Virus-Associated RNA I–Deleted Adenovirus, a Potential Oncolytic Agent Targeting EBV-Associated Tumors

Research Article

Yaohe Wang,1 Shao-An Xue,2 Gunnel Hallden,1 Jennelle Francis,1 Ming Yuan,1 Beverly E. Griffin,2,3 and Nick R. Lemoine1

1Cancer Research UK Molecular Oncology Unit, Institute of Cancer, Barts and the London School of Medicine and Dentistry, Queen Mary University of London; 2Division of Medicine, Hammersmith Hospital, Imperial College London; and 3Imperial College London at St. Mary’s, London, United Kingdom

Abstract More recent reports have linked EBV with conventional epithelial Given the growing number of tumor types recognizably cancers of other sites, including breast (3–5), lung (6–9), prostate associated with EBV infection, it is critically important that (7), liver (10), colon (7), and also with lymphoepithelioma-like therapeutic strategies are developed to treat such tumors. carcinoma of the esophagus (11). Given the ever-growing Replication-selective oncolytic adenoviruses represent number of tumor types associated with EBV infection, thera- a promising new platform for anticancer therapy. Virus- peutic strategies to treat EBV-associated tumors may be associated I (VAI) RNAs of adenoviruses are required for considered a high priority in oncology. efficient translation of viral mRNAs. When the VAI gene is Replication-selective, oncolytic viruses provide a new platform deleted, adenovirus replication is impeded in most cells to treat cancer. Promising clinical trial data with mutant (including HEK 293 cells). EBV-encoded small RNA1 is adenoviruses have shown both their antitumor potency and uniformly expressed in most EBV-associated human tumors safety (12, 13). Two main approaches are currently being used to and can functionally substitute for the VAI RNAs of engineer adenoviruses with tumor-selective replication. The first adenovirus. It enables replication to proceed through strategy limits the expression of the E1A gene to tumor tissues complementation of VAI-deletion mutants. We hypothesized through the use of tumor- and/or tissue-specific promoters; that VAI-deleted adenovirus would selectively replicate in consequently, E1A-induced S phase entry and activation of viral EBV-positive tumor cells due to the presence of EBV-encoded and cellular genes will only occur in tumor tissues. A second small RNA1 with no (or poor) replication in normal or EBV- strategy optimizes tumor selectivity by deletion of viral genes negative tumor cells. In this report, we show that high levels that are critical for efficient replication in normal cells but of replication occurred in the VAI-deleted mutant in the EBV- expendable in tumor cells (14). The most commonly described positive tumor cells compared with low (or negligible) levels oncolytic viruses explored over the past 10 years have been in EBV-negative and normal human primary cells. Corre- mutant adenoviruses where at least 25 different ones have been spondingly, high toxicity levels were observed in EBV-positive described for use in cancer treatments to date (15). tumor cells but not in EBV-negative tumor or normal human Several studies have shown similarities in the function of primary cells. In vivo, VAI-deleted adenovirus showed adenovirus and EBV genes (16–20). Based on the similarities superior antitumoral efficacy to wild-type adenovirus in described, we believe that a window of opportunity exists in the EBV-positive tumor xenografts, with lower hepatotoxicity design of replication-selective adenovirus for EBV-associated than wild-type adenovirus. Our data suggest that VAI-deleted tumors. The adenovirus genome encodes two RNA polymerase f adenovirus is a promising replication-selective oncolytic III-directed, 160-nucleotide-long RNAs, the so-called virus- virus with targeting specificity for EBV-associated tumors. associated RNA I and RNA II. These accumulate to high levels (Cancer Res 2005; 65(4): 1523-31) during the late stages of viral infection (21–23). VAI RNA is obligatory for efficient translation of viral and cellular mRNAs (24, 25). It binds to and blocks the activation of cellular double- Introduction stranded RNA-dependent serine/threonine protein kinase (PKR), EBV, a ubiquitous human herpesvirus found latently expressed which phosphorylates protein translation initiation factor eIF-2a, in 90% of the human population, is the causative agent of enabling protein synthesis to proceed at normal levels (26–29). A infectious mononucleosis and posttransplant immunoprolifera- mutant that fails to produce VAI RNA (dl331) grows more poorly tive disorders in immunocompromised patients. It is closely than its parent in human 293 cells (25). associated with the development of a variety of malignant EBV also expresses two low-molecular-weight RNAs, designat- diseases, including endemic Burkitt’s lymphoma, B-cell lympho- ed EBV-encoded RNAs (EBER) 1 and 2, transcribed by RNA ma of immunocompromised patients, nasopharyngeal carcinoma, polymerase III (30). Although there is no striking nucleotide Hodgkin’s disease, T-cell lymphoma, natural killer cell leukemia/ sequence homology between virus-associated RNAs and EBERs, lymphoma, smooth-muscle tumors, and gastric cancer (1, 2). the RNAs are similar in size, genomic organization, and degree of secondary structure; they have similar functions (19, 20, 31). In particular, the two small RNAs encoded by EBV can efficiently complement the VAI RNA–mediated translational defect in Requests for reprints: Nick R. Lemoine, Cancer Research UK Molecular Oncology Unit, Institute of Cancer, Barts and the London School of Medicine and adenovirus-infected cells, as shown by constructing an Ad5 Dentistry, Queen Mary University of London, John Vane Science Building, substitution mutant in which the two virus-associated RNA Charterhouse Square, London EC1 6BQ, United Kingdom. Phone: 44-20-7014-0420; E-mail: [email protected]. genes have been deleted and replaced by an EBV DNA segment I2005 American Association for Cancer Research. encoding the two EBERs (32, 33). Given the uniform expression www.aacrjournals.org 1523 Cancer Res 2005; 65: (4). February 15, 2005

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. Cancer Research of EBERs in human EBV-associated tumors, generally at high viruses. Cell survival was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3- levels, and the similar functions between VAI RNA and EBER1, carboxymethoxy-phenyl)-2-(4-sulfony)-2H-tetrazolium assay (MTS, Prom- we hypothesized that VAI RNA–defective adenovirus could be ega, Madison, WI ) 6 days postinfection, and the EC50 values of different complemented by EBER1 in EBV-positive tumors, thus restricting adenoviruses obtained as previously described (39). For NHBE and PrEC cells, 100 AL fresh medium was added to each well 3 days postinfection. virus-induced cell killing and replication to EBV-positive tumors. All assays were done at least thrice with each concentration of virus In the present report, we tested this hypothesis. We show that in sextuplicate. VAI-deleted adenovirus is a potential therapeutic agent that can Detection of EBER1 Expression by Reverse Transcription-PCR. Total specifically target EBV-associated tumors. RNA was extracted from different cells, using an RNA extraction kit (Qiagen, Crawley, United Kingdom) according to the manufacturer’s instructions. To synthesize cDNA, 1 Ag of total RNA was treated with Materials and Methods 10 units of DNase I (Roche, Lewes, United Kingdom) at 37jC for 30 Cell Lines and Cell Culture. Human Burkitt’s lymphoma (Raji), minutes, as described (40). After purification, DNase I-treated RNA was epidermoid carcinoma of larynx (Hep-2), lung cancer (A549), and reverse transcribed by Superscript II reverse transcriptase (Invitrogen, embryonic kidney (HEK; a subclone named JH-293) cell lines were all Renfrew, United Kingdom) with 50 pmol pd(N)6 random hexamer primers obtained from the Cancer Research UK Central Cell Service (Clare Hall, (Amersham Biosciences, Chalfont St. Giles, United Kingdom) in 20 AL. For Middlesex, United Kingdom). The Jijoye cell line was obtained from PCR amplification, 1 AL cDNA was added to 49 AL PCR reaction mixture American Type Culture Collection (Manassas, VA) and the human [75 mM Tris-HCl (pH 8.8), 20 mM (NH4)2SO4, 0.01% (vol/vol) Tween 20, nasopharyngeal carcinoma line C666-1 from Prof. Dorly P. Huang (The 1.5 mM MgCl2, 0.16 mM (each) deoxynucleoside triphosphate, and 30 Chinese University of Hong Kong, Hong Kong, China). The presence of pmol of each primer] containing 1.5 units of TaqDNA polymerase EBV has been consistently shown in C666-1 (34). Raji, Jijoye, Hep-2, and (ABgene, Epsom, United Kingdom). To analyze expression of EBER1, the C666 cells were maintained in RPMI containing 10% fetal bovine serum, primer pairs used were 5V-AGGACCTACGCTGCCCTAGA-3V (forward) and 2% L-glutamine, and antibiotics. JH-293 and A549 cells were maintained 5V-CCCTAGTGGTTTCGGACACAC-3V (reverse). After 30 cycles of amplifi- in DMEM supplemented with 10% fetal bovine serum and antibiotics. cation, PCR products were separated by electrophoresis on 1% agarose The human gastric cancer cell lines AGS and AGS-EBV were a generous gels containing ethidium bromide. The human b-actin gene was used as gift from Prof. Lindsey Hutt-Fletcher (Louisiana State University, an internal control. Shreveport, LA). The AGS-EBV cell line was obtained by G418 selection Western Blotting. Control and infected cells were lysed at indicated of AGS cells that were infected with a recombinant Akata virus in which times with lysis buffer [20 mmol/L Tris-HCl (pH 7.6), 400 mmol/L NaCl, 50 a neomycin resistance cassette had been inserted into the nonessential mmol/L KCl, 5 mmol/L h-mercaptoethanol, 1% Triton X-100, 20% glycerol, BDLF3 open reading frame. Both the AGS and AGS-EBV cell lines were 100 Ag/mL phenylmethylsulfonyl fluoride, 1 Ag/mL leupeptin, and 10 Ag/mL maintained in Ham’s F12 medium, supplemented with 10% fetal bovine aprotinin]. Protein lysates were quantitated by the bicinchoninic acid assay serum, antibiotics, and G418 at 500 Ag/mL (AGS-EBV cells only). Normal (Sigma, Poole, United Kingdom). Proteins were separated by reducing SDS- human bronchial epithelial cells (NHBE) and normal prostate epithelial 10% polyacrylamide gel electrophoresis, electrotransferred onto nitrocellu- cells (PrEC) were obtained from Clonetics Corp. (San Diego, CA) and lose membranes and probed with a mouse anti-total PKR antibody (Santa were propagated as recommended by the manufacturer. All cells were Cruz Biotechnology, Santa Cruz, CA), or a rabbit anti-phospho-PKR maintained at 37jC in a humidified atmosphere containing 10% CO2. (Biosource, Camarillo, CA), or rabbit anti-eIF-2a[pS52] phosphospecific Viruses. VAI-deleted adenovirus dl331 (25), wild-type Ad5 and E3B- antibody (Biosource), or mouse anti-human proliferating cell nuclear deleted adenoviral mutant dl309 (35) were gifts from Prof. Bayar antigen antibody (Research Monoclonal Antibody Service, Cancer Research Thimmappaya (Northwestern University, Chicago, IL) and Dr. Thomas UK, London, United Kingdom). Immunocomplexes were detected by Shenk (Princeton University, Princeton, NJ). The replication-defective Ad5 incubation with a horseradish peroxidase-conjugated secondary antibody mutant dl312 was inactivated by psoralen-UV, as previously described, to (DAKO, Glostrup, Denmark) and visualized by enhanced chemiluminescence serve as a nonreplicating particle control for in vitro cell survival and in reagent (Amersham Biosciences). vivo efficacy studies. Ad5, dl309, and dl312 adenoviruses were amplified In vivo Efficacy, Replication, and Gene Expression. Two approaches in the human embryonic kidney cell line HEK 293; dl331 was generated were used to evaluate the antitumor efficacy of VAI-deleted adenovirus and in A549 cells. All adenoviruses were purified and titered as previously wild-type Ad5 in vivo as described below. All animal experiments were described (36). approved by the Cancer Research UK Animal and Safety Committee and the Viral Infection and Replication Assay. Cells were seeded in six-well United Kingdom Home Office. plates (3 Â 105-1 Â 106 cells/well, according to their growth properties) and In the first protocol, cultured C666-1 cells were infected with vehicle buffer, infected with viruses at 100 particles per cell when 70% to 80% confluence or psoralen-UV-inactivated dl312, wild-type Ad5, and dl331, for 2 hours at 1,000 had been reached. For all human tumor cells, viral infections were done in particles per cell. Fourteen hours postinfection, 10% preinfected C666-1 cells standard growth medium without serum. Primary human cells were mixed with 90% uninfected cells, and 1.5 Â 107 mixed cells, in 200 AL of PBS, infected in predefined growth medium, with no alterations to growth factor were injected s.c. into the flanks of 6 BALB/c nu/nu mice (4-6 weeks old) for components. Infections were done in a low volume of medium for 2 hours each group. Tumor growth, as measured by volume, was monitored twice at 37jC/10% CO2. Medium was then replaced either with fresh medium weekly. containing 2% fetal bovine serum for tumor cells or with growth factors for In the second protocol, 2 Â 107 C666-1 cells were suspended in 200 AL primary human cells. Experiments were repeated under each condition in normal saline and injected s.c. into the flanks of athymic (nu/nu) mice (4-6 triplicate. Cells and media were collected at 48 and 72 hours postinfection. weeks old) and allowed to grow. Volume was estimated twice weekly using The collected samples were freeze-thawed thrice, and replicating virus was the formula: volume = (length Â width2)(3.142/6). Once tumors reached the determined by the limiting dilution assay as described (36) with the use of size range of 33.5 to 117 AL, mice were stratified by tumor size and then A549 and 293-T cells (293 cells transformed with SV40 T antigen) as randomly allocated to treatment groups; treatment groups were balanced by indicator cells. A549 supports VAI-deleted adenovirus replication to an tumor size at the time of treatment initiation in all cases (P > 0.8, t test for equivalent level to wild-type adenovirus (37), and SV40 T antigen (in 293-T) tumor volumes). To treat the established tumors, 100 AL of PBS without or could rescue the translational defect resulting from infection with VAI- with virus (Ad5, dl331, or replication-incompetent control adenovirus, deleted adenovirus (38). PUVdl312) were directly injected into them. Injections were introduced In vitro Cell Survival Assay. For cell survival assays, 1 Â 104 cells (1 Â through a single central tumor puncture site and three to four needle tracts 103 cells of Hep-2) were seeded in each well of 96-well plates in 100 AL made radially from the center. For efficacy experiments, tumors were injected medium and infected 24 hours later with serial dilutions of different (1 Â 1010 particles/d) on 5 consecutive days, and for biological end point

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Table 1. Human cells evaluated and EBER1 expression Results EBER1 Expression in Normal Human Primary and Trans- Cell name Derived tissues EBER1 status formed Cells. To determine the selectivity of replication of VAI- deleted adenovirus targeting EBV-associated tumors and cell killing, NHBE Normal human À a panel of human cell lines including normal primary and EBV- bronchial epithelial cells negative and EBV-positive tumor cell lines was used, as listed in PrEC Normal human À Table 1. Given that EBV resides in 90% of the human population, the prostate epithelial cells expression pattern of EBER1 in all cells was predetermined by HEK Human embryonal À semiquantitative reverse transcription-PCR before testing our kidney cells complementation hypothesis. Raji, Jijoye, AGS-EBV, and C666-1 A549 Human lung À expressed varying levels of EBER1 RNA whereas other cells were cancer cells Panc-1 Human pancreatic À negative (Fig. 1; Table 1). Among the four EBV-positive cell lines, the cancer cells highest EBER1 RNA expression levels were observed in the C666-1 Hep-2 Human epidermoid À and Raji cells, whereas slightly lower levels were detected in the carcinoma of larynx AGS-EBV cells previously stably transfected with the EBV genome. AGS Human stomach À Selectivity of EBER1-Positive and EBER1-Negative Cells to cancer cells Oncolysis by VAI-Deleted Adenovirus. EC50 values were deter- AGS-EBV AGS-transfected + mined in each cell to compare the cell killing potencies of VAI- with EBV deleted and wild-type adenovirus in normal and tumor cells with Jiyoye African Burkitt’s + different EBER1 status (Table 1). The ratios of EC value for dl331 lymphoma cells 50 and wild-type Ad were compared and correlated to EBER1 status in Raji African Burkitt’s + lymphoma cells each cell line (Fig. 2A). In the panel of human cells examined, the C666-1 Human nasopharyngeal + dl331 mutant showed a cell killing potency that was equivalent or carcinoma cells superior to wild-type adenovirus in the EBV-positive AGS-EBV, Raji, Jijoye, and C666-1 cells. In contrast, the dl331 mutant was markedly attenuated in the EBV-negative human cells tested both in tumor cell lines and normal primary human cells. Surprisingly, Panc-1 cells, a studies, tumors were injected once only, on day 1. Tumors were harvested at human ras mutant pancreatic cancer cell line previously reported to stated time points after injection for analysis of viral gene expression, be susceptible to dl331 (41), showed a very low level of cytopathic replication, and general histopathology as described (15). Tumor size and effect to dl331 in the present study. These results show that the VAI- animal survival were monitored. Animals were sacrificed when tumor deleted adenovirus could selectively kill EBER1-positive cell lines, reached the size limited by Home Office regulations or after 3 months. whereas they have little effect on EBV-negative and normal cells. In Survival analysis was done according to the method of Kaplan-Meier. addition, the VAI-deleted adenovirus was as potent as wild-type 6 Viral Hepatoxicity. Murine lung adenocarcinoma CMT-64 (5 Â 10 ) cells, adenovirus in the EBER1-positive tumor cell lines. were injected s.c. into both flanks of immunocompetent C57/BL6 mice Viral Replication of VAI-Deleted Adenovirus in Normal (obtained from Cancer Research UK Biological Resources). The animals were Primary Human, EBV-Negative and EBV-Positive Tumor Cells. randomized into four groups (n = 9 per group) and treated with PBS, To determine whether dl331-induced selective cell killing was PUVdl312, wild-type Ad5, and dl331. I.v. injection of viruses (1 Â 1010 indicative of replication induction in EBV-positive cells, replica- particles in a volume of 100 AL) via the tail vein was initiated once the tumor size reached 40 to 80 mm3. At 24, 48, and 72 hours postinjection, mice were tion of mutant and wild-type adenovirus was compared in sacrificed (three mice per time point), liver samples were collected and individual cell lines. The EBV-positive (AGS-EBV, Jijoye, Raji, and processed for general histopathology, and viral protein expression was C666-1), EBV-negative (JH-293, Panc-1, Hep-2, and AGS-ATCC) analyzed as described (15). and normal human primary (NHBE and PrEC) cells were infected Statistical Analysis. Unpaired t test or one-way ANOVA were used for with VAI-deleted and wild-type adenoviruses, and cells were all in vitro studies and in vivo toxicity studies. Kaplan-Meier curves were harvested at 48 or 72 hours. The VAI-deleted mutant replicated to compared using the log-rank test. levels of 20% to 73% that observed for wild-type Ad5 in all four

Figure 1. EBER1 RNA expression in a panel of human cells by reverse transcription-PCR. Total RNA extraction, reverse transcription of RNA, and PCR amplification were described in Materials and Methods. PCR products were separated on 1% agarose gels and visualized by ethidium bromide staining. Lane M, 100 bp ladder; lane 1, DNase I–treated EBV-C15 NPC DNA; lane 2, NHBE; lane 3, PrEC; lane 4, JH-293; lane 5, A549; lane 6, Panc-1; lane 7, HeLa; lane 8, Hep-2; lane 9, AGS-ATCC; lane 10, AGS-EBV; lane 11, Jijoye; lane 12, Raji, lane 13, C666-1 late passage; lane 14, C666-1 early passage; lane 15, primers only.

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Figure 2. Selective cell killing and replication of VAI-deleted adenovirus in EBV-positive and EBV-negative cells. A, EC50 value comparison for wild-type Ad5 and VAI deletion mutant. The EC50 values of Ad5 and mutant dl331 (VAI-deleted) in different human transformed cell lines were obtained by a cell death assay (MTS assay) as described in Materials and Methods. The EC50 values of the mutant virus were compared with the wild-type control and an EC50 ratio was calculated as Ad5/dl331. A ratio >1.0 shows that the potency of mutant virus is greater than that of wild-type virus. The data represent triplicate experiments. B, comparison of viral production by Ad5 and dl331 adenovirus in normal human primary cells and EBV-positive and EBV-negative tumor cells. Measurements were done by tissue-culture infectious dose-50% assay on A549 cells as described in Materials and Methods. Viral production by wild-type Ad5 and dl331 in different cells was calculated as plaque-forming unit per cell (n = 3), compared and expressed as the ratio of dl331/Ad5. A ratio of 1 would indicate that the mutant replicates as well as wild-type virus.

EBV-positive tumor cell lines (Fig. 2B). The highest level of Because in each cell line, viral infection, replication, lysis, and replication of dl331 was observed in C666-1 cells, which also spread are dependent on their intrinsic properties, it may not be express the highest level of EBER1 RNA among these cells. Viral reasonable to compare two tumor cell lines derived from different production by VAI-deleted adenovirus in EBV-negative cells was sources. Therefore, data from matched cell lines, such as AGS and lower than in EBV-positive cells. To our surprise, viral production AGS-EBV, could be more meaningful. The viral replication ratio of by VAI-deleted adenovirus in normal primary cells was f4,000 dl331/Ad5 in AGS-EBV at 48 hours proved to be 16 times (P <0.01) times less than wild-type adenovirus in NHBE, and 500 to 1,000 higher than in AGS cells, and after 72 hours increased to 33 times times less in PrEC cells, respectively. Of note, among EBV- (P <0.001;Fig.2B). negative human tumor cell lines, only Panc-1 showed a relative These results show that VAI-deleted adenovirus can selectively level of replication, that is f20% that of wild-type Ad5 at replicate in EBV-positive tumor cells with no significant replication 72 hours. being observed in normal primary cells.

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Changes in PKR and eIF-2A Activation in Response to Viral Ad5- or dl331-preinfected cells up to 205 days postimplantation Infection. VAI RNA regulates mRNA translation and controls with the mixed tumor cells. Statistical analysis confirmed viral replication through the PKR/eIF-2a pathway; EBER1 has a significant differences between both of wild-type Ad5- and similar function (19, 42, 43). To test whether the selectivity of dl331-treated groups and each control group (P < 0.01). the VAI mutant for EBV-positive cells occurs through a blocked The antitumor efficacy of dl331 was also confirmed in preestab- PKR-eIF-2a pathway, total PKR, phosphorylated-PKR, and lished s.c. C666-1 tumors treated by intratumoral virus administration phosphorylated-eIF-2a were analyzed by immunoblotting in as described in Materials and Methods. Tumor growth and survival EBV-positive cells (C666-1) and EBV-negative cells (Hep-2 and rate over time were monitored (Fig. 4A and B). The dl331 mutant Panc-1) at 24 hours postinfection with wild-type Ad5, dl309, and significantly inhibited tumor growth compared with the two control dl331 (Fig. 3). Viral infection resulted in increased expression groups, and was superior to wild-type Ad5 (Fig. 4A). Significantly levels of total PKR in all these cell lines and there was no prolonged survival was also observed in the dl331-treated animals difference in the level of total PKR between viruses (Fig. 3, 1). In compared with Ad5-treated animals (Fig. 4B). The dl331 and Ad5 normal cells, PKR is active through a mechanism involving treated groups had 75% and 50%, respectively, animals alive after autophosphorylation in response to viral infection. Changes in 75 days compared with controls with no animals surviving phosphorylated PKR levels were therefore determined in the after 50 days. three cells following infection with different adenoviruses To confirm cell death and active viral replication in the tumor (Fig. 3, 2). As expected, dl331 infection of EBV-negative cells mass, H&E and immunohistochemical staining with antiviral showed a significant elevation of phosphorylated PKR compared protein E1A and hexon antibodies were done on cross sections with wild-type adenovirus infection. However, EBV-positive taken on day 5 after single viral injections (Fig. 4C). H&E C666-1 cells still showed elevated levels of phosphorylated staining confirmed the killing of tumor cells from live virus- PKR following dl331 treatment, whereas infection with wild- treated tumors, with dl331 resulting in more severe cytopathic type Ad5 and dl309 mutant blocked PKR phosphorylation (Fig. effects than wild-type adenovirus (Fig. 4C, 1). Equivalent levels of 3, 2). We further investigated phosphorylation of the substrate viral proteins E1A and hexon were observed in EBV-positive tumors a a of PKR, eIF-2 , using a rabbit anti-eIF-2 [pS52] phosphospe- after single treatments with dl331 and wild-type adenovirus (Fig. 4C, 2 cific antibody. As observed in Fig. 3, 3,infectionwithdl331 and 3), demonstrating that viral replication had occurred within the resulted in a minor increase in the level of phosphorylated eIF- tumor cells. a 2 in comparison with wild-type adenovirus infection in EBV- VAI-Deleted Mutant Results in Reduced Hepatotoxicity positive cells; however, dl331 produced a significantly elevated In vivo. Intratumoral treatment with replication-selective onco- a level of phosphorylated eIF-2 compared with wild-type lytic adenovirus is well tolerated in both mice and humans (44). adenovirus in EBV-negative cells. These results indicate that However, systemic administration might have higher toxicity a EBER 1 may complement the regulation of eIF-2 by a route risks in organs such as liver. The systemic delivery of not exclusively dependent on PKR. In addition, no differences replication-competent adenoviruses, even when modified for a in phosphorylated PKR and phosphorylated eIF-2 levels were tumor-selective replication, has previously been shown to cause observed in normal human primary cells following wild-type or dose-limiting toxicity in mice, associated with liver necrosis VAI-deleted adenovirus infection (data not shown) although the resulting from direct exposure to adenoviral coat proteins, viral cells showed a significant difference in viral replication with early gene expression, viral replication, and/or de novo late gene low or no replication occurring with VAI mutant. expression (45). The host immune response may also play a role Antitumoral Efficacy of the VAI-Deleted Adenovirus In vivo in mediating this dose-limiting toxicity. To explore this topic, the Is Equivalent or Superior to that of Wild-type Adenovirus in acute hepatotoxicity of dl331 and wild-type adenoviruses in the EBV-Positive Tumors. To evaluate whether dl331 could inhibit recently developed CMT-64 immunocompetent mouse tumor tumor growth in vivo, preinfected C666-1 cells were implanted model (36) was investigated, as described in Materials and s.c. in BALB/C nu/nu mice as described in Materials and Methods. Histopathologic observations showed that cytopathic Methods. Tumor formation and growth were monitored over effects of hepatocytes following dl331 treatment decreased with time. Tumors formed in all PBS-treated control animals and in time, whereas those of wild-type adenovirus were consistently 83.3% (5 of 6) of animals that received inactivated, nonreplicating higher at three time points. Viral late gene (hexon) expression control adenovirus at 27 days postimplantation. No tumor and cytopathic effects in the liver after dl331 treatment were formed in any of the animals that received 10% of wild-type significantly lower than that found with wild-type adenovirus

Figure 3. PKR and eIF-2-a alterations in EBV-positive and EBV-negative tumor cell lines after infection with dl331 and wild-type adenovirus. Cells were infected with Ad5, dl309, or dl331 at 100 particles per cell as described in Materials and Methods. Twenty-four hours later, proteins were extracted, separated by gel-electrophoresis, and immunoblotted for total PKR, phosphorylated PKR (P-PKR), phosphorylated eIF2-a (P-eIF2-a), and proliferating cell nuclear antigen (PCNA). Lane 1, uninfected-cells; lane 2, wild-type Ad5-infected cells; lane 3, dl309-infected cells; lane 4, dl331-infected cells. Data are from duplicate experiments. www.aacrjournals.org 1527 Cancer Res 2005; 65: (4). February 15, 2005

Figure 4. Antitumoral efficacy of VAI deletion mutant (dl331) in established C666-1 (EBV-positive) tumors in vivo. C666-1 human nasopharyngeal carcinoma xenografts implanted s.c. in C57BL/6 nu/nu mice as described in Materials and Methods. PBS or viruses (1 Â 1010 particles) were injected intratumorally on 5 consecutive days. Single injections were done for the biological time-points study. A, tumor growth curves; animals treated with dl331 (n = 7) and wild-type Ad5 (n = 6) showed significant delays in tumor growth compared with the control groups injected with PBS (n = 7) or the inactivated particles control group (n =7;P < 0.01). B, animal survival curves. dl331 and wild-type Ad5 treatment produced significantly higher survival rates than the control group (P < 0.001); the survival rate of dl331-treated mice was superior to Ad5. C, representative histologic changes and viral protein expression in C666-1 xenografts on day 5 after treatment with different adenoviruses, as indicated. H&E staining (original magnification, Â200) shows that Ad5 and dl331 produce significant cytopathic effects in tumor cells (i.e., cell death, eosinophilic cytoplasm, enlarged nuclei, and inclusion bodies). Control tumors show no significant change. Immunohistochemical analysis of such sections (original magnification, Â200) shows that the remaining tumor cells stain positively for viral proteins E1A and hexon (brown), whereas the PBS-treated tumors show no immunoreactivity; the PUVdl312-treated tumors show only weak nuclear staining for the hexon coat protein and no E1A staining.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. Oncolytic VAI-Deleted Mutant Adenovirus after 48 hours: There was no difference of E1A expression inactivating PKR, it was recently reported that a VAI mutant between wild-type and VAI-deleted adenoviruses infection. The adenovirus could be used selectively to treat tumors with an viral gene hexon expression was consistent with the cytopathic activated ras oncogene (41). In the present study, VAI-deleted effect alteration. Figure 5 represents examples of histopathology adenovirus showed selective cell killing and replication in EBV- and adenovirus hexon protein staining in liver after viral positive tumor cells but not significant in a tumor cell line treatment. (Panc-1) with activated Ras pathway. Our results show that in the EBV-positive tumor-derived cell line C666-1, dl331 induced a higher level of phosphorylated-PKR but a minor increase level of Discussion phosphorylated-eIF-2a when compared with wild-type adenovi- The presence and expression of the EBV genome undoubtedly rus. These findings suggest that EBER may act through another contributes to the development of many, if not all, tumors pathway independent of PKR to affect host protein translation. associated with this virus, opening up an unique opportunity to It is known that the phosphorylation of eIF2a is mediated by develop novel anticancer treatments directed against viral, rather at least three different protein kinases: the IFN-inducible dsRNA- than cellular, targets. A number of therapies have been proposed activated kinase PKR, the erythroid cell-specific, heme-regulated to target EBV-associated tumors based on expression of viral kinase HCR (HRI) and the nutrient-regulated protein kinase GCN2 genes, as recently reviewed (46). Some examples are those (47). A recent report (48) showed that EBER-1 could enhance inhibiting the expression of EBV-encoded oncogenes or induction protein synthesis by a PKR-independent mechanism, strongly of EBV episome loss. Others involve the use of EBV-dependent supporting our data. In addition, SV40 antigen can rescue the promoters and replication origins to express toxic genes, translational defect in monkey and human cells that result from preferentially within EBV-positive tumors, the deliberate induc- infection of VAI-deleted adenovirus. However, the large T antigen tion of the lytic EBV replication in tumor cells, or various of SV40 does not prevent the activation of PKR and the immune therapies targeting EBV viral proteins. In the present complementation appears to occur at a step downstream of PKR study, we exploited the fact that EBERs are uniformly expressed activation, maybe by dephosphorylation of eIF-2a (38), and EBER1 in most human EBV-associated tumors, engineering a comple- may act through a similar pathway. In the EBV-negative tumor cell mentary mutation in a replication-competent adenovirus to lines, Hep-2 and Panc-1, dl331 induced higher levels of phosphor- produce a selective oncolytic viral therapy for these tumors. ylated-PKR and phosphorylated-eIF2a than wild-type adenovirus Indeed, a VAI-deleted mutant, dl331, showed selectivity for EBV- although Panc-1 has a mutant ras oncogene that could be expected positive cells with the therapeutic index being especially to inactivate PKR. These results are consistent with the poor cell advantageous for these cells as opposed to normal primary killing and viral replication observed in response to dl331 in these human cells. The data presented here show that this mutant cells. However, 72 hours after infection with dl331, viral replication in could be used to treat EBV-associated human tumors. Panc-1 cells approached 20% of that of wild-type adenovirus, VAI RNA–PKR-eIF-2a has been recognized as a major pathway comparable with the observation in some EBV-positive cells. This in host defense against viral infection and replication. Based on suggests that partial complementation occurred in the cells but there common functions exhibited by adenovirus VAI RNA and Ras for was very low cytotoxicity with dl331 in Panc-1 cells compared with

Figure 5. Histopathologic changes and viral protein expression in liver after adenovirus treatment. Livers were harvested at the same time points after treatment with different viruses and histopathologic changes and viral protein expression was examined as described in Materials and Methods. Top, representative of H&E staining (original magnification, Â200), 48 hours after i.v. injection of different viruses. Black arrows, cytopathic effects, showing cells with enlarged nuclei and inclusion bodies as well as eosinophilic cytoplasm. Bottom, representative of viral hexon protein staining (original magnification, Â200) at 48 hours after virus injection, with strong staining in Ad5-treated group. www.aacrjournals.org 1529 Cancer Res 2005; 65: (4). February 15, 2005

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2005 American Association for Cancer Research. Cancer Research the EBV-positive cells. The mechanism is not clear at this time. treatment alone was still low. It would therefore be desirable to Of note, in the normal human primary cells tested, dl331 showed develop other strategies that maintain selectivity but enhance good selectivity of viral replication compared with wild-type the efficacy of the adenovirus. Based on our findings (15, 55), adenovirus; however, no differences were observed in the level of deletion of the E1B 19K gene but retention of E3B seems phosphorylated PKR and phosphorylated eIF2a after dl331 and Ad5 promising in this regard. In addition, it is conceivable that treatments in these cells (data not shown). This suggests that VAI combination of adenovirus and conventional approach, such as RNA apparently has other functions that control viral replication in chemotherapy, radiotherapy, and immunotherapy, will improve these cells. Indeed, VAI RNA of adenovirus, like EBER-1, can activate antitumor efficacy. the 2-5 (A) synthetases, which also affect viral replication (20, 31). Lei Replication-selective, oncolytic adenoviruses have been evalu- et al. (49) reported that adenovirus VAI RNA may affect viral and ated extensively in epithelial tumors but relatively little in cellular gene expression by modulation of RNA editing by lymphoid malignancies mainly owing to the perceived resistance antagonizing the RNA-editing activity of the ADAP adenosine of the latter to adenovirus infection. However, in the present deaminase. Further investigation will be required to explore the study, we found that this resistance is not absolute and that, molecular mechanism for this. whereas most lines are refractory, adenovirus can infect some In the present study, one most interesting result is that dl331 B-cell lymphoma lines, such as the Burkitt’s lymphoma derived showed superior cell killing in vitro and antitumoral efficacy lines Raji and Jijoye, resulting in cytotoxicity to these cells. This is in vivo compared with wild-type adenovirus, in EBV-positive consistent with observations described elsewhere using primary cells, although the absolute level of viral replication of dl331 was chronic lymphocytic leukemia and B-cell lymphoma lines (56). lower than wild-type adenovirus. Our data indicate that EBV- Taken together, our findings suggest that oncolytic adenovirus is positive C666-1 cells still have a relatively high level of also a potential agent to treat at least a proportion of phosphorylated PKR after dl331 infection. PKR has been lymphomas. We found that the infectivity of Burkitt’s lymphoma reported to be indispensable for cell apoptosis by apoptotic lines is associated with the expression of adenovirus receptors, stimulators such as E2F-1 and tumor necrosis factor-a (50, 51), such as coxsackievirus adenovirus receptor and heparan sulfate and activation of PKR can induce the expression of Fas and glycosaminoglycans, but that genetic alterations also play an trigger apoptosis through the Fas-associated death domain important role in the cytotoxicity of adenoviruses in lymphoma protein/caspase-8 death signaling pathway (52, 53). Thus, it is cells (57); the mechanism behind the latter finding is not reasonable to postulate that higher PKR levels in dl331-infected understood at present. cells may promote adenovirus-induced cell death and this would In conclusion, our data suggest that a VAI-deleted adenovirus can explain the lower level of replication in these cells. In addition, induce replication and cell lysis selectively in EBV-positive tumor an increasing production of IFN-h in the absence of VAI RNA cells. The replication-selective, oncolytic adenovirus showed may also explain why dl331 showed superior cell killing and efficient tumor killing in vitro and in vivo for EBV-positive tumor antitumoral efficacy compared with wild-type adenovirus. VAI cells, and lower hepatotoxicity than wild-type adenovirus. It RNA also has similar functions to E3L of vaccinia virus. Deletion represents a potential therapeutic agent for targeting human EBV- of the E3L has been shown to activate IFN regulatory factor 3 associated tumors, an increasingly important group of malignant (IRF3) and increase IFN-h production in cells infected by diseases. vaccinia virus lacking this gene (54). Of note, dl331 was derived from dl327, which has a large deletion in the adenoviral E3 Acknowledgments region (25). This deletion may be another factor that affects the Received 8/27/2004; revised 11/10/2004; accepted 11/21/2004. antitumoral efficacy because we have found that E3B deletion of Grant support: Cancer Research United Kingdom. adenovirus can enhance potency compared with wild-type The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance adenovirus in vitro and in vivo in nude mice but not in with 18 U.S.C. Section 1734 solely to indicate this fact. immunocompetent mouse tumor models (15). We thank Profs. Bayar Thimmappaya, Lindsey Hutt-Fletcher, and Dorly P. Huang Although dl331 has superior antitumoral efficacy compared for the generous gifts of valuable materials; Gary Martin (Biological Resources Unit and the Central Cell Service of Cancer Research UK) for valuable support; and with wild-type adenoviruses in vivo, the proportion of cases Drs. Iain McNeish and Georges Vassaux for helpful insights and reading of the (2 of 7) showing complete regression of tumor after viral manuscript.

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Yaohe Wang, Shao-An Xue, Gunnel Hallden, et al.

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