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179. Coxsackievirus A11 Displays Remarkable Oncolytic Activity Against Oxaliplatin-Resistant Human Colorectal Cancer Cells
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 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 do 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 2 of 3 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 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
1,2
1
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 6 of 7 (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<0.05). Furthermore, our results of xenograft models of nude mice transplanted with L-OHP-resistant WiDr and HT29 cells showed that intratumoral CVA11 administrations at low dose (3×103 TCID50) significantly suppressed the outgrowth of preestablished subcutaneous xenografts and prolonged the survival of the tumor-bearing mice compared with untreated mice (p<0.01). Of importance, repeated experiments revealed that all of mice that received CVA11 treatment elicited no significant change of body weight during the treatment. Lastly, our result of caspase-3 staining assays showed that CVA11 infection induced robust caspase 3-postive cells (18%) in colorectal cancer cells as indicative of apoptotsis, while L-OHP alone displayed less caspase-3 activity (6%). Of note, combination treatment of z-VAD pan-caspase inhibitor with CVA11 infection exhibited significantly less oncolytic activity in CRC cells compared with CVA11 treatment alone (p<0.001). Taken together, our findings for the first time showed that CVA11 displayed remarkable oncolytic activity against oxaliplatin-resistant human colorectal cancer cells both in vitro and in vivo, and that both of the PI3K/Akt pathway and caspase-dependent apoptosis contributed to the process of CVA11-driven oncolysis, providing a new avenue to improve therapeutic outcomes for CRC patients.
180. Vstat120 Reduces the Innate Inflammatory Response To Infection and Enhances Oncolytic Viral Therapy for Glioblastoma
Walter H. Meisen,1 Jayson Hardcastle,2 Jeffrey Wojton,1 Eric Wohleb,3 Christopher Alvarez-Breckenridge,1 Pete PowAwpongkul,1 Jonathan Godbout,3 Balveen Kaur.1 1 Neurological Surgery, Ohio State Wexner Medical Center, Columbus; 2Molecular Medicine, Mayo Clinic, Rochester; 3 Neuroscience, Ohio State Wexner Medical Center, Columbus.
Objectives: A major barrier to oncolytic virus (OV) efficacy for the treatment of Glioblastoma is the innate immune response to OV infection; this response limits virus replication and reduces tumor cell destruction. Our lab developed a novel OV, RAMBO, which expresses the anti-angiogenic fragment Vasculostatin (Vstat120). In addition to its anti-angiogenic effects, we observe the RAMBO virus replicates more efficiently and persists longer in vivo than a control virus (rHSVQ1). The primary objective of this study is to investigate the effects RAMBO directed Vstat120 expression on microglia/ macrophage activation and OV efficacy. Methodology: Mice bearing intracranial tumors were treated with RAMBO, rHSVQ1, or PBS. Treated tumors were analyzed by flow cytometry to examine monocyte/microglia activation/recruitment. Coculture experiments were established with microglia and human glioma cells infected with RAMBO or rHSVQ1 virus. Cytokines Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
178. Targeting Glioblastoma Stem Cells with Oncolytic HSV and PARP Inhibitors
Jianfang Ning,1 Hiroaki Wakimoto, Robert L. Martuza,1 Samuel D. Rabkin.1 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 do 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 2 of 3 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 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
1,2
1
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 6 of 7 (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<0.05). Furthermore, our results of xenograft models of nude mice transplanted with L-OHP-resistant WiDr and HT29 cells showed that intratumoral CVA11 administrations at low dose (3×103 TCID50) significantly suppressed the outgrowth of preestablished subcutaneous xenografts and prolonged the survival of the tumor-bearing mice compared with untreated mice (p<0.01). Of importance, repeated experiments revealed that all of mice that received CVA11 treatment elicited no significant change of body weight during the treatment. Lastly, our result of caspase-3 staining assays showed that CVA11 infection induced robust caspase 3-postive cells (18%) in colorectal cancer cells as indicative of apoptotsis, while L-OHP alone displayed less caspase-3 activity (6%). Of note, combination treatment of z-VAD pan-caspase inhibitor with CVA11 infection exhibited significantly less oncolytic activity in CRC cells compared with CVA11 treatment alone (p<0.001). Taken together, our findings for the first time showed that CVA11 displayed remarkable oncolytic activity against oxaliplatin-resistant human colorectal cancer cells both in vitro and in vivo, and that both of the PI3K/Akt pathway and caspase-dependent apoptosis contributed to the process of CVA11-driven oncolysis, providing a new avenue to improve therapeutic outcomes for CRC patients.
180. Vstat120 Reduces the Innate Inflammatory Response To Infection and Enhances Oncolytic Viral Therapy for Glioblastoma
Walter H. Meisen,1 Jayson Hardcastle,2 Jeffrey Wojton,1 Eric Wohleb,3 Christopher Alvarez-Breckenridge,1 Pete PowAwpongkul,1 Jonathan Godbout,3 Balveen Kaur.1 1 Neurological Surgery, Ohio State Wexner Medical Center, Columbus; 2Molecular Medicine, Mayo Clinic, Rochester; 3 Neuroscience, Ohio State Wexner Medical Center, Columbus.
Objectives: A major barrier to oncolytic virus (OV) efficacy for the treatment of Glioblastoma is the innate immune response to OV infection; this response limits virus replication and reduces tumor cell destruction. Our lab developed a novel OV, RAMBO, which expresses the anti-angiogenic fragment Vasculostatin (Vstat120). In addition to its anti-angiogenic effects, we observe the RAMBO virus replicates more efficiently and persists longer in vivo than a control virus (rHSVQ1). The primary objective of this study is to investigate the effects RAMBO directed Vstat120 expression on microglia/ macrophage activation and OV efficacy. Methodology: Mice bearing intracranial tumors were treated with RAMBO, rHSVQ1, or PBS. Treated tumors were analyzed by flow cytometry to examine monocyte/microglia activation/recruitment. Coculture experiments were established with microglia and human glioma cells infected with RAMBO or rHSVQ1 virus. Cytokines Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy