Abstract
The persistence of latent HIV-1 reservoirs throughout combination antiretroviral therapy (cART) is a major barrier on the path to achieving a cure for AIDS. It has been shown that bromodomain and extra-terminal (BET) inhibitors could reactivate HIV-1 latency, but restrained from clinical application due to their toxicity and side effects. Thus, identifying a new type of BET inhibitor with high degrees of selectivity and safety is urgently needed. Apabetalone is a small-molecule selective BET inhibitor specific for second bromodomains, and has been evaluated in phase III clinical trials that enrolled patients with high-risk cardiovascular disorders, dyslipidemia, and low HDL cholesterol. In the current study, we examined the impact of apabetalone on HIV-1 latency. We showed that apabetalone (10−50 μmol/L) dose-dependently reactivated latent HIV-1 in 4 types of HIV-1 latency cells in vitro and in primary human CD4+ T cells ex vivo. In ACH2 cells, we further demonstrated that apabetalone activated latent HIV-1 through Tat-dependent P-TEFB pathway, i.e., dissociating bromodomain 4 (BDR4) from the HIV-1 promoter and recruiting Tat for stimulating HIV-1 elongation. Furthermore, we showed that apabetalone (10−30 μmol/L) caused dose-dependent cell cycle arrest at the G1/G0 phase in ACH2 cells, and thereby induced the preferential apoptosis of HIV-1 latent cells to promote the death of reactivated reservoir cells. Notably, cardiovascular diseases and low HDL cholesterol are known as the major side effects of cART, which should be prevented by apabetalone. In conclusion, apabetalone should be an ideal bifunctional latency-reversing agent for advancing HIV-1 eradication and reducing the side effects of BET inhibitors.
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Introduction
Human immunodeficiency virus type 1 (HIV-1) is a viral antigen that causes incurable AIDS. Although combination antiretroviral therapy (cART) suppresses HIV-1 to undetectable levels and partially restores immune function in infected individuals [1], it produces serious side effects, such as lipid metabolism disorders and cardiovascular diseases [2]. Moreover, interruption of cART causes the virus to rapidly rebound to its pretreatment levels [3, 4]. The main cause of treatment failure is the existence of latent HIV-1 reservoirs [5,6,7,8]. Recent studies have explored a strategy named “shock and kill,” which would eradicate HIV-1 in latent HIV-1 reservoirs by activating HIV-1 transcription and viral antigen expression in the presence of cART [9,10,11,12]. When attempting to use this strategy, the first challenge is to find latency reversing agents (LRAs) to efficiently reactivate latent HIV-1. Several agents have been explored [13] and two types of LRAs have reached human testing. The histone deacetylase (HDAC) inhibitors romidepsin [14], vorinostat (SAHA) [15], and panobinostat [16, 17] transcriptionally reactivate latent HIV-1 but do so in a nonspecific manner [18, 19]. A second candidate, disulfiram, was proven effective as an LRA but failed to reduce the reservoir size [20, 26]. Notably, apabetalone is a highly selective BET inhibitor that specifically targets the BD2 domain of BET proteins [28]. A microarray study showed that JQ1 strongly affects gene transcription with almost a 10-fold difference when compared with the BD2-specific inhibitor apabetalone. Apabetalone is being developed by Resverlogix Corporation for the treatment of cardiovascular diseases associated with atherosclerosis. In addition, apabetalone has recently entered clinical studies for the treatment of LDL dyslipidemia and recent phase III clinical data concerning the use apabetalone as an ApoA1 modulator have been encouraging [27, 38]. These disorders are all known as side effects of cART and might be prevented by apabetalone.
A recent study preliminarily showed that apabetalone could reactivate HIV-1 latency by increasing CDK9 Thr-186 phosphorylation [39]. However, our team has investigated the effects of apabetalone on latent HIV-1 and found that apabetalone could induce the preferential apoptosis of HIV-1 latent cells to promote the death of reactivated reservoir cells following viral reactivation, which makes apabetalone an ideal bifunctional LRA for HIV-1 eradication.
In the current study, the results indicated that apabetalone could significantly promote HIV-1 expression in various types of HIV-1 latency cells. Notably, the degrees of HIV-1 reactivation in the Tat-dependent latency cell models J-Lat A2 (Fig. 1c) and ACH2 (Fig. 1d) were significantly higher than the degrees of activation in the J-Lat 10.6 (Fig. 1e) and U1 cell models (Fig. 1f), suggesting that the effect of apabetalone on latent HIV-1 depends on Tat. Remarkably, we found that apabetalone induced the latent HIV-1 5’-LTR transcripts in primary CD4+ T cells from infected individuals receiving suppressive cART (Fig. 1g). Importantly, unlike other LRAs, such as Phorbol-12-myristate-13-acetate (PMA), prostratin, and bryostatin 1, which effectively reverse HIV-1 latency ex vivo but induce global T-cell activation and are too toxic for clinical use, apabetalone had no effect on T-cell activation (Fig. 2a,b) and pro-inflammatory cytokines (Fig. 2c), and showed no evidence of toxicity (Table 1). These traits may be related to data in previous reports that showed that apabetalone can act as an immunosuppressive agent and a chemokine inhibitor. All of these results support further investigations of apabetalone as a potential agent that can be incorporated into an HIV-1 curative strategy.
HIV-1 treatment strategies that employ combinational LRAs are widely assumed to be capable of efficiently reactivating latent HIV reservoirs [40, 41]. Prostratin and SAHA are conventional LRAs that work via different mechanisms. Apabetalone significantly promoted the reactivation effect of SAHA or prostratin in J-Lat A2 cells (Fig. 3a), ACH2 cells (Fig. 3b), and primary CD4+ T cells obtained from HIV-1-infected individuals receiving suppressive cART (Fig. 3c). The above results indicate that apabetalone is an efficacious and safe candidate for development as an HIV-1 LRA.
Notably, the role of cART drugs on viral clearance in the “shock and kill” strategy are crucial for preventing the spread of HIV-1 infection and achieving an HIV-1 cure [9, 42]. In this study, we combined apabetalone with different types of cART drugs with high antiviral activities including Zidovudine (nucleoside reverse transcriptase inhibitors (NRTI)), Raltegravir (HIV-1 integrase inhibitor), Nevirapine (non-NRTI), Maraviroc (CCR5-HIV-1 entry inhibitor), and Plerixafor (CXCR4-HIV-1 entry inhibitor). Our results showed that the infection of activated HIV-1 by apabetalone could significantly decrease after treatments of all tested cART drugs (Fig. 3d) and the combination of cART with apabetalone does not interfere with cART drugs’ antiretroviral activity (Table 2). This indicated that the combination of apabetalone with cART could protect uninfected target cells from becoming reinfected and is favorable to eliminating latent HIV-1 reservoirs.
Recent studies have reported that Tat has a critical role in JQ1 or OTX015-mediated reactivation of latent HIV-1 [52]. Further investigations in this area are warranted.
In summary, we have provided strong evidence that the BET inhibitor apabetalone is a potent antagonist of HIV-1 latency. Apabetalone acts by activating P-TEFb via dissociating BDR4 from the HIV-1 promoter and recruiting Tat for stimulating HIV-1 elongation. Furthermore, apabetalone can down-regulate cyclin D1 expression, upregulate p21waf1/Cip1, and induce G1/G0 phase cell cycle arrest. It was particularly interesting that apabetalone induced the preferential apoptosis of HIV-1 latent cells and further promoted the death of reactivated reservoir cells. In conclusion, its low degree of cytotoxicity, coupled with its abilities to reactivate latent HIV-1 reservoirs, induce HIV-1 latent cell apoptosis, and reduce the side effects of cART, all make apabetalone worth investigating for development as a possible LRA for use in accelerating HIV-1 eradication.
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Acknowledgements
We thank Dr. Shibo Jiang and Dr. Lu Lu at Fudan University, China, for generously providing HIV-1 latent cell models and reagents. This study was financially supported by grants from the Natural Science Foundation of China (81773787 to SL and 81673481 to LL) and National Science and Technology Major Project (2018ZX10301101 to SL).
Author contributions
XZ, LL, and SL conceived the project and wrote the manuscript. XZ, JL, and TL performed the experiments. HD and BX analyzed the data. XT provided the clinical samples. All authors reviewed the manuscript. LL and SL supervised the project and revised the manuscript.
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Zhang, Xx., Lin, J., Liang, Tz. et al. The BET bromodomain inhibitor apabetalone induces apoptosis of latent HIV-1 reservoir cells following viral reactivation. Acta Pharmacol Sin 40, 98–110 (2019). https://doi.org/10.1038/s41401-018-0027-5
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DOI: https://doi.org/10.1038/s41401-018-0027-5
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