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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.

Jiang G, Mendes EA, Kaiser P, Wong DP, Tang Y, Cai I, Fenton A, Melcher GP, Hildreth JE, Thompson GR, Wong JK, Dandekar S - PLoS Pathog. (2015)

Bottom Line: Therefore, effective strategies for eradication of the HIV reservoirs are urgently needed.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.This anti-cancer compound is a potential candidate for advancing HIV eradication strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of California, Davis, Davis, California, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

PEP005-induced HIV reactivation is mediated through PKCδ/θ-IκBα/ε-NF-ĸB signaling.(A) PEP005-induced HIV reactivation is suppressed by inhibition of the PKCδ/θ. J-Lat A1 cells were treated with 6 nM of PEP005 in the presence of 1, 2 or 5 μM of PKC inhibitor (PKCθ/δ inhibitor; Milipore/Calbiochem (539649)) and evaluated for GFP expression by RT-qPCR. (B) NF-ĸB inhibition partially suppresses PEP005-induced HIV reactivation in J-Lat A1 cells. J-Lat A1 cells were treated with 6 nM of PEP005 in the presence of 2.5 μM Bay 11–7082, an NF-ĸB inhibitor, and were evaluated for GFP expression by RT-qPCR. (C) J-Lat A1 cells were treated with PEP005 or alternatively with PKC inhibitor alone or in the presence of PEP005 and the relative binding of NF-ĸB to the HIV LTR was determined using ChIP-qPCR.
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ppat.1005066.g004: PEP005-induced HIV reactivation is mediated through PKCδ/θ-IκBα/ε-NF-ĸB signaling.(A) PEP005-induced HIV reactivation is suppressed by inhibition of the PKCδ/θ. J-Lat A1 cells were treated with 6 nM of PEP005 in the presence of 1, 2 or 5 μM of PKC inhibitor (PKCθ/δ inhibitor; Milipore/Calbiochem (539649)) and evaluated for GFP expression by RT-qPCR. (B) NF-ĸB inhibition partially suppresses PEP005-induced HIV reactivation in J-Lat A1 cells. J-Lat A1 cells were treated with 6 nM of PEP005 in the presence of 2.5 μM Bay 11–7082, an NF-ĸB inhibitor, and were evaluated for GFP expression by RT-qPCR. (C) J-Lat A1 cells were treated with PEP005 or alternatively with PKC inhibitor alone or in the presence of PEP005 and the relative binding of NF-ĸB to the HIV LTR was determined using ChIP-qPCR.

Mentions: Although it was shown that PEP005 can activate the PKC-NF-κB pathway, it is not known at which step of the PKC-NF-κB pathway is modulated during the reactivation of latent HIV. Therefore, we examined protein expression of several components of the PKC pathway in J-Lat A1 cells by Western blot analysis using antibodies specific for four PKC super families, including PKCμ/D, PKCα, PKCδ, and PKCθ. There was no significant induction of expression of these PKC proteins except for a modest up-regulation of PKCδ that was detected at 1 hr following 12nM PEP005 treatment. Moreover, rapid phosphorylation of Ser643/Ser676 in PKCδ/θ was induced by PEP005 with greater than a two-fold increase as early as 30 minutes post treatment (Fig 3A, 3B and 3C). These findings were further validated in PEP005 treated PBMCs from healthy donors. To investigate the involvement of the up-stream kinases in PKC-NF-κB signaling, Western blot analysis was performed using anti-phospho-IκB antibodies. Our data showed that PEP005 treatment induced phosphorylation of IκBα and IκBε, but not of IκBβ (Fig 3A and 3B). Interestingly, expression of NF-κB/p65 did not change in the presence of PEP005 (Fig 3A). This is clearly different from the effects of IngB treatment which involved an increased expression of NF-κB/p65 protein [24]. To further confirm the role of PKC-NF-κB signaling in reactivation of HIV latency, J-Lat A1 cells were treated with a PKCθ/δ inhibitor (Fig 4A). Our data showed that inhibition of PKCθ/δ resulted in a reduction of latent HIV reactivation by more than 65%. The addition of the NF-κB inhibitor, Bay-11-7082, to J-Lat A1 cells resulted in an approximately 50% reduction in PEP005-induced disruption of HIV latency (Fig 4B). To determine whether PEP005 reactivates latent HIV by promoting NFκB/p65 binding to the HIV LTR, ChIP-qPCR assays were performed with J-Lat A1 cells treated with 12 nM of PEP005 with or without PKCδ/θ inhibitor. PEP005 treatment resulted in a 6-fold increase in NF-κB/p65 binding to HIV LTR region (Fig 4C). This increase was reduced by more than 70% following the addition of PKC inhibitor. Collectively, our findings indicate that PEP005-induced reactivation of latent HIV most likely occurs through the PKCδ/θ-NF-κB signaling pathway. However, this does not exclude a possibility that other PKC isoforms are also potentially involved.


Synergistic Reactivation of Latent HIV Expression by Ingenol-3-Angelate, PEP005, Targeted NF-kB Signaling in Combination with JQ1 Induced p-TEFb Activation.

Jiang G, Mendes EA, Kaiser P, Wong DP, Tang Y, Cai I, Fenton A, Melcher GP, Hildreth JE, Thompson GR, Wong JK, Dandekar S - PLoS Pathog. (2015)

PEP005-induced HIV reactivation is mediated through PKCδ/θ-IκBα/ε-NF-ĸB signaling.(A) PEP005-induced HIV reactivation is suppressed by inhibition of the PKCδ/θ. J-Lat A1 cells were treated with 6 nM of PEP005 in the presence of 1, 2 or 5 μM of PKC inhibitor (PKCθ/δ inhibitor; Milipore/Calbiochem (539649)) and evaluated for GFP expression by RT-qPCR. (B) NF-ĸB inhibition partially suppresses PEP005-induced HIV reactivation in J-Lat A1 cells. J-Lat A1 cells were treated with 6 nM of PEP005 in the presence of 2.5 μM Bay 11–7082, an NF-ĸB inhibitor, and were evaluated for GFP expression by RT-qPCR. (C) J-Lat A1 cells were treated with PEP005 or alternatively with PKC inhibitor alone or in the presence of PEP005 and the relative binding of NF-ĸB to the HIV LTR was determined using ChIP-qPCR.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4520526&req=5

ppat.1005066.g004: PEP005-induced HIV reactivation is mediated through PKCδ/θ-IκBα/ε-NF-ĸB signaling.(A) PEP005-induced HIV reactivation is suppressed by inhibition of the PKCδ/θ. J-Lat A1 cells were treated with 6 nM of PEP005 in the presence of 1, 2 or 5 μM of PKC inhibitor (PKCθ/δ inhibitor; Milipore/Calbiochem (539649)) and evaluated for GFP expression by RT-qPCR. (B) NF-ĸB inhibition partially suppresses PEP005-induced HIV reactivation in J-Lat A1 cells. J-Lat A1 cells were treated with 6 nM of PEP005 in the presence of 2.5 μM Bay 11–7082, an NF-ĸB inhibitor, and were evaluated for GFP expression by RT-qPCR. (C) J-Lat A1 cells were treated with PEP005 or alternatively with PKC inhibitor alone or in the presence of PEP005 and the relative binding of NF-ĸB to the HIV LTR was determined using ChIP-qPCR.
Mentions: Although it was shown that PEP005 can activate the PKC-NF-κB pathway, it is not known at which step of the PKC-NF-κB pathway is modulated during the reactivation of latent HIV. Therefore, we examined protein expression of several components of the PKC pathway in J-Lat A1 cells by Western blot analysis using antibodies specific for four PKC super families, including PKCμ/D, PKCα, PKCδ, and PKCθ. There was no significant induction of expression of these PKC proteins except for a modest up-regulation of PKCδ that was detected at 1 hr following 12nM PEP005 treatment. Moreover, rapid phosphorylation of Ser643/Ser676 in PKCδ/θ was induced by PEP005 with greater than a two-fold increase as early as 30 minutes post treatment (Fig 3A, 3B and 3C). These findings were further validated in PEP005 treated PBMCs from healthy donors. To investigate the involvement of the up-stream kinases in PKC-NF-κB signaling, Western blot analysis was performed using anti-phospho-IκB antibodies. Our data showed that PEP005 treatment induced phosphorylation of IκBα and IκBε, but not of IκBβ (Fig 3A and 3B). Interestingly, expression of NF-κB/p65 did not change in the presence of PEP005 (Fig 3A). This is clearly different from the effects of IngB treatment which involved an increased expression of NF-κB/p65 protein [24]. To further confirm the role of PKC-NF-κB signaling in reactivation of HIV latency, J-Lat A1 cells were treated with a PKCθ/δ inhibitor (Fig 4A). Our data showed that inhibition of PKCθ/δ resulted in a reduction of latent HIV reactivation by more than 65%. The addition of the NF-κB inhibitor, Bay-11-7082, to J-Lat A1 cells resulted in an approximately 50% reduction in PEP005-induced disruption of HIV latency (Fig 4B). To determine whether PEP005 reactivates latent HIV by promoting NFκB/p65 binding to the HIV LTR, ChIP-qPCR assays were performed with J-Lat A1 cells treated with 12 nM of PEP005 with or without PKCδ/θ inhibitor. PEP005 treatment resulted in a 6-fold increase in NF-κB/p65 binding to HIV LTR region (Fig 4C). This increase was reduced by more than 70% following the addition of PKC inhibitor. Collectively, our findings indicate that PEP005-induced reactivation of latent HIV most likely occurs through the PKCδ/θ-NF-κB signaling pathway. However, this does not exclude a possibility that other PKC isoforms are also potentially involved.

Bottom Line: Therefore, effective strategies for eradication of the HIV reservoirs are urgently needed.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.This anti-cancer compound is a potential candidate for advancing HIV eradication strategies.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, University of California, Davis, Davis, California, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus