Epstein-Barr Virus Thymidine Kinase Research Articles

A phase Ib/II study of oral nanatinostat (N) and valganciclovir (VG) in subjects with Epstein-Barr virus (EBV)-associated lymphomas.

7551 Background: Nanatinostat (N; VRx-3996) is a Class I-selective oral hydroxamate histone deacetylase (HDAC) inhibitor active against HDAC 1-3, but not HDAC 6. N induces expression of EBV thymidine kinase (TK, BXLF1) and protein kinase (PK, BGLF4) in EBV+ lymphomas. This study examines if N+VG results in tumor sensitization to antivirals (i.e., VG) and impacts EBV-induced T-cell exhaustion. Methods: The study employs a 3+3 design with expansion in patients (pts) with relapsed/refractory EBV+ lymphomas (EBER-ISH). Objectives: Maximal Tolerated Dose (MTD), Recommended Phase 2 Dose (RP2D), and efficacy. Primary endpoint: safety. Secondary endpoints: PK, response rate. Exploratory endpoints: PBMC histone H3 acetylation (Ac), quantitative plasma EBV DNA (pEBVd), and immune biomarkers. Responses (investigator-assessed and centrally-reviewed) are based on the Lugano Classification. Early safety and efficacy data are reported. Results: As of 10-Jan-2019, 15 pts (4F/11M, median age 60 yrs [19-79]) were enrolled with a median of 2 prior therapies and various lymphoma subtypes. Cohort 1 (N 10 mg BID/VG 900 mg BID) exceeded MTD (DLTs: leukopenia, neutropenia and thrombocytopenia). PK demonstrated ~2-fold higher N exposure compared to a prior study. VG levels were consistent with published data. Doses were reduced in Cohort 2 (N 5 mg BID/VG 450 mg BID) with no DLTs to date and 3 active pts. Most non-hematologic treatment-related AEs (TRAEs) were G1-2. Most G3+ TRAEs were hematologic (neutropenia, leukopenia, thrombocytopenia). Increases in creatinine occurred early and responded to reductions in VG. Among 7 evaluable pts, 4 responses were seen (2 CR, 2 PR) in PTLD, DLBCL, AITL, and plasmablastic lymphoma. Two pts had SD, and 1 PD. Of 8 pts with detectable baseline pEBVd, 7 demonstrated a reduction (median -54% [17 to -83%]). PBMC H3 Ac levels, multi-cytokine expression panel, and functional assessments in CD8+ T cells are in progress. Conclusions: N+VG appears well-tolerated at 5 mg and 450 mg BID, respectively with hematologic DLTs in a higher dose cohort. Responses are observed at all doses in both B and T cell lymphomas, with reductions in pEBVd levels and changes in T cell functionality. A RP2D will be defined prior to dose expansion. Clinical trial information: NCT03397706.

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

AbstractAbstract 1831Strong 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. Disclosures:Faller:HemaQuest Pharmaceuticals: Consultancy, Equity Ownership. Perrine:HemaQuest Pharmaceuticals, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Williams:HemaQuest Pharmaceuticals: Consultancy, Equity Ownership. Berenson:HemaQuest Pharmaceuticals, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

High-Dose Methotrexate and Azidothymidine in Combination with Alternating DA-EPOCH Is An Effective Regimen for the Treatment of EBV-Related Plasmablastic Lymphoma.

Abstract 4753 IntroductionPlasmablastic lymphoma (PBL) is an aggressive and generally fatal, rare type of B-cell non-Hodgkin's lymphoma (NHL) with predilection to sino-oral mucosa. PBL is usually associated with human immunodeficiency virus (HIV) and displays an unusual phenotype lacking the expression of B-cell surface antigens. PBLs are infected by Epstein Barr virus (EBV) in approximately 80% of cases and exhibit a restricted pattern of EBV gene expression as they do not typically express latent membrane proteins (LMPs), which enforce viral latency. Azidothymidine (AZT), a thymidine analogue, is an excellent substrate for EBV thymidine kinase, and is capable of inducing EBV lytic gene expression and apoptosis in primary Type I latency EBV+ Burkitt lymphoma (BL) cell lines. AZT was initially developed as an antineoplastic agent but was found to have low efficacy due to poor affinity to human DNA polymerase and low incorporation into DNA. The chemotherapy drug methotrexate (MTX), which also induces gamma-herpes virus lytic induction, inhibits thymidylate synthase thus blocking de novo synthesis of dTMP increasing the likelihood of AZT incorporation into DNA. Indeed, the combination of high-dose MTX and AZT was found to be clinically efficacious in HIV-infected patients with aggressive relapsed NHL. Similarly, at the University of Miami, we have found this drug combination to be highly effective when used in patients with aggressive gamma-herpes virus lymphomas including EBV+ BL and human herpes virus 8 (HHV-8) related primary effusion lymphoma (solid PEL). We report here the role of high dose MTX plus intravenous AZT with alternating infusional EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, and adriamycin) in the treatment of HIV associated EBV+ plasmablastic lymphoma. MethodsThree HIV+ patients with biopsy proven, EBV-encoded RNA (EBER)+ PBL in sino-oral mucosa were treated with high dose methotrexate (4.5 g/m2 IV on day 1) and AZT 1.5 g IV infusion q12 hours (days 1-3) alternating with dose-adjusted (DA) EPOCH chemotherapy for 6-8 cycles as first line therapy. Patients were started/kept on HAART and were administered prophylactic intrathecal MTX±cytarabine. Responses were evaluated by oral exam and CT scans. ResultsSelected characteristics of patients are demonstrated in Table. Only Patient 2 was on HAART at the time of diagnosis. Patient 1 had intracranial extension of the large left maxillary lesion through sphenoid sinus with negative CSF cytology. All patients treated with HD-MTX/AZT and alternating EPOCH as above achieved a complete remission (CR). Patient 1 achieved CR after the 3rd cycle. However, 19 days after the 4th cycle he was found to have local recurrence, which was thought to be secondary to poor chemotherapy penetration to necrotic oral palate tissue, and was administered a total of 4620 cGy intensity-modulated radiotherapy to head and neck region followed by consolidation with the same chemotherapy regimen for 4 more cycles. He remains alive and free of disease after 14 months. Patient 2 and 3 received a total of 6 cycles of chemotherapy and remain disease-free after 12 and 13 months, respectively. No grade ≥3 toxicities were encountered during the treatment. ConclusionThe combination of high dose MTX and AZT plus alternating DA-EPOCH chemotherapy is a tolerable and effective treatment for HIV related EBV+ plasmablastic lymphomas. The combination of MTX and AZT is a highly active, EBV lytic inducing and targeted regimen which deserves further investigation in the treatment of gamma-herpes virus associated malignancies.NoSexAge at dxCD4 at dxLDH (normal<618)Stage# of cyclesDFS (from dx)1M52571300IIB4 + IMRT + 4>14 mos2F33102638IIA6>12 mos3M44339450IIA6>13mos Disclosures:No relevant conflicts of interest to declare.

Bortezomib-induced enzyme-targeted radiation therapy in herpesvirus-associated tumors

We investigated the possibility of using a pharmacologic agent to modulate viral gene expression to target radiotherapy to tumor tissue. In a mouse xenograft model, we had previously shown targeting of [(125)I]2'-fluoro-2'-deoxy-beta-D-5-iodouracil-arabinofuranoside ([(125)I]FIAU) to tumors engineered to express the Epstein-Barr virus thymidine kinase (EBV-TK). Here we extend those results to targeting of a therapeutic radiopharmaceutical [(131)I]FIAU to slow or stop tumor growth or to achieve tumor regression. These outcomes were achieved in xenografts with tumors that constitutively expressed the EBV-TK. With naturally infected EBV tumor cell lines (Burkitt's lymphoma and gastric carcinoma), activation of viral gene expression by pretreatment with bortezomib was required. Marked changes in tumor growth could also be achieved in naturally infected Kaposi's sarcoma herpesvirus tumors after pretreatment with bortezomib. Bortezomib-induced enzyme-targeted radiation therapy illustrates the possibility of pharmacologically modulating tumor gene expression to result in targeted radiotherapy.

Epstein-Barr Virus Thymidine Kinase Is a Centrosomal Resident Precisely Localized to the Periphery of Centrioles

The thymidine kinase (TK) encoded by Epstein-Barr virus (EBV) differs not only from that of the alphaherpesviruses but also from that of the gamma-2 herpesvirus subfamily. Because cellular location is frequently a determinant of regulatory function, to gain insight into additional role(s) of EBV TK and to uncover how the lymphocryptovirus and rhadinovirus enzymes differ, the subcellular localizations of EBV TK and the related cercopithecine herpesvirus-15 TK were investigated. We show that in contrast to those of the other family members, the gamma-1 herpesvirus TKs localize to the centrosome and even more precisely to the periphery of the centriole, tightly encircling the tubulin-rich centrioles in a microtubule-independent fashion. Centrosomal localization is observed in diverse cell types and occurs whether the protein is expressed independently or in the context of lytic EBV infection. Surprisingly, analysis of mutants revealed that the unique N-terminal domain was not critical for targeting to the centrosome, but rather, peptide sequences located C terminal to this domain were key. This is the first herpesvirus protein documented to reside in the centrosome, or microtubule-organizing center, an amembranous organelle that regulates the structural biology of the cell cycle through control of chromosome separation and cytokinesis. More recently, proteasome-mediated degradation of cell cycle regulatory proteins, production and loading of antigenic peptides onto HLA molecules, and transient homing of diverse virion proteins required for entry and/or egress have been shown to be coordinated at the centrosome. Potential implications of centrosomal localization for EBV TK function are discussed.

Enzymatically-targeted 131I therapy for herpesvirus-associated malignancies

3010 Background: Epstein-Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV) are associated with tumors including AIDS-related lymphoma, Burkitt’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma and Kaposi’s sarcoma. Both viruses encode kinases that selectively phosphorylate nucleoside analogs such as ganciclovir and FIAU. We hypothesized that it might be possible to use the viral enzymes to specifically concentrate 131I-FIAU in tumor cells harboring virus and thus deliver therapeutic radiation. Bortezomib is a potent stimulator of viral kinase expression in gammaherpesvirus tumor cell lines. Methods: We evaluated the effect of 131I-FIAU on human cancer xenografts in SCID mice. These included a tumor line engineered to constitutively express the EBV thymidine kinase (EBVTK), and a control engineered with a sham vector (SHAM), as well 2 EBV(+) Burkitt’s lymphoma (BL) lines, and 1 KSHV(+) primary effusion lymphoma (PEL) cell line. Mice were treated with buffer, bortezomib 2 ug/gm, or I-FIAU, or I-FIAU and bortezomib in combination. For imaging, mice, 125I -FIAU and SPECT were used. For therapy, 131I-FIAU was used and tumor dimensions were monitored with calipers. Results: Treatment with buffer had no effect on on 3 EBVTK tumors and 3 SHAM tumors all of which increased in volume. Treatment with 1.4 mCi 131I-FIAU alone led to tumor responses (&gt;90% volume reduction at 10 days) in 3/3 mice with EBVTK tumors and 0/3 mice with SHAM tumors. Treatment with 131I-FIAU alone had no effect on BL (0/3) or PEL (0/9) xenografts and all tumors increased in volume. Treatment with bortezomib induced modest responses in all tumors but had no greater effect on EBVTK tumors than SHAM tumors. However, treatment with bortezomib and 131I-FIAU led to marked tumor regression (&gt;95%) in each of the virus-associated tumors (3/3 BL, 9/9 PEL). SPECT imaging with 125I-FIAU showed selective concentration of radiolabel in tumor tissue in the EBVTK tumor (2/2) and in viral tumors (6/6) when bortezomib was administered. There was no selective concentration in the absence of bortezomib treatment in the viral tumors (0/8). Conclusions: Treatment with bortezomib leads to selective concentration of labeled FIAU in the herpesvirus-associated tumor xenografts evaluated and to regression of tumor when the isotope is 131I. No significant financial relationships to disclose.

Epstein–Barr virus‐associated B‐cell lymphoma in the setting of iatrogenic immune dysregulation presenting initially in the skin

Epstein-Barr virus (EBV) has been implicated in B-cell lymphoma associated with iatrogenic immune dysregulation, primarily in the context of extracutaneous lymphoma. We describe six patients, five transplant recipients receiving cyclosporine and one patient with rheumatoid arthritis receiving methotrexate, who developed cutaneous presentations of EBV-associated B-cell lymphoma. Human herpesvirus 8 (HHV8) and EBV thymidine kinase (vTK) expression were also explored. The cases comprised plasmablastic lymphoma (one case), plasmacytic marginal zone lymphoma (two cases), and diffuse large B-cell lymphoma (three cases). There was a monoclonal gammopathy in one and concurrent extracutaneous disease in two of the six patients. EBV-associated latent small nuclear RNA was detected in all cases with coexpression of HHV8 in one of the five cases and of vTK in three of the six cases. Three patients responded to a reduction in the immunosuppressive regimen and antiviral therapy. Recurrent disease developed in two, with one patient succumbing to multiorgan dissemination. EBV-associated cutaneous B-cell lymphoma is characterized by a long interval between the initiation of immunosuppression and the development of lymphoma. Although previous reports have reported an indolent clinical course, an aggressive clinical course may occur. HHV8 and lytic phase EBV antigens are detected in some cases, possibly suggesting a pathogenetic role.

Characterization of Three Essential Residues in the Conserved ATP-Binding Region of Epstein-Barr Virus Thymidine Kinase

The thymidine kinase encoded by Epstein-Barr virus (EBV TK) is an important target for antiviral therapy and the treatment of EBV-associated malignancies. Through computer-assisted alignment with other human herpesviral TK proteins, EBV TK was shown to contain a conserved ATP-binding motif as for the other TK enzymes. To investigate functional roles of three highly conserved residues (G294, K297, T298) within this region, site-directed mutagenesis was employed to generate various mutants. The TK enzyme activity and ATP-binding ability of these mutant TK enzymes were determined and compared with EBV wild-type TK (wtTK). Mutant G294V lost its ATP-binding ability and was inactive in enzyme activity assay. As the enzyme activity of G294A was reduced to 20% of that of wtTK, the K(m) for ATP binding of G294A was 48.7 microM as compared with 30.0 microM of EBV wtTK. These results suggested that G294 participates in ATP binding and contributes to maintenance of structure. EBV TK mutants K297E, K297Q, and K297R lost their ATP-binding ability and enzyme activity. However, K297R was shown to have a preference for usage of GTP (K(m): 43.0 microM) instead of ATP (K(m): 87.6 microM) as the phosphate donor. This implies that, in addition to nucleotide binding, K297 was involved in the selection of phosphate donor. While EBV TK mutant T298S retained approximately 80% of wtTK enzyme activity, T298A lost its enzyme activity, suggesting that a hydroxyl group at this position is important for the enzyme activity. Interestingly, T298A retained its ATP-binding ability, suggesting a role of T298 in the catalytic process but not in the coordination of ATP. This study demonstrated that amino acid residues G294, K297, and T298 in the ATP-binding motif of EBV TK enzyme are essential for the enzymatic activity but are involved in different aspects of its action.

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase.

Thymidine kinase (TK), encoded by EBV (Epstein-Barr virus), is an attractive target for antiviral therapy and provides a novel approach to the treatment of EBV-associated malignancies. Despite the extensive use of nucleoside analogues for the treatment of viral infections and cancer, the structure-function relationship of EBV TK has been addressed rarely. In the absence of any structural information, we sought to identify and elucidate the functional roles of amino acids in the nucleoside-binding site using site-directed mutagenesis. Through alignment with other human herpesviral TK protein sequences, we predicted that certain conserved regions comprise the nucleoside-binding site of EBV TK and, through site-directed mutagenesis, showed significant changes in activity and binding affinity for thymidine of site 3 (-DRH-) and 4 (-VFP-) mutants. For site 3, only mutants D392E (Asp392-->Glu) and R393H retain activity, indicating that a negative charge is important for Asp392 and a positive charge is required for Arg393. The increased binding affinities of these two mutants for 3'-deoxy-2',3'-didehydrothymidine suggest that the two residues are also important for substrate selection. Interestingly, the changed metal-ion usage pattern of D392E reveals that Asp392 plays multiple roles in this region. His394 cannot be compensated by other amino acids, also indicating a crucial role. In site 4, the F402Y mutant retains full activity; however, F402S retains only 60% relative activity. Strikingly, when Phe402 is substituted with serine residue, the original preferred pyrimidine substrates, such as 3'-azido-3'-deoxythymidine, iododeoxyuridine and beta-L-5-iododioxolane uracil (L-form substrate), have decreased competitiveness with thymidine, suggesting that Phe402 plays a crucial role in substrate specificity and that the aromatic ring is important for function.

Antiviral treatment of Epstein-Barr virus-associated lymphoproliferations.

Epstein-Barr virus (EBV)-associated lymphoproliferations may arise in individuals with hereditary or acquired immunodeficiencies. T-cell dysfunction and resulting insufficient control of EBV infection is common to all these patients in whom EBV-associated lymphoproliferations develop. EBV is an oncogenic virus which induces proliferation and transformation of B-lymphocytes. Antiviral treatment may represent a causal treatment option with relatively low toxicity. Among the different antiviral drugs aciclovir and ganciclovir are not the drugs of choice, because in EBV-associated lymphoproliferations the viral thymidine kinase enzyme is not encoded regularly. The agent arginine butyrate has the ability to selectively activate EBV thymidine kinase genes in EBV-infected lymphoma cells. In combination with ganciclovir it has demonstrated efficacy in patients with EBV-associated lymphoproliferations after solid organ transplantation. The action of foscarnet, another antiviral agent, is directed against the viral DNA, independent of the presence of the viral thymidine kinase. In our experience treatment with foscarnet resulted in continuous complete remissions in patients with EBV-associated lymphoproliferations. These clinical experiences demonstrate the efficacy of antiviral treatment in EBV-associated lymphoproliferations.

Induction of the Epstein-Barr virus thymidine kinase gene with concomitant nucleoside antivirals as a therapeutic strategy for Epstein-Barr virus-associated malignancies.

Lymphoproliferative diseases (LPDs) associated with the Epstein-Barr virus (EBV) include non-Hodgkin lymphomas, which occur in the setting of immunosuppression, including that induced by human immunodeficiency virus, and posttransplant lymphoproliferative disorders. These LPDs are characterized by actively proliferating, latently infected EBV-positive B lymphocytes and often follow a rapidly progressive fatal clinical course. Pharmacologic treatment for herpesvirus infections has targeted the virus-specific enzyme, thymidine kinase (TK), with nucleoside analogs. The lack of viral TK expression in EBV-positive tumors, caused by viral latency, however, makes antiviral therapy alone ineffective as an antineoplastic therapy. Arginine butyrate selectively activates the EBV TK gene in latently infected EBV-positive tumor cells. We have developed a strategy for treatment of EBV-associated lymphomas using pharmacologic induction of the latent viral TK gene and enzyme in tumor cells using arginine butyrate, followed by treatment with ganciclovir. A phase I/II trial, using an intrapatient dose escalation of arginine butyrate combined with ganciclovir, is underway. This combination therapy has produced complete clinical responses in 5 of 10 previously refractory patients, with partial responses occurring in 2 additional patients. This virus-targeted antitumor strategy may provide a new therapeutic approach to EBV-associated neoplasms.

Epstein--Barr virus post-transplant lymphoproliferative disease and virus-specific therapy: pharmacological re-activation of viral target genes with arginine butyrate.

Lymphoproliferative disorders associated with the Epstein-Barr virus (EBV) include non-Hodgkin's lymphoma, Hodgkin's lymphoma, and "post-transplant lymphoproliferative disorders" (PTLD), which occur with immunosuppression after marrow and organ transplantation. PTLD is characterized by actively proliferating, latently infected EBV(+) B-lymphocytes, and often manifests a rapidly progressive fatal clinical course if the immunosuppression cannot be reversed. Lung transplant recipients are a subset of patients at special risk for developing PTLD. The incidence of PTLD development in these patients has been estimated at 5--10%. Whereas immunologic and antiviral therapy have been moderately effective for treating EBV-associated infections in the lytic phase, they have been less useful in the more common latent phase of the disease. One common treatment for herpesvirus infections has targeted the virus-specific enzyme thymidine kinase (TK). The lack of viral TK expression in EBV(+) tumor cells, due to viral latency, makes anti-viral therapy alone ineffective as an anti-neoplastic therapy, however. We have developed a strategy for the treatment of EBV-associated lymphomas/PTLD using pharmacologic induction of the latent viral TK gene and enzyme in the tumor cells, followed by treatment with ganciclovir. Arginine butyrate selectively activates the EBV TK gene in latently EBV-infected human lymphoid cells and tumor cells. A Phase I/II trial has been initiated, employing an intra-patient dose escalation of arginine butyrate combined with ganciclovir. In six patients with EBV-associated lymphomas or PTLD, all of which were resistant to conventional radiation and/or chemotherapy, this combination produced complete clinical responses in four of six patients, with a partial response occurring in a fifth patient. Pathologic examination in two of three patients demonstrated complete necrosis of the EBV lymphoma, with no residual disease, following a single three-week course of the combination therapy. Possible side-effects of the therapy included nausea and reversible lethargy at the highest doses. One patient suffered acute liver failure, thought to be secondary to release of FasL from the necrotic tumor. Analysis of patient-derived tumor cells in culture demonstrated that arginine butyrate produced selective induction of the EBV TK gene, which then conferred sensitivity to ganciclovir, resulting in tumor apoptosis. Additional patient accrual is sought for further evaluation of this therapy.

Induction of Epstein-Barr virus kinases to sensitize tumor cells to nucleoside analogues.

The presence of Epstein-Barr virus (EBV) in the tumor cells of some EBV-associated malignancies may facilitate selective killing of these tumor cells. We show that treatment of an EBV(+) Burkitt's lymphoma cell line with 5-azacytidine led to a dose-dependent induction of EBV lytic antigen expression, including expression of the viral thymidine kinase (TK) and phosphotransferase (PT). Azacytidine treatment for 24 h modestly sensitized the cell line to all nucleosides tested. To better characterize EBV TK with regard to various nucleoside analogues, we expressed EBV TK in stable cell clones. Two EBV TK-expressing clones were moderately sensitive to high doses of acyclovir and penciclovir (PCV) (62.5 to 500 microM) and to lower doses of ganciclovir (GCV) and bromovinyldeoxyuridine (BVdU) (10 to 100 microM) compared to a control clone and were shown to phosphorylate GCV. Similar experiments in a transient overexpression system showed more killing of cells transfected with the EBV TK expression vector than of cells transfected with the control mutant vector (50 microM GCV for 4 days). A putative PT was also studied in the transient transfection system and appeared similar to the TK in phosphorylating GCV and conferring sensitivity to GCV, but not in BVdU- or PCV-mediated cell killing. Induction of EBV kinases in combination with agents such as GCV merits further evaluation as an alternative strategy to gene therapy for selective killing of EBV-infected cells.

Antibodies to Epstein-Barr virus thymidine kinase: a characteristic marker for the serological detection of nasopharyngeal carcinoma.

Patients suffering from nasopharyngeal carcinoma (NPC) generally exhibit elevated serum IgA antibody titres to Epstein-Barr virus (EBV) early antigen (EA) and virus capsid antigen (VCA). This property is frequently used as a diagnostic aid. Preliminary experiments suggested that an ELISA for IgA antibodies against the EBV-encoded thymidine kinase (TK) could form the basis of a more reliable diagnostic test. Here, we describe the construction of a recombinant baculovirus that expresses the EBV TK and present a full analysis of its use in serological surveys of NPC patients. Baculovirus-derived TK was used to develop a simple ELISA for serum IgA against this antigen. ELISA reactivity was strongly associated with NPC compared with an EBV-positive, normal control population. Comparison with the existing IgA-VCA and EA assays showed that the TK ELISA had higher sensitivity whilst the specificity was similar or higher. We conclude that the TK ELISA presents a strong predictor of NPC and, in its refined form, has improved pickup rates. In addition, results from patients with chronic nasopharyngitis (CNP) suggest that individuals with both symptoms of CNP and an elevated TK ELISA value may be at increased risk for the development of head-and-neck cancer.

Arginine Butyrate-Induced Susceptibility to Ganciclovir in an Epstein–Barr-Virus-Associated Lymphoma

ABSTRACT: Lymphoproliferative disorders associated with Epstein-Barr virus (EBV) infections can occur in the setting of immunosuppression. In some patients, the lymphoproliferative disorder can resemble an aggressive monoclonal non-Hodgkins lymphoma (NHL). These NHL are poorly responsive to conventional therapy. Similarly, antiviral therapy with synthetic nucleosides such as ganciclovir are ineffective because the genes that render the virus susceptible to therapy are not expressed in EBV+lymphomas. Using a cell line derived from a lung transplant recipient with an EBV+immunoblastic NHL, we studied the ability of arginine butyrate to induce the expression of EBV thymidine kinase. Arginine butyrate was not only effective in inducing EBV thymidine kinase transcription, but also acted synergistically with the antiviral agent ganciclovir to inhibit cell proliferation and decrease cell viability. Based on these findings, the patient from whom the cell line was derived was treated with arginine butyrate/ganciclovir as well as conventional cytotoxic chemotherapy. No additional toxicity was observed with the arginine butyrate/ganciclovir therapy. Histologic examination of the tumor showed substantial necrosis. These observations suggest the feasibility of arginine butyrate induction of ganciclovir susceptibility in patients with EBV-associated lymphomas.

Functional analysis of C-terminal deletion mutants of Epstein-Barr virus thymidine kinase.

Thymidine kinase (TK) activity was detected following expression of the TK gene of Epstein-Barr virus (EBV) using the pET expression plasmid and E. coli BL21 (DE3)pLysS. To study the amino acid residues required at the C terminus of the EBV TK protein for enzymatic activity, a series of C-terminal deletion mutants was generated by direct truncation, linker insertion or PCR mutagenesis to create stop codons at particular sites. Deletion of nine residues from the C terminus caused a 35% reduction in TK activity, while a ten-residue deletion completely abolished the activity. A single point mutation at residue Cys570, corresponding to Cys336 of herpes simplex virus TK, did not alter the TK activity. Single amino acid changes within the last seven to ten residues also did not affect activity. The results indicate that maintenance of the conformation of the C terminus is important for enzyme activity.

Substrate specificity of Epstein-Barr virus thymidine kinase.

Purified recombinant protein encoded by the BXLF-I open reading frame of the Epstein-Barr virus genome has thymidine kinase activity. The substrate behaviors of various nucleosides toward this enzyme were tested. Halogenated deoxyuridines, zidovudine, and bromovinyldeoxyuridine are efficient substrates, while acyclovir and dihydroxypropylmethylguanine are relatively poor substrates for the Epstein-Barr virus thymidine kinase.

Cloning and expression of a cDNA encoding the Epstein-Barr virus thymidine kinase gene

A clone of the Epstein-Barr virus (EBV) thymidine kinase (TK) gene was derived from a cDNA library of P3HR1 cells. The gene product was expressed as a fusion protein in a procaryotic system by using T7 RNA polymerase. The recombinant TK showed a molecular mass of 67 kDa and was biologically active. Antiserum raised in mice immunized with partially purified TK recognized an antigen present in EBV-superinfected Raji cells using an indirect immunofluorescence assay.

Diagnosis of nasopharyngeal carcinoma by means of recombinant Epstein-Barr virus proteins

The immune response of patients with nasopharyngeal carcinoma to Epstein-Barr virus (EBV) antigens is diagnostic of the tumour. Existing tests use EBV antigens produced in EBV-infected lymphoblastoid cells, but the virus replicates poorly in these cells. Serum samples from 18 patients diagnosed as having nasopharyngeal carcinoma were screened by western blot analysis, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence tests for antibodies to the EBV-coded alkaline deoxyribonuclease (DNase), thymidine kinase, and membrane antigen (gp340/ 220) produced in recombinant baculovirus or bovine papillomavirus systems. Each protein was a useful diagnostic marker for nasopharyngeal carcinoma, although in the gp340/220 ELISAs there was substantial overlap for both IgG and IgA antibodies between serum samples from nasopharyngeal carcinoma patients and those from healthy donors seropositive for EBV. The EBV thymidine kinase was the most sensitive predictor of nasopharyngeal carcinoma; all such samples showed both IgG and IgA antibody responses to this protein and all gave clearly distinct titres from those of the EBV-seropositive donors in the IgA test. Each of the recombinant systems described is suitable for use in large-scale screening programmes for the early diagnosis of nasopharyngeal carcinoma.

Characterization of the Epstein-Barr virus-encoded thymidine kinase expressed in heterologous eucaryotic and procaryotic systems.

The establishment of mammalian and procaryotic systems which express the Epstein-Barr virus (EBV) thymidine kinase (TK) has been reported previously (E. Littler, J. Zeuthen, A. A. McBride, E. Trøst-Sørensen, K. L. Powell, J. E. Walsh-Arrand, and J. R. Arrand, EMBO J. 5:1959-1966, 1986). The EBV TK activity expressed in both of these systems was characterized by in vitro assays and found to resemble that of the herpes simplex virus TK both in its broad range of nucleoside and nucleotide utilization and also in its ability to accept antiviral nucleoside analogs as substrates. Further results are presented which suggest that these in vitro systems may prove suitable for studying the potential anti-EBV activity of other candidate antiviral compounds.