Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial

doi:10.1016/S2352-3018(14)70014-1

The Lancet HIV

Volume 1, Issue 1, October 2014, Pages e13-e21

https://doi.org/10.1016/S2352-3018(14)70014-1 Get rights and content

Summary

Background

Activating the expression of latent virus is an approach that might form part of an HIV cure. We assessed the ability of the histone deacetylase inhibitor panobinostat to disrupt HIV-1 latency and the safety of this strategy.

Methods

In this phase 1/2 clinical trial, we included aviraemic adults with HIV treated at Aarhus University Hospital, Denmark. Participants received oral panobinostat (20 mg) three times per week every other week for 8 weeks while maintaining combination antiretroviral therapy. The primary outcome was change from baseline of cell-associated unspliced HIV RNA. Secondary endpoints were safety, plasma HIV RNA, total and integrated HIV DNA, infectious units per million CD4 T cells, and time to viral rebound during an optional analytical treatment interruption of antiretroviral therapy. This trial is registered with ClinicalTrial.gov, number NCT01680094.

Findings

We enrolled 15 patients. The level of cell-associated unspliced HIV RNA increased significantly at all timepoints when patients were taking panobinostat (p<0·0001). The median maximum increase in cell-associated unspliced HIV RNA during panobinostat treatment was 3·5-fold (range 2·1–14·4). Panobinostat induced plasma viraemia with an odds ratio of 10·5 (95% CI 2·2–50·3; p=0·0002) compared with baseline. We recorded a transient decrease in total HIV DNA, but no cohort-wide reduction in total HIV DNA, integrated HIV DNA, or infectious units per million. Nine patients participated in the analytical treatment interruption, median time to viral rebound was 17 days (range 14–56). Panobinostat was well tolerated. 45 adverse events were reported, but only 16 (all grade 1) were presumed related to panobinostat.

Interpretation

Panobinostat effectively disrupts HIV latency in vivo and is a promising candidate for future combination clinical trials aimed at HIV eradication. However, panobinostat did not reduce the number of latently infected cells and this approach may need to be combined with others to significantly affect the latent HIV reservoir.

Funding

The Danish Council for Strategic Research and Aarhus University.

Introduction

Combination antiretroviral therapy effectively suppresses replication of HIV but persisting virus fuels rebound viraemia when treatment ceases.¹ Thus, lifelong adherence to antiretroviral therapy is necessary for disease control, raising concerns of long-term adverse effects and the financial burden of treatment. The development of treatments that cure infection, therefore, is a fundamental goal in HIV research.

The most significant obstacle to eradication of HIV infection is the presence of replication-competent provirus in long-lived resting CD4 T cells.2, 3, 4 In the transcriptionally silent resting state, viral proteins are not expressed and, therefore, the infected status of resting cells remains invisible to the immune system and unresponsive to antiretroviral therapy. This reversibly non-productive state of infection is referred to as HIV latency.⁵ Drugs aimed at activating HIV from latency should ideally induce viral transcription, lead to viral protein production, and release of viral particles—essential prerequisites for immune-mediated elimination.⁶ However, the effect of this approach is subject to ongoing controversy with an ex-vivo study suggesting that none of the leading candidate drugs can alone disrupt the latent HIV reservoir.⁷ Clinical trials are the only experimental approaches to investigate whether drugs aimed at disrupting HIV latency in vivo are effective or not.

One of several mechanisms controlling HIV latency is the activity of histone deacetylases, which repress proviral transcription by promoting histone deacetylation.8, 9 Several studies have shown that histone deacetylase inhibitors disrupt HIV latency in vitro.10, 11, 12 Additionally, administration of a single dose of vorinostat—a histone deacetylase inhibitor approved by the US Food and Drug Administration—led to significant increases in cell-associated HIV RNA,¹³ as did multiple doses of vorinostat over 14 days.¹⁴ By contrast, three doses per week of vorinostat over 8 weeks only increased amounts of cell-associated HIV RNA above baseline for three of five participants.¹⁵ Increased plasma viraemia or reduction of the latent HIV reservoir have not been shown in studies of vorinostat. These findings have led researchers to question the ability of histone deacetylase inhibitors to disrupt latency to the extent that HIV proteins are expressed on the surface of infected cells, thus enabling immune-mediated elimination to occur.7, 16, 17

Panobinostat is a highly potent hydroxamic acid pan-histone deacetylase inhibitor in clinical development for the treatment of multiple myeloma.¹⁸ Therapeutic concentrations of panobinostat induce viral production in latently infected cells in vitro.¹⁹ On the basis of these data, we designed an interventional study to assess whether 8 weeks of cyclic panobinostat treatment added to antiretroviral therapy would increase HIV transcription, increase plasma viraemia, and affect the latent HIV reservoir.

Section snippets

Study design and participants

We did a single group, phase 1/2 trial at Aarhus University Hospital, Denmark, between Oct 1, 2012 and Jan 16, 2014. We enrolled HIV-infected adults taking antiretroviral therapy with virological suppression (<50 copies per mL, at least two measurements per year) for at least 2 years and CD4 counts above 500 cells per μL. Exclusion criteria included co-infection with hepatitis B or C viruses, clinically significant cardiac disease including QTc prolongation, and current use of a protease

Results

We enrolled 15 patients and all completed full panobinostat dosing (table 1). The amount of cell-associated unspliced HIV RNA increased during panobinostat treatment (p<0·0001; repeated measurement ANOVA incorporating all data from baseline and on panobinostat); with significant increases at all assayed timepoints compared with baseline (figure 1A, appendix p 3). Amounts of cell-associated unspliced HIV RNA increased rapidly, with a mean increase of 2·4-fold (95% CI 1·8–3·3; p<0·0001) measured

Discussion

8 weeks of cyclic treatment with panobinostat was safe, well tolerated, and effectively increased HIV transcription in patients taking antiretroviral therapy. Furthermore, panobinostat-induced HIV transcription was temporally associated with increased detection of plasma HIV RNA. Viral rebound followed treatment interruption, although the time to rebound varied between individuals. Together, these results provide evidence that panobinostat can effectively activate HIV from latency in vivo and

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Selection of epigenetically privileged HIV-1 proviruses during treatment with panobinostat and interferon-α2a
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CD4⁺ T cells with latent HIV-1 infection persist despite treatment with antiretroviral agents and represent the main barrier to a cure of HIV-1 infection. Pharmacological disruption of viral latency may expose HIV-1-infected cells to host immune activity, but the clinical efficacy of latency-reversing agents for reducing HIV-1 persistence remains to be proven. Here, we show in a randomized-controlled human clinical trial that the histone deacetylase inhibitor panobinostat, when administered in combination with pegylated interferon-α2a, induces a structural transformation of the HIV-1 reservoir cell pool, characterized by a disproportionate overrepresentation of HIV-1 proviruses integrated in ZNF genes and in chromatin regions with reduced H3K27ac marks, the molecular target sites for panobinostat. By contrast, proviruses near H3K27ac marks were actively selected against, likely due to increased susceptibility to panobinostat. These data suggest that latency-reversing treatment can increase the immunological vulnerability of HIV-1 reservoir cells and accelerate the selection of epigenetically privileged HIV-1 proviruses.
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Tremendous advances have been made in the discovery and use of combination antiretroviral treatment (cART) for HIV-1 infection over the past three decades. ART suppresses active viral infection in most of the body compartments and keeps the plasma viral load to an undetectable level while the patient continues to take the medication. Despite significant advances in the treatment of HIV-1, there remains no cure. Certain immune cells can be infected with HIV but lie dormant without actively producing virus, even in the presence of highly active combinatorial antiretroviral drugs. These immune cells carrying the dormant virus get reactivated and subsequently start producing new virus once the ART is withdrawn or discontinued and spread the virus all over the body. This state of dormancy is known as HIV latency, and the infected cells bearing the integrated provirus are labeled as the latent reservoir. Immune cell types (lymphocytes, macrophages, and microglia) carrying latent HIV in the central nervous system present unique challenges for potential HIV elimination, because most of the ART drugs available for treatment cannot penetrate well into the CNS due to the presence of blood–brain barrier. This leads to continuous low-level viral replication in the CNS and the emergence of HIV-1-associated neurocognitive disorders. Thus, eliminating HIV from CNS reservoir compartments could speed up HIV eradication efforts. Extensive research was published focusing on different methods for HIV-1 elimination, such as “shock-and-kill” latency reversal agents, CRISPR-Cas9-mediated gene editing, broadly neutralizing antibody therapy, innate immune regulators, “block-and-lock” strategies, and CAR T cell therapies. These methods have had varied success in vitro and, to some extent, in animal models. In this chapter, we summarized the approaches used by our group and other research groups for HIV-1 elimination and elaborated on the advantage for the sequential use of cART and CRISPR-based gene editing to be pursued for an ultimate HIV cure.
A histone deacetylase network regulates epigenetic reprogramming and viral silencing in HIV-infected cells
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A long-lived latent reservoir of HIV-1-infected CD4 T cells persists with antiretroviral therapy and prevents cure. We report that the emergence of latently infected primary CD4 T cells requires the activity of histone deacetylase enzymes HDAC1/2 and HDAC3. Data from targeted HDAC molecules, an HDAC3-directed PROTAC, and CRISPR-Cas9 knockout experiments converge on a model where either HDAC1/2 or HDAC3 targeting can prevent latency, whereas all three enzymes must be targeted to achieve latency reversal. Furthermore, HDACi treatment targets features of memory T cells that are linked to proviral latency and persistence. Latency prevention is associated with increased H3K9ac at the proviral LTR promoter region and decreased H3K9me3, suggesting that this epigenetic switch is a key proviral silencing mechanism that depends on HDAC activity. These findings support further mechanistic work on latency initiation and eventual clinical studies of HDAC inhibitors to interfere with latency initiation.
AP-1/c-Fos supports SIV and HIV-1 latency in CD4 T cells infected in vivo
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Persistent HIV-1 reservoirs of infected CD4 T cells are a major barrier to HIV-1 cure, although the mechanisms by which they are established and maintained in vivo remain poorly characterized. To elucidate host cell gene expression patterns that govern virus gene expression, we analyzed viral RNA+ (vRNA) CD4 T cells of untreated simian immunodeficiency virus (SIV)-infected macaques by single-cell RNA sequencing. A subset of vRNA+ cells distinguished by spliced and high total vRNA (7–10% of reads) expressed diminished FOS, a component of the Activator protein 1 (AP-1) transcription factor, relative to vRNA-low and -negative cells. Conversely, FOS and JUN, another AP-1 component, were upregulated in HIV DNA+ infected cells compared to uninfected cells from people with HIV-1 on suppressive therapy. Inhibiting c-Fos in latently infected primary cells augmented reactivatable HIV-1 infection. These findings implicate AP-1 in latency establishment and maintenance and as a potential therapeutic target to limit HIV-1 reservoirs.
Venetoclax, alone and in combination with the BH3 mimetic S63845, depletes HIV-1 latently infected cells and delays rebound in humanized mice
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HIV-1 persists indefinitely in people living with HIV (PLWH) on antiretroviral therapy (ART). If ART is stopped, the virus rapidly rebounds from long-lived latently infected cells. Using a humanized mouse model of HIV-1 infection and CD4⁺ T cells from PLWH on ART, we investigate whether antagonizing host pro-survival proteins can prime latent cells to die and facilitate HIV-1 clearance. Venetoclax, a pro-apoptotic inhibitor of Bcl-2, depletes total and intact HIV-1 DNA in CD4⁺ T cells from PLWH ex vivo. This venetoclax-sensitive population is enriched for cells with transcriptionally higher levels of pro-apoptotic BH3-only proteins. Furthermore, venetoclax delays viral rebound in a mouse model of persistent HIV-1 infection, and the combination of venetoclax with the Mcl-1 inhibitor S63845 achieves a longer delay in rebound compared with either intervention alone. Thus, selective inhibition of pro-survival proteins can induce death of HIV-1-infected cells that persist on ART, extending time to viral rebound.
The efficacy and tolerability of latency-reversing agents in reactivating the HIV-1 reservoir in clinical studies: a systematic review
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A literature search was performed using medical databases focusing on studies with adults living with HIV-1 receiving LRAs. Eligibility criteria required participants from prospective clinical studies, a studied compound hypothesised as LRA, and reactivation or tolerability assessments. Relevant demographical data, LRA reactivation capacity, reservoir size, and adverse events were extracted. A study quality assessment with analysis of bias was performed by RoB 2 and ROBINS-I tools. The primary endpoints were HIV-1 reservoir reactivation after LRA treatment quantified by cell-associated unspliced HIV-1 RNA, and LRA tolerability defined by adverse events. Secondary outcomes were reservoir size and the effect of LRAs on analytical treatment interruption (ATI) duration.
After excluding duplicates, 5182 publications were screened. In total 45 publications fulfilled eligibility criteria including 26 intervention studies and 16 randomised trials. The risk of bias was evaluated as high. Chromatin modulators were the main investigated LRA class in 24 studies. Participants were mostly males (90.1%). Where reported, HIV-1 subtype B was most frequently observed. Reactivation after LRA treatment occurred in 78% of studies and was observed with nearly all chromatin modulators. When measured, reactivation mostly occurred within 24 h after treatment initiation. Combination LRA strategies have been infrequently studied and were without synergistic reactivation. Adverse events, where reported, were mostly low grade, yet occurred frequently. Seven studies had individuals who discontinued LRAs for related adverse events. The reservoir size was assessed by HIV-1 DNA in 80% of studies. A small decrease in reservoir was observed in three studies on immune checkpoint inhibitors and the histone deacetylase inhibitors romidepsin and chidamide. No clear effect of LRAs on ATI duration was observed.
This systematic review provides a summary of the reactivation of LRAs used in current clinical trials whilst highlighting the importance of pharmacovigilance. Highly heterogeneous study designs and underrepresentation of relevant patient groups are to be considered when interpreting these results. The observed reactivation did not lead to cure or a significant reduction in the size of the reservoir. Finding more effective LRAs by including well-designed studies are needed to define the required reactivation level to reduce the HIV-1 reservoir.

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ArticlesPanobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial

Summary

Background

Methods

Findings

Interpretation

Funding

Introduction

Section snippets

Study design and participants

Results

Discussion

Immunity

Immunity

Lancet

Immunity

HIV-1 and T cell dynamics after interruption of highly active antiretroviral therapy (HAART) in patients with a history of sustained viral suppression

Proc Natl Acad Sci USA

Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy

Proc Natl Acad Sci USA

Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection

Nature

Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy

Science

HIV: shock and kill

Nature

New ex vivo approaches distinguish effective and ineffective single agents for reversing HIV-1 latency in vivo

Nat Med

Transcriptional activation and chromatin remodeling of the HIV-1 promoter in response to histone acetylation

Embo J

NF-kappaB p50 promotes HIV latency through HDAC recruitment and repression of transcriptional initiation

Embo J

Expression of latent HIV induced by the potent HDAC inhibitor suberoylanilide hydroxamic acid

AIDS Res Hum Retroviruses

Entinostat is a histone deacetylase inhibitor selective for class 1 histone deacetylases and activates HIV production from latently infected primary T cells

AIDS

Unique characteristics of histone deacetylase inhibitors in reactivation of latent HIV-1 in Bcl-2-transduced primary resting CD4+ T cells

J Antimicrob Chemother

Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy

Nature

Articles
Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial