The Histone Deacetylase Inhibitor ITF2357 Decreases Surface CXCR4 and CCR5 Expression on CD4+ T-Cells and Monocytes and is Superior to Valproic Acid for Latent HIV-1 Expression in Vitro

Introduction

Since the introduction of highly active anti-retroviral therapy (HAART) more than a decade ago, HIV-1 infection can be well controlled, with HIV-1 viremia below detectable levels. However, eradication of HIV-1 infection with prolonged HAART therapy is still not feasible. Following discontinuation of HAART, rebound viremia occurs, typically after two weeks ^1-4. The source of viral re-emergence is from a long-lived pool, most likely the latently infected memory T-cell reservoir, harboring integrated HIV-1 proviral DNA ². Well established techniques are available to quantify the latent pool and it is estimated that one in every million memory cells of an HIV-1 positive patient bears a replication competent integrated provirus^{5, 6}.

The latent reservoir of HIV-1 within resting CD4+ cells is established early after acute infection and the initiation of HAART during this period does not prevent its establishment ^6-8. The latent pool is an extremely stable reservoir, having a half-life of 6-44 months even in treated patients who are continuously aviremic for long periods of time^{6, 9-12}. Having this prolonged half-life, a complete decay of the reservoir is not expected before 70 years of treatment making eradication improbable. These time frames might be somewhat shorter by starting HAART early during acute infection or by intensifying HAART but not sufficient as a practical method for eradication ^{13, 14}.

Mechanisms that maintain the proviral DNA transcriptionally inactive in the quiescent cells (see review in ^{15, 16}) include chromatin-associated regulation. In the latent cell, the integrated proviral DNA is densely organized in nucleosomes. The HIV-1 5′ long terminal repeat (LTR), containing the promoter and enhancer elements, binds several transcription factors and is arranged in two nucleosomes (nuc-0 and nuc-1)¹⁷. The NFκB p50 homodimer, as well as AP-4, YY1 and LSF1 recruit histone deacetylase (HDAC)-1 to the LTR, which in turn results in deacetylation of local histones, compaction of the chromatin and prevention of RNA polymerase-II binding^18-21. In vitro studies have demonstrated that activation of the latent cell pool by different stimuli would reverse the repressive effect of the p50 homodimer-HDAC-1 complex by the binding of cytosolic NFκB p50-RelA heterodimer ^{19, 22}. This would enable the recruitment of histones acetyltransferase (HAT), acetylaton of the local histones, relaxation of the chromatin and initiation of transcription ^{19, 23, 24}.

Inhibition of the enzymatic activity of HDAC-1 and likely HDAC-2 and 3 by synthetic inhibitors of HDACs (HDACi) leads to activation of the HIV-1 LTR and HIV-1 gene expression. Moreover, unlike cell activators of NFκB, such as IL-2, OKT3 or TNFα, HDACi facilitate gene expression without general activation cytokines and the T-cell^{25, 26}.

In vitro, various HDACi induce HIV-1 gene expression from latently infected cells line^27-29. Valproic acid (VPA), a carboxilate HDACi prescribed for seizures and psychiatric disorders, has been combined with HAART in small clinical trials but without the desirable significant decrease of the latent reservoir ^{26, 30-32}. The studies with VPA, a non-specific weak HDACi, have not resolved the potential of HDACi to purge the virus.

ITF2357 is a hydroxamic acid-containing HDACi that has anti-inflammatory and anti-tumor properties in vitro and in vivo^33-36. At therapeutic plasma levels of 125-250nM, there is no cell-toxicity in vitro and only minor reversible thrombocytopenia occurs in patients³⁷. As an anti-inflammatory agent, twelve weeks of daily ITF2357 has been given to children with systemic onset juvenile idiopathic arthritis (SoJIA) with no safety issues and promising clinical improvement ³⁸. Since ITF2357 was shown to be a potent anti-inflammatory drug that is effective in nano-molar concentrations for cytokines suppression, we hypothesized it would be a potent stimulator of HIV-1 gene expression in latently infected cell lines. We also examined the effects of three analogues of ITF2357, with a higher affinity and specificity for HDAC-1. Because of the importance of the chemokine co-receptors for HIV-1 cell entry, we evaluated surface expression of CCR5 and CXCR4 on primary human mononuclear blood cells.

Materials and Methods

Results

Discussion

The reservoir of latently infected memory CD4+ T-cells represents a major obstacle in eliminating HIV-1 infection. Although consistent with reversing viral latency, HDAC inhibitors have not been fully exploited to purge the virus from the quiescent pool. In the present report, we demonstrate that ITF2357 exhibits a greater induction of HIV-1 than VPA, the only HDAC inhibitor that has been evaluated in vivo for HIV-1 purging. ITF2357 increased p24 production in a macrophage as well as T-cell line. Based on the present study, we propose that ITF2357 would be superior to VPA in clinical trials. Clearly, the next step is to compare viral induction of resting CD4+ T-cells from aviremic HIV-1 positive donors when exposed to ITF2357 or VPA in vitro.

There are three major advantages of ITF2357. First, at clinically achievable blood levels, ITF2357 induces at least a 10-fold increase in HIV-1 from latently infected cell in vitro compared to less than 2-fold for VPA. Second, oral ITF2357 is well tolerated in humans, including children³⁸. Third, ITF2357 did not increase CCR5 surface expression on CD4+ T-cells, despite the fact that HDAC inhibitors often increase gene expression. It would be counter-productive if agents stimulating latent virus up-regulated CCR5 and CXCR4 surface expression. In fact, surface expression of CXCR4 on CD4+ T-cells and CCR5 on monocytes was reduced by 50% by ITF2357. Although downregulation of CXCR4 by HDAC inhibitors has been observed in leukemic cells ⁴³, this property of ITF2357 was unexpected in primary blood monocytes and T-cells from healthy donors. Consistent with decreasing co-receptors surface expression, ITF2357 reduced steady-state mRNA levels of CXCR4 and CCR5 in PBMC, as measured by RT-PCR and also by gene chip expression, respectively. To our knowledge, this is the first time that this inhibitory role of HDAC inhibitors on CD4+ T-cells and monocytes has been demonstrated

One possible explanation for the decreased co-receptor gene expression is that ITF2357 directly inhibits transcription. Another possibility is that ITF2357 up-regulates transcriptional repressors. Although several pro-inflammatory cytokines such as tumor necrosis factor-α, interferon-γ, and interleukin-1β negatively regulate CXCR4 transcription⁴⁴, a cytokine-mediated mechanism is unlikely since ITF2357 decreases the synthesis of these cytokines induced by Toll-like receptor ligands in PBMCs³⁶.

Clinically, the cell surface density of co-receptors has a major role in HIV-1 infection. CCR5 density on CD4+ T-cells is an important driver of R5 HIV-1 replication ⁴⁵ and positively correlated with viral load ⁴⁶and disease progression⁴⁷. The density of CCR5 can also determine the susceptibility of macrophages and monocytes to infection by R5 strains^{48, 49}. The emergence of X4 strain is associated with disease progression and a rapid decline in CD4+ T-cells ⁵⁰ and a link between CXCR4 density and X4 emergence has been suggested. There is an ongoing issue whether residual viremia in HIV-1 patients under HAART represents new cycles of viral replication¹³ or virus output from stable reservoirs ⁵¹. These studies are based on viremia under steady state conditions. However, although treated, HIV-1 patients often have transient rebounds of viremia. Therefore, the reduction in co-receptor surface expression by ITF2357 is valuable if added to the HAART regimen.

Attractive as the concept of HIV-1 purging by HDAC inhibitors is, clinical trials using VPA did not reduce the size of the latent pool within resting CD4+ T cells. As the probability of side effects and particularly severe thrombocytopenia increases significantly at VPA plasma concentrations above 110μg/mL in females and 135μg/mL in males (see ⁵²for review). To induce more viral expression with higher concentrations of VPA would be impractical. As shown in the present study, ITF2357 is significantly more effective than VPA in inducing HIV-1 production from U1 and ACH2 cells. Another hydroxamic acid containing HDACi, SAHA, is also more potent than VPA for HIV-1 induction in vitro²⁷. The comparison of HDAC inhibition between ITF2357 and SAHA is shown in Table 1. In unpublished data from our laboratory, ITF2357 is 50-100 times more potent than SAHA in HIV-1 expression from U1 and ACH2 cells. Also, we describe here that two hydroxamic acid-containing analogues of ITF2357, ITFa and ITFc, at comparable nanomolar concentrations, induced p24 to higher levels compared to the parent compound.

HDACs are divided into 3 major classes. Class I is comprised of HDAC-1, 2, 3 and 8. Class II is comprised of HDAC 4, 5, 6, 7 and 10. HDAC-11 has properties of both class I and class II. The class III HDAC are not affected by HDAC inhibitors. Various pharmacological agents such as pyrrole-imidazole polyamides, trichostatin A, SAHA or phorbol esters effectively prevent the recruitment of HDAC-1 to the HIV-1 promoter allowing hyperacetylation of H3 and H4^{23, 53, 54}. In addition, a link between HDAC-2 and HDAC-3 and the HIV-1 LTR has been suggested^{55, 56}. As shown in Table 1, we compared the ability of four hydroxamic acid-containing HDAC inhibitors to reduce the enzymatic activity of HDACs by 50%. In addition, the ability of VPA to inhibit the same HDACs was determined in the same assays. In each case, the IC50 of each the four hydroxamic acid-containing HDAC inhibitors and VPA were compared to induction of HIV-1 in U1 and ACH2 cells.

In general, there was greater induction of HIV-1 expression with analogues with the lowest IC50 for Class I HDACs 1, 2 and 3. These observations are consistent with the molecular association between the HIV-1 LTR and HDACs 1, 2 and 3. In contrast, VPA is impressively a weak inhibitor of each HDAC compared to ITF2357 and analogues (Table 1). In fact, the IC50 for VPA for HDAC inhibition is not achievable clinically and therefore not surprisingly, VPA failed to efficiently induce HIV-1 expression in vitro in the present study. Thus testing the hypothesis of HDAC inhibition for HIV-1 purging from latently infected cells should be re-visited using HDAC inhibitors such as ITF2357 or its analogues with specificity and potency for class I HDACs.

Consistent with the present observations, Archin et al demonstrated that HDAC inhibitors specifically targeting class I HDACs induced HIV-1 gene expression from cell lines and resting CD4+ T cells of aviremic patients⁵⁷. An additive effect was seen with inhibition of HDAC-6. Our findings support those data regarding the selectivity of the class I HDACs 1, 2 and 3 inhibition and the potency of induction of HIV-1 gene expression. For example, in ACH2 cells, ITFc, a specific inhibitor of HDAC-1, 2 and 3 was twice as active in inducing p24 (35 fold) compared to ITF2357 (15-fold). However, inhibiton of HDAC-6 is inconsistent with the effectiveness of the three analogues, which were poor inhibitors of HDAC-6, particularly ITFc. Importantly, preserving the deacetylated status of HDAC-6 might protect CD4+ cells from HIV-1 infection and Env-mediated syncytia formation⁵⁸.

In the study by Archin et al, out of four HDAC inhibitors that were active in inducing HIV-1 gene expression in cells lines, only two recovered replication competent virus from resting CD4+ T cells of infected patients. We propose that from the data of the present study, ITF2357 or its analogues would be effective inducers of virus outgrowth from resting CD4+ cells of aviremic HIV-1 infected patients. The analogues have minor antiproliferative properties, which Archin et al suggested may interfere with maintaining the cell line in vitro potency in resting CD4+ of infected patients. However, since cell lines do not reflect the status HIV-1 of resting CD4+ T cells, measuring recovery of replication competent virus from these cells would clearly be necessary.

Safety is always a consideration when evaluating a drug in a disease with no immediate danger to the patient. In testing the hypothesis that HDAC inhibition will purge the latent pool of HIV-1, VPA falls short due to a low level of expression compared to ITF2357. Oral ITF2357 is safe and effective in humans. In healthy human subjects in a Phase I trial, a single dose of ITF2357 of 1.5mg/Kg resulted in a peak plasma level of 200nM ³⁷. In a Phase II trial in children with active SoJIA, a daily oral dose of ITF2357 at 1.5 mg/kg for 12 weeks exhibited no organ toxicity and achieved significant (p<0.01) reduction in parameters of systemic disease as well as the number of painful joints ³⁸.

In conclusion, this present study demonstrates that at clinically relevant doses, ITF2357 induces HIV-1 production from latently infected cell lines, and at the same time reduces the MFI of HIV-1 entry co-receptors. For targeting clearance of HIV-1 infection, ITF2357 might be an effective addition to the HAART regimen of HIV-1 patients.