Neddylation inhibitor MLN4924 has anti‐HBV activity via modulating the ERK‐HNF1α‐C/EBPα‐HNF4α axis

Abstract Hepatitis B virus (HBV) infection is a major public health problem. The high levels of HBV DNA and HBsAg are positively associated with the development of secondary liver diseases, including hepatocellular carcinoma (HCC). Current treatment with nucleos(t)ide analogues mainly reduces viral DNA, but has minimal, if any, inhibitory effect on the viral antigen. Although IFN reduces both HBV DNA and HBsAg, the serious associated side effects limit its use in clinic. Thus, there is an urgent demanding for novel anti‐HBV therapy. In our study, viral parameters were determined in the supernatant of HepG2.2.15 cells, HBV‐expressing Huh7 and HepG2 cells which transfected with HBV plasmids and in the serum of HBV mouse models with hydrodynamic injection of pAAV‐HBV1.2 plasmid. RT‐qPCR and Southern blot were performed to detect 35kb mRNA and cccDNA. RT‐qPCR, Luciferase assay and Western blot were used to determine anti‐HBV effects of MLN4924 and the underlying mechanisms. We found that treatment with MLN4924, the first‐in‐class neddylation inhibitor currently in several phase II clinical trials for anti‐cancer application, effectively suppressed production of HBV DNA, HBsAg, 3.5kb HBV RNA as well as cccDNA. Mechanistically, MLN4924 blocks cullin neddylation and activates ERK to suppress the expression of several transcription factors required for HBV replication, including HNF1α, C/EBPα and HNF4α, leading to an effective blockage in the production of cccDNA and HBV antigen. Our study revealed that neddylation inhibitor MLN4924 has impressive anti‐HBV activity by inhibiting HBV replication, thus providing sound rationale for future MLN4924 clinical trial as a novel anti‐HBV therapy.


| INTRODUC TI ON
HBV infection is one of the major public health problem with 364 million people being chronically infected by this distinct pathogen worldwide. 1,2 About 25% of population with chronically infection of HBV in childhood develop serious liver diseases, such as hepatitis, cirrhosis and even HCC. 3,4 Nearly 1 million persons die from cirrhosis and HCC secondary to HBV infection each year. 5,6 Current treatment strategy for chronic Hepatitis B (CHB) is to improve life quality and extend survival by slowing the deteriorative process to hepatitis, cirrhosis and HCC. As circulation hallmarks of CHB, high blood levels of viral load (HBV DNA) and HBsAg are responsible for liver cirrhosis and HCC. 7,8 Currently, the standard nucleos(t)ide analogues therapy only decreases HBV DNA and has minimal, if any, inhibitory effect on HBsAg. Although Interferon inhibits both viral DNA and antigen, the side effects associated limit its use in clinic. 1,9,10 Therefore, potential new anti-viral strategies that effectively reduce both HBV DNA and HBsAg levels are highly desirable.
Neddylation, one type of post-translational modifications of a given protein, is catalysed by three sequential enzymatic reactions.
These enzymes are E1 NEDD8-activating enzyme (NAE), E2 neddylation conjugating enzymes and E3 neddylation ligase. In mammalian cells, there is only one E1, containing a heterodimer of regulatory subunit, NAE1/APPBP1, and a catalytic subunit, UBA3/NAEβ; two E2s, UBE2M and UBE2F; and little over 10 E3s, along with limited number of neddylation substrates. 11 A well-characterized physiological substrate of neddylation is a family of Cullin-RING Ligase (CRL), which is the largest family of the E3 ubiquitin ligase, and responsible for ubiquitination of 20% cellular proteins for degradation by proteasome system. 12,13 CRL is a multi-component E3 ligase, consisting of a scaffold protein, cullin (with 8 family members, Cullins 1-3, 4A, 4B, 5, 7 and 9), an adapt protein (such as S-phase kinase-associated protein 1(SKP1)), substrate-recognizing subunit (eg an F-box protein), and a RING protein family member, ROC1/RBX1 or ROC2/RBX2/ SAG. [14][15][16][17][18] Activity of CRLs requires a) the RING component, ROC1 or SAG, which binds to a ubiquitin-loaded E2, and b) cullin neddylation, which prevents inhibitory binding of CAND1. 13,19 MLN4924, also known as pevonedistat, is a small-molecule inhibitor of E1 NAE, discovered 10 years ago. 12 By inhibiting E1, MLN4924 blocks the entire neddylation modification and inactivates all family members of CRLs. 19 As CRLs are frequently overexpressed in many types of human cancers, 19 MLN4924 has shown impressive anti-cancer activity in extensive preclinical settings against a variety of human cancer cells by inducing growth arrest, apoptosis, autophagy and senescence. 11 Currently, MLN4924 is in several phases II clinical trials for the treatment of haematological malignancies and solid tumours, mainly in combination with conventional chemotherapies. 11,12,[20][21][22] Interestingly, in addition to anti-cancer application, MLN4924 has been shown to have broad activity against various viruses, 23 including HIV, [24][25][26][27][28][29][30] influenza A virus, 31 and most recently, HCV, 27 mainly through inactivation of CRLs to cause accumulation of anti-viral proteins.
Two groups reported that MLN4924 has anti-HBV activity with mechanism involving restoration of Smc5/6 protein levels to suppress viral replication. 32,33 However, the detailed mechanism underlying the function of the MLN4924 in silencing HBV replication remains elusive.
In this study, we systematically investigated anti-HBV activity of MLN4924 and the underlying mechanism. We found that in both cell culture and animal models, HBV infection activates cullin neddylation, and MLN4924 effectively suppressed HBV replication and HBsAg production. Our mechanistic study revealed that MLN4924 activates ERK to block expression of transcription factors HNF1α, C/EBPα and HNF4α, which is required for viral replication. Our results suggest the potential use of MLN4924 as an alternative therapeutic strategy for HBV treatment.

| Animals
C57BL/6 mice (male, 6-8 weeks old) in animal tests were purchased from Shanghai Laboratory Animal Center (Shanghai, China). Ten micrograms of pAAV-HBV1.2 plasmid DNA in a volume of PBS equivalent to 8% of mouse weight were injected via tail vein in 5s according to the previous method. 36 The mice were divided into MLN4924 group and vehicle (10% hydroxypropyl-beta-cyclodextrin (HPβ-CD)) group 5 days after injection.Mice then injected MLN4924  U0126 (HY-12031, MedChemExpress USA) was purchased from F I G U R E 1 HBV activates cullin neddylation in hepatoma cells and liver tissues. (A) HepAD38 cells were cultured in 6 well plates and treated with tetracycline (1μg/μL) or not until confluent. The cell lysates were harvested for Western blotting for NEDD8-Cullins. (B) HepG2 cells and (C) Huh7 cells were transfected with pHBV1.37 plasmids. Cells were collected at indicated times after transfection and subjected to Western blotting using indicated Abs.(D) HepG2 cells and Huh7 cells were transfected with pHBV1.37 plasmids in a time-dependent manner(the concentration of plasmids were 1μg/mL) as well as with different dose of plasmids(culture mediums were collected for HBV detection after 48h transfection) separately. Culture mediums were collected for RT-qPCR to determine the HBV DNA levels and subjected to ELISA to measure the levels of HBsAg and HBeAg. (E) 10 μg of pAAV-HBV1.2 plasmid, along with empty vector control, were injected into C57BL/6 mice through the tail vein. The mice livers were collected from 4 controls and 6 experimental mice 4 days after injection, followed by Western blotting with indicated antibodies. Cells were then harvested for protein lysate preparation, followed by Western blotting with indicated Abs

| Transfection
Lipofectamine 3000 (L3000015, Invitrogen, USA) was used to transfect plasmid. The procedure was carried out according to the instructions.

| Immunohistochemistry
Mice Liver tissues were collected and kept in OCT.
Immunohistochemical was used to detect intrahepatic HBcAg

| HBV cccDNA detection
Huh7 transfected with pHBV1.37 were lysed in lysis buffer within proteinase K (QIAGEN), total DNA was extracted according to a standard phenol-chloroform extraction protocol. The total DNA was digested with plasmid-safe adenosine triphosphate (ATP)-dependent deoxyribonuclease DNase (PSAD) (Epicentre F I G U R E 2 MLN4924 suppresses HBV in HBV-expressing liver cells. (A) Cytotoxicity of MLN4924. HepG2.2.15 cells were treated with MLN4924 at the indicated concentrations for 48h, followed by CCK8 growth assay. (B-D) Effect of MLN4924 on HBV replication: HepG2.2.15 cells were treated with MLN4924 at indicated concentration for 48h. Culture mediums were collected for RT-qPCR to determine the HBV DNA levels (B) and subjected to ELISA to measure the levels of HBsAg (C) and HBeAg (D  slides from Fujifilm, Tokyo, Japan).

| Western blot
Proteins extracted from cells and tissues were boiled for 10 min at   39 Hirt method was used to detect cccDNA, as described previously. 40,41 The isolated DNA samples were separated on 0.9%agarose gel then transferred to nylon membrane following hybridized with HBV-specific probe according to the instructions. 40 The primers for HBV-specific probe: 5'AATTCC ACAACCTTTCACCAAACTC3'(Forward);5'CACTGCATGGCCTGAG GATGAGT'(Reverse).

| Statistical analysis
Results were analysed by GraphPad Prism v7.0a (GraphPad Software, Inc, SanDiego, USA). Data were presented as mean ± SEM. Results of significance were using Student t test and P < .05 was considered statistically significant.

| HBV activates cullin neddylation
Previous studies have shown that MLN4924 has anti-viral activity in various virus models by inactivating CRLs (Cullin-RING ligases).
To determine potential involvement of CRLs in HBV expression, we used HBV stable-expression cell line HepAD38. As tetracycline can completely control HBV replication in HepAD38 cells and the viral replication can be greatly inhibited in the presence of tetracycline, 42 we treated HepAD38 cells with tetracycline or not. We found that the levels of NEDD8-Cullins are higher in tetracycline-absent group ( Figure 1A). We also transiently transfected HBV-expressing plasmid pHBV1.37 into hepatoma HepG2 and Huh7 cells and detected again elevated levels of neddylated cullins in both lines ( Figure 1B&C).
We show the HBV replication in a time-dependent manner as well as with different dose of transfection of HBV plasmid in the meanwhile ( Figure 1D). More importantly, using in vivo mouse model, we detected increased levels of neddylated cullins in liver tissues derived from mice 4 days after tail-vein injection of pAAV-HBV1.2 plasmids ( Figure 1E). Taken together, our results showed that HBV viral plasmids elevated the levels of neddylated cullins both in vitro and in vivo models, suggesting a potential involvement of CRLs in HBV infection.

| MLN4924 inhibits the HBV replication and antigen production in vitro
We then investigated whether MLN4924, a potent inhibitor of neddylation E1-activating enzyme, 12 can inhibit HBV replication in in vitro cell culture setting. We first tested MLN4924 cytotoxicity and found that in HepG2.2.15 cells, MLN4924 at 500 nM caused less than 20% of growth inhibition (Figure 2A). We found that MLN4924 significantly reduced the levels of secreted HBV DNA and HBsAg, as well as HBeAg (to a less extent) in culture supernatants in a dosedependent manner (Figure 2 B-D). Using these doses, we showed the concentration of NEDD8-Cullins ( Figure 2E). We further confirmed the inhibitory effect of MLN4924 on HepG2 ( Figure 2F) and Huh7 ( Figure 2F) cells after transiently transfection of plasmid F I G U R E 5 MLN4924 suppressed the expression of HNF1α, HNF4α and C/EBPα, required for HBV transcription. HepG2.2.15 cells were treated with 250 nM MLN4924, along with DMSO control for 48 h. Cells were then harvested for total RNA isolation or protein lysate preparation, followed by RT-PCR analysis for indicated transcription factor (A-D) and Western blotting with indicated Abs (E). Shown are mean ± SEM from three independent experiments ** P < .01, *** P < .001, NS, no significance encoding pHBV1.37.Tenofovir Disoproxil Fumarate (TDF) has been widely used as first-line agents for the treatment of infection of HBV in clinic. 43 It was shown that TDF significantly inhibited HBV DNA ( Figure S1A), but it had no obvious effect on the production of HBsAg and HBeAg ( Figure S1B-C). Collectively, MLN4924 showed significant anti-HBV activity in cell culture settings. Given that antiviral effect of MLN4924 is similar between concentrations of 250nM and 500nM, we used MLN4924 at 250nM for the rest of study.

| MLN4924 inhibits HBV particles and the levels of HBV antigen in vivo
We further explored whether MLN4924 has anti-HBV activity in vivo. C57BL/6 mice were injected via the tail vein with 10 μg of pAAV-HBV1.2 plasmid. 36 The mice were subsequently injected i.p. HBcAg expression were decreased in the liver of MLN4924-treated mice ( Figure 3G). These results indicated that MLN4924 indeed has anti-HBV activity at non-toxic dose in vivo.

| MLN4924 inhibits production of HBV 3.5kb RNA and cccDNA and blocks HBV promoter activity
To explore how MLN4924 suppressed HBV, we used RT-qPCR to measure the levels of HBV 3.5 kb RNA in HepG2.2.15 cells (with stable HBV expression) and found a significant reduction upon MLN4924 treatment ( Figure 4A). Significantly, MLN4924 treatment also reduced the levels of the cccDNA in Huh7 cells transfected with pHBV 1.37 plasmid, as measured by RT-qPCR ( Figure 4B) and Southern blot ( Figure 4C). Finally, we determined the effects of MLN4924 on HBV promoter activities, using luciferase-based reporters driven by XP, preS1P, preS2P and CP which represent respectively the promoters that drive the expression of the genes encoding HBV X protein, HBV large surface protein, HBV middle and small surface protein, HBV core protein in HepG2 ( Figure 4D) and Huh7 ( Figure 4E) cells. Again, MLN4924 significantly inhibited the activities of these HBV promoters. Taken together, HBV replication and transcription were significantly inhibited by MLN4924.

| MLN4924 anti-HBV activity is mediated by activation of MAPK signal
We have previously shown that MLN4924 triggers EGFR dimerization to activate ERK (pERK), 46 whereas pERK was reported to suppress HBV 46-48 and negatively regulates HNF1α, C/EBPα and HNF4α. 49 We, therefore, determined whether the pERK was involved in MLN4924-induced HBV suppression. We first confirmed that MLN4924 treatment indeed activated ERK in HepG2.2.15 cells, as evidenced by increased phosphor-ERK (pERK), which was blocked by MEK inhibitor, U0126 ( Figure 6A). We then used all three cellular HBV-expressing models and found that MLN4924 reduced the levels of transcription factors HNF1α, C/EBPα and HNF4α, which can be rescued by MEK inhibitor U0126, while U0126 had no effect if acting alone ( Figure 6A-C). Finally, MLN4924-mediated anti-HBV activity can be largely rescued by U0126 ( Figure 6D), strongly suggests a causal role of pERK in mediating MLN4924 suppression of HBV.

| D ISCUSS I ON
Previous studies have shown that MLN4924 has broad anti-virus activity, mainly by inactivation of CRLs. For example, MLN4924 anti-HIV activity was mediated by inhibiting CRL5-induced degradation of APOBEC3G 24 or CRL4-induced degradation of SAMHD1. 25 The F I G U R E 6 pERK plays a critical role in mediating MLN4924 anti-HBV activity. The HepG2.2.15 cells were treated with MLN4924 (250 nM), U0126 (10 uM), alone or in combination for 48 h, followed by Western blotting with indicated Ab (A). HepG2 (B) or Huh7 (C) cells were transfected with pHBV1.37 plasmid, treated with MLN4924 or U0126 alone or in combination and followed by Western blot analysis using indicated Ab. Supernatants collected in HepG2.2.15 (D) were subjected to RT-qPCR and ELSA measurement of HBV DNA and HBsAg levels separately. Densitometry quantification of Western blots was conducted using Image J. *P < .05, ** P < .01, *** P < .001, NS: no significance anti-influenza virus activity of MLN4924 was mediated by blocking NFκB nuclear translocation to reduce secretion of pro-inflammatory cytokines, 31 whereas anti-HCV activity was achieved by impairing the function of VPR. 27 In the case of HBV, two studies reported that X protein of hepatitis B virus promotes degradation of SMC5/6 via CRL4 to enhance HBV replication. 32,33 A most recent study showed that MLN4924 has anti-HBV activity by restoring SMC5/6 levels via inactivating CRL4. 50 Whether the anti-virus activity of MLN4924 can also be mediated by a mechanism other than CRLs inactivation is previously unknown.
In this study, with a portion of it reported last year in an international symposium, 51 we used both HBV-infected in vitro cell culture and in vivo mouse models to test anti-HBV activity of MLN4924. We first found that HBV infection activated cullin neddylation in all three cellular models, which is effectively inhibited by MLN4924 (Figure 1). We further showed that MLN4924 What is the possible mechanism by which MLN4924 down-regulated the promoter activity of HBV? We turned our attention to four liver-enriched transcription factors, HNF1α, HNF4α, C/EBPα and PPARα, which are not only important regulators for liver metabolic homeostasis, 45 but also shown to bind HBV promoter/enhancer elements to activate HBV transcription. [52][53][54] Specifically, HNF1α enhances viral transcription by activating a) HBV preS1P activity via binding to its enhancer/promoter, 55,56 and b) HBV CP activity via combining HBV Enh II B2 region. 57 While HNF4α overexpression increases activities of preS1P, preS2P and CP, 48,58,59 C/EBPα binds and F I G U R E 7 MLN4924 anti-HBV model. MLN4924 has two distinct mechanisms of action. On one hand, MLN4924 inactivates CRLs to restore the levels of SMC5/6 to suppress cccDNA, 50 and on the other hand, it activates ERK via EGFR signals to down-regulate the expression of HNF1α, HNF4α and C/EBPα. Together, MLN4924 inhibits activities of various HBV promoters, leading to reduction of HBV RNA, HBsAg and rcDNA and finally virion production activates the HBV Enh II, CP and preS2P. 52 We found that MLN4924 effectively reduced the levels of HNF1α, HNF4α and C/EBPα without affecting PPARα (Figure 5), providing a molecular explanation of how MLN4924 suppresses viral promoter activity.
We further pursued how MLN4924 reduces the protein levels of these cellular transcription factors. The effect is unlikely due to direct inhibition of CRLs, since CRLs inactivation would cause an increase, not decrease of substrates. A previous study has showed that in human hepatoma cells, activation of MAPK signal down-regulates HNF-4 expression and completely inhibits C/EBPα expression with compromised recruitment of HNF-3β and HNF-1α to the HNF-4 enhancer, and RNA polymerase II to the proximal HNF-4 promoter, 49 indicating MPAK signal is a negative regulator of these liver transcription factors. Furthermore, RAS-MAPK activation by external stimuli has previously shown to suppress HBV replication in both Huh7 and HepG2 cells. 60 Is there any connection between MLN4924 and MAPK activation? Indeed, we recently found that in addition to blocking cullin neddylation as a potent NAE inhibitor, MLN4924 also activates EGFR and downstream AKT1 and ERK1/2 signals by triggering EGFR dimerization in lung, breast and colon cancer cells. 46 Here, we showed that in all three EBV-infected liver cell models, MLN4924 activates MAPK signal leading to increased ERK1/2 phosphorylation, and ERK1/2 activation inhibits protein levels of HNF1α, HNF4α and C/EBPα ( Figure 6). More importantly, inactivation of pERK1/2 by a MEK inhibitor U0126 rescued MLN4924 effects, as evidenced by restoring the levels of these three transcription factors, and abrogating inhibition in the production of HBV DNA and HBsAg ( Figure 6).
Taken together, we conclude that MAPK activation plays a causal role in anti-HBV activity of MLN4924.
In summary, we made two novel observations in this study: First, HBV infection in all three cellular models activates CRLs by enhancing cullin neddylation. The underlying mechanism is unknown at the present time, but it is certainly an interesting subject for future investigation; and second, anti-HBV activity of MLN4924 can also be achieved by activation of MAPK signal, which suppresses few transcription factors that drive HBV transcription. Our study, along with a recent publication, 50 supports the following model for MLN4924 anti-HBV activity. MLN4924, on one hand, inactivates CRL4 to restore the levels of SMC5/6 to block cccDNA synthesis, and on the other hand, activates MAPK signals to suppress transcriptional activity of HFN1α, HFN4α and C/EPBα, leading to inhibition of viral promoters of S1p, S2p, Cp and Xp to reduce the levels of 3.5 kb HBV RNA, and eventually reduced HBV rcDNA and HBsAg and HBcAg (Figure 7). It has been reported that CHB patients with the high levels of HBV DNA and HBsAg are more frequently progressed to HCC. 8 Thus, simultaneous inhibition of viral DNA and antigens will be an ideal approach for anti-HBV therapy. MLN4924 is a highly selective small-molecule inhibitor of NEDD8 and can block the entire neddylation modification cascade effectively. 61 In addition to well-characterized anti-neddylation activity, recent studies showed that MLN4924 has several neddylation-independent activities including the ERK activation we found in this work. 46,61 So the anti-HBV activation of MLN4924 may involve neddylation inhibition as well as other mechanisms. Therefore, the degree of NEDD8 inhibition after medication may not be parallel to the inhibition efficiency of HBV and the detailed mechanisms need further research. Taken together, our study showed that MLN4924 is an effective anti-HBV agent by blocking both viral DNA and antigen, thus providing a sound rational for future clinical trial of this new application.

ACK N OWLED G EM ENTS
We thank Pei-Jer Chen from the Department of Internal Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine for providing pAAV-HBV1.2 and Min Chen from the Chongqing Medical University for providing HepAD38 cells.
We thank Yong jun Li from the First Affiliated Hospital, Zhejiang University for excellent technical support.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.