Apabetalone, a Clinical-Stage, Selective BET Inhibitor, Opposes DUX4 Target Gene Expression in Primary Human FSHD Muscle Cells.

Facioscapulohumeral dystrophy (FSHD) is a muscle disease caused by inappropriate expression of the double homeobox 4 (DUX4) gene in skeletal muscle, and its downstream activation of pro-apoptotic transcriptional programs. Inhibitors of DUX4 expression have the potential to treat FSHD. Apabetalone is a clinical-stage bromodomain and extra-terminal (BET) inhibitor, selective for the second bromodomain on BET proteins. Using primary human skeletal muscle cells from FSHD type 1 patients, we evaluated apabetalone for its ability to counter DUX4's deleterious effects and compared it with the pan-BET inhibitor JQ1, and the p38 MAPK inhibitor-and DUX4 transcriptional repressor-losmapimod. We applied RNA-sequencing and bioinformatic analysis to detect treatment-associated impacts on the transcriptome of these cells. Apabetalone inhibited the expression of DUX4 downstream markers, reversing hallmarks of FSHD gene expression in differentiated muscle cells. JQ1, but not apabetalone, was found to induce apoptosis. While both BET inhibitors modestly impacted differentiation marker expression, they did not affect myotube fusion. Losmapimod also reduced expression of DUX4 target genes but differed in its impact on FSHD-associated pathways. These findings demonstrate that apabetalone inhibits DUX4 target gene expression and reverses transcriptional programs that contribute to FSHD pathology, making this drug a promising candidate therapeutic for FSHD.

Apabetalone Downregulates Fibrotic, Inflammatory and Calcific Processes in Renal Mesangial Cells and Patients with Renal Impairment.

Epigenetic mechanisms are implicated in transcriptional programs driving chronic kidney disease (CKD). Apabetalone is an orally available inhibitor of bromodomain and extraterminal (BET) proteins, which are epigenetic readers that modulate gene expression. In the phase 3 BETonMACE trial, apabetalone reduced risk of major adverse cardiac events (MACE) by 50% in the CKD subpopulation, indicating favorable effects along the kidney-heart axis. Activation of human renal mesangial cells (HRMCs) to a contractile phenotype that overproduces extracellular matrix (ECM) and inflammatory cytokines, and promotes calcification, frequently accompanies CKD to drive pathology. Here, we show apabetalone downregulated HRMC activation with TGF-ß1 stimulation by suppressing TGF-ß1-induced α-smooth muscle actin (α-SMA) expression, α-SMA assembly into stress fibers, enhanced contraction, collagen overproduction, and expression of key drivers of fibrosis, inflammation, or calcification including thrombospondin, fibronectin, periostin, SPARC, interleukin 6, and alkaline phosphatase. Lipopolysaccharide-stimulated expression of inflammatory genes IL6, IL1B, and PTGS2 was also suppressed. Transcriptomics confirmed apabetalone affected gene sets of ECM remodeling and integrins. Clinical translation of in vitro results was indicated in CKD patients where a single dose of apabetalone reduced plasma levels of key pro-fibrotic and inflammatory markers, and indicated inhibition of TGF-ß1 signaling. While plasma proteins cannot be traced to the kidney alone, anti-fibrotic and anti-inflammatory effects of apabetalone identified in this study are consistent with the observed decrease in cardiovascular risk in CKD patients.

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.

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.

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.

Metabotropic NMDA receptor signaling couples Src family kinases to pannexin-1 during excitotoxicity.

Overactivation of neuronal N-methyl-D-aspartate receptors (NMDARs) causes excitotoxicity and is necessary for neuronal death. In the classical view, these ligand-gated Ca(2+)-permeable ionotropic receptors require co-agonists and membrane depolarization for activation. We report that NMDARs signal during ligand binding without activation of their ion conduction pore. Pharmacological pore block with MK-801, physiological pore block with Mg(2+) or a Ca(2+)-impermeable NMDAR variant prevented NMDAR currents, but did not block excitotoxic dendritic blebbing and secondary currents induced by exogenous NMDA. NMDARs, Src kinase and Panx1 form a signaling complex, and activation of Panx1 required phosphorylation at Y308. Disruption of this NMDAR-Src-Panx1 signaling complex in vitro or in vivo by administration of an interfering peptide either before or 2 h after ischemia or stroke was neuroprotective. Our observations provide insights into a new signaling modality of NMDARs that has broad-reaching implications for brain physiology and pathology.

Survival of adult generated hippocampal neurons is altered in circadian arrhythmic mice.

The subgranular zone of the hippocampal formation gives rise to new neurons that populate the dentate gyrus throughout life. Cells in the hippocampus exhibit rhythmic clock gene expression and the circadian clock is known to regulate the cycle of cell division in other areas of the body. These facts suggest that the circadian clock may regulate adult neurogenesis in the hippocampus as well. In the present study, neurogenesis in the hippocampal subgranular zone was examined in arrhythmic Bmal1 knockout (-KO) mice and their rhythmic heterozygous and wildtype littermates. Proliferation and survival of newly generated subgranular zone cells were examined using bromodeoxyuridine labelling, while pyknosis (a measure of cell death) and hippocampal volume were examined in cresyl violet stained sections. There was no significant difference in cellular proliferation between any of the groups, yet survival of proliferating cells, 6 weeks after the bromodeoxyuridine injection, was significantly greater in the BMAL1-KO animals. The number of pyknotic cells was significantly decreased in Bmal1-KO animals, yet hippocampal volume remained the same across genotypes. These findings suggest that while a functional circadian clock is not necessary for normal proliferation of neuronal precursor cells, the normal pruning of newly generated neurons in the hippocampus may require a functional circadian clock.

Lesion size and behavioral deficits after endothelin-1-induced ischemia are not dependent on time of day.

BACKGROUND: The occurrence of stroke exhibits a strong circadian pattern with a peak in the morning hours after waking. The factors that influence this pattern of stroke prevalence may confer varying degrees of neuroprotection and therefore influence stroke severity. This question is difficult to address in clinical cases because of the variability in the location and duration of the ischemic event. METHODS: The purpose of this study was to determine if time of day affected the severity of stroke targeting the motor cortex in rats. Strokes were produced using topical application of the vasoconstrictor endothelin-1 to motor cortex of unanesthetized animals at 2 time points: early day and early night. Behavioral deficits were measured using reaching, cylinder, and horizontal ladder tasks, and the volume of the lesion was quantified. RESULTS: Behavior on reaching and horizontal ladder tasks were both severely impaired by endothelin-1 treatment compared to vehicle-treated animals, but deficits did not differ according to time of treatment. Similarly, while endothelin-1 produced larger lesions of the motor cortex than did vehicle treatment, the size of the lesion did not differ according to time of treatment. CONCLUSIONS: These results suggest that while many factors under circadian control can influence the prevalence of stroke, the magnitude of lesion and behavioral deficit resulting from an ischemic event may not be influenced by time of day.

Physiological responses of the circadian clock to acute light exposure at night.

Circadian rhythms in physiological, endocrine and metabolic functioning are controlled by a neural clock located in the suprachiasmatic nucleus (SCN). This structure is endogenously rhythmic and the phase of this rhythm can be reset by light information from the eye. A key feature of the SCN is that while it is a small structure containing on the order of about 20,000 cells, it is amazingly heterogeneous. It is likely that anatomical heterogeneity reflects an underlying functional heterogeneity. In this review, we examine the physiological responses of cells in the SCN to light stimuli that reset the phase of the circadian clock, highlighting where possible the spatial pattern of such responses. Increases in intracellular calcium are an important signal in response to light, and this increase triggers many biochemical cascades that mediate responses to light. Furthermore, only some cells in the SCN are actually endogenously rhythmic, and these cells likely do not receive strong direct input from the retina. Therefore, this review also considers how light information is conveyed from the retinorecipient cells to the endogenously rhythmic cells that track circadian phase. A number of neuropeptides, including vasoactive intestinal polypeptide, gastrin-releasing peptide and substance P, may be particularly important in relaying such signals, but other neurochemicals such as GABA and nitric oxide may participate as well. A thorough understanding of the intracellular and intercellular responses to light, as well as the spatial arrangements of such responses may help identify important pharmacological targets for therapeutic interventions to treat sleep and circadian disorders.