An attractive option for treating PEL and other γ-herpesvirus–induced cancers is targeting endogenous latent viruses with drugs that reactivate their lytic replication, thereby eradicating virally infected reservoirs. In this study, using a direct xenograft PEL model, we demonstrated that the combination of the antineoplastic agents Btz and SAHA synergized to induce KSHV lytic replication, while leading to extensive apoptosis and a significant survival advantage in PEL-bearing mice. Importantly, this potent killing effect occurred in the absence of infectious KSHV production. Btz and SAHA are FDA-approved drugs that are clinically available and currently under investigation for the treatment of HIV and γ-herpesvirus–related lymphomas in National Cancer Institute–sponsored AIDS Malignancies Consortium clinical trials (AMC-053 and AMC-075).
Considering that all PEL tumors are infected with latent KSHV, the antineoplastic effect observed by the combined use of Btz and SAHA might be in part accomplished by their ability to target latency and induce KSHV lytic replication. While the mechanism by which Btz induces viral reactivation remains unclear, HDIs like SAHA are thought to induce γ-herpesvirus lytic reactivation through chromatin remodeling. HDACs regulate the transcriptional activity of KSHV RTA (9
). Viral lytic induction is known to cause G0
cell cycle arrest, which can lead to cytotoxicity (37
). Indeed, in UM-PEL-1 cells the percentage of apoptotic cells closely correlated with drug-induced lytic reactivation (vGPCR-positive cells), indicating that KSHV lytic replication could be causally associated with cytotoxicity. While single drugs alone tend not to induce robust KSHV lytic reactivation in PEL (38
), the combination of Btz and SAHA synergized to induce KSHV lytic replication and enhanced apoptosis of PEL cells, an effect that translated into prolonged survival in vivo.
The powerful induction of lytic KSHV replication with concomitant inhibition of virus production was an unexpected yet clinically desirable outcome of the Btz/SAHA combination. To understand the nature of this inhibition, we analyzed viral DNA loads in vitro and found that Btz-treated UM-PEL-1c cells harbored nearly 4 fold the number of viral DNA copies compared with the control and SAHA-treated cells. We reasoned that since Btz was inhibiting late lytic gene expression, the accumulation of intracellular viral DNA could be a reflection of DNA replication, along with failure to complete the lytic replicative cycle. The results from viral infection assays support this hypothesis, as PEL cells stimulated with Btz or Btz/SAHA produced fewer infectious virions. This was confirmed in vivo: mice treated with Btz had markedly less encapsidated viral DNA in the ascites than those treated with SAHA alone.
The inhibition of infectious KSHV production by Btz is supported by a previous in vitro study describing Btz inhibition of virion production (39
). This antiviral effect likely results from the dependency of KSHV on the proteasome throughout the viral replicative cycle, which has been described in the context of other herpesvirus (40
). Here, while Btz induced the expression of many KSHV lytic genes, the transcription of several key genes was affected negatively, indicating that the proteasome inhibition has gene-specific effects on viral lytic transcription. For instance, transcription of RTA and ORF45 was synergistically enhanced by combining Btz and SAHA, while SAHA-induced K8 expression was inhibited by Btz. This may be a significant event, as the K8 protein coordinately activates, along with RTA, the expression of some KSHV lytic genes (42
, a late lytic gene that encodes a key glycoprotein and is transcribed from the same locus as K8, was similarly found to be inhibited at the mRNA and protein levels. While we did not interrogate all ORFs encoded by KSHV, it is likely that there are other loci that are similarly inhibited by proteasome inhibition. As shown in Figures and , we observed a distinct pattern of lytic activation between in vitro and in vivo experiments. This is most likely the consequence of differences between in vitro and host microenvironments, which are known to alter KSHV permissibility and viral gene expression in PEL (43
). Finally, while lytic herpesviruses are canonically cytopathic, whether KSHV reactivation contributes to apoptosis in PEL when mature virion production is blocked remains to be elucidated. Although it has been previously reported that Btz can reactivate EBV expression (44
), we did not observe reactivation of EBV, making it unlikely that EBV contributed to Btz/SAHA–induced cell death.
In addition to viral lytic induction, Btz and SAHA likely promote PEL cell apoptosis via other mechanisms. While the exact mechanisms of cell death by proteasome inhibitors remain controversial, studies have demonstrated that induction of cell death in B cell lymphomas by proteasome inhibitors is mediated by p53 (45
). Similarly, a possible explanation for the Btz-induced apoptosis seen in our PEL model was posttranslational stabilization of phosphorylated and acetylated p53 protein along with the accumulation of its targets, p21 and Bax. The observed increase in phosphorylated p53 and γH2AX proteins by Btz is consistent with a DNA damage response (Figure A). The partial shRNA-mediated abrogation of p53 expression in PEL xenografts, resulting in decreased cell death and blunted caspase activation, supports a role for p53 in mediating Btz-induced apoptosis in PEL. Additionally, the SAHA-induced p53 acetylation resulting in decreased p53-MDM2 interaction and augmented p21 transcription serves as evidence that p53 may also contribute to the antitumor effects of Btz/SAHA combination. While p53 acetylation is indispensable for its activation (35
), the exact early events induced by SAHA and critical p53 acetylation sites in PEL remain to be determined in future studies. Last, histone hyperacetylation has been shown to have proapoptotic effects in other neoplastic models (30
). The SAHA-induced acetylation and Btz-mediated accumulation of acetylated histones that we observed likely contributed to chromatin remodeling and the activation of silenced viral and cellular genes.
In summary, the results from this study point to a novel treatment strategy for KSHV-infected PEL (Figure D). Using the Btz/SAHA combination allows for robust viral induction while concurrently blocking infectious virus production, thus ensuring destruction of PEL cells. Given the observed anti-KSHV effect of Btz in stalling full lytic replication and virion production and the enhanced effect of Btz and SAHA on apoptotic pathways, this study provides a strong rationale for combining these drugs as a potent PEL therapy, especially in the setting of HIV and immunosuppression. Based on our findings, the clinical use of the combination of proteasome inhibitors and HDIs is clearly feasible for the treatment of PEL and potentially other γ-herpesvirus–related malignancies.