Limits...
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 and JQ1 synergistically induce HIV transcription in primary CD4+ T cells from HIV infected individuals on suppressive ART.Primary CD4+ T cells were isolated from the peripheral blood of HIV positive individuals on suppressive ART and treated with 12 nM PEP005 alone or in combination with JQ1 for 6 or 48 hours, and HIV transcription was quantified using RT-qPCR for the 5’ LTR region or Poly A region of the virus. (A) Copy number of HIV RNA in one μg total RNA in the primary CD4+ T cells after reactivation with 12 nM PEP005. *, p<0.05. (B) PEP005 synergizes with JQ1 to significantly increase HIV mRNA expression in primary CD4+ T cells isolated from patients under suppressive ART. The Bliss independence model was utilized for calculation of synergy for LRA combinations [35] (See materials and method). Dotted horizontal line signifies pure additive effect (∆faxy = 0). For these analyses, we included all data for which every parameter for the synergy analysis was available and excluded individual cases where the parameters were not met. Synergy is defined as ∆faxy>0 while ∆faxy<0 indicates antagonism. Statistical significance was determined using a one tailed paired ratio t-test comparing predicted and observed drug combination effects. *p < 0.05; **p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4520526&req=5

ppat.1005066.g011: PEP005 and JQ1 synergistically induce HIV transcription in primary CD4+ T cells from HIV infected individuals on suppressive ART.Primary CD4+ T cells were isolated from the peripheral blood of HIV positive individuals on suppressive ART and treated with 12 nM PEP005 alone or in combination with JQ1 for 6 or 48 hours, and HIV transcription was quantified using RT-qPCR for the 5’ LTR region or Poly A region of the virus. (A) Copy number of HIV RNA in one μg total RNA in the primary CD4+ T cells after reactivation with 12 nM PEP005. *, p<0.05. (B) PEP005 synergizes with JQ1 to significantly increase HIV mRNA expression in primary CD4+ T cells isolated from patients under suppressive ART. The Bliss independence model was utilized for calculation of synergy for LRA combinations [35] (See materials and method). Dotted horizontal line signifies pure additive effect (∆faxy = 0). For these analyses, we included all data for which every parameter for the synergy analysis was available and excluded individual cases where the parameters were not met. Synergy is defined as ∆faxy>0 while ∆faxy<0 indicates antagonism. Statistical significance was determined using a one tailed paired ratio t-test comparing predicted and observed drug combination effects. *p < 0.05; **p < 0.01.

Mentions: Our data showed that PEP005 used in combination with JQ1 resulted in synergic reactivation of HIV expression in both the J-Lat A1 cells and the U1 cell models of HIV latency in vitro (Fig 2). This prompted us to examine whether the combination of JQ1 and PEP005 would significantly enhance the ex vivo induction of latent HIV expression in primary CD4+ T cells from HIV infected individuals on suppressive ART. HIV transcription was measured by RT-qPCR following the treatment of cells with 12 nM PEP005 alone, 2 μM JQ1 alone, or 12 nM PEP005 plus 2 μM JQ1 for 6 hrs or 48 hrs. After 6 hrs of stimulation, a strong combined effect of PEP005 and JQ1 induced transcription of HIV RNA using 5’ LTR assay was seen in CD4+ T cell samples from all individuals except one. When transcripts were measured with assay for the HIV poly A region, an enhanced combination effect was seen in 6 of 8 HIV infected individuals. In donor 1 and donor 9, the amount of cDNA was only sufficient to measure HIV RNA with either the Poly A region or LTR region assay (Fig 9). The combination treatment at 48 hrs was similarly more potent compared to PEP005 treatment alone in 6 of 7 patient samples when assessed by assay of HIV 5’ LTR region (up to 10 fold increase) and in all 6 patient samples by assay of the Poly A region of the HIV genome (1.6 to 14 fold increase). In donor 9, the amount of cDNA was only sufficient for the Poly A region assay (Fig 10). When analyzed as HIV RNA copy number in 1 μg of total RNA from CD4+ T cells, PEP005 significantly induced latent HIV reactivation compared with control treatment after 6 hr incubation, and combined treatment of PEP005 with JQ1 further significantly induced full-length latent HIV reactivation compared with PEP005 treatment alone after 6 or 48 hr incubation (*, p<0.05; ** p<0.01, Fig 11A). Applying the Bliss independence model for combined drug effects, criteria for synergic ex vivo reactivation of full length latent HIV transcription by PEP005 and JQ1 were met with the caveat that this assessment does not determine whether these viral transcripts were translated into viral proteins of viral particles (Fig 11B). Taken together, PEP005 exerted a synergic effect with JQ1 on reactivation of HIV transcription from latency in both cell line models and from CD4+ T cells obtained from patients on suppressive ART that included expression of fully elongated and processed HIV RNAs.


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 and JQ1 synergistically induce HIV transcription in primary CD4+ T cells from HIV infected individuals on suppressive ART.Primary CD4+ T cells were isolated from the peripheral blood of HIV positive individuals on suppressive ART and treated with 12 nM PEP005 alone or in combination with JQ1 for 6 or 48 hours, and HIV transcription was quantified using RT-qPCR for the 5’ LTR region or Poly A region of the virus. (A) Copy number of HIV RNA in one μg total RNA in the primary CD4+ T cells after reactivation with 12 nM PEP005. *, p<0.05. (B) PEP005 synergizes with JQ1 to significantly increase HIV mRNA expression in primary CD4+ T cells isolated from patients under suppressive ART. The Bliss independence model was utilized for calculation of synergy for LRA combinations [35] (See materials and method). Dotted horizontal line signifies pure additive effect (∆faxy = 0). For these analyses, we included all data for which every parameter for the synergy analysis was available and excluded individual cases where the parameters were not met. Synergy is defined as ∆faxy>0 while ∆faxy<0 indicates antagonism. Statistical significance was determined using a one tailed paired ratio t-test comparing predicted and observed drug combination effects. *p < 0.05; **p < 0.01.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4520526&req=5

ppat.1005066.g011: PEP005 and JQ1 synergistically induce HIV transcription in primary CD4+ T cells from HIV infected individuals on suppressive ART.Primary CD4+ T cells were isolated from the peripheral blood of HIV positive individuals on suppressive ART and treated with 12 nM PEP005 alone or in combination with JQ1 for 6 or 48 hours, and HIV transcription was quantified using RT-qPCR for the 5’ LTR region or Poly A region of the virus. (A) Copy number of HIV RNA in one μg total RNA in the primary CD4+ T cells after reactivation with 12 nM PEP005. *, p<0.05. (B) PEP005 synergizes with JQ1 to significantly increase HIV mRNA expression in primary CD4+ T cells isolated from patients under suppressive ART. The Bliss independence model was utilized for calculation of synergy for LRA combinations [35] (See materials and method). Dotted horizontal line signifies pure additive effect (∆faxy = 0). For these analyses, we included all data for which every parameter for the synergy analysis was available and excluded individual cases where the parameters were not met. Synergy is defined as ∆faxy>0 while ∆faxy<0 indicates antagonism. Statistical significance was determined using a one tailed paired ratio t-test comparing predicted and observed drug combination effects. *p < 0.05; **p < 0.01.
Mentions: Our data showed that PEP005 used in combination with JQ1 resulted in synergic reactivation of HIV expression in both the J-Lat A1 cells and the U1 cell models of HIV latency in vitro (Fig 2). This prompted us to examine whether the combination of JQ1 and PEP005 would significantly enhance the ex vivo induction of latent HIV expression in primary CD4+ T cells from HIV infected individuals on suppressive ART. HIV transcription was measured by RT-qPCR following the treatment of cells with 12 nM PEP005 alone, 2 μM JQ1 alone, or 12 nM PEP005 plus 2 μM JQ1 for 6 hrs or 48 hrs. After 6 hrs of stimulation, a strong combined effect of PEP005 and JQ1 induced transcription of HIV RNA using 5’ LTR assay was seen in CD4+ T cell samples from all individuals except one. When transcripts were measured with assay for the HIV poly A region, an enhanced combination effect was seen in 6 of 8 HIV infected individuals. In donor 1 and donor 9, the amount of cDNA was only sufficient to measure HIV RNA with either the Poly A region or LTR region assay (Fig 9). The combination treatment at 48 hrs was similarly more potent compared to PEP005 treatment alone in 6 of 7 patient samples when assessed by assay of HIV 5’ LTR region (up to 10 fold increase) and in all 6 patient samples by assay of the Poly A region of the HIV genome (1.6 to 14 fold increase). In donor 9, the amount of cDNA was only sufficient for the Poly A region assay (Fig 10). When analyzed as HIV RNA copy number in 1 μg of total RNA from CD4+ T cells, PEP005 significantly induced latent HIV reactivation compared with control treatment after 6 hr incubation, and combined treatment of PEP005 with JQ1 further significantly induced full-length latent HIV reactivation compared with PEP005 treatment alone after 6 or 48 hr incubation (*, p<0.05; ** p<0.01, Fig 11A). Applying the Bliss independence model for combined drug effects, criteria for synergic ex vivo reactivation of full length latent HIV transcription by PEP005 and JQ1 were met with the caveat that this assessment does not determine whether these viral transcripts were translated into viral proteins of viral particles (Fig 11B). Taken together, PEP005 exerted a synergic effect with JQ1 on reactivation of HIV transcription from latency in both cell line models and from CD4+ T cells obtained from patients on suppressive ART that included expression of fully elongated and processed HIV RNAs.

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