Hopes raised by HDAC inhibitor, but uncertainties remain
Richard Jefferys, TAG
In the 26 July 2012 issue of the journal Nature, Nancy Archin and colleagues from the laboratory of David Margolis published results from a trial investigating whether the approved cancer drug vorinostat can reverse HIV latency. 
Vorinostat (also known as SAHA, trade name Zolinza) belongs to a group of compounds called histone deacetlyase (HDAC) inhibitors. Histone deacetylases are a class of cellular enzymes involved in condensing DNA and repressing gene expression. In laboratory experiments, inhibiting histone deacetylases appears to free latent HIV from lockdown, causing viral RNA to be produced. HDAC inhibitors have therefore emerged as lead candidates for depleting latent HIV reservoirs, a task that is widely believed to be an important step along the path to a cure.
Margolis has previously presented preliminary results from the trial, first at a meeting in St. Maarten in December 2011, then at CROI and Keystone in March of this year. Each time the number of participants with available data increased, from four initially to six at CROI and seven at Keystone. The published Nature paper brings the final count to eight. I wrote about the data when it was initially presented, and below is an updated version of that report.
The first step of the protocol involved screening potential participants to assess whether vorinostat could reactivate latent HIV from their CD4 T cells ex vivo. Sixteen individuals had lymphocytes extracted by leukopheresis, then sorted into discrete pools of 1 million purified resting CD4 cells each (ending up with 12-48 pools per participant). These pools were exposed to either vorinostat or no drug, and a mean level of HIV RNA per million cells (and a standard deviation) was calculated for each person (the assay used can measure down to 10 copies per million cells). Margolis noted in St. Maarten that the statistical approach used to calculate the mean RNA levels is robust but complicated, and a paper explaining it is currently in press at a statistics journal. Eleven of the sixteen people screened showed an upregulation of HIV RNA expression in this analysis and eight agreed to participate in the next step of the trial.
A 200 mg dose of vorinostat was given first for safety, followed by a 400 mg dose to study pharmacokinetics and for analyses of histone acetylation and acetylation of the p21 gene (in other words, analyses of the effects of the drug on cellular genetic machinery and not HIV). The pharmacokinetic data mirrored reports from cancer studies, with peak levels occurring a mean two hours after dosing. A significant increase in the acetylation of cellular histone H3 was observed, along with a trend towards increased acetylation of histones at the p21 gene in five participants who had sufficient cells available for this analysis.
A final 400 mg dose of vorinostat was then administered with leukopheresis performed 4-6 hours afterward based on the pharmacokinetic data indicating this would be around the time of maximum activity. No grade 1 or greater toxicities were seen, and cell-associated unspliced HIV gag RNA expression increased significantly compared to baseline in all eight individuals by a mean of 4.8-fold (range: 1.5-10-fold). HIV RNA in peripheral blood was assessed using a single copy assay but no change was detected. The researchers also state that “a limited evaluation did not reveal a substantial reduction in the frequency of replication-competent HIV within resting CD4 T cells” but the data is not included in the paper, and they note that this would not be unexpected given that only a single dose was administered. The primary import of the results, they conclude, is the proof-of-concept: “these findings demonstrate that therapy targeted at persistent, latent infection within resting CD4 T cells is feasible, and open the way for the development of HDAC inhibitors with improved specificity, potency and safety profiles for the selective targeting of latent proviral genomes.”
When he presented the early results in December 2011, Margolis offered a list of questions that remain to be answered.
- Is there an equal effect from multiple doses or does it become attenuated?
- How much drug exposure is needed?
- Should drug be administered continuously or pulsed?
- Will toxicities emerge?
- What number of cells is needed to measure relatively rare reactivation events?
- Are additional inducers needed?
- Does RNA expression lead to virion production or clearance of the infected cell?
- Are additional interventions needed to clear the latently cells that have been induced to express HIV RNA?
Since that time, data addressing some of these questions has been presented and published. Tae-Wook Chun’s laboratory at NIAID has shown that the induction of RNA expression by vorinostat does not lead to significant virion production or clearance of the infected cell.  Robert Siliciano’s group also documented a lack of clearance of infected cells, and furthermore demonstrated that functional HIV-specific CD8 T cells are needed to perform this task. 
More recently, at the AIDS 2012 conference that took place in July in Washington DC, Jeff Lifson presented data indicating that multiple doses of vorinostat do not have consistent effects on the viral reservoir in the SIV/macaque model.  The relevance of these findings to humans should be revealed when results become available from a clinical trial involving a 10-day course of vorinostat, which is currently being conducted by Sharon Lewin in Australia. 
In addition to the caveat that HDAC inhibitors alone appear unlikely to be sufficient to achieve a cure of HIV infection, there is also a question that wasn’t on Margolis’s list: are there latently infected cells that cannot be reactivated by these drugs? At a symposium on cure research that took place immediately prior to AIDS 2012,  Robert Siliciano presented evidence that, while the majority of resting CD4 T cells containing HIV DNA harbor viral genomes that are compromised by various genetic alterations (such as deletions) some—16.8% in his study (range: 6-36%)—contain replication-competent HIV that is not induced by normal methods. It is as yet unclear if this virus can be induced to replicate in vivo.
In a commentary accompanying the Archin paper, Steve Deeks highlights the work that remains and the likely need for combination approaches, but also emphasises that these results represent an important first step beyond antiretroviral therapy and into new territory, with a cure for HIV infection the ultimate destination.
Source: TAG Basic Science Blog (27 July 2012).
- Archin NM et al. Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy. Nature 487, 482–485 (26 July 2012) doi:10.1038/nature11286.
- Blazkova J, Chun T-W et al. Effect of Histone Deacetylase Inhibitors on HIV Production in Latently Infected, Resting CD4+ T Cells from Infected Individuals Receiving Effective Antiretroviral Therapy. J Infect Dis. (2012) doi: 10.1093/infdis/jis412, First published online: June 25, 2012.
- Sham L et a;. Stimulation of HIV-1-Specific Cytolytic T Lymphocytes Facilitates Elimination of Latent Viral Reservoir after Virus Reactivation. Immunity, Volume 36, Issue 3, 491-501, 23 March 2012. doi 10.1016/j.immuni.2012.01.014.
- Lifson J. Evaluation of treatment with the histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid; SAHA) in antiretroviral drug treated, SIVmac239-infected rhesus macaques. 19th International AIDS Conference, 22-27 July 2012, Washington DC. Late breaker oral abstract MOLBA02.
- Safety and Effect on HIV Transcription of Vorinostat in Patients Receiving Suppressive Combination Anti-retroviral Therapy.
- Towards an HIV Cure: AIDS 2012 pre-conference symposium, 20 – 21 July 2012. Washington DC.
- Deeks SG. HIV: Shock and Kill. Nature 487, 439–440 (26 July 2012) doi:10.1038/487439a