Combination Therapy with Reovirus and ATM Inhibitor Enhances Cell Death and Virus Replication in Canine Melanoma

Journal Pre-proof Combination Therapy with Reovirus and ATM Inhibitor Enhances Cell Death and Virus Replication in Canine Melanoma Masaya Igase, Shusaku Shibutani, Yosuke Kurogouchi, Noriyuki Fujiki, Chung Chew Hwang, Matt Coffey, Shunsuke Noguchi, Yuki Nemoto, Takuya Mizuno PII:

S2372-7705(19)30074-9

DOI:

https://doi.org/10.1016/j.omto.2019.08.003

Reference:

OMTO 132

To appear in:

Molecular Therapy: Oncolytics

Received Date: 13 November 2018 Accepted Date: 16 August 2019

Please cite this article as: Igase M, Shibutani S, Kurogouchi Y, Fujiki N, Hwang CC, Coffey M, Noguchi S, Nemoto Y, Mizuno T, Combination Therapy with Reovirus and ATM Inhibitor Enhances Cell Death and Virus Replication in Canine Melanoma, Molecular Therapy: Oncolytics (2019), doi: https:// doi.org/10.1016/j.omto.2019.08.003. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019

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Original article

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Combination Therapy with Reovirus and ATM Inhibitor Enhances Cell Death and Virus

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Replication in Canine Melanoma

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Masaya Igase1, Shusaku Shibutani2, Yosuke Kurogouchi3, Noriyuki Fujiki3, Chung

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Chew Hwang1, Matt Coffey4, Shunsuke Noguchi5, Yuki Nemoto1,3 and Takuya

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Mizuno1,3

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of Veterinary Science, Yamaguchi University, Yamaguchi, Japan

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University, Yamaguchi, Japan

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Medicine, Yamaguchi University, Yamaguchi, Japan

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4

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Sciences, Osaka Prefecture University, Osaka, Japan

Laboratory of Molecular Diagnostics and Therapeutics, The United Graduate School

Laboratory of Veterinary Hygiene, Joint Faculty of Veterinary Medicine, Yamaguchi

Laboratory of Molecular Diagnostics and Therapeutics, Joint Faculty of Veterinary

Oncolytics Biotech Inc., Calgary, Alberta, Canada Laboratory of Veterinary Radiology, Graduate School of Life and Environmental

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Correspondence should be addressed to T. M. ([email protected]).

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Takuya Mizuno, D.V.M., Ph.D., Professor

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Laboratory of Molecular Diagnostics and Therapeutics, Joint Faculty of Veterinary

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Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan

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Tel & Fax: +81-83-933-5894. E-mail: [email protected]

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Short title: Combined therapy with reovirus and ATM inhibitor

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27

ABSTRACT

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Oncolytic virotherapy using reovirus is a promising new anti-cancer treatment with

29

potential for use in humans and dogs. Because reovirus monotherapy shows limited

30

efficacy in human and canine cancer patients, the clinical development of a combination

31

therapy is necessary. To identify candidate components of such a combination, we

32

screened a 285-compound drug library for those that enhanced reovirus cytotoxicity in a

33

canine melanoma cell line. Here, we show that exposure to an inhibitor of the ataxia

34

telangiectasia mutated protein (ATM) enhances the oncolytic potential of reovirus in 5

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of 6 tested canine melanoma cell lines. Specifically, the ATM inhibitor potentiated

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reovirus replication in cancer cells along with promoting the lysosomal activity,

37

resulting in an increased proportion of caspase-dependent apoptosis and cell cycle arrest

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at G2/M compared to those observed with reovirus alone. Overall, our study suggests

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that the combination of reovirus and the ATM inhibitor may be an attractive option in

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cancer therapy.

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INTRODUCTION

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Canine melanoma is among the most aggressive cancers, exhibiting the ability to

43

readily metastasize to other organs such as the lung, lymph nodes, and liver. Naturally

44

occurring canine melanoma is considered a preclinical model of human melanoma

45

Therefore, new therapeutic approaches with efficacy against canine cancers have

46

potential for application in the treatment of human cancers.

1,2

.

47

Oncolytic virotherapy has been studied extensively in human cancers. In 2015, a

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gene-modified herpes simplex virus-1, talimogene laherparepvec (T-VEC), was

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approved by the U.S. Food and Drug Administration (FDA) as a treatment for

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melanoma in patients with unresectable lesions of the skin and lymph nodes 3.

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Oncolytic viruses are considered attractive treatment options because such reagents are

52

known to activate anti-tumor innate and/or adaptive immune effector cells

53

multiple clinical trials have examined the efficacy against human cancers of oncolytic

54

viruses in combination with chemotherapeutic drugs, radiation, and small molecule

55

inhibitors

56

therapy with oncolytic viruses for treating human cancers.

4-7

. Indeed,

8-10

. There still needs to identify new candidate drugs for use in combination

57

In the veterinary field, there have been only a limited number of reports regarding

58

the use of oncolytic viruses against canine cancer, but several pre-clinical trials

59

suggesting possible application in human medicine have been reported recently

60

Reovirus is a double-stranded RNA virus with low pathogenicity in humans. Our

61

laboratory has previously reported that REOLYSIN®, a GMP-grade non-modified

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reovirus serotype 3 Dearing strain reovirus, exhibits efficacy in the treatment of canine

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cancers including mast cell tumors, lymphoma, mammary gland tumors, and melanoma

64

when tested in vitro and in vivo in mouse xenograft models

4

15-18

11-14

.

. We also have carried

65

out pilot clinical studies using REOLYSIN® to treat 19 dogs with spontaneously

66

occurring tumors, demonstrating that reovirus therapy was safe and well-tolerated in

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tumor-bearing dogs 19. Although decreased tumor volume was observed in some of the

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reovirus-treated dogs, complete tumor regression was not seen in any of the enrolled

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dogs. REOLYSIN® has been used in multiple clinical trials in human cancer patients,

70

primarily in combination with chemotherapeutic agents, with the intent of enhancing the

71

efficacy of oncolytic therapy

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enhance reovirus oncolysis for the treatment of dogs and humans with tumors.

10,20

. In general, other therapeutic options are needed to

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Therefore, the objective of our current study was to develop a new combination

74

approach for oncolytic virotherapy using reovirus in canine cancers. By screening a

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large number of small molecule inhibitors in combination with reovirus, we

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successfully identified a novel inhibitor of the ataxia telangiectasia mutated protein

77

(ATM). Here, we report the first evidence to our knowledge that the cytotoxicity of

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reovirus is potentiated by inhibition of ATM in canine melanoma cell lines. We also

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show that ATM inhibition increases reovirus replication, the endosomal acidification

80

and the cathepsin B activity. Notably, reovirus was able to induce the phosphorylation

81

of ATM without inducing DNA damage. Thus, our study demonstrated that the

82

combination of reovirus and an ATM inhibitor may be an attractive option in cancer

83

therapy.

5

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RESULTS

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The combination of an ATM inhibitor and reovirus enhances anti-tumor effects in

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cell lines

87

To identify drugs that enhance reovirus-induced anti-tumor effects, we screened a

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285-compound signaling pathway inhibitor library for activity in the CMeC1 canine

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melanoma cell line (Figure S1). This screen revealed that the ATM inhibitor KU55933

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showed no effect on cell proliferation by itself, but potentiated the cytotoxicity of

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reovirus when used in combination with reovirus. Moreover, the combination of

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KU55933 and reovirus yielded dose-dependent suppression of CMeC1 cell growth

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(Figure S2). For subsequent experiments, a higher specificity inhibitor of the ATM,

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KU60019 21 was used in place of KU55933.

95

To confirm if KU60019 enhances reovirus-induced anti-tumor effects in other

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types of canine melanoma cell lines, we also examined in vitro cell survival using

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another 5 canine melanoma cell lines (Figure 1). KU60019 combined with reovirus

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(multiplicity of infection (MOI) 100) significantly suppressed cell proliferation in

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CMeC1, KMeC, CMM12, LMeC, and CMM10 cell lines, as shown with KU55933.

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These results indicated that the combination of KU60019 and reovirus yielded

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significant cell growth inhibition compared to compound or virus alone in 5 of 6 tested

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canine melanoma cell lines excepting CMGD2. These data provided evidence that

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combination treatment with reovirus and ATM inhibitor potentiated anti-tumor activity

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in canine melanomas.

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To confirm the specificity of the ATM inhibitor, the ATM-encoding gene was

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knocked out in several canine melanoma cell lines (CMeC1, KMeC, CMM10, and

107

CMM12) using a CRISPR/Cas9-based system (Figure 2A). At 48 hr after infection with

6

108

reovirus, ATM knock-out cells showed greater inhibition of cell viability than did the

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respective ATM+ cells (Figure 2B). Whereas the ATM knock-out did not influence on the

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cell growth in CMeC1 and CMM10 compared to ATM+ (wild type) cells, but the growth

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of KMeC and CMM12 were suppressed to some extent (Figure S3). These results

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supported that inhibition of ATM sensitizes canine melanoma cell lines to

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reovirus-induced cytotoxicity.

114 115

KU60019 enhances reovirus-induced apoptosis and cell cycle arrest

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Next, we assessed the potential mechanism by which the combination of KU60019

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and reovirus induces anti-tumor effects. In addition to cell proliferation assay, cell

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viability was examined in 4 of these cell lines (CMeC1, KMeC, LMeC, and CMGD2)

119

by means of a trypan blue exclusion test (Figure 3A). Compared to reovirus alone, the

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combination of KU60019 and reovirus strongly potentiated cell death in 3 of the 4

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canine melanoma cell lines, excepting CMGD2. At the earlier time point (24 hr),

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cleaved caspase-3 accumulated to higher levels in CMeC1 cells subjected to the

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combination treatment than in the same cell line treated with reovirus alone (Figure 3B).

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As expected, the percentage of sub-G1 cells increased from 24 hr to 48 hr in CMeC1

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cells subjected to the combination treatment compared to the percentage in cells treated

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with reovirus alone (Figure 3C). Notably, pre-treatment of CMeC1 and KMeC with 50

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µM Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone,

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an irreversible caspase inhibitor), before exposure to the combination of reovirus and

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KU60019, resulted in the complete inhibition of reovirus-induced cell death, as assessed

130

by the trypan blue exclusion test (Figure 3D). However, in a cell proliferation assay, the

131

combination treatment was not sufficient to completely abrogate cell growth in either of

7

132

these cell lines (Figure 3E). Therefore, we inferred that the combination of reovirus and

133

KU60019 also affects cell cycle progression in canine melanoma cells.

134

Consistent with previous studies showing that reovirus infection results in cell 22-26

135

cycle arrest at G2/ M in several cancer cell lines

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reovirus alone induced cell cycle arrest at G2/ M in CMeC1. Moreover, treatment of

137

CMeC1 with the combination of KU60019 and reovirus resulted in strong cell cycle

138

arrest at G2/ M and a reduction of the G1 interval (Figure 4A). In a previous report, the

139

viral nonstructural protein sigma 1s regulated the activity of CDK1 (the G2/ M

140

cyclin-dependent kinase; cdc2), which was required for cell cycle arrest at G2/ M 23. We

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sought to extend that analysis by using immunoblotting to test treated cells for their

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levels of CDK1 and p21 (the cyclin-dependent kinase inhibitor) (Figure 4B). After 24 hr,

143

cells treated with the combination of KU60019 and reovirus accumulated significantly

144

lower levels of CDK1 than cells treated with either reagent alone. In contrast, the 24-hr

145

levels of p21 did not differ significantly among cells treated with the combination or

146

with KU60019 or reovirus alone. Together, these results indicated that the treatment of

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canine melanoma cells with the combination of KU60019 and reovirus induces both

148

caspase-dependent apoptosis and cell cycle arrest at G2/M.

, we observed that treatment with

149 150

KU60019 increases reovirus replication and progeny virus number

151

Next, we assessed the correlation between reovirus replication and anti-tumor

152

effect in canine melanoma cell lines. Western blotting detected reovirus proteins in

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reovirus-infected CMeC1 and KMeC cells, and further revealed that combination

154

treatment (i.e., reovirus infection in the presence of KU60019) yielded increased levels

155

of these proteins at each time point (Figure S4). This analysis was extended by

8

156

quantifying the progeny virus in the supernatant using the TCID50 assay. In 3 of 4 tested

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canine melanoma cell lines (CMeC1, KMeC, and CMGD2, but not in LMeC), the

158

combination treatment yielded apparent increases in the number of progeny viruses

159

compared to the input titer (Figure 5A). There was no statistical significance between

160

reovirus alone and combination of KU60019 because the fold increase of virus titer was

161

modest. To prove that viral replication is involved in the cell death effect of the

162

combination of reovirus and KU60019, we used western blotting to assess the

163

accumulation of reovirus structural proteins and cleavage of caspase-3 in CMeC1

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subjected to the combination treatment in the presence of ribavirin, an anti-viral reagent

165

27

166

caspase-3 cleavage (Figure 5B).

. Notably, ribavirin treatment completely suppressed both reovirus replication and

167 168

The combination of KU60019 and reovirus dose not enhance reovirus entry

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To investigate the biological roles and functions of ATM in the reovirus replication

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cycle, each step of the replication cycle was analyzed during the treatment with reovirus

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and ATM inhibitor. The upregulation of JAM-A, the reovirus receptor, might be

172

associated with sensitivity to virus-induced cell death 28,29. The surface expression level

173

of JAM-A was stable on cells treated with KU60019 for 1 hr and 24 hr by flow

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cytometric analysis (Figure S5). This data indicated that KU60019 did not influence the

175

step of virus entry.

176

In addition to test whether the effect of ATM inhibition was mediated via the

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proteolytic step of reovirus replication, we examined the enhancement of viral protein

178

expressions using protease-stripped reovirus virions (infectious subviral particles,

179

ISVPs), which are capable of infecting cells lacking virus receptors and cells that have

9

30-32

180

lost their ability to un-coat incoming virions

181

treatment of reovirus and KU60019 enhanced the expression of reoviral proteins at 12

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and 24 hr compared to cells exposed to reovirus alone. In contrast, subsequent western

183

blotting (Figure 5D) demonstrated that KU60019 showed no effect on reoviral protein

184

expressions after addition of ISVPs at early time points (at 6 hr and 12 hr), indicating

185

that KU60019 influences an event before viral disassembly. However, at 24 hr, reovirus

186

protein levels were elevated in CMeC1 cells treated with the combination of KU60019

187

and ISVPs; at 48 hr, the combination treatment yielded significant potentiation of cell

188

growth inhibition compared to either component alone (Figure S6), in parallel to the

189

results of Figure 1. We infer that this delayed effect (at 24 hr and after) reflects the

190

production of and infection by intact virus after one replication cycle (approximately 18

191

hr

192

treatment. These results suggested that outer capsid proteins and the ATM inhibition

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during the reovirus life-cycle might trigger the enhancement of virus replication.

33

. As shown in Figure 5C, combination

), given that the production of intact virus was potentiated by the combination

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Reovirus infection induces the phosphorylation of ATM without phosphorylation of

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DNA damage-related proteins

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Infection by some viruses has been shown to induce phosphorylation of ATM, 34-36

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resulting in the induction of DNA damage

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activity to replication of mammalian reovirus remains unclear. At an early stage of

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infection (12 hr), reovirus induced phosphorylation of ATM in cell line CMeC1, but this

201

modification was abrogated by KU60019 (Figure 6). In addition, we examined whether

202

reovirus induced DNA damage in cancer cells by determining the phosphorylation state

203

of p53, that is known to be regulated by ATM and are known to detect DNA damage 37.

10

; however, the mechanism linking ATM

204

Notably, reovirus did not induce phosphorylation of p53 in a canine melanoma cell line

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(Figures S7). This observation suggested that reovirus infection leads to increases in the

206

phosphorylation of ATM in cancer cells without associated DNA damage, leading to the

207

enhancement of reovirus replication.

208 209

Endosomal acidification and protease activity by the combination of KU60019 and

210

reovirus

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For early phase of infection, disassembly of the reovirus particle in the endosome

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is required for the replication cycle following internalization of the virus and depends

213

on acidification of the endosome 38,39. To analyze the effect of the ATM inhibitor on the

214

pH of the endosome, we used LysoTracker-Red, a dye which exhibits increased

215

fluorescence at the low pH typical of endosomes, lysosomes or authophagosomes

216

(Figure 7A and 7B). Although the fluorescence intensity of LysoTracker-Red was not

217

significantly altered compared to mock-treated cells following treatment with either

218

reovirus or KU60019 alone, fluorescence was significantly increased with the

219

combination treatment (Figure 7B). Confocal microscopy confirmed this result,

220

showing that the number of LysoTracker-positive dots was elevated in cells treated with

221

combination of reovirus and KU60019 (Figure 7B). These data suggested that the

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combination treatment potentiated the acidification of endosomes/lysosomes.

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The endosomal acidification modulates the activity of some proteases such as

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cathepsins 39-41. Therefore, we evaluated the cathepsin B activity in CMeC1 cells treated

225

as in the above experiments. As unexpected, the cathepsin B activity in cells treated

226

with KU60019 for 1 hr was little difference as compared to combination treatment of

227

reovirus (data not shown). Cells exposed to reovirus alone for 24 hr incubation

11

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exhibited nominal decreases in cathepsin B activity, although the effect fell short of

229

significance (p = 0.06 compared to mock-treated cells; Figure 7C). However,

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combination treatment restored the level of cathepsin B activity to a value similar to that

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seen with mock treatment. Even though the effect of the simultaneous treatment with

232

reovirus and KU60019 on cathepsin B activity remains unclear, these results implied

233

that the effect of KU60019 treatment is mediated by enhanced the lysosomal activity in

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the late phase of infection. Thus, combination treatment potentiated the decreased pH in

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endosomes and lysosomes, followed by activating proteases, but these effects did not

236

commit to enhance viral proteolysis during virus entry step of first viral infection.

12

237

DISCUSSION

238

In this study, we screened a drug library to identify new drugs that enhance

239

reovirus cytotoxicity. As a result, we identified an ATM inhibitor. Previous reports

240

demonstrated that many kinds of anti-cancer drugs have synergistic anti-tumor effects

241

on cancer cells when combined with reovirus

242

represents the first report to our knowledge that an ATM inhibitor can be used as a

243

combination drug with reovirus treatment. Based on our observations, the combinatorial

244

effects of reovirus and the ATM inhibitor resulted in reovirus-related apoptosis and cell

245

cycle arrest at G2/M in most canine melanoma cell lines, with the exception of CMGD2

246

(a canine melanoma line). The specificity of the ATM inhibitor in our study was

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supported by a cell survival assay that treated ATM knock-out canine melanoma cell

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lines with reovirus. Canine melanoma knock-out cell lines exhibited higher

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susceptibility to reovirus compared to the isogenic wild-type (ATM+) cell lines.

250

However, the cell growth inhibition of reovirus combined with KU60019 were higher

251

than that of reovirus with ATM knock-out. We hypothesize that KU60019 inhibits not

252

only ATM but also other kinases (members of the phosphatidylinositol 3-kinase

253

(PI3K)-related kinase (PIKK) family))

254

high-efficiency virus replication and cytotoxicity.

6,26,42-45

. However, the present work

21

, in a dose-dependent manner, leading to

255

We further examined the effect of the combination treatment on the ATM

256

phosphorylation that is associated with reovirus replication in cancer cells. These

257

experiments were inspired by observations of the effects of human herpes simplex

258

virus-1 (HSV-1), hepatitis C virus and human immunodeficiency virus-1 infection in

259

other cell lines

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supports viral replication, such that the inhibition of ATM negatively regulates

46-49

. For those viruses, ATM activation along with DNA damage

13

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replication. In contrast, it was unknown whether ATM activation is associated with

262

mammalian reovirus replication. We are aware of only one relevant report, which

263

demonstrated that two ATM-deficient cell lines (L3 lymphoblastoid cells and Granta

264

mantle cell lymphoma cells) were more susceptible to reovirus than were other cell lines

265

50

266

deficiency was not proven in that study. Therefore, in the present work, we identified a

267

putative mechanism for the potentiation of reoviral oncolysis by the normal status and

268

knock-out of ATM. Interestingly, p53 phosphorylation, a marker of DNA damage, was

269

not observed in canine melanoma cells even though reovirus infection might trigger

270

unknown nuclear damage followed by activation of ATM. To confirm the biological

271

roles and functions of ATM in the reovirus replication cycle, each step of the replication

272

cycle was analyzed. We found that inhibition of ATM enhanced the endosomal

273

acidification and re-activation of protease only in late phase of reovirus infection.

274

Previous work has shown that endosomal acidification and protease activity are

275

essential for reovirus replication in early time points

276

another mechanism. Reoviridae family members including mammalian reovirus

277

facilitate the induction of autophagy and its autophagy elevates the virus replication 52-55.

278

To assess the effect of combination treatment of KU60019 and reovirus on autophagy,

279

we have examined the expression levels of the autophagy marker LC3B-II (the lipidated

280

form of LC3B) in CMeC1 and KMeC by the western blotting (data not shown). As a

281

result, the accumulation of LC3B-II was observed in canine melanoma cells treated with

282

reovirus alone at 24-hr and 48-hr, and then treatment of reovirus with KU60019

283

increased the LC3B-II expression. Although the overall mechanism of virus replication

284

induced by ATM inhibition remains unclear, these data suggested that, at late phase of

. However, the relationship between reovirus replication, oncolysis, and ATM

14

31,39,51

, therefore there might be

285

infection, combination of reovirus and KU60019 facilitates the induction of autophagy

286

(ie: the upregulation of cathepsin B activity, the acidification of ensdosomes/lysosomes

287

and the LC3B-II accumulation), which might be associated with enhancement of virus

288

replication.

289

Compared to KU55933 (the library-identified compound), KU60019 has greater

290

specificity and is considered a much improved ATM kinase inhibitor. However, neither

291

KU55933 or KU60019 are suitable for in vivo studies, given the low aqueous solubility

292

and low bioavailability of these compounds

293

recently reported

294

for oral dosing; this compound is the subject of an ongoing phase I clinical trial being

295

performed by AstraZeneca. We hope to evaluate the combination of reovirus and

296

AZD0156 in xenograft mice harboring canine melanoma cell lines.

57

56-58

. The ATM inhibitor AZD0156 was

and shown to exhibit superior pharmacokinetics with the potential

297

Taken together, our results suggested that the ATM inhibitor KU60019 enhances

298

reovirus-induced anti-tumor effects in canine cancers (Figure 8). This synergy may

299

provide a route to resolving the limited efficacy of reovirus treatment of cancers.

300

Although the mechanism linking ATM activity, endosomal acidification and reovirus

301

replication remains unknown, we confirmed that treatment with the ATM inhibitor

302

up-regulated viral replication, resulting in increased the cell death. Given that the

303

incidence of adverse events may be increased in combination treatment, in vivo

304

experiments (e.g., mouse xenograft studies) will be needed to test this combination

305

therapy. Nonetheless, these findings support further investigation of this combination

306

therapy for the treatment of canine cancer patients.

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307

MATERIALS AND METHODS

308

Cell, reovirus, and inhibitors Seven canine malignant melanoma cell lines (CMeC1, KMeC, LMeC 59, CMGD2

309 310

60

311

(CMeC1, KMeC, LMeC, CMM10 and CMM12) were kindly provided by Dr. T.

312

Nakagawa (The University of Tokyo, Tokyo, Japan). CMGD2 was kindly provided by

313

Dr. J. Modiano (University of Minnesota, Minneapolis, MN). A mouse L929

314

fibroblastic cell line (used for the titration of viral progeny) was obtained from the Cell

315

Resource Center for Biomedical Research (Institute of Development, Aging and Cancer,

316

Tohoku University, Sendai, Japan). All cell lines were grown in R10 complete medium

317

(RPMI1640 supplemented with 10% Fetal Bovine Serum (FBS), 100 U/ml penicillin,

318

100 µg/ml streptomycin, and 55 µM 2-mercaptoethanol) and maintained at 37 °C in a

319

humidified 5 % CO2 incubator.

, CMM10, and CMM12

61

) were used in this study. Five of the canine cell lines

320

The Dearing strain of reovirus serotype 3 (Reolysin®; GMP-grade reovirus) was

321

obtained from Oncolytics Biotech, Inc. (Calgary, Canada). Infectious subviral particles

322

(ISVPs) were produced just before use, and were generated according to the protocol of

323

Alain et al. (2006)

324

digested with chymotrypsin-HCl (2 µg; Roche Diagnostics K.K., Tokyo, Japan) for 30

325

min at 37 °C. The digestion was terminated by adding phenymethylsulphonyl fluoride

326

(5 mM in ethanol) and shifting the reaction to 4 °C.

327 328

30

. Briefly, reovirus (2.835 x108 Plaque-forming unit (PFU)) was

The ATM inhibitors KU55933 and KU60019 were obtained from AdooQ Bioscience (Irvine, CA).

329 330

Drug screening

16

331

To identify the new combination drug, we screened the SCADS inhibitor kit (The

332

Ministry of Education, Culture, Sports, Science and Technology, Tokyo, Japan), which

333

includes 285 signaling pathway inhibitors. The canine melanoma cell line CMeC1

334

(5,000 cells/ well) was plated in 96-well plates containing each inhibitor, with or

335

without reovirus at a MOI of 100 PFU per cell, and the plates were incubated for 48 hr.

336

After treatment, cell growth inhibition was evaluated by use of the Cell Counting Kit-8

337

(CCK-8; Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions.

338 339

Cell survival assay

340

Each cell line was seeded at the density of 5,000 cells (CMeC1, KMeC, LMeC,

341

CMGD2, and CMM12) or 10,000 cells (CMM10) in triplicates in 96-well plates and

342

mock-infected or infected with reovirus at a MOI of 10 or 100 PFU per cell in the

343

presence of the ATM inhibitor KU60019 at 1.25, 2.5, 5.0 or 10 µM; assays were

344

performed in triplicate wells. Cells were cultured for 48 hr before adding the CCK-8

345

reagent. The cell proliferation rate was calculated by measuring absorbance. In the

346

cytotoxicity assay, each cell line was treated with reovirus at a MOI of 100 PFU per cell

347

and KU60019 at 2.5 or 10 µM; assays were performed in triplicate wells. Cells were

348

cultured for 48 hr before performing the trypan blue exclusion test.

349

To assess the inhibition of apoptosis induced by treatment with reovirus and

350

inhibitor, canine melanoma cell lines (CMeC1 and KMeC) were seeded at 5,000 cells

351

per well in 96-well plates. The irreversible pan-caspase inhibitor, Z-VAD-FMK

352

(Calbiochem, Billerica, MA) was added to triplicate wells at a concentration of 50 µM

353

for 1 hr before the cells were treated with reovirus (MOI 100) and KU60019 (indicated

354

concentration). After 48 hr, cell proliferation and cell viability were evaluated by

17

355 356

CCK-8 and trypan blue exclusion test, respectively. All experiments were repeated at least three times.

357 358 359

Reovirus replication and progeny virus Reovirus replication was examined by western blotting analysis using anti-reovirus 15-18

). Cell lines were seeded at 3 x 105 cells per

360

antibody (produced by our laboratory

361

well in 6-well plates and treated with reovirus (MOI 100) and KU60019 (2.5 µM) for 24

362

and 48 hr. In some experiments examining the reovirus proteins at early stages, wells

363

were rinsed to remove unbound reovirus following adsorption. Briefly, CMeC1 was

364

plated at 5 x 105 cells per well in 6-well plates and cultured before use. On the

365

following day, cells were exposed to reovirus (MOI 100) or ISVPs (MOI 10) for 1 hr at

366

4 °C. Then, cells were washed and cultured in the presence of KU60019 (2.5 µM) for 6,

367

12, or 24 hr at 37 °C. Cells were harvested at the indicated time points and stored at

368

-80 °C pending use for western blotting.

369

Supernatants of the samples from the cytotoxicity assay were collected and stored

370

at -80°C pending use. Viral progeny in the supernatants were measured using the 50%

371

tissue culture dose (TCID50) assay on L929 cells, as previously described 15-18. Titration

372

of the viral progeny was performed on all the supernatants obtained from three

373

independent cytotoxicity assays.

374 375 376

Genome editing A

small

guide

RNA

(sgRNA)

targeting

canine

ATM,

5'-

377

TAGTTTCAGGATCCCGAATC -3', was designed through the online CRISPR design

378

tool (http://crispr.mit.edu/); the resulting sgRNA was synthesized and subcloned into the

18

62

379

lentiCRISPRv2 plasmid which was gift from Dr. Feng Zhang

(obtained from

380

Addgene (Cambridge, MA) as accession #52961). The plasmid and packaging vectors

381

were transfected to HEK293T to produce the lentivirus expressing an sgRNA targeting

382

ATM. After transduction with the lentivirus, canine melanoma cell lines were cultured in

383

the presence of puromycin (Sigma Aldrich Japan K.K., Tokyo, Japan; 0.6 – 5.0 µg/ml)

384

to select for stably transduced cells. Individual clones were isolated by the limiting

385

dilution method and further analyzed by western blotting to confirm that the cells no

386

longer produced ATM protein. The resulting clones were used for further experiments.

387 388

Flow cytometric analysis

389

To assess the cell cycle distribution, canine melanoma cells (3 x105 cells/ well in

390

6-well plates) were treated with reovirus (MOI 100) and KU60019 (2.5 µM) for 24 and

391

48 hr. Cells were trypsinized and washed in Phosphate-buffered saline (PBS) and fixed

392

in 70 % ice cold ethanol at -30 °C for at least 24 hr. The fixed cells were re-suspended

393

in PBS supplemented with 0.1 mg/ml of RNase (Nacalai Tesque, Kyoto, Japan) and

394

incubated for 30 min at room temperature. The digested cells then were incubated with

395

0.1 mg/ml of propidium iodide (PI; Nacalai Tesque) for 5 min and analyzed.

396

To evaluate surface expression of reovirus receptor Junction adhesion molecule

397

(JAM)-A, a canine melanoma cell line (CMeC1) were seeded at 2 x105 cells per well in

398

12-well plates and treated with mock and KU60019 (2.5 µM) for 1 hr or 24 hr.

399

Incubated cells were stained with primary antibodies including mouse IgG1 isotype

400

control (eBioscience) and mouse anti-human JAM-A monoclonal antibody (Santa cruz),

401

followed by incubation with Dylight488-conjugated goat anti-mouse IgG antibody

402

(BioLegend). Those cells were stained with PI to gate out dead cells.

19

403

All stained samples were analyzed using a BD Accuri C6 Flow Cytometer (BD

404

Biosciences). All given data were analyzed using FlowJo software ver. 10.1 (FlowJo

405

LLC, Ashland, OR).

406 407

Western blotting

408

Cells were plated at 3 x105 cells per well in 6-well plates and treated with reovirus

409

(MOI 100) and KU60019 (2.5 µM) for 24 and 48 hr. For short-term incubation, CMeC1

410

cells were plated at 5 x105 cells per well in 6-well plates, and on the following day, cells

411

were treated as shown above (see Reovirus replication and progeny viruses). Harvested

412

cells were lysed at 4 °C with NP40 lysis buffer (1 % NP40, 10 mM Tris-HCl (pH 7.5),

413

150 mM NaCl, 1 mM EDTA) supplemented with protease inhibitor cocktails (Nacalai

414

Tesque), 1 mM Na3VO4, and 50 mM NaF. The resulting supernatants were collected and

415

used as cell lysates. These lysates were subjected to SDS-PAGE, and proteins then were

416

transferred to Hybond PVDF membrane (GE Healthcare Bio-Sciences, Pittsburgh, PA).

417

The membrane was blocked with Tris buffered saline (TBS) containing with 0.05 %

418

Tween20 (Polyoxyethylene sorbitane monolaureate) and 5 % skim milk, followed by

419

addition of the primary antibodies. The primary antibodies are listed in Table S1.

420

Horseradish peroxidase (HRP) -conjugated antibodies were used as the secondary

421

antibodies. The labeled proteins were detected using the ECL reagent (PerkinElmer).

422

The intensity of the bands was determined by ImageJ software ver. 1.48.

423 424

LysoTracker-Red staining

425

CMeC1 cells (1 x105 cells) were cultured on microcover glasses (Matunami

426

Garasu Kogyo, Osaka, Japan) in the wells of a 12-well plate. On the following day, cells

20

427

were exposed to reovirus (MOI 100) for 1 hr at 4 °C. Then, cells were washed and

428

cultured in the presence of KU60019 (2.5 µM) for 24 hr at 37 °C. After treatment, cells

429

were incubated with LysoTracker-Red DND-99 (Invitrogen, Carlsbad, CA) for 30 min

430

at 37 °C, followed by fixation with 4 % paraformaldehyde. The coverslips were

431

mounted onto glass slides with Molecular Probes Prolong Gold with DAPI (Invitrogen)

432

for staining of nuclei. Samples were visualized at a 100x magnification using LSM710

433

confocal microscope (Carl Zeiss, Oberkochen, Germany). Images were analyzed by

434

using ZEN software 2012 (Carl Zeiss). The percentage of LysoTracker-Red-positive

435

CMeC1 cells was quantified by flow cytometric analysis as described above.

436 437

Measurement of cathepsin B activity

438

Cathepsin activity was determined with a Cathepsin B Activity Assay Kit

439

(PromoCell, Heidelberg, Germany), according to the manufacturer's instructions.

440

CMeC1 cells were cultured at the density of 5 x105 cells per well in 6-well plates. Then,

441

cells were treated with reovirus (MOI 100) and KU60019 (2.5 µM) for 24 hr. After

442

treatments, cells were lysed with CB Cell Lysis Buffer, followed by mixing with the CB

443

Reaction Buffer. The cell lysate was incubated at 37 °C for 1 hr with 200 µM CB

444

Substrate Ac-RR-AFC. Cathepsin activity was measured in an ARVO X4 fluorometer

445

(PerkinElmer). The fluorescence values were normalized to values obtained for a

446

mock-treated sample after subtraction of background autofluorescence.

447 448

Statistical analysis

449

The mean values and standard deviation (SD) in each assay were calculated from the

450

mean of at least three biological independent experiments. The significance of

21

451

differences between samples were determined by the one-way factorial ANOVA test

452

followed by multiple comparison using Tukey-Kramer test. Differences were

453

considered statistically significant if the p values were less than 0.05. Multiple

454

comparison tests were performed using JMP software ver. 9.0 (SAS Institute Japan,

455

Tokyo, Japan).

22

456 457

CONFLICTS OF INTEREST

458

M.C. is president/chief executive officer and has financial interests with Oncolytics

459

Biotech, Inc. The other authors declare no conflict of interest.

460 461

ACKNOWLEDGMENTS

462

This study was supported by JSPS KAKENHI Grant Number 15H04598 and 16J06655.

463

The SCADS Inhibitor Kit was provided by The Screening Committee of Anticancer

464

Drugs supported by a Grant-in-Aid for Scientific Research on Innovative Areas,

465

Scientific Support Programs for Cancer Research, from The Ministry of Education,

466

Culture, Sports, Science and Technology, Japan. Dr. Takayuki Nakagawa (The

467

University of Tokyo) and Dr. Jaime Modiano (University of Michigan) kindly provided

468

canine melanoma cell lines to the authors. The authors would like to acknowledge the

469

technical expertise of the DNA Core facility of the Center for Gene Research,

470

Yamaguchi University, supported by a grant-in-aid from the Ministry of Education,

471

Science, Sports and Culture of Japan.

472 473

AUTHOR CONTRIBUTIONS

474

M.I. and T.M. conceived the study design and performed all experiments, in addition to

475

writing the paper. S.S and M.C. provided essential resources and developed methods.

476

Y.K. and N.F. obtained additional data for this study. C.C.H., S.N., and Y.N. analyzed

477

the data and provided critical interpretation. All authors read and approved the final

478

manuscript.

23

479

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29

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Figure legends Figure 1. ATM inhibitor KU60019 enhances reovirus-induced cell growth inhibition in canine melanoma cell lines. To evaluate cell proliferation, canine melanoma cell lines (CMeC1, KMeC, LMeC, CMM10, CMM12, and CMGD2) were treated with reovirus (MOI 100 for all cell lines except CMGD2 at MOI 10) and KU60019 (indicated concentration) for 48 hr before adding CCK-8 reagent. Data are expressed as the mean ± SD from at least three independent experiments. p values were calculated for the comparison between reovirus alone and reovirus combined with KU60019. To focus on the additional effects provided by KU60019, significance was tested only where no significant difference was observed between mock control and KU60019 alone. Tukey-Kramer test, *p < 0.05, **p < 0.01. Figure 2. Reovirus susceptibility is enhanced by ATM knock-out. (A) The accumulation of ATM in the indicated melanoma cell lines was examined by western blotting. Vinculin was used as a protein loading control. (B) To evaluate cell viability, ATM knock-out canine melanoma cell lines (CMeC1, KMeC, CMM10, and CMM12) were treated with reovirus (MOI 100) for 48 hr before the trypan blue exclusion test. Viable cells and dead cells were counted, and the percentage of viable cells were calculated. Data are expressed as the mean + SD derived from three independent experiments. Tukey-Kramer test, *p < 0.05, **p < 0.01, ns: not significant. Figure 3. The combination of KU60019 and reovirus induces apoptotic cell death. (A) CMeC1, KMeC, and LMeC were treated with reovirus (MOI 100) and KU60019 (2.5 and 10 µM), and CMGD2 was treated with reovirus (MOI 10) and KU60019 (2.5 and 10 µM). After a 48-hr incubation, cell viability was quantified by the trypan blue exclusion test. Data are expressed as the mean ± SD from at least three independent experiments. p values were calculated for the comparison to reovirus alone. Tukey-Kramer test, *p < 0.05, **p < 0.01. (B) Cleaved caspase-3 levels in CMeC1 treated with reovirus (MOI 100) and KU60019 (2.5 µM) for the indicated time are shown in the upper panel. The relative level of cleaved caspase-3 normalized to actin was quantified from three independent experiments. Data are expressed as the mean + SD. Tukey-Kramer test, *p < 0.05, **p < 0.01. (C) The sub-G1 population was analyzed in CMeC1 treated with reovirus (MOI 100) and KU60019 (2.5 µM) for 24 and 48 hr. The cell numbers and stages were quantified using flow cytometry. The percentage of sub-G1 was calculated in total cells. Data are expressed as the mean + SD derived from three independent experiments. Tukey-Kramer test , *p < 0.05, **p < 0.01. Histogram of PI staining of each sample at 48-hr is shown for representative data from one of three independent experiments. (D, E) The effects of co-treatment for 48 hr with Z-VAD-FMK (50 µM) along with the combination of reovirus (MOI 100) and KU60019 (2.5 µM or indicated various concentrations) were quantified using the trypan blue exclusion test (D) or the CCK-8 cell viability assay (E). Data are expressed as the mean ± SD from at least three independent experiments. p values were calculated for the comparison to the vehicle sample. Tukey-Kramer test , **p < 0.01. Figure 4. The combination of KU60019 and reovirus induces cell cycle arrest at G2/M and increases sub-G1 populations in canine melanoma cells.

30

724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777

(A) CMeC1 was treated with reovirus (MOI 100) and KU60019 (2.5 µM). After 24-hr or 48-hr incubations, the cell cycle of CMeC1 cells was assessed with propidium iodide (PI) staining, as shown in Fig. 3C. Bar graphs indicate the percentages of each cell cycles (G1, S, and G2/M) at 24-hr and 48-hr in the total cells except sub-G1 populations. p values shown were calculated for the comparison between "Reo" and "KU+Reo".Tukey-Kramer test, *p < 0.05, **p < 0.01. In addition, p values shown were calculated for the comparison between "Mock" and "Reo".Tukey-Kramer test, †p < 0.01. (B) cdc2 and p21 protein expression levels in CMeC1 treated as described in (A) are shown in the upper panel. The relative level of each protein normalized to actin was quantified from three independent experiments. Mean + SD are shown. Tukey-Kramer test, *p < 0.05, **p < 0.01. The membrane in Figure 3B was reprobed and used for this panel. Figure 5. Reovirus replication is enhanced by KU60019 treatment. (A) Supernatants of reovirus (MOI 100 for CMeC1, KMeC, and LMeC, or MOI 10 for CMGD2) -infected cell lines treated with KU60019 (2.5 µM) were harvested after a 48-hr incubation, and virus titers were determined by the TCID50 assay. The fold increase of reovirus titer represents values calculated from the titer of progeny virus divided by the titer of input virus. The mean + SD was calculated from three independent experiments. p values were calculated by Tukey-Kramer test, *p < 0.05, **p < 0.01, ns: not significant. (B) Effects of ribavirin (200 µM) in CMeC1 treated with combination of reovirus (MOI 100) and KU60019 (2.5 µM) for 24 hr were assessed by western blotting using anti-reovirus antibody and anti-cleaved caspase-3 antibody. Actin was used as a protein loading control. (C, D) CMeC1 cells were pre-treated with reovirus virions (MOI 100) or ISVPs (MOI 10), and washed-out unattached virus followed by treatment with KU60019 (2.5 µM) for the indicated time points. Reovirus structural proteins were detected by western blotting using anti-reovirus polyclonal antibody. Actin was used as a protein loading control. The relative level of reovirus protein normalized to actin was quantified from at least three independent experiments. Mean + SD are shown. Tukey-Kramer test, *p < 0.05, **p < 0.01. Figure 6. Reovirus infection induces the phosphorylation of ATM in cancer cells. Phospho-ATM and total ATM expression levels were assessed by western blotting of lysates from CMeC1 cells treated with reovirus (MOI 100) and KU60019 (2.5 µM) for the indicated times. Vinculin was used as a protein loading control. The graph shows the relative level of each protein (normalized to vinculin) quantified from four independent experiments. The fold change of phospho-ATM divided by total ATM was normalized to the value of Mock at 0 hr. Mean + SD are shown. Tukey-Kramer test, *p < 0.05. Figure 7. The combination of reovirus and KU60019 promotes the endosomal acidification and activation of cathepsin B. (A, B) The acidification of endosomes/lysosomes was determined by LysoTracker-Red staining. CMeC1 cells were treated with reovirus (MOI 100) and KU60019 (2.5 µM) for 24 hr before staining. (A) Histogram overlay of LysoTracker-Red staining of each sample is shown for representative data from one of four independent experiments. Relative median fluorescence intensity (MFI) values relative to mock treatment are indicated. Mean + SD are shown from four independent experiments. Tukey-Kramer test, *p < 0.05, **p < 0.01. (B) Representative images of samples from each treatment

31

778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799

groups are visualized using confocal microscopy at a 100x magnification. Nuclei were stained with DAPI. Scale bar: 10 µm. (C) The enzyme activity of cathepsin B in CMeC1 cells treated as described above was measured and normalized to the value obtained with mock treatment. Mean + SD are shown from five independent experiments. Tukey-Kramer test, *p < 0.05.

Figure 8. Model of anti-tumor effects of reovirus combined with KU60019. In general, the phosphorylation of ATM protein is induced when DNA double-strand breaks are caused by UV or radiation. We newly found that reovirus infection activated the ATM protein unaccompanied with DNA double-strand breaks, at the late time points of reovirus infection (after 12 hr)(A) (Figure 6). This indicates that reovirus may cause an atypical ATM signaling pathways, which is abrogated by KU60019 (B). The combination treatment of reovirus and KU60019 leads to enhancement the virus replication (C), followed by cell cycle arrest at G2/ M and caspase-dependent apoptosis, potentiating anti-tumor effects. However, the mechanism between upregulation of lysosomal activities and reovirus cytotoxicity is still unknown.

32

Figure 7 A

2.5 Mock KU Reo KU + Reo Sample Name

250

**

Count

Tube - 9_Data Source - 1.fcs DsRed-Monomer-A subset 15656 Tube - 8_Data Source - 1.fcs Lymphocytes

17177

Tube - 7_Data Source - 1.fcs Lymphocytes

17129

Tube - 6_Data Source - 1.fcs Lymphocytes

17439

Count

150

100

50

0 10

3

10

4

10

5

LysoTracker-Red FL3-A :: DsRed-Monomer-A

Relative MFI ( /Mock )

200

Count

Subset Name

*

2

1.5 1 0.5 0

Mock

KU60019

KU

Reo

KU + Reo

C 160

Reovirus

KU + Reo

Cathepsin B activity (% of Mock)

B

Mock

*

140 120 100 80 60 40 20 0

Mock

KU

Reo

KU + Reo

Figure 8

DNA damage

Reovirus

X

X X

(A)

ATM (B)

p-ATM

(C)

Virus replication Cell cycle arrest at G2/M Caspase-dependent apoptosis

KU60019

?

Up-regulation of lysosomal activity

Figure 1 KMeC

100

100

100

60 **

40

** **

20

0

1.25 2.5 5 KU60019 (μM)

**

60 **

40 20 0 1.25 2.5 5 KU60019 (μM)

10

**

20

0

% of cell proliferation

80

0

**

40

1.25 2.5 5 KU60019 (μM)

80 **

60 40 20 0

10

0

CMM10

120

100

**

60

0

10

CMGD2

120

80

1.25 2.5 5 KU60019 (μM)

10

CMM12 120

100 80 60

**

40 20

% of cell proliferation

80

% of cell proliferation

120

0

% of cell proliferation

LMeC

120 % of cell proliferation

% of cell proliferation

CMeC1 120

100 80 60

KU60019 *

40

**

20

KU60019 + Reov irus **

**

0

0 0

1.25 2.5 5 KU60019 (μM)

10

0

1.25 2.5 5 KU60019 (μM)

10

Figure 2 A KMeC

CMeC1

CMM10

WT 1C10 1D11 WT 2C10 3G9 WT 1D9 3D7

CMM12 WT 3C12 3E9

ATM Vinculin

B

KMeC

CMeC1 120

** **

80 60 40 20

ns

80 60 40 20

0

0 WT

1C10

1D11

WT

CMM10 120

2C10

**

** ns

% of cell viability

100

80 60 40

3G9

CMM12

120

**

100 % of cell viability

**

100 % of cell viability

100 % of cell viability

120

80 Mock Reovirus

60 40 20

20

0

0 WT

1D9

3D7

WT

3C12

3E9

15M

15M

15M

800

200

200

0

0 5.0M

10M

15M

0

*

0

0

5.0M

*

A02.fcs

10M 3 10

2

10

15M 4

10

60

10

6

10

400

100

200

0 15M

0

0 10

5.0M

2 10M

FSC-A :: FSC-A FSC-A :: FSC-A

C 70A02.fcs

A01.fcs Live 8598

Ungated 10000

A01.fcs Live 8598

A03.fcs Ungated 15096

10

0 10

5

6 10 5.0M

2 10M 10

10

3

Mock

15M10 4

0 0 10

0 5.0M 5.0M 6 10

5

KU60 019 Reo viru s KU + Reo B01.fcs A04.fcs Ungated Ungated 7410 31343

A02.fcs Live 8504

A02.fcs Live 8504

300

10

0

Count

Count

SSC-A :: SSC-A

B02.fcs Ungated 7023

10M 3 10

2

10

15M 4

10

5

10

6

10

100

0

0

Count

SSC-A :: SSC-A

100

15M

200

400

Live 72.3

600

10M

0

5

B02.fcs Live 5077

0

40

10

5

2 10 10M

5.0M 6 10

6

10

2 10M

10

FSC-A :: FSC-A #1 #2

FL2-A subset 3.13

Live 30.0

3 15M

10

0 4

0

10

5

FL2-A :: FL2-A

DMSO 300

B02.fcs Ungated 7023

Live 72.3

100

10M

FL2-A subset 34.7

80

3

4 10 15M

5

10

6

Actin

FL2-A subset 20.3

0

5 100

10

0 6 5.0M 10

FL2-A :: FL2-A

#3

10M

2

10

#5

B03.fcs Ungated 20172

10

15M 4

3

10

FSC-A :: FSC-A

#4

10

5

10

#6 FL2-A ::#7 FL2-A

2

10

3

6

#8

10

4

10

5

10

6

FL2-A :: FL2-A

#9 B03.fcs Live 6056

B02.fcs Live 5077

Reovirus - 48 hr

KU + Reo - 48 hr 400

15M

Live 9.37

200

FL2-A subset 43.9

Count

Live 30.0

300

FL2-A subset 20.3

10M

Count

SSC-A :: SSC-A

FL2-A subset 3.13

400

200

5.0M

200

100

10M 2 10

10

2

10

FSC-A :: FSC-A

3

2 3 10 10M 10

5 6 6 10 10 5.0M 10

FL2-A FL2-A :: FL2-A :: FL2-A

B01.fcsA04.fcs Live Live 5096 7238

0

0

0 4 3 15M 4 5 0 10 10 10 10

10M2 3 10 10

4 10 15M

100

Live 30.0

FL2-A subset 10M 3.13

200

10

5

10

10

6

2

10M

10

FSC-A :: FSC-A

FL2-A :: FL2-A

B03.fcs Ungated 20172

A04.fcs Live 7238

0

10M

2

10

15M 4 10

3

10

5

10

6

10

2

10

400

3

** FL2-A subset 20.3

200

Live 9.37

3

15M 4 10

0 05 10

6 10 5.0M

FL2-A :: FL2-A

B02.fcs Live 5077

DMSO

400 15M

300

2 10M

10

3

4 10 15M

FL2-A :: FL2-A

FSC-A :: FSC-A

B03.fcs Live 6056

B04.fcs Ungated 88008

KU60019

10

Reovirus 400

KU + Reo

10

5

4

10

CMeC1

400

120

100

100

300

** FL2-A subset 43.9 **

80

**

200

**

**

60

100

40

10

6

0 10

2

20 0

10

3

10

4

10

5

10

6

FL2-A :: FL2-A

B04.fcs Live 8245

0

5

10

6

B04.fcs Live 8245

120

300

10

FL2-A :: FL2-A

B03.fcs Live 6056

E

Ungated 88008

100

5.0M

0 5.0M

0

10

FSC-A :: FSC-A FL2-A :: FL2-A

0

40

0

5.0M

6

15M

400

0

15M 4 10

10

DNA content B04.fcs

B02.fcs Live 5077

KMeC

50

FL2-A :: FL2-A

FSC-A :: FSC-A

B03.fcs Ungated 20172

0

10

60

KU60019 Reovirus KU + Reo 15M

10

100

KU60019 - 48 hr

20

A03.fcs Live 5746

A04.fcs Ungated 31343

6 0 10 5.0M

4

200

200

B01.fcs Live 5096

**

5.0M

FSC-A :: FSC-A

20 0

10

10

**

0

10 5.0M

5.0M

600

120

100

4 0

3

**

400

5.0M

200

Count

Live 72.3

FL2-A FL2-A subsetsubset 2.39 34.710M

Z-VAD

**

5.0M

0

10

300

FL2-A subset 2.39

15M

FSC-A :: FSC-A

B02.fcs Ungated 7023

60 10

5.0M 6 10

5

15M 300

Cleaved Caspase-3

2

FL2-A :: FL2-A

B01.fcs Live 5096

600

B02.fcs Ungated 7023

SSC-A :: SSC-A

Count

Count

200

FL2-A :: FL2-A

A03.fcs Live 5746

FL2-A subset 14.5 10M Live 23.1

80

10

Vehicle

300

200

0

15M 15M 4 10

15M

FL2-A :: FL2-A

A03.fcs Ungated 15096

0

10M 2 10M 3 10 10

FSC-A :: FSC-A FSC-A :: FSC-A

FL2-A :: FL2-A

FSC-A :: FSC-A

Count

Count

SSC-A :: SSC-A

FSC-A :: FSC-A

3

200

400

5.0M

0 0

0

0

100

15M

10

Live Live 68.8 23.1

Count

200

FL2-A subset10M 14.5

100

FL2-A subset 10M 1.95

10M 2

10

200

A03.fcs Ungated 15096

% of cell viability

0

24

600

5.0M 5.0M

20

5.0M

5.0M

48

KU60019 (2.5 μM) Reovirus (MOI 100) 48 Time (hr)

FSC-A :: FSC-A FL2-A :: FL2-A

600

SSC-A :: SSC-A

Live 400 38.1

**

0

24

+ +

200

300

30

D

200

0 4 10

A03.fcs Live 5746

15M

10M FL2-A subset 10M 1.95

15M

400

10M

3 15M

+ +

0

5.0M

FSC-A :: FSC-A

Mock - 48 hr

15M 15M

600

120

Live 38.1

10

FSC-A :: FSC-A

B01.fcs Ungated 7410

A02.fcs Live 8504

CMeC1

600

10M2 10

300

40

0 FL2-A :: FL2-A

800

10

0

15M

Count

A02.fcs Ungated 10000

10

6

10M

− +

0

% of cell proliferation

FSC-A :: FSC-A

5.0M

5

300

15M 4 10

3

10

5.0M 6 10

5

Count

10

10

4

48

− +

400

SSC-A :: SSC-A

2 10M

15M

3

Count

10

10

0

0

CMGD2

10M

2

SSC-A :: SSC-A

5.0M

6 0 10 5.0M

FSC-A :: FSC-A

24 hr

10

0

0

KMeC

5

10

FL2-A :: FL2-A

5.0M

200

0 10

FL2-A :: FL2-A

800

Count

SSC-A :: SSC-A

Count 400

4

% of cell viability

% of sub-G1 cells

50

FL2-A subset 1.40 10M

Live 85.0

10 15M

0

+ −

400

15M

600

LMeC

3

48 hr

60 800

10

SSC-A :: SSC-A SSC-A :: SSC-A

A01.fcs Ungated 10000

0

0

CMeC1

0

Count

10M

4

5.0M

200

5.0M

10

1.4 1.2 FL2-A subset 1 14.5 0.8 0.6 0.4 0.2 0 FL2-A :: FL2-A

Live 68.8

200

5.0M

5.0M

5.0M

0

3

200

24

15M

10M

FL2-A subset 1.95

Live 38.1

Count

Live 85.0

400

20

SSC-A :: SSC-A

Count

10M

600

48

B01.fcs Ungated 7410

300

Count

FL2-A subset 10M 1.40

600

10

*

15M

**

40

2

A02.fcs Live 8504

**

800

800

24

+ −

5.0M

0

FL2-A :: FL2-A

** 15M

15M

5

A01.fcs Live 8598

**Ungated 10000

SSC-A :: SSC-A

% of cell viability

A01.fcs Ungated 10000

SSC-A :: SSC-A

Live 86.0

10

FSC-A :: FSC-A FL2-A :: FL2-A

FSC-A :: FSC-A

80

0

− −

0

SSC-A :: SSC-A

0

− −

FL2-A subset 3.13

400

% of cell proliferation

100

− −

B 5.0M

Reovirus + KU 10 μM

FL2-A subset 2.39

Live 72.3

400

Count

Reovirus + KU 2.5 μM

400

Live 68.8

10M

SSC-A :: SSC-A

Reovirus

400 5.0M

5.0M

Count

Live 85.0

Relative expression levels of cleaved casp-3 (/Actin)

Mock

10M

SSC-A :: SSC-A

Live 86.0

FL2-A subset 10M 1.95

Count

A

10M

600 600

FL2-A subset 1.40

600

Count

SSC-A :: SSC-A

Figure 3

SSC-A :: SSC-A

800

Count

15M

10

**

** **

80

** **

60

**

**

**

Vehicle+KU Vehicle+KU+Reo Z-VAD+KU

40

Z-VAD+KU+Reo

20 0

1.25 2.5 5 KU60019 (μM)

KMeC

0

1.25 2.5 5 10 KU60019 (μM)

Figure 4 A

24 hr

120% 120

*

†

100 100%

Percentages of cells

100% 100 80% 80

% of cells

% of cells

S

40% 40

20 20%

**

20% 20

0%0

0%0 M

k oc KU

0 60

19 R

s ru vi o e

KU

+

R

eo

M

− −

− −

− −

+ −

+ −

− +

− +

+ +

0

24

48

24

48

24

48

24

k oc KU

+ +

01 60

9

s eo ru R vi + o e R KU

KU60019 (2.5 μM)

Reovirus (MOI 100) 48 Time (hr)

cdc2/CDK1 p21

Relative expression levels of p21 (/Actin)

Relative expression levels of cdc2/CDK1 (/Actin)

Actin 2.5 *

2 1.5 1 0.5

1.5

#1

#2

#3

#4

#5

#6

#7

#8

#9

#1

#2

#3

#4

#5

#6

#7

#8

#9

1

0.5

0

G2/M

†

60 60%

40 40%

0

**

80 80%

60 60%

B

48 hr

120 120%

G1

Figure 5

**

35

A

*

**

ns

B

ns

ns

Fold increase of virus titer (/Input)

30

–

–

–

–

+

+

+

– −

– +

+ −

+ +

– −

– +

+ −

25 20

Reovirus Protein

Input Reovirus + DMSO

15

Reovirus + KU60019 2.5μM 10

Cleaved Caspase-3

5

Actin

0

CMeC1

LMeC

KMeC

CMGD2

C

D − −

− +

− +

− +

+ +

+ +

+ +

0

6

12

24

6

12

24

− KU60019 (2.5 μM) − Reovirus (MOI 100) 24 Time (hr)

− −

− +

− +

− +

+ +

+ +

+ +

0

6

12

24

6

12

24

Actin

Actin

**

p > 0.05

2 Relative expression levels of reovirus protein (/Actin)

2.5

* 2

1.5

1

0.5

p > 0.05

1.5

1

0.5

0

0 o,

r 6h

,1

r 2h

,2

r 4h

, +r

r 6h

r,

1

r 2h

r,

2

r 4h

− KU60019 (2.5 μM) − ISVPs (MOI 10) 24 Time (hr)

Reovirus Protein

Reovirus Protein

Relative expression levels of reovirus protein (/Actin)

+ Ribavirin (200 μM) + Reovirus (MOI 100) + KU60019 (2.5 μM)

o,

6h

r o,

12

hr o,

24

hr

, +r

6h

r r,

12

hr r,

24

hr

Figure 6 – – 0

– – 12

– – 6

– – 24

– + 6

– + 12

– + 24

+ + 6

+ + 12

+ KU60019 (2.5 μM) + Reovirus (MOI 100) 24 Time (hr)

p-ATM (Ser1981)

ATM

Vinculin 12 Fold change of p-ATM/ATM (Normalized to Mock at 0 hr)

*

10 8

*

6 4 2 0

#1

#2

#3

#4

#5

#6

#7

#8

#9

#10