2014 “ Towards an HIV Cure ” symposium Melbourne

1. 2014 “Towards an HIV Cure” symposiumMelbourne 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

2. Inducers of HIV-1 gene expression ? Strategies aimed at reducing the size of the persistent reservoirs of latent HIV-1 by forcing viral gene expression cART + „purging strategy”

3. HMBA P-TEFb Brd4 Tat P-TEFb P-TEFb 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 Cytoplasm Nucleus 3’ 5’ NF-kB sites nuc-1 JQ1 I-BET I-BET151 TAR Cellular genes

4. Five recent publications show that BET inhibitors reactivate latent HIV-1 BETi Cellular model Function

5. Compounds targeting P-TEFb • JQ1 • IBET • IBET151 • HMBA PKC agonists • Prostratine • Bryostatin-1 • Ingenol

6. Co-treatment with P-TEFb and NF-B inducers leads to strong synergistic activation of HIV-1 production (I) Similar results obtained for prostratin

7. Co-treatment with P-TEFb and NF-B inducers leads to strong synergistic activation of HIV-1 production (II) Similar results obtained for prostratin

8. vpr tat 5’ LTR gag 3’ LTR vif 5’ 3’ rev pol env GFP vpu The combination NF-B inducers + PTEFb inducer activates HIV-1 expression in a greater proportion of cells than each compound alone (I)

9. Microglial cells The combination NF-B inducers + PTEFb inducer activates HIV-1 expression in a greater proportion of cells than each compound alone (II) ** p<0.005

10. Evaluation of HIV-1 recovery in CD8(+)-depleted PBMCs from virally suppressed patients (I)

11. Evaluation of HIV-1 recovery in CD8(+)-depleted PBMCs from virally suppressed patients (II)

12. JQ1 combinatory treatments with PKC activators synergistically activate latent HIV ex-vivo in resting CD4 T cells from virally suppressed patients

13. 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 relevantforreducing/eliminating the cellular reservoirs of latent HIV-1.

14. 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, France • 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 Thanks to L.Gama and to Amazônia Fitomedicamentos, Brazil for providing Ingenol.

15. P-TEFb inducers increase HIV-1 expression in a dose-dependent manner without cytotoxicity

16. NF-B inducers increase HIV-1 expression in a dose-dependent manner without cytotoxicity mock mock mock mock mock mock mock mock

17. The combination Bryostatin-1/Prostratin + PTEFb inducer activates HIV-1 transcription

18. Down-regulation of CD4 receptor expressed on resting CD4+ T cells (I)

19. Global T cell activation (II)

20. Global T cell activation (I)

21. 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.

22. 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.

23. * * * * * 250 * 119 108 100 101 103 124 111 110 107 200 P1 150 P2 100 81 60 35 31 P3 P4 100 P5 50 0 Evaluation of cellular viability in CD8(+)-depleted PBMCs treated with P-TEFb and NF-B inducers % cellular viability mock Prostratin Bryostatin-1 JQ1 I-BET I-BET151 HMBA CD8(+)-depleted PBMCs isolated from 5 healthy donors

24. 250 100 103 117 120 64 119 67 125 69 71 200 P1 150 P2 % cellular viability P3 P4 100 P5 50 0 Mock JQ1 I-BET I-BET151 HMBA - JQ1 I-BET I-BET151 HMBA Bryostatin - - - - - + + + + + 200 100 100 103 111 107 60 83 84 75 122 180 160 140 P1 120 P2 % cellular viability 100 P3 P4 80 P5 60 40 20 0 Mock JQ1 I-BET I-BET151 HMBA - JQ1 I-BET I-BET151 HMBA Prostratin - - - - - + + + + + Evaluation of cellular viability of the combination P-TEFb + NF-B inducer in CD8(+)-depleted PBMCs

25. + cART Limit of detection 50 RNA copies/ml of plasma Persistent residual low-level viremia (1 to 5 viral RNA copies /ml) cART Combination antiretroviral therapy (cART) is potent and life-prolongingbut does not eradicate HIV infection Viral rebound Plasma viral RNA (copies/ml) Blips Latently-infected resting CD4+ T cells (and/or other cellular reservoirs)

26. cART Cellular stimuli (antigens, phorbol esters, mitogens, cytokines,…) HIV-1 latent reservoirs are not eliminated by cART Viral cytopathic effects Host immune response Productively infected cells most common - cell death (days) 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