Combination antiretroviral therapy (cART) has led to a substantial reduction in morbidity and mortality in HIV-infected patients; however, cART alone is unable to cure HIV and therapy is life-long. The main barrier to eradication of HIV is the persistence of long-lived latently infected resting memory CD4+ T cells [1,2]. One approach being explored to eliminate latency is to stimulate virus production from latently infected cells using compounds such as histone deacetylase inhibitors (HDACi) (reviewed in ). A recent clinical trial of the HDACi vorinostat in eight HIV-infected patients on suppressive cART confirmed that vorinostat can activate virus transcription in resting CD4+ T cells in vivo .
HDACi increase acetylation of both cellular and viral genes and are in advanced clinical development for the treatment of malignancy [5,6]. There are multiple HDACs expressed in resting CD4+ T cells, which include class I (HDAC 1, 2, 3 and 8) and class II HDACs (HDAC4, 5, 6, 7, 9 and 10) . In latently infected cells lines, it has been shown that HDAC1, HDAC2 and HDAC3 are the major HDACs involved in maintaining latency [8,9], but this has not been well defined in primary T cells. Inhibition of Class I but not Class II HDACs was shown to induce viral production in latently infected resting CD4 T cells isolated from patients on suppressive cART [8–10]. Evaluation of newer HDACi using latently infected primary T cells is critical to identify more potent, less toxic and more selective compounds that could potentially move into clinical trials.
Entinostat is an HDACi selective for class I HDAC [11,12]. Entinostat has the highest potency against HDAC1 (nanomolar range) and significantly less potency against HDAC2 and HDAC3 (micromolar range)  and no reported activity against HDAC8 or any class II HDACs . Greater potency for HDAC1 than other Class 1 HDACs has been confirmed by others . Entinostat is currently being evaluated in 23 Phase I or II trials for a range of malignant conditions, including myeloid and lymphocytic leukaemia and nonsmall cell lung cancer; breast and colorectal cancer [clinicaltrials.gov database]. Although no specific activity against malignancy in vivo has been published to date, entinostat was well tolerated, reports a negative Ames test , increased histone acetylation and extracellular signal-related kinase protein expression in tumour tissue [14,15]. In a mouse renal cancer model, entinostat also suppressed regulatory T-cell function , which may be an additional beneficial associated effect when pursuing a ‘shock and kill’ approach to eliminating HIV latency .
In this study, we aimed to determine the relative potency and toxicity of a panel of HDACi that are either pan HDACi [e.g. panobinostat, vorinostat and metacept-3 (MCT-3)] or a class I HDAC-selective HDACi (e.g. entinostat) using latently infected primary T cells [18,19]. Our previously reported model of chemokine-induced HIV latency is highly reproducible leading to consistent high rates of HIV integration, limited viral production, production of multiply spliced RNA that is retained within the nucleus (as described in patient-derived cells ) and no evidence of T-cell activation [18,19,21]. Therefore, this is an ideal model to assess the potency, toxicity and mechanism of action of HDACi in stimulating HIV production from latently infected cells . In addition, we sought to demonstrate which specific HDACs were expressed in resting CD4+ T cells and which of these were critical for maintenance of HIV latency. We show different expression of HDACs in cell lines and primary cells and considerable variation in the potency and toxicity of HDACi in latently infected cell lines and primary CD4+ T cells. Furthermore, the HDACi entinostat that is selective for class I HDAC induced virus production in latently infected primary CD4+ T cells, making this compound an attractive option for future clinical trials.
Materials and methods
Isolation of CD4+ T cells and establishment of latency in vitro
Resting CD4+ T cells were isolated from buffy coats (Australian Red Cross Blood Service, Southbank Australia), treated with chemokine CCL19 and infected as previously [18,19,21].
HDACi (0.01–5 μmol/l) were added to cell cultures, and after 24 h, cultures were analysed for cell viability with MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, Wisconsin, USA). LD50 curves were determined using nonlinear regression analysis (Graphpad, La Jolla, California, USA).
Virus production from latently infected cells
CCL19-treated latently infected CD4+ T cells were incubated with phorbyl myristoacetate (PMA; 10 nmol/l, Sigma-Aldrich, St. Louis, Missouri, USA) and phytohemagluttin (PHA; 10 μg/ml, Sigma-Aldrich); IL-7 (5 ng/ml, R&D Systems, Minneapolis, Minnesota, USA); or the HDACi vorinostat [suberoylhydroxamic acid (SAHA); 0.5 μmol/l], panabinostat (LBH589; 0.05 μmol/l), entinostat (MS-275; 0.5 μmol/l) (all Selleck Chemicals, Houston, Texas, USA) and MCT-3 (0.5 μmol/l) (Australian Centre for Blood Diseases, Monash University, Australia) on day 4 postinfection and left in cultures for the duration. HDACi were dissolved in dimethyl sulfoxide (DMSO) and 0.1% DMSO was used in all conditions, including the negative control in which no drug was added. PHA-activated feeder peripheral blood mononuclear cells (PBMCs) were added to primary cell cultures 24 h after activation to amplify virus replication. Virus production was measured in supernatant by reverse transcriptase (RT) production. (Fig. 2a) .
Stimuli were also assessed in latently infected cell lines ACH2 , U1  and J-Lat 6.3  (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH) at day 1, 2 and 3 poststimulation.
Flow cytometry for activation markers
Purified resting CD4+ T cells were treated with drug for 24 h and then stained with CD69-FITC, CD38-PE, HLA-DR-APC, CCR5-PE, CXCR4-FITC, IgG-PE or IgG-FITC (all Becton Dickinson (BD) Biosciences, San Jose, California, USA) for analysis on a FACSCalibur (BD Biosciences).
Microarray analysis of histone deacetylase expression in primary T cells
Resting CD4+ T cells were treated with CCL19 (24 nmol/l) or media alone and cultured for 6 or 18 h. Gene expression was quantified as previously .
Histone deacetylase RNA expression using real-time PCR
Expression of RNA for all HDACs was determined as previously [25,26]. Results were normalized to GAPDH using ΔCt method with data showing the ratio of (Ct(HDAC)/Ct(GAPDH)) in PBMC: (Ct(HDAC)/Ct(GAPDH)) in the ACH2 cells.
Latently infected primary CD4+ T cells were treated for 4 h with 0.5 μmol/l vorinostat or entinostat or left in media, fixed and chromatin immunoprecipitation (ChIP) performed using a ChIP assay kit (Millipore, Billerica, Massachusetts, USA) as previously described . Control cells were pretreated 24 h prior to infection with the integrase inhibitor L870812 (kind gift from Merck, Darmstadt, Germany).
A Kruskal–Wallis one-way analysis of variance (ANOVA) test was used to determine whether there was an overall difference between activity of each HDACi. Significance of pairwise comparisons of each HDACi was determined by paired t-tests.
Effect of histone deacetylase inhibitor on cytotoxicity and viral production in latently infected cell lines and primary cells
Cytotoxicity was observed in both cell lines and resting PBMC following treatment with all HDACi. Of the HDACi tested, panabinostat was most toxic, with an LD50 value of 0.09 μmol/l in PBMC. LD50 values were higher in resting PBMC than in the rapidly dividing cell lines (Fig. 1). For subsequent experiments to assess potency of inducing HIV production from latently infected cell lines (ACH2 and J-Lat 6.3) and CCL19-treated latently infected primary CD4+ T cells, we used each HDACi at a concentration below that of the LD50 in PBMC (0.5 μmol/l except for panabinostat 0.05 μmol/l). This gave a mean viability of 89% for vorinostat, 81% for panabinostat, 91% for entinostat and 92% for MCT-3. The selected concentration was at the range of clinically relevant concentrations for vorinostat (Cmax at 400 mg/day 1.2 ± 0.53 μmol/l ) and entinostat (0.14 ± 0.24–0.3 ± 0.22 μmol/l [14,28]) and lower than clinically achievable levels for panabinostat (Cmax 0.78 ± 0.12–1.4 ± 0.4 μmol/l ).
In latently infected CCL19-treated primary CD4+ T cells, the mean (range) of virus production, measured at day 3 posttreatment and expressed as a percentage of maximal stimulation with PHA/PMA, was entinostat equal to 22.5% (15–31%); MCT-3 equal to 17% (12–22%); vorinostat equal to 10% (6–13%) and panobinostat equal to 11% (6–25%) (P = 0.028, Kruskal–Wallis for all drugs; Fig. 2b, upper row, n = 4). Entinostat and MCT-3 led to significantly higher virus production than vorinostat (P = 0.02 and P = 0.04, respectively, paired t-test). At day 6, the differences in RT in supernatant between entinostat and vorinostat were no longer significant (P = 0.32).
In the ACH2 cell line, virus production was similar following incubation with entinostat [14% (11–17%)] and panobinostat [14% (11–16%)] while vorinostat [5% (3–8%)] and MCT-3 [4.5% (2–8%] were less potent (P = 0.038, Kruskal–Wallis; Fig. 2b, second row, n = 3). There was no significant difference in potency between each drug and vorinostat [entinostat and panobinostat (P = 0.1) and MCT-3 (P = 0.8; paired t-tests)]. In the J-lat 6.3 line, virus production was measured by enhanced green fluorescent protein (EGFP) expression and again expressed as a percentage of the maximum expression induced by PMA/PHA stimulation (Fig. 2b, third row, n = 3). We found similar expression of EGFP with entinostat [12% (11–15%)] and vorinostat [11% (9–18%)] but lower levels following MCT-3 [8% (7–12%)] and panobinostat [6% (4–6%); P = 0.06, Kruskal–Wallis)]. There was no significant difference in potency between each drug and vorinostat.
We also asked whether virus production could be enhanced by activation with an HDACi and another agent that activated latent HIV by a different pathway, such as IL-7 [21,30]. Virus production following combination treatment with vorinostat and the cytokine IL-7 of latently infected CCL19-treated primary CD4+ T cells resulted in an increased production of RT [36% (22–38%)] as compared with vorinostat [11% (8–12.5%)] or IL-7 alone [27% (15–32%)], although this increase did not reach statistical significance (P = 0.07; two-way ANOVA test; Fig. 2c; n = 3). There was therefore some evidence of an additive but not synergistic response with the combination of vorinostat and IL-7.
Potency of each HDACi in latently infected primary T cells was not related to induction of cell activation or expression of HIV coreceptors, as there was no significant change in expression of the activation markers HLA-DR, CD69 or CD38 or the coreceptors CXCR4 or CCR5 in the presence of each HDACi (n = 3; Fig. 3).
Taken together, these data demonstrate that the relative potency of HDACi differed in latently infected primary T cells and cell lines, that there was no cellular activation following HDACi treatment and that the Class I HDACi, entinostat, activated virus production in latently infected primary T cells.
Expression of histone deacetylases in primary CD4+ T cells
To determine whether differences in the response to HDACi in latently infected primary cells and cell lines were in part due to different expression of cellular HDACs, we assessed which HDACs were expressed in resting CD4+ T cells and whether there were changes in the RNA expression of HDACs in response to CCL19 treatment. Using an Illumina bead microarray analysis, we first generated heat maps on the basis of normalized data and found that in resting CD4+ T cells, HDAC1 was expressed the highest, followed by HDAC 2, 4 and 3 (in that order), which were expressed at similar but lower levels (Fig. 4a). All other HDACs were also detected. CCL19 did not alter the expression of the HDACs, as similar changes were found in resting CD4+ T cells that had been incubated with CCL19 for 6 and 18 h (Fig. 4a). Expression of the class I HDACs was similar to a previous study of resting CD4+ T cells from HIV-infected patients on cART .
The expression of RNA for HDACs 1–11 in both primary CD4+ T cells and the ACH2 cell line was confirmed using RT-PCR (Fig. 4b). There was significant differential expression of each HDAC RNA in primary T cells and the cell line ACH2 with four to five-fold greater expression of HDAC 3, 4 and 6 in primary CD4+ T cells (Fig. 4b). We also showed that HDAC1, 2, 3 and 4 proteins were all detected by western blot in CD4+ T cells and the ACH2 cell line, with no change in expression observed in CD4+ T cells treated with CCL19 (Fig. 4c). The relative expression of HDAC 1–3 in ACH2 and primary T cells was similar for RNA and protein expression with reduced expression of HDAC3 protein in ACH2 cells. However, for HDAC4, we noted higher expression of HDAC4 RNA but lower HDAC4 protein expression in primary T cells than ACH2. This discrepancy may be related to the complex splicing and posttranslational modification of HDAC4 . We only show here the most highly expressed isoform (with a molecular weight of 140 kDa).
Association of histone deacetylases with the HIV-long-terminal repeat in latently infected primary CD4+ T cells
The ability of more selective HDACi to reverse HIV latency is dependent not just on the presence of the specific HDAC in the cell but on the association of the HDAC with the HIV LTR. To determine which HDACs were present at the LTR in latently infected CCL19-treated primary CD4+ T cells, we performed ChIP assays specific for HDAC1, 2 and 3 and acetylated histone 3 (H3). Latently infected CCL19-treated primary CD4+ T cells were either left untreated or treated with 0.5 μmol/l vorinostat or entinostat for 4 h prior to fixation and subsequent ChIP analysis. Vorinostat led to a reduced amount of HDAC1 (P = 0.007) and 2 (P = 0.03) bound to the LTR (n = 3; Fig. 5a). There was a significant reduction in binding of only HDAC1 following treatment with entinostat (P = 0.03; n = 3; Fig. 5a). HDAC3 was bound to the LTR but at low levels and there was little change following incubation with each HDACi, although this was difficult to measure given the very low levels of binding in the absence of an HDACi. Acetylation of H3 significantly increased following incubation with vorinostat and entinostat.
In the presence of the integrase inhibitor L8, integration was successfully inhibited (Fig. 5b); however, we still detected HDACs 1, 2 and 3 bound to the HIV LTR at low levels. This most likely represented association of these HDACs with either 1 or 2-LTR circles or unintegrated HIV DNA. When we subtracted the signal identified in the presence of an integrase inhibitor, although the total signal was reduced, the overall pattern was unchanged (data not shown) demonstrating that HDAC1, 2 and 3 were all associated with integrated HIV in this model of HIV latency.
Activating latent infection with HDACi may be a potential strategy to eliminate latently infected cells in HIV-infected patients on cART. We demonstrate here that multiple HDACi in varying stages of preclinical and clinical development can induce viral production in latently infected primary CD4+ T cells and latently infected cell lines. The HDACi entinostat selective for Class I HDACs was associated with low levels of toxicity in PBMC, inhibition of binding of HDAC1 to the HIV LTR, enhanced acetylation of H3 and induced virus production from latently infected primary CD4+ T cells. These features support further investigation of selective HDAC inhibitors and potential future clinical trials of entinostat in HIV-infected patients on cART.
We observed significant differences in efficacy in inducing virus production from latency between cell line and primary cell models, with the greatest difference seen in the response to panobinostat, which was the most effective HDACi at inducing viral production in the ACH2 cell line and least effective in latently infected primary CD4+ T cells. The differences between cell lines and primary CD4+ T cells in response to HDACis, at least to pan HDACi, may potentially be explained by differential expression of HDACs. HDAC1, 2 and 3 are thought to be the major HDACs that maintain latency [8–10]. However, we did not identify any significant differential expression of HDAC1 or 2. We did observe a decrease in HDAC3 expression in ACH2 cells, which may have potentially explained some differences in response to individual HDACi. Potency may also be related to the capacity of an HDACi to induce apoptosis and cell cycle arrest in a rapidly dividing cell compared with a resting cell [32,33]. We clearly demonstrated a far lower LD50 for toxicity (or cell death) in resting PBMC than in the ACH2 cell line.
HDAC4 has recently been shown to be critical for repression of HIV expression in the latently expressed cell line ACH2 but not in the latently infected monocyte cell line U1 . The importance of HDAC4 in latently infected primary T cells is unknown, although we demonstrated significant expression of HDAC4 protein in both ACH2 and resting CD4+ T cells. Given that entinostat has no effects on HDAC4, our data would argue that HDAC4 may not be critical to silencing of virus expression in latently infected primary T cells, although to specifically address this, inhibiting HDAC4 with siRNA in latently infected primary T cells should be performed.
Several other studies have also identified differing responses to HDACi in different cell lines  compared with primary T cells isolated from patients on suppressive cART , but there has been no other systematic comparisons of the activity of HDACi in in-vitro models of latently infected primary T cells. Given the differences identified in latently infected cell lines and latently infected primary T cells, our data further confirm that assessment of the activity of HDACi using latently infected cell lines alone should be interpreted with caution. The gold standard in-vitro model to identify or quantify activation of HIV transcription from latency, that is the model that best mimics activity in vivo, remains to be determined. This could potentially be evaluated in the current clinical trials of vorinostat (NCT01319383; NCT01365065), panobinostat (NCT01680094) and romedepsin that are planned or already underway in HIV-infected patients on cART.
Entinostat, a selective inhibitor of Class I HDACs, induced virus production from latently infected primary T cells with similar, if not greater potency than observed with vorinostat. We were specifically interested to compare potency and toxicity of each compound with vorinostat, given vorinostat was the first HDACi to be licensed for the treatment of cutaneous T-cell lymphoma; it is already in clinical trials in HIV-infected patients on ART [4,6]; and a single dose of vorinostat can induce HIV production from latently infected cells in vivo in patients on suppressive cART . Therefore, it is highly likely that vorinostat will be the standard for evaluation of newer HDACi in clinical trials. The real question, however, is whether our measure of in-vitro ‘potency’ has any relevance to in-vivo ‘potency’, specifically capacity to eliminate latently infected cells. Other factors such as proportion of latently cells activated, response to repeated dosing, drug permeability in infected cells and tissue, differing toxicity in target and nontarget cells and death of recently activated infected cells following stimulation by an HDACi will also be important measures of efficacy of latency-activating agents.
Previous studies in CD4+ T cells from HIV-infected patients on cART have suggested that inhibition of binding of HDAC1, 2 and 3  was needed to activate virus production from latency . In our model of latently infected CCL19-treated primary T cells, we showed that inhibition of binding of HDAC1 alone was sufficient to activate virus production from latency. Therefore, the primary activity of entinostat is largely mediated through HDAC1 at the concentrations used in this study.
HDACi can induce a variety of immunomodulatory effects in vitro and in vivo (reviewed in ). These include upregulation of surface molecules including HLA-DR, changes in signal transducer and activator of transcription (STAT) signalling and cytokine responses. We found no evidence in vitro of T-cell activation by HDACi at the concentrations tested in our study. Specifically, we also saw no change in the expression levels of CCR5 and CXCR4 in this model. Belinostat (ITF2357), an HDACi that inhibits both class I and II HDACs (reviewed in ), has been shown to decrease surface expression of CXCR4 in CD3+CD4+ T cells in vitro at 4 h posttreatment . In contrast, with the panel of HDACi used in this study, we saw no change in CXCR4 expression suggesting that any downregulation of CXCR4 expression may be unique to belinostat and not common to all HDACi.
Several groups have already demonstrated that activation of virus production from latently infected cells can be enhanced in vitro with various combinations of drugs, including protein kinase C (PKC) modulators bryostatin and prostratin with the HDACi vorinostat, entinostat, trichostatin A or valproic acid, and methyltransferase inhibitors with vorinostat [36,40–43]. Although we saw an increase in viral production with the combination of IL-7 and vorinostat, this effect was not synergistic. However, additive effects may still be advantageous when using activation strategies. It is likely that a specific HDACi may only activate a subset of latently infected cells and may not activate virus production from all latently infected resting CD4+ T cells in all patients [37,43,44]. Indeed, vorinostat activated virus production ex vivo from PBMC from just over half of HIV-infected patients on cART [43,44] A similar frequency of response was observed when vorinostat activity was evaluated ex vivo using purified resting CD4+ T cells . Although we saw a modest additive effect of IL-7 and vorinostat, we were unable to distinguish between the possibilities of either both compounds activating the same latently infected cell or each compound activating different populations of latently infected cells. This is an important distinction that could potentially be examined using an EGFP reporter virus in our model, which we are currently developing.
In conclusion, we have shown variation in the potency and toxicity of HDACi in latently infected cell lines and primary CD4+ T cells and this was not explained by major differences in constitutive expression of key HDACs involved in maintaining HIV latency. Given that the HDACi entinostat activates virus production from latently infected primary CD4+ T cells, is selective for Class I HDACs and therefore likely to have fewer ‘off-target’ effects, and has some unique immunoregulatory effects, this compound should be considered as an attractive option for future clinical trials in HIV-infected patients on suppressive cART.
We acknowledge David Margolis, Nancie Archin and Kirston Barton for their assistance with ChIP assays and for helpful comments on this manuscript. F.W. is supported by an Australian National Health and Medical Research Council (NHMRC) Biomedical postgraduate scholarship. S.R.L. is an NHMRC practitioner fellow. This work was supported by an NHMRC project grant 1009533 and National Institutes of Health Delaney AIDS Research Enterprise (DARE) to find a cure U19 AI096109.
Conflicts of interest
S.R.L. has received grant support from Merck and Gilead Sciences and has received payment for participation in educational activities from Merck, Gilead, Bristol Myers Squib, Janssen and Viiv. All payments were made to her institution.
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