Identification of the ENT1 Antagonists Dipyridamole and Dilazep As Amplifiers of Oncolytic Herpes Simplex Virus-1 Replication

Published OnlineFirst April 27, 2010; DOI: 10.1158/0008-5472.CAN-10-0155

Priority Report Cancer Research Identification of the ENT1 Antagonists Dipyridamole and Dilazep as Amplifiers of Oncolytic Herpes Simplex Virus-1 Replication

Brent J. Passer1, Tooba Cheema1, Bingsen Zhou3, Hiroaki Wakimoto1, Cecile Zaupa1, Mani Razmjoo1, Jason Sarte1, Shulin Wu2, Chin-lee Wu2, James W. Noah4, Qianjun Li4, John K. Buolamwini5, Yun Yen3, Samuel D. Rabkin1, and Robert L. Martuza1

Abstract Oncolytic herpes simplex virus-1 (oHSV) vectors selectively replicate in tumor cells, where they kill through oncolysis while sparing normal cells. One of the drawbacks of oHSV vectors is their limited replication and spread to neighboring cancer cells. Here, we report the outcome of a high-throughput chemical library screen to identify small-molecule compounds that augment the replication of oHSV G47Δ. Of the 2,640-screened bioactives, 6 compounds were identified and subsequently validated for enhanced G47Δ replication. Two of these compounds, dipyridamole and dilazep, interfered with nucleotide metabolism by potently and directly inhibiting the equilibrative nucleoside transporter-1 (ENT1). Replicative amplification promoted by dipyridamole and dilazep were dependent on HSV mutations in ICP6, the large subunit of ribonucleotide reductase. Our results indicate that ENT1 antagonists augment oHSV replication in tumor cells by increasing cellular ribonucleoside activity. Cancer Res; 70(10); 3890–5. ©2010 AACR.

Introduction Materials and Methods

Viral vectors genetically engineered for cancer cell– High-throughput screen. Piloting and primary screen restricted replication represent an attractive strategy for tu- was conducted at the Institute of Chemistry and Cell mor therapy because these viruses can replicate and spread Biology-Longwood core facility. Z′-factors were used to in situ, exhibiting oncolytic activity through direct cytopathic normalize for plate-to-plate variation (6). PC3 cells were effects (1, 2). We and others have previously shown that seeded in 384-well cell culture plates; the following day, appropriately selected pharmaceuticals can synergize with the compounds were added in duplicate and incubated oncolytic herpes simplex virus-1 (oHSV) to increase oncolytic for 6 hours before infection with G47Δ-GFP [multiplicity efficacy (3–5). To identify new agents and mechanisms that of infection (MOI), 0.05]. Forty-eight hours postinfection, would increase G47Δ replication in cancer cells, we under- PC3 cells were imaged on an automated Image-Xpress took an unbiased high-throughput screen of known bioactive inverted fluorescent microscope (Molecular Devices) molecules. We have identified dipyridamole and dilazep as using two wavelengths, 488 nm to detect G47Δ-GFP potent enhancers of G47Δ replication, revealing a previously infected cells and 350 nm for nuclear DNA bound by unidentified function for two well-characterized inhibitors of Hoechst-33342. the equilibrative nucleoside transporter-1 (ENT1). Viruses. G47Δ-GFP (G47Δ-BAC) contains a cytomegalo- virus promoter–driven enhanced green fluorescent protein (GFP) in place of lacZ in G47Δ (7). R3616 contains 1-kb γ34.5 Δ 1 2 deletions of both copies of (8). G47 was derived Authors' Affiliations: Departments of Neurosurgery and Pathology, α47 US11 Massachusetts General Hospital and Harvard Medical School, Boston, from G207 by deleting and the promoter (9). Massachusetts; 3Department of Clinical and Molecular Pharmacology, FΔ6isastrainF–derived recombinant with an ICP6- City of Hope National Medical Center, Duarte, California; 4Biochemistry lacZ and Molecular Biology, Southern Research Institute, Birmingham, inactivating insertion (10). Alabama; and 5Department of Pharmaceutical Sciences, University of Compounds. Dipyridamole, dialazep, nitrobenzylthioino- Tennessee Health Science Center, Memphis, Tennessee sine (NBMPR), zaprinast, uridine, thymidine, and adeno- Note: Supplementary data for this article are available at Cancer sine were purchased from Sigma. SB203580, GF109203X, Research Online (http://cancerres.aacrjournals.org/). EHNA, AG1517 (PD153035), and I-methyl-3-isobutyl- Corresponding Author: Brent J. Passer, Brain Tumor Research Center, xanthine were purchased from Calbiochem-Novabiochem. Neurosurgery Department, Massachusetts General Hospital, Simches Research Building CRPZN 3800, 185 Cambridge Street, Boston, MA Gene expression analysis. Reverse transcription-PCR 02114. Phone: 617-726-6798; Fax: 617-643-3422; E-mail: bpasser@ (RT-PCR) was used to verify human ENT1, RR1, RR2, and partners.org. GAPDH mRNA levels in tumor cells. For quantitative RT- doi: 10.1158/0008-5472.CAN-10-0155 PCR analysis, PCR was performed using the 7000 Real-Time ©2010 American Association for Cancer Research. PCR Sequence Detection System (Applied Biosystems).

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Figure 1. High-throughput screen of chemical amplifiers of G47Δ. A, scatter plot analysis of the 2,640 small-molecule bioactives tested that either increased (□), decreased (○), or had no effect▵ ( ) on viral replication or spread. B, validation of augmented G47Δ replication by dipyridamole (DP) or dilazep (DL) in PC3 cells. C, dipyridamole and dilazep augment viral replication in a panel of tumor cell lines. Virus titers are expressed as the mean pfu/mL ± SEM and represent one of three independent experiments. D, left, G47Δ-mediated DU145 or HCT-116 tumor cell killing is enhanced by dipyridamole or dilazep. * and **, statistical significance at P < 0.05 and P = 0.01, respectively. Right, fluorescent images depicting enhanced plaque formation by G47Δ-GFP as a function of treatment with either dipyridamole or dilazep.

Multistep growth assays. Tumor cells were pretreated consecutive days starting 2 days before the first intratumoral for 4 to 6 hours with a dose range of compounds fol- injection of G47Δ (2 × 106 pfu). lowed by virus infection at an MOI of 0.05. Forty-eight Organ culture assay. G47Δ titers were assessed in the hours after infection, virus titers [plaque forming units presence or absence of dipyridamole or dilazep in prostate (pfu)/mL] were determined by standard plaque assay on organ cultures as previously described (12). Vero cells. Statistical analysis. For cell susceptibility assays and Ribonucleotide reductase assay. Ribonucleotide reduc- in vivo efficacy studies, Student's t test (two-tailed) was used tase activity was measured using the CDP assay method as to analyze significance between two treatment groups using previously described (11). GraphPad Prism v.4. In vivo studies. Du145 cells (5 × 106) were implanted s.c. into the flanks of athymic male mice (National Cancer Results and Discussion Institute). Mice were given dipyridamole (dissolved in DMSO and diluted in 0.1 N HCl plus 0.9% NaCl) or dilazep (dissolved We screened 2,640 compounds of known bioactives de- in H2O and diluted in PBS) i.p. (40 mg/kg/injection) for 14 rived from three pharmacologically active libraries (NINDS,

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Biomol, and Prestwick1-collection). PC3 prostate cancer cells enhanced by dipyridamole and dilazep from viruses lacking were pretreated with compounds and subsequently infected ICP6 (G47Δ and FΔ6), whereas minimal changes were seen with G47Δ-expressing GFP at a MOI of 0.05 for an additional in transcripts from ICP6-intact strain F (Fig. 2B). 48 hours (Supplementary Fig. S1). Compounds that amplified Dipyridamole and dilazep are also known to function as G47Δ-GFP (as measured by GFP+ cells) at least 3 SD above phosphodiesterase (PDE) antagonists as well as protein the overall plate average were considered strong “hits.” kinase (PK) inhibitors (14, 15). Therefore, we tested represen- Scatter plot analysis showed that 15 (0.57%) of the library tative inhibitors of PDE and PK activity using virus yield compounds reflected potential “amplifiers” of viral spread assays and fluorescent imaging (Fig. 2C; Supplementary (Fig. 1A). Many of the small-molecule compounds that fit Fig. S4). Inhibitors of PDE (Zaprinast, IBMX, and EHNA) or these criteria were antimetabolites: two antifolates (pyri- PK (SB203580, GF109203x, and AG1517) had minimal effects methamine and methotrexate) and two fluoropyrimidines on increasing G47Δ replication relative to virus alone. Fur- [fluorodeoxyuridine (FdUrd) and carmofur; Table 1]. FdUrd thermore, increasing adenosine, uridine, or thymidine con- has been reported to enhance the spread of oncolytic G207 centrations had no effect. This is in contrast to the action (13), a HSV vector related to G47Δ; therefore, its identifica- of NBMPR, a potent ENT1 antagonist (16), which increased tion validated the effectiveness of the chemical library screen. G47Δ virus replication to a similar degree as dipyridamole or Dipyridamole and dilazep have been clinically used as vaso- dilazep (Fig. 2C). We next examined the effects on G47Δ rep- dilators and fall into another pharmacologic group, termed lication of a panel of dipyridamole analogues that have been ENT1 inhibitors (14). We focused our efforts on dipyridamole previously shown to exhibit a range of binding affinities to- and dilazep because they represent a class of compounds ward ENT1 based on structure-activity relationship studies that have not been studied in the context of virotherapy (17). A number of dipyridamole derivatives reported to exhib- and they could be readily translated to clinical trial. Multi- it strong binding affinities toward ENT1 (compounds 2, 4, 11, step growth curve assays (herein referred to as plaque assays) 15, 52, 58, and 64) also promoted notable increases in G47Δ were performed in PC3 cells to validate the amplifying- replication in PC3 cells (Supplementary Fig. S5). These data promoting activities of these compounds (Fig. 1B). Pretreat- show that dipyridamole and dilazep likely act on ENT1 to ment of PC3 cells with dipyridamole or dilazep resulted in a augment the replication of ICP6-deficient HSV vectors. dose-dependent increase in G47Δ production 48 hours Ribonucleotide reductase, which is composed of two sub- postinfection. Fluorescent imaging of G47Δ-GFP–infected units, RR1 and RR2, plays a critical role in the synthesis of PC3 cells showed that dipyridamole and dilazep increased DNA by reducing ribonucleotides to their corresponding GFP+ cell numbers (Fig. 1B). deoxyribonucleotides, thereby providing an essential reser- The effects of dipyridamole or dilazep on oHSV replication voir of precursors for DNA synthesis and repair (18). As di- were also tested in other human tumor cell lines (Fig. 1C). pyridamole, dilazep, and NBMPR inhibit nucleoside import/ HCT-116, Du145, and HT-1080 were particularly sensitive to- export through binding to ENT1 (16), we hypothesized that ward the effects of dipyridamole and dilazep on G47Δ repli- the actions of these compounds could possibly affect RR1 cation, whereas SK-Mel-2 and T98 cell lines (Supplementary and RR2 mRNAlevels.RT-PCRanalysisindicatedthatRR1 Fig. S2) were less sensitive and PANC-1 seemed to be resis- and RR2 mRNA levels were indistinguishable from dipyrida- tant. These cell lines expressed relatively similar levels of mole- or dilazep-treated PC3 cells compared with untreated ENT1 mRNA and protein (Supplementary Fig. S3). Dipyrida- controls (Supplementary Fig. S6). Next, we investigated mole and dilazep did not increase viral production of G47Δ whether treatment with either dipyridamole or dilazep in normal prostate epithelial cells (Fig. 2C). Cell cytotoxicity results in increased ribonucleotide reductase activity. assays showed that cell killing mediated by G47Δ was significantly increased by dipyridamole and dilazep (P <0.05). These data were further supported by fluorescence imaging Table 1. List of bioactives that significantly of 4′,6-diamidino-2-phenylindole–stained nuclei, which also increased G47D titers revealed that dipyridamole and dilazep enhanced tumor cell z killing mediated by G47Δ (Fig. 1D). Overall, these data show Class/name Mode of action -Score* that dipyridamole and dilazep potentiate G47Δ replication Antimetabolites and tumor cell killing. Methotrexate Inhibits folic acid metabolism 3.3 To determine whether one or more of the deletion muta- Pyrimethamine Inhibits folic acid synthesis 3.6 tions within G47Δ confers augmented viral replication by di- − Carmofur Inhibits thymidylate synthase 3.1 pyridamole or dilazep, we tested HSV-1 viruses FΔ6(ICP6 ), − − − Floxuridine Inhibits thymidylate synthase 4.5 R3616 (γ34.5 ), and G207 (γ34.5 , ICP6 ), and wild-type strain Vasodilators F in the presence or absence of dipyridamole or dilazep. † Dipyridamole Inhibits ENTs 3.6 Plaque assays consistently revealed that oHSVs lacking Dilazep Inhibits ENTs 4.0 ICP6, a gene that encodes the large subunit of ribonucleotide reductase, were associated with augmented viral replication *z-Score value indicates SD above the plate mean for each by dipyridamole and dilazep (Fig. 2A). Furthermore, quanti- readout value. tative RT-PCR showed that viral transcripts representing im- †Identified in all three libraries with z-score ≥3.0. mediate early (ICP4), early (UL23), or late (UL44) genes were

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Figure 2D shows that by 6 hours after treatment with dipyr- day 45, statistically significant differences in tumor weights idamole or dilazep, ribonucleotide reductase activity in- were also observed between untreated control and G47Δ creased by 30% and 36%, respectively, over baseline levels treatment groups, as well as for G47Δ alone and in combina- in PC3 cells. By contrast, ribonucleotide reductase activity in tion with dipyridamole or dilazep (Fig. 3B). Recently, we PANC-1 cells was not increased with treatment of either com- reported on the use of prostate organ cultures derived from pound and, moreover, was minimally detected (Fig. 2D). This radical prostatectomies to assess oHSV target specificity and observation strongly parallels the data derived in Fig. 1B, illus- replication competence (13). We exploited this system to trating that neither drug augmented G47Δ in PANC-1 cells. address whether dipyridamole and dilazep enhances G47Δ Last, we evaluated the in vivo antitumor efficacy of G47Δ replication in primary human prostate cancer specimens. in combination with either dipyridamole or dilazep in s.c. These results show that treatment of organ cultures with Du145 tumors. By day 45 after tumor implantation, a statis- dipyridamole or dilazep over a 3-day period increased G47Δ tically significant decrease in tumor volume was observed be- titers by 2- to 4-fold over the no-treatment control (Fig. 3C). tween control (882 ± 86 mm3, n = 7) and all of the treatment Using an unbiased chemical library screen, we have iden- groups (Fig. 3A). As compared with mock, treatment with tified a novel application for the ENT1 antagonists, dipyrida- G47Δ resulted in a notable reduction in tumor size (625 ± mole and dilazep (and NBMPR), namely to “amplify” the 76 mm3; n = 7), whereas the combination of G47Δ with di- replication of G47Δ in cancer cells. Our results reveal that pyridamole (426 ± 37 mm3; n = 6) or dilazep (377 ± 56 mm3; these ENT1 antagonists augment HSV vectors specifically n = 6) resulted in a statistically significant tumor regression lacking ICP6, which is the viral homologue of the human compared with G47Δ alone. Neither compound by itself RR1 gene, and that ENT1 antagonists may predispose some reduced or delayed tumor growth (data not shown). At cancer cells to augmented oHSV replication by increasing

Figure 2. HSV ICP6-negative mutants are responsive to the amplifying-activities of dipyridamole and dilazep. A, effects of dipyridamole (red columns) and dilazep (blue columns) on wild-type HSV-1 or oHSVs carrying deletion mutations present in G47Δ. Values are the average ± SEM and represent three independent experiments. B, quantitative RT-PCR analysis of ICP4, UL23,orUL44 viral transcript levels in PC3 cells untreated or treated with dipyridamole or dilazep and infected with G47Δ (left), FΔ6 (middle), or strain F (right) for 24 or 48 hours. GAPDH was used to normalize for mRNA levels. C, pharmacologic inhibitors of PDE, PK, or ENT1 and nucleosides (ns) were assessed for their ability to promote increased G47Δ replication by plaque assays. D, ribonucleotide reductase activity is transiently elevated by dipyridamole and dilazep in PC3 (left) but not PANC-1 (right) cells. Results are expressed as a percentage of the control value [relative ribonucleotide reductase (RR) activity].

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Figure 3. Dipyridamole and dilazep enhances the in vivo antitumor efficacy of G47Δ. A, assessment of Du145 tumor growth. *, P < 0.05 for mock (n =7) or G47Δ only (n = 7) versus G47Δ + dipyridamole (n = 6) or + dilazep (n = 6) at day 28; **, P < 0.05 for G47Δ only versus G47Δ in combination with either dipyridamole or dilazep at days 32 and 36; ***, P < 0.05 for mock versus G47Δ only; G47Δ versus G47Δ in combination with either dipyridamole or dilazep at day 45. †, P < 0.01 for mock versus G47Δ in combination with either dipyridamole or dilazep at days 32, 36, and 45. B, Du145 tumor weights were assessed for the indicated treatment groups 45 days postimplantation. P values are indicated. C, evaluation of dipyridamole and dilazep augmented G47Δ replication in prostate cancer surgical specimens. Each color dot indicates a different prostate surgical specimen (n = 5).

cellular ribonucleotide reductase activity. From a translational Acknowledgments perspective, dipyridamole has been used to potentiate the effects of chemotherapeutic agents in phase II clinical trials in We thank the screening staff of the Institute of Chemistry and Cell Biology- Longwood (Harvard Medical School) for their technical support: Caroline solid tumors (19). Although the therapeutic dosing for these Shamu, Stewart Rudnicki, Katrina Rudnicki, Melody Tsui, David Fletcher, studies was less than what was used in the present study, dipyr- DavidWrobel,andSeanJohnston;Dr.DonaldCoen(HarvardMedical idamole is well tolerated at higher doses with minimal adverse School) for his helpful advice on setting up the conditions for the reactions in animals (20). These data suggest that the combina- high-throughput screen; and Melissa Marinelli for laboratory assistance. Wild-type strain F and its derivative R3616 were both obtained from tion of dipyridamole and oHSV represents a clinically relevant Dr. B. Roizman (The University of Chicago, Chicago, IL). treatment paradigm and should merit consideration for clinical studies. In this context, blocking nucleoside import and/or ex- Grant Support port via ENT1 antagonists represents a new strategy toward enhancing the efficacy of oHSV vectors for cancer therapy. Grants R01 CA10139 and NS032677 (R.L. Martuza), R03 MH083258 (B.J. Passer), and P30 NS045776 (S.D. Rabkin) for the real-time PCR core. The costs of publication of this article were defrayed in part by the payment Disclosure of Potential Conflicts of Interest of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. R.L. Martuza and S.D. Rabkin are consultants to MediGene Ag, which has a license from Georgetown University for G207. The other authors disclosed no Received 01/13/2010; revised 03/12/2010; accepted 03/16/2010; published potential conflicts of interest. OnlineFirst 04/27/2010.

References 1. Martuza RL, Malick A, Markert JM, Ruffner KL, Coen DM. Experi- 6. Zhang JH, Chung TD, Oldenburg KR. A simple statistical parameter mental therapy of human glioma by means of a genetically engi- for use in evaluation and validation of high throughput screening as- neered virus mutant. Science 1991;252:854–6. says. J Biomol Screen 1999;4:67–73. 2. Liu TC, Hwang TH, Bell JC, Kirn DH. Development of targeted onco- 7. Fukuhara H, Ino Y, Kuroda T, Martuza RL, Todo T. Triple gene-deleted lytic virotherapeutics through translational research. Expert Opin Biol oncolytic herpes simplex virus vector double-armed with interleukin Ther 2008;9:1381–91. 18 and soluble B7–1 constructed by bacterial artificial chromosome- 3. Passer BJ, Castelo-Branco P, Buhrman JS, et al. Oncolytic herpes mediated system. Cancer Res 2005;65:10663–8. simplex virus vectors and taxanes synergize to promote killing of 8. Chou J, Kern ER, Whitley RJ, Roizman B. Mapping of herpes simplex prostate cancer cells. Cancer Gene Ther 2009;16:551–60. virus-1 neurovirulence to γ134.5, a gene nonessential for growth in 4. Bennett JJ, Adusumilli P, Petrowsky H, et al. Up-regulation of culture. Science 1990;250:1262–6. GADD34 mediates the synergistic anticancer activity of mitomycin 9. Todo T, Martuza RL, Rabkin SD, Johnson PA. Oncolytic herpes Candaγ134.5 deleted oncolytic herpes virus (G207). J FASEB simplex virus vector with enhanced MHC class I presentation 2004;18:1001–3. and tumor cell killing. Proc Natl Acad Sci U S A 2001;98: 5. Lin SF, Gao SP, Price DL, et al. Synergy of a herpes oncolytic virus 6396–401. and paclitaxel for anaplastic thyroid cancer. Clin Cancer Res 2008; 10. Wakimoto H, Kesari S, Farrell CJ, et al. Human glioblastoma-derived 14:151928. cancer stem cells: establishment of invasive glioma models and

3894 Cancer Res; 70(10) May 15, 2010 Cancer Research

ENT1 Antagonists Augment Oncolytic HSV-1 Replication

treatment with oncolytic herpes simplex virus vectors. Cancer Res 16. Griffiths M, Beaumont N, Yao SY, et al. Cloning of a human nucleo- 2009;69:3472–81. side transporter implicated in the cellular uptake of adenosine and 11. Zhou B, Liu X, Mo X, et al. The human ribonucleotide reductase sub- chemotherapeutic drugs. Nat Med 1997;3:89–93. unit hRRM2 complements p53R2 in response to UV-induced DNA 17. Wenwei WL, Buolamwini JK. Synthesis, flow cytometric evaluation, repair in cells with mutant p53. Cancer Res 2003;63:6383–94. and identification of highly potent dipyridamole analogues as equili- 12. Passer BJ, Wu CL, Wu S, Rabkin SD, Martuza RL. Analysis of genet- brative nucleoside transporter 1 inhibitors. J Med Chem 2007;50: ically engineered oncolytic herpes simplex viruses in human prostate 3906–20. cancer organotypic cultures. Gene Ther 2009;12:1477–82. 18. Elledge SJ, Zhou Z, Allen JB. Ribonucleotide reductase: regulation, 13. Petrowsky H, Roberts GD, Kooby DA, et al. Functional interaction regulation, regulation. Trends Biol Sci 1992;17:119–23. between fluorodeoxyuridine-induced cellular alterations and replica- 19. Isacoff WH, Bendetti JK, Barstis JJ, Jazieh AR, Macdonald JS, Philip tion of a ribonucleotide reductase-negative herpes simplex virus. PA. Phase II trial of infusional fluorouracil, leucovorin, mitomycin, and J Virol 2001;75:7050–8. dipyridamole in locally advanced unresectable pancreatic adenocar- 14. King AE, Ackley MA, Cass CE, Young JD, Baldwin SA. Nucleoside cinoma: SWOG S9700. J Clin Oncol 2007;25:1665–9. transporters: from scavengers to novel therapeutic targets. Trends 20. Venkatesh PK, Pattillo CB, Branch B, et al. Dipyridamole en- Pharmacol Sci 2006;27:416–25. hances ischaemia-induced arteriogenesis through an endocrine 15. Kim HH, Liao JK. Translational therapeutics of dipyridamole. Arter- nitrite/nitric oxide-dependent pathway. Cardiovasc Res 2010;85: ioscler Thromb Vasc Biol 2008;28:s39–42. 661–70.

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Identification of the ENT1 Antagonists Dipyridamole and Dilazep as Amplifiers of Oncolytic Herpes Simplex Virus-1 Replication

Brent J. Passer, Tooba Cheema, Bingsen Zhou, et al.

Cancer Res 2010;70:3890-3895. Published OnlineFirst April 27, 2010.

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