Small Molecule Inhibitors of BAF; A Promising Family of Compounds in HIV-1 Latency Reversal

Abstract

Persistence of latently infected cells in presence of Anti-Retroviral Therapy presents the main obstacle to HIV-1 eradication. Much effort is thus placed on identification of compounds capable of HIV-1 latency reversal in order to render infected cells susceptible to viral cytopathic effects and immune clearance. We identified the BAF chromatin remodeling complex as a key player required for maintenance of HIV-1 latency, highlighting its potential as a molecular target for inhibition in latency reversal. Here, we screened a recently identified panel of small molecule inhibitors of BAF (BAFi's) for potential to activate latent HIV-1. Latency reversal was strongly induced by BAFi's Caffeic Acid Phenethyl Ester and Pyrimethamine, two molecules previously characterized for clinical application. BAFi's reversed HIV-1 latency in cell line based latency models, in two ex vivo infected primary cell models of latency, as well as in HIV-1 infected patient's CD4 + T cells, without inducing T cell proliferation or activation. BAFi-induced HIV-1 latency reversal was synergistically enhanced upon PKC pathway activation and HDAC-inhibition. Therefore BAFi's constitute a promising family of molecules for inclusion in therapeutic combinatorial HIV-1 latency reversal.

Keywords: BAF complex; BAF, BRG-Brahma Associated Factors; BAF250a, BAF Associated Factor 250 a; BAFi, BAF inhibitor; BRG-1, Brahma Related Gene 1; CAPE, caffeic acid phenetyl esther; ChIP, Chromatin Immunoprecipitation; Chromatin remodeling; CycA, Cyclophilin A; DHS-1, DNase Hypersensitive Site 1; ES cells, embryonic stem cells; FAIRE, Formaldehyde Assisted Isolation of Regulatory Elements; FBS, Fetal Bovine Serum; GFP, Green Fluorescent Protein; HDAC, histone deacetylase; HIV; HIV-1, human immunodeficiency virus type 1; IFNß, Interferon beta; IL10, Interleukin 10; IL12, Interleukin 12; IL4, Interleukin 4; IL6, Interleukin 6; INI-1, Integrase Interactor 1; IRES, Internal Ribosome Entry Site; IκB-α, Inhibitor of NF-κB – alpha; LRA, latency reversal agent; LTR, Long Terminal Repeat; Latency; MIP26, Major Intrinsic Protein; MMP9, Matrix Metallopeptidase 9; NF-κB, Nuclear Factor Kappa-light-chain-enhancer of activated B cells; PBMC, peripheral blood mononuclear cell; PBS, Phosphate Buffered Saline; PKC, Protein Kinase C; PYR, Pyrimethamine; RT-qPCR, Reverse Transcriptase, quantitative Polymerase Chain Reaction; SAHA, Suberoylanilide Hydroxamic Acid; SD, Standard Deviation; SOCS3, Suppressor Of Cytokine Signaling 3; TGF-ß, Transforming Growth Factor beta; TLR2, Toll-like Receptor 2; bp, base pairs; cART, combination Antiretroviral Therapy; latency reversal agents; nuc, nucleosome; siRNA, small interfering RNA.

Fig. 1

Small molecule Inhibitors of the BAF complex (BAFi's) re-activate latent HIV. J-Lat A2 (Panel a) and 11.1 (Panel b) cells were treated with BAFi's at 1 μM and 5 μM concentrations and re-activation quantitated at 24 and 48 h post treatment. Percent of GFP positive cells (left axes, dark colored bars), corresponding to the level of HIV-1 activation, and cell viability (right axes light colored bars) were evaluated by FACS analysis. GFP levels of samples in which cell viability was below 50% are not shown. HIV-1 activation was considered significant when GFP levels exceeded the average + 2SD of untreated controls (red horizontal line). Red squares identify BAFi's which induced activation of HIV-1 without significantly affecting cell viability. These compounds were further investigated for their ability to activate HIV-1 transcription in 11.1 cells (Panel c). Data are presented as mean ± SD.

Fig. 2

Treatment with BAFi's specifically modulates BAF target genes, and displaces the BAF complex from the latent HIV-1 LTR. (Panel a) Knockdown of BAF complex was obtained by nucleofecting J-Lat 11.1 cells with siRNA targeting the BAF complex specific subunit BAF250a. RNA levels of BAF250a were quantitated by RT PCR analysis, whereas protein levels of BAF250a as well as of the other subunits of the complex were determined by Western blot. RT-PCR analysis of J-Lat 11.1 cells after BAF250a knockdown (Panel b) or treatment with BAFi's A01 (1 μM), A11 (1 μM) and C09 (1 μM) for 18 h (Panel c). The effect of BAFi's treatment was monitored analyzing the changes in gene expression of HIV-1 LTR driven genes (GFP, GAG) and BAF target genes (TLR2, MMP9, IFNß, IL6, IL10, SOCS3). A set of BAF independent genes (CYCA2, MIP26, ß-ACTIN, ß-2-MICROGLOBULIN) was used as control. RT-qPCR data are presented as mean ± SD. (Panel d) BAFi treatment results in displacement of the BAF complex-specific subunit BAF250a from DHS1 and nuc-1 regions of the HIV-1 LTR. J-Lat 11.1 cells untreated or treated with BAFi's at indicated concentrations were subjected to ChIP using antibodies specific for the BAF250a subunit. Input and immunoprecipitated DNA were analyzed by PCR using primer pairs specific for the HIV-1 LTR nuc-0, DHS1, and nuc-1 regions and for a control region located upstream of the Axin gene promoter. ChIP results are presented as percent of immunoprecipitated material over input, error bars represent the standard deviation of three independent experiments. * indicates the level of significance at p < 0.05.

Fig. 3

Re-activation of latent HIV-1 by BAF inhibition is synergistically enhanced with co-treatment with HDACi's and PKC agonists. (Panel a) Percent of GFP positive cells was evaluated in BAF complex depleted J-Lat 11.1 cells 24 and 48 h after treatment with suboptimal concentrations of Prostratin and SAHA. (Panel b and c) J-Lat 11.1 cells were treated with BAFi's A01, A11 and C09 alone or together with SAHA and-or Prostratin. HIV-1 re-activation was monitored by measuring the percentage of cells expressing GFP: colored bars show the percent of GFP positive cells after treatment, whereas gray bars show cell viability. (Panel d) Levels of nucleosome occupancy of the HIV-1 5'LTR Nuc-1 region following treatment with BAFi's alone and in combination with Prostratin were analyzed using FAIRE assay. Data are presented as mean ± SD. The (^) symbol indicates a synergistic interaction between treatments.

Fig. 4

BAFi treatment re-activates latent HIV-1 in two distinct primary model systems of HIV-1 latency. Latency reversal activity of the BAFi's A11 and C09 was tested on two models of HIV-1 latency. Panel a (Lassen and Greene) and b (Bosque and Planelles) depict the protocols for latency establishment in primary human CD4 + T cells. FACS plots show the characteristic of the cells population at the moment of reactivation for each method. Dotplots (Panels c and d) show the fold increase in luciferase activity after treatment with different concentrations of A11 and C09. Each dot represents a single measurement, black horizontal lines show the average fold increase for each treatment. Experiments were performed in duplicate using cells obtained from 6 healthy blood donors. Panel e shows the levels of cell associated HIV-1 RNA in CD4 + T cells isolated from three virologically suppressed HIV-infected patients after treatment with the BAFi's C09 and A11. Cells obtained from Patient 1 and Patient 2 were further analyzed and treated with BAFi's in combination with Prostratin (Panel f). Cell associated HIV-1 RNA was measured as the number of copies of HIV-1 POL per μg of input RNA; for each condition, bars represent the average of experiments performed at least in triplicate. Statistical significance was calculated within patient, based on at least three independent replicates performed for each condition. Asterisks indicate the level of significance (* p < 0.05, ** p < 0.01, *** p < 0.001).

Fig. 5

BAF inhibitors do not induce T cell activation or proliferation. Percentage of cells expressing the markers of cell activation (CD25, panel a) and proliferation (KI67, panel b) in primary CD4 + T cells treated with A11 and C09. Treatment with PMA/Ionomycin was used as a positive control. Bars represent the average ± SD of experiments performed on samples deriving from three healthy blood donors. Images on the figure shows representative FACS plots for each marker.

Fig. 6

Inhibition of BAF prevents the establishment of latent HIV + infections. (Panel a) FACS plots show the protocol for quantification of latently infected cells. GFP-negative cells were sorted (left panel) five days after infections. Following sorting, the GFP-negative population (middle panel) was stimulated with PMA and the percentage of reactivated cells was measured 48 h after reactivation (right panel). (Panel b) Infection efficiency in untreated vs BAFi's treated samples was determined by FACS. Bars represent the percent of GFP positive cells (i.e. productive infections) 5 days after infection. (Panel c) Proviral load from the infected population was measured by Alu-PCR. Bars represent the relative amount of HIV-1 DNA compared to untreated control. (Panel d) GFP-negative population (containing uninfected as well as latently infected cells) was treated with PMA for 48 h. Bars represent the percentage of GFP positive cells (i.e. latently infected cells) for each treatment. Data are presented as mean ± SD, * indicates the level of significance at p < 0.05.