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

Abstract

Objectives: Chromatin-associated repression is one mechanism that maintains HIV-1 latency. Inhibition of histone deacetylases (HDAC) reverses this repression resulting in viral expression from quiescently infected cells. Clinical studies with the HDAC inhibitor valproic acid (VPA) failed to substantially decrease the latent pool within resting CD4(+) cells. Here we compared the efficacy of ITF2357, an orally active and safe HDAC inhibitor, with VPA for HIV-1 expression from latently infected cells in vitro. We also evaluated the effect of ITF2357 on the surface expression of CXCR4 and CCR5.

Methods: Latently infected cell lines were incubated with either ITF2357 or VPA and p24 levels were measured. Peripheral blood mononuclear cells of uninfected donors were treated with ITF2357 and HIV-1 coreceptors expression was assessed by flow cytometry.

Results: At clinically relevant concentrations, ITF2357 increased p24 by 15-fold in ACH2 cells and by 9-fold in U1 cells, whereas VPA increased expression less than 2-fold. Analogues of ITF2357 primarily targeting HDAC-1 increased p24 up to 30-fold. In CD4(+) T cells treated with ITF2357, CXCR4 expression decreased by 54% (P < 0.001).

Conclusion: ITF2357 is superior to VPA in inducing HIV-1 from latently infected cells. Safely used in humans, ITF2357 is an attractive candidate for HIV-1 clinical purging.

Figure 1. HIV-1 expression in ACH2 and U1 cells stimulated by ITF2357 or VPA

(A) Mean ±SEM p24 pg/mL in ACH2 cells of 20 separate experiments. (B) Mean ±SEM p24 pg/mL in U1 cells of 22 separate experiments. Numbers above error bars indicate the mean fold change of cell death as determined by LDH cytotoxicity assay. The levels of LDH for each experiment without HDAC inhibitors were set at 1.0 and fold increases calculated. The brackets above the error bars indicate the range of therapeutic plasma levels for each HDAC inhibitors.

Figure 2. Time-dependent HIV-1 production in ACH2 cells exposed to VPA or ITF2357

Mean ±SEM fold-increase in levels of p24. The level of p24 for each experiment without HDAC inhibitors was set at 1.0 and fold increase values calculated. All experiments were performed in triplicate in three independent experiments. Dashed line represents control cultures set at 1.0. The brackets indicate the range of therapeutic plasma levels for each HDAC inhibitor. (A) p24 fold increase after incubation of 6 hours with either ITF2357 or VPA. (B) p24 fold increase after incubation of 12 hours. (C) p24 after 24 hours of incubation.

Figure 3. HIV-1 expression in ACH2 and U1 cells by ITF2357 and three analogues

p24 (A) Mean ±SEM p24 in pg/ml/mL in ACH2 cells of 17 separate experiments. (B) Mean ±SEM p24 in pg/ml/mL in U1 cells of 16 separate experiments. Numbers above error bars indicate the mean fold change of cell death as determined by LDH cytotoxicity assay. The levels of LDH for each experiment without HDAC inhibitors were set at 1.0 and mean fold increases calculated.

Figure 4. CXCR4 expression on peripheral blood lymphocytes

PBMC were treated with ITF2357 and VPA for either 4 or 24 hours. Percent of CXCR4 expressing cells was determined by four-color flow cytometry. The concentration for each HDACi are shown under each bar (I- ITF2357; V- VPA) . (A) Mean MFI ± SEM of CXCR4 on CD4+ T-cells after 4 hours (left) and 24 hours (right) incubation. The data are from seven different donors. (B) A representative experiment showing percent of maximal CXCR4 MFI of control cultures compared to 125nM and 250nM of ITF2357 at 4 (left) and 24 hours. * P<0.05; ** P<0.01. *** P<0.001 compared to control expression level.

Figure 5. Effect of HDACi on MFI of CCR5 in mononcytes

PBMC were treated with ITF2357 and VPA for 24 hours and then subjected to flow cytometry for CCR5 expression. The concentration of each HDACi are shown under each bar (I- ITF2357; V- VPA). (A) MFI ±SEM in 7 donors. (B) A representative experiment showing MFI of CCR5 in isotype control, control culture, ITF2357 125nM and 250nM. * P<0.05. ** P<0.01