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.

Pulmonary hypertension is a co-morbidity, which strongly participates in morbi-mortality in patients with chronic obstructive pulmonary disease (COPD). Recent findings showed that bromodomain-containing proteins, in charge of reading histone acetylation, could be involved in pulmonary arterial hypertension. Our aim was to study the effect of I-BET151, an inhibitor of bromodomain and extra-terminal domain (BET), on the right ventricle hypertrophy and pulmonary hypertension, induced by a combination of chronic hypoxia and pulmonary inflammation, as the two main stimuli encountered in COPD. Adult Wistar male rats, exposed to chronic hypoxia plus pulmonary inflammation (CHPI), showed a significant right ventricle hypertrophy (+57%, p < 0.001), an increase in systolic pressure (+46%, p < 0.001) and in contraction speed (+36%, p < 0.001), when compared to control animals. I-BET151 treated animals (CHPI-iB) showed restored hemodynamic parameters to levels similar to control animals, despite chronic hypoxia plus exposure to pulmonary inflammation. They displayed lower right ventricle hypertrophy and hematocrit compared to the CHPI group (respectively −16%, p < 0.001; and −9%, p < 0.05). Our descriptive study shows a valuable effect of the inhibition of bromodomain and extra-terminal domain proteins on hemodynamic parameters, despite the presence of chronic hypoxia and pulmonary inflammation. This suggests that such inhibition could be of potential interest for COPD patients with pulmonary hypertension. Further studies are needed to unravel the underlying mechanisms involved and the net benefits of inhibiting adaptations to chronic hypoxia.

Background: Myeloid cells are critical determinants of the sustained inflammation in Crohn’s Disease (CD). Targeting such cells may be an effective therapeutic approach for refractory CD patients. Bromodomain and extra-terminal domain protein inhibitors (iBET) are potent anti-inflammatory agents; however, they also possess wide-ranging toxicities. In the current study, we make use of a BET inhibitor containing an esterase sensitive motif (ESM-iBET), which is cleaved by carboxylesterase-1 (CES1), a highly expressed esterase in mononuclear myeloid cells. Methods: We profiled CES1 protein expression in the intestinal biopsies, peripheral blood, and CD fistula tract (fCD) cells of CD patients using mass cytometry. The anti-inflammatory effect of ESM-iBET or its control (iBET) were evaluated in healthy donor CD14⁺ monocytes and fCD cells, using cytometric beads assay or RNA-sequencing. Results: CES1 was specifically expressed in monocyte, macrophage, and dendritic cell populations in the intestinal tissue, peripheral blood, and fCD cells of CD patients. ESM-iBET inhibited IL1β, IL6, and TNFα secretion from healthy donor CD14⁺ monocytes and fCD immune cells, with 10- to 26-fold more potency over iBET in isolated CD14⁺ monocytes. Transcriptomic analysis revealed that ESM-iBET inhibited multiple inflammatory pathways, including TNF, JAK-STAT, NF-kB, NOD2, and AKT signaling, with superior potency over iBET. Conclusions: We demonstrate specific CES1 expression in mononuclear myeloid cell subsets in peripheral blood and inflamed tissues of CD patients. We report that low dose ESM-iBET accumulates in CES1-expressing cells and exerts robust anti-inflammatory effects, which could be beneficial in refractory CD patients.

Aims: To improve the tolerability and therapeutic application of histone deacetylase inhibitors (HDACi), by application of an esterase‐sensitive motif (ESM), to target pharmacological activity directly to mononuclear myeloid cells expressing the processing enzyme carboxylesterase‐1 (CES1).Methods: This first‐in‐human study comprised single and multiple ascending dose cohorts to determine safety and tolerability. Pharmacodynamic parameters included acetylation, cytokine inhibition and intracellular concentrations of processed acid metabolite in isolated monocytes. Mechanistic work was conducted in vitro and in a CES1/Es1e^lo mouse strain.Results: ESM‐HDAC391 showed transient systemic exposure (plasma half‐life of 21–30 min) but selective retention of processed acid for at least 12 hours, resulting in robust targeted mechanistic engagement (increased acetylation in monocytes plus inhibition of ex vivo stimulated cytokine production). ESM‐HDAC391 was well tolerated and clinical toxicities common to non‐targeted HDACi were not observed. ESM‐HDAC391 treatment was accompanied by the novel finding of a dose‐dependent monocyte depletion that was transient and reversible and which plateaued at 0.06 × 10⁹ monocytes/L after repeat dosing with 20 or 40 mg. Characterisation of monocyte depletion in transgenic mice (CES1/Es1e^lo) suggested that colony stimulating factor 1 receptor loss on circulating cells contributed to ESM‐HDAC‐mediated depletion. Further mechanistic investigations using human monocytes in vitro demonstrated HDACi‐mediated change in myeloid fate through modulation of colony stimulating factor 1 receptor and downstream effects on cell differentiation.Conclusion: These findings demonstrate selective targeting of monocytes in humans using the ESM approach and identify monocytopaenia as a novel outcome of ESM‐HDACi treatment, with implications for potential benefit of these molecules in myeloid‐driven diseases.

AIM: To improve the tolerability and therapeutic application of histone deacetylase inhibitors (HDACi), by application of an esterase-sensitive motif (ESM), to target pharmacological activity directly to mononuclear myeloid cells expressing the processing enzyme carboxylesterase-1 (CES1). METHODS. This first-in-human study comprised of single and multiple ascending dose cohorts to determine safety and tolerability. Pharmacodynamic parameters included acetylation, cytokine inhibition and intracellular concentrations of processed acid metabolite in isolated monocytes. Mechanistic work was conducted in vitro and in a CES1/Es1e ^lo mouse strain. RESULTS. ESM-HDAC391 was well tolerated whilst showing robust targeted mechanistic engagement, as demonstrated by selective retention of compound and increased acetylation in monocytes plus inhibition of ex vivo stimulated cytokine production. Importantly, common clinical HDACi toxicities were not observed. ESM-HDAC391 treatment was accompanied by the novel finding of a dose-dependent monocyte depletion that was transient and reversible. In-depth characterisation of monocyte depletion in a transgenic mouse model ( CES1/Es1e ^lo ) suggested that CSF1R loss on circulating cells contributes to ESM-HDAC-mediated depletion. Further mechanistic investigations using human monocytes in vitro demonstrated HDACi-mediated change in myeloid fate through modulation of CSF1R and downstream effects on cell differentiation. CONCLUSION. These findings demonstrate selective targeting of monocytes in humans using the ESM approach and identify monocytopaenia as a novel outcome of ESM-HDACi treatment, with implications for the potential benefit of these molecules in myeloid-driven diseases.