Histone Deacetylase Inhibitors: Potent Inducers of Tumor Latent EBV Thymidine Kinase Induction

Abstract 1831

Strong epidemiological association of Epstein-Barr Virus (EBV) with various human lymphoid malignancies and in vitro studies demonstrating tumorigenic activity of many EBV latent gene products suggest a causal relationship between EBV and these diseases. However, as EBV maintains a latent state of infection in these lymphomas, typical anti-herpesviral drugs, such as the nucleoside analogs ganciclovir (GCV) or acyclovir, are ineffective as these pro-drugs require expression of a lytic phase EBV protein, thymidine kinase (TK), for their activity. Therefore, selective induction of EBV lytic-phase gene expression in lymphoma cells that harbor latent EBV, coupled with simultaneous exposure to anti-herpesviral drugs, has been advanced as promising targeted therapy, because of resulting targeting of cytotoxicity to the EBV-infected tumor cells. A variety of agents including short-chain fatty acids and chemotherapeutic drugs, have been used to induce EBV lytic-phase infection in cultured cells, but these in vitro studies have generally not translated into clinical application. We have successfully used arginine butyrate and GCV to treat EBV-positive lymphoid malignancies in a recent Phase I/II clinical trial. In this study of 15 patients with relapsed or refractory EBV-positive lymphoid tumors, 4 patients achieved complete tumor remissions and 6 patients partial tumor remissions. However, the rapid metabolism of butyrate requires continuous IV administration of high doses. Butyrate has pan-HDAC inhibitory activity, and we have established that this activity is responsible for the induction of the EBV-TK protein. In recent years, several potent HDAC inhibitors (HDACi) have been tested in the clinic as anti-cancer agents. In the current study, we have investigated a number of HDACi, including some new, highly-potent compounds, for their potential to induce EBV lytic phase gene expression and to kill EBV-infected cells in combination with anti-herpesviral drugs. Our study included short-chain fatty acids (sodium butyrate and valproic acid); hydroxamic acids [Oxamflatin, Scriptaid, Suberoyl anilide hydroxamic acid (SAHA), Panobinostat (LBH589) and Belinostat (PXD101)]; the benzamide MS275; cyclic tetrapeptide Apicidin, and newly-identified HDAC inhibitor Largazole, which was originally isolated from a marine cyanobacterium. We assayed the induction of lytic phase in EBV-positive lymphoma cell lines exposed to different HDACi for 24–48 hrs, then quantitated the expression of EBV TK and other EBV transcripts by RT-PCR analysis. To determine tumor cytotoxic activity of the combination of HDACi and GCV, EBV+ lymphoma cells were exposed to a range of concentrations of HDACi and GCV for 3 days and then to GCV alone for another 3 days. Efficacy of a particular HDACi in the combination treatment approach was then determined by enumerating living cells. With the exception of SAHA and PXD101, the other HDACi had synergistic activity with anti-viral agents in killing EBV+ lymphoma cells. The hydroxamic acid LBH589, the benzamide MS275, and synthetic largazole derivatives 234a and 234b were 10⁴ to 10⁵-times more potent in killing EBV+ lymphoma cells in presence of GCV, compared to sodium butyrate. The effective concentration of LBH589 was in the range of 50–100 nM, MS275 at 200–500 nM and Largazole 234a and 234b at 100–200 nM. Of note, at these concentrations, the drugs as single agents produced no growth inhibitory activity in the tumor cells. LBH589, MS275 and Largazole 234a and 234b also strongly induced EBV-TK expression in the tumor cells. The effectiveness of these HDACi compounds at such low concentrations makes them potentially applicable as sensitizers to anti-viral therapeutics for the treatment of EBV-associated lymphomas. Our finding therefore provides an intriguing possibility that these novel HDACi may be used as an alternative therapeutic option, in combination with nucleoside antivirals, for the treatment of EBV-associated tumors.