Lytic Induction Therapy against Epstein-Barr Virus-Associated Malignancies: Past, Present, and Future.

Epstein-Barr virus (EBV) lytic induction therapy is an emerging virus-targeted therapeutic approach that exploits the presence of EBV in tumor cells to confer specific killing effects against EBV-associated malignancies. Efforts have been made in the past years to uncover the mechanisms of EBV latent-lytic switch and discover different classes of chemical compounds that can reactivate the EBV lytic cycle. Despite the growing list of compounds showing potential to be used in the lytic induction therapy, only a few are being tested in clinical trials, with varying degrees of success. This review will summarize the current knowledge on EBV lytic reactivation, the major hurdles of translating the lytic induction therapy into clinical settings, and highlight some potential strategies in the future development of this therapy for EBV-related lymphoid and epithelial malignancies.

Autophagy-Dependent Reactivation of Epstein-Barr Virus Lytic Cycle and Combinatorial Effects of Autophagy-Dependent and Independent Lytic Inducers in Nasopharyngeal Carcinoma.

Autophagy, a conserved cellular mechanism, is manipulated by a number of viruses for different purposes. We previously demonstrated that an iron-chelator-like small compound, C7, reactivates Epstein-Barr virus (EBV) lytic cycle by activating the ERK1/2-autophagy axis in epithelial cancers. Here, we aim to identify the specific stage of autophagy required for EBV lytic reactivation, determine the autophagy dependency of EBV lytic inducers including histone deacetylase inhibitor (HDACi) and C7/iron chelators, for EBV lytic reactivation and measure the combinatorial effects of these types of lytic inducers in nasopharyngeal carcinoma (NPC). Inhibition of autophagy initiation by 3-MA and autolysosome formation by chloroquine demonstrated that only autophagy initiation is required for EBV lytic reactivation. Gene knockdown of various autophagic proteins such as beclin-1, ATG5, ATG12, ATG7, LC3B, ATG10, ATG3 and Rab9, revealed the importance of ATG5 in EBV lytic reactivation. 3-MA could only abrogate lytic cycle induction by C7/iron chelators but not by HDACi, providing evidence for autophagy-dependent and independent mechanisms in EBV lytic reactivation. Finally, the combination of C7 and SAHA at their corresponding reactivation kinetics enhanced EBV lytic reactivation. These findings render new insights in the mechanisms of EBV lytic cycle reactivation and stimulate a rational design of combination drug therapy against EBV-associated cancers.

Viral-Targeted Strategies Against EBV-Associated Lymphoproliferative Diseases.

Epstein-Barr virus (EBV) is strongly associated with a spectrum of EBV-associated lymphoproliferative diseases (EBV-LPDs) ranging from post-transplant lymphoproliferative disorder, B cell lymphomas (e.g., endemic Burkitt lymphoma, Hodgkin lymphoma, and diffuse large B cell lymphoma) to NK or T cell lymphoma (e.g., nasal NK/T-cell lymphoma). The virus expresses a number of latent viral proteins which are able to manipulate cell cycle and cell death processes to promote survival of the tumor cells. Several FDA-approved drugs or novel compounds have been shown to induce killing of some of the EBV-LPDs by inhibiting the function of latent viral proteins or activating the viral lytic cycle from latency. Here, we aim to provide an overview on the mechanisms by which EBV employs to drive the pathogenesis of various EBV-LPDs and to maintain the survival of the tumor cells followed by a discussion on the development of viral-targeted strategies based on the understanding of the patho-mechanisms.

High risk Epstein-Barr virus variants characterized by distinct polymorphisms in the EBER locus are strongly associated with nasopharyngeal carcinoma.

Whether certain variants of Epstein-Barr virus (EBV) are linked to the pathogenesis of nasopharyngeal carcinoma (NPC), which shows a marked geographic restriction, remains an unresolved issue. We performed a case-control study comparing genomic sequences of EBV isolated from saliva samples of 142 population carriers with those from primary tumour biopsies derived from 62 patients with NPC of Hong Kong. Cluster analysis discovered five EBV subgroups 1A-C and 2A-B amongst the population carriers in contrast to the predominance of 1A and -B in the majority of NPC. Genome-wide association study (GWAS) identified a panel of NPC-associated single nucleotide polymorphisms (SNPs) and indels in the EBER locus. The most significant polymorphism, which can be found in 96.8% NPC cases and 40.1% population carriers of Hong Kong, is a four-base-deletion polymorphism downstream of EBER2 (EBER-del) from coordinates 7188-7191 (p = 1.91 × 10-7 ). In addition, the predicted secondary structure of EBER2 is altered with likely functional consequence in nearly all NPC cases. Using the SNPs and indels associated with NPC, genetic risk score is assigned for each EBV variant. EBV variants with high genetic risk score are found to be much more prevalent in Hong Kong Chinese than individuals of other geographic regions and in NPC than other EBV-associated cancers. We conclude that high risk EBV variants with polymorphisms in the EBER locus, designated as HKNPC-EBERvar, are strongly associated with NPC. Further investigation of the biological function and potential clinical application of these newly identified polymorphisms in NPC and other EBV-associated cancers is warranted.

Intracellular Iron Chelation by a Novel Compound, C7, Reactivates Epstein⁻Barr Virus (EBV) Lytic Cycle via the ERK-Autophagy Axis in EBV-Positive Epithelial Cancers.

Pharmaceutical reactivation of lytic cycle of Epstein⁻Barr virus (EBV) represents a potential therapeutic strategy against EBV-associated epithelial malignancies, e.g., gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC). A novel lytic-inducing compound, C7, which exhibits structural similarity to Di-2-Pyridyl Ketone 4, 4-Dimethyl-3-Thiosemicarbazone (Dp44mT), a known chelator of intracellular iron, is found to reactivate EBV lytic cycle in GC and NPC. This study aims to investigate the role of intracellular iron chelation by C7 and other iron chelators in lytic reactivation of EBV in GC and NPC. Testing of six structural analogs of C7 revealed only those which have high affinity towards transition metals could induce EBV lytic cycle. Precomplexing C7 and iron chelators to iron prior to treatment of the cells abolished EBV lytic reactivation. Though hypoxia signaling pathway was activated, it was not the only pathway associated with EBV reactivation. Specifically, C7 and iron chelators initiated autophagy by activating extracellular signal-regulated kinase (ERK1/2) to reactivate EBV lytic cycle since autophagy and EBV lytic reactivation were abolished in cells treated with ERK1/2 blockers whilst inhibition of autophagy by 3-Methyladenine (3-MA) and atg5 knockdown significantly abolished EBV lytic reactivation. In summary, we discovered a novel mechanism of reactivation of the EBV lytic cycle through intracellular iron chelation and induction of ERK-autophagy axis in EBV-positive epithelial malignancies, raising the question whether clinically available iron chelators can be incorporated into existing therapeutic regimens to treat these cancers.

Establishment and characterization of new tumor xenografts and cancer cell lines from EBV-positive nasopharyngeal carcinoma.

The lack of representative nasopharyngeal carcinoma (NPC) models has seriously hampered research on EBV carcinogenesis and preclinical studies in NPC. Here we report the successful growth of five NPC patient-derived xenografts (PDXs) from fifty-eight attempts of transplantation of NPC specimens into NOD/SCID mice. The take rates for primary and recurrent NPC are 4.9% and 17.6%, respectively. Successful establishment of a new EBV-positive NPC cell line, NPC43, is achieved directly from patient NPC tissues by including Rho-associated coiled-coil containing kinases inhibitor (Y-27632) in culture medium. Spontaneous lytic reactivation of EBV can be observed in NPC43 upon withdrawal of Y-27632. Whole-exome sequencing (WES) reveals a close similarity in mutational profiles of these NPC PDXs with their corresponding patient NPC. Whole-genome sequencing (WGS) further delineates the genomic landscape and sequences of EBV genomes in these newly established NPC models, which supports their potential use in future studies of NPC.

Counteracting survival functions of EBNA3C in Epstein-Barr virus (EBV)-driven lymphoproliferative diseases by combination of SAHA and bortezomib.

Combination of suberoylanilide hydroxamic acid (SAHA) and bortezomib (SAHA/bortezomib) was shown to synergistically induce killing of lymphoblastoid cell lines (LCL) and Burkitt lymphoma (BL) of type III or Wp-restricted latency, both of which express EBNA3A, -3B and -3C proteins. We hypothesize that SAHA/bortezomib can counteract the survival functions conferred by the EBNA3 proteins. We tested the effect of SAHA/bortezomib on the survival of BL cell lines containing EBNA3A, -3B or -3C knockout EBV with or without the respective revertant EBNA3 genes. Isobologram analysis showed that SAHA/bortezomib induced significantly greater synergistic killing of EBNA3C-revertant cells when compared with EBNA3C-knockout cells. Such differential response was not observed in either EBNA3A or -3B revertant versus their knockout pairs. Interestingly, EBNA3C-knockout cells showed significant G2/M arrest whilst EBNA3C-revertant cells and LCLs escaped G2/M arrest induced by SAHA/bortezomib and became more susceptible to the induction of apoptosis. In parallel, SAHA/bortezomib induced stronger expression of p21WAF1 but weaker expression of p-cdc25c, an M-phase inducer phosphatase, in EBNA3C-expressing cells when compared with EBNA3C-knockout cells. SAHA/bortezomib also induced greater growth suppression of EBNA3C-expressing xenografts (EBNA3C-revertant and LCL) than that of EBNA3C-knockout xenografts in SCID mice. In conclusion, our data showed that SAHA/bortezomib could synergistically induce killing of BL and LCL through counteracting the survival functions of EBNA3C, providing a strong basis for clinical testing of this drug combination in patients with EBV-associated lymphoproliferative diseases.

Therapeutic Strategies against Epstein-Barr Virus-Associated Cancers Using Proteasome Inhibitors.

Epstein-Barr virus (EBV) is closely associated with several lymphomas (endemic Burkitt lymphoma, Hodgkin lymphoma and nasal NK/T-cell lymphoma) and epithelial cancers (nasopharyngeal carcinoma and gastric carcinoma). To maintain its persistence in the host cells, the virus manipulates the ubiquitin-proteasome system to regulate viral lytic reactivation, modify cell cycle checkpoints, prevent apoptosis and evade immune surveillance. In this review, we aim to provide an overview of the mechanisms by which the virus manipulates the ubiquitin-proteasome system in EBV-associated lymphoid and epithelial malignancies, to evaluate the efficacy of proteasome inhibitors on the treatment of these cancers and discuss potential novel viral-targeted treatment strategies against the EBV-associated cancers.

Inhibition of class I histone deacetylases by romidepsin potently induces Epstein-Barr virus lytic cycle and mediates enhanced cell death with ganciclovir.

Pan-histone deacetylase (HDAC) inhibitors, which inhibit 11 HDAC isoforms, are widely used to induce Epstein-Barr virus (EBV) lytic cycle in EBV-associated cancers in vitro and in clinical trials. Here, we hypothesized that inhibition of one or several specific HDAC isoforms by selective HDAC inhibitors could potently induce EBV lytic cycle in EBV-associated malignancies such as nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). We found that inhibition of class I HDACs, particularly HDAC-1, -2 and -3, was sufficient to induce EBV lytic cycle in NPC and GC cells in vitro and in vivo. Among a panel of selective HDAC inhibitors, the FDA-approved HDAC inhibitor romidepsin was found to be the most potent lytic inducer, which could activate EBV lytic cycle at ∼0.5 to 5 nM (versus ∼800 nM achievable concentration in patients' plasma) in more than 75% of cells. Upregulation of p21(WAF1) , which is negatively regulated by class I HDACs, was observed before the induction of EBV lytic cycle. The upregulation of p21(WAF1) and induction of lytic cycle were abrogated by a specific inhibitor of PKC-δ but not the inhibitors of PI3K, MEK, p38 MAPK, JNK or ATM pathways. Interestingly, inhibition of HDAC-1, -2 and -3 by romidepsin or shRNA knockdown could confer susceptibility of EBV-positive epithelial cells to the treatment with ganciclovir (GCV). In conclusion, we demonstrated that inhibition of class I HDACs by romidepsin could potently induce EBV lytic cycle and mediate enhanced cell death with GCV, suggesting potential application of romidepsin for the treatment of EBV-associated cancers.

Induction of MAPK- and ROS-dependent autophagy and apoptosis in gastric carcinoma by combination of romidepsin and bortezomib.

Proteasome inhibitors and histone deacetylase (HDAC) inhibitors can synergistically induce apoptotic cell death in certain cancer cell types but their combinatorial effect on the induction of autophagy remains unknown. Here, we investigated the combinatorial effects of a proteasome inhibitor, bortezomib, and an HDAC inhibitor, romidepsin, on the induction of apoptotic and autophagic cell death in gastric carcinoma (GC) cells. Isobologram analysis showed that low nanomolar concentrations of bortezomib/romidepsin could synergistically induce killing of GC cells. The synergistic killing was due to the summative effect of caspase-dependent intrinsic apoptosis and caspase-independent autophagy. The autophagic cell death was dependent on the activation of MAPK family members (ERK1/2 and JNK), and generation of reactive oxygen species (ROS), but was independent of Epstein-Barr virus infection. In vivo, bortezomib/romidepsin also significantly induced apoptosis and autophagy in GC xenografts in nude mice. This is the first report demonstrating the potent effect of combination of HDAC and proteasome inhibitors on the induction of MAPK- and ROS-dependent autophagy in addition to caspase-dependent apoptosis in a cancer type.

Identification of Novel Small Organic Compounds with Diverse Structures for the Induction of Epstein-Barr Virus (EBV) Lytic Cycle in EBV-Positive Epithelial Malignancies.

Phorbol esters, which are protein kinase C (PKC) activators, and histone deacetylase (HDAC) inhibitors, which cause enhanced acetylation of cellular proteins, are the main classes of chemical inducers of Epstein-Barr virus (EBV) lytic cycle in latently EBV-infected cells acting through the PKC pathway. Chemical inducers which induce EBV lytic cycle through alternative cellular pathways may aid in defining the mechanisms leading to lytic cycle reactivation and improve cells' responsiveness towards lytic induction. We performed a phenotypic screening on a chemical library of 50,240 novel small organic compounds to identify novel class(es) of strong inducer(s) of EBV lytic cycle in gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC) cells. Five hit compounds were selected after three successive rounds of increasingly stringent screening. All five compounds are structurally diverse from each other and distinct from phorbol esters or HDAC inhibitors. They neither cause hyperacetylation of histone proteins nor significant PKC activation at their working concentrations, suggesting that their biological mode of action are distinct from that of the known chemical inducers. Two of the five compounds with rapid lytic-inducing action were further studied for their mechanisms of induction of EBV lytic cycle. Unlike HDAC inhibitors, lytic induction by both compounds was not inhibited by rottlerin, a specific inhibitor of PKCδ. Interestingly, both compounds could cooperate with HDAC inhibitors to enhance EBV lytic cycle induction in EBV-positive epithelial cancer cells, paving way for the development of strategies to increase cells' responsiveness towards lytic reactivation. One of the two compounds bears structural resemblance to iron chelators and the other strongly activates the MAPK pathways. These structurally diverse novel organic compounds may represent potential new classes of chemicals that can be used to investigate any alternative mechanism(s) leading to EBV lytic cycle reactivation from latency.

Combination of SAHA and bortezomib up-regulates CDKN2A and CDKN1A and induces apoptosis of Epstein-Barr virus-positive Wp-restricted Burkitt lymphoma and lymphoblastoid cell lines.

Epstein-Barr virus (EBV) latent proteins exert anti-apoptotic effects on EBV-transformed lymphoid cells by down-regulating BCL2L11 (BIM), CDKN2A (p16(INK4A) ) and CDKN1A (p21(WAF1) ). However, the potential therapeutic effects of targeting these anti-apoptotic mechanisms remain unexplored. Here, we tested both in vitro and in vivo effects of the combination of histone deacetylase (HDAC) and proteasome inhibitors on the apoptosis of six endemic Burkitt lymphoma (BL) lines of different latency patterns (types I and III and Wp-restricted) and three lymphoblastoid cell lines (LCLs). We found that the combination of HDAC and proteasome inhibitors (e.g. SAHA/bortezomib) synergistically induced the killing of Wp-restricted and latency III BL and LCLs but not latency I BL cells. The synergistic killing was due to apoptosis, as evidenced by the high percentage of annexin V positivity and strong cleavage of PARP1 (PARP) and CASP3 (caspase-3). Concomitantly, SAHA/bortezomib up-regulated the expression of CDKN2A and CDKN1A but did not affect the level of BCL2L11 or BHRF1 (viral homologue of BCL2). The apoptotic effects were dependent on reactive oxygen species generation. Furthermore, SAHA/bortezomib suppressed the growth of Wp-restricted BL xenografts in nude mice. This study provides the rationale to test the novel application of SAHA/bortezomib on the treatment of EBV-associated Wp-restricted BL and post-transplant lymphoproliferative disorder.

Combination of proteasome and class I HDAC inhibitors induces apoptosis of NPC cells through an HDAC6-independent ER stress-induced mechanism.

The current paradigm stipulates that inhibition of histone deacetylase (HDAC) 6 is essential for the combinatorial effect of proteasome and HDAC inhibitors for the treatment of cancers. Our study aims to investigate the effect of combining different class I HDAC inhibitors (without HDAC6 action) with a proteasome inhibitor on apoptosis of nasopharyngeal carcinoma (NPC). We found that combination of a proteasome inhibitor, bortezomib, and several class I HDAC inhibitors, including MS-275, apicidin and romidepsin, potently induced killing of NPC cells both in vitro and in vivo. Among the drug pairs, combination of bortezomib and romidepsin (bort/romidepsin) was the most potent and could induce apoptosis at low nanomolar concentrations. The apoptosis of NPC cells was reactive oxygen species (ROS)- and caspase-dependent but was independent of HDAC6 inhibition. Of note, bort/romidepsin might directly suppress the formation of aggresome through the downregulation of c-myc. In addition, two markers of endoplasmic reticulum (ER) stress-induced apoptosis, ATF-4 and CHOP/GADD153, were upregulated, whereas a specific inhibitor of caspase-4 (an initiator of ER stress-induced apoptosis) could suppress the apoptosis. When ROS level in the NPC cells was reduced to the untreated level, ER stress-induced caspase activation was abrogated. Collectively, our data demonstrate a model of synergism between proteasome and class I HDAC inhibitors in the induction of ROS-dependent ER stress-induced apoptosis of NPC cells, independent of HDAC6 inhibition, and provide the rationale to combine the more specific and potent class I HDAC inhibitors with proteasome inhibitors for the treatment of cancers.

Bortezomib and SAHA synergistically induce ROS-driven caspase-dependent apoptosis of nasopharyngeal carcinoma and block replication of Epstein-Barr virus.

A novel drug combination of a proteasome inhibitor, bortezomib, and a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), was tested in nasopharyngeal carcinoma (NPC), both in vitro and in vivo. Dose-response of different concentrations of bortezomib and SAHA on inhibition of cell proliferation of NPC was determined. Mechanisms of apoptosis and effects on lytic cycle activation of Epstein-Barr virus (EBV) were investigated. Combination of bortezomib and SAHA (bortezomib/SAHA) synergistically induced killing of a panel of NPC cell lines. Pronounced increase in sub-G1, Annexin V-positive, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cell populations were detected after treatment with bortezomib/SAHA when compared with either drug alone. Concomitantly, markedly augmented proteolytic cleavage of PARP, caspase-3, -7, -8, and -9, reactive oxygen species (ROS) generation, and caspase-8-dependent histone acetylation were observed. ROS scavenger, N-acetyl cysteine, diminished the apoptotic effects of bortezomib/SAHA, whereas caspase inhibitor Z-VAD-FMK significantly suppressed the apoptosis without decreasing the generation of ROS. Bortezomib inhibited SAHA's induction of EBV replication and abrogated production of infectious viral particles in NPC cells. Furthermore, bortezomib/SAHA potently induced apoptosis and suppressed the growth of NPC xenografts in nude mice. In conclusion, the novel drug combination of bortezomib and SAHA is highly synergistic in the killing of NPC cells in vitro and in vivo. The major mechanism of cell death is ROS-driven caspase-dependent apoptosis. Bortezomib antagonizes SAHA's activation of EBV lytic cycle in NPC cells. This study provides a strong basis for clinical testing of the combination drug regimen in patients with NPC.