BET inhibitor downregulates components of complement cascade
Downregulation of the Complement Cascade In Vitro, in Mice and in Patients with Cardiovascular Disease by the BET Protein Inhibitor Apabetalone (RVX-208)
Apabetalone (RVX-208) is an epigenetic regulator that affects transcription, thereby modulating pathways that underline cardiovascular disease (CVD), such as reverse cholesterol transport, vascular inflammation, coagulation and complement.
The complement system is an essential component of the innate immunity and a major initiator of host inflammatory responses. This system consists of more than 30 proteins, of which some are secreted into the bloodstream by hepatocytes and others associate with the plasma membrane of many other cell types. It can be activated through 3 distinct pathways: the classical, lectin and alternative pathway, each ending into the C5-driven pathway. These pathways can also be downregulated by multiple proteins. Complement expression and activity are tightly regulated to avoid immune dysregulation and host tissue damage . Dysregulation is associated with increased susceptibility to infectious diseases and non-infectious disorders with an autoimmune and chronic inflammatory component, such as cardiometabolic disease including atherosclerosis, diabetes, metabolic syndrome and acute coronary syndrome .
RVX-208 is an orally available experimental small molecule that targets bromodomain and extraterminal (BET) proteins BRD2, BRD3 and BRD4, which regulate epigenetics. It is developed to treat CVD . In contrast to the pan-BET inhibitors that preferably bind to BD1 and BD2 domains of BET proteins, RVX-208 rather binds to BD2 . Post hoc analyses of pooled data from phase IIb SUSTAIN and ASSURRE trials showed that RVX-208 enhances HDL-c and ApoA-1, while lowering the incidence of MACE in CVD patients .
In this current study, the beneficial effects of RVX-208 beyond the improvement of the lipid profile were investigated. For this, in vitro experiments were performed using primary human hepatocytes or cell lines as well as in vivo experiments in urokinase-type plasminogen activator (uPA)/severe combined immunodeficient (SCID) mice with livers repopulated with human hepatocytes. Furthermore, proteomic analyses, including complement components, were performed using plasma from coronary artery disease (CAD) patients treated with 200 mg RVS-208 daily (n=77) or placebo (n=72) in the ASSERT and ASSURRE trials.
In vitro experiments
- Comparing microarray data from primary human hepatocytes from donor child 1 treated in vitro with RVX-208 (48hrs) and these of adult donor 2 to vehicle, revealed the complement system as one of the top canonical pathways affected (downregulated) by RVX-208 (using Ingenuity pathway analysis).
- Not only complement cascade components were affected but also complement activators, including soluble PRRs CRP, serum amyloid P (APCS), and collectin-11 (COLEC11); coagulation cascade components factor II (thrombin), factor IX (F9), factor X (F10), factor XI (F11), factor XII (F12), and kallikrein B1; complement inhibitors, including factor I (CFI), factor H (CFH), C4-binding protein (C4BPA and C4BPB), and carboxypeptidase N (CPN) were downregulated.
- Similar changes in expression profile were observed upon treatment with JQ1, another BET inhibitor with a chemical scaffold distinct from RVX-208.
- Central complement genes were confirmed with real-time PCR in three primary human hepatocyte donors, and downregulation of MBL2, C3, C4, C5 and C9 were dose- and time-dependent. Protein analyses showed similar results.
- Also in Huh-7 hepatocarcinoma cells, RVX-208 treatment suppressed MBL2, C1S, C3, C4 and C5 gene expression, as well as dose-dependent C3, C4 and C5 proteins. Downregulation of complement expression in Huh-7 cells and primary human hepatocytes was also observed for JQ1 treatment.
- IL-6 and INFγ treatment of primary human hepatocytes induced C1s, C2 and C4 gene expression and IL-6 also induced MBL2, C3, C5 and C9 expression. This cytokine-induced complement expression was reduced when co-treating these cells with RVX-208. On protein level, C3, C4, C5 and C9 expression were alleviated with co-treatment.
- In Huh-7 cells, co-treatment with RVX-208 strongly repressed cytokine-induced expression of MBL2, CIS, C2, C3, C4 and C5, often towards baseline or below.
In vivo experiments
- C4, C9 and MBL2 liver gene expression were significantly reduced by 36, 45 and 61% respectively, upon treatment of mice with 150 mg/kg bid RVX-208.
Patient plasma protein expression
- Based on a protein assay (~40 proteins/activated fragments that compose or regulate the complement cascade), multiple cascade components (factor B, C2, C3, C4, C5, C6, C8, C9), cascade inhibitors (factor I, CD55, C1INH, vitronectin) and cascade activators (CRP, APCS, collectin-11, properdin) were significantly (P<0.05) or trending towards significance (P<0.1) downregulated upon treatment with RVX-208 compared to placebo.
- Moreover, also a substantial reduction of cleaved complement fragments that reflect complement activation, was observed, including C3b (32.2%, P=0.06, C5a (51.4%, P=0.0001) and C5b-C6 (10.4%, P=0.002).
BET inhibitor RVX-208 shows a repressive effect on expression of multiple complement cascade components and regulators. As these effects were also observed with a different BET inhibitor, JQ1, this seems to be BET inhibitor-specific rather than RVX-208-specific. This was also observed when complement was induced upon cytokine treatment. In vitro and in vivo experiments were confirmed with CAD patient analyses, which showed a reduction in circulating levels of multiple complement components and regulators with RVX-208, versus placebo. Thus, in addition to improving the lipoprotein and inflammatory profile, RVX-208 may benefit CVD patients by modulating the complement cascade.