The inhibitor apoptosis protein antagonist Debio 1143 Is an attractive HIV-1 latency reversal candidate - PubMed (original) (raw)
The inhibitor apoptosis protein antagonist Debio 1143 Is an attractive HIV-1 latency reversal candidate
Michael Bobardt et al. PLoS One. 2019.
Abstract
Antiretroviral therapy (ART) suppresses HIV replication, but does not cure the infection because replication-competent virus persists within latently infected CD4+ T cells throughout years of therapy. These reservoirs contain integrated HIV-1 genomes and can resupply active virus. Thus, the development of strategies to eliminate the reservoir of latently infected cells is a research priority of global significance. In this study, we tested efficacy of a new inhibitor of apoptosis protein antagonist (IAPa) called Debio 1143 at reversing HIV latency and investigated its mechanisms of action. Debio 1143 activates HIV transcription via NF-kB signaling by degrading the ubiquitin ligase baculoviral IAP repeat-containing 2 (BIRC2), a repressor of the non-canonical NF-kB pathway. Debio 1143-induced BIRC2 degradation results in the accumulation of NF-κB-inducing kinase (NIK) and proteolytic cleavage of p100 into p52, leading to nuclear translocation of p52 and RELB. Debio 1143 greatly enhances the binding of RELB to the HIV-1 LTR. These data indicate that Debio 1143 activates the non-canonical NF-kB signaling pathway by promoting the binding of RELB:p52 complexes to the HIV-1 LTR, resulting in the activation of the LTR-dependent HIV-1 transcription. Importantly, Debio 1143 reverses viral latency in HIV-1 latent T cell lines. Using knockdown (siRNA BIRC2), knockout (CRIPSR NIK) and proteasome machinery neutralization (MG132) approaches, we found that Debio 1143-mediated HIV latency reversal is BIRC2 degradation- and NIK stabilization-dependent. Debio 1143 also reverses HIV-1 latency in resting CD4+ T cells derived from ART-treated patients or HIV-1-infected humanized mice under ART. Interestingly, daily oral administration of Debio 1143 in cancer patients at well-tolerated doses elicited BIRC2 target engagement in PBMCs and induced a moderate increase in cytokines and chemokines mechanistically related to NF-kB signaling. In conclusion, we provide strong evidences that the IAPa Debio 1143, by initially activating the non-canonical NF-kB signaling and subsequently reactivating HIV-1 transcription, represents a new attractive viral latency reversal agent (LRA).
Conflict of interest statement
Author PAG received funding from Debiopharm International. Authors NW and GV are employees of Debiopharm International. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Figures
Fig 1
Model for canonical (left) and non-canonical (right) NF-kB pathways and site of action of Debio 1143 (adapted from Pache et al. [33]). A. The non-canonical NF-κB pathway occurs through a subset of TNFRs. In the absence of stimulation, a complex of BIRC2, BIRC3 (cIAP2), TRAF2, and TRAF3 degrades NIK. Upon receptor activation, BIRC2 and BIRC3 promote ubiquitination and degradation of TRAF3, permitting an accumulation of NIK. In turn, NIK activation results in the phosphorylation of IKK alpha, leading to the proteolytic processing of p100 into p52. p52 forms a heterodimer with the RELB transcription factor and translocates to the nucleus, inducing the expression of target genes. B. Debio 1143 induces the E3 ubiquitin ligase activity of BIRC2, leading to its auto-ubiquitination and degradation.
Fig 2. Analysis of the mechanisms of action of Debio 1143.
CD4+ T-lymphocytes (100,000) (triplicate) were incubated with VSVG-NL4.3-GFP and Debio 1143 (0 to 10 μM) for 24 h and analyzed for infection by FACS (A) and for cytotoxicity by LDH assay (B). C. NF-κB reporter (Luc)-3T3 cells (BPS Bioscience) (100,000) (triplicate) were treated with Debio 1143 and luciferase activity in cell lysates was quantified after 6 h. D. CD4+ T-lymphocytes (1,000,000) were treated (from 15 min to 2 h) with DMSO, Debio 1143 (1 μM) and TNF alpha (10 ng/mL) and cell lysates analyzed by Western blotting for BIRC2, IkB alpha and CypA expression. E. Same as D except that cells were treated for 15 min with 0, 0.1, 1 and 10 μM of Debio 1143. F. Same as D except that cells were treated for 24 h with DMSO or 1 μM of Debio 1143, and cell lysates analyzed by Western blotting for BIRC2, p100/52, NIK and CypA expression. G. Same as D except that cells were treated for 24 h with DMSO, 1 μM of Debio 1143 or 10 ng/mL of TNF alpha, and cytosolic and nuclear extracts analyzed by Western blotting for the expression of various components of the NF-kB signaling pathways and cytosolic and nuclear markers. H. 2D10 cells (500,000) (triplicate) were treated with 1 μM Debio 1143 for 10 h prior to ChIP analysis using control, anti-RELB or anti-RELA IgG. RELB- and RELA-specific association with the HIV-1 LTR and the IkB alpha gene promoter region, or an intergenic region upstream of the PABPC1 gene not known to contain NF-kB binding sites as negative control, was analyzed by qPCR and is presented as fold enrichment over control IgG. Data from A to F are each representative of two independent experiments. P-values are presented. ANOVA and Bonferroni’s multiple comparison tests were used to compare means of different groups. Bar graphs indicate each sample point, mean, and SEM. Key for adjusted p-values from Bonferroni’s multiple comparison tests on graphs: ns = no significance, * = 0.01≤p<0.05, ** = 0.001 ≤p<0.01, *** = 0.0001≤p<0.001, and **** = p<0.0001.
Fig 3. Debio 1143 reverses HIV-1 latency.
A. Typical FACS graphs for the percentage of GFP+ HIV-1 latent reporter cells. The population on the forward scatter/side scatter dot plot was placed by the automatic scaling feature of the cytometer. Minor variability between reporter cell replicates was observed as shown by representative graphs of 2D8 cells treated with control DMSO or Debio 1143 (1 μM). B. Latently infected JLat 10.6, 2D10 and 5A8 GFP reporter cells (250,000) (duplicate) were treated for 48 h with LCL161 (1 μM), Debio 1143 (1 μM) or in combination with panobinostat (10 nM) and vorinostat (500 nM). GFP expression was quantified by FACS. Data are expressed as % of GFP levels. Two distinct experiments were conducted in triplicate and averaged data are presented. C. Same as B except that the capacity of Debio 1143 at reversing HIV-1 latency in 2D10 cells was compared with that of other IAPa. D. CD4+ T-lymphocytes (250,000) (triplicate) were incubated with the LRAs and cytotoxicity was quantified after 48 h by LDH assay as above (Fig 2). E. 2D10 cells were treated with control (siCTL) and BIRC2 (siBIRC2) siRNA for 24 h, then incubated with DMSO or Debio 1143 (1 μM) and analyzed for GFP expression after 48 h (left panel) and cell lysates analyzed by Western blotting for BIRC2, NIK and CypA expression. F. 2D10 cells (triplicate) were first incubated with or without MG132 (25 μM) for 30 min, subsequently exposed to DMSO or Debio 1143 (1 μM) for 48 h, and analyzed by FACS for GFP content. G. Wild-type or NIK knockout (ΔNIK) 2D10 cells were exposed to DMSO or Debio 1143 (1 μM) for 48 h, and analyzed by FACS for GFP content. Two distinct experiments were conducted in triplicate and averaged data are presented. P-values are presented. ANOVA and Bonferroni’s multiple comparison tests were used to compare means of different groups. Bar graphs indicate each sample point, mean, and SEM. Key for adjusted p-values from Bonferroni’s multiple comparison tests on graphs: ns = no significance, * = 0.01≤p<0.05, ** = 0.001 ≤p<0.01, *** = 0.0001≤p<0.001, and **** = p<0.0001.
Fig 4. Debio 1143 reverses HIV-1 latency in resting CD4+ T cells from ART-treated patients.
Purified resting CD4+ T cells from two ART-treated patients were serially diluted fivefold from 1,000,000 cells per well to 1,600 cells per well and seeded into individual wells of 48-well plates (5 replicates at each dilution). Each dilution was then treated with DMSO, anti-CD3/CD28 IgG-coated microbeads or 1 μM of Debio 1143. At day 2, highly permissive MOLT-4-CCR5 cells were added to each dilution to propagate released virions after latency reversal by LRAs. The ratio of target cells added was 107 MOLT-4-CCR5 cells to the 1x106 resting CD4+ T cell dilution, and 106 MOLT-4-CCR5 cells to all other resting CD4+ T cell dilutions. To increase infection, MOLT-4-CCR5 cells and released virions were spinoculated. Supernatants were collected at
day 7
and split for HIV-1 RNA quantification (A) and HIV-1 infection (B) in triplicate. HIV-1 RNA was measured by RT-qPCR. Control experiments showed that >95% of HIV-1 RNA in supernatants could be pelleted by centrifugation at 24,000 g for 1 h and were resistant to DNase I treatment. Infectivity of released virions was scored using TZM indicator cells after spinoculation. TZM infection was scored after 48 h by β-galactosidase activity in cell lysates. P-values are presented. ANOVA and Bonferroni’s multiple comparison tests were used to compare means of different groups. Bar graphs indicate each sample point, mean, and SEM. Key for adjusted p-values from Bonferroni’s multiple comparison tests on graphs: ns = no significance, * = 0.01≤p<0.05, ** = 0.001 ≤p<0.01, *** = 0.0001≤p<0.001, and **** = p<0.0001.
Fig 5. Debio 1143 reverses ex vivo HIV-1 latency in resting CD4+ T cells isolated from BLT mice.
A. Experimental design for the HIV-1 latency model in BLT mice. When ART is interrupted, viral rebound occurs B. Same as A except that ART was maintained until isolation of resting CD4+ T cells from blood, thymic organoid, lung, spleen, bone marrow, lymph nodes and liver of HIV-1-infected BLT mice (5 mice per treatment). Isolated resting CD4+ T cells from each group were pooled, counted, split in triplicate and treated with vehicle, PMA (20 ng/mL) + ionomycin (1 μg/mL), Debio 1143 (1 μM), LCL161 (1 μM), panobinostat (1 μM) or vorinostat (1 μM). De novo released virions from supernatants were purified as above and quantified by RT-qPCR. Data are expressed in copies of HIV-1 RNA/mL of supernatant. P-values are presented. ANOVA and Bonferroni’s multiple comparison tests were used to compare means of different groups. Bar graphs indicate each sample point, mean, and SEM. Key for adjusted p-values from Bonferroni’s multiple comparison tests on graphs: ns = no significance, * = 0.01≤p<0.05, ** = 0.001 ≤p<0.01, *** = 0.0001≤p<0.001, and **** = p<0.0001.
Fig 6. Debio 1143 does not stimulate the production of pro-inflammatory cytokines.
A. PBMCs from an ART-treated patient were incubated with DMSO, anti-CD3 (200 ng/mL)/CD28 (500 ng/mL) antibodies (mAbs) or Debio 1143 (1 μM). Supernatants were collected after 24 h, and cytokine concentrations were determined by BioPlex analysis. B. Cytokine mRNAs levels from PBMCs were quantified by PCR using β-actin as control. C. Blood of HIV-1-infected BLT mice under ART untreated (n = 10) or treated with Debio 1143 (100 mg/kg, p.o.) (n = 10) was collected 48 h post-drug treatment and human cytokine levels were quantified by BioPlex analysis. P-values are presented. ANOVA and Bonferroni’s multiple comparison tests were used to compare means of different groups. Bar graphs indicate each sample point, mean, and SEM. Key for adjusted p-values from Bonferroni’s multiple comparison tests on graphs: ns = no significance, * = 0.01≤p<0.05, ** = 0.001 ≤p<0.01, *** = 0.0001≤p<0.001, and **** = p<0.0001.
References
- UNAIDS (2016) UNAIDS 2016 Global Fact Sheet.
- Wong JK, Hezareh M, Gunthard HF, Havlir DV, Ignacio CC, Spina C, et al. (1997) Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 278: 1291–1295. -PubMed
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