J. of Cardiovasc. Trans. Res. DOI 10.1007/s12265-017-9755-z ORIGINAL ARTICLE Downregulation of the Complement Cascade In Vitro, in Mice and in Patients with Cardiovascular Disease by the BET Protein Inhibitor Apabetalone (RVX-208) Sylwia Wasiak 1 & Dean Gilham1 & Laura M. Tsujikawa1 & Christopher Halliday1 & Cyrus Calosing1 & Ravi Jahagirdar1 & Jan Johansson2 & Michael Sweeney2 & Norman C. Wong1 & Ewelina Kulikowski1 Received: 21 December 2016 /Accepted: 17 May 2017 # The Author(s) 2017. This article is an open access publication Abstract Apabetalone (RVX-208) is an epigenetic regulator Abbreviations developed to treat cardiovascular disease (CVD) that targets APCS Amyloid P component serum BET proteins. Through transcriptional regulation RVX-208 BET Bromodomain and extraterminal modulates pathways that underlie CVD including reverse cho- BETi BET inhibitor lesterol transport, vascular inflammation, coagulation, and C4BP C4b-binding protein complement. Using transcriptomics and proteomics we show CAD Coronaryarterydisease that complement is one of the top pathways downregulated by CVD Cardiovascular disease RVX-208 in primary human hepatocytes (PHH) and in plasma CPN Carboxipeptidase N from CVD patients. RVX-208 reduces basal and cytokine- CRP C-reactive protein driven expression of complement factors in PHH and in chi- FB Factor B meric mice with humanized livers. Plasma proteomics of FD Factor D CVD patients shows that RVX-208 decreases complement HDL High-density lipoprotein proteins and regulators, including complement activators FH Factor H SAP and CRP. Circulating activated fragments C5a, C3b, FI Factor I and C5b-C6 are reduced by 51, 32, and 10%, respectively, IL-1 Interleukin-1 indicating decreased activity of complement in patients. As IL-6 Interleukin-6 complement components are linked to CVD and metabolic INFγ Interferon γ syndrome, including major acute cardiac events, modulating MACE Major adverse cardiac event their levels and activity by RVX-208 may alleviate risks asso- MASPs MBL-associated serine proteases ciated with these diseases. MBL Mannose-binding lectin PHH Primary human hepatocytes Keywords Epigenetics . Bromodomain . Bromodomain and PRRs Pattern recognition receptors extraterminal protein . Biomarker . Inflammation . TNFα Tumor necrosis factor α Complement cascade . Innate immunity . Cardiovascular disease; major acute cardiac event Associate Editor Emanuele Barbato oversaw the review of this article Introduction Electronic supplementary material The online version of this article The complement system is an essential component of the in- (doi:10.1007/s12265-017-9755-z) contains supplementary material, which is available to authorized users. nate immunity and a major trigger of host inflammatory re- sponses. As such, it helps immune cells fight infections and * Ewelina Kulikowski maintains homeostasis by eliminating pathogens, immune [email protected] complexes, debris, and apoptotic cells. The complement sys- tem is composed of more than 30 proteins, some of which are 1 Resverlogix Corp., Suite 300, 4820 Richard Road SW, secreted into the bloodstream by hepatocytes, while others are Calgary, AB T3E 6L1, Canada associated with plasma membrane of many cell types. Most of 2 Resverlogix Inc., San Francisco, CA, USA circulating complement proteins participate in activation of J. of Cardiovasc. Trans. Res. the complement cascade, serving as enzymes, enzyme cofac- component. Specifically, excessive complement expression tors, or precursors of biologically active fragments (reviewed and activity are implicated in several aspects of human in [1]; see Fig. 1c). cardiometabolic disease, including atherosclerosis, diabe- Depending on the context the complement system can be tes, metabolic syndrome, and acute coronary syndrome activated through three distinct pathways: classical, lectin, and (reviewedin[4]). alternative, each leading to a common terminal C5-driven path- Apabetalone (RVX-208) is an orally available small mole- way (reviewed in [1]; see Fig. 1c). The classical pathway is cule developed for the treatment of cardiovascular disease activated by C1q which recognizes antigen-antibody com- (CVD) that targets epigenetic regulators bromodomain and plexes, microbial and apoptotic cells, and soluble pattern rec- extraterminal (BET) proteins BRD2, BRD3, and BRD4 [5]. ognition receptors (PRRs) such as the C-reactive protein Mechanistically, RVX-208 precludes interactions of BET pro- (CRP). In association with C1s and C1r, C1q propagates sig- teins with acetylated lysines on histone tails that normally naling through C4 and C2, leading to C3 cleavage and activa- promote active transcription [5, 6]. This inhibition is mediated tion. The lectin pathway is activated by soluble PRR mannose- through binding of RVX-208 to bromodomains 1 and 2 of binding lectins (MBLs), ficolins, and collectins. Subsequently, BET proteins [5, 6]. Of note, RVX-208 binds preferentially activation of MBL-associated serine proteases (MASPs) leads to BD2, which differentiates it from pan-BET inhibitors that to cleavage and activation of C3. Lastly, the alternative path- target BD1 and BD2 with equal affinity [6]. RVX-208 is the way involves a spontaneous cleavage of C3 in conjunction with first BET inhibitor (BETi) to enter human clinical trials for factor B, factor D, and properdin, making C3 an integrator of treatment of chronic disease, and analysis of its in vivo activity all the signals that stimulate the complement cascade. Cleavage is ongoing [7–10]. A post hoc analysis of pooled data from of C3 generates the C3b fragment which in turn cleaves and phase IIb trials SUSTAIN and ASSURE showed that RVX- activates C5, the initiator of the terminal pathway. The cleavage 208 treatment moderately enhances the plasma lipid profile by fragment C5b associates with C6, C7, C8, and C9 to form C5b- increasing high-density lipoprotein cholesterol (HDLc) and 9 on the membrane of target cells, leading to cell perme- ApoA-I while substantially lowering the incidence of major abilization and lysis. Soluble C5b-containing end products of adverse cardiac events (MACE) in CVD patients [10]. These complement activation are eliminated by clusterin and data prompted an investigation of beneficial effects of RVX- vitronectin to avoid unwanted association with non-target 208 beyond the improvement of the lipid profile. Of note, membranes. Cleavage of C3 and C5 not only promotes cell BET inhibition by RVX-208 reduces atherosclerosis and ex- lysis but also contributes to inflammation and leukocyte recruit- pression of inflammatory markers in the ApoE mouse knock- ment through anaphylatoxic activity of the C3a and C5a frag- out model [11 , 12]. In cultured primary human hepatocytes ments. These are inactivated by carboxypeptidase N that RVX-208 modulates expression of genes and pathways that cleaves their C-terminal arginine residue to yield C3a-desArg underlie CVD, including reverse cholesterol transport, vascu- and C5a-desArg. There are multiple proteins which downreg- lar inflammation, coagulation, and complement [10]. Here, ulate complement activity, including regulators of the classical using transcriptomics, real-time PCR, ELISA assays, and pro- and lectin pathways C1 inhibitor and C4b-binding protein, and teomics, we demonstrate that the complement cascade is regulators of the alternative pathway Factor H and Factor I. broadly and coordinately downregulated by RVX-208 in pri- Extensive cross talk between the coagulation and complement mary human hepatocytes (PHH), in mice and in CVD patients. pathways also leads to activation of C3 and C5 via extrinsic Our studies clearly indicate that RVX-208 impacts comple- proteases of the coagulation system (reviewed in [2]). ment protein abundance and activation in vivo, and suggest Specifically, thrombin and activated coagulation factors IXa, that complement repression by RVX-208 may contribute to Xa, XIa, and plasmin can directly cleave C3 and C5 to generate the decreased CVD risk and lower incidence of MACE ob- pro-inflammatory C3a and C5a. Factor XIIa has also been served in RVX-208 clinical trials. demonstrated to initiate classical pathway activation via cleav- age of C1r. Conversely, activated complement fragments C3a, C5a, and C5b-C9 are known to enhance platelet activation and Methods aggregation and promote thrombin generation on the platelet surface and fibrin formation, suggesting that complement acti- Tissue Culture vation may contribute to thrombus formation [3]. Complement expression and activity are tightly regulated Cryopreserved primary human hepatocytes (CellzDirect/Life to avoid immune dysregulation and host tissue damage. Both Technologies) were plated and then overlaid with Matrigel™ inefficient activation and overstimulation of complement can as recommended by the supplier. Cells were treated with com- be detrimental for the host, and is associated with increased pounds dissolved in DMSO (0.1% final concentration) and/or susceptibility to infectious diseases and non-infectious disor- cytokines for the indicated time in media recommended by the ders with an autoimmune and chronic inflammatory supplier supplemented with 10% fetal bovine serum (v/v). J. of Cardiovasc. Trans. Res. ac Complement RVX-208 JQ1 System Rank p-value Rank p-value PHH Donor 1 1/628 4.5E-7 8/628 2.4E-5 PHH Donor 2 22/628 2.0E-4 38/628 2.3E-3 b PHH Donor 1 PHH Donor 2 Gene Gene Name Symbol RVX-208 JQ1 RVX-208 JQ1 mannose-binding lecn (protein C) 2, soluble MBL2 0.09 0.07 0.25 0.25 complement component 9 C9 0.11