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SYNERGISTIC REACTIVATION OF LATENT HIV EXPRESSION BY INGENOL-3-ANGELATE, PEP005, TARGETED NF-kB SIGNALING IN COMBINATION WITH JQ1 INDUCED p-TEFB ACTIVATION

Sunday, 2nd of August 2015

“Our findings identify this anti-cancer drug, PEP005, as having a distinct mechanism of molecular signaling and as a potential candidate for advancing to future HIV eradication studies.”

SYNERGISTIC REACTIVATION OF LATENT HIV EXPRESSION BY INGENOL-3-ANGELATE, PEP005, TARGETED NF-kB SIGNALING IN COMBINATION WITH JQ1 INDUCED p-TEFB ACTIVATION

Excerpts below. Full text, with figures, is at http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1005066

Guochun Jiang et al.,

Published: July 30, 2015

Abstract

Although anti-retroviral therapy (ART) is highly effective in suppressing HIV replication, it fails to eradicate the virus from HIV-infected individuals. Stable latent HIV reservoirs are rapidly established early after HIV infection. Therefore, effective strategies for eradication of the HIV reservoirs are urgently needed. We report that ingenol-3-angelate (PEP005), the only active component in a previously FDA approved drug (PICATO) for the topical treatment of precancerous actinic keratosis, can effectively reactivate latent HIV in vitro and ex vivo with relatively low cellular toxicity. Biochemical analysis showed that PEP005 reactivated latent HIV through the induction of the pS643/S676-PKCδ/θ-IκBα/ε-NF-κB signaling pathway. Importantly, PEP005 alone was sufficient to induce expression of fully elongated and processed HIV RNAs in primary CD4+ T cells from HIV infected individuals receiving suppressive ART. Furthermore, PEP005 and the P-TEFb agonist, JQ1, exhibited synergism in reactivation of latent HIV with a combined effect that is 7.5-fold higher than the effect of PEP005 alone. Conversely, PEP005 suppressed HIV infection of primary CD4+ T cells through down-modulation of cell surface expression of HIV co-receptors. This anti-cancer compound is a potential candidate for advancing HIV eradication strategies.

Author Summary

Stable latent viral reservoirs in HIV infected individuals are rapidly reactivated following the interruption of anti-retroviral therapy (ART). Despite an early initiation of ART, viral reservoirs are established and persist as demonstrated in the case of the Mississippi baby and from recent studies of the SIV model of AIDS. Therefore, new strategies are needed for the eradication of the latent HIV reservoirs. We found that ingenol-3-angelate (PEP005), a member of the new class of anti-cancer ingenol compounds, effectively reactivated HIV from latency in primary CD4+ T cells from HIV infected individuals receiving ART. Importantly, a combination of PEP005 and JQ1, a p-TEFb agonist, reactivated HIV from latency at level on average 7.5-fold higher compared to PEP005 alone. The potency of synergistic effects of PEP005 and JQ1 provide novel opportunities for advancing HIV eradication strategies in the future. In summary, ingenols represent a new group of lead compounds for combating HIV latency.

Citation: Jiang G, Mendes EA, Kaiser P, Wong DP, Tang Y, Cai I, et al. (2015) Synergistic Reactivation of Latent HIV Expression by Ingenol-3-Angelate, PEP005, Targeted NF-kB Signaling in Combination with JQ1 Induced p-TEFb Activation. PLoS Pathog 11(7): e1005066. doi:10.1371/journal.ppat.1005066

Editor: Guido Silvestri, Emory University, UNITED STATES

Received: January 3, 2015; Accepted: July 6, 2015; Published: July 30, 2015

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This study is supported by NIH grants DK61297, AI43274, and UC Davis Research Investments in Science and Engineering (RISE) grant to SD and a post-doctoral fellowship from CAPES/Brazil (BEX 2951/12-6) to EAM. PK is supported by the Swiss National Science Foundation (PBZHP3_147260). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Anti-retroviral therapy (ART) is effective in suppressing HIV replication but it fails to eliminate latent viral reservoirs in HIV infected resting CD4+ T cells which, in blood, consist mainly of central and transitional memory CD4+ T cells [1–4]. Current ART options do not eradicate HIV from infected cells. In addition, these cells are invisible to the virus-specific immune responses in the setting of viral latency [5,6]. The viral reservoir is rapidly seeded and HIV latency might be established immediately after virus infection [7,8]. Despite initiation of ART in infants within hours of birth to HIV infected mothers, stable viral reservoirs were established and viral rebound occurred when therapy was interrupted [9]. In the simian immunodeficiency virus (SIV) model of AIDS, stable viral reservoirs are established within 2.5 days of infection [10]. The viral reactivation was detected in rhesus macaques following therapy interruption despite the initiation of ART at 3 days post SIV infection [10,11]. Collectively, these studies demonstrate that a very early initiation of ART may not be sufficient to prevent nor eliminate latent virus reservoirs [9,11,12]. It has been observed that the morbidity of HIV persistence in HIV-positive individuals on long-term ART includes drug toxicities and a higher risk of developing complications including dyslipidemia, cardiovascular disease and insulin resistance [13–15]. Therefore, a therapeutic cure of HIV is urgently needed that leads to viral eradication and experimental strategies for directly targeting HIV latent reservoirs are warranted.

Recent studies have explored an experimental strategy for viral eradication of HIV infected CD4+ T cells by activating HIV transcription and viral antigen expression from the latent viral reservoirs in the presence of ART [6]. This would lead to the detection and clearance of infected cells by the virus-specific host immune responses while the ART prevents new rounds of infection. Cytopathic effects of the viral reactivation would further increase the clearance of the latent viral reservoir. This “shock and kill” strategy was applied in a pilot clinical trial using the histone deacetylase (HDAC) inhibitor, vorinostat, in patients receiving suppressive ART [16–18]. The findings from these studies showed some promise but failed to result in significant clearance of residual HIV reservoirs. Potential mechanisms of this failure include the modest induction of HIV by this earlier generation of latency reversing agents (LRAs) used singly and due to immune defects in clearance of infected cells in spite of the reactivation of viral expression [19,20]. These studies demonstrate an urgent need for the development of new strategies both for disrupting HIV latency and facilitating elimination of infected cells after HIV expression is reactivated.

Several cell signaling pathways are critical for the establishment and maintenance of HIV latency [6,21,22]. Disruption of one or more of these pathways could lead to effective reactivation of HIV from latency. Various compounds have been tested for the disruption of HIV latency, and those inducing HIV reactivation from the viral long terminal repeat (LTR) through the stimulation of the protein kinase C (PKC)-NF-κB pathway showed high potency. These include phorbol esters (PMA and prostratin) and non-phorbol ester diterpenes (bryostatin and gnidimacrin) that induce NF-κB nuclear translocation and activation through the PKC pathway [22,23]. Some of these compounds effectively induce latent HIV reactivation in vitro at picomolar levels [24,25]. The LRAs, functioning through the PKC-NF-κB signaling, are able to reactivate latent HIV across a broad range of HIV latency models [20]. A recent study showed that LRAs stimulating PKC-NF-κB signaling may be most effective in inducing complete transcription of HIV from resting CD4+T cells of HIV infected individuals on suppressive ART [26]. Moreover, these compounds cause down-modulation of the expression of cell surface receptors, CD4, CXCR4 or CCR5, and protect cells against HIV infection [22]. Therefore, LRAs that activate PKC-NF-κB signaling are potential candidates for HIV cure studies. We previously reported that an ingenol ester, ingenol-3-hexanoate or IngB, is an excellent candidate for the reactivation of HIV from latency [24]. The modified ingenol-3-hexanoate was originally isolated from an Amazonian plant, Euphorbia tirucalli. It exerts low toxicity in CD4+ T cells and does not induce global T-cell activation. It caused reactivation of latent HIV at nanomolar levels [24]. However, since IngB induces expression and activation of both NF-κB and CyclinT1/CDK9, and stimulate IFNγ expression in primary CD4+ T cells, further search for new ingenol compounds with better HIV reactivation potential and lower cellular toxicity is needed [24,27,28].

Among previously identified ingenol compounds, ingenol-3-angelate (PEP005) is currently approved for clinical use. A recently FDA approved drug, PICATO, for the topical treatment of precancerous actinic keratosis contains ingenol-3-angelate as an active component [29]. A prior study suggested that ingenol-3-angelate could induce HIV expression from the U1 monocyte cell line harboring HIV genome [30]. In the current study, we report that PEP005 can effectively reactivate latent HIV through the activation of the pS643/S676-PKCδ/θ-IκBα/ε-NF-κB pathway in an HIV latency model in vitro but does not induce or increase NF-κB protein production by itself. It also reactivated full-length HIV transcription based on an assay targeting the poly A tail region of HIV transcripts in cells from ART-suppressed HIV-positive individuals, while exerting minimal toxicity and effects on T cell activation ex vivo [26]. Importantly, the effect of PEP005 was synergistic with JQ1, a p-TEFb activator, and the combination was highly potent in reactivating latent HIV expression both in vitro and ex vivo. Our findings identify this anti-cancer drug, PEP005, as having a distinct mechanism of molecular signaling and as a potential candidate for advancing to future HIV eradication studies.