BET bromodomain inhibition as a novel strategy for reactivation of HIV-1

Abstract

The persistence of latent HIV-1 remains a major challenge in therapeutic efforts to eradicate infection. We report the capacity for HIV reactivation by a selective small molecule inhibitor of BET family bromodomains, JQ1, a promising therapeutic agent with antioncogenic properties. JQ1 reactivated HIV transcription in models of latent T cell infection and latent monocyte infection. We also tested the effect of exposure to JQ1 to allow recovery of replication-competent HIV from pools of resting CD4(+) T cells isolated from HIV-infected, ART-treated patients. In one of three patients, JQ1 allowed recovery of virus at a frequency above unstimulated conditions. JQ1 potently suppressed T cell proliferation with minimal cytotoxic effect. Transcriptional profiling of T cells with JQ1 showed potent down-regulation of T cell activation genes, including CD3, CD28, and CXCR4, similar to HDAC inhibitors, but JQ1 also showed potent up-regulation of chromatin modification genes, including SIRT1, HDAC6, and multiple lysine demethylases (KDMs). Thus, JQ1 reactivates HIV-1 while suppressing T cell activation genes and up-regulating histone modification genes predicted to favor increased Tat activity. Thus, JQ1 may be useful in studies of potentially novel mechanisms for transcriptional control as well as in translational efforts to identify therapeutic molecules to achieve viral eradication.

Figure 1.. JQ1 reactivates HIV transcription in Ach2 T cells and U1 promonocytes.

(A) Two enantiomers of JQ1 have been described: the S and R forms [31]. Asterisks indicate the stereocenter at C6. (B) Ach2 T cells were treated with JQ1S at 500 nM for 24 h, followed by measurement of viral transcription with qRT-PCR. In all cases, the specific inhibitor JQ1 (S, 500 nM) but not the stereoisomer control JQ1 (R, 500 nM) dose-responsively reactivated HIV-1 RNA transcription (B and Supplemental Fig. 1 [37]). (C) Representative time course experiment indicating that maximal JQ1 stimulation (500 nM) of Ach2 cells occurred within 24 h. Similar results were seen with U1 cells (data not shown). All experiments were repeated three times. Error bars on graphs show sd between samples. All differences were found to be significant using a Student's t test to P < 0.05 for bar graphs.

Figure 2.. JQ1 reactivates HIV transcription in J-Lat T cell line.

The latent J-Lat 10.6 cell line was treated with mock (complete media), 500 nM JQ1S, or PHA/IL-2 as a reference control for 24 h, followed by analysis using flow cytometry. As indicated, stimulation with PHA and IL-2 or JQ1S resulted in an increase in GFP expression. Flow data are representative results of n = 5 experiments. SSC, Side-scatter.

Figure 3.. JQ1 effects in vitro and ex vivo using stimulated and resting CD4⁺ T cells.

(A) Ach2 T cells were treated with 0.5 ng/ml TNF-α for 1 h followed by treatment with JQ1S at 500 nM for 24 h. HIV transcription was augmented when JQ1S was added compared with TNF treatment alone (similar results were observed with PMA; data not shown). (B) Primary CD4 T cells were treated with PHA/IL-2 and virus for 3 days before being stimulated with JQ1S for an additional 24 h. JQ1 reproducibly augmented expression of HIV transcription as indicated. All in vitro experiments were repeated at least three times. Error bars on graphs show sd between samples. All differences were found to be significant using a Student's t test to P < 0.05 for bar graphs. (C) JQ1-mediated recovery of virus from pools of resting CD4⁺ T cells isolated from HIV-infected, ART-treated patients. In one of three patients, exposure to 1 μM JQ1 allowed recovery of virus above that seen in unstimulated culture conditions (IL-2, 20 U/ml).

Figure 4.. Influence of JQ1 on CD4⁺ T cell cytotoxicity and proliferation.

Cytoxicity was measured by flow cytometry analysis of 7AAD and Annexin V staining after primary T cell treatment with mock (complete media) or 500 nM JQ1S for 24 h. For proliferation measurements based on MTT assay, cells were mock-treated or treated with varying concentrations of JQ1S (500 nM, 50 nM, 5 nM). (A) Flow cytometric gating for uniform cells indicate that nearly all cells purified from whole blood by magnetic beads were CD4-positive. FSC-A, Forward-scatter-area. (B) Cellular gating strategy to identify apoptotic and necrotic cells. (C) Based on quadrant analysis, JQ1 negligibly increased early apoptosis (7AAD−Annexin V+ cells) and modestly increased late apoptosis/necrosis (7AAD+Annexin V+ cells) compared with a reference control staurosporine. (D) Primary T cell proliferation was dose-responsively suppressed by JQ1, consistent with previous reports of JQ1 in other cell types [31, 41]. For these experiments, n = 5; error bars show sd between samples, and all differences were found to be significant using ANOVA to P < 0.05.

Figure 5.. The top gene categories for induction and repression by α CD3/α CD28 and JQ1 in J-Lat T cells.

Clusters A and B, respectively, indicate the top-induced GO category “leukocyte activation” and top-repressed GO category “membrane-enclosed lumen” with αCD3/αCD28 stimulation. Clusters C and D, respectively, indicate the top-induced GO category “chromatin organization” and top-repressed GO category “lymphocyte activation” with JQ1S stimulation. Cluster E indicates selected HIV-associated genes that were influenced by JQ1S treatment. Transcripts validated by qRT-PCR in J-Lat 10.6 cells are indicated with a dark oval (CD28, MYC, HEXIM1).