Inhibition of HDAC3 induces outgrowth of HIV-1 from latently infected patient cells ex vivo.

<p>A. HIV-1 outgrowth from latently infected CD4+ T cells following overnight exposure to 15 µM BRD3308 or 335 nM SAHA. The frequency of viral outgrowth following mitogen stimulation of HDAC inhibitor exposure is expressed as infected cells per million resting CD4+ T cells (IUPM). In four patients (Pt 1, Pt 2, Pt 3, and Pt 4) exposure to BRD3308 induced a comparable frequency of outgrowth to that observed following exposure to SAHA. *IUPM for PHA for Pt.3 is obtained from three prior assays; all other assays are contemporaneous. B. PBMCs were exposed to 5, 10, 15, or 30 µM of BRD3308, and viability was measured. The results are expressed as the viability relative to the control DMSO condition. BRD3308 does not significantly affect viability of PBMCs.</p

IC50 values of BRD3308 and SAHA for HDACs 1–9.

<p>IC50 values of BRD3308 and SAHA for HDACs 1–9.</p

Chemical inhibition of HDACs following depletion of HDAC3 significantly increases expression from the HIV-1 promoter.

<p>A. 2D10 cell that had been depleted of HDAC1, −2, or −3 or had been infected with the scrambled control lentivirus were exposed to 0.015% DMSO as the vehicle control for 24 hours. B. Chemical inhibition of HDAC1 and −2 using Mrk 12 (20 µM) does not result in a significant increase in the percentage of 2D10 cells expressing GFP following depletion of HDAC1 or −2. However, similar to depletion of HDAC3 alone, a significant increase in the percent of cells expressing GFP was observed when Mrk 12 was added to cells depleted of HDAC3. C. Chemical inhibition of HDAC1, −2 and −3 with Mrk 13 (200 nM) resulted in a significant increase in the percent of GFP positive 2D10 cells in cells depleted of HDAC2 or −3, but not HDAC1. D. A submaximal (250 nM) concentration of SAHA resulted in a significant increase in the percent of cells expressing GFP following depletion of HDAC2 and −3. E. A maximal concentration of SAHA (500 nM) resulted in a significant increase in the percent of cells expressing GFP in cells depleted of HDAC3. However, in D. or E. depletion of HDAC1 did not increase the percent of cells expressing GFP. F. Chemical inhibition of HDACs with the non-selective inhibitor TSA (25 nM) resulted in a significant increase in the percent of GFP expressing 2D10 cells in cells depleted of HDAC3, but not HDAC1 or HDAC2. G. Chemical inhibition of HDAC3, −6, and −8 with the selective HDAC inhibitor Droxinostat (2 µM) resulted in a significant increase in the percent of 2D10 cells expressing GFP in cells that had been depleted of HDAC3 but not HDAC1 or −2. (*p<0.05).</p

Depletion of HDAC1, −2, and −3 significantly increases expression from the HIV-1 promoter.

<p>A. Fold change of HDAC1, −2, and −3 mRNA expression as compared to 2D10 cells transduced with the scrambled shRNA control. A significant reduction in the mRNA expression of all three HDACs was observed. B. Cell viability and proliferation as a percentage of the 2D10 cells transduced with the scrambled shRNA control. Depletion of HDAC1, −2, and −3 did not significantly affect cell viability and proliferation. C. A significant increase in the percentage of cells expressing GFP protein from the HIV-1 promoter was observed following depletion of HDAC1, −2, and −3. Furthermore, a significant increase in expression of GFP mRNA from the HIV-1 promoter was observed following depletion of HDAC1, −2, and −3. (*p<0.05).</p