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 5

Pan or selective targeting of BET protein BD domains alone or in combination with IAPi.

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Pan or selective targeting of BET protein BD domains alone or in combina...
(A) Dose-response curves for pan-BD and BD-selective BETi in the triple Jurkat model across n = 3 independent experiments. (B–D) Combination activity of IAPi and (B) pan-BETi, (C) iBET-BD1, or (D) iBET-BD2 in the triple Jurkat model. Representative of n = 4 independent experiments. Conditions without IAPi treatment were plotted as (A) 5 × 10–9 M or (B–D) 1 × 10–11 M for visualization on the log10 x axis. (E) Resting CD4+ T cell HIV gag caRNA (TBP normalized, except for PMA/i due to known TBP upregulation following PMA/i exposure, ref. 78, I) with parallel measurements of cell-associated p24 protein (F), and (G) culture medium p24 protein induction following 40 hours exposure of IAPi (100 nM AZD5582), pan-BETi (1 μM I-BET151), BD1- or BD2-selective BETi (2 μM), or combinations thereof compared with the positive control PMA/i. Horizontal black lines indicate (E) mean ± SEM or (F and G) geometric mean across all donors. (H) Total cellular ATP levels and (I) cellular viability following 40 hours drug exposure. Note that PMA/i viability with AO/PI staining may be an underestimate of viability due to the formation of large clusters of proliferating viable cells. (J) QVOA following IAPi and pan-BETi exposure relative to the positive control PHA/IL-2. Infectious unit per million resting CD4+ T cells (IUPM) for each condition represented as a percentage of the PHA IUPM for each donor (different shapes). Open shapes indicate no positive wells were detected. For QVOA, resting CD4+ T cells from donors E-2, G, and D-2 were evaluated (Supplemental Table 1). All error bars represent mean ± SEM. FDR-corrected P values for pairwise comparisons using Wilcoxon’s signed-rank tests are indicated as follows: *P < 0.05; **P < 0.01.