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BET Epigenetic Reader Proteins in Cardiovascular Transcriptional Programs

Originally publishedhttps://doi.org/10.1161/CIRCRESAHA.120.315929Circulation Research. 2020;126:1190–1208

    Abstract

    Epigenetic mechanisms involve the placing (writing) or removal (erasing) of histone modifications that allow heterochromatin to transition to the open, activated euchromatin state necessary for transcription. A third, less studied epigenetic pathway involves the reading of these specific histone marks once placed. The BETs (bromodomain and extraterminal-containing protein family), which includes BRD2, BRD3, and BRD4 and the testis-restricted BRDT, are epigenetic reader proteins that bind to specific acetylated lysine residues on histone tails where they facilitate the assembly of transcription complexes including transcription factors and transcriptional machinery like RNA Polymerase II. As reviewed here, considerable recent data establishes BETs as novel determinants of induced transcriptional programs in vascular cells, like endothelial cells and vascular smooth muscle cells, cardiac myocytes and inflammatory cells, like monocyte/macrophages, cellular settings where these epigenetic reader proteins couple proximal stimuli to chromatin, acting at super-enhancer regulatory regions to direct gene expression. BET inhibition, including the use of specific chemical BET inhibitors like JQ-1, has many reported effects in vivo in the cardiovascular setting, like decreasing atherosclerosis, angiogenesis, intimal hyperplasia, pulmonary arterial hypertension, and cardiac hypertrophy. At the same time, data in endothelial cells, adipocytes, and elsewhere suggest BETs also help regulate gene expression under basal conditions. Studies in the cardiovascular setting have highlighted BET action as a means of controlling gene expression in differentiation, cell identity, and cell state transitions, whether physiological or pathological, adaptive, or maladaptive. While distinct BET inhibitors are being pursued as therapies in oncology, a large prospective clinical cardiovascular outcome study investigating the BET inhibitor RVX-208 (now called apabetalone) has already been completed. Independent of this specific agent and this one trial or the numerous unanswered questions that remain, BETs have emerged as novel epigenetic players involved in the execution of coordinated transcriptional programs in cardiovascular health and disease.

    BETs and the Reading of the Epigenetic Code

    The term genetic code has reached common parlance—a broad reference to universal rules that explain how DNA sequence defines protein structure, function and hence, phenotype.1 The genetic code’s power is manifest with disease-causing single DNA nucleotide variants and increasingly revealed, as in large population genome-wide studies linking DNA variants to distinct clinical conditions, including cardiovascular disease.2,3 Advances in understanding DNA since the double helix structure 66 years ago also enabled the intense, ongoing work into how noncoding DNA sequences, like promoters and enhancers control gene expression.4

    The genetic code offers context for the more recent recognition of a distinct, elaborate, highly regulated system that governs transcription but independent of (from the Greek, epi- or above) gene sequence that governs transcription: the epigenetic code. The massive DNA expanses present in every nucleus creates a space problem, one that is solved by DNA compaction—tightly winding chromosomal material around spool-like histones.5 However, transcription requires histone-wound DNA, or euchromatin, to open up and transition to chromatin, which allows transcription factors (TFs) and transcriptional machinery, like RNA Polymerase II (RNAPII), to access relevant DNA regulatory regions (Figure 1). Chemical modifications of specific amino acid residues on histone tails can help enable or repress gene expression. In simple but helpful terms, epigenetics refers to enzymatic processes that write (place), as achieved through methylation or acetylation of lysine, or erase (remove) as with de-methylation or de-acetylation of these same histone marks. Similarly, epigenetic modifications in the DNA methylation at the 5′ carbon on the pyrimidine ring of cytosine nucleotides can also be observed as an important mechanism that controls gene expression without affecting DNA sequence. The prospects entailed by understanding and controlling transcription through studies on histone and DNA modifications have received extensive attention.6 However, a third critical process exists in epigenetic communication via chromatin remodeling—the reading of histone marks once placed.7 Although most attention has focused on enzymatic placement or removal of histone marks, the essential role for epigenetic reader proteins in executing gene expression has often been overlooked, including in the vasculature.8 By binding to modified histone tails, epigenetic reader proteins (Table 1) allow for the engines of transcription, like RNAPII, and specific transcription drivers, like master proximal TFs—which often control the regulatory activity of other TFs and related genes—to be coupled in the regulatory regions of defined gene cassettes. The bromodomain extraterminal (BET)-containing family of epigenetic reader proteins, including BRD2, BRD3, and BRD4, and the testis-restricted BRDT (referred to as BETs throughout), provides a robust example of how epigenetic reader proteins can orchestrate transcriptional programs, provide new insight into physiological and pathological cellular states and offer potentially novel therapeutic strategies. This compendium review considers the recent and rapidly expanding evidence for BETs in vascular biology and atherosclerosis, after beginning with a conceptual framework for epigenetic reader proteins as a prelude to considering basic BET biology.

    Table 1. Summary of Principal Epigenetic Reader Proteins Grouped by Chromatin Modification and Protein Binding Site Domain

    ModificationSites of ModificationBinding DomainProtein Reader
    Acetylated histonesH3 (K9, K14, K18, K27, K56, K122), H4 (K5, K8, K13, K16)BDsBRD2, BRD3, BRD4, BRDT
    Methylated CpGCpGMBDMeCP-2, MBDs1-6
    MBD/SETSETDB-1, SETDB-2
    MBD/DDT/PHD/BDBAZ2A/B
    Methylated histonesH3K4ChromoCHD-1
    PHDBPTF, TAF-3, ING-4, CFP-1, SPIN-1, PHF-2/23, PYGO-2
    TudorSgf-29
    H3K9ChromoHP-1α/β/γ
    TudorUHRF-1
    H3K27ChromoCBX-7
    WD-40EED
    BAHBAHD-1
    PWWPNSD-2
    H3K36PWWPDNMT-3A, LEDGF, ZMYND-11
    ChromoMRG-15
    NBS-1, Ku-70
    H3K79TudorTP53BP-1
    H4K20TudorTP53BP-1
    MBTL3MBTL-1
    BAHORC-1
    WD repeatORCA
    PWWPPDP-1

    BAH indicates bromo-adjacent homology; BAHD-1, bromo adjacent homology domain-containing protein 1; BAZ-2A/B, bromodomain adjacent to zinc finger domain 2A/B; BPTF, bromodomain PHD finger transcription factor; CBX-7, chromobox 7; CFP-1, CxxC zinc finger protein 1; CHD-1, chromodomain helicase DNA binding protein 1; DDT, domain; DNMT-3A, DNA methyltransferase 3 alpha; EED, embryonic ectoderm development; HP-1α/β/γ, heterochromatin protein 1 α/β/γ; ING-4, inhibitor of growth 4; Ku-70, Ku autoantigen 70; L3MBTL1, lethal(3) malignant brain tumor-like protein 1; LEDGF, lens epithelium-derived growth factor; MBD, methyl-CpG binding domain; MBD1-6, methyl-CpG binding domain protein 1-6; MBT, malignant brain tumor domain; MeCP-2, methyl-CpG binding protein 2; MRG-15, MORF-related gene on chromosome 15; NBS-1, nijmegen breakage syndrome; NSD-2, nuclear receptor binding SET domain protein 2; ORC-1, origin recognition complex subunit 1; ORCA, origin recognition complex associated; PDP-1, pyruvate dehyrogenase phosphatase; PHD, plant homeodomain; PHF-2/23, PHD finger protein 2/23; PYGO-2, pygopus 2; SET, su(var)3-9, enhancer-of-zeste, and trithorax; SETDB-1-2, SET domain bifurcated histone lysine methyltransferase 1-2; Sgf-29, SAGA complex associated factor 29; SPIN-1, spindlin 1; TAF-3, TATA-box binding protein associated factor 3; TP53BP1, tumor protein P53 binding protein 1; UHRF-1, ubiquitin like with PHD and ring finger domains 1; WD-40, tryptophan-aspartic acid 40; and ZMYND-11, zinc finger MYND-type containing 11.

    Figure 1.

    Figure 1. The BET (bromodomain and extraterminal) family of epigenetic reader proteins in transcriptional regulation.A, The BET family members BRD2, BRD3, and BRD4, and testis-restricted BRDT, all possess 2 bromodomains BD-1 and BD-2, as well as an extraterminal (ET) domain. BRD4 unique c-terminal domain (CTD) may facilitate its interaction with P-TEFb and RNA Polymerase II (RNAPII). B, BET bromodomain module predicted ribbon structure includes a hydrophobic pocket that binds to acetylated lysine residues on the 5′ end of histone tails. The pan-BETi (BET inhibitors) JQ-1 fits tightly in this pocket, disrupting BETs association with chromatin and regulation of transcription. C, BET action is coupled to epigenetic mechanisms involved in the transition from heterochromatin to euchromatin. Heterochromatin consists of DNA tightly wound around histone spools, which results in DNA compaction but precludes transcription factor (TF) access to regulatory regions of DNA, repressing gene expression. For transcription to proceed, chromatin transitions to the euchromatin state, which opens DNA regulatory regions, allowing access for transcriptional machinery. BETs, in this case, BRD4, binds to acetylated lysines on histone tails and facilitates assembly of TFs and RNAPII. Reprinted from Croken et al157 with permission. Copyright ©2012, Elsevier. D, Upper, Schematic representation of BRD4 binding to a histone tail and allowing for assembly of key players of the transcriptional apparatus, including RNAPII and P-TEFb. Mediator involves 30 protein subunits involved in the preinitiation complex. Lower, In the presence of a BETi, BRD4 histone tail binding is disrupted, preventing BRD4 from its function as a transcriptional scaffold and mediator. Reprinted from Luo et al158 with permission. Copyright ©2012, Springer Nature. E, Browser views of chromatin immunoprecipitation and high throughput sequencing (ChIP-Seq) data in human umbilical vein endothelial cells (ECs) at baseline and after TNF (tumor necrosis factor)-α stimulation serves as an example of BRD4 regulation of vascular cell gene expression. E, Left: Immunoprecipitation of individual proteins amassing at the 5′ end of the chemokine CCL-2 gene body (arrow, chr17:29,622,217). Data are shown in pairs, without (upper) or with (lower) TNF-α stimulation. At the top, in blue, absent TNF-α stimulation, minimal immunoprecipitated p65 is found proximal to the CCL-2 start site. As expected, TNF-α stimulation recruits p65 to the CCL-2 promoter region. In red, BRD4 protein accumulates upstream of the CCL-2 start site after TNF-α stimulation but was not present under basal conditions. In orange, H3K27Ac indicates an activated promoter after TNF-α stimulation. In black, RNAPII is present only after TNF-α stimulation and is evident walking down the CCL-2 gene body, consistent with transcription. JQ-1 treatment of ECs blocks BRD4 and blocks these responses (not shown). E, Right: The ChIP-Seq browser view data for Sox-18 (arrow) is shown. In contrast to CCL-2 and other TNF-α-induced, BRD4-regulated genes, Sox-18 shows BRD4 and RNAPII association under basal conditions that are lost upon TNF-α stimulation.73F, Left: Multiple TNF-α-stimulated proatherosclerotic target genes involve BRD4 action at super-enhancer regions. Right: BRD4 accumulation is shown where TNF-α either increased (red, gained) or decreased (green, lost) BRD4 at respective super-enhancers. Each horizontal line corresponds to a distinct gene, including e-selectin (SELE), VCAM (vascular cell adhesion molecule 1)-1, CCL-2, and Sox-18. Panels E and F reprinted from Brown et al73 with permission. Copyright ©2014, Elsevier. For additional details, see text and associated references.

    Proximal master TFs induce or repress expression of multiple genes, helping explain coordinated transcriptional programs, as seen with NF-κB (nuclear factor-κB) activation directing complex inflammatory programs or PPARs (peroxisome proliferator-activated receptors) directing energy balance. Epigenetic reader proteins provide another distinct way of controlling integrated transcriptional programs, coupling proximal signals to TFs and chromatin marks. The placement of histone marks, and their subsequent reading, can account for biologic memory - cellular recording of stimuli and exposures as a way of facilitating key transcriptional responses, as might be expected in host defenses,9 energy storage,10 or coagulation,11 all being examples of programs essential to survival. Biologic memory may also direct maladaptive responses, as in chronic disease states or understanding the long-term impact of prior cigarette use,12 genetic hypercholesterolemia,13 or gestational diabetes mellitus.14 Epigenetic reader proteins can also help explain gene expression as a means of defining cell state transitions as required in response to dynamic environments involving multiple stimuli. One fundamental cell state change is stem cell differentiation, as master TFs stimulate pluripotent stem cells to acquire a specific biologic identity and function, as typically defined by marker expression.15 Mature cells must also mount functional responses to stimuli, whether mechanical, like hemodynamic pressure, or humoral, as with inflammatory cytokines. Data already implicate epigenetic reader proteins in both stem cell differentiation and mature cell function. Although the focus here is on BET regulation of transcription, which has received more attention in the cardiovascular setting in this nascent area, it is worth noting that other epigenetic reader proteins exist (Table 1), with no doubt data to come regarding their part in vascular responses. Understanding basic aspects of BET biology will facilitate consideration of the evidence for how this specific epigenetic reader protein family directs gene expression in multiple cardiovascular settings and may emerge as a therapeutic target, as has already been explored in clinical cardiovascular trials.

    BET Biology: Directing Transcriptional Choreography

    A bromodomain is a conserved ≈110 amino acid module found in some 60 different proteins in the human genome, including the BET subfamily. The bromodomain structure, characterized by 4 alpha helices (A, B, C, and Z) connected by 2 loops between their helices BC and ZA, is the only protein domain capable of binding to acetylated lysine residues.16 The bromodomain module forms a deep hydrophobic pocket that recognizes and binds to ε-N-acetylated lysines on the 5′ end of histone tails. Although most bromodomain-containing proteins have only one such domain, all 4 BET family members have an extraterminal domain, in addition to 2 tandem N-terminal bromodomains (BD, specifically BD-1 and BD-2). Differential BD affinity among BET inhibitors may determine their functional effects. By binding to acetylated lysine marks, BETs serve as a scaffold for assembling TFs and essential transcriptional machinery, including RNAPII, which occurs at active promoter sites or cis-regulatory elements that can facilitate transcription from a distance. Similar BET actions also produce noncoding RNAs as another means of altering functional responses. High-resolution protein crystallography reveals that the affinity for acetylated lysines among BET family BDs is modest for monoacetylated lysine but increases significantly when multiple acetylated sites exist within a span of one to 5 amino acids.17,18 The accumulation of BRD4 at enhancer elements can account for the dominant transcriptional activity in specific cell states.19,20 BRD2 and BRD3 have also been identified in genomic enhancer regions in cell lines.21–23 The terms super-enhancer or stretch enhancer has been used to refer to this concentrated transcriptional action at discrete cis-regulatory domains that involve BETs, as potential pioneer signals, acetylated histones, DNA-bound TFs and co-activators like mediator 1.19,20 BET localization is also linked to formation of enhancer transcripts, which can use bromodomains cooperatively as docking sites to increase BRD4 affinity for acetylated lysine residues while augmenting other cofactor recruitment and increasing transcriptional activity.24 BET proteins can also interact with acetylated TFs, such as ERG (ETS-related gene) and NF-κB, through BD-dependent and independent mechanisms, a potential contributor to context-specific effects of BET inhibition. Indeed, suppressing BETs action represses downstream targets genes while DNA-bound TFs remain unaffected.25 Although beyond the focus of this review, BETs may also exert nontranscriptional effects, for example, in DNA stress responses.26,27

    BET action in controlling gene expression depends on their function as both scaffolds for localizing transcriptional machinery and as players that actively help initiate transcription and RNA production. Both BRD4 and BRDT include a c-terminal domain, which facilitates transcription activation and elongation by recruiting and interacting with the P-TEFb (positive-transcriptional elongation factor b), a complex composed of the CDK-9 (cyclin-dependent kinase 9) and a regulatory subunit cyclin T1 or T2.28,29 In this regard, BETs, and their role in cardiovascular gene expression, can be understood as integral to complex, fundamental transcriptional mechanisms that continue to be uncovered. After RNAPII promoter recruitment, the multiprotein preinitiation complex forms, which include the GTF (general transcriptional factors) and TFII (transcriptional factor human II)—composed of 10 subunits.30 This complex not only allows RNAPII to initiate transcription but also establishes the pause state, in which recruited, promoter-associated RNAPII halts transcription and RNA elongation when phosphorylated on serine 5 by the CDK7 (cyclin-dependent kinase 7) subunit.31 RNAPII pausing is regulated mainly by 2 complexes: DSIF (DRB sensitivity-inducing factor) and NELF (negative elongation factor).32 RNAPII release depends on DSIF, NELF, and P-TEFb-induced RNAPII phosphorylation on serine 2, which in turn activates transcriptional elongation at the promoter-proximal region,33 a process referred to as pause release.34 BRD4 association with P-TEFb is essential for RNAPII activation since this interaction stimulates P-TEFb kinase activity, and prevents P-TEFb interaction with 7SK/HEXIM, a ribonucleoprotein that segregates P-TEFb into its kinase-inactive form.35 BRD4 may also promote elongation in a P-TEFb-independent manner since acute BRD4 degradation abrogates RNAPII serine 2 phosphorylation and subsequent elongation, without altering genomic occupancy of P-TEFb catalytic subunit CDK-9.36 BRD4 may also phosphorylate RNAPII at serine 2, through an atypical kinase activity, further contributing to transcription initiation.35 Thus, BRD4 is critically involved in several steps that rapidly increase transcription after signal-induced activation is triggered, as with activation, recruitment, and execution of TF programs.

    More recent studies have extended mechanisms through which BETs can regulate transcription. The extraterminal domain in BRD4 may also promote gene expression by independently recruiting histone modifiers that increase transcriptional activity, including the lysine methyltransferase NSD-3 (nuclear receptor binding set domain protein 3) and the arginine demethylase JMJD-6 (jmjC domain-containing protein 6).37,38 Furthermore, the extraterminal domain can also interact with ATP-dependent chromatin modifiers like SWI-SNF and CHD-2.37 These interactions reveal that BRD4 controls gene expression not only by regulating transcription initiation and elongation through RNAPII and P-TEFb, as outlined above, but also by directing these other chromatin modifiers, which can drive chromatin de-compaction when BRD4 occupies active transcription sites. BETs can also regulate gene expression by associating with the mediator complex,39 a group of ≈30 subunits that interact with TFs and helps recruit RNAPII.40 Although the precise interaction site between BETs and the mediator complex is still unclear, several studies indicate that BRD4 and the mediator complex colocalize at several binding sites, including super-enhancers, with this BRD4-mediator interaction stabilizing these proteins at specific genomic locations.41,42

    Recent reports also indicate those BET family proteins, and in particular, BRD4, may exhibit intrinsic HAT (histone acetyltransferase) activity, providing another means for BETs to direct gene expression. Devaiah et al24 reported that different lysine residues in histone H3 and H4 can be directly acetylated by BRD4 through an activity distinct from classical HAT proteins. Furthermore, these authors identified that BRD4-mediated acetylation on H3K122 leads to nucleosome eviction and chromatin de-compaction, thus increasing gene transcription.24 Tian et al43 also found intrinsic BRD4 HAT activity and its induction of transcription, observing that pharmacological BRD4 inhibition reduced acetylation of H3K122 and decreased viral-induced airway inflammation. In addition, BRD4 HAT activity and its extraterminal domains have been reported to regulate RNA splicing by interacting with HnRNPM, a key alternative splicing regulatory factor, with BRD4 interacting with alterative exons independent of acetylated lysine binding.44 Although this rapidly expanding data continues to unfold, the evidence establishes BETs as potent determinants of transcription, by bringing master TFs and essential transcriptional machinery like RNAPII and P-TEFb to specific histone sites with acetylated lysine residues. Moreover, BET action in the choreography of transcription is multifaceted—coordinating localization, allowing interaction among the assembled players, and exerting activity that all help promote gene expression at these epigenetically determined sites.

    BET Inhibitors: Chemical Tools, Clinical Promise?

    Insight into the role of BETs on gene expression and subsequent cellular effects has been greatly advanced by the discovery and development of small molecule BETi (BET inhibitors; Table 2).45,46 Although these compounds have reached clinical development for therapeutic use in various settings, including cardiovascular disease, as further discussed below, they served initially as valuable chemical tools for probing BET function, as evident in studies in models of atherosclerosis and other cardiovascular diseases. BETi was first discovered employing chemical shift mapping and nuclear magnetic resonance from the BD acetyl-binding pocket structure in complex with putative ligands.59 Although the first molecules exhibited low affinity for the BD pocket, they provided proof-of-principle that BDs from the BET family could be inhibited.60 Subsequently, several small molecules with higher BETs BD affinity were identified, in part, through further chemical design based on earlier BET ligand structures.61 The thienotriazolodiazepine JQ-1 exhibits a shape that is highly complementary to the tandem BDs in BRD2, BRD3, and BRD4, binding to the conserved asparagine residue present in their hydrophobic pocket. As such, JQ-1 mimics the interaction between BETs and the acetyl-lysine on histones.62 By binding at this location, JQ-1 displaces BETs from its location on histone tails, disrupting the assembled transcriptional scaffolding and blocking expression of BET-controlled target genes. Other chemical probes that inhibit BETs, like I-BET762 were also developed but identified through other means, as discussed below. Unlike JQ-1, I-BET762 has a dimethylisoxazole ring scaffold that serves as an acetyl-lysine mimic.63 Further optimization of I-BET762 yielded I-BET151, which showed enhanced pharmacokinetics and a longer terminal half-life for use in in vivo studies.54 RVX-208, now called apabetalone, is a BETi in clinical trials for cardiovascular benefit, as discussed below.64 Finally, several different distinct BETi structures have been identified that are either in preclinical or clinical development for studying and treating cancer.65

    Table 2. Summary of Drugs Targeting BET Proteins

    ClassCompoundSelectivityStageIndicationReference
    BET inhibitorsJQ-1BD-1 and BD-2 from BRD2/3/4 and BRDTPreclinicalHematologic malignanciesAbedin et al47
    Lung cancerGao et al48
    Breast cancerOcana et al49
    Prostate cancerAsangani et al50 and Tan et al51
    Colon cancerZhang et al52
    Hepatocellular cancerLi et al53
    RVX-208 (RVX000222)BD-2 > affinity BRD2/3; BD-2 < affinity BRD4Phase IIDyslipidemia, coronary artery diseaseNCT01423188; NCT01067820
    Phase IIDiabetesNCT02586155
    Phase I/IIFabry diseaseNCT03228940
    I-BET 762 (GSK525762)BD-1 and BD-2; BRD2/3/4 and BRDTPhase INeoplasmNCT01943851
    Phase ICarcinoma, MidlineNCT01587703
    I-BET 151 (GSK1210151A)BD-1 and BD-2; BRD2/3/4 and BRDTPreclinicalMixed-lineage leukemiaDawson et al54
    MelanomaGallagher et al55
    MyelomaChaidos et al56
    MK-8628/OTXO15BRD2/3/4 not tested for BRDTPhase INUT midline carcinomaNCT02259114
    Triple-negative breast cancer
    Castrate-resistant prostate cancer
    Lung cancer
    FT-1101BRD2/3/4 and BRDTPhase IAcute myeloid leukemiaNCT02543879
    Non-Hodgkin lymphoma
    CPI-0610BD-1 of BRD2/4 and BRDTPhase IMultiple myelomaNCT02157636
    ABBV-075BRD2/3/4Phase IBreast cancerNCT02391480
    Prostate cancer
    Non-Hodgkin lymphoma
    Acute myeloid leukemia
    Multiple myeloma
    BMS-986158UndisclosedPhase I/IIAdvanced tumorsNCT02419417
    BET degradersBET-PROTAC (ARV-771)BRD4PreclinicalProstate cancerRaina et al57
    Non-Hodgkin lymphomaSun et al58
    BET-PROTAC (ARV-825)BRD2/3/4PreclinicalNon-Hodgkin lymphomaSun et al58

    The compounds are listed according to their (1) mechanism of action in blocking BETs function; (2) selectivity for BET family proteins; (3) stage of research; and (4) condition or diseases for which they are being studied. BET indicates bromodomain extraterminal.

    Relevant to BETi, the BET bromodomains BD-1 and BD-2 may exhibit differential functions, including distinct cellular responses to agents with BD-selective versus equal BD targeting as well as individual preferential BET inhibition. Most BETis, including JQ-1, ABBV-075, and OTX015, target both BDs equally, as JQ-1 does while RVX-208 is reportedly BD-2-selective.66 Deeper understanding about BD selectivity might improve clinical efficacy and tolerability.67 Increased antitumor activity and reduced gastrointestinal toxicity was reported with the more BRD4-specific BD-2-selective inhibitor ABBV-774 as compared to equal BD inhibition.68 For RVX-208, whose BD-2-selectivity may be greater for BRD2 and BRD3 than BRD4, decreased potency was seen in cancer cell lines compared to JQ-1.66 As discussed subsequently, BD selectivity is implicated in vascular cell biology69 and may be relevant to RVX-208 clinical effects. Further investigation is needed to clarify specific BD function, distinct BETs family member roles, and specific inhibitor pharmacology to optimize BETi approaches.

    More recently, the system of proteolysis targeting chimerics (PROTACs) has been applied to BETs to develop a completely novel class of BETi known as BET-PROTACs—ligands that target specific BETs for proteolytic degradation.70 These heterobifunctional molecules possess 2 binding regions connected by a linker. One ligand binds specifically to the target protein of interest, as with JQ-1 interacting with BETs while the other binding region recruits a modifier protein like the E3-ubiquitin ligase, which causes polyubiquitination of the target protein and proteasomal degradation. PROTACs can be designed for BET protein specificity, for example, degrading all BETs, as with BET-PROTAC ARV-771 and ARV-825 or targeting mainly BRD4, as with BD-2-selective MZ-1 and AT-170 or BD-1-selective dBET-57 and dBET-23.58 Recent studies indicate that lower doses of BET degraders might improve therapeutic responses compared with BETi. The BET-PROTACs ARV-825 and ARV-771 showed enhanced antitumoral activity in vitro and in vivo, respectively, as compared to BETi, which paralleled changes in global transcription.71 Completely distinct responses between BET degraders versus BET inhibitors establish the need for further study.72 Despite impressive preclinical tumor model effects, no BET-PROTACs have reached clinical trials, although their potential use continues to be pursued and potentially relevant for cardiovascular disease. The interest in harnessing BET action for therapeutic use in the cardiovascular setting derives from preclinical data seen with BET action and inhibition in endothelial, vascular smooth muscle cells (SMCs), leukocytes, and myocardial settings.

    BETs in the Endothelium

    BET family members vary in their relative endothelial expression in mouse and human endothelial cells (ECs). We reported siRNA knockdown of BRD2, BRD3, or BRD4 expression in HUVECs decreased TNF-α (tumor necrosis factor α) induced gene expression, with BRD4 as the key player in this response. Given embryonic lethality of BET deficiency, JQ-1 was used to investigate BET effects in vitro and in vivo, with this chemical strategy also offering the advantage of avoiding effects that might occur with genetic BRD4 deficiency from birth.73 JQ-1 selectivity and specificity for BRD2, BRD3, and BRD4 among other bromodomain-containing proteins has been established.62 Treatment of ECs with either JQ-1 or the BETi I-BET blocked TNF-α induction of multiple proinflammatory and proatherosclerotic endothelial genes like VCAM-1 (vascular cell adhesion molecule 1) and functional EC responses such as vascular adhesion and trans-endothelial migration of leukocytes in vitro and ex vivo.73 As these findings would predict, administering BETi in vivo decreased atherosclerosis in the LDL (low-density lipoprotein)-receptor deficient mouse model.73 Similar effects were seen with RVX-208 treatment of ApoE-deficient mice.74 BET regulation of endothelial responses prompted consideration of global gene expression as well as genome-wide analysis of BET association and action in the endothelial transcriptome using chromatin immunoprecipitation and high throughput sequencing (ChIP-Seq).75 ChIP-Seq involves stimulating cells before immunoprecipitating relevant proteins, like BRD4 and RNAPII, and the DNA fragments attached to these proteins; subsequent massive parallel sequencing and alignment of the DNA coimmunoprecipitated with these proteins fixes where that protein, like BRD4, was located across the entire genome under defined conditions.

    Applying global ChIP-Seq approaches to BRD4 in human ECs yielded insights relevant to BET action in vascular biology, atherosclerosis, and other cardiovascular settings. First, after cytokine stimulation, BRD4 facilitated expression of multiple proinflammatory and proatherosclerotic targets involved in pathways like thrombosis, leukocyte adhesion, and endothelial barrier function, thus identifying that BRD4 helps orchestrate a coordinated transcriptional program relevant to atherosclerosis. Second, BRD4 activity was concentrated at specific regulatory super-enhancer regions. This BET-restricted pattern of gene expression suggests the complex state like atherosclerosis may be reduced to a more tractable gene set, as proposed for BRD4 in multiple myeloma.41 Of note, relatively little overlap existed between BRD4-associated super-enhancers in macrophages and ECs.73 It is worth noting that the gene identified as regulated by a given BRD4-associated enhancer is a predictive exercise, typically based on the nearest gene’s start site. Given transcriptional regulation over significant distances, the predicted transcriptional regulation of any specific gene requires more direct confirmation. In response to TNF-α, BRD4 location aligned closely with p65 across the genome, strongly supporting BRD4 as coupling this key NF-κB component to chromatin and the execution of the TNF-α transcriptional program. ChIP-Seq data also revealed that while TNF-α stimulation induced BRD4 chromatin association and increased expression of canonical proinflammatory, proatherosclerotic endothelial gene targets, for example, VCAM-1, BRD4 association and expression of another distinct set of endothelial genes were lost in response to this inflammatory cytokine, for example, Sox-18 (sex-determining region Y-box 18; Figure 1). This finding implicates BRD4 in basal EC gene expression and that TNF-α stimulation prompts BRD4 redeployment to newly formed enhancers that defines a new inflammatory cell state through this induced transcriptional program (Figure 2). Similar basal versus stimulated regulation by BETs have been seen elsewhere, including adipogenesis.76

    Figure 2.

    Figure 2. BET (bromodomain and extraterminal) epigenetic reader proteins in cell state transitions and cellular identity. BETs are known to direct stem cell differentiation, cellular identity, and transitions in mature cell states, as relevant in the cardiovascular system. Endothelial cell (EC) changes are used here to illustrate concepts applicable to BETs in other vascular and inflammatory cells. Multiple inputs, including physical forces like pressure, wall stress, fluid dynamics, and chemical stimuli, for example, inflammatory cytokines, stimulate cellular responses that include chromatin remodeling. BETs link these inputs to coordinated transcriptional programs by serving as a recruited scaffold for transcriptional mediators, for example, RNAPII. BET involvement in stem cell differentiation may influence mature vascular and inflammatory cells characteristics, function, and identity. In response to inputs, mature ECs undergo dynamic cell state transitions as defined by distinct expression patterns, as with resting vs inflammatory stimulated endothelium. BETs govern transcriptional programs at rest and after stimulation, as shown in response to cytokines, VEGF (vascular endothelial growth factor) and conditions like hypoxia. Persistent or recorded, via histone marks, exposures may direct pathological, maladaptive BET-regulated transcriptional programs, promoting chronic conditions like atherosclerosis. BETs are also involved in changes in mature cell identity, as shown for endothelial mesenchymal transition. Although BET mechanisms must be able to be terminated or reset, including removal of histone lysine acetylation marks, much less is known about these potentially protective processes. Similar cell identity and state issues apply to BET action in smooth muscle cells, leukocytes, and cardiomyocytes, each with their own distinctive stimuli and cell states, as well as other settings, including adipogenesis. References as per text.

    These BRD4 actions relate to other studies identifying BET involvement in endothelial biology. BETs help coordinate VEGF (vascular endothelial growth factor) responses in angiogenesis and hypoxia, effects first identified in oncology. For example, BET inhibition was found to decrease hypoxia’s potent induction of carbonic anhydrase IX expression, which may promote tumor heterogeneity predict worse therapeutic responses,77 as well as limit vascularization and tumor growth in childhood sarcoma and breast cancer models.78,79 BET inhibition in HUVECs (human umbilival vein endothelial cells) through JQ-1 treatment or shRNA to BRD2 or BRD4 repressed VEGF-mediated angiogenesis and vascular permeability, decreasing endothelial nitric oxide activation and phosphorylation of VEGFR-2 (VEGF receptor 2) and PAK-1 (p21-activated kinase 1) but without altering VEGFR-2 expression while in vivo, JQ-1 decreased Matrigel angiogenesis and retinal neovascularization.80 In detailed studies, Pu et al identified that VEGF stimulation increased ETS-1 (V-Ets avian erythroblastosis virus E26 oncogene homolog 1) acetylation, prompting its association with BRD4 and the induction of various genes that promote angiogenesis.81

    ECs may be a unique setting that offers insight into BET action. The endothelium must rapidly and effectively transduce multiple stimuli, whether sensing mechanical forces, detecting a loss of vascular integrity to initiate coagulation, marshaling inflammatory responses, or any one of the other known roles for this dynamic organ. The endothelium is essential for initiating and often propagating such responses in both health and disease. The EC response to these stimuli represents cell state transitions. Cell state changes can also be understood as contributing to pathogenesis, as with chronic inflammation, hypertension, and many other scenarios. BETs facilitate the rapid execution of transcriptional programs that define these cell states, including their part in reading epigenetic marks recorded from prior exposures. From this perspective, the failure to reset BET action can help explain chronic disease states and highlights the importance of missing insight into how BET signals are terminated.

    BETs in Vascular SMCs

    Despite SMCs constituting the integrity, strength, and contractility of the vascular wall, even highly differentiated SMCs demonstrate remarkable plasticity, reactivity, and contributions to vascular pathology, in keeping with BET-governed cell state transitions.82 Indeed, such shifts in phenotype of cells with a set genome are by definition epigenetic in nature.83 Well-established SMC changes, whether in response to mechanical forces, injury, and cytokine stimulation, which can induce the proliferative intimal hyperplasia (IH) found in atherosclerosis, (re)stenosis and other vascular pathologies, have all been suggested to involve BETs, and in particular, BRD4, as a key determinant of SMC phenotype and pathological states.84,85

    Wang et al86 observed pronounced increased BRD4 expression in the neointima of human artery and vein samples and in the rat carotid artery IH model of balloon angioplasty injury. In cultured rat primary SMCs, the BETi JQ-1(+), but not its inactive enantiomer, abrogated cell proliferation and migration; siRNA to BRD4 but not siBRD2 or siBRD3, as well as JQ-1-containing perivascular hydrogel, recapitulated these SMC effects.86 Further studies suggest BET inhibition can have unique effects in vascular cells. For example, in balloon-injured rat carotid arteries, JQ-1 delivered intravenously via biomimetic, platelet membrane-coated nanoclusters decreased IH without impairing re-endothelialization of the injury site while similarly delivered rapamycin also reduced IH but decreased re-endothelialization,87 consistent with BET inhibition protecting against EC growth impairment, inflammation, and apoptosis.73 The effects seen with BET inhibition suggest this as a means of circumventing the offsetting prothrombotic EC effects of current rapamycin- or paclitaxel-eluting stents.88

    BETs as epigenetic reader proteins orchestrating transcriptional programs may involve another dimension especially relevant to the vasculature: intracellular communications. Endothelial function and dysfunction can direct SMC responses in a paracrine and endocrine manner, producing biologically active mediators like nitric oxide, controlling the uptake of metabolites and nutrients like fatty acids and glucose, responding to and releasing inflammatory mediators, all of which can contribute to changes in SMC phenotype. As such, BET effects in either ECs or SMCs may modulate SMC responses. BETs are also involved in another mechanism that spans EC and SMC biology known as endothelial-to-mesenchymal transition (EndMT), a variation of epithelial to mesenchymal transition discussed in oncology and other settings.89 In EndMT, ECs begin expressing mesenchymal markers like smooth muscle alpha actin and TAGLIN (Transghelin), while concurrently losing expression of EC-restricted proteins like CD31—a definitive example of cell state transition. EndMT contributes directly to IH in atherosclerosis90 and vein graft loss91 in humans. BRD4 has been shown to govern EndMT in human and rodent ECs.69,92 Interestingly, in a deeper analysis of bromodomain function, the decrease in IH seen with BET inhibition appeared to depend on the BD-2 but not BD-1 domain of BRD4.69 Although if this effect derived from inhibiting BRD4 in ECs or SMCs could not be established in vivo, these findings provide strong evidence for BRD4 role in SMC phenotypic transitions.93

    Pulmonary arterial hypertension (PAH) is a distinct vascular disease state that has been reported to involve disruption of normal EC and SMC biology, EndMT, and BET involvement.94 Bonnet et al have reported increased BRD4 levels in lungs and specifically pulmonary artery SMCs (PASMCs) of patients with PAH, with evidence that BET inhibition decreases PASMC proliferation and enhances apoptosis in a BRD4-dependent manner.94,95 Mechanisms proposed for these BET-mediated effects involved decreased expression of three major PAH-associated oncogenes that occurred with BET inhibition or specific siBRD4 knockdown: nuclear factor of activated T cells (Nfatc2), B-cell lymphoma 2 (Bcl-2), and survivin. The increase in BRD4 in PASMCS during PAH was found to depend on microRNA-204, which was a previously reported contributor to PAH. In the established Sugen hypoxia rat model, JQ-1 treatment reversed PAH severity.94 Intriguingly, these same investigators reported that IL-6 (interleukin 6) increased BRD4 expression, which might then promote both PAH and the association between PAH and increased coronary artery disease. Higher BRD4 levels were found in SMCs from both pulmonary and coronary PAH patient arteries while in rat PAH models, IL-6 stimulation increased both BRD4 levels in SMCs and BRD4-dependent proliferation.95 In vivo experiments with JQ-1 or siBRD4 produced similar responses in the rat Sugen hypoxia PAH model. This data prompted the hypothesis that systemic inflammation increases BRD4, propelling SMC proliferation in both pulmonary and coronary arteries and helping explain the increased coronary artery disease observed in PAH.96 BET inhibition as a means of improving PAH is supported by the proposed BETi apabetalone (RVX-208) effects in microvascular ECs and SMCs from patients with PAH and in other preclinical studies as well as pooled data in human studies.97

    While the studies noted above support a key role for BRD4 in SMC proliferation/migration and vascular wall thickening, the underlying molecular mechanisms remained unclear. Natarajan et al97a investigated BRD4 in mediating SMC changes in response to Ang II (angiotensin II). Ang II was found to induce BRD4 activity at enhancers/super-enhancer associated with key cell-signaling TFs, including AP-1 (activating protein-1), ETS, and specific kinases like Jun. This coordinated Ang II–activated SMC transcriptional program was abolished by either specific enhancer deletion or JQ-1 treatment. Long noncoding RNAs that overlapped with the relevant identified enhancer regions, namely lnc-Ang184 and lnc-Ang383, were also found to be involved in Ang II stimulation, an example of the novel concept that long noncoding RNAs may contribute to BET action at specific enhancers. Finally, intraperitoneal JQ-1 treatment of mice ameliorated Ang II–induced hypertension, arterial wall medial hypertrophy, and inflammation. These findings further extend BETs in integrating complex SMC transcriptional programs, linking these epigenetic readers to Ang II–induced gene expression, long noncoding RNAs, and hypertension.

    Other reports identify BET involvement in PAH and in other pulmonary settings like airway SMCs. Chemical library screening for modulators of PASMC proliferation identified emetine, a principal alkaloid extracted from the Brazilian ipecac root in use as an emetic and antiprotozoal inhibited PASMC proliferation, ultimately finding that this occurred through decreased expression of BRD4 and survivin.98 Separate studies report JQ-1 can inhibit airway SMC proliferation,99 inflammation,100 and enhance antioxidant gene expression.101 Whether these effects derived from BRD2, BRD3, or BRD4 remains unclear. While airway SMCs differ considerably from vascular SMCs, these reports may help further identify BET-directed effects and functional responses in vascular SMCs and elsewhere. Early evidence that BET inhibition may limit aneurysm formation and expansion may also involve SMC effects.102

    BETs in Leukocyte Biology: Monocytes, Macrophages, T Cells

    The evidence for BETs in directing pathological responses has included a common theme of BET inhibition decreasing transcriptional programs in inflammation. Such findings build upon earlier studies demonstrating BET involvement in responses to extracellular pathogens and sepsis.63 The evidence for BET coupling TNF-α signaling to NF-κB activation in ECs aligns with and would predict effects in other settings regarding BET inhibition decreasing p65 signaling in other settings, in keeping with the overlap between acute inflammatory responses essential for host defenses and the low-grade, chronic inflammation involved in atherosclerosis.103 Many prior reports established a proinflammatory role for BETs in various in vitro and in vivo pathogenic conditions, as considered in recent reviews.104–106 Herein, we focus mainly on new progress regarding BETs in leukocyte-related function.

    Inflammatory cell modulation by BET inhibition was among the first reports implicating this epigenetic reader protein family outside of oncology. In genome-wide analyses in bone marrow-derived macrophages (BMDM), the the BETi known as I-BET suppressed lipopolysaacharide-induced inflammatory gene expression, including IL-6, IL-1-beta, and chemokines like CXC-9 and CCL-12, all of which are mediators also involved in atherosclerosis.63 Particularly, impressive was the finding that I-BET administration to mice tempered sepsis and improved animal survival. Subsequent reports using BETi or individual BET knockdowns extended these findings to proinflammatory activation or differentiation of monocytes,107,108 macrophages,109–111 T cells,112,113 natural killer cells,114 dendritic cells,115,116 B cells,116 and microglia.117,118 Issues remain, however, including some divergence among studies and individual roles among BRD2, BRD3, or BRD4.

    While the BMDM isolated from hypomorphic heterozygous BRD2-deficient mice (Brd2lo), which have lower BRD2 expression versus wild type, were found to produce less inflammatory cytokines, despite having intact and unchanged BRD3 and BRD4, silencing each BET in vitro in wild-type BMDM reduced inflammatory cytokine expression to a similar extent.119 In another study, the use of a novel PROTAC to delete BRD2 and BRD4 proteins, lipopolysaacharide-stimulated microglia activation was dampened,120 suggesting a limited role for BRD3 in lipopolysaacharide-induced inflammation. In the N9 microglia cell line, siRNA to BRD2 blocked lipopolysaacharide-induced inflammatory cytokine gene expression most potently, with siBRD4 and siBRD3 showing less and no effect, respectively.121 Despite this, BRD3 deficiency in mouse RAW246.7 macrophages inhibited virus-triggered IFN (interferon)-β production.122 Bao et al123 demonstrated that myeloid-specific Brd4 deficiency resulted in resistance to lipopolysaacharide-induced sepsis, with much more marked diminution of inflammatory gene expression in Brd4-deficient versus wild-type BMDMs. Interestingly, more recently, using cell-type–specific BRD4 deficiency and subsequent ChIP-Seq/RNAseq analysis, Dey et al124 found a limited role for BRD4 in BMDM development and lipopolysaacharide-induced inflammation. These investigators reported that IL-1β, TNF-α, and Ccl-5 were actually BRD4-independent, and p65 binding was enhanced rather than decreased after BRD4 depletion. The divergence between these studies and reports in other settings suggests BET action may have cell context-dependency in inflammation, sensitivity to potential variables among specific models and compensatory changes. Indeed, increased BRD2 and BRD3 levels were observed in BRD4-deficient BMDMs.124 The nature of specific inhibitors may also be a factor, given differences among inhibitors regarding preferential BD-1 versus BD-2 targeting. While blocking BD-2 (in all BETs) more effectively repressed lipopolysaacharide-induced inflammation in mouse N9 cells,121 BD-1 was found to exert a more dominant role in the murine oligodendrocyte125 and Th17 cell126 differentiation.

    To date, BET-directed control over proinflammatory responses in leukocytes has been most often attributed to BRD4 associating with the master TF NF-κB73,124 and the P-TEFb transcription elongation complex, which then activates RNAPII.116,124Brd4-deficient mouse BMDM also showed increased IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) expression, which reduced NF-κB binding to inflammatory gene promoters.123 Such counter-regulatory responses have been seen in other BET settings, for example, with TNF-α stimulation of ECs resulting in BRD4-mediated increases in inflammatory target gene expression while also inducing targets that limit NF-κB activation.73 Such findings support BETs as coordinating balanced gene expression to maintain biologic equipoise. In another study, glucocorticoid-receptor activation inhibited BMDM responses by countering p300 and BRD4 recruitment.127 Interactions have also been reported between BRD4 and the TF IRF-1 (interferon regulatory factor-1)124 and between BRD3 and IRF-3/p300.122 Although BRD4 is considered a prominent super-enhancer mediator, BRD4 has been reported to be unnecessary for some lipopolysaacharide-induced super-enhancer function in BMDMs.124 In contrast, others have identified separate yet interdependent BRD2 and BRD4 function in Th17 cell development and adaptive immunity.21 Although BRD2 was involved in localizing STAT3 to active enhancers occupied by TFs IRF-4 and BATF, it was BRD4 that controlled RNAPII pause release, doing so in a temporal manner.21 More recently, BRD4 in thymocytes and T cells was reported to interact with RNA splicing machinery, regulating alternative splicing.44 Using pan-BETi, additional BET-regulated responses have been identified in distinct inflammatory cell responses—increasing autophagy in human macrophages,128 augmenting antioxidant gene expression in a human monocyte cell line101 and inhibiting PD-1/PD-L1 immune-checkpoint responses in human and mouse B cells.129 Although many aspects of these and other BET actions require further delineation, like how specific cis and trans elements perform BET responses and how individual BETs are involved in different leukocyte subtypes, differentiation pathways, and in response to different stimuli, the importance of BETs in leukocyte biology and its relevance to atherosclerosis and other cardiovascular pathologies is already apparent and provides a basis for pursuing such studies.

    BETs in Myocardial Function

    The myocardium is another setting in which cell state changes, shifting cell identities, and even stem cell differentiation have all been invoked, as with cardiomyocyte changes and cardiac remodeling from either pressure or volume overload, in responses to exercise or injury and with induced myocardial fibrosis, among others. We identified BRD4 as regulating key TFs involved in heart failure, as seen in response to phenylephrine stimulation; in vivo, BET inhibition altered cardiomyocyte responses to ventricular pressure overload caused by trans-aortic constriction and prevented cardiac hypertrophy.130 BETs were found to act as pause-release factors for these master myocardial TFs. In response to trans-aortic constriction, BRD4 was involved in TFs controlling expression of myocardial hypertrophy genes with active gene body elongation via RNAPII while in the setting of BET inhibition, these TFs were more often found at transcription start sites but without movement down the gene body, consistent with RNAPII pausing. These BET-mediated shifts controlled cardiac gene expression involved in cytoskeletal reorganization, extracellular matrix production, cell-cycling, cell growth through paracrine and autocrine stimuli and inflammation, all of which are relevant forces in heart failure. Despite BET control of MYC (myelocytomatosis oncogene) signaling in cancer and MYCs established myocardial role, BET action in cardiac myocytes was MYC-independent, further supporting distinct BET effects in different cellular settings. Subsequently, BRD4 was reported to control a fibrotic and inflammatory transcriptional network in different murine heart failure models and in agonist-induced hypertrophic changes in human iPSC (induced pluripotent stem cells)-derived cardiomyocytes.131 BRD4 may control cardiac fibrosis and heart failure by targeting genes that induce quiescent cardiac fibroblasts to convert into cells producing extracellular matrix components through TGF-β (transforming growth factor-β) and p38 MAPK (mitogen-activated protein kinase) expression.132 In subsequent work consistent with BET-mediated regulation of EndMT reviewed earlier, BET inhibition was reported to limit cardiac fibrosis induced by trans-aortic constriction or TGF-β, with effects on EndMT-associated transcription factors, the SMAD pathway and TGF-β receptor I.92 Controversy exists regarding trans-aortic constriction, EndMT and differences among murine models of cardiac fibrosis133 but simply overexpressing BRD4 increased EndMT responses in the absence of TGF-β stimulation, suggesting a fundamental shift towards a profibrotic cell state.

    BETs as a Therapeutic Target for Cardiovascular Diseases

    The prospect of interrupting the execution of a coordinated transcriptional program that underlies complex patterns in diseases like atherosclerosis, PAH, or heart failure is appealing (Figure 3). The relationship between BETs and clinical phenotypes is evident in the finding that NUT midline carcinoma, a poorly differentiated squamous cell carcinoma with multiple abnormalities, derives from a fusion protein involving BRD4 and the NUT gene.134 Although most attention in epigenetic therapeutics has focused on blocking histone mark placement or directing their removal, the prospect of keeping previously placed marks that promote atherosclerosis or other cardiovascular disorders left unread is also compelling. Such an approach may be even more appealing given that histone marks may be placed early in life, even in utero, be long-lived, involve maladaptive responses and not required for normal biologic function. Based, in part, on the evidence reviewed here, clinical therapeutic strategies targeting BETs have been pursued, even reaching largescale prospective clinical cardiovascular trials.

    Figure 3.

    Figure 3. BRD4 controls transcriptional responses in vascular and inflammatory cells. Examples of stimuli, regulated gene targets, and pathological responses involving specifically BRD4 in endothelial cells (ECs), smooth muscle cells (SMCs), monocyte/macrophages (Mo/MPs), and cardiomyocytes as reviewed here are shown. These BRD4 effects have been demonstrated through the use of gene expression, BETi (bromodomain and extraterminal inhibitors) responses and global profiling tools of RNA-Seq and chromatin immunoprecipitation and high throughput sequencing (ChIP-Seq). This data reveals BRD4 exerts broad, integrated transcriptional effects executed in large through its association with super-enhancer regions to determine functional cellular responses and associated disease states. An example that supports BET action as directing transcriptional programs to achieve a coordinated output is evident in BET inhibition repressing NF-κB (nuclear factor-κB)-directed gene expression while concurrently increasing translation of IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), which also decreases NF-κB activity. BRD2 and BRD3 have also been implicated in similar, although often distinct responses relevant to the vasculature. Abbreviations, references as per text.

    Therapeutic BET inhibition has been most explored in cancer, given the BRD4-NUT oncogene and BET involvement in MYC oncogene effects in hematologic malignancies like mixed-lineage leukemia, acute myeloid leukemia, Burkitt’s and Burkitt-like lymphoma.135 Distinct BETi have been developed and are under study in clinical trials for various cancers (Table 2), based on encouraging preclinical and sometimes translational data.136 Some common adverse events have been observed among BETi, including thrombocytopenia, anemia, neutropenia, gastrointestinal issues, increased bilirubin, and fatigue.137 Preclinical studies suggest that combining BETi with other epigenetic modulators may have a synergistic antitumor activity, allowing for lower doses and perhaps fewer adverse events, while other strategies involve combining BETi with immune-modulators and hormone therapy for greater efficacy.138 In terms of cardiovascular responses, BETi trials in cancer will provide insight into the safety and tolerability of specific BET agents and perhaps indirect evidence on cardiovascular responses. One emerging issue is BETi resistance—the induction or existence of counter-regulatory responses that may limit longer-term effectiveness of BET inhibition in cancer treatment.139 Examples include specific AML cell subsets that are resistant to BETi-induced apoptosis, compensatory induction of MYC through WNT signaling and increased post-translational BRD4 modifications like phosphorylation.140–142 Whether resistance to BET inhibition might influence cardiovascular responses is unknown but BET involvement in basal cellular function, their wide expression and their regulation of genes that offset stimulated TF responses may limit BET efficacy.

    Another intriguing clinical angle has been the discovery that BET inhibition can reactivate latent HIV infection. A significant hurdle in HIV eradication is HIV latency, which is the virus’ ability to maintain itself in cellular reservoirs like T cells, thus avoiding exposure to antiviral therapies.143 Although the mechanism(s) for how BET inhibition activates HIV remains unclear, the hypotheses under study adds insight into how BETs and their inhibitors operate.144–147 HIV replication depends on RNAPII activation, which is induced by the viral Tat protein. Related to earlier mechanistic discussions, after HIV initiates transcription, RNAPII pauses, a consequence of 2 negative elongation factors that inhibit RNAPII activity. For elongation to begin, the P-TEFb complex, consisting of CDK-9 and the regulatory subunit cyclin T1, is recruited, which eliminates the negative elongation factors; the HIV Tat protein recruits P-TEFb. Hypotheses for why BETi may cause HIV re-activation include increases in P-TEFb levels and activity, alterations in T-cell gene expression and removal of BRD4 from its position on the HIV promoter that keeps Tat from allowing transcription to proceed.

    The BETi farthest along as a cardiovascular therapeutic agent is RVX-208 (previously RVX000222) and as noted, now called apabetalone.64 Initially, this quinazolone compound with BD-2-selectivity was identified during small molecule screening for ApoA1 (apolipoprotein A-1) expression inducers.66,148,149 A similar ApoA1 transcription assay screen identified the I-BET inhibitors.150 Interestingly, neither of these screens were designed to identify inhibition of BETs; it remains unclear if increased ApoA1 contributes to RVX-208 or other BETi effects.

    Early studies supported RVX-208 effects on HDL-C (high-density lipoprotein-cholesterol) levels and particle size as well as cholesterol efflux, including in primates.148 RVX-208 decreased atherosclerosis in ApoE-deficient model,74 similar to JQ-1 effects in LDLR-deficient mice.73 RVX-208 decreased atherosclerosis without major lipid level changes, suggesting possible anti-inflammatory effects. The 26-week phase II Apo A-I synthesis stimulation and intravascular ultrasound for coronary atheroma regression evaluation showed RVX-208 failed to significantly decrease the primary end point of percent change in atheroma volume versus placebo although total atheroma volume from baseline declined.151 RVX-208 treated patients had lower inflammatory markers but also elevated liver function tests (7.1% versus 0%, P=0.009). The phase II ASSERT trial (ApoA1 Synthesis Stimulation Evaluation in Patients Requiring Treatment for Coronary Artery Disease) studied 299 statin-treated patients with stable coronary artery disease receiving placebo or one of 4 RVX-208 doses.152 A dose-dependent but not statistically significant increase in ApoA1 levels occurred; HDL particle number was significantly increased (≈1%). RVX-208 was also associated with transient, reversible increases in alanine transaminase and aspartate transaminase, as previously observed, without bilirubin or creatinine changes; adverse effects seen with other BETi were not evaluated. Pooling phase 2 trial data suggested stronger evidence for RVX-208 clinical benefits on death, myocardial infarction, coronary revascularization, or cardiovascular hospitalization (5.9% versus 10.4%, P=0.02), especially in higher-risk patients with elevated hs-CRP (high sensitivity C-reactive protein levels; >2 mg/dL, 5.4 versus 14.2%; P=0.02), lower baseline HDL (5.5% versus 12.8%, P=0.01), and type 2 diabetes mellitus (5.4% versus 12.7%, P=0.02). Given the prior negative intravascular ultrasound data, plaque stabilization could be postulated as a contributing mechanism. A phase 3 trial was undertaken (BETonMACE) testing apabetalone (RVX-208) versus placebo on first major adverse cardiovascular events in high-risk patients with diabetes mellitus, low HDL-C, and a recent (7–90 days) acute coronary syndrome event.153 Carried out at 195 sites in 13 countries, BETonMACE data are not yet published but have been presented.154 These presented results are considered here given relevance to BET mechanisms and further BETi development and clinical trials.

    In BETonMACE, apabetalone showed no significant difference versus placebo on the primary end point (combined cardiovascular death, myocardial infarction, or stroke) in 2425 subjects with T2D, recent acute coronary syndrome, and low HDL-C (10.3% versus 12.4%, hazard ratio of 0.82, P = 0.11) over the average 26-month follow-up.154,155 Participants (mean age 62 years, 26% female) excluded those with a prior or current diagnosis of heart failure, recent coronary bypass (90 days), planned coronary revascularization, advanced kidney disease and all underwent mandatory high-intensity statin run-in (atorvastatin 40 or 80 mg or rosuvastatin 20 or 40 mg). At study initiation, subjects had a median LDL-C 65 mg/dL, HDL-C 33 mg/dL and an A1C of 7.3%. A primary end point MACE (major adverse cardiovascular events) sensitivity analysis showed a hazard ratio of 0.79 (P=0.06). Nominal but insignificant decreases were observed with apabetalone on cardiovascular death and myocardial infarction (9.2% versus 11.5%, P>0.05) and all-cause mortality (5.0% versus 5.7%, P>0.05). While HDL-C increased significantly from baseline (16.2% versus 10.4%, P=0.001) there was no change in hs-CRP. Although involving smaller numbers, prespecified subgroup analyses posted by the sponsor suggests possible greater benefit on heart failure hospitalizations and in those with decreased kidney function and lower LDL-C levels. Adverse events were seen more common in patients receiving apabetalone including discontinuations (114, 9.4% versus 69, 5.7%) and increased liver function tests (78, 6.4% versus 18, 1.5%), all versus placebo. Analysis of BETonMACE and insight into the trial’s implications on BET inhibition as a strategy for atherosclerotic complications awaits full publication. The Food and Drug Administration recently granted Breakthrough Therapy Designation for apabetalone, a status designed for expediting drugs that have preliminary clinical evidence indicating a potential substantial improvement over available therapy on clinically significant end point.156

    More germane to this discussion are the scientific implications from BETonMACE. While clinical trial data analysis and interpretation require defined rigor, mechanistically, the BETonMACE trends suggest BET inhibition may limit cardiovascular events, aligning with preclinical data. Apabetalone is but one chemical entity aimed at BET inhibition; the prospects for improving cardiovascular outcomes by disrupting BET action as an epigenetic reader cannot be reduced to a single agent or trial. Indeed, in oncology, multiple distinct BETi is being pursued in different cancer types. Apabetalone is a less potent and BD-2-selective BETi that may target BRD2 and BRD3 more than BRD4.66 How BD selectivity, potency of BET inhibition or selectivity/potency against individual BET family members translates into clinical cardiovascular efficacy remains unknown. Of particular interest will be how BETonMACE data aligns with the reported basic science effects of BET inhibition, for example, in terms of inflammatory markers/mediators, cardiac hypertrophy/heart failure, and hypertension. Analysis of global gene expression patterns, mediator levels, or ChIP-Seq in patient samples, like circulating leukocytes, is possible but unfortunately not done in BETonMACE. Such deeper analyses could shed light on responders and nonresponders, especially given the obvious complexity of BET biology.

    Independent of this one trial, targeting BETs for cardiovascular benefit entails both great promise and inherent hurdles. The powerful nature of BET action in integrating expression of multiple targets in response to distinct proximal stimuli via an epigenetic mechanism identifies BETs as warranting further study in the cardiovascular system. At the same time, the extent of BET control of transcriptional programs raises cautionary notes. Systemic BET inhibition may cause loss of necessary adaptive responses while evidence for BET activity under basal conditions and their wide expression patterns suggest their involvement in transcriptional programs directing normal physiology. Perhaps more selective BET interventions, like delivery to specific cells or clinical circumstances, or agents with less potent BET inhibition may be necessary. No doubt such paths forward are ultimately dependent on better-understanding BET biology in vascular and inflammatory cells.

    Future BETs: Directions and Conclusions

    The elucidation of epigenetic mechanisms, which operate above the genome, has revealed a completely new dimension to how control over gene expression can dictate phenotypes in health and disease (Figure 3). Chromatin remodeling adds a dynamic component to fixed genotypes, with processes that can record prior exposures, circumstances or stimuli, with functional effects. While the writing and erasing of histone marks require attention in cardiovascular diseases, we now know that the BET bromodomain-containing epigenetic reader proteins are critical determinants of executing this epigenetic code. As evident in the data reviewed here, BETs help orchestrate transcriptional programs involved in the endothelium, SMCs, inflammatory cells and the myocardium, directing differentiation, cellular identity and cell state transitions in response to forces commonly encountered in the heart, lungs, and vasculature.

    Despite exciting progress regarding BET action, essential information is missing about how these epigenetic reader proteins determine transcription in cardiovascular settings. What are the distinct roles of individual BETs, how is that selectivity determined and how does this or other mechanisms account for gene selectivity for BET regulation as well as BET inhibitors? Do changes in expression of BETs themselves impact transcriptional output, as suggested in some vascular settings, or do other forces modulate BET activity or enhancer association? What common features define those genes that do, or do not, involve BET transcriptional control? How do BETs drive basal cellular function in vascular and inflammatory cells? Although BETs bind to acetylated lysines on histone tails, what determines which acetylated lysines interact with BETs and how is this integrated into the 3-dimensional space of chromatin? Perhaps particularly important given the chronicity of cardiovascular diseases, what determines temporal aspects of BET action, including signal termination? These and many other questions are prompted by the startling and relatively recent recognition of BETs as epigenetic reader proteins in coordinating gene expression in the cardiovascular setting. Despite rapid advances in this area, even reaching clinical cardiovascular BETi trials, continued progress will require answers to these and other questions, especially if the predictive and therapeutic potential of BET biology is to be fully realized.

    Nonstandard Abbreviations and Acronyms

    Ang II

    angiotensin II

    ApoA1

    apolipoprotein A-1

    BET

    bromodomain extraterminal

    BETi

    BET inhibitors

    BMDM

    bone marrow-derived macrophages

    CDK-9

    cyclin-dependent kinase 9

    ChIP-Seq

    chromatin immunoprecipitation and high throughput sequencing

    DSIF

    DRB sensitivity-inducing factor

    EC

    endothelial cell

    EndMT

    endothelial-to-mesenchymal transition

    ERG

    ETS-related gene

    GTF

    general transcriptional factors

    HAT

    histone acetyltransferase

    HDL-C

    high-density lipoprotein-cholesterol

    IFN

    interferon

    IH

    intimal hyperplasia

    IL-6

    interleukin 6

    JMJD-6

    jmjC domain-containing protein 6

    LDL

    low-density lipoprotein

    MAPK

    mitogen-activated protein kinase

    NF-κB

    nuclear factor-κB

    NSD-3

    nuclear receptor binding set domain protein 3

    PAH

    pulmonary arterial hypertension

    PASMCs

    pulmonary artery SMCs

    PPARs

    peroxisome proliferator-activated receptors

    PROTACs

    proteolysis targeting chimerics

    P-TEFb

    positive-transcriptional elongation factor b

    RNAPII

    RNA polymerase II

    SMC

    smooth muscle cell

    TFII

    transcriptional factor human II

    TFs

    transcription factors

    TGF-β

    transforming growth factor beta

    TNF-α

    tumor necrosis factor α

    VCAM-1

    vascular cell adhesion molecule 1

    VEGF

    vascular endothelial growth factor

    VEGFR-2

    VEGF receptor 2

    Footnotes

    For Sources of Funding and Disclosures, see page 1204.

    Correspondence to: Jorge Plutzky, MD, Cardiovascular Medicine, Brigham and Women’s Hospital, Harvard Medical School, 77 Ave Louis Pasteur NRB742, Boston, MA 02115. Email

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