177 Deletions of Both Vaccinia Growth Factor and O1 Protein Genes Enhance Therapeutic Index of Oncolytic Vaccinia Virus Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Soc[.]
CANCER-ONCOLYTIC VIRUSES I of xenograft models using nude mice transplanted with NSCLC tumors showed that intratumoral CVA11 administrations significantly suppressed the outgrowth of preestablished xenografts and prolonged the survival of the CVA11-treated mice Of note, all mice treated with CVA11 elicited no significant change of body weight during the treatment To dissect the role of differential modalities of cancer cell death in CVA11-mediated oncolysis, we assessed the impact of pretreatment with Ac-YVAD-cmk, a pan-caspase inhibitor which prevents apoptosis, necrostatin-1, a RIP-1 kinase inhibitor which prevents necroptosis, and Ac-YVAD-cmk, a caspase-1 inhibitor which prevents pyroptosis, on the CVA11-driven cytotoxicity Apoptosis as well as necroptosis partially but significantly contributed to the CVA11-driven oncolysis in NSCLC cell lines, whereas pyroptosis did not Altogether, we showed that CVA11 displayed remarkable oncolytic activity against human NSCLC both in vitro and in vivo with tolerability, presumably via the receptor ICAM-1 and resultant cell death machinery of apoptosis and necroptosis, offering an attractive alternative for the treatment of NSCLC 176 Systemic Treatment with Fiber-Redesigned Oncolytic Adenovirus Eliminates Tumors in Pancreatic Cancer In Vivo Model Yoshiaki Miura,1 Mizuho Sato,1 Julia Davydova,1 Masato Yamamoto.1 Department of Surgery, University of Minnesota, Minneapolis, MN Many cancer patients are diagnosed at unresectable stage in various cancers, and most of these patients require treatment of metastatic and/ or locally advanced diseases However, efficacy of current systemic therapies is quite limited to date Oncolytic virus has high potential for systemic therapy because of low requirement of initial local concentration for antitumor effect, and adenovirus (Ad) has been one of such platforms for oncolytic virus Unlike loco-regional therapy, systemic application of cancer gene therapy mandates different level of selectivity of delivery, and lack of tumor selectivity at the stage of infection has hampered the in vivo efficacy of systemic therapy The improvement of vector distribution and cancer selective transduction would overcome the obstacles for systemic delivery and enable efficient systemic treatment of cancer AdML-VTIN was identified as a mesothelin (MSLN) targeted Ad by high-throughput screening of 109 order-diversity Ad-fiber library This AB-loop-redesigned Ad yielded a potent and selective infectivity to MSLN-positive pancreatic cancer (Panc-1) in vitro Moreover, intra-tumor injection of AdML-VTIN (1010vp/tumor) showed selective and significant anti-tumor effect against Panc-1 tumors, and about half of the Panc-1 tumor disappeared while the same virus showed no anti-tumor effect in MiaPaCa-2 (MSLN-negative) In order to assess the potential of VTIN virus for systemic application, we compared organ distribution after intravenous injection to the nude mice with subcutaneous Panc-1 xenograft among AdML-VTIN, AdML-5WT (wild type Ad5 fiber), and AdMLGERS (against PC-3, prostate cancer cell) The liver sequestration of both AB-loop redesigned Ads were lowered more than one order of magnitude compared to AdML-5WT at both hr and 48 hrs after injection At day 7, the viral copy number of AdML-VTIN in the tumor was more than orders of magnitude higher that those with AdML-5WT Next, systemic therapeutic effect against Panc-1 tumors was compared among AdML-VTIN, AdML-5WT, and AdML-GERS In the single intravenous (i.v.) injection, tumor volume was significantly decreased only in AdML-VTIN treated group while Ad with wildtype fiber didn’t show any tumor suppression Four out of ten tumors were eliminated at 15 days after the injection of low amount of virus (109vp/mouse), which was significant lower than that of intra-tumor Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy injection Then, six out of ten AdML-VTIN treated tumors showed the regrowth at day 47, and the multiple i.v treatments (day 47, 61, 68, and 75) of AdML-VTIN were applied to them Even though the multiple i.v treatment was started after tumor volume reached 500mm3, all mice survived until the end of experiment (day 75) and four out of six regrown tumors shrunk In this study, systemic injection of the AB-loop redesigned oncolytic Ad with VTIN motif showed remarkable anti-tumor effect with low viral amount Furthermore, multiple i.v treatment suppressed the regrown tumors The systemic therapy with this new oncolytic Ad may embody efficient treatment for cancers which are mostly found with spread or metastatic lesions 177 Deletions of Both Vaccinia Growth Factor and O1 Protein Genes Enhance Therapeutic Index of Oncolytic Vaccinia Virus Ikumi Goto,1 Naoyoshi Nitta,1 Motomu Nakatake,1 Nao Okada,1 Masato Yamane,1 Hajime Kurosaki,1 Takafumi Nakamura.1 Department of Biomedical Science, Graduate School of Medical Sciences, Tottori University, Yonago, Japan Vaccinia virus, once widely used for smallpox vaccine, has been engineered and used as an oncolytic virus for cancer virotherapy Epidermal growth factor (EGF)-like vaccinia growth factor (VGF) is a secreted protein produced early in viral infection and contributes to viral spread and replication via EGFR-dependent MAPK/ERK1/2 activation O1 protein is another activator of the pathway located downstream of the EGFR, complementing the function of VGF Thus, the deletion of such vaccinia virus genes would inhibit pathogenic viral replication in normal cells, while retaining its therapeutic replication in tumor cells that have the constitutive ERK1/2 activation Our study is the first to develop a combined VGF-deleted (VGF-) and O1-deleted (O1-) vaccinia virus and investigate its properties in vitro and in vivo The expression cassette encoding luciferase and EGFP was inserted into the HA, VGF or O1 locus of an LC16mO strain of vaccinia virus, resulting in VGF+/O1+VV, VGF-VV or O1-VV respectively Furthermore, the expression cassette encoding DsRed was inserted into O1 locus of the VGF-VV, resulting in VGF-/ O1-VV All the recombinant viruses at 30hr after infection equally induced abundant cytopathic effect (CPE) following EGFP expression in not only human tumor cell lines (A549, BxPC3 and SKOV3) during both proliferation and serum starvation but normal human lung fibroblasts (NHLF) during proliferation In contrast, VGF-/ O1-VV caused very little in serum-starved NHLF cells, although VGF+/O1+VV induced massive CPE, and VGF-VV or O1-VV still did moderate CPE Furthermore, these results were correlated with the ERK1/2 activity The levels of phosphorylated ERK1/2 (pERK1/2) were much more dramatically reduced by serum starvation in the normal cells compared with the tumor cells In serum-starved NHLF cells, VGF-/O1-VV infection did not upregulate the pERK1/2 levels compared with mock infection, while VGF+/O1+VV did extremely significantly, and VGF-VV or O1-VV did significantly SCID mice were intraperitoneally injected with 106 pfu of each recombinant virus VGF+/O1+VV spread from the abdomen to several areas of their body at a much earlier point in time than O1-VV did All of the mice treated with VGF+/O1+VV or O1-VV died on days 35–49 or days 49–77 respectively, due to the virus-associated toxicity Interestingly, all the VGF-VV-treated mice finally died around 36 weeks after injection, although VGF-VV did not spread from the abdomen within at least weeks after injection In contrast, VGF-/O1-VV have lived >50 weeks Furthermore, the oncolytic activity was evaluated by a single intraperitoneal injection of each virus (106 pfu) in SCID mice bearing peritoneally disseminated BxPC3 xenografts VGF-/O1-VV significantly prolonged survival compared with VGF-VV (P=0.031), O1-VV (P=0.0018), VGF+/ O1+VV (P=0.0023) and mock therapy (P=0.0047) Importantly, the S67 CANCER-ONCOLYTIC VIRUSES I viral replication of VGF-/O1-VV was detected in the tumor tissue, but not in normal tissues Our study demonstrated that the MAPKdependent oncolytic vaccinia virus VGF-/O1-VV enhances safety profile, tumor specificity and the therapeutic index 178 Targeting Glioblastoma Stem Cells with Oncolytic HSV and PARP Inhibitors Jianfang Ning,1 Hiroaki Wakimoto, Robert L Martuza,1 Samuel D Rabkin.1 Neurosurgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA Glioblastoma, the most common primary brain tumor in adults, is invariably fatal despite all current therapies, with a median survival of about 15 months One possible reason is that most therapeutic strategies have not targeted the cancer stem cell subpopulation We have isolated glioblastoma stem cells (GSCs) from primary glioblastoma specimens using neurosphere culture in media containing EGF and FGF, in place of serum GSCs differ genetically and phenotypically, as the patient tumors, and form xenografts that histologically resemble the patient’s tumors from which they were derived Oncolytic herpes simplex viruses (oHSVs) are geneticallyengineered to selectively replicate in cancer cells We recently constructed oHSV MG18L, containing a deletion in Us3, apoptosis and Akt inhibitor, and an inactivating insertion of LacZ in ICP6, ribonucleotide reductase As opposed to G207, MG18L replicates in and kills GSCs, but like G207 is safe after intracerebral injection GSCs vary in their sensitivity to killing by either MG18L or G47D (deletion of γ34.5 and inactivating insertion of LacZ in ICP6), but none were resistant It has been reported that PTEN loss sensitizes cancer and glial cells to poly (ADP-ribose) polymerase inhibitors (PARPi) Therefore, we examined the sensitivity of a panel of GSCs (PTEN-positive and PTEN-null) to PARPi olaparid (AZD2281), veliparid (ABT-888), and rucaparid (AG-014699) Half of the tested GSCs were sensitive to PARPi, with of PTEN-null GSCs resistant Moderate synergistic effects were observed in MGG4 cells (PTEN-positive, PARPi sensitive) between PARPi and MG18L or G47Δ, but not other GSCs examined In an in vivo model, with MGG4-derived intracerebrally tumors, olaparib alone had a small, but not significant, effect and MG18L alone had a significant effect, whereas the combination greatly extended survival These studies indicate that about half of GSCs were sensitive to PARPi in vitro, while the effect in vivo was limited In contrast, all the GSCs were sensitive to both MG18L and G47Δ in vitro, and a single injection of MG18L extended the survival of tumor-bearing mice in a GSC-derived glioma model, while the combination with olaparid significantly extended survival compared either treatment alone 179 Coxsackievirus A11 Displays Remarkable Oncolytic Activity Against Oxaliplatin-Resistant Human Colorectal Cancer Cells Beibei Wang, Hiroyuki Inoue, Shohei Miyamoto, Yuki Nakano,1 Chika Sakamoto,1 Megumi Narusawa,1 Koichi Takayama,2 Hiroyuki Shimizu,3 Yoichi Nakanishi,2 Kenzaburo Tani.1 Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; 2Research Institute of Diseases of the Chest, Kyushu University, Fukuoka, Japan; 3Department of Virology II, National Institute of Infections Diseases, Tokyo, Japan 1,2 Colorectal cancer (CRC) is one of the most common malignant tumors worldwide Although systemic chemotherapy with oxaliplatin (L-OHP) provides a response rate of 50%, resistance develops in nearly all metastatic patients Development of novel treatment S68 modalities is therefore required to improve their overall survival rates Oncolytic virotherapy using enteroviruses is a promising new strategy to treat various human cancers From a safety point of view, we focused on enterovirus strain of coxsackievirus A11 (CVA11) because it has low pathogenicity but possesses broad oncolytic activity against solid cancers in our first screening assay We found that CVA11 displayed potent oncolytic activities in of (87%) various human CRC cell lines including L-OHP-resistant WiDr and HT29 cells even at the very low MOI of 0.001 Notably, pretreatment with L-OHP 12 hours beforehand enhanced (sensitized) the oncolytic activity of CVA11 against WiDr and HT29 cells, whereas treatment with either L-OHP or CVA11 alone caused no or slight oncolytic activity As the activity of phosphatidylinositol 3-kinase (PI3K)/Akt pathway known to be involved in is shown to be chemoresistance of CRC cells to L-OHP, we examined whether PI3K/Akt inhibition had an effect on the CVA11-mediated cytotoxicity in WiDr cells and found that the oncolytic activity was significantly impaired when LY294002, a highly selective inhibitor of PI3K, was added to the CVA11 (p