Epigenetic drug Gar1041 (auranofin) in combination with antiretroviral therapy (ART) reduces the proviral DNA reservoir

1. Epigenetic drug Gar1041 (auranofin) in combination with antiretroviral therapy (ART) reduces the proviral DNA reservoir in SIVmac251-infected macaques Andrea Savarino

2. Barriers to cure • Latently infected T-cells • Residual viral replication • Anatomical reservoirs

3. Main latent virus reservoirs: central memory (TCM) and transitional memory (TTM) CD4+ T-cells

5. “Classic” strategies for eradication of virus reservoirs • Prevent any virus spread by intensified ART • Target multiple sites of viral inhibition • Induce latently infected cells to replicate virus • Histone deacetylase inhibitors • Eliminate infected cells • Virus cytotopathogenicity • Immune response • Passive therapies • Drugs Van Lint et al., 2004 Hamer, 2004 Archin et al., 2005 Savarino et al., 2009 Margolis, 2010

6. Our novel strategy for eradication of virus reservoirs “Sabotage” • Aimed at reducing the capacity of the viral reservoir to expand • Aimed at decreasing the half-life of the latently infected reservoir persisting during antiretroviral therapy (ART)

7. Auranofin (Gar1041) • A gold-based compound, • Orally available, • Used for years in the treatment of rheumatoid arthritis (“drug repositioning”), • Capable of inducing oxidative stress (generation of intracellular peroxide through a superoxide dismutase-mimicking reaction), • Adopted in the experimental treatment of certain cancers due to its pro-differentiating properties.

8. Reactive oxygen species (ROS) induction ACH2 ctrl 0.6 μM auranofin Auranofin (Gar1041) Hydrogen peroxide ↓ Dihydrorhodamine 123 → rhodamine 123 (FLUORESCENT) Hydrogen peroxide accumulation plays a major role in cellular differentiation!

9. Auranofin induces CD27 downregulation TCM TTM TEM Mock treated 50nM auranofin CD27 expression

10. Working hypothesis: CD27 down-modulation in central and transitional memory CD4+ T cells may decrease the half-life of the latently infected viral reservoir and contribute to its depletion.

11. Response of SIVmac251-infected macaques to antiretroviral therapy RAL + PMPA + FTC RAL RAL + PMPA + FTC RAL RAL- 100mg BID, oral PMPA - 20mg/kg, SQ FTC – 50mg/kg, SQ Lewis M. Norelli S., et al., Retrovirology 2010.

12. Auranofin decreases the central memory T cell compartment in vivo but does not affect total CD4 counts P < 0.05 P < 0.05

14. Darunavir as a tool for ART intensification in SIV+ macaques Darunavir (DRV) complexed with HIV-2 protease DRV complexed with SIVmac251 protease Kovalevsky et al., 2008 Barreca ML, Norelli S, and Savarino A, unpublished

15. Auranofin (Gar1041) significantly decreases proviral DNA in PBMC from SIV+ macaques treated with in ART intensified withritonavir-boosted darunavir (iART) iART/Gar1041 (average trend over time: P = 0.0207; t-test for regression). iART alone (average trend over time: P = 0.8878; t-test for regression).

16. Stimulation of viral replication by histone deacetylase inhibitor, SAHA in SIVmac251-infected monkeys treated with intensified ART alone but not in monkeys SIVmac251-infected monkeys treated with iART and auranofin *P < 0.05 **P < 0.01 intensified ART alone intensified ART plus auranofin

17. Addition of auranofin to intensified ART delayed re-appearance of viral load following treatment suspension Intensified ART alone Intensified ART plus auranofin * *P < 0.05; Gehan-Breslow-Wilcoxon test for survival

18. Upon re-appearance of viral load following therapy suspension, auranofin-treated monkeys, but not intensified ART (iART)-only controls, acquire the ability to control viral load peak When viral load re-appeared in monkeys that had been treated with auranofin, an immediate peak was observed that was followed by progressive declines in viral RNA iART plus Gar1041 decrease iART alone …viral RNA eventually declined to levels lower than those presented before ART initiation *P < 0.05; paired Student’s t-test

19. Upon re-appearance of viral load following therapy suspension, auranofin-treated monkeys, but not intensified ART (iART)-only controls, maintain high CD4 counts iART alone iART plus Gar1041 Drug free remission… eventually?

20. Conclusions Oxidative stress inducing agent auranofin impacts the proviral DNA reservoir in SIVmac251-infected macaques under treatment with ART, likely acting by an entirely novel mechanism (CD28 downmodulation in long-lived and proliferation-competent central and transitional memory T cells → generation of short-lived phenotypes) Our findings following ART intensification indicate that a drug-free remission is possible, at least for the follow up period adopted in this study, and represent a milestone towards the identification of future drugs capable of curing HIV/AIDS or inducing a drug-free remission. Our results also highlight the need for ART intensification when adopting drugs capable of inducing HIV-1 replication.

21. Acknowledgments Bioqual Matt Collins Jake Yalley-Ogunro University of Rome “Tor Vergata” Jack Greenhouse Enrico Garaci Wendy Wagner ICGEB Istituto Superiore di Sanità, RomeMarina Lusic Sandro Norelli Barbara Chirullo University of Rome, La Sapienza Marco Sgarbanti Rossella Sgarbanti Andrea Savarino Anna Teresa Palamara VTGI, Florida Nicolas Chomont Sandrina DaFonseca Rafick-Pierre Sékaly