Hypoxia Enhances the Phosphorylation and Cytotoxicity of Ganciclovir and Zidovudine in Kaposi's Sarcoma-Associated Herpesvirus–Infected Cells

Abstract

Primary effusion lymphoma (PEL) is a rare B-cell lymphoma caused by Kaposi's sarcoma-associated herpesvirus (KSHV). PEL is poorly responsive to standard cytotoxic chemotherapy and portends a poor survival. Consequently, new effective treatment options are urgently needed. It is known that KSHV encodes two lytic genes, ORF36 (phosphotransferase) and KSHV ORF21 (thymidine kinase), which can phosphorylate ganciclovir and azidothymidine, respectively. Here, we have explored whether these genes can be used as therapeutic targets for PEL. PEL arises in pleural spaces and other effusions that provide a hypoxic environment. Based on Northern blot analysis, exposure of PEL cells to hypoxia up-regulated the expression of both ORF36 and ORF21. Using a newly developed nonradioactive reverse-phase high-performance liquid chromatography/mass spectrometry method to separate and quantify the phosphorylated forms of ganciclovir and azidothymidine, we found that PEL cells exposed to hypoxia produced increased amounts of the toxic triphosphates of these drugs. Moreover, we found that hypoxia increased the cell toxicity of ganciclovir and azidothymidine in PEL cells but had no significant effect on the herpesvirus-negative cell line CA46. These findings may have clinical applicability in the development of effective therapies for PEL or other KSHV-related malignancies.

High-dose zidovudine plus valganciclovir for Kaposi sarcoma herpesvirus-associated multicentric Castleman disease: a pilot study of virus-activated cytotoxic therapy

“…27 AZT at a dose of 10 m/L induces cell toxicity in PEL cells induced to lytic KSHV activation; such AZT levels are attained with an intravenous dose of 5 mg/kg. 25,43 One hypothesis is that the KS responses that the original AZT trial were at least partially the result of KSHV ORF21-induced accumulation of toxic AZT triphosphate, and associated cytotoxic activity in a subset of KS tumor cells with lytic KSHV replication. However, we cannot exclude the possibility that the KS responses in that study were related to control of HIV viremia and resulting enhanced immune function, as is currently observed in some patients receiving HAART.…”

“…25 One KSHV lytic gene, ORF36, encodes a phosphotransferase that activates ganciclovir to a toxic triphosphate moiety, and another, ORF21, encodes a thymidine kinase that phosphorylates zidovudine (AZT). [25][26][27] We previously showed that PEL cells in which KSHV was lytically activated produced increased amounts of AZT and ganciclovir triphosphate moieties. AZT and ganciclovir had synergistic toxicity at doses attainable in patients, in these PEL lines.…”

“…AZT and ganciclovir had synergistic toxicity at doses attainable in patients, in these PEL lines. 25 We hypothesized that the combination of AZT and ganciclovir would selectively target the KSHV-infected plasmablasts in KSHV-MCD and have utility in the treatment of symptomatic disease. To translate these findings, we conducted a pilot study of high-dose AZT and valganciclovir (VGC), an orally available prodrug of ganciclovir, in patients with symptomatic KSHV-MCD.…”

A viral kinase mimics S6 kinase to enhance cell proliferation

“…Glycolysis is a frequent bioenergetic adaptation of cancer cells, both in aerobic conditions and in hypoxic environments unfavorable for oxidative phosphorylation. Notably, vPK is induced by hypoxia (13,14), and, furthermore, KSHV is known to up-regulate glycolysis in both lytic and latent conditions (33,34). Thus, vPK may modulate the induction of the glycolytic phenotype.…”

“…This wide repertoire of targets results from vPK localizing within both the cytoplasmic and nuclear compartments (12). Within the KSHV genome, vPK is located within the ORF 34-37 cluster, which contains a hypoxia-inducible factor (HIF) response element within the promoter region (13,14). Thus, vPK expression may be induced in hypoxic environments, independent of lytic replication.…”