Background: Histone deacetylase inhibitors (HDACi) induce cancer cell death through mitochondrial apoptosis, a pathway regulated by the Bcl-2 family of proteins. The extent to which HDACi alter apoptosis priming in non-neoplastic cells is unknown.
Methods: We used BH3 profiling, a flow cytometry-based mitochondrial phenotyping assay, to investigate the mitochondrial priming effects of activation stimuli on peripheral blood mononuclear cell (PBMC) subpopulations. PBMC isolated from HIV-uninfected and HIV-infected, treated, virologically suppressed participants – defined as HIV-1 plasma RNA levels <50 copies/mL for ≥1 year – were studied. Cells were incubated in the presence or absence of anti(a)-CD3/anti(a)-CD28 beads, vorinostat (SAHA), panobinostat (PNB), or romidepsin (RMD). Dose-ranging studies were performed and, after treatment with a standardized panel of death peptides, BH3 profiles were generated. To determine the selectivity of priming for latently HIV-infected cells, we used the J89 cell line in a series of dual HDACi and small-molecule Bcl-2 inhibitor experiments.
Results: Marked changes in the magnitude and phenotype of CD4+ T cell mitochondrial priming were observed after immune activation or exposure to PNB and RMD, compared to untreated HIV-infected PBMC. BH3 profiles varied as a function of drug type, dose, and duration of exposure. Increased priming in response to the peptide BAD and decreased permeabilization to the peptide NOXAA was observed with both transcriptional and immune activating stimuli, signifying an increased dependence on the anti-apoptotic Bcl-2 protein. The rank order of increased mitochondrial priming was RMD > PNB > a-CD3/a-CD28 beads >> SAHA. Similar magnitudes of priming occurred in central and transitional memory CD4+ T cell subsets. Increasing concentrations of the Bcl-2 inhibitors ABT-263 or -199 in the presence of RMD 20nM led to progressive losses of HIV-expressing J89 cells.
Conclusions: Histone deacetylase inhibition increased primary CD4+ T cell mitochondrial priming and cellular reliance on the anti-apoptotic protein Bcl-2. The mitochondrial phenotypes that we observed were specific to the immune activation or transcriptional re-activation stimulus being used. Combination HDACi and Bcl-2-inhibition selectively eliminated HIV-expressing cells in a latency cell line. Small molecule Bcl-2 inhibition merits further investigation as a targeted HIV eradication strategy.