Combined noncanonical NF-κB agonism and targeted BET bromodomain inhibition reverse HIV latency ex vivo
Shane D. Falcinelli, … , Nancie M. Archin, David M. Margolis
Shane D. Falcinelli, … , Nancie M. Archin, David M. Margolis
Published April 15, 2022
Citation Information: J Clin Invest. 2022;132(8):e157281. https://doi.org/10.1172/JCI157281.
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Research Article AIDS/HIV Infectious disease

Combined noncanonical NF-κB agonism and targeted BET bromodomain inhibition reverse HIV latency ex vivo

Abstract

Latency reversal strategies for HIV cure using inhibitor of apoptosis protein (IAP) antagonists (IAPi) induce unprecedented levels of latent reservoir expression without immunotoxicity during suppressive antiretroviral therapy (ART). However, full targeting of the reservoir may require combinatorial approaches. A Jurkat latency model screen for IAPi combination partners demonstrated synergistic latency reversal with bromodomain (BD) and extraterminal domain protein inhibitors (BETi). Mechanistic investigations using CRISPR-CAS9 and single-cell RNA-Seq informed comprehensive ex vivo evaluations of IAPi plus pan-BET, bD-selective BET, or selective BET isoform targeting in CD4+ T cells from ART-suppressed donors. IAPi+BETi treatment resulted in striking induction of cell-associated HIV gag RNA, but lesser induction of fully elongated and tat-rev RNA compared with T cell activation–positive controls. IAPi+BETi resulted in HIV protein induction in bulk cultures of CD4+ T cells using an ultrasensitive p24 assay, but did not result in enhanced viral outgrowth frequency using a standard quantitative viral outgrowth assay. This study defines HIV transcriptional elongation and splicing as important barriers to latent HIV protein expression following latency reversal, delineates the roles of BET proteins and their BDs in HIV latency, and provides a rationale for exploration of IAPi+BETi in animal models of HIV latency.

Authors

Shane D. Falcinelli, Jackson J. Peterson, Anne-Marie W. Turner, David Irlbeck, Jenna Read, Samuel L.M. Raines, Katherine S. James, Cameron Sutton, Anthony Sanchez, Ann Emery, Gavin Sampey, Robert Ferris, Brigitte Allard, Simon Ghofrani, Jennifer L. Kirchherr, Caroline Baker, JoAnn D. Kuruc, Cynthia L. Gay, Lindsey I. James, Guoxin Wu, Paul Zuck, Inmaculada Rioja, Rebecca C. Furze, Rab K. Prinjha, Bonnie J. Howell, Ronald Swanstrom, Edward P. Browne, Brian D. Strahl, Richard M. Dunham, Nancie M. Archin, David M. Margolis

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Figure 6

26.

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26. HIV transcript profiling and p24 protein induction following IAPi an...
HIV transcript profiling and p24 protein induction following IAPi and/or BETi exposure in primary CD4+ T cells from aviremic donors. (A) Fold change values for different HIV transcripts normalized to μg RNA input in total CD4+ T cells following 8 hours of LRA stimulation. Each dot represents the average fold change over DMSO-treated cells for an individual donor. Donors (A-2, D-3, H-2, J, K-2) were tested for IAPi/pan-BETi (I-BET151) and single agents; 4 donors were tested for IAPi, pan-, and BD-selective-BETi (D-3, H-2, J, K-2) (Supplemental Table 1). For 1 donor there was a likely PCR amplification failure for the nef transcript. In 2 donors, there were insufficient cells to evaluate the HDACi suberoylanilide hydroxamic acid (SAHA) in parallel. Filled symbols represent conditions where there were no detectable transcripts above background and data were left censored at 5 copies/μg RNA input, based on the background digital droplet signal observed in no reverse transcriptase and no template control wells run for each donor on each plate for each primer/probe set. Horizontal bars indicate the median fold change across the donors tested for an indicated transcript/drug condition. (B) Bliss synergy indexes for IAPi+BETi drug combinations across different transcripts. Horizontal bars indicate the median index calculated across 4 to 5 donors depending on the transcript/drug condition. (C) Ultrasensitive p24 measurements in culture supernatants following 8 hours drug exposure and washout for 4 different donors (D-3, H-2, J, K-2).