A phase 1/2 trial of arginine butyrate and ganciclovir in patients with Epstein-Barr virus-associated lymphoid malignancies

Malignancies associated with latent Epstein-Barr virus (EBV) are resistant to nucleoside-type antiviral agents because the viral enzyme target of these antiviral drugs, thymidine kinase (TK), is not expressed. Short-chain fatty acids, such as butyrate, induce EBV-TK expression in latently infected B cells. As butyrate has been shown to sensitize EBV(+) lymphoma cells in vitro to apoptosis induced by ganciclovir, arginine butyrate in combination with ganciclovir was administered in 15 patients with refractory EBV(+) lymphoid malignancies to evaluate the drug combination for toxicity, pharmacokinetics, and clinical responses. Ganciclovir was administered twice daily at standard doses, and arginine butyrate was administered by continuous infusion in an intrapatient dose escalation, from 500 mg/(kg/day) escalating to 2000 mg/(kg/day), as tolerated, for a 21-day cycle. The MTD for arginine butyrate in combination with ganciclovir was established as 1000 mg/(kg/day). Ten of 15 patients showed significant antitumor responses, with 4 CRs and 6 PRs within one treatment cycle. Complications from rapid tumor lysis occurred in 3 patients. Reversible somnolence or stupor occurred in 3 patients at arginine butyrate doses of greater than 1000 mg/(kg/day). The combination of arginine butyrate and ganciclovir was reasonably well-tolerated and appears to have significant biologic activity in vivo in EBV(+) lymphoid malignancies which are refractory to other regimens.     

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

Abstract: 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.     

Histone deacetylase inhibitors are potent inducers of gene expression in latent EBV and sensitize lymphoma cells to nucleoside antiviral agents

Induction of EBV lytic-phase gene expression, combined with exposure to an antiherpes viral drug, represents a promising targeted therapeutic approach to EBV-associated lymphomas. Short-chain fatty acids or certain chemotherapeutics have been used to induce EBV lytic-phase gene expression in cultured cells and mouse models, but these studies generally have not translated into clinical application. The recent success of a clinical trial with the pan-histone deacetylase (pan-HDAC) inhibitor arginine butyrate and the antiherpes viral drug ganciclovir in the treatment of EBV lymphomas prompted us to investigate the potential of several HDAC inhibitors, including some new, highly potent compounds, to sensitize EBV(+) human lymphoma cells to antiviral agents in vitro. Our study included short-chain fatty acids (sodium butyrate and valproic acid); hydroxamic acids (oxamflatin, Scriptaid, suberoyl anilide hydroxamic acid, panobinostat [LBH589], and belinostat [PXD101]); the benzamide MS275; the cyclic tetrapeptide apicidin; and the recently discovered HDAC inhibitor largazole. With the exception of suberoyl anilide hydroxamic acid and PXD101, all of the other HDAC inhibitors effectively sensitized EBV(+) lymphoma cells to ganciclovir. LBH589, MS275, and largazole were effective at nanomolar concentrations and were 10(4) to 10(5) times more potent than butyrate. The effectiveness and potency of these HDAC inhibitors make them potentially applicable as sensitizers to antivirals for the treatment of EBV-associated lymphomas.     

Successful treatment of posttransplantation lymphoproliferative disorder (PTLD) following renal allografting is associated with sustained CD8(+) T-cell restoration

Posttransplantation lymphoproliferative disorder (PTLD) is a life-threatening Epstein-Barr virus (EBV)-associated B-cell malignancy occurring in 1% to 2% of renal transplantation patients. Host- and PTLD-related factors determining the likelihood of tumor response following reduction of immune suppression (IS) and antiviral therapy remain largely unknown. Standard therapy for PTLD is not well established. Eleven consecutive renal transplantation patients who developed EBV-positive PTLD 8 to 94 months after allografting were uniformly treated with acyclovir and IS reduction. All PTLDs were EBV-positive diffuse large B-cell lymphomas. Ten patients (91%) obtained a durable complete response (CR), and 9 (82%) have remained in continuous CR with a median follow-up of 29 months. Five patients (45%) lost their allograft. Of these, 4 patients had PTLD affecting the transplanted kidney. Peripheral blood CD8(+) T cells increased significantly (P =.0078) from baseline in 8 responders available for analysis. One of 2 patients whose absolute CD8(+) T-cell count subsequently dropped to baseline after IS reduction relapsed. The expanded CD8(+) T cells from 2 responders specifically recognized an immunodominant peptide from the EBV lytic gene BZLF-1. Another lytic EBV gene, thymidine kinase, was expressed in all 8 PTLDs tested. IS reduction and antiviral therapy for PTLD after renal transplantation is a highly successful therapeutic combination, but the risk of graft rejection is significant, particularly in patients with PTLD involving the renal allograft. A sustained expansion of CD8(+) T cells and a cellular immune response to EBV lytic antigens may be important for PTLD clearance in renal transplantation patients.     

Azidothymidine inhibits NF-kappaB and induces Epstein-Barr virus gene expression in Burkitt lymphoma

The antiviral compound azidothymidine (AZT), alone or in combination with other agents, induces apoptosis in early-passage, Epstein-Barr virus-positive Burkitt lymphoma (EBV+ BL) lines and has clinical activity in EBV+ BL. We report here a mechanism of AZT's antitumor activity. The nuclei of these cells contain activated nuclear factor-kappaB (NF-kappaB) subunits p50, c-Rel, RelB, and p52, but not p65. Treatment of primary EBV+ BL lines with AZT inhibited NF-kappaB within 1 to 2 hours. This was followed by up-regulation of EBV gene expression including viral thymidine kinase (vTK) and apoptosis. Subclones of EBV+ BL cells that demonstrated activated p65 were resistant to AZT. In EBV+ BLs, AZT but not ganciclovir (GCV) was highly phosphorylated to its monophosphate form (AZT-MP). Phosphorylation, as well as apoptosis, was markedly enhanced in the presence of hydroxyurea. AZT inhibits NF-kappaB and up-regulates EBV gene expression in primary EBV+ BLs. AZT with hydroxyurea may represent an inexpensive, targeted regimen for endemic BL.     

Antisense oligodeoxynucleotides to latent membrane protein 1 induce growth inhibition, apoptosis and Bcl-2 suppression in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cells, but not in EBV-positive natural killer cell lymphoma cells

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) is essential for immortalization of B cells by EBV, protects the infected cells from apoptotic cell death and induces Bcl-2 expression. Suppression of LMP-1 expression by antisense oligodeoxynucleotides (AS-oligo) to LMP-1 inhibits proliferation, promotes apoptosis and suppresses Bcl-2 expression in EBV-transformed B cells. However, the function of LMP-1 expression in EBV-positive natural killer (NK) cell lymphoma cells has not been reported previously. We examined the function of LMP-1 in two EBV-positive NK cell lymphoma cell lines (NK-YS and YT) through suppressing LMP-1 expression by AS-oligo to LMP-1. The AS-oligo to LMP-1 suppressed LMP-1 mRNA and protein expression in two EBV-positive NK cell lymphoma cell lines, as well as in an EBV-transformed B-cell line (CMG-1). Proliferation was inhibited, apoptosis was induced and Bcl-2 expression was suppressed in CMG-1 cells, but none of these events were observed in NK-YS or YT cells. These results suggest that proliferation, inhibition of apoptosis and Bcl-2 expression in EBV-positive NK cell lymphoma cells are not directly regulated by LMP-1 as in EBV-transformed B-cell lines, but are probably mediated through other signal transducing systems.     

Endoplasmic reticulum stress causes EBV lytic replication

Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)-specific phosphatase inhibitor Salubrinal (SAL) synergized with TG to induce EBV lytic genes; however, TG treatment alone was sufficient to activate EBV lytic replication. SAL showed ER-stress-dependent and -independent antiviral effects, preventing virus release in human LCLs and abrogating gp350 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated B95-8 cells. TG resulted in sustained BCL6 but not BLIMP1 or CD138 expression, which is consistent with maintenance of a germinal center B-cell, rather than plasma-cell, phenotype. Microarray analysis identified candidate genes governing lytic replication in LCLs undergoing ER stress.     

Cells producing recombinant retrovirus with thymidine kinase gene from Herpes simplex virus suitable for human cancer gene therapy

Therapeutic cells producing amphotropic retrovirus, which are able to transduce in vivo thymidine kinase gene of Herpes simplex virus were prepared. Single-cell clone cells with high virus productivity (PA-3 17JH5c113) were obtained by cell cloning. The cells were found free of replication competent retrovirus, they were non-tumorigenic in xenogeneic host and highly sensitive to ganciclovir treatment in vitro and in vivo. The therapeutic efficacy of PA-317JH5c113 cells was tested in rat brain tumor model. Increase in survival in the group of treated versus untreated rats was observed. Therefore, these cells are suitable for application in human clinical trial.     

A candidate live inactivatable attenuated vaccine for AIDS.

The recent discovery of long term AIDS nonprogressors who harbor nef-attenuated HIV suggests that a naturally occurring live vaccine for AIDS may already exist. Animal models have shown that a live vaccine for AIDS, attenuated in nef, is the best candidate vaccine. There are considerable risks, real and perceived, with the use of live HIV vaccines. We have introduced a conditional lethal genetic element into HIV-1 and simian immunodeficiency virus (SIV) molecular clones deleted in nef. The antiviral strategy we employed targets both virus replication and the survival of the infected cell. The suicide gene, herpes simplex virus thymidine kinase (tk), was expressed and maintained in HIV over long periods of time. Herpes simplex virus tk confers sensitivity to the antiviral activity of acyclic nucleosides such as ganciclovir (GCV). HIV-tk and SIV-tk replication were sensitive to GCV at subtoxic concentrations, and virus-infected cells were eliminated from tumor cell lines as well as primary cell cultures. We found the HIV-tk virus to be remarkably stable even after being cultured in media containing a low concentration of GCV and then challenged with the higher dose and that while GCV resistant escape mutants did arise, a significant fraction of the virus remained sensitive to GCV.     

Epstein-Barr virus provides a survival factor to Burkitt's lymphomas

Epstein-Barr virus (EBV) has been causally associated with at least five human malignancies. The exact contributions made by EBV to these cancers remain unknown. We demonstrate that one viral protein found in all EBV-associated malignancies, Epstein-Barr nuclear antigen 1 (EBNA-1), is required for survival of one of these cancers, EBV-positive Burkitt's lymphoma. Inhibition of EBNA-1 decreases survival of these tumor cells by inducing apoptosis. Expression of EBNA-1 in uninfected cells also can inhibit apoptosis induced by expression of p53 in the absence of the EBV genome. Our findings demonstrate that EBNA-1 is critical for the continued survival of EBV-associated Burkitt's lymphoma, and, by extension, for the other B cell tumors with which EBV is associated. Efficient inhibitors of EBNA-1's functions would likely prove useful in the therapy of EBV-associated malignancies.     

Suppression of Epstein-Barr nuclear antigen 1 (EBNA1) by RNA interference inhibits proliferation of EBV-positive Burkitt’s lymphoma cells

Purpose: Epstein-Barr virus (EBV) is associated with the development of several lymphoid and epithelial malignancies, including Burkitt’s lymphoma. The EBV latent protein, EBV Nuclear Antigen 1 (EBNA1), is detectable in almost all types of EBV-associated tumors and is essential for replication and maintenance of the latent episome of EBV. We here examined whether the RNA interference (RNAi) technique could be employed to suppress expression of EBNA1 in EBV-positive Burkitt’s lymphoma cells. Methods: A Raji cell line expressing small hairpin RNAs (shRNAs) against EBNA1 was established and EBNA1 mRNA level was determined by real-time RT-PCR analysis. We investigated the effects of EBNA1 silence on lymphoma cell growth and cell cycle progression. Results: Transfection of an EBNA1 RNAi plasmid resulted in substantial loss of EBNA1 mRNA and significantly inhibited proliferation of Raji cells relative to the control plasmid case. Suppression of EBNA1 was also associated with downregulation of EBV oncogene EBNA2, a decreased PCNA labeling index and increased G0/G1 fraction in cell cycle analysis. Conclusions: These findings point to potential therapeutic applications for vector-mediated siRNA delivery to control EBV-associated malignant disorders.     

Epstein-Barr virus infection in Richter's transformation

Chronic lymphocytic leukemia (CLL) may convert to a diffuse large cell lymphoma (Richter's syndrome) over time. In occasional cases of Richter's transformation, Epstein-Barr virus (EBV) has been identified in the lymphoma cells. To evaluate the association of EBV infection with Richter's syndrome, the biopsy specimens and clinical records of 25 patients who were seen at the Mayo Clinic between 1984–1996 were retrospectively evaluated for the presence of EBV by immunoperoxidase staining for expression of EBV latent membrane protein (LMP), as well as the expression of EBV RNA and DNA in the cells by in situ hybridization. Four of the 25 patients showed evidence of EBV in the diffuse large cell lymphoma cells—three patients with a B-cell phenotype were positive for LMP, EBV DNA, and RNA; and one patient with a T-cell phenotype had positive EBV RNA in the large cell lymphoma cells. The Richter's syndrome was treated with combination chemotherapy in 15 patients, three received radiotherapy, three were followed without further therapy after a splenectomy, two died before treatment could be started, and one patient had insufficient follow-up. One patient with evidence of EBV in large cell lymphoma cells was treated with acyclovir as initial therapy. The median survival of EBV-positive patients was three months compared with nine months for EBV-negative patients, but this difference was not statistically significant (P = 0.385). Evidence for EBV infection related to Richter's transformation was present in 16% of the patients in this study and may be associated with a poorer outcome. Primary therapy with acyclovir in one patient did not seem to be beneficial and other therapeutic modalities in patients with EBV-positive Richter's transformation need to be explored. Am J. Hematol. 60:99–104, 1999. © 1999 Wiley-Liss, Inc.     

Transactivation of CCL20 gene by Epstein-Barr virus latent membrane protein 1

CCL20 is expected to play a crucial role in the initiation of immune responses and tumour growth. However, expression of CCL20 in Epstein-Barr virus (EBV)-associated diseases has not been studied. We examined the contribution of EBV infection and EBV-encoded latent membrane protein (LMP)-1 to CCL20 expression. EBV infection and LMP-1 induced CCL20 mRNA expression in the EBV-negative Burkitt lymphoma (BL) cell lines and the embryonic kidney cell line. Histone deacetylase inhibitor-stimulated endogenous LMP-1 also induced CCL20 expression in an EBV-positive BL cell line. Analysis of the CCL20 promoter showed that it was activated by LMP-1 C-terminal activation region (CTAR)-1 and CTAR-2. Co-expression of IkappaB alpha, IkappaB beta, IkappaB kinase (IKK)alpha, IKKbeta, IKKgamma, nuclear factor (NF)-kappaB-inducing kinase and tumour necrosis factor receptor-associated factor 2 dominant-negative constructs with LMP-1 inhibited the activation of the CCL20 promoter by LMP-1, suggesting that LMP-1 induces CCL20 via NF-kappaB signalling. The requirement for the NF-kappaB-binding site in the CCL20 promoter in LMP-1 responsiveness was established. Our results indicate that activation of the NF-kappaB pathway by LMP-1 is required for the activation of CCL20 expression.     

Construction of gene therapy vectors targeting thyroid cells: enhancement of activity and specificity with histone deacetylase inhibitors and agents modulating the cyclic adenosine 3',5'-monophosphate pathway and demonstration of activity in follicular and anaplastic thyroid carcinoma cells

Thyroid carcinoma accounts for the majority of deaths from endocrine cancers. Although effective therapies exist for well differentiated tumors, the treatment options for poorly differentiated and anaplastic tumors are much less effective. In the present study we demonstrate that the thyroglobulin (Tg) promoter can be used to direct specific expression of either luciferase or thymidine kinase in thyroid cancer cells. Furthermore, using a putative enhancer element for the Tg gene, the activity of the Tg promoter in and its specificity for thyroid cells were enhanced. In transient transfectants or in stably transfected thyroid carcinoma cells, treatment with the histone deacetylase inhibitors, depsipeptide (FR9012228) and sodium butyrate, alone or in combination with 8-bromo-cAMP, resulted in further enhancement. In experiments in which the herpes simplex virus thymidine kinase (HSV-TK) gene was driven by the Tg promoter and the putative enhancer, HSV-TK expression and ganciclovir sensitivity were augmented. Similar results were obtained in two cell lines derived from a follicular thyroid carcinoma and in two anaplastic thyroid carcinoma cell lines. In summary, we report the construction of a suicide HSV-TK vector with preferential toxicity for thyroid cells. The results in anaplastic thyroid carcinoma cells suggest that it may be of use in the full spectrum of thyroid malignancies.     

Glycoprotein gp110 of Epstein-Barr virus determines viral tropism and efficiency of infection

The Epstein-Barr virus (EBV) genome has been detected in lymphomas and in tumors of epithelial or mesenchymal origin such as nasopharyngeal carcinoma or leiomyosarcoma. Thus, there is little doubt that EBV can infect cells of numerous lineages in vivo, in contrast to its in vitro infectious spectrum, which appears restricted predominantly to B lymphocytes. We show here that the EBV BALF4 gene product, the glycoprotein gp110, dramatically enhances the ability of EBV to infect human cells. gp110(high) viruses were up to 100 times more efficient than their gp110(low) counterparts in infecting lymphoid or epithelial cells. In addition, gp110(high) viruses infected the carcinoma cell line HeLa and the T cell lymphoma cell line Molt-4, both previously thought to be refractory to EBV infection. Analysis of several virus isolates showed that the amount of BALF4 present within mature virions markedly differed among these strains. In some strains, gp110 was found expressed during lytic replication not only at the nuclear but also at the cellular membrane. Heterologous expression of gp110 during the virus lytic phase neither altered virus concentration nor affected virus binding to cells. It appears that gp110 plays a crucial role after the virus has adhered to its cellular target. gp110 constitutes an important virulence factor that determines infection of non-B cells by EBV. Therefore, the use of gp110(high) viruses will help to determine the range of the target cells of EBV beyond B lymphocytes and provide a useful in vitro model to assess the oncogenic potential of EBV in these cells.     

Bortezomib induction of C/EBPβ mediates Epstein-Barr virus lytic activation in Burkitt lymphoma

Epstein-Barr virus (EBV) is associated with a variety of lymphoid malignancies. Bortezomib activates EBV lytic gene expression. Bortezomib, a proteasome inhibitor, leads to increased levels of CCAAT/enhancer-binding proteinβ (C/EBPβ) in a variety of tumor cell lines. C/EBPβ activates the promoter of the EBV lytic switch gene ZTA. Bortezomib treatment leads to increased binding of C/EBP to previously recognized binding sites in the ZTA promoter. Knockdown of C/EBPβ inhibits bortezomib activation of EBV lytic gene expression. Bortezomib also induces the unfolded protein response (UPR), as evidenced by increases in ATF4, CHOP10, and XBP1s and cleavage of ATF6. Thapsigargin, an inducer of the UPR that does not interfere with proteasome function, also induces EBV lytic gene expression. The effects of thapsigargin on EBV lytic gene expression are also inhibited by C/EBPβ knock-down. Therefore, C/EBPβ mediates the activation of EBV lytic gene expression associated with bortezomib and another UPR inducer.     

Adenovirus-mediated targeted expression of toxic genes to adrenocorticotropin-producing pituitary tumors using the proopiomelanocortin promoter.

Management of Cushing's disease remains challenging, despite advances in its diagnosis and treatment. Here, we describe a strategy for targeting the expression of toxic genes to ACTH-producing tumor cells using adenoviral vectors. The POMC promoter was used to express either a marker gene (beta-galactosidase) or a toxic gene [herpes simplex virus thymidine kinase (TK)]. In ACTH-producing AtT20 cells, infection with recombinant adenoviruses containing the POMC promoter (AdPOMCGal; AdPOMCTK) led to high-level gene expression. Stereotactic injection of AdPOMCGal into the rat pituitary resulted in localized expression of the beta-galactosidase transgene in corticotrope cells. Cytotoxicity studies were performed using the TK-containing vectors and treatment with ganciclovir. AdPOMCTK caused greater than 95% cytotoxicity of AtT20 cells at a viral dose (multiplicity of infection, 5 plaque-forming units/cell) that induced minimal toxicity using control viruses. No cellular toxicity was seen using a nonpituitary cell line (T47D breast tumor cells). AtT20 cells transplanted into nude mice induced features of Cushing's syndrome and were used as an in vivo model of ACTH-producing tumors. Injection of the AdPOMCTK virus caused significant regression of the transplanted AtT20 tumors. These studies suggest that the POMC promoter may provide a useful gene therapy strategy for the adjunctive treatment of pituitary tumors causing ACTH-dependent Cushing's syndrome.