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• W793746453 abstract "Research Article17 December 2015Open Access Sequential treatment with 5-aza-2′-deoxycytidine and deacetylase inhibitors reactivates HIV-1 Sophie Bouchat Sophie Bouchat Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Nadège Delacourt Nadège Delacourt Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Anna Kula Anna Kula Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Gilles Darcis Gilles Darcis Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Service des Maladies Infectieuses, Centre Hospitalier Universitaire (CHU) de Liège, Domaine Universitaire du Sart-Tilman, Université de Liège, Liège, Belgium Search for more papers by this author Benoit Van Driessche Benoit Van Driessche Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Francis Corazza Francis Corazza Laboratory of Immunology, IRISLab, CHU-Brugmann, Université Libre de Bruxelles (ULB), Brussels, Belgium Search for more papers by this author Jean-Stéphane Gatot Jean-Stéphane Gatot Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Adeline Melard Adeline Melard Service de Virologie, EA7327, AP-HP, Hôpital Necker-Enfants-Malades, Université Paris-Descartes, Paris, France Search for more papers by this author Caroline Vanhulle Caroline Vanhulle Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Kabamba Kabeya Kabamba Kabeya Service des Maladies Infectieuses, CHU St-Pierre, Université Libre de Bruxelles (ULB), Brussels, Belgium Search for more papers by this author Marion Pardons Marion Pardons Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Véronique Avettand-Fenoel Véronique Avettand-Fenoel Service de Virologie, EA7327, AP-HP, Hôpital Necker-Enfants-Malades, Université Paris-Descartes, Paris, France Search for more papers by this author Nathan Clumeck Nathan Clumeck Service des Maladies Infectieuses, CHU St-Pierre, Université Libre de Bruxelles (ULB), Brussels, Belgium Search for more papers by this author Stéphane De Wit Stéphane De Wit Service des Maladies Infectieuses, CHU St-Pierre, Université Libre de Bruxelles (ULB), Brussels, Belgium Search for more papers by this author Olivier Rohr Olivier Rohr IUT Louis Pasteur de Schiltigheim, University of Strasbourg, Schiltigheim, France Institut Universitaire de France (IUF), Paris, France Search for more papers by this author Christine Rouzioux Christine Rouzioux Service de Virologie, EA7327, AP-HP, Hôpital Necker-Enfants-Malades, Université Paris-Descartes, Paris, France Search for more papers by this author Carine Van Lint Corresponding Author Carine Van Lint Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Sophie Bouchat Sophie Bouchat Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Nadège Delacourt Nadège Delacourt Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Anna Kula Anna Kula Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Gilles Darcis Gilles Darcis Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Service des Maladies Infectieuses, Centre Hospitalier Universitaire (CHU) de Liège, Domaine Universitaire du Sart-Tilman, Université de Liège, Liège, Belgium Search for more papers by this author Benoit Van Driessche Benoit Van Driessche Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Francis Corazza Francis Corazza Laboratory of Immunology, IRISLab, CHU-Brugmann, Université Libre de Bruxelles (ULB), Brussels, Belgium Search for more papers by this author Jean-Stéphane Gatot Jean-Stéphane Gatot Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Adeline Melard Adeline Melard Service de Virologie, EA7327, AP-HP, Hôpital Necker-Enfants-Malades, Université Paris-Descartes, Paris, France Search for more papers by this author Caroline Vanhulle Caroline Vanhulle Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Kabamba Kabeya Kabamba Kabeya Service des Maladies Infectieuses, CHU St-Pierre, Université Libre de Bruxelles (ULB), Brussels, Belgium Search for more papers by this author Marion Pardons Marion Pardons Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Véronique Avettand-Fenoel Véronique Avettand-Fenoel Service de Virologie, EA7327, AP-HP, Hôpital Necker-Enfants-Malades, Université Paris-Descartes, Paris, France Search for more papers by this author Nathan Clumeck Nathan Clumeck Service des Maladies Infectieuses, CHU St-Pierre, Université Libre de Bruxelles (ULB), Brussels, Belgium Search for more papers by this author Stéphane De Wit Stéphane De Wit Service des Maladies Infectieuses, CHU St-Pierre, Université Libre de Bruxelles (ULB), Brussels, Belgium Search for more papers by this author Olivier Rohr Olivier Rohr IUT Louis Pasteur de Schiltigheim, University of Strasbourg, Schiltigheim, France Institut Universitaire de France (IUF), Paris, France Search for more papers by this author Christine Rouzioux Christine Rouzioux Service de Virologie, EA7327, AP-HP, Hôpital Necker-Enfants-Malades, Université Paris-Descartes, Paris, France Search for more papers by this author Carine Van Lint Corresponding Author Carine Van Lint Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium Search for more papers by this author Author Information Sophie Bouchat1, Nadège Delacourt1, Anna Kula1, Gilles Darcis1,2, Benoit Van Driessche1, Francis Corazza3, Jean-Stéphane Gatot1,8, Adeline Melard4, Caroline Vanhulle1, Kabamba Kabeya5, Marion Pardons1, Véronique Avettand-Fenoel4, Nathan Clumeck5, Stéphane De Wit5, Olivier Rohr6,7, Christine Rouzioux4 and Carine Van Lint 1 1Service of Molecular Virology, Department of Molecular Biology (DBM), Université Libre de Bruxelles (ULB), Gosselies, Belgium 2Service des Maladies Infectieuses, Centre Hospitalier Universitaire (CHU) de Liège, Domaine Universitaire du Sart-Tilman, Université de Liège, Liège, Belgium 3Laboratory of Immunology, IRISLab, CHU-Brugmann, Université Libre de Bruxelles (ULB), Brussels, Belgium 4Service de Virologie, EA7327, AP-HP, Hôpital Necker-Enfants-Malades, Université Paris-Descartes, Paris, France 5Service des Maladies Infectieuses, CHU St-Pierre, Université Libre de Bruxelles (ULB), Brussels, Belgium 6IUT Louis Pasteur de Schiltigheim, University of Strasbourg, Schiltigheim, France 7Institut Universitaire de France (IUF), Paris, France 8Present address: Service de Génétique, Centre Hospitalier Universitaire (CHU) de Liège, Domaine Universitaire du Sart-Tilman, Liège, Belgium *Corresponding author. Tel: +32 2 650 98 07; Fax: +32 2 650 98 00; E-mail: [email protected] EMBO Mol Med (2016)8:117-138https://doi.org/10.15252/emmm.201505557 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Reactivation of HIV gene expression in latently infected cells together with an efficient cART has been proposed as an adjuvant therapy aimed at eliminating/decreasing the reservoir size. Results from HIV clinical trials using deacetylase inhibitors (HDACIs) question the efficiency of these latency-reversing agents (LRAs) used alone and underline the need to evaluate other LRAs in combination with HDACIs. Here, we evaluated the therapeutic potential of a demethylating agent (5-AzadC) in combination with clinically tolerable HDACIs in reactivating HIV-1 from latency first in vitro and next ex vivo. We showed that a sequential treatment with 5-AzadC and HDACIs was more effective than the corresponding simultaneous treatment both in vitro and ex vivo. Interestingly, only two of the sequential LRA combinatory treatments tested induced HIV-1 particle recovery in a higher manner than the drugs alone ex vivo and at concentrations lower than the human tolerable plasmatic concentrations. Taken together, our data reveal the benefit of using combinations of 5-AzadC with an HDACI and, for the first time, the importance of treatment time schedule for LRA combinations in order to reactivate HIV. Synopsis Sequential administration of latency-reversing agents (LRAs), namely deacetylase inhibitors (HDACIs) and demethylating agents, together with an efficient cART, could represent an adjuvant anti-HIV-1 therapy to induce viral production and possibly reduce the size of latent HIV-1 reservoirs. A sequential treatment with 5-AzadC and HDACIs is more effective both in vitro and ex vivo at inducing HIV gene expression than the corresponding simultaneous treatments, highlighting for the first time the importance of treatment schedule for LRAs combinations. Combining 5-AzadC + panobinostat and 5-AzadC + romidepsin show reactivation potentials at concentrations lower than the human tolerable plasmatic concentrations. These findings are valuable for designing future anti-latency therapeutic strategies and thereby constitute a step forward in achieving HIV remission. Introduction Thirty years after its discovery, human immunodeficiency virus type 1 (HIV-1) remains a major problem of public health. Combination antiretroviral therapy (cART) is potent but not curative. cART requires lifelong adherence and does not fully restore health or a normal immune status in HIV-1-infected individuals. Although multiple reservoirs may exist, the HIV-1 reservoirs containing stably integrated, transcriptionally silent but replication-competent proviruses are recognized to predominate among infected CD4+ T cells (Eisele & Siliciano, 2012). They are therefore a permanent source for virus reactivation and could be responsible for the rebound of plasma viremia observed after cART interruption (Tyagi & Bukrinsky, 2012). Persistence of truly latent (i.e. non-defective) HIV-1 proviruses represents a major obstacle to eradication, as suggested by the failure of cART intensification strategies at clearing the viral reservoirs (Dinoso et al, 2009; Gandhi et al, 2010; Yukl et al, 2010). Indeed, the levels of HIV-1 reservoirs appear as one of the critical factors influencing the duration of a remission after cART cessation (Saez-Cirion et al, 2013). Consequently, a decline of the HIV-1 latent reservoirs to a level sufficient to permit an efficient control of the infection by the host immune system might allow interruptions in therapy (“treatment-free windows”). Reactivation of HIV gene expression in latently infected cells together with an efficient or intensified cART could serve as an adjuvant therapy aimed at eliminating/decreasing the pool of latent viral reservoirs. The chromatin organization and the epigenetic control of the HIV-1 promoter are key elements in transcriptional silencing (Van Lint et al, 2013). The repressive nucleosome nuc-1, located immediately downstream of the transcription start site, is maintained hypoacetylated by histone deacetylases (HDACs) in latent conditions (Verdin et al, 1993; Van Lint et al, 1996). The use of HDAC inhibitors (HDACIs) as latency-reversing agents (LRAs) has been well characterized in several latency models and in ex vivo cART-treated HIV-1+ patient cell cultures (Quivy et al, 2002; Archin et al, 2009; Contreras et al, 2009; Reuse et al, 2009; Matalon et al, 2010). Several anti-HIV latency clinical studies and trials using HDACIs have been reported in the HIV field [VPA (Lehrman et al, 2005; Siliciano et al, 2007; Archin et al, 2008, 2010; Sagot-Lerolle et al, 2008; Routy et al, 2012a,b), SAHA (Archin et al, 2012, 2014; Elliott et al, 2014), panobinostat (Rasmussen et al, 2015), and romidepsin (Sogaard et al, 2015)]. Altogether, these studies are encouraging but question the efficiency of HDACIs used alone to reduce the size of the HIV-1 reservoirs and underline the need to evaluate other classes of LRAs, alone or in combination with HDACIs. Targeting simultaneously different mechanisms of latency should be more efficient when viral eradication/remission is the objective since the combination of different classes of compounds could synergize (i.e. result in a higher reactivation level than the sum of the reactivations produced by each compound individually) to reactivate HIV expression in latently infected cells. In this regard, we have previously demonstrated proof-of-concepts for the coadministration of two different classes of promising LRAs [an NF-κB inducer + an HDACI (Quivy et al, 2002; Adam et al, 2003; Reuse et al, 2009), an NF-κB inducer + a P-TEFb-releasing agent (Darcis et al, 2015), a histone methyltransferase inhibitor (HMTI) + an HDACI (Bouchat et al, 2012), an HMTI + an NF-κB inducer (Bouchat et al, 2012)] as a therapeutic perspective to decrease the pool of latent HIV-1 reservoirs in the presence of efficient cART. Epigenetically, it is known that DNA methylation and histone deacetylation cooperate to establish and maintain a heterochromatin environment. In the case of HIV, the HIV-1 promoter has been previously shown to be hypermethylated ex vivo and resistant to reactivation in the latent reservoirs from cART-treated aviremic HIV-1 infected individuals, as opposed to the hypomethylated 5′ LTR of integrated proviruses present in viremic patients (Blazkova et al, 2009). Although controversy remains about the level of DNA methylation in patient cells in vivo (Blazkova et al, 2009, 2012; Palacios et al, 2012; Ho et al, 2013), the DNA methylation status of the HIV-1 promoter could contribute to “lock” the silent state of the provirus in cooperation with histone repressive post-translational modifications such as histone deacetylation, thereby making the return of the provirus to an active state more difficult (Blazkova et al, 2009). In this view, demethylating agents could represent promising candidate drugs in combination with HDACIs for reducing the pool of latent HIV reservoirs. Two well-characterized nucleoside analog DNA methylation inhibitors, 5-azacytidine (5-AzaC, marketed as Vidaza) and 5-aza-2′-deoxycytidine (5-AzadC, marketed as Dacogen), are currently FDA-approved to treat myelodysplastic syndrome and used in cancer therapies (Kantarjian et al, 2006). Few studies have already tested the HIV-1 reactivation potential of 5-AzadC + HDACI combinatory treatments using latently infected cell lines but have failed to show any synergistic effect in vitro (Blazkova et al, 2009; Kauder et al, 2009; Fernandez & Zeichner, 2010). In this report, we thoroughly studied the sequential aspect of cellular treatments combining demethylating agents with clinically tolerable HDACIs in latently infected T-cell lines and in ex vivo cultures of CD8+-depleted PBMCs or resting CD4+ T cells from cART-treated aviremic HIV-1+ patients. We demonstrated that these two classes of LRAs synergistically reactivated HIV in the context of sequential treatments. Moreover, we determined their metabolic activity profiles and their impact on global T-cell activation. Taken together, our data reveal the benefit of using combinations of a demethylating agent and an HDACI and, for the first time, the importance of treatment time schedule for LRA combinations in order to reactivate HIV. Results The DNA methylation inhibitor 5-AzadC induces HIV-1 transcription and production in a latently infected T-cell line Several postintegration latency models exist to study the mechanisms of transcriptional reactivation and the pathogenesis of HIV-1. In order to test the HIV-1 reactivation potential of 5-AzaC and 5-AzadC DNA methylation inhibitors, we used the HIV-1 latently infected J-Lat 8.4 cell line since the Verdin's laboratory has previously reported that two CpG islands flanking the transcription start site are hypermethylated in several latently infected J-Lat cell lines (Kauder et al, 2009). As these drugs are nucleoside analogs and are incorporated into DNA or RNA, we performed stimulation kinetics (24, 48 and 72 h) and only obtained viral production in culture supernatants after 72 h of treatment. Therefore, Fig 1A shows only the data for the 72-h time point. At 72 h post-treatment, we observed that 5-AzadC, but not 5-AzaC at the same doses, induced viral production in a dose-dependent manner from 400 nM to 6.25 μM (Fig 1A). WST-1 assays revealed that metabolic activities decreased in a dose-dependent manner from 400 nM ranging from 66.3% to 39.3% when using increasing 5-AzadC concentrations (Fig 1B). We confirmed the potency of 5-AzadC in comparison with 5-AzaC (observed by quantification of p24 viral production in Fig 1A) by showing that 5-AzadC increased the number of GFP-positive cells (assessed by FACS in Fig 1C) and increased viral mRNA expression (assessed by RT–qPCR in Fig 1D). Of note, relative levels of initiated (TAR) transcripts were lower than those of elongated (tat) transcripts for all conditions as compared to mock-treated condition. This phenomenon can be explained by the fact that more TAR transcripts are detected in mock-treated condition due to RNA polymerase II pausing present in latency condition. We also analyzed the mean fluorescence intensities (MFI) of the GFP-positive cell populations following increasing concentrations of 5-AzadC (Appendix Fig S1), and we showed that the amount of GFP produced per cell was also increased, indicating an enhanced HIV-1 gene expression. Figure 1. The DNA methylation inhibitor 5-AzadC induces HIV-1 expression in latently infected T cells A–D. J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment, viral production was measured by quantifying p24 antigen production in culture supernatants (A); metabolic activity was assessed by a WST-1 assay (B); viral protein expression was analyzed by FACS (C); and initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT–qPCR (D). The selected dose was indicated by an arrow. E. J-Lat 8.4 cells were mock-treated or treated with 5-AzadC (400 nM) or TNF-α (10 ng/ml) as a positive control. At 24, 48 or 72 h post-treatment, initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT–qPCR. Data information: For (D, E), results were normalized using β-actin gene primers and are presented as histograms indicating the fold inductions compared to mock-treated condition for each time period. For (A–E), means and standard errors of the means from three independent biological duplicates (n = 6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value of 1 (D, E) and 100% (B). Download figure Download PowerPoint As 5-AzadC reactivated HIV-1 from latency at a concentration of 400 nM (Fig 1A) that is lower than the tolerable peak of plasmatic concentration (Cmax) after usual dosage (20 mg/m²) in human anticancer therapy [around 650 nM (Inc E (2014) Dacogen (decitabine) for injection, full prescribing information)], we decided to use 400 nM of 5-AzadC as working concentration in our next experiments (indicated by an arrow, Fig 1A). Study of the 5-AzaC reactivation potential at higher doses than the ones we used had no interest because the Cmax after usual dosage (75 mg/m²) in human anticancer therapy is around 3 μM for 5-AzaC (Laille et al, 2014). The higher reactivation potential of 5-AzadC compared to 5-AzaC can be explained by the different intracellular metabolisms of these two drugs (Li et al, 1970). Indeed, in contrast to its reduced analog 5-AzadC, 5-AzaC is a ribonucleoside and has to be first reduced in a deoxynucleoside via a limiting enzymatic step before being incorporated into DNA. Moreover, while 5-AzadC incorporates exclusively into DNA, only a small percentage (10–20%) of 5-AzaC is incorporated into DNA, the remainder being incorporated into RNA (Li et al, 1970). Since a 72-h treatment with 5-AzadC was required to observe an increase in HIV-1 expression (see here above), we performed kinetics studies to follow the viral mRNA level increase in response to 5-AzadC treatment. As shown in Fig 1E, 5-AzadC caused an increased expression of both initiated (TAR) and elongated (tat) HIV-1 transcripts, which was the highest at 72 h post-treatment, whereas a 24-h treatment was sufficient to observe the effect of the NF-κB inducer TNF-α on viral transcriptional activity (Fig 1E). In conclusion, we demonstrated for the first time that 5-AzadC, in contrast to 5-AzaC, reactivated HIV-1 expression from latency in latently infected J-Lat 8.4 cells treated for 72 h, time needed to obtain an effective removal of the viral transcription block, probably due, at least partially, to DNA demethylation as previously shown by Verdin and colleagues (Kauder et al, 2009). Consequently, Dacogen (5-AzadC), but not Vidaza (5-AzaC), used at concentrations lower than that generally achieved in human cancers could be a promising LRA and be used in combination with other LRAs in reactivation strategies aimed at reducing the HIV-1 reservoirs. Determination of an optimal concentration of several clinically tolerable HDACIs used in human therapy to induce HIV-1 production in a latently infected T-cell line In order to highlight new therapeutic approaches to purge latent HIV reservoirs, we selected some clinically tolerable HDACIs that could be administrated in future HIV clinical trials. We compared the reactivation potentials of HDACIs previously extensively tested in several HIV-1 reactivation studies (VPA, NaBut, MS-275, and SAHA) (reviewed in Van Lint et al, 2013) to those of three promising and more recently tested HDACIs (belinostat, panobinostat, and romidepsin) (Rasmussen et al, 2013; Wei et al, 2014). Table 1 describes the characteristics of these HDACIs. Table 1. Characteristics of used HDACIs Acronym in this study Name Marketed as Approved for Treatment corresponding to Cmax Human tolerable plasmatic concentration (Cmax) Selected doses in this study Clinical trials in HIV field MS-275 Entinostat n.a. Ongoing clinical trials for the treatment of various cancers For an usual dosage 0.14 ± 0.24–0.3 ± 0.22 μM (Wightman et al, 2013) 0.5 μM n.a. NaBut Sodium butyrate Buphenyl Sickle cell anemia and beta-thalassemia For 27 and 36 g/day 1.225 and 1.605 mM (Phuphanich et al, 2005) 1.5 mM n.a. SAHA Vorinostat, suberoylanilide hydroxamic acid Zolinza Cutaneous T-cell lymphoma For an usual dosage two times/day during 2–3 weeks 0.3–1.7 μM (Merck (2013) Zolinza (vorinostat) prescribing information) 1.25 μM Archin et al (2014, 2012), Elliott et al (2014) VPA Valproic acid Depakine Chronic neurological and psychiatric disorders For an usual dosage 0.25–0.5 mM (AbbVie (2014) Depakote prescribing information) 2.5 mM Archin et al (2010, 2008), Lehrman et al (2005), Routy et al (2012a,b), Sagot-Lerolle et al (2008), Siliciano et al (2007) Beli Belinostat, PXD101 Beleodaq Relapsed or refractory peripheral T-cell lymphoma 1,000 mg/m² for five consecutive days > 1 μM (Steele et al, 2011) 1 μM n.a. Pano Panobinostat, LBH-589 Faridak Myeloma therapy For one dose with an usual dosage 0.6–1.4 μM (Rathkopf et al, 2010) 0.15 μM Rasmussen et al (2015) Romi Romidepsin, FK228 Istodax Peripheral T-cell lymphoma or cutaneous T-cell lymphoma 14 mg/m² 0.112 μM (Celgene (2014) Istodax prescribing information) 0.0175 μM Sogaard et al (2015) As shown in Fig 2, all selected HDACIs, except MS-275, induced viral production after 24 h in a dose-dependent manner in the latently infected J-Lat 8.4 cell line (Fig 2A and B). This measurement is commonly performed 24 h post-treatment for HDACIs in in vitro HIV reactivation experiments (Reuse et al, 2009). The absence of reactivation with MS-275 was in agreement with results previously reported (Reuse et al, 2009; Wightman et al, 2013). Of note, in the Lewin's study (Wightman et al, 2013), a significant viral production has been detected following MS-275 treatment after 48 h of stimulation in the ACH2 cell line and after 3 days of stimulation in a primary CD4+ T-cell model for HIV-1 latency. Figure 2. Determination of an optimal concentration for each HDACI to induce HIV-1 production in latently infected cells A–D. J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of HDACIs. At 24 h post-treatment, viral production was measured by quantifying p24 antigen production in culture supernatants (A, B) and metabolic activity was assessed by a WST-1 assay (C, D). Means and standard errors of the means from three independent biological duplicates (n = 6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value of 100% (C, D). The selected doses are indicated by an arrow. Plasmatic concentrations after usual dosage (Cmax) of each drug in human therapy are indicated below the LRA names and by a box in the graph. Download figure Download PowerPoint As shown in Fig 2A, NaBut presented a higher reactivation potential than those observed with VPA, MS-275, and SAHA. However, the HDACIs belinostat, panobinostat, and romidepsin induced higher HIV-1 production than VPA, NaBut, MS-275, and SAHA (Fig 2B, compared to Fig 2A). Moreover, if we only considered the HDACI concentrations close to their Cmax (indicated by a box in Fig 2A and B), panobinostat and romidepsin were more potent than the other HDACIs (including belinostat). Taken together, we confirmed and extended previous works (Rasmussen et al, 2013; Wei et al, 2014) by comparing the HIV-1 reactivation potentials of several HDACIs. This allowed us to determine for each HDACI an optimal concentration (outlined by an arrow in Fig 2A and B) in terms of both their HIV-1 reactivation potential and their Cmax (mentioned at the bottom of Fig 2A and B). In our next experiments, we selected the following HDACI concentrations for combinatory treatments with 5-AzadC: (i) MS-275 at a concentration of 0.5 μM in agreement with Cmax; (ii) NaBut at 1.5 mM consistent with Cmax; (iii) SAHA at 1.25 μM in agreement with Cmax; (iv) VPA at 2.5 mM, which is higher than Cmax but allowed HIV reactivation as opposed to VPA reactivation incapacity at Cmax used in clinical trials (Lehrman et al, 2005; Siliciano et al, 2007; Archin et al, 2008, 2010; Sagot-Lerolle et al, 2008; Routy et al, 2012a,b); (v) belinostat at 1 μM consistent with Cmax; (vi) panobinostat at 0.15 μM corresponding to the dose initiating the plateau phase; and (vii) romidepsin at 0.0175 μM corresponding to an intermediate dose between the concentration initiating the plateau phase and Cmax. In parallel, we assessed cellular metabolic activities after treatment with increasing HDACI concentrations and observed dose-dependent decreases (Fig 2C and D). Those decreases were drastic for belinostat, panobinostat, and romidepsin after the second tested dose despite their approval in human therapy (Fig 2D). Metabolic activity decrease did not seem to be due to viral cytopathic effects but only to intrinsic toxicity of the drugs since the same profiles were observed after LRA treatment of the uninfected Jurkat cell line [the parental cell line of the J-Lat cell clones (Jordan et al, 2003)] (Appendix Fig S2). In conclusion, we determined for each HDACI an optimal concentration based on its HIV-1 reactivation potential in a latently infected T-cell line and on its Cmax. We demonstrated that panobinostat and romidepsin were more potent than the other HDACIs tested at a dose 4- to 9.3-fold and 6.4-fold inferior to their corresponding Cmax, respectively. Sequential treatment with the DNA methylation inhibitor 5-AzadC and several HDACIs synergistically activates HIV-1 gene expression and production in latently infected T-cell lines DNA methylation and histone deacetylation contribute to gene silencing. Consequently, both mechanisms could also cooperate to establish a heterochromatin environment. To evaluate the reactivation potential of treatments combining an HDACI and 5-AzadC in latently infected J-Lat 8.4 cells, we measured HIV-1 production after sequential or simultaneous treatment in order to determine whether 5-AzadC treatment could have a favorable effect on viral reactivation induced by HDACIs. To this end, we progressively increased the time of 5-Az" @default.
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• W793746453 title "Sequential treatment with 5‐aza‐2′‐deoxycytidine and deacetylase inhibitors reactivates <scp>HIV</scp> ‐1" @default.
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