinflammatory activation in vitro and in a mouse model of systemic inflammation.

Brain vascular inflammation is characterized by endothelial activation and immune cell recruitment to the blood vessel wall, potentially causing a breach in the blood - brain barrier, brain parenchyma inflammation, and a decline of cognitive function. The clinical-stage small molecule, apabetalone, reduces circulating vascular endothelial inflammation markers and improves cognitive scores in elderly patients by targeting epigenetic regulators of gene transcription, bromodomain and extraterminal proteins. However, the effect of apabetalone on cytokine-activated brain vascular endothelial cells (BMVECs) is unknown. Here, we show that apabetalone treatment of BMVECs reduces hallmarks of in vitro endothelial activation, including monocyte chemoattractant protein-1 (MCP-1) and RANTES chemokine secretion, cell surface expression of endothelial cell adhesion molecule VCAM-1, as well as endothelial capture of THP-1 monocytes in static and shear stress conditions. Apabetalone pretreatment of THP-1 downregulates cell surface expression of chemokine receptors CCR1, CCR2, and CCR5, and of the VCAM-1 cognate receptor, integrin α4. Consequently, apabetalone reduces THP-1 chemoattraction towards soluble CCR ligands MCP-1 and RANTES, and THP-1 adhesion to activated BMVECs. In a mouse model of brain inflammation, apabetalone counters lipopolysaccharide-induced transcription of endothelial and myeloid cell markers, consistent with decreased neuroendothelial inflammation. In conclusion, apabetalone decreases proinflammatory activation of brain endothelial cells and monocytes in vitro and in the mouse brain during systemic inflammation.

Reduction in the risk of major adverse cardiovascular events with the BET protein inhibitor apabetalone in patients with recent acute coronary syndrome, type 2 diabetes, and moderate to high likelihood of non-alcoholic fatty liver disease.

Background: Nonalcoholic fatty liver disease (NAFLD) is common among patients with type 2 diabetes mellitus (T2DM) and is associated with increased risk for coronary atherosclerosis and acute cardiovascular (CV) events. We employed the validated, non-invasive Angulo NAFLD fibrosis score (FS) in an intervention study in patients with T2DM and recent acute coronary syndrome (ACS) to determine the association of FS with CV risk and treatment response to apabetalone. Apabetalone is a novel selective inhibitor of the second bromodomain of bromodomain and extra-terminal (BET) proteins, epigenetic regulators of gene expression. Methods: The Phase 3 BETonMACE trial compared apabetalone with placebo in 2,425 patients with T2DM and recent ACS. In this post hoc analysis, we evaluated the impact of apabetalone therapy on CV risk, defined as a composite of major adverse cardiovascular events (MACE: CV death, non-fatal myocardial infarction [MI], or stroke) and hospitalization for heart failure (HHF) in two patient categories of FS that reflect the likelihood of underlying NAFLD. Patients were initially classified into three mutually exclusive categories according to a baseline Angulo FS <-1.455 (F0-F2), -1.455 to 0.675 (indeterminant), and >0.675 (F3-F4), where F0 through F4 connote fibrosis severity none, mild, moderate, severe, and cirrhosis, respectively. The composite of ischemic MACE and HHF in the placebo group was higher in indeterminant and F3-F4 categories compared to the F0-F2 category (17.2% vs 15.0% vs 9.7%). Therefore, for the present analysis, the former two categories were combined into an elevated NAFLD CVD risk group (FS+) that was compared with the F0-F2 group (lower NAFLD risk, FS0-2). Results: In 73.7% of patients, FS was elevated and consistent with a moderate-to-high likelihood of advanced liver fibrosis (FS+); 26.3% of patients had a lower FS (FS0-2). In the placebo group, FS+ patients had a higher incidence of ischemic MACE and HHF (15.4%) than FS0-2 patients (9.7%). In FS+ patients, addition of apabetalone to standard of care treatment lowered the rate of ischemic MACE compared with placebo (HR = 0.79; 95% CI 0.60-1.05; p=0.10), HHF (HR = 0.53; 95% CI 0.33-0.86; p=0.01), and the composite of ischemic MACE and HHF (HR = 0.76; 95% CI 0.59-0.98; p=0.03). In contrast, there was no apparent benefit of apabetalone in FS0-2 patients (HR 1.24; 95% CI 0.75-2.07; p=0.40; HR 1.12; 95% CI 0.30-4.14; p=0.87; and HR 1.13; 95% CI 0.69-1.86; p=0.62, respectively). Over a median duration of 26.5 months, FS increased from baseline in both treatment groups, but the increase was smaller in patients assigned to apabetalone than to placebo (p=0.04). Conclusions: Amongst patients with T2DM, recent ACS, and a moderate-to-high likelihood of advanced liver fibrosis, apabetalone was associated with a significantly lower rate of ischemic MACE and HHF and attenuated the increase in hepatic FS over time.

Breaking boundaries: Pan BETi disrupt 3D chromatin structure, BD2-selective BETi are strictly epigenetic transcriptional regulators.

BACKGROUND: Bromodomain and extraterminal proteins (BETs) are more than just epigenetic regulators of transcription. Here we highlight a new role for the BET protein BRD4 in the maintenance of higher order chromatin structure at Topologically Associating Domain Boundaries (TADBs). BD2-selective and pan (non-selective) BET inhibitors (BETi) differentially support chromatin structure, selectively affecting transcription and cell viability. METHODS: Using RNA-seq and BRD4 ChIP-seq, the differential effect of BETi treatment on the transcriptome and BRD4 chromatin occupancy of human aortic endothelial cells from diabetic patients (dHAECs) stimulated with TNFα was evaluated. Chromatin decondensation and DNA fragmentation was assessed by immunofluorescence imaging and quantification. Key dHAEC findings were verified in proliferating monocyte-like THP-1 cells using real time-PCR, BRD4 co-immunoprecipitation studies, western blots, proliferation and apoptosis assays. FINDINGS: We discovered that 1) BRD4 co-localizes with Ying-Yang 1 (YY1) at TADBs, critical chromatin structure complexes proximal to many DNA repair genes. 2) BD2-selective BETi enrich BRD4/YY1 associations, while pan-BETi do not. 3) Failure to support chromatin structures through BRD4/YY1 enrichment inhibits DNA repair gene transcription, which induces DNA damage responses, and causes widespread chromatin decondensation, DNA fragmentation, and apoptosis. 4) BD2-selective BETi maintain high order chromatin structure and cell viability, while reducing deleterious pro-inflammatory transcription. INTERPRETATION: BRD4 plays a previously unrecognized role at TADBs. BETi differentially impact TADB stability. Our results provide translational insight for the development of BETi as therapeutics for a range of diseases including CVD, chronic kidney disease, cancer, and COVID-19.

Inhibition of epigenetic reader proteins by apabetalone counters inflammation in activated innate immune cells from Fabry disease patients receiving enzyme replacement therapy.

Fabry disease (FD) is a rare X-linked disorder of lipid metabolism, characterized by the accumulation of globotriaosylceramide (Gb3) due to defective the lysosomal enzyme, α-galactosidase. Gb3 deposits activate immune-mediated systemic inflammation, ultimately leading to life-threatening consequences in multiple organs such as the heart and kidneys. Enzyme replacement therapy (ERT), the standard of care, is less effective with advanced tissue injury and inflammation in patients with FD. Here, we showed that MCP-1 and TNF-α cytokine levels were almost doubled in plasma from ERT-treated FD patients. Chemokine receptor CCR2 surface expression was increased by twofold on monocytes from patients with low eGFR. We also observed an increase in IL12B transcripts in unstimulated peripheral blood mononuclear cells (PBMCs) over a 2-year period of continuous ERT. Apabetalone is a clinical-stage oral bromodomain and extra terminal protein inhibitor (BETi), which has beneficial effects on cardiovascular and kidney disease related pathways including inflammation. Here, we demonstrate that apabetalone, a BD2-selective BETi, dose dependently reduced the production of MCP-1 and IL-12 in stimulated PBMCs through transcriptional regulation of their encoding genes. Reactive oxygen species production was diminished by up to 80% in stimulated neutrophils following apabetalone treatment, corresponding with inhibition of NOX2 transcription. This study elucidates that inhibition of BET proteins by BD2-selective apabetalone alleviates inflammatory processes and oxidative stress in innate immune cells in general and in FD. These results suggest potential benefit of BD2-selective apabetalone in controlling inflammation and oxidative stress in FD, which will be further investigated in clinical trials.

Insulin mimetic effect of D-allulose on apolipoprotein A-I gene.

Several nutrients modulate the transcriptional activity of the apolipoprotein A-I (apo A-I) gene. To determine the influence of rare sugars on apo A-I expression in hepatic (HepG2) and intestinal derived (Caco-2) cell lines, apo A-I, albumin, and SP1 were quantified with enzyme immunoassay and Western blots while mRNA levels were quantified with real-time polymerase chain reaction. The promoter activity was measured using transient transfection assays with plasmids containing various segments and mutations in the promoter. D-allulose and D-tagatose, increased apo A-I concentration in culture media while D-sorbose and D-allose did not have any measurable effects. D-allulose did not increase apo A-I levels in Caco-2 cells. These changes paralleled the increased mRNA levels and promoter activity. D-allulose-response was mapped at the insulin response core element (IRCE). Mutation of the IRCE decreased the ability of D-allulose and insulin to activate the promoter. Treatment of HepG2 cells, but not Caco-2 cells, with D-alluose and insulin increased SP1 expression relative to control cells. D-allulose augmented the expression and IRCE binding of SP1, an essential transcription factor for the insulin on apo A-I promoter activity. D-allulose can modulate some insulin-responsive genes and may have anti-atherogenic properties, in part due to increasing apo A-I production. PRACTICAL APPLICATIONS: Coronary artery disease (CAD) is the number one cause of mortality in industrialized countries. A risk factor associated with CAD is low high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apo A-I) concentrations in plasma. Thus, novel therapeutic agents or nutrients that upregulate apo A-I production should be identified. D-allulose and D-tagatose are used as sweeteners and may have favorable effects on insulin resistance and diabetes. This study shows that D-allulose and D-tagatose increases apo A-I production through increased transcription factor SP1-binding to insulin response element of the promoter. These sweeteners modulate some insulin responsive genes, increase the production of apo-A-I, and therefore may have anti-atherogenic properties.

Cognitive Effects of the BET Protein Inhibitor Apabetalone: A Prespecified Montreal Cognitive Assessment Analysis Nested in the BETonMACE Randomized Controlled Trial.

BACKGROUND: Epigenetic changes may contribute importantly to cognitive decline in late life including Alzheimer's disease (AD) and vascular dementia (VaD). Bromodomain and extra-terminal (BET) proteins are epigenetic "readers" that may distort normal gene expression and contribute to chronic disorders. OBJECTIVE: To assess the effects of apabetalone, a small molecule BET protein inhibitor, on cognitive performance of patients 70 years or older participating in a randomized trial of patients at high risk for major cardiovascular events (MACE). METHODS: The Montreal Cognitive Assessment (MoCA) was performed on all patients 70 years or older at the time of randomization. 464 participants were randomized to apabetalone or placebo in the cognition sub-study. In a prespecified analysis, participants were assigned to one of three groups: MoCA score≥26 (normal performance), MoCA score 25-22 (mild cognitive impairment), and MoCA score≤21 (dementia). Exposure to apabetalone was equivalent in the treatment groups in each MoCA-defined group. RESULTS: Apabetalone was associated with an increased total MoCA score in participants with baseline MoCA score of≤21 (p = 0.02). There was no significant difference in change from baseline in the treatment groups with higher MoCA scores. In the cognition study, more patients randomized to apabetalone discontinued study drug for adverse effects (11.3% versus 7.9%). CONCLUSION: In this randomized controlled study, apabetalone was associated with improved cognition as measured by MoCA scores in those with baseline scores of 21 or less. BET protein inhibitors warrant further investigation for late life cognitive disorders.

Relation of insulin treatment for type 2 diabetes to the risk of major adverse cardiovascular events after acute coronary syndrome: an analysis of the BETonMACE randomized clinical trial.

BACKGROUND: In stable patients with type 2 diabetes (T2D), insulin treatment is associated with elevated risk for major adverse cardiovascular events (MACE). Patients with acute coronary syndrome (ACS) and T2D are at particularly high risk for recurrent MACE despite evidence-based therapies. It is uncertain to what extent this risk is further magnified in patients with recent ACS who are treated with insulin. We examined the relationship of insulin use to risk of MACE and modification of that risk by apabetalone, a bromodomain and extra-terminal (BET) protein inhibitor. METHODS: The analysis utilized data from the BETonMACE phase 3 trial that compared apabetalone to placebo in patients with T2D, low HDL cholesterol, andACS. The primary MACE outcome (cardiovascular death, myocardial infarction, or stroke) was examined according to insulin treatment and assigned study treatment. Multivariable Cox regression was used to determine whether insulin use was independently associated with the risk of MACE. RESULTS: Among 2418 patients followed for median 26.5 months, 829 (34.2%) were treated with insulin. Despite high utilization of evidence-based treatments including coronary revascularization, intensive statin treatment, and dual antiplatelet therapy, the 3-year incidence of MACE in the placebo group was elevated among insulin-treated patients (20.4%) compared to those not-treated with insulin (12.8%, P = 0.0001). Insulin treatment remained strongly associated with the risk of MACE (HR 2.10, 95% CI 1.42-3.10, P = 0.0002) after adjustment for demographic, clinical, and treatment variables. Apabetalone had a consistent, favorable effect on MACE in insulin-treated and not insulin-treated patients. CONCLUSION: Insulin-treated patients with T2D, low HDL cholesterol, and ACS are at high risk for recurrent MACE despite the use of evidence-based, contemporary therapies. A strong association of insulin treatment with risk of MACE persists after adjustment for other characteristics associated with MACE. There is unmet need for additional treatments to mitigate this risk. Trial registration ClinicalTrials.gov NCT02586155, registered October 26, 2015.

Bromodomain and Extraterminal Protein Inhibitor, Apabetalone (RVX-208), Reduces ACE2 Expression and Attenuates SARS-Cov-2 Infection In Vitro.

Effective therapeutics are urgently needed to counter infection and improve outcomes for patients suffering from COVID-19 and to combat this pandemic. Manipulation of epigenetic machinery to influence viral infectivity of host cells is a relatively unexplored area. The bromodomain and extraterminal (BET) family of epigenetic readers have been reported to modulate SARS-CoV-2 infection. Herein, we demonstrate apabetalone, the most clinical advanced BET inhibitor, downregulates expression of cell surface receptors involved in SARS-CoV-2 entry, including angiotensin-converting enzyme 2 (ACE2) and dipeptidyl-peptidase 4 (DPP4 or CD26) in SARS-CoV-2 permissive cells. Moreover, we show that apabetalone inhibits SARS-CoV-2 infection in vitro to levels comparable to those of antiviral agents. Taken together, our study supports further evaluation of apabetalone to treat COVID-19, either alone or in combination with emerging therapeutics.

BET inhibition blocks inflammation-induced cardiac dysfunction and SARS-CoV-2 infection.

Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined but could be through direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory "cytokine-storm", a cocktail of interferon gamma, interleukin 1ß, and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids (hCOs) and hearts of SARS-CoV-2-infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCOs and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression, and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the Food and Drug Administration (FDA) breakthrough designated drug, apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage.

Inhibitors of bromodomain and extra-terminal proteins for treating multiple human diseases.

Clinical development of bromodomain and extra-terminal (BET) protein inhibitors differs from the traditional course of drug development. These drugs are simultaneously being evaluated for treating a wide spectrum of human diseases due to their novel mechanism of action. BET proteins are epigenetic "readers," which play a primary role in transcription. Here, we briefly describe the BET family of proteins, of which BRD4 has been studied most extensively. We discuss BRD4 activity at latent enhancers as an example of BET protein function. We examine BRD4 redistribution and enhancer reprogramming in embryonic development, cancer, cardiovascular, autoimmune, and metabolic diseases, presenting hallmark studies that highlight BET proteins as attractive targets for therapeutic intervention. We review the currently available approaches to targeting BET proteins, methods of selectively targeting individual bromodomains, and review studies that compare the effects of selective BET inhibition to those of pan-BET inhibition. Lastly, we examine the current clinical landscape of BET inhibitor development.

BET protein inhibitor apabetalone (RVX-208) suppresses pro-inflammatory hyper-activation of monocytes from patients with cardiovascular disease and type 2 diabetes.

BACKGROUND: Patients with cardiovascular disease (CVD) and type 2 diabetes (DM2) have a high residual risk for experiencing a major adverse cardiac event. Dysregulation of epigenetic mechanisms of gene transcription in innate immune cells contributes to CVD development but is currently not targeted by therapies. Apabetalone (RVX-208) is a small molecule inhibitor of bromodomain and extra-terminal (BET) proteins-histone acetylation readers that drive pro-inflammatory and pro-atherosclerotic gene transcription. Here, we assess the impact of apabetalone on ex vivo inflammatory responses of monocytes from DM2 + CVD patients. RESULTS: Monocytes isolated from DM2 + CVD patients and matched controls were treated ex vivo with apabetalone, interferon γ (IFNγ), IFNγ + apabetalone or vehicle and phenotyped for gene expression and protein secretion. Unstimulated DM2 + CVD monocytes had higher baseline IL-1α, IL-1ß and IL-8 cytokine gene expression and Toll-like receptor (TLR) 2 surface abundance than control monocytes, indicating pro-inflammatory activation. Further, DM2 + CVD monocytes were hyper-responsive to stimulation with IFNγ, upregulating genes within cytokine and NF-κB pathways > 30% more than control monocytes (p < 0.05). Ex vivo apabetalone treatment countered cytokine secretion by DM2 + CVD monocytes at baseline (GROα and IL-8) and during IFNγ stimulation (IL-1ß and TNFα). Apabetalone abolished pro-inflammatory hyper-activation by reducing TLR and cytokine gene signatures more robustly in DM2 + CVD versus control monocytes. CONCLUSIONS: Monocytes isolated from DM2 + CVD patients receiving standard of care therapies are in a hyper-inflammatory state and hyperactive upon IFNγ stimulation. Apabetalone treatment diminishes this pro-inflammatory phenotype, providing mechanistic insight into how BET protein inhibition may reduce CVD risk in DM2 patients.

Epigenetic Modulation by Apabetalone Counters Cytokine-Driven Acute Phase Response In Vitro, in Mice and in Patients with Cardiovascular Disease.

Chronic systemic inflammation contributes to cardiovascular disease (CVD) and correlates with the abundance of acute phase response (APR) proteins in the liver and plasma. Bromodomain and extraterminal (BET) proteins are epigenetic readers that regulate inflammatory gene transcription. We show that BET inhibition by the small molecule apabetalone reduces APR gene and protein expression in human hepatocytes, mouse models, and plasma from CVD patients. Steady-state expression of serum amyloid P, plasminogen activator inhibitor 1, and ceruloplasmin, APR proteins linked to CVD risk, is reduced by apabetalone in cultured hepatocytes and in humanized mouse liver. In cytokine-stimulated hepatocytes, apabetalone reduces the expression of C-reactive protein (CRP), alpha-2-macroglobulin, and serum amyloid P. The latter two are also reduced by apabetalone in the liver of endotoxemic mice. BET knockdown in vitro also counters cytokine-mediated induction of the CRP gene. Mechanistically, apabetalone reduces the cytokine-driven increase in BRD4 BET occupancy at the CRP promoter, confirming that transcription of CRP is BET-dependent. In patients with stable coronary disease, plasma APR proteins CRP, IL-1 receptor antagonist, and fibrinogen γ decrease after apabetalone treatment versus placebo, resulting in a predicted downregulation of the APR pathway and cytokine targets. We conclude that CRP and components of the APR pathway are regulated by BET proteins and that apabetalone counters chronic cytokine signaling in patients.

Apabetalone (RVX-208) reduces vascular inflammation in vitro and in CVD patients by a BET-dependent epigenetic mechanism.

BACKGROUND: Apabetalone (RVX-208) is a bromodomain and extraterminal protein inhibitor (BETi) that in phase II trials reduced the relative risk (RR) of major adverse cardiac events (MACE) in patients with cardiovascular disease (CVD) by 44% and in diabetic CVD patients by 57% on top of statins. A phase III trial, BETonMACE, is currently assessing apabetalone's ability to reduce MACE in statin-treated post-acute coronary syndrome type 2 diabetic CVD patients with low high-density lipoprotein C. The leading cause of MACE is atherosclerosis, driven by dysfunctional lipid metabolism and chronic vascular inflammation (VI). In vitro studies have implicated the BET protein BRD4 as an epigenetic driver of inflammation and atherogenesis, suggesting that BETi may be clinically effective in combating VI. Here, we assessed apabetalone's ability to regulate inflammation-driven gene expression and cell adhesion in vitro and investigated the mechanism by which apabetalone suppresses expression. The clinical impact of apabetalone on mediators of VI was assessed with proteomic analysis of phase II CVD patient plasma. RESULTS: In vitro, apabetalone prevented inflammatory (TNFα, LPS, or IL-1ß) induction of key factors that drive endothelial activation, monocyte recruitment, adhesion, and plaque destabilization. BRD4 abundance on inflammatory and adhesion gene promoters and enhancers was reduced by apabetalone. BRD2-4 degradation by MZ-1 also prevented TNFα-induced transcription of monocyte and endothelial cell adhesion molecules and inflammatory mediators, confirming BET-dependent regulation. Transcriptional regulation by apabetalone translated into a reduction in monocyte adhesion to an endothelial monolayer. In a phase II trial, apabetalone treatment reduced the abundance of multiple VI mediators in the plasma of CVD patients (SOMAscan® 1.3 k). These proteins correlate with CVD risk and include adhesion molecules, cytokines, and metalloproteinases. Ingenuity® Pathway Analysis (IPA®) predicted that apabetalone inhibits pro-atherogenic regulators and pathways and prevents disease states arising from leukocyte recruitment. CONCLUSIONS: Apabetalone suppressed gene expression of VI mediators in monocytes and endothelial cells by inhibiting BET-dependent transcription induced by multiple inflammatory stimuli. In CVD patients, apabetalone treatment reduced circulating levels of VI mediators, an outcome conducive with atherosclerotic plaque stabilization and MACE reduction. Inhibition of inflammatory and adhesion molecule gene expression by apabetalone is predicted to contribute to MACE reduction in the phase III BETonMACE trial.

The Effect of Bromodomain and Extra-Terminal Inhibitor Apabetalone on Attenuated Coronary Atherosclerotic Plaque: Insights from the ASSURE Trial.

BACKGROUND: Apabetalone is a selective bromodomain and extra-terminal (BET) inhibitor which modulates lipid and inflammatory pathways implicated in atherosclerosis. The impact of apabetalone on attenuated coronary atherosclerotic plaque (AP), a measure of vulnerability, is unknown. METHODS: The ApoA-1 Synthesis Stimulation and intravascular Ultrasound for coronary atheroma Regression Evaluation (ASSURE; NCT01067820) study employed serial intravascular ultrasound (IVUS) measures of coronary atheroma in 281 patients treated with apabetalone or placebo for 26 weeks. AP was measured at baseline and follow-up. Factors associated with changes in AP were investigated. RESULTS: AP was observed in 31 patients (11%) [27 (13.0%) in the apabetalone group and four (5.5%) in the placebo group]. The apabetalone group demonstrated reductions in AP length by - 1 mm [interquartile range (IQR) - 4, 1] (p = 0.03), AP arc by - 37.0° (IQR - 59.2, 8.2) (p = 0.003) and the AP index by - 34.6 mm° (IQR - 52.6, 10.1) (p = 0.003) from baseline. The change in AP index correlated with on-treatment concentration of high-density lipoprotein (HDL) particles (r = - 0.52, p = 0.006), but not HDL cholesterol (r = - 0.11, p = 0.60) or apolipoprotein A-1 (r = - 0.16, p = 0.43). Multivariable analysis revealed that on-treatment concentrations of HDL particles (p = 0.03) and very low-density lipoprotein particles (p = 0.01) were independently associated with changes in AP index. CONCLUSIONS: Apabetalone favorably modulated ultrasonic measures of plaque vulnerability in the population studied, which may relate to an increase in HDL particle concentrations. The clinical implications are currently being investigated in the phase 3 major adverse cardiac event outcomes trial BETonMACE.

Apabetalone downregulates factors and pathways associated with vascular calcification.

BACKGROUND AND AIMS: Apabetalone is an inhibitor of bromodomain and extraterminal (BET) proteins. In clinical trials, apabetalone reduced the incidence of major adverse cardiac events (MACE) in patients with cardiovascular disease and reduced circulating factors that promote vascular calcification (VC). Because VC contributes to MACE, effects of apabetalone on pro-calcific processes were examined. METHODS AND RESULTS: Apabetalone inhibited extracellular calcium deposition and opposed induction of transdifferentiation markers in human coronary artery vascular smooth muscle cells (VSMCs) under osteogenic culture conditions. Tissue-nonspecific alkaline phosphatase (TNAP) is a key contributor to VC, and apabetalone suppressed osteogenic induction of the mRNA, protein and enzyme activity. The liver is a major source of circulating TNAP, and apabetalone also downregulated TNAP expression in primary human hepatocytes. BRD4, a transcriptional regulator and target of apabetalone, has been linked to calcification. Osteogenic transdifferentiation of VSMCs resulted in disassembly of 100 BRD4-rich enhancers, with concomitant enlargement of remaining enhancers. Apabetalone reduced the size of BRD4-rich enhancers, consistent with disrupting BRD4 association with chromatin. 38 genes were uniquely associated with BRD4-rich enhancers in osteogenic conditions; 11 were previously associated with calcification. Apabetalone reduced levels of BRD4 on many of these enhancers, which correlated with decreased expression of the associated gene. Bioinformatics revealed BRD4 may cooperate with 7 specific transcription factors to promote transdifferentiation and calcification. CONCLUSIONS: Apabetalone counters transdifferentiation and calcification of VSMCs via an epigenetic mechanism involving specific transcription factors. The mechanistic findings, combined with evidence from clinical trials, support further development of apabetalone as a therapeutic for VC.

Regulation of apolipoprotein A-I gene expression by the histamine H1 receptor: Requirement for NF-κB.

AIMS: Earlier it had been found by us that apolipoprotein A-I (apo A-I) is suppressed by histamine in HepG2 cells. Histamine has been shown to regulate NF-κB activity, though not in hepatocytes. Therefore we examined the role of the histamine receptors and NF-κB in histamine-mediated apo A-I gene expression in HepG2 liver cells. MAIN METHODS: The effect of histamine on histamine H1 receptor expression, and NF-κB p65 and p50 subunits was examined by Western blot. Histamine H1 receptor involvement was examined by loss-of-function (via siRNA) and gain-of-function studies overexpressing the histamine H1 receptor. The requirement for the p65 subunit of NF-κB for histamines effect was elucidated by loss-of-function studies (siRNA). Finally, the effect of histamine on NF-κB binding to the apo A-I gene promoter was examined by chromatin immunoprecipitation. KEY FINDINGS: Treatment of HepG2 cells with histamine had no effect on histamine H1 receptor expression. However, treatment with histamine increased NF-κB p65 and p50 subunit expression significantly. At low levels, the exogenous histamine H1 receptor plasmid suppressed apo A-I gene promoter activity while addition of higher levels of plasmid DNA actually increased apo A-I gene promoter activity. Inhibition of NF-κB activity with SN50 prevented histamine from repressing apo A-I promoter activity as did silencing p65 expression via siRNA. Finally, treatment with histamine increased binding of the p65 subunit of NF-κB to the apo A-I gene promoter. SIGNIFICANCE: Histamine suppresses apo A-I gene expression in hepatocytes via the histamine H1 receptor by elevating NF-κB expression and binding to the apo A-I promoter.

Inhibition of hepatic apolipoprotein A-I gene expression by histamine.

In a recent high throughput analysis to identify drugs that alter hepatic apolipoprotein A-I (apo A-I) expression, histamine receptor one (H1) antagonists emerged as potential apo A-1 inducing drugs. Thus the present study was undertaken to identify some of the underlying molecular mechanisms of the effect of antihistaminic drugs on apo AI production. Apo A-I levels were measured by enzyme immunoassay and Western blots. Apo A-I mRNA levels were measured by reverse transcription real-time PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as the internal control. The effects of histamine and antihistamines on apo A-I gene were determined by transient transfection of plasmids containing the apo A-I gene promoter. Histamine repressed while (H1) receptor antagonist azelastine increased apo A-I protein and mRNA levels within 48 h in a dose-dependent manner. Azelastine and histamine increased and suppressed, respectively, apo A-I gene promoter activity through a peroxisome proliferator activated receptor α response element. Treatment of HepG2 cells with other H1 receptor antagonists including fexofenadine, cetirizine, and diphenhydramine increased apo A-I levels in a dose-dependent manner while treatment with H2 receptor antagonists including cimetidine, famotidine, and ranitidine had no effect. We conclude that H1 receptor signaling is a novel pathway of apo A1 gene expression and therefore could be an important therapeutic target for enhancing de-novo apo A-1 synthesis.

RVX-297, a BET Bromodomain Inhibitor, Has Therapeutic Effects in Preclinical Models of Acute Inflammation and Autoimmune Disease.

Bromodomain (BD) and extra-terminal domain containing proteins (BET) are chromatin adapters that bind acetylated histone marks via two tandem BDs, BD1 and BD2, to regulate gene transcription. BET proteins are involved in transcriptional reprogramming in response to inflammatory stimuli. BET BD inhibitors (BETis) that are nonselective for BD1 or BD2 have recognized anti-inflammatory properties in vitro and counter pathology in models of inflammation or autoimmune disease. Although both BD1 and BD2 bind acetylated histone residues, they may independently regulate the expression of BET-sensitive genes. Here we characterized the ability of RVX-297, a novel orally active BETi with selectivity for BD2, to modulate inflammatory processes in vitro, in vivo, and ex vivo. RVX-297 suppressed inflammatory gene expression in multiple immune cell types in culture. Mechanistically, RVX-297 displaced BET proteins from the promoters of sensitive genes and disrupted recruitment of active RNA polymerase II, a property shared with pan-BETis that nonselectively bind BET BDs. In the lipopolysaccharide model of inflammation, RVX-297 reduced proinflammatory mediators assessed in splenic gene expression and serum proteins. RVX-297 also countered pathology in three rodent models of polyarthritis: rat and mouse collagen-induced arthritis, and mouse collagen antibody-induced arthritis. Further, RVX-297 prevented murine experimental autoimmune encephalomyelitis (a model of human multiple sclerosis) disease development when administered prophylactically and reduced hallmarks of pathology when administered therapeutically. We show for the first time that a BD2-selective BETi maintains anti-inflammatory properties and is effective in preclinical models of acute inflammation and autoimmunity.