EZH2 Inhibition by DS3201 Triggers the Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycle and Potentiates the Effects Induced by SAHA in Primary Effusion Lymphoma Cells

Abstract

Primary Effusion Lymphoma (PEL) cells carry Kaposi's sarcoma-associated herpesvirus (KSHV) in a latent state, except for a small number of cells in which the virus replicates to ensure its persistence into the infected host. However, the lytic cycle can be reactivated in vitro by exposing these lymphoma cells to various treatments, leading to cell lysis. To restrict viral antigen expression, KSHV induces repressive epigenetic changes, including DNA methylation and histone modifications. Among the latter, histone deacetylation and tri-methylation of Histone H3 lisyne-27 (H3K27me3) have been reported to play a role. Here, we found that the inhibition of H3K27 tri-methylation by valemetostat DS3201 (DS), a small molecule that inhibits Enhancer of Zeste Homolog 2 (EZH2) methyltransferase, induced the KSHV lytic cycle in PEL cells, and that this effect involved the activation of the wtp53-p21 axis and autophagic dysregulation. DS also potentiated the lytic cycle activation mediated by the Histone deacetylases (HDAC) inhibitor Suberoylanilide hydroxamic acid (SAHA) and reinforced its cytotoxic effect, suggesting that such a combination could be used to unbalance the latent/lytic cycle and further impair the survival of PEL cells.

Keywords: KSHV; PEL cells; autophagy; lytic cycle; p53.

Figure 1

Valemetostat DS3201 (DS) activates Kaposi sarcoma associated herpesvirus (KSHV) lytic cycle in BC3 and BCBL-1 PEL cells. BC3 and BCBL-1 were exposed to DS3201 (DS) for 24 h, and (A) H3K27 trimethylation and (B) basic region leucine-zipper protein (KbZIP) expression were evaluated by Western blot analysis. Bar-graphs represent the mean plus SD of proteins of interest on house-keeping proteins calculated based on three different experiments; (C) KbZIP and (D) gp64 expression, as evaluated by IFA in BC3 and BCBL-1 cells treated by DS, Bar = 50 μM. The percentage of positive cells was 10% for KbZIP in both BC3 and BCBL-1, and 12% and 15% for gp64 in BC3 and BCBL-1, respectively); (E,F) Replication transactivation activator (RTA) and K8.1 mRNA expression as evaluated by Real Time quantitative PCR (RT-qPCR). The difference was considered statistically significant when the p-value was: * < 0.05; ** < 0.01.

Figure 2

EZH2 inhibition activates wtp53, which contributes to lytic cycle activation and inhibits EZH2 activity. (A) p53 and p21 expression levels as evaluated by Western blot in BC3 and BCBL-1 cells treated by DS; (B) expression of KbZIP in PEL cells pretreated or not with Pifithrin-α p53 inhibitor (ip53) before exposure to DS; (C) H3K27me3 expression in BC3 cells treated by DS or Pifithrin/DS (ip53+DS) combination. Bar-graphs represent the mean plus SD of proteins of interest on house-keeping proteins calculated based on three different experiments. The difference was considered statistically significant when the p-value was: * < 0.05; ** < 0.01.

Figure 3

DS triggers autophagy and inhibits its final phases, which promotes the KSHV lytic cycle. (A,B) Microtubule associated protein 1A/1B light chain 3 (LC3)I/II accumulation in BC3 and BCBL-1 cells treated by DS in the presence or absence of Bafilomycin A (BAF), as evaluated by Western blotting; (C) KbZIP expression in BC3 PEL cells treated by DS in the presence or absence of 3-methyladenine (3-MA) or Bafilomycin (BAF). Bar-graphs represent the mean plus SD of proteins of interest on house-keeping proteins calculated based on three different experiments. (D) Expression of RTA, as evaluated by RT-qPCR in BC3 cells treated or not with DS in the presence or absence of 3-MA or Bafilomycin (BAF). The difference was considered statistically significant when the p-value was: * < 0.05.

Figure 4

DS increases the activation of the viral lytic cycle and the cytotoxic effect induced by suberoylanilide hydroxamic acid (SAHA). BC3 PEL cells, treated by DS, SAHA or combination of both were investigated (A) for the expression of KbZIP by Western blot analysis; (B) for the expression of RTA by RT-qPCR, (C) for the expression of KbZIP, and (D) gp64 by IFA, following treatment by DS in the presence or absence of SAHA; bar = 50 μM. (E) Cytotoxic effect of DS, SAHA or combination of both as evaluated by trypan-blue assay in BC3 cells after 24 h of treatment, in the absence or presence of phosphonoacetic acid PAA; (F) Poly adenosine diphosphate-ribose polymerase (PARP) cleavage in BC3 and BCBL-1 cells treated by DS, SAHA or DS/SAHA combination investigated by Western blotting. Bar-graphs represent the mean plus SD of proteins of interest on house-keeping proteins calculated based on three different experiments. The difference was considered statistically significant when the p-value was: * < 0.05; ** < 0.01.

Figure 5

Scheme recapitulating the involvement of EZH2 in KSHV lytic cycle.