Reassessing the role of HDAC inhibitors in cure research
Gareth Hardy, HIV i-Base
Two recent papers present conflicting data on the efficacy of the histone deacteylase (HDAC) inhibitors, the leading class of latency reversing agents (LRAs).
Korin Bullen, from Robert Siliciano’s group at Johns Hopkins University in Baltimore, USA and Datsen Wei, from Gilead and working with John Mellor’s group at University of Pittsburgh, tested a panel of HDAC inhibitors and other LRAs for their ability to induce activation of latently infected CD4 T cells from patients on ART. The two groups report contradictory findings.
HDACs are enzymes involved in repression of gene expression and are thought to actively repress HIV expression during viral latency. Clinical trials have suggested that the HDAC inhibitor vorinostat increases HIV RNA expression in CD4 T cells by an average of 4.8 fold, when used in people on ART . Despite this, it is not known if HDAC inhibitors can induce replication-competent virus from patients’ CD4 T cells, leading to an increased decay-rate of the latent-reservoir.
Bullen et al, describe their results in Nature Medicine . Using clinically relevant concentrations of each agent, they assessed the HDAC inhibitors vorinostat, romidepsin and panobinostat, as well as agents from other classes of LRAs: disulfiram; JQ1 and; bryostatin-1. In order to allow reactivated HIV to sufficiently grow from cultures of patient CD4 T cells, they first treated resting CD4 T cells from 13 ART-treated patients with LRAs for 18 hours and then added lab-grown T cells for a further 14 days to allow viral propagation. In previous versions of this assay, the use of T cell blasts for propogation may have caused mixed lymphocyte reactions leading to background T cell activation, which could have caused a false positive signal for viral induction by LRAs. To prevent this they used the lab-adapted T cell line, MOLT-4. Surprisingly, using this new assay, HDAC inhibitors did not induce latent HIV as measured by p24 ELISA of supernatants. These results were confirmed by assessment of supernatant HIV mRNA in five of these patients, for which one demonstrated induced virus, in response to bryostatin-1. The PMA/I positive control induced HIV p24 expression in CD4 T cells of 11 of 13 patients.
Most HIV mRNA PCR assays target gag or gag and LTR together. Bullen and colleagues considered that the sequences detected by these assays may not represent bona fide HIV RNA, as “HIV integrates into host genes that are actively transcribed in resting CD4 T cells, allowing for the production of chimeric host-HIV transcripts”. These viral transcripts will not lead to production of functional virus but would contain sequences of HIV gag and be indistinguishable from HIV-LTR-initiated transcripts when using conventional HIV PCR assays. In order to address this, Bullen and colleagues designed a PCR assay to detect a portion of the HIV LTR gene that should not be present in LTR-initiated transcripts, but would be present in host gene-initiated HIV ‘readthroughs’.
Using their new PCR assay they treated resting CD4 T cells from five HIV infected patients receiving ART, with vorinostat and determined that while vorinostat did not induce complete HIV RNA, it did induce a two fold increase in host-initiated ‘readthrough’ transcripts, which were comparable to the induction of gag-containing transcripts. The authors conclude: “although not every potential LRA will induce readthrough transcription by activating a host gene, our data show that chimeric host-HIV transcripts can have a confounding effect on the RT-qPCR signal obtained using standard gag primers. Such an effect should be taken into consideration when evaluating LRAs using conventional gag RT-qPCR assays”.
These results have two implications. Firstly, HDAC-inhibitors may not be effective at inducing latent HIV in vivo; and secondly, the mechanisms by which HIV latency naturally occurs, are not captured by laboratory latency models.
In contrast to these results, Wei at al describe their findings in PLoS Pathogens , showing that vorinostat and romidepsin induce expression of latent HIV from CD4 T cells of patients receiving ART. Using an in vitro latency model, they screened the activity of the clinically tested HDAC inhibitors: vorinostat, romidepsin, panobinostat, givinostat, mocetinostat, and pracinostat. Wei et al showed dose-dependent increases in activity for all HDAC inhibitors, with superior activity for romidepsin. Wei et al then assessed the ability of each of the HDAC inhibitors to induce expression of HIV from purified resting CD4 T cells of HIV infected patients receiving ART. Unlike the viral outgrowth approach taken by Bullen et al, described above, Wei and colleagues treated purified resting CD4 T cells from patients with vorinostat or romidepsin and then assessed HIV RNA expression by PCR using the Roche COBAS HIV test after 6, 12, 24 and 48 hours of culture. This assay would not be as dependent on production of replication-competent HIV as that used by Bullen. Using this method Wei et al found that vorinostat induced 2- to 4- fold increases in HIV RNA expression, which peaked at 6 hours and the more potent romidepsin induced 5- to 6- fold increases that peaked after 24 – 48 hours.
In order to demonstrate that vorinostat or romidepsin could induce extracellular virions from resting CD4 T cells, supernatants were tested for HIV RNA after 6 days culture. HIV RNA could be detected in the supernatants of romidepsin-treated cells, but not vorinostat-treated cells. The authors add that HIV RNA released into supernatants following treatment with romidepsin, could be pelleted by high-speed centrifugation, suggesting that the RNA must be encapsulated in virions, rather than having been released during cell death potentially caused by HDAC inhibitor toxicity.
The authors conclude: “We observed reproducible ex vivo activation of HIV by romidepsin… Given these results… clinical testing is warranted to assess whether romidepsin can activate latent HIV and potentially reduce the size of the latent reservoir in HIV-infected patients on suppressive ART”.
There are two key differences in the approaches taken by the Bullen and Wei groups that could explain their discrepant results. First, Bullen used a viral outgrowth assay that depended on propogation of infectious virus (over 14 days), whereas Wei measured shorter-term viral RNA expression (24 hours – 7 days) that may have represented a partial phase of the viral life cycle. The former method used by Bullen is likely to be more sensitive to the production of replication-competent virus, yet only Wei showed viral induction by HDAC inhibitors. The inability of the more robust method used by Bullen to detect HIV activation would suggest that the viral expression detected by Wei may not have been replication competent.
The second important difference was Bullen’s PCR assay for host-initiated HIV readthrough transcripts. Conventional HIV PCR assays that target gag and LTR are likely to measure viral transcripts initiated by host genes. These will not reflect properly spliced viral transcripts, required for functional virions. This is another important difference between the approaches taken by the two groups. Wei’s detection of an effect of HDAC inhibitors, relies on Roche’s COBAS HIV PCR assay. According to Roche , this assay uses primers for gag and LTR that would also, presumably, measure HIV transcripts initiated by host genes. As the mechanism of action of HDAC inhibitors is to release genes from a silent state, enabling their expression, HDAC inhibitors will activate host genes. Therefore HDAC-inhibitors are liable to initiate HIV transcription by activating the promotors of host genes, into which HIV has integrated. The implication of this is that Wei’s detection of HIV mRNA sequences in HDAC-treated cells could conceivably be a false signal.
Wei et al say that HIV virions from the supernatants of HDAC-inhibitor treated CD4 cells can be pelleted by centrifugation, as further evidence that supernatant HIV RNA is encapsulated in virions (rather than RNA released from dying cells due to HDAC inhibitor toxicity). However, host cell-derived extracellular RNA occurs in normal biology, in the form of extracellular vesicles. These can be the same size and density as HIV virions and so very hard to distinguish by centrifugation. If the centrifuged particles are extracellular vesicles or cell debris containing host-initiated viral read-through RNA, it is possible that Wei’s data does not confirm viral induction by HDAC inhibitors and Bullen’s conclusion that HDAC inhibitors do not induce latent HIV may stand. Some of these questions could be resolved by the use of Bullen’s readthrough PCR assay, to measure the RNA in the cells and supernatants that Wei et al have generated.
- Archin NM et al. Adminsitration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy. Nature (2012), 487: 482-485.
- Bullen CK et al. New ex vivo approach to distinguish effective and ineffective single agents for reversing HIV-1 latency in vivo. Nature Medicine (2014).
- Wei DG et al. Histone deacetylase inhibitor romidepsin induces HIV expression in CD4 T cells from patients on suppressive antiretroviral therapy at concentrations achieved by clinical dosing. PLoS Pathogens (2014), 10 (4).
- Roche Molecular Diagnostics. COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test, v2.0. (Accessed 16 May 2014).