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.

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.

Apabetalone Mediated Epigenetic Modulation is Associated with Favorable Kidney Function and Alkaline Phosphatase Profile in Patients with Chronic Kidney Disease.

BACKGROUND/AIMS: The association between serum alkaline phosphatase (ALP) with adverse cardiovascular outcomes, in Chronic Kidney Disease (CKD) patients has previously been reported and may be a result of increased vascular calcification and inflammation. Here we report, for the first time, the effects of pharmacologic epigenetic modulation on levels of ALP and kidney function via a novel oral small molecule BET inhibitor, apabetalone, in CKD patients. METHODS: A post-hoc analysis evaluated patients with estimated glomerular filtration rate (eGFR) <60 mL/min/1.73m2, who participated in the apabetalone phase 2 randomized controlled trials (SUSTAIN and ASSURE). 48 CKD subjects with a history of cardiovascular disease (CVD) were treated with 100mg twice-daily of 24 and 26 weeks of apabetalone or placebo. ALP and eGFR were measured prior to randomization and at final visits. RESULTS: Patients who received apabetalone (n=35) versus placebo (n=13) over 6 months showed significantly (p=0.02) lowered serum ALP -14.0% (p<0.0001 versus baseline) versus -6.3% (p=0.9 versus baseline). The eGFR in the apabetalone group increased by 3.4% (1.7 mL/min/1.73 m2) (p=0.04 versus baseline) and decreased by 5.8% (2.9 mL/min/1.73 m2) (p=0.6 versus baseline) in the placebo group. Apabetalone was well tolerated. CONCLUSION: A post-hoc analysis of CKD subjects from the SUSTAIN and ASSURE randomized controlled trials demonstrated favorable effects of apabetalone on ALP and eGFR, and generated the hypothesis that epigenetic modulation by BET inhibition may potentially offer a novel therapeutic strategy to treat CVD and progressive kidney function loss in CKD patients. This is being examined in the phase III trial BETonMACE.

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 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.

Apabetalone lowers serum alkaline phosphatase and improves cardiovascular risk in patients with cardiovascular disease.

BACKGROUND AND AIMS: In patients with cardiovascular disease, considerable residual risk remains despite evidence-based secondary prevention measures. Alkaline phosphatase (ALP) has been suggested as a modifiable cardiovascular risk factor. We sought to determine whether cardiovascular risk reduction by the bromodomain and extra-terminal (BET) protein inhibitor apabetalone is associated with the concomitant lowering of serum ALP. METHODS: In a post-hoc analysis of 795 patients with established coronary heart disease and statin treatment, who participated in phase 2 placebo-controlled trials of apabetalone, we determined the effect of assigned treatment for up to 24 weeks on the incidence of major adverse cardiovascular events (MACE) and serum ALP. RESULTS: Baseline ALP (median 72 U/L) predicted MACE (death, non-fatal myocardial infarction, coronary revascularization, or hospitalization for cardiovascular causes), independent of high-sensitivity C-reactive protein (hsCRP), sex, age, race, study, cardiovascular risk factors, chronic kidney disease (CKD), liver function markers and treatment allocation (hazard ratio [HR] per standard deviation [SD] 1.6, 95% CI 1.19-2.16, p = 0.002). Mean placebo-corrected decreases in ALP from baseline were 9.2% (p < 0.001) after 12-14 weeks and 7.7% (p < 0.001) after 24-26 weeks of apabetalone treatment. In the apabetalone group, a 1-SD reduction in ALP was associated with a HR for MACE of 0.64 (95% CI 0.46-0.90, p = 0.009). CONCLUSIONS: Serum ALP predicts residual cardiovascular risk, independent of hsCRP, established cardiovascular risk factors and CKD, in patients with cardiovascular disease on statin treatment. Apabetalone lowers serum ALP, which was associated with a lower risk of cardiovascular events. Whether the beneficial cardiovascular effects of apabetalone are causally related to ALP reduction remains undetermined.

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.

Benefit of Apabetalone on Plasma Proteins in Renal Disease.

INTRODUCTION: Apabetalone, a small molecule inhibitor, targets epigenetic readers termed BET proteins that contribute to gene dysregulation in human disorders. Apabetalone has in vitro and in vivo anti-inflammatory and antiatherosclerotic properties. In phase 2 clinical trials, this drug reduced the incidence of major adverse cardiac events in patients with cardiovascular disease. Chronic kidney disease is associated with a progressive loss of renal function and a high risk of cardiovascular disease. We studied the impact of apabetalone on the plasma proteome in patients with impaired kidney function. METHODS: Subjects with stage 4 or 5 chronic kidney disease and matched controls received a single dose of apabetalone. Plasma was collected for pharmacokinetic analysis and for proteomics profiling using the SOMAscan 1.3k platform. Proteomics data were analyzed with Ingenuity Pathway Analysis to identify dysregulated pathways in diseased patients, which were targeted by apabetalone. RESULTS: At baseline, 169 plasma proteins (adjusted P value <0.05) were differentially enriched in renally impaired patients versus control subjects, including cystatin C and ß2 microglobulin, which correlate with renal function. Bioinformatics analysis of the plasma proteome revealed a significant activation of 42 pathways that control immunity and inflammation, oxidative stress, endothelial dysfunction, vascular calcification, and coagulation. At 12 hours postdose, apabetalone countered the activation of pathways associated with renal disease and reduced the abundance of disease markers, including interleukin-6, plasminogen activator inhibitor-1, and osteopontin. CONCLUSION: These data demonstrated plasma proteome dysregulation in renally impaired patients and the beneficial impact of apabetalone on pathways linked to chronic kidney disease and its cardiovascular complications.

Downregulation of the Complement Cascade In Vitro, in Mice and in Patients with Cardiovascular Disease by the BET Protein Inhibitor Apabetalone (RVX-208).

Apabetalone (RVX-208) is an epigenetic regulator developed to treat cardiovascular disease (CVD) that targets BET proteins. Through transcriptional regulation RVX-208 modulates pathways that underlie CVD including reverse cholesterol transport, vascular inflammation, coagulation, and complement. Using transcriptomics and proteomics we show that complement is one of the top pathways downregulated by RVX-208 in primary human hepatocytes (PHH) and in plasma from CVD patients. RVX-208 reduces basal and cytokine-driven expression of complement factors in PHH and in chimeric mice with humanized livers. Plasma proteomics of CVD patients shows that RVX-208 decreases complement proteins and regulators, including complement activators SAP and CRP. Circulating activated fragments C5a, C3b, and C5b-C6 are reduced by 51, 32, and 10%, respectively, indicating decreased activity of complement in patients. As complement components are linked to CVD and metabolic syndrome, including major acute cardiac events, modulating their levels and activity by RVX-208 may alleviate risks associated with these diseases.

RVX-208, a BET-inhibitor for treating atherosclerotic cardiovascular disease, raises ApoA-I/HDL and represses pathways that contribute to cardiovascular disease.

High density lipoproteins (HDL), through activity of the main protein component apolipoprotein A-I (ApoA-I), can reduce the risk of cardiovascular disease (CVD) by removing excess cholesterol from atherosclerotic plaque. In this study, we demonstrate that the bromodomain and extraterminal domain (BET) inhibitor RVX-208 increases ApoA-I gene transcription and protein production in human and primate primary hepatocytes. Accordingly, RVX-208 also significantly increases levels of ApoA-I, HDL-associated cholesterol, and HDL particle number in patients who received the compound in recently completed phase 2b trials SUSTAIN and ASSURE. Moreover, a post-hoc analysis showed lower instances of major adverse cardiac events in patients receiving RVX-208. To understand the effects of RVX-208 on biological processes underlying cardiovascular risk, we performed microarray analyses of human primary hepatocytes and whole blood treated ex vivo. Overall, data showed that RVX-208 raises ApoA-I/HDL and represses pro-inflammatory, pro-atherosclerotic and pro-thrombotic pathways that can contribute to CVD risk.

Data on gene and protein expression changes induced by apabetalone (RVX-208) in ex vivo treated human whole blood and primary hepatocytes.

Apabetalone (RVX-208) inhibits the interaction between epigenetic regulators known as bromodomain and extraterminal (BET) proteins and acetyl-lysine marks on histone tails. Data presented here supports the manuscript published in Atherosclerosis "RVX-208, a BET-inhibitor for Treating Atherosclerotic Cardiovascular Disease, Raises ApoA-I/HDL and Represses Pathways that Contribute to Cardiovascular Disease" (Gilham et al., 2016) [1]. It shows that RVX-208 and a comparator BET inhibitor (BETi) JQ1 increase mRNA expression and production of apolipoprotein A-I (ApoA-I), the main protein component of high density lipoproteins, in primary human and African green monkey hepatocytes. In addition, reported here are gene expression changes from a microarray-based analysis of human whole blood and of primary human hepatocytes treated with RVX-208.

Avian flu: the creation of expectations in the interplay between science and the media.

This paper examines the emerging cultural patterns and interpretative repertoires in reports of an impending pandemic of avian flu in the UK mass media and scientific journals at the beginning of 2005, paying particular attention to metaphors, pragmatic markers ('risk signals'), symbolic dates and scare statistics used by scientists and the media to create expectations and elicit actions. This study complements other work on the metaphorical framing of infectious disease, such as foot and mouth disease and SARS, tries to link it to developments in the sociology of expectations and applies insights from pragmatics both to the sociology of metaphor and the sociology of expectations.