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2014 “Towards an HIV Cure” symposium Melbourne
Synergistic activation of HIV-1 expression by compounds targeting the positive transcription elongation factor b (P-TEFb) and by inducers of the NF-B signaling pathway Gilles Darcis Carine Van Lint lab Good morning, First, I would like to thank the
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cART + „purging strategy”
Strategies aimed at reducing the size of the persistent reservoirs of latent HIV-1 by forcing viral gene expression cART + „purging strategy” Inducers of HIV-1 gene expression ? In this regard, activation of HIV-1 expression in latently-infected cells, while maintaining cART in order to prevent new spreading infection, has been proposed as an adjuvant therapy, aimed at reducing the pool of latent reservoirs to a level bearable by the host immune system. (Recent studies have suggested that the latently-infected cells may not die following activation of the reservoirs. Therefore, additional immunotherapeutic interventions such as vaccines may be required.) This kind of strategy would allow latently-infected cells to die from viral cytopathic effects or host immune response. 2
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Postintegration latency is a multifactorial phenomenon
Chromatin structure and epigenetic modifications histone posttranslational modifications DNA methylation Absence of cellular inducible transcription factor Sequestration of P-TEFb TF (such as NF-KB) Prostratin Bryostatin Ingenol HMBA P-TEFb Cytoplasm Nucleus P-TEFb TAR Tat Multiple mechanisms are involved in maintaining HIV-1 latency, mostly through suppression of transcription. Here we will focus on two of this mechanisms : The first one is the absence of a crucial inducible host transcription factor :NF-B . Prostratin and bryostatin activate the PKC pathway, thereby allowing active NF-kB to migrate to the nucleus and to activate HIV expression. The second mechanism is the sequestration of an essential cellular elongation factor : P-TEFb. Nucleosom1 imposes a block to transcriptional elongation. To overcome this restriction, the HIV protein Tat recruits PTEFb to the LTR. Unlike HIV that relies on Tat to recruit P-TEFb , many cellular genes use the bromodomain protein Brd4 to recruit P-TEFb to their own promoters. Thus, Brd4 directly competes with Tat for binding to P-TEFb. Compounds named Bromodomain and Extraterminal inhibitors, the BET inhibitors (JQ1, I-BET, I-BET151) have recently been identified as able to reactivate HIV-1 from latency by releasing PTEF-b from Brd4. HMBA is another compound which act also on PTFEb by releasing this factor from an inactive complex. I will later use the term PTEF-b inducers for HMBA and BET inhibitors. The work that I will now present to you consists in investigating the reactivation potential of these PTFE-b inducers alone or in combination with these two NF-kB activators. 3’ 5’ NF-kB sites nuc-1 JQ1 I-BET I-BET151 P-TEFb Cellular genes Brd4 3
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BETi Cellular model Function
Five recent publications show that BET inhibitors reactivate latent HIV-1 BETi Cellular model Function Banerjee C, et al. J Leukoc Biol 2012 JQ1 Ach2, U1, J-Lat 10.6 resting CD4+ Tcells isolated from ART treated patients JQ1 reactivates HIV-1 in several cellular systems and in 1 out of 3 patients. Li Z, et al. Nucleic Acids Res 2013 JQ1+Pro J-Lat A2, 2D10, Jurkats 1G5 Hela JQ1 activates HIV transcription in latently infected Jurkat Tcells. JQ1 dissasociates BRD4 from chromatin and increases Tat binding to HIV-1 LTR Bartholomeeusen K, et al. J Biol Chem 2012 JQ1+SAHA JΔK Jurkat cell line (lack both NFkB binding sites) JQ1 activates HIV transcription in JΔK cells JQ1 activates HIV-1 transcription via the release of free P-TEFb from the 7SK snRNP Zhu J, et al. Cell Rep 2012 JQ1+PHA HeLa, U1, J-Lats, J-Lat A2, 293T CD8+-depleted PBMCs and resting CD4+ Tcells isolated from HIV-1 negative or from ART-treated patients Enhanced viral reactivation in NL4-3-GFP -HIV-1 infected PBMCs isolated from 6 negative donors. Enhanced viral replication when JQ1 + Prostratin or JQ1+ PHA in 7 out of 19 ART-treated patients. Boehm D, et al. Cell Cycle 2012 I-BET I-BET151 Jurkats, J-LatA2, J-LatA72 Primary T- cell models (Bcl-2 transduced resting Cd4+ T cells isolated from healthy donors) BETi reactivate HIV from latency in Jurkat cells and in T-cell model of HIV latency. This activation is depenedent on P-TEFb but independent from Tat.
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Compounds targeting P-TEFb
JQ1 IBET IBET151 HMBA PKC agonists Prostratine Bryostatin-1 Ingenol
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Co-treatment with P-TEFb and NF- B inducers leads to strong synergistic activation of HIV-1 production (I) Then, we wanted to assess if PTEFb inducers synergistically reactivated HIV-1 production when combined with bryostatin and prostratin. We performed P24 experiment in the same latently infected T cell line. A synergistic action means that the effect obtained by both coumpounds when jointly used is better than the somm of the effect of the same compounds when separatly used. So in simple words, in our case, A is one of PTFEb inducers and B is either Prostratin or Bryostatin. The synergistic action clearly appears for both bryostatin concentration but is better with this one (I show). That is the reason why we have adopted it for our further experiments. Similar results obtained for prostratin
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Co-treatment with P-TEFb and NF- B inducers leads to strong synergistic activation of HIV-1 production (II) The same conclusions can be drown when promonocytic cells are used instead of lymphocytic cells. In this case, the synergy is even more visible. Furthermore, similar results were obtained with Prostratin. Similar results obtained for prostratin
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The combination NF- B inducers + PTEFb inducer activates HIV-1 expression in a greater proportion of cells than each compound alone (I) tat rev 5’ 3’ vpr 5’ LTR gag pol vif env GFP 3’ LTR vpu In order to determin wether the synergystic action, observed by P24 experiment, is due , to a more potent activation of the responsive cells ,or to the recruitment of unresponsive cells, we performed flow cytometry experiment. We have used again the J-Lat cells since the HIV-1 genome, integrated in their DNA, contains the GFP gene. On the left we can see the non induced sample, followed by the compounds alone and finally the combinations of Bryostatin with PTFE-b inducers. The graph clearly shows that the synergy previously observed is due, at least partially, to the activation of a greater proportion of cells. 8
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The combination NF- B inducers + PTEFb inducer activates HIV-1 expression in a greater proportion of cells than each compound alone (II) Microglial cells ** p<0.005
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% of activated patients (>150 cop/ml)
Evaluation of HIV-1 recovery in CD8(+)-depleted PBMCs from virally suppressed patients (I) Patients HIV DNA copies/ 106 PBMCs Mock JQ1 I-BET I-BET151 HMBA Pro Bryo Prostratin Bryostatin-1 C+ P1 680 - 422 584 1016 669 449 3591 141 4200 282 318 P2 1217 237 P3 70 717 5359 657 694 585 435 691 830 191 1017 P4 350 729 // 1707 944 394 271 531 316 187 670 14741 308 7027 P5 1782 812 3870 2381 6857 3328 5876 5563 6307 2496 10158 3142 20524 P6 2418 5579 2505 1582 1336 1918 774 1666 472 3936 5741 936 8798 39773 P7 25034 586 459 296 536 797 566 274 2274 36562 P8 782 576 964 650 2377 665 512 253 851 329 526 P9 847 490 257 265 273 247 261 6468 P10 345 278 619 2153 644 334 264 P11 1435 604 784 2380 1304 1699 24068 485 4099 962 3274 1007 16036 P12 947 357 325 627 967 5251 2636 273634 P13 381 181 185 224 3194 P14 1823 789 766 193 206 380 634 382 P15 390 167 1594 377 370 790 172 P16 340 901 608 6835 403 4506 P17 263 849 3920 898 9752 4119 P18 333 1064 1579 953 451 883 2834 1002 517 1502 4161 3171 1190 2056 P19 1330 520 4880 428 79344 P20 1236 481 914 1949 2669 3393 783 6281 2597 655 212 916 19029 P21 600 693 954 846 1039 918 814 2717 P22 737 319 869 14170 965 1855 1395 4230 7879 P23 1239 647 2106 1702 109 1032 1870 6074 3833 1092 7267 P24 203 815 430 662 1956 813 402 464 % of activated patients (>150 cop/ml) 8 58 48 54 42 46 67 63 52 79 33 75
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% of activated patients (>150 cop/ml)
Evaluation of HIV-1 recovery in CD8(+)-depleted PBMCs from virally suppressed patients (II) Patients HIV DNA copies/ 106 PBMCs Mock JQ1 Ingenol Ingenol + JQ1 C+ P21 600 693 954 713 // 2717 P22 737 3468 7879 P23 380 1239 7267 P24 203 768 464 P25 11 902 453 2313 373 P26 548 181 888 10407 P27 4486 2433 3417 9412 13199 31132 P28 2552 1911 920 834 6751 2348 % of activated patients (>150 cop/ml) 38 75 100
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JQ1 combinatory treatments with PKC activators synergistically activate latent HIV ex-vivo in resting CD4 T cells from virally suppressed patients Patients HIV DNA cop/106 resting CD4+ T cells Age CD4+ T cell count Last treatment Aviremic for (years) Mock JQ1 Pro Bryo Ing C+ P1 1200 65 869 TRU EFV 8 - 483 764 1002 P2 1179 48 670 CBV NVP 15 4982 648 460 P3 112 41 848 TRU KLT 4 649 5022 676 180 1007 4131 1337 P4 327 38 367 TRU NVP 6 P5 4414 418 ATR 7 503 545 741 540 7687 P6 1777 44 401 403 860 1077 P7 5220 2 497 642 643 1399 // 7868 P8 911 736 KVX NVP 3 522 268 P9 3841 58 818 RTV DRV ETV MVC 9 607 369 624 538 8858 P10 946 49 663 405 333 392 4155 453 P11 525 46 928 TZV 10 400 7880 P12 1523 66 817 0.5 423 235 P13 9248 63 1091 994 3148 261 2677 2018 P14 4849 45 686 KVX RTV ATV 1 377 609 521 412 3839 5497 P15 1177 47 845 221 210 218 543 2452 P16 758 39 561 533 1259 % of activated patients (>150 cop/ml) 19 56 50 43 69 % of patients with combinatory beneficial effect (out of activated patients) 71 75 100
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Conclusion and perspectives
We have identified combinations of compounds exhibiting real potential of reactivation in several different post-integration latency cellular models. Some of these combinations might be clinically relevant for reducing/eliminating the cellular reservoirs of latent HIV-1. (In conclusion, BET inhibitors, HMBA and NF-kB inducers activate HIV-1 expression in a dose-dependent manner in latently-infected J-Lat and U1 promonocytic cell lines with minimal cytotoxicity. Co-treatment leads to strong synergistic activation of HIV-1 expression these cell lines with minimal cytotoxicity. Co-treatment synergistically increases the proportion of reactivated cells in lymphocytic and microglial latency cellular models. The combination P-TEFb inducers + prostratin synergistically increases transcriptional activity of viral LTR. This induction requires intact NF-kB binding sites.) Therefore, we have identified combination of compounds provided with a real power of reactivation in many cellular models. Some of them, especially Bryostatin in combination with BET inhibitors, since these compounds are already tested in cancer therapy, might be useful in clinical trials aimed at purging the HIV-1 reservoirs. However, we are conscious that much remains to be done. For example, we intend ,in further experiments to evaluate the physiological relevance of the synergy observed in ex-vivo cultures of PBMCs from cART-treated patients.
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Laboratory of Molecular Virology, University of Brussels, Belgium
Carine Van Lint Anna Kula Arsène Burny Sophie Bouchat Christelle Cardona Jean-Stéphane Gatot Nadège Delacourt Caroline Vanhulle Acknowledegments University of Strasbourg, France Olivier Rohr University of Franche-Comté, Georges Herbein St Pierre Hospital, Belgium Nathan Clumeck Stéphane De Wit Kabamba Kabeya Necker Hospital, Paris, France Christine Rouzioux Adeline Melard University of Liège Michel Moutschen Dolores Vaira Finally, I ‘d like to express my gratitude to everyone whose help has made this work possible, especially my promoters Carine Van Lint and Michel Moutschen, and Ania , my indispensable co-worker. And now, I would be happy to answer any questions. Thanks to L.Gama and to Amazônia Fitomedicamentos, Brazil for providing Ingenol.
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P-TEFb inducers increase HIV-1 expression in a dose-dependent manner without cytotoxicity
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NF-B inducers increase HIV-1 expression in a dose-dependent manner without cytotoxicity
mock mock mock mock Next we tested the reactivation potential of NF-kB pathways activators- prostratin and bryostatin-1. As you can see prostratin and bryostatin are also able to increase the p24 production in a dose dependent manner in the same cellular model. No cellular toxicity has been observed. Highlighted concentrations were used for further experiments. mock mock mock mock
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The combination Bryostatin-1/Prostratin + PTEFb inducer activates HIV-1 transcription
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Down-regulation of CD4 receptor expressed on resting CD4+ T cells (I)
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Global T cell activation (II)
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Global T cell activation (I)
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BET BET proteins (BRD2, BRD3, BRD4 and BRDT in human) contain two conserved N-terminal bromodomains (BRDs), small helical modules that specifically recognize acetylated lysine sites in proteins (Muller et al., 2011), an extra terminal domain (ET) and a more divergent C-terminal recruitment domain (CT motif or CTM). They bind to P-TEFb via their CT motif, tethering the complex to acetylated histone tails via their two N-terminal BRDs, resulting in assembly of the transcriptional machinery.
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NF-kB Inducible transcription factor made up of homo- and heterodimers of P50, P65, P52, re1B, et c-rel subunits that interact with a family of inhibitory IkB proteins, of which IkB alpha is the best characterized. Phosphorylation of IkBalpha at serines 32 and 36 is a key step involved in the activation of NF-kB complexes. This event is mediated par IKK, activated by several upstream kinases including some members of the PKC family.
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Evaluation of cellular viability in CD8(+)-depleted PBMCs treated with P-TEFb and NF-B inducers
50 100 150 200 250 P1 P2 P3 P4 P5 * % cellular viability 81 60 35 31 100 108 119 100 101 103 124 111 110 107 Then , we wanted to select the concentration of each compound that we will use to test their reactivation potential in (ex-vivo cultures of) PBMCs from cART-treated patients. We first tested their cytoxicity in 5 healthy patients and we selected one (I show) concentration for each compound. mock Prostratin Bryostatin-1 JQ1 I-BET I-BET151 HMBA CD8(+)-depleted PBMCs isolated from 5 healthy donors
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Evaluation of cellular viability of the combination P-TEFb + NF-B inducer in CD8(+)-depleted PBMCs
Mock JQ1 I-BET I-BET151 HMBA JQ1 I-BET I-BET151 HMBA Bryostatin 50 100 150 200 250 P1 P2 P3 P4 P5 % cellular viability 103 117 120 64 100 67 125 69 71 119 Mock JQ1 I-BET I-BET151 HMBA JQ1 I-BET I-BET151 HMBA Prostratin 20 40 60 80 100 120 140 160 180 200 P1 P2 P3 P4 P5 % cellular viability 100 103 111 107 83 84 75 122 60 We tested later the cytotoxicity of PTEFb inducers in combination with bryostatin and prostatin in these 5healthy patients. We can see that despite some heterogeneity, the overall cytoxicity is low.
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50 RNA copies/ml of plasma
Combination antiretroviral therapy (cART) is potent and life-prolonging but does not eradicate HIV infection 50 RNA copies/ml of plasma Blips Viral rebound Limit of detection + cART cART Persistent residual low-level viremia (1 to 5 viral RNA copies /ml) Plasma viral RNA (copies/ml) You very well know that during cART, the plasma virus levels falls below the level of detection of classical assays. However, cART is not curative since interruption of treatment results in a rapid viral rebound. During this constant phase, occasional viremic episodes are detected. Moreover, a perssistent residual low-level viremia can be detected in most patients using ultrasensitive RT-PCR assays. Latently-infected resting CD4+ T cells (and/or other cellular reservoirs) 25
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HIV-1 latent reservoirs are not eliminated by cART
Viral cytopathic effects Host immune response Productively infected cells most common - cell death (days) Cellular stimuli (antigens, phorbol esters, mitogens, cytokines,…) Latently-infected cells, HIV-1 reservoir rare event (0.1-1 per 106 resting CD4+ T cells) long duration (months) Uninfected Infected CD4+ T lymphocytes 26
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