Histone deacetylase inhibitors are potent inducers of gene expression in latent EBV and sensitize lymphoma cells to nucleoside antiviral agents

Abstract

Induction of EBV lytic-phase gene expression, combined with exposure to an antiherpes viral drug, represents a promising targeted therapeutic approach to EBV-associated lymphomas. Short-chain fatty acids or certain chemotherapeutics have been used to induce EBV lytic-phase gene expression in cultured cells and mouse models, but these studies generally have not translated into clinical application. The recent success of a clinical trial with the pan-histone deacetylase (pan-HDAC) inhibitor arginine butyrate and the antiherpes viral drug ganciclovir in the treatment of EBV lymphomas prompted us to investigate the potential of several HDAC inhibitors, including some new, highly potent compounds, to sensitize EBV(+) human lymphoma cells to antiviral agents in vitro. Our study included short-chain fatty acids (sodium butyrate and valproic acid); hydroxamic acids (oxamflatin, Scriptaid, suberoyl anilide hydroxamic acid, panobinostat [LBH589], and belinostat [PXD101]); the benzamide MS275; the cyclic tetrapeptide apicidin; and the recently discovered HDAC inhibitor largazole. With the exception of suberoyl anilide hydroxamic acid and PXD101, all of the other HDAC inhibitors effectively sensitized EBV(+) lymphoma cells to ganciclovir. LBH589, MS275, and largazole were effective at nanomolar concentrations and were 10(4) to 10(5) times more potent than butyrate. The effectiveness and potency of these HDAC inhibitors make them potentially applicable as sensitizers to antivirals for the treatment of EBV-associated lymphomas.

Figures

Figure 1

Chemical structures and chemical classes of the HDAC inhibitors used.

Figure 2

HDAC inhibitor–mediated induction of TK transcript in EBV+ lymphoma cells. Three million P3HR1 cells in 3 mL of RPMI 1640 medium were exposed to individual HDAC inhibitors for 48 hours. HDAC inhibitors used include short-chain fatty acids (butyrate and valproate), cyclic tetrapeptide (apicidin), cyclic depsipeptide (parent largazole and analogs A and B), benzamide (MS275), and hydroxamic acids (oxamflatin, LBH589, SAHA, PXD101, and Scriptaid). Inhibitor concentrations were determined empirically so that cytotoxicity remained minimal. Total RNA extraction, reverse transcription, and real-time PCR analysis were performed as described in “Analysis of lytic gene expression.” Real-time PCR was performed in triplicate on each HDAC inhibitor–treated sample for both TK mRNA and β-actin mRNA, and these values were used to determine respective ΔCt and the fold induction. RNA from P3HR1 cells treated with 1.0 and 2.5mM sodium butyrate were used as internal controls in each experiment (not shown for each inhibitor). Each assay was repeated 3 times and error bars in each individual figure represent SDs.

Figure 3

Cytotoxic activity of HDAC inhibitors in the presence of an antiherpes virus drug. (A) Three hundred thousand P3HR1 cells were exposed to either 40μM GCV or vehicle, and the indicated concentrations of individual HDAC inhibitors in a 1-mL volume in 24-well plates in triplicate. Three days later, 800 μL of the medium was removed without disturbing the settled cells, 1 mL of fresh growth medium containing GCV (40μM) was added, and the cells were allowed to grow for another 3 days. HDAC inhibitors used included butyrate, valproate, apicidin, largazole and its analogs, MS275, oxamflatin, LBH589, SAHA, and Scriptaid. The number above the HDAC + GCV bar represents the percentage of cells surviving relative to the cultures exposed to that particular HDAC inhibitor alone (assigned a value of 100%). Error bars represent SDs in individual experiments. (B) Cytotoxic activity of selected HDAC inhibitors (butyrate, MS275, and LBH589) in the presence of GCV in the EBV− B-lymphoma lines BJAB and Toledo. Experiments were carried out essentially as in panel A. The right panel shows detection of EBER1- and β-actin–specific PCR products generated from cellular DNA of BJAB, P3HR1, and Toledo cells analyzed in a 2% agarose gel. A vertical line has been inserted to indicate a repositioned gel lane.

Figure 4

HDAC inhibitor–mediated induction of EBV lytic-phase gene expression. (A) Comparison of TK and BGLF4 transcript expression in P3HR1 cells after exposure to different HDAC inhibitors. Cells were treated with individual HDAC inhibitors as indicated and total RNA was analyzed by reverse transcription and real-time PCR analysis as described in the legend to Figure 2. Expression of β-actin mRNA under similar treatment conditions was used to normalize the dataset. (B) Immunoblot analysis for EA-D protein. Thirty micrograms of whole-cell lysates from individual HDAC inhibitor–treated cells (for 48 hours) or untreated cells were separated in 10% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with a 1:2000 dilution of mouse anti-EBV EA-D Ab (Millipore). Equal loading of proteins was verified by immunoblotting with 1:15 000 dilution of mouse anti–β-actin Ab (Sigma-Aldrich). Each assay was repeated 3 times and error bars in each individual figure represent SDs.

Figure 5

Sensitization of EBV lymphoma cells to GCV-mediated killing by brief exposure to HDAC inhibitors. (A) Three hundred thousand P3HR1 cells in a 1-mL volume were treated with 20nM LBH589 for the indicated period of time in the presence or absence of 40μM GCV. The culture medium was completely removed after centrifugation at the end of incubation with LBH589 and replenished with fresh growth medium with or without GCV, as indicated. Media for all cells were replaced again at 72 hours. Cells were counted at 144 hours (day 6). (B) Similar protocol as in panel A, but MS275 was evaluated at 3 different concentrations. The overwhelming toxicity after exposure to MS275 at 5μM for 48 or 72 hours precluded any meaningful cell count. Cells exposed to sodium butyrate at 1mM were used as an internal control. The number above the HDAC + GCV bar represents the percentage of cells surviving relative to the cell count after exposure to the HDAC inhibitor alone (assigned a value of 100%). Experiments were repeated 3 times and error bars represent SDs in individual experiments.

Figure 6

Effect of HDAC inhibitor and GCV combination treatment on other EBV+ lymphoma cells. (A) Analysis of promoter use by the 3 different EBV lymphoma cell lines used in the study. Reverse transcription and PCR analysis of total RNA was carried out using primers that specifically detected the Qp, Wp, or Cp transcripts (Table 2). Products were analyzed on a 2% agarose gel with a 100-bp DNA ladder as a marker. A vertical line has been inserted to indicate a repositioned gel lane. (B) Effect of combination treatment on the BL line Daudi. Four hundred thousand cells/mL/well were used along with 60μM GCV in the appropriate wells. Assay parameters were as described in the legend for Figure 2. (C) Effect of combination treatment on the EBV-transformed lymphoblastoid cell line JY. In this case, 200 000 cells/mL/well were used, along with 60μM GCV as appropriate. Experiments were repeated 3 times and error bars represent SDs in individual experiments.