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.

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.

Effect of the ABCA1 agonist CS-6253 on amyloid-ß and lipoprotein metabolism in cynomolgus monkeys.

BACKGROUND: Inducing brain ATP-binding cassette 1 (ABCA1) activity in Alzheimer's disease (AD) mouse models is associated with improvement in AD pathology. The purpose of this study was to investigate the effects of the ABCA1 agonist peptide CS-6253 on amyloid-ß peptides (Aß) and lipoproteins in plasma and cerebrospinal fluid (CSF) of cynomolgus monkeys, a species with amyloid and lipoprotein metabolism similar to humans. METHODS: CS-6253 peptide was injected intravenously into cynomolgus monkeys at various doses in three different studies. Plasma and CSF samples were collected at several time points before and after treatment. Levels of cholesterol, triglyceride (TG), lipoprotein particles, apolipoproteins, and Aß were measured using ELISA, ion-mobility analysis, and asymmetric-flow field-flow fractionation (AF4). The relationship between the change in levels of these biomarkers was analyzed using multiple linear regression models and linear mixed-effects models. RESULTS: Following CS-6253 intravenous injection, within minutes, small plasma high-density lipoprotein (HDL) particles were increased. In two independent experiments, plasma TG, apolipoprotein E (apoE), and Aß42/40 ratio were transiently increased following CS-6253 intravenous injection. This change was associated with a non-significant decrease in CSF Aß42. Both plasma total cholesterol and HDL-cholesterol levels were reduced following treatment. AF4 fractionation revealed that CS-6253 treatment displaced apoE from HDL to intermediate-density- and low density-lipoprotein (IDL/LDL)-sized particles in plasma. In contrast to plasma, CS-6253 had no effect on the assessed CSF apolipoproteins or lipids. CONCLUSIONS: Treatment with the ABCA1 agonist CS-6253 appears to favor Aß clearance from the brain.

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.

ABCA1 Agonist Mimetic Peptide CS-6253 Induces Microparticles Release From Different Cell Types by ABCA1-Efflux-Dependent Mechanism.

BACKGROUND: Small peptides based on the C-terminal domain of apo E have recently been proposed as ATP-binding cassette transporter A1 (ABCA1) agonist with therapeutic potential. Previous work has shown that a novel synthetic peptide, CS-6253, acts synergistically with apolipoprotein A-I or alone to generate high-density lipoprotein (HDL) particles; we have also shown that cells can release microparticles (50-350 nm in apparent diameter) in an ABCA1- and apolipoprotein A-I-dependent manner. The purpose of this study was to explore the ability of a novel synthetic peptide CS-6253 to induce microparticle release from various cell lines in the process of HDL biogenesis. METHODS: The effects of CS-6253 on microparticle formation through the ABCA1 transporter were examined in vitro using cell-based systems and pharmacologic manipulations. RESULTS: In cell-based systems combined with fast performance liquid chromatography and nano-sight-tracking analysis, we show that ABCA1 and CS-6253 mediate and increase the production of microparticles containing cholesterol. CS-6253 in baby hamster kidney cells not expressing ABCA1 (baby hamster kidney mock cells) did not alter cholesterol removal across the plasma membrane in the absence of ABCA1 expression even at high concentrations. We report that CS-6253 is not cytotoxic. CONCLUSIONS: The present study shows that CS-6253 generates cholesterol containing microparticles with size heterogeneity (100-350 nm) in an ABCA1-dependent manner. We show that microparticles contribute to cell cholesterol efflux from monocyte-macrophage cells. At high doses, CS-6253 is not able to extract cholesterol from cells not expressing ABCA1, indicating that CS-6253 requires ABCA1 cooperation for cholesterol mobilization. We conclude that CS-6253 is an ABCA1 agonist peptide that promotes cellular cholesterol efflux through HDL biogenesis and microparticle formation.

Novel ABCA1 peptide agonists with antidiabetic action.

Previously, apoE-derived ABCA1 agonist peptides have been shown to possess anti-atherosclerotic and possibly antidiabetic properties. Here we assessed the in vitro and in vivo actions of a second generation of ABCA1 peptide agonists, CS6253 and T6991-2, on glucose homeostasis. The results show that these two peptides improve glucose tolerance in a prediabetic diet-induced obesity mouse model by enhancing insulin secretion. It was further demonstrated that T6991-2 also improved glucose tolerance in leptin-deficient (ob/ob) mice. CS6253 increased insulin secretion both under basal conditions and in response to high glucose stimulation in pancreatic INS-1 ß-cells rendered leptin receptor deficient with specific siRNA. Additional in vitro cell studies suggest that the CS6253 agonist attenuates hepatic gluconeogenesis and glucose transport. It also potentiates insulin-stimulated glucose uptake and utilization. These observed anti-diabetic actions suggest additional benefits of the CS6253 and T6991-2 ABCA1 peptide agonists for cardiovascular disease beyond their direct anti-atherosclerosis properties previously described.

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.

Differential Effects of apoE4 and Activation of ABCA1 on Brain and Plasma Lipoproteins.

Apolipoprotein E4 (apoE4), the leading genetic risk factor for Alzheimer's disease (AD), is less lipidated compared to the most common and AD-benign allele, apoE3. We have recently shown that i.p. injections of the ATP-binding cassette A1 (ABCA1) agonist peptide CS-6253 to apoE mice reverse the hypolipidation of apoE4 and the associated brain pathology and behavioral deficits. While in the brain apoE is the main cholesterol transporter, in the periphery apoE and apoA-I both serve as the major cholesterol transporters. We presently investigated the extent to which apoE genotype and CS-6253 treatment to apoE3 and apoE4-targeted replacement mice affects the plasma levels and lipid particle distribution of apoE, and those of plasma and brain apoA-I and apoJ. This revealed that plasma levels of apoE4 were lower and eluted faster following FPLC than plasma apoE3. Treatment with CS-6253 increased the levels of plasma apoE4 and rendered the elution profile of apoE4 similar to that of apoE3. Similarly, the levels of plasma apoA-I were lower in the apoE4 mice compared to apoE3 mice, and this effect was partially reversed by CS-6253. Conversely, the levels of apoA-I in the brain which were higher in the apoE4 mice, were unaffected by CS-6253. The plasma levels of apoJ were higher in apoE4 mice than apoE3 mice and this effect was abolished by CS-6253. Similar but less pronounced effects were obtained in the brain. In conclusion, these results suggest that apoE4 affects the levels of apoA-I and apoJ and that the anti-apoE4 beneficial effects of CS-6253 may be related to both central and peripheral mechanisms.

ABCA1 Agonist Reverses the ApoE4-Driven Cognitive and Brain Pathologies.

The allele ɛ4 of apolipoprotein E (apoE4) is the most prevalent genetic risk factor for Alzheimer's disease (AD) and is therefore a promising therapeutic target. Human and animal model studies suggest that apoE4 is hypolipidated; accordingly, we have previously shown that the retinoid X receptor (RXR) agonist bexarotene upregulates ABCA1, the main apoE-lipidating protein, resulting in increased lipidation of apoE4, and the subsequent reversal of the pathological effects of apoE4, namely: accumulation of Aß42 and hyperphosphorylated tau, as well as reduction in the levels of synaptic markers and cognitive deficits. Since the RXR system has numerous other targets, it is important to devise the means of activating ABCA1 selectively. We presently utilized CS-6253, a peptide shown to directly activate ABCA1 in vitro, and examined the extent to which it can affect the degree of lipidation of apoE4 in vivo and counteract the associated brain and behavioral pathologies. This revealed that treatment of young apoE4-targeted replacement mice with CS-6253 increases the lipidation of apoE4. This was associated with a reversal of the apoE4-driven Aß42 accumulation and tau hyperphosphorylation in hippocampal neurons, as well as of the synaptic impairments and cognitive deficits. These findings suggest that the pathological effects of apoE4 in vivo are associated with decreased activation of ABCA1 and impaired lipidation of apoE4 and that the downstream brain-related pathology and cognitive deficits can be counteracted by treatment with the ABCA1 agonist CS-6253. These findings have important clinical ramifications and put forward ABCA1 as a promising target for apoE4-related treatment of AD.

Novel Apo E-Derived ABCA1 Agonist Peptide (CS-6253) Promotes Reverse Cholesterol Transport and Induces Formation of preß-1 HDL In Vitro.

Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from the carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preß-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preß-1 HDL with increase in the cycling of apo A-I between the preß and α-HDL particles in-vitro. These mechanisms are potentially anti-atherogenic.