Effect Of OBP-301 Research Articles

Abstract 4852: Boosting immunity against pancreatic cancer by OBP-702 (Pfifteloxin), telomerase-specific replicative adenovirus armed with wild-type p53 gene

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal disease with a 5-year survival rate of less than 10%. Although immune checkpoint blockade has recently emerged as a novel antitumor therapy, PDAC is less sensitive to immunotherapy due to small number of tumor-infiltrating T cells. Recently, oncolytic virotherapy has been shown to stimulate the immune system as an immunogenic antitumor therapy. Activation of p53 has been also known to enhance antitumor immunity. In this study, we investigated the potential of telomerases-specific p53-expressing oncolytic adenovirus (OBP-702, Pfifeteloxin) for inducing the immunogenic cell death in PDAC cells. Methods: OBP-702 (Pfifteloxin) is a telomerase-specific oncolytic adenovirus, in which the human telomerase reverse transcriptase (hTERT) promoter drives the expression of the viral E1 gene for tumor-specific replication, that expresses the wild-type p53 by inserting the human p53 cDNA at the deleted E3. OBP-301 (Telomelysin) is an original telomerase-specific adenovirus lacking p53. OBP-502 is an RGD mutant fiber-containing OBP-301. We used 4 human PDAC cell lines (MIA PaCa-2, Capan-1, BxPC-3, Panc-1) and mouse PDAC cell line (Pan02). In vitro antitumor effect of these viruses was evaluated using a XTT assay. The molecular mechanism of virus-mediated cell death was investigated by western blot analysis. The virus-induced immunogenic cell death was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1) using ELISA assay. The comparative in vivo antitumor efficacy was also investigated using a syngeneic Pan02 subcutaneous tumor model. Results: Telomerase-specific oncolytic adenoviruses induced the antitumor effect in all PDAC cells in a dose-dependent manner. The antitumor effect of OBP-702 was superior to that of OBP-301 or OBP-502. OBP-301 and OBP-502 mainly induced autophagy, whereas OBP-702 induced autophagy and apoptosis at 72 h after infection. The concentration of extracellular ATP and HMGB1 was significantly increased in OBP-702-infected PDAC cells compared to OBP-301- or OBP-502-infected cells at 24 and 48 h after infection. In mice carrying subcutaneous Pan02 murine PDAC tumors, intratumoral injection of OBP-702 resulted in a significant inhibition of tumor growth. Moreover, the number of tumor-infiltrating CD8+ T cells was significantly increased in OBP-702-treated groups compared to mock-treated group. Conclusion: Our data suggest that telomerases-specific p53-expressing oncolytic adenovirus OBP-702 (Pfifteloxin) induces profound immunogenic cell death to boost the immune responses in PDAC. Clinical trials with OBP-702 and immune checkpoint inhibitors for PDAC are warranted. Citation Format: Hiroyuki Araki, Hiroshi Tazawa, Takuro Fushimi, Takeyoshi Nishiyama, Satotu Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Boosting immunity against pancreatic cancer by OBP-702 (Pfifteloxin), telomerase-specific replicative adenovirus armed with wild-type p53 gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4852.

Abstract 4823: Elimination of invasive pancreatic cancer cells by p53-activating oncolytic virotherapy as novel precision medicine

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal disease due to early onset of local recurrence and distant metastasis. Invasive PDAC cells are the main causes of recurrence and metastasis after curative surgery. The precision medicine based on the genetic alterations in the KRAS, p53, CDKN2A, and SMAD4 genes has been recently expected as a promising strategy for the treatment of PDAC patients; however, therapeutic strategy for targeting these genetic alterations in PDAC has not been developed yet. To eliminate p53-inactivated malignant tumor cells, we have developed telomerase-specific replication-competent oncolytic adenovirus OBP-702 that expresses tumor suppressor p53. In this study, we investigated the in vitro and in vivo therapeutic potential of OBP-702 against PDAC cells. Methods: Four human PDAC cell lines (Capan-1, MIA PaCa-2, BxPC-3, Panc-1) with different invasive property were used. The therapeutic effect of OBP-702 and p53-nonexpressing OBP-301 was assessed in the proliferation, migration and invasion abilities of PDAC cells. The underlying mechanism of virus-mediated therapeutic effect was analyzed on the modulation of p53 signaling and KRAS-MAPK signaling. Subcutaneous and orthotopic BxPC-3 xenograft tumor models were used to evaluate the virus-mediated antitumor efficacy. Results: OBP-702 induced antitumor effect in association with autophagy and apoptosis more strongly compared to OBP-301 in PDAC cells through activation of p53 expression. OBP-702 inhibited the migration and invasion properties of PDAC cells more efficiently compared to OBP-301 through suppression of KRAS-ERK1/2 signaling, even when ERK signaling is enhanced by nerves and neurosecretory factors. Similar with OBP-702, treatment with ERK1/2 inhibitor or siRNA significantly reduced migration and invasion abilities. Moreover, OBP-702 significantly suppressed tumor growth in subcutaneous and orthotopic BxPC-3 xenograft tumor models. Conclusions: Our results suggest that OBP-702 is a promising antitumor reagent to eliminate invasive PDAC cells through p53 activation and ERK suppression. Further clinical study is warranted to evaluate the safety and feasibility of OBP-702 as a novel precision medicine based on genetic alterations in KRAS and p53 genes. Citation Format: Takuro Fushimi, Hiroshi Tazawa, Takeshi Koujima, Hiroyuki Araki, Takeyoshi Nishiyama, Satoru Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Elimination of invasive pancreatic cancer cells by p53-activating oncolytic virotherapy as novel precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4823.

Abstract 4851: Involvement of extracellular vesicles in the abscopal effect induced by oncolytic adenovirotherapy

Abstract Background: The abscopal effect is a phenomenon that shrinkage of metastatic tumors occurs concurrently with shrinkage of primary tumors by local treatments such as radiotherapy, and is generally considered to be caused through activation of the immune system. Extracellular vesicles (EVs) are recent interesting topics in scientific fields and are known to play important roles in a variety of intercellular communication processes including immune response. We previously developed a telomerase specific oncolytic adenovirus, Telomelysin (OBP-301), which is currently being tested in clinical trials, and have sometimes observed therapeutic effects on metastatic tumors after local injection to primary tumors in immunodeficient mouse models. Here, we hypothesized that EVs derived from OBP-301-treated tumors may be associated with the abscopal effect of OBP-301. Methods: A human colon carcinoma cell line HCT116 and HCT116 expressing RFP (HCT116-RFP) were used in this study. EVs were isolated by ultracentrifugation from HCT116 cells treated with OBP-301 (Exo-301). Intercellular communication and cytotoxic activity via EVs after OBP-301 treatment were assessed in a non-contact co-culture system in vitro and a bilateral subcutaneous tumor mouse model in vivo. Results: Exo-301 showed potent cytotoxic effects on HCT116 cells and this cytotoxicity was significantly blocked in the presence of anti-CD9 antibody, which suggested that Exo-301, not OBP-301 itself, was involved in this cytotoxicity. In non-contact co-culture experiments, more RFP was taken up into HCT116 cells after OBP-301 treatment on HCT116-RFP cells than control treatments, which meant that EVs including RFP reached HCT116 cells through membrane after OBP-301 treatment on HCT116-RFP cells. In a HCT116 bilateral subcutaneous tumor model in BALB/c nude mice, intratumoral injection of OBP-301 significantly suppressed the growth of untreated tumors as well as treated tumors, and in a bilateral tumor model with HCT116 on one side and HCT116-RFP on the opposite side, immunohistochemical staining showed the presence of RFP in untreated HCT116 tumors after OBP-301 treatment on HCT116-RFP tumors. These findings suggested that EVs derived from OBP-301-treated tumors was possibly involved in some cytotoxic effects on the opposite untreated tumors. Conclusion: EVs may be an important factor in the abscopal effect brought by local treatment of OBP-301 besides immune activation. Citation Format: Yoshihiko Kakiuchi, Shinji Kuroda, Nobuhiko Kanaya, Kento Kumon, Tomoko Tsumura, Satoru Kikuchi, Masahiko Nishizaki, Shunsuke Kagawa, Hiroshi Tazawa, Yasuo Urata, Toshiyoshi Fujiwara. Involvement of extracellular vesicles in the abscopal effect induced by oncolytic adenovirotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4851.

Abstract 5915: Overexpression of tumor suppressor p53 gene by oncolytic adenovirus boosts immune responses in human pancreatic ductal adenocarcinoma cells

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the worst prognosis disease with an overall 5-year survival rate of less than 5%. Although immune checkpoint therapy has been emerged as a novel antitumor therapy, PDAC is less sensitive to immunotherapy due to small number of infiltrating T cells. Recently, oncolytic virotherapy has been shown to promote intratumoral T cell infiltration as an immunogenic antitumor therapy. In the current study, we explored whether overexpression of the wild-type p53 tumor suppressor gene could synergize with oncolysis for induction of immunogenic cell death in human PDAC cells. Methods: OBP-702 is a tumor-specific replication-competent oncolytic adenovirus, in which the hTERT promoter drives the expression of the viral E1A and E1B genes for tumor-specific viral replication and the Egr-1 promoter induces wild-type p53 expression. We used 4 PDAC cell lines (MIA PaCa-2, Capan-1, BxPC-3, Panc-1). Molecular mechanisms of OBP-702-mediated cell death was investigated. Migration and invasion properties were assessed using transwell chamber assay. In addition, virus-induced immunogenic cell death was assessed by analyzing the level of extracellular ATP and high-mobility group box protein B1 (HMGB1). In vivo antitumor effect of OBP-702 was assessed using an orthotopic BxPC-3 xenograft tumor model. Results: At 72 hours after virus infection, OBP-702 induced profound antitumor effect in all human PDAC cells. OBP-702 induced autophagy- and apoptosis-related cell deaths. In migration and invasion assays, MIA Paca-2 and Capan-1 cells were low-invasive type, and Panc-1 and BxPC-3 cells were high-invasive type. OBP-702 inhibited migration and invasion properties of high-invasive type PDAC cells At 24 and 48 hours after virus infection, the secretion of ATP was significantly higher in OBP-702-infected PDAC cells compared to p53-null virus-infected cells. Moreover, OBP-702 significantly induced the higher amount of extracellular HMGB1 in all PDAC cells. Finally, OBP-702 significantly suppressed tumor growth in an orthotopic BxPC-3 xenograft tumor model compared to p53-null virus. Conclusions: Our data suggest that oncolytic adenovirus-mediated p53 overexpression induces more profound immunogenic cell death to boost the immune responses in PDAC. This additional immunological function of p53 is quite attractive and warrants clinical trials combined with immune checkpoint inhibitors. Citation Format: Hiroyuki Araki, Hiroshi Tazawa, Takeshi Ieda, Takuro Fushimi, Shinji Kuroda, Ryuichi Yoshida, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Overexpression of tumor suppressor p53 gene by oncolytic adenovirus boosts immune responses in human pancreatic ductal adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5915.

Abstract 5188: Molecular radiosensitization in soft tissue sarcomas by telomerase-specific oncolytic adenovirus

Abstract INTRODUCTION:Treatment options for soft tissue sarcoma (STS) include surgical resection and adjuvant chemotherapy and radiotherapy. Despite the development of combined modality treatments in recent years, a significant proportion of patients with sarcomas respond poorly to adjuvant therapy, leading to local recurrence or distant metastasis. Therefore, there is an urgent need to develop novel therapeutic strategies for improvement of patient prognoses. We previously reported the therapeutic potential of OBP-301, a telomerase-specific oncolytic adenoviruses, and OBP-702, an armed OBP-301 expressing the wild-type p53 tumor suppressor gene against bone and soft tissue sarcoma cells. Furthermore, the synergistic anti-tumor effect of OBP-301 in combination with radiation has been observed. While OBP-702 suppressed the viability of OBP-301-sensitive and -resistant osteosarcoma cells more efficiently, the combination effect of OBP-702 and radiotherapy has been unknown. In this study, we investigated the radiosensitizing effect of OBP-702 against human STS cells. METHODS:We used four human STS cell lines, HT1080 (fibrosarcoma), NMS-2 (malignant peripheral nerve sheath tumor), and SYO-1 (synovial sarcoma). Cells were irradiated 24 h after infection with OBP-301 and OBP-702, and cell viability was assessed by XTT assay 4 days after irradiation. Combined effect of radiation with OBP-301 and OBP-702 was analyzed with the CalcuSyn software (BioSoft). These cells were also analyzed for apoptosis and DNA damage using Western blot analysis. RESULTS:While OBP-301 and OBP-702 showed anti-tumor effect for STS cell lines respectively, HT1080 and NMS-2 were highly resistant to radiation. When combined with radiation, not only OBP-301 but also OBP-702 enhanced the inhibitory anti-tumor effect in all STS cell lines. The calculation of combination index demonstrated additive or synergistic anti-tumor effect in combination therapy. Further analysis revealed that OBP-301 and OBP-702 increased radiation-induced apoptosis in STS cells. Notably, the radio-sensitizing effect of OBP-702 was associated with increase of p53 expression and interruption of anti-apoptotic myeloid cell leukemia 1 (MCL1) expression. DISCUSSION:Our study demonstrated that OBP-702 had much stronger anti-tumor effect compared to OBP-301, and sensitized radiotherapy to various types of osteosarcoma cell lines. Currently, the clinical trial of OBP-301 is performed, and preclinically OBP-702 accumulates good therapeutic results in various tumors. Thus, OBP-702 may provide a novel treatment strategy for STSs and wide application of radiotherapy for localized as well as advanced STSs. Citation Format: Tadashi Komatsubara, Toshinori Omori, Hiroshi Tazawa, Kazuhisa Sugiu, Yusuke Mochizuki, Yasuaki Yamakawa, Shuhei Osaki, Joe Hasei, Tomohiro Fujiwara, Toshiyuki Kunisada, Yasuo Urata, Tsoshifumi Ozaki, Toshiyoshi Fujiwara. Molecular radiosensitization in soft tissue sarcomas by telomerase-specific oncolytic adenovirus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5188. doi:10.1158/1538-7445.AM2017-5188

Abstract 4299: Chemosensitizing effect of telomerase-dependent oncolytic adenovirus through virally induced microRNA-29-mediated MCL1 downregulation in osteosarcoma cells

Abstract Osteosarcoma is a rare highly malignant tumor. It is generally refractory to chemotherapy, which contributes to its poor prognosis. The enhancement of chemosensitivity is a potential approach to improve the prognostic outcome of osteosarcoma patients. We developed a telomerase-specific replication-competent oncolytic adenovirus, OBP-301. We recently confirmed the antitumor effect of OBP-301 monotherapy in human osteosarcoma cells. We also revealed the synergistic chemosensitizing effect of OBP-301 in human epithelial malignant tumor cells. In this study, we investigated the chemosensitizing effect of OBP-301 and its underlying mechanisms in human osteosarcoma cells. We used four human osteosarcoma cell lines, SaOS-2, MNNG/HOS, HOS and 143B. OBP-301 is an attenuated adenovirus, in which the hTERT promoter drives the expression of E1 gene, and causes tumor-selective lysis in a variety of human malignant tumor cells with telomerase activity. OBP-301 infection enhanced the cytotoxic effect of chemotherapeutic agents (cisplatin and doxorubicin). The calculation of combination index revealed the synergistic effects in all four human osteosarcoma cell lines. Combination of OBP-301 increased chemotherapy-induced apoptosis. To clarify the molecular mechanism underlying the chemosensitizing effect of OBP-301, we investigated whether OBP-301 affects the expression of anti-apoptotic BCL2 family proteins. In SaOS-2 and MNNG/HOS cells, the expression level of MCL1 was markedly decreased by OBP-301 infection. MCL1 knockdown by siRNA enhanced the chemotherapy-induced apoptosis as well as OBP-301. We recently revealed that OBP-301 increase the expression of cellular miRNAs in human cancer cells. To investigate the underlying mechanism of OBP-301-mediated MCL1 suppression, we determined whether OBP-301 upregulates MCL1-targeted miRNAs. OBP-301 dose-dependently upregulated the expression of MCL1 targeted miR-29 in SaOS-2 and MNNG/HOS cells. Exogenously introduced miR-29 efficiently suppressed MCL1 expression and enhanced the chemotherapy-induced apoptosis. Moreover, combination treatment significantly inhibited tumor growth, as compared to monotherapy, in a subcutaneous xenograft tumor model. MCL1 is overexpressed in various types of human tumor cells. Overexpression of anti-apoptotic proteins is an important factor to prevent chemotherapy-induced apoptosis in cancer. We demonstrated OBP-301 efficiently kills human osteosarcoma cells and markedly sensitizes them to chemotherapy. MCL1 suppression via OBP-301-induced miR-29 is critical as the underlying mechanism of the OBP-301 mediated chemosensitizing effect. These results suggest that replicative virus-mediated tumor specific MCL1 ablation may be a promising strategy to attenuate chemoresistance in osteosarcoma patients. Citation Format: Shuhei Osaki, Hiroshi Tazawa, Joe Hasei, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Chemosensitizing effect of telomerase-dependent oncolytic adenovirus through virally induced microRNA-29-mediated MCL1 downregulation in osteosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4299. doi:10.1158/1538-7445.AM2017-4299

Abstract 5816: Ablation of oncogenic MYCN expression by hTERT-driven oncolytic adenovirus induces cell death in MYCN-amplified neuroblastoma

Abstract Background: Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. Amplification and overexpression of the MYCN proto-oncogene occur in approximately 20% of NB. Since MYCN activation is highly associated with poor prognosis, MYCN is an attractive target for the treatment of MYCN-amplified NB. MYCN is a transcriptional activator that induces the expression of many down-stream target genes including human telomerase reverse transcriptase (hTERT). We developed two types of hTERT-driven oncolytic adenoviruses, OBP-301 and OBP-702, in which the hTERT promoter drives the expression of the viral E1A and E1B genes for tumor-specific virus replication, and OBP-702 further expresses tumor suppressor p53 protein. Although hTERT-driven oncolytic viruses are expected to be effective against MYCN-amplified NB with hTERT activation, the therapeutic potential of these viruses in MYCN-amplified NB remains unclear. In this study, we investigated whether OBP-301 and OBP-702 induce cell death and modulate MYCN expression in MYCN-amplified NB cells. Methods: We used 3 human NB cell lines with MYCN amplification, including IMR-32, CHP-134 and LA-N-5. The antitumor effect of OBP-301 and OBP-702 was evaluated using XTT assay. Virus-mediated cell death and modulation of MYCN and E2F1 expression were analyzed by Western blot analysis. The mRNA expression of hTERT and MYCN was analyzed by real-time RT-PCR analysis. To explore the role of E2F1 in the virus-mediated MYCN modulation, Ad-E2F1, a replication-defective adenovirus expressing E2F1 gene, was further used. Results: All NB cell lines showed high hTERT mRNA expression. OBP-301 and OBP-702 showed profound antitumor effect through autophagy-related cell death in all NB cell lines. Both viruses induced E1A and its target mediator E2F1 expression in these cell lines. Expression of MYCN mRNA and protein was downregulated by these viruses. Replication-deficient Ad-E2F1 infection also downregulated the expression of MYCN. Conclusions: These results suggest that hTERT-driven oncolytic adenoviruses are promising antitumor agent for the treatment of MYCN-amplified NB. These viruses induce profound autophagy through hTERT-dependent viral replication. E2F1 upregulation by viral infection is supposed to be one of the causes for MYCN downregulation and autophagy-related cell death. In vivo experiments are under way to investigate the antitumor effect of these viruses against xenograft NB tumors. Citation Format: Terutaka Tanimoto, Hiroshi Tazawa, Hiroshi Noso, Takanori Oyama, Yasuo Urata, Shunsuke Kagawa, Takuo Noda, Toshiyoshi Fujiwara. Ablation of oncogenic MYCN expression by hTERT-driven oncolytic adenovirus induces cell death in MYCN-amplified neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5816. doi:10.1158/1538-7445.AM2017-5816

Abstract 2448: Suppression of MYCN-driven neuroblastoma malignant phenotype by telomerase-targeted tumor suppressor p53 transactivation

Abstract Background: Neuroblastoma (NB) is a primary malignant tumor in the peripheral sympathetic nervous system. High-risk group of NB has an unfavorable clinical course, even after treatment with current chemotherapy regimens and aggressive surgical resection. Therefore, a novel therapeutic strategy is needed for the treatment of high-risk group of NB. Recent accumulating evidences in whole-genome sequencing analysis have shown that human telomerase reverse transcriptase (hTERT) gene rearrangement and MYCN gene amplification are predictive biomarkers for the high-risk group of NB, in which massive transcriptional activation of hTERT mRNA was frequently observed. We recently developed two types of hTERT-driven oncolytic adenoviruses, OBP-301 and OBP-702, in which the hTERT promoter drives the expression of the viral E1A and E1B genes for tumor-specific virus replication, and OBP-702 further expresses tumor suppressor p53 protein. In this study, we investigated whether OBP-301 and OBP-702 inhibit cell viability and MYCN protein expression in human NB cells with different MYCN status. Methods: We used 3 human NB cell lines with or without MYCN amplification, including IMR-32 (MYCN-amplified), LA-N-5 (MYCN-amplified), SK-N-SH (MYCN-not amplified). The antitumor effects of OBP-301, OBP-702, and Ad-p53, a replication-defective adenovirus expressing p53 gene, were evaluated using XTT assay. Virus-induced apoptosis and MYCN suppression were analyzed by Western blot analysis. Results: OBP-301, OBP-702, and Ad-p53 suppressed the viability of all 3 NB cell lines, whereas OBP-702 showed the most profound anti-tumor effect especially in MYCN-amplified NB cells. OBP-702 induced higher p53 expression than Ad-p53, resulting in the p53-mediated apoptotic cell death. Although MYCN expression in NB cells with MYCN amplification was downregulated after infection with all types of adenoviruses, the inhibitory effect of OBP-702 was the strongest among these adenoviruses. Conclusions: These results suggest that a hTERT-driven oncolytic adenovirus OBP-702 is a promising antitumor agent to induce profound apoptotic cell death through p53 transactivation and MYCN suppression in human NB cells with or without MYCN amplification. In vivo experiments are under way to investigate the antitumor effect of OBP-702 against xenograft NB tumors. Citation Format: Terutaka Tanimoto, Hiroshi Tazawa, Hiroshi Noso, Takanori Oyama, Yasuo Urata, Shunsuke Kagawa, Takuo Noda, Toshiyoshi Fujiwara. Suppression of MYCN-driven neuroblastoma malignant phenotype by telomerase-targeted tumor suppressor p53 transactivation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2448.

Abstract 3748: Virally enhanced p53 reactivation impairs KRAS-driven pancreatic cancer invasion

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the worst prognosis disease with an overall 5-year survival rate of less than 5%. More than 80% of patients are not eligible for curative surgical resection due to extensive local tumor invasion and early systemic metastasis at the time of diagnosis. Moreover, even after curative surgery, PDAC patients still have poor prognosis due to local recurrence and systemic metastasis. Although activation of oncogenic KRAS is implicated in the initiation, promotion and progression of pancreatic cancer, the development of cancer treatment targeting KRAS remains unsuccessful. Moreover, tumor suppressor p53 is also frequently inactivated in PDAC patients. We recently developed two types of tumor-specific replication-competent oncolytic adenoviruses, OBP-301 and OBP-702, which is modified OBP-301 to express tumor suppressor p53 protein. In this study, we investigated whether oncolytic adenoviruses inhibit cell viability, migration, invasion and KRAS-driven signaling pathway in PDAC cells. Methods: We used 4 PDAC cells (MIA PaCa-2, Capan-1, Panc-1, BxPC-3). The cell viability was examined by XTT assay. Migration and invasion properties were assessed using transwell chamber assay. The modulation of KRAS-driven signaling pathway was investigated by Western blot analysis. Results: OBP-301 induced moderate antitumor effect in MIA Paca-2, Capan-1 and BxPC-3 cells and strong antitumor effect in Panc-1 cells in a dose-dependent manner, whereas OBP-702 induced profound antitumor effect in all human PDAC cells, suggesting the broad spectrum of OBP-702's efficacy. OBP-301 induced autophagy-related cell death, whereas OBP-702 induced autophagy- and apoptosis-related cell deaths. In migration and invasion assays, MIA Paca-2 and Capan-1 cells were low-invasive type, and Panc-1 and BxPC-3 cells were high-invasive type. OBP-301 and OBP-702 inhibited migration and invasion properties of high-invasive type PDAC cells, and the inhibitory effect of OBP-702 was stronger than that of OBP-301. We also found that oncolytic adenoviruses inhibited the expression of KRAS and KRAS-downstream target MEK/ERK proteins in high-invasive type PDAC cells. Conclusions: Our results suggest that oncolytic adenoviruses, OBP-301 and OBP-702, inhibit the survival and invasive phenotype of pancreatic cancer by suppressing KRAS-driven signaling pathway and reactivating p53-driven signaling pathway. In vivo experiments are under way to investigate the anti-tumor and anti-invasive effects of oncolytic adenoviruses using BxPC-3 xenograft tumors. The clinical trial of intratumoral administration of oncolytic adenoviruses in patients with invasive PDAC should be also warranted. Citation Format: Takeshi Koujima, Hiroshi Tazawa, Takeshi Ieda, Kazuya Kuwada, Terutaka Tanimoto, Shinji Kuroda, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Virally enhanced p53 reactivation impairs KRAS-driven pancreatic cancer invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3748.

Abstract 1794: Preclinical evaluation of radiotherapy in combination with radio-sensitizing telomerase-specific oncolytic virus for human bone and soft tissue sarcomas

Abstract Background: Despite major advances in the treatment of bone and soft tissue sarcomas, including surgery, chemotherapy, and radiation, some sarcoma patients show poor prognosis due to resistance to conventional therapy. Therefore, the development of a novel therapeutic strategy for sarcoma patients is needed. We recently revealed that a telomerase-specific, replication-competent oncolytic adenovirus OBP-301 efficiently killed human sarcoma cells. Moreover, combination therapy with OBP-301 and radiation has been confirmed to show synergistic antitumor effect in epithelial malignant tumor cells; however, its effect in human sarcoma cells remains elusive. In this study, we investigated the antitumor effect of OBP-301 in combination with radiation against human sarcoma cells. Methods: We used five human sarcoma cell lines, SK-ES-1 (Ewing sarcoma), RD-ES (Ewing sarcoma) SYO-1 (synovial sarcoma), U2OS (osteosarcoma) and HOS (osteosarcoma). Cells were irradiated 24 h after infection with OBP-301, and cell viability was assessed by XTT assay 4 days after irradiation. Combined effect of radiation with OBP-301 was analyzed with the CalcuSyn software (BioSoft). These cells were analyzed for apoptosis using western blot analysis. To analyze the effect of OBP-301 in DNA repair process, immunofluorescence staining was performed after treatment of irradiation with or without OBP-301 infection on SK-ES-1 cell. We further investigated the in vivo combined effect of OBP-301 and radiation. The SK-ES-1 tumor-bearing mice were irradiated at a dosage of 1 Gy/tumor once per week for three cycles, and OBP-301 (1 × 108 plaque-forming units per tumor), or PBS was injected into the tumor 3 times per week for three cycles. Results: Combination treatment with OBP-301 and radiation showed synergistic or additive antitumor effect in all human sarcoma cells. Western blot analysis showed that combination treatment increased the expression of cleaved-PARP, and prolonged the level of γH2AX protein than radiation treatment. Combination therapy of OBP-301 with radiation showed antitumor effect more significantly than monotherapy in SK-ES-1 xenograft tumor model. Conclusions: These results suggest that combination therapy of OBP-301 with radiation is a promising antitumor strategy for bone and soft tissue sarcomas. Citation Format: Toshinori Omori, Yasuaki Yamakawa, Joe Hasei, Hiroshi Tazawa, Shuhei Osaki, Tusyoshi Sasaki, Kazuhisa Sugiu, Tomohiro Fujiwara, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Preclinical evaluation of radiotherapy in combination with radio-sensitizing telomerase-specific oncolytic virus for human bone and soft tissue sarcomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1794. doi:10.1158/1538-7445.AM2015-1794

Abstract 5338: Preclinical study of telomerase-specific p53 tumor suppressor gene overexpression in human scirrhous gastric cancer cells with different p53 status

Abstract Background: Despite multimodal therapy for scirrhous gastric cancer, many patients show refractory to conventional therapy and rapid progression, leading to poor prognosis. Therefore, the development of novel therapeutic strategy is required to improve the clinical outcome in patients with scirrhous gastric cancers. As novel therapeutic strategies, Ad-p53, a replication-defective adenovirus expressing a tumor suppressor p53 gene, is currently under clinical evaluation for various types of cancers. We recently developed a novel p53-expressing telomerase-specific replication-competent oncolytic adenovirus OBP-702, which induces the p53 gene expression in a telomerase-dependent manner. In this study, we investigated the antitumor effects of Ad-p53 and OBP-702 in human scirrhous gastric cancer cells with different p53 status. Methods: We used 3 human scirrhous gastric cancer cell lines with different p53 status, NUGC4 (p53 wild-type), GCIY (p53 mutant-type), and KATOIII (p53 null). The antitumor effects of Ad-p53 and OBP-702 were evaluated using XTT assay. The 50% inhibiting dose (ID50) value of Ad-p53 and OBP-702 for each cell was calculated using cell viability data obtained 3 days after virus infection. We evaluated the virus-mediated cell death in the sphere-forming cells by LIVE/DEAD assay. The apoptosis- and autophagy-related cell death was assessed by western blot analysis for cleaved poly (ADP-ribose) polymerase (PARP) and p62. Results: Ad-p53 suppressed the cell viability in p53-inactivated scirrhous gastric cancer cells (KATO III, GCIY), whereas p53-intact NUGC4 cells were resistant to Ad-p53. OBP-702 efficiently suppressed the cell viability in all scirrhous gastric cancer cells with different p53 function. The ID50 value of OBP-702 was lower than that of Ad-p53 in all cell lines. LIVE/DEAD assay showed that OBP-702 induced cell death more efficiently than Ad-p53 in the sphere-forming cells. OBP-702 induced more profound p53 expression and apoptotic cell death than Ad-p53. Moreover, only OBP-702 induced not only apoptosis but also autophagy. Conclusions: These results suggest that p53-expressing tumor-specific oncolytic adenovirus OBP-702 is a promising antitumor agent to induce profound cell death in scirrhous gastric cancers with different p53 status. Now, in vivo experiments are under way to investigate the antitumor effect of OBP-702 in the peritoneal dissemination of scirrhous gastric cancer cells. Citation Format: Naoto Hori, Hiroshi Tazawa, Masahiko Nishizaki, Satoru Kikuchi, Shuya Yano, Michihiro Ishida, Megumi Watanabe, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Preclinical study of telomerase-specific p53 tumor suppressor gene overexpression in human scirrhous gastric cancer cells with different p53 status. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5338. doi:10.1158/1538-7445.AM2015-5338

Abstract 3535: A novel tumor-specific oncolytic biological therapy against invasive pancreatic cancer

Abstract Pancreatic cancer is one of the worst prognosis disease all over the world and has an overall 5-year survival rate of less than 5%. At the time of diagnosis, more than 80% of patients are not eligible for curative surgical resection due to extensive local tumor invasion and early systemic metastasis. Moreover, even after curative surgery, pancreatic cancers still show poor prognosis due to local recurrence and systemic metastasis. Gemcitabine is the standard chemotherapeutic agent to treat advanced pancreatic cancers, but median overall survival ranged from 5.0 to 7.2 months, suggesting that standard chemotherapy is less effective to most of pancreatic cancer patients. Therefore, the development of novel therapeutic strategy is needed for the treatment of pancreatic cancers. We developed a novel oncolytic adenovirus, OBP-702, in which a human telomerase reverse transcriptase gene promoter drives viral E1 gene for virus replication, and armed with the wild-type tumor suppressor p53 gene. Precise antitumor effects of OBP-702 have been evaluated in human pancreatic cancer cells. We used four pancreatic cancer cell lines with different invasion ability, including non-invasive type (MIA PaCa-2, Panc-1) and invasive type (BxPC-3, Capan-1). The antitumor effect of OBP-702 for each cell line was assessed using XTT assay. OBP-702 induced profound anti-tumor effect in all human pancreatic cancer cells, whereas OBP-301 lacking the p53 tumor suppressor gene showed differential effects among the cell types. OBP-301 induced moderate anti-tumor effect in MIA Paca-2, BxPC-3 and Capan-1 cells and strong anti-tumor effect in Panc-1 cells in a dose-dependent manner, suggesting the broad spectrum of OBP-702's efficacy. The molecular mechanism of anti-tumor effect was assessed by Western blot analysis. OBP-301 induced autophagy-related cell death, whereas OBP-702 induced autophagy- and apoptosis-related cell deaths. These results suggest that an oncolytic adenovirus, OBP-702, is a promising antitumor agent to induce profound cell death in invasive human pancreatic cancer cells. The clinical trial of intratumoral administration of OBP-702 in patients with invasive pancreatic cancer is warranted. Citation Format: Takeshi Koujima, Hiroshi Tazawa, Naoto Hori, Shuta Tamura, Shinji Kuroda, Hiroyuki Kishimoto, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. A novel tumor-specific oncolytic biological therapy against invasive pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3535. doi:10.1158/1538-7445.AM2015-3535

Abstract 3531: Companion diagnostics-based telomerase-specific oncolytic virotherapy: preclinical evaluation in human colorectal cancer cell lines differentially affected in the RAS/RAF/MEK signaling pathway

Abstract Colorectal cancers harboring KRAS or BRAF mutations often show resistance to anti-cancer drugs, and are poor prognostic disease that causes metastasis and recurrence compared to those with wild type KRAS and BRAF. KRAS/BRAF-wild type colorectal cancers are sensitive to both cetuximab and panitumumab that are epidermal growth factor receptor (EGFR)-targeting agents. However, KRAS/BRAF-mutant colorectal cancers are resistant to EGFR-targeting agents because of constitutive activation of the EGFR-downstream RAS/RAF/MEK signaling pathway. Therefore, the development of novel therapeutic strategy is required to improve the clinical outcome in patients with KRAS/BRAF-mutant colorectal cancers. We developed two types of telomerase-specific replication-competent oncolytic adenoviruses, OBP-301 and OBP-702 armed with the wild-type p53 tumor suppressor gene. In this study, we established the companion diagnostics-based selection algorithm for our oncolytic viruses. We compared the antitumor effects of OBP-301 and OBP-702 in human colorectal cancer cells with wild type KRAS/BRAF (SW48, Colo320DM), mutated KRAS (DLD-1, SW620) and mutated BRAF (RKO, HT29). We evaluated the antitumor effect of OBP-301 and OBP-702 in human colorectal cancer cells using XTT assay. Oncolytic adenovirus-mediated induction of apoptosis- and autophagy-related cell death was analyzed by Western blot analysis. KRAS-mutant colorectal cancer cells were sensitive to both OBP-301 and OBP-702 as well as KRAS/BRAF-wild type cells. In contrast, BRAF-mutant colorectal cancer cells were sensitive to OBP-702 rather than OBP-301. Western blot analysis showed that OBP-301 induced autophagy, whereas OBP-702 induced both autophagy and apoptosis. Taken together, our data suggest that oncolytic viruses should be used according to the genetic background of the RAS/RAF/MEK signaling pathway, especially in patients with colorectal cancer. Citation Format: Shuta Tamura, Hiroshi Tazawa, Naoto Hori, Takeshi Koujima, Satoru Kikuchi, Shinji Kuroda, Hiroyuki Kishimoto, Takeshi Nagasaka, Masahiko Nishizaki, Yasuo Urata, Shunsuke Kagawa, Toshiyoshi Fujiwara. Companion diagnostics-based telomerase-specific oncolytic virotherapy: preclinical evaluation in human colorectal cancer cell lines differentially affected in the RAS/RAF/MEK signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3531. doi:10.1158/1538-7445.AM2015-3531

Abstract 3316: Oncolytic adenovirus-armed p53 induces apoptosis significantly through upregulating miR-93 and 106b in human osteosarcoma cells..

Abstract Bone and soft tissue sarcomasare the third most common cancer in children and account for 15.4% of all childhood malignancies. About one fourth of the patients show a poor response to conventional chemo-radiotherapy, resulting in subsequent recurrence and leading to a poor prognosis. Therefore, the development of a novel therapeutic strategy is required. We previously developed an oncolytic adenovirus, OBP-301 (Telomelysin), in which the hTERT gene promoter drives the expression of the E1A and E1B genes linked to an internal ribosome entry site (IRES). Recently, we revealed that OBP-301 shows the cytopathic activity in human bone and soft tissue sarcoma cells. However, some human osteosarcoma cells (MNNG/HOS, SaOS-2) were less sensitive to cytopathic activity of OBP-301. Therefore, we developed a telomerase-specific p53-armed oncolytic adenovirus (OBP-702). In this study, we examined the antitumor effect of OBP-702 in OBP-301-resistant osteosarcoma cells, and revealed the mechanism. We compared the antitumor effects of OBP-702 and OBP-301 using XTT assay. The 50% inhibiting dose (ID50) value of OBP-702 and OBP-301 for each cell was calculated using cell viability data obtained on day 5 after virus infection. We further evaluated the expression of p53, p21 and cleaved-PARP proteins using western blot analysis in OBP-301-resistant osteosarcoma cells after infection with OBP-702 or Ad-p53, which is a p53-expressing replication-defective adenovirus. OBP-702 showed more cytopathic activity compared to OBP-301. The ID50 value of OBP-702 was much lower than that of OBP-301 in all cell lines. Western blot analysis showed OBP-702 infection induced the cleavage of PARP in a dose-dependent manner and cell cycle analysis also showed that OBP-702 induced the highest percentages of sub-G1 population in MNNG/HOS cells compared to Ad-p53 or OBP-301. These results suggest that OBP-702 induces increased apoptosis compared to Ad-p53 or OBP-301. OBP-702 infection induced more profound p53 expression than Ad-p53, but p53-downstream target p21 proteins were not activated by OBP-702. The expression levels of miR-93 and miR-106b increased after OBP-702 infection. When SaOS-2 cells were infected with Ad-p53 at an MOI of 10 for 48 hours, pre-transfection with miR-93, miR-106b or both efficiently suppressed Ad-p53-induced p21 expression. These results suggest that OBP-702 suppresses p21 expression through E1A-dependent upregulation of miR-93 and miR-106b. In conclusion, we have clearly demonstrated that the p53-expressing oncolytic adenovirus OBP-702 has a much stronger antitumor effect than OBP-301 and Ad-p53. Oncolytic adenovirus-mediated p53 gene transduction would induce profound p53 expression without p21 activation regulated by miR-93 and 106b, resulting in the enhancement of antitumor effect. Citation Format: Joe Hasei, Tsuyoshi Sasaki, Hiroshi Tazawa, Yuuri Hashimoto, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Oncolytic adenovirus-armed p53 induces apoptosis significantly through upregulating miR-93 and 106b in human osteosarcoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3316. doi:10.1158/1538-7445.AM2013-3316

Abstract 584: Enhanced chemosensitivity of osteosarcoma cells by telomerase-specific oncolytic adenovirus in combination therapy.

Abstract Osteosarcomas are the most common malignant bone tumors. Although the multi-agent chemotherapy has improved the long-term survival rate of osteosarcoma patients, some patients show a poor response to chemotherapy, leading to recurrence and poor prognosis. Therefore, the enhancement of chemosensitivity is required to cure patients with osteosarcomas. We recently developed a telomerase-specific replication-competent oncolytic adenovirus, Telomelysin (OBP-301), and confirmed the antitumor effect of OBP-301 in human osteosarcoma cells. A phase I clinical trial in US has also shown the safety of OBP-301 in cancer patients. In this study, we investigated whether OBP-301 enhances the antitumor effect of chemotherapeutic agents, doxorubicin and cisplatin, that are used for the treatment of osteosarcomas, and the molecular mechanism of chemotherapy- and OBP-301-mediated cell death pathway, autophagy and apoptosis, in human osteosarcoma cell lines. We used four human osteosarcoma cell lines, HOS, MNNG/HOS, 143B and SaOS2. OBP-301 is an attenuated adenovirus, in which the human telomerase reverse transcriptase (hTERT) promoter element drives expression of E1A and E1B genes, and causes tumor-selective lysis in a variety of human malignant tumor cells with high telomerase activity. OBP-301 infection enhanced the cytotoxic effect of chemotherapeutic agents and the calculation of combination index revealed the synergistic effects in all human osteosarcoma cell lines. To analyze the molecular mechanism in the synergistic effect induced by combination therapy, western blot analysis for apoptosis (PARP) and autophagy (LC3, p62) was performed. Combination of OBP-301 increased both apoptosis (the cleavage of PARP) and autophagy (conversion of LC3-I to LC3-II and p62 downregulation) in the chemotherapeutic agents-treated human osteosarcoma cells. In contrast, the replication of OBP-301 was not suppressed by chemotherapeutic agents. Moreover, combination therapy of chemotherapeutic agents with OBP-301 significantly suppressed tumor growth in a subcutaneous xenograft tumor model compared to monotherapy with chemotherapeutic agents or OBP-301. These results suggest that combination therapy of chemotherapeutic agents with OBP-301 provides a novel therapeutic strategy for human osteosarcomas. Citation Format: Shuhei Osaki, Tsuyoshi Sasaki, Hiroshi Tazawa, Joe Hasei, Yasuaki Yamakawa, Yuuri Hashimoto, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Enhanced chemosensitivity of osteosarcoma cells by telomerase-specific oncolytic adenovirus in combination therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 584. doi:10.1158/1538-7445.AM2013-584

Abstract 4973: Telomerase-specific replication-selective oncolytic viruses for adenoid cystic carcinoma cell lines

Abstract There is no effective treatment of adenoid cystic carcinoma (ACC) in oral region, which low sensitivity of chemotherapy and radiation therapy. We previously showed that telomerase-specific replication-competent adenovirus (Telomelysin, OBP-301), in which the human telomerase reverse transcriptase (hTERT) promoter controls the adenoviral E1 gene expression, induces a selective antitumor effect in oral squamous cell carcinoma (OSCC) cells in vitro and in vivo orthotopic graft model. We conducted to evaluate the effectiveness of OBP301 against ACC. First, we could confirm that the expression of hTERT in two types of ACC cell lines (ACC2, ACCM) by Western blotting. Then, we examined the antitumor effect of OBP301 in human ACC cells in vitro. OBP-301 infection induced cell death in human ACC cells in a dose-dependent manner. We also examined the expression levels of coxsackievirus and adenovirus receptor (CAR) on the cell surface of each type of cell by flow cytometric analysis. Apparent amounts of CAR expression were detected on human ACC cells. Relative hTERT mRNA expression in ACC was analyzed by real-time RT-PCR, which showed high expression time dependent. In addition, OBP-401 is a genetically engineered adenovirus that expresses GFP by inserting the GFP gene under the control of the cytomegalovirus promoter at the deleted E3 region of OBP-301. Human ACC cells expressed bright GFP fluorescence as early as 12 h after OBP-401 infection. The fluorescence intensity gradually increased in a dose-dependent manner, followed by rapid cell death due to the cytopathic effect of OBP-401, as evidenced by floating, highly light-refractile cells under phase-contrast photomicrographs. In conclusion, our data clearly indicate that telomerase-specific oncolytic adenoviruses have a significant therapeutic potential against human ACC in vitro. These results suggested that treatment of OBP-301 and OBP-401 are possible to improvement of QOL of oral cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4973. doi:1538-7445.AM2012-4973

Abstract 5651: p53-mediated apoptotic signaling overcomes the resistance to oncolytic adenovirus in human osteosarcoma cells

Abstract Despite major advances in the treatment of bone and soft tissue sarcomas, about one fourth of the patients show a poor response to conventional therapy, resulting in subsequent recurrence and leading to a poor prognosis. Therefore, the development of a novel therapeutic strategy is required to cure patients with bone and soft tissue sarcomas. We previously developed an oncolytic adenovirus, OBP-301, in which human telomerase reverse transcriptase (hTERT) gene promoter drives viral E1 gene for replication. OBP-301 induces tumor-specific oncolytic cell death in a telomerase-dependent manner. Recently, we revealed that OBP-301 shows cytopathic activity in human bone and soft tissue sarcoma cells. However, some human osteosarcoma cells were less sensitive to cytopathic activity of OBP-301. To enhance the cytopathic activity of OBP-301, we recently developed a novel telomerase-specific oncolytic adenovirus, OBP-702, which expresses tumor suppressor p53 gene. In this study, we investigated the antitumor effect of OBP-702 in OBP-301-sensitive (OST, HOS and U2OS) and OBP-301-resistant (MNNG/HOS and SaOS-2) human osteosarcoma cells. We compared the antitumor effects of OBP-702 and OBP-301 using XTT assay. The 50% inhibiting dose (ID50) value of OBP-702 and OBP-301 for each cell was calculated using cell viability data obtained on day 5 after virus infection. We further evaluated the expression of p53, p21 and cleaved poly (ADP-ribose) polymerase (PARP) proteins using western blot analysis in both OBP-301-sensitive and OBP-301-resistant osteosarcoma cells after infection with OBP-702 or Ad-p53, which is a p53-expressing replication-defective adenovirus. OBP-702 showed more cytopathic activity compared to OBP-301 in both OBP-301-sensitive and OBP-301-resistant osteosarcoma cells. The ID50 value of OBP-702 was lower than that of OBP-301 in all cell lines. OBP-702 induced not only oncolysis, but also apoptotic cell death with the cleavage of PARP. OBP-702 infection induced more profound p53 expression than Ad-p53. However, p53-downstream target p21 proteins were not activated by OBP-702. Taken together, we demonstrated that the p53-expressing oncolytic adenovirus OBP-702 has a much stronger antitumor effect than OBP-301 in human osteosarcoma cells. Oncolytic adenovirus-mediated p53 gene transduction would induce profound apoptosis through p53 upregulation without p21 activation, resulting in the enhancement of antitumor effect by OBP-301. OBP-702 would be a promising antitumor reagent for the treatment of OBP-301-resistant human osteosarcoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5651. doi:1538-7445.AM2012-5651

Abstract 343: Inhibitory effect of oncolytic adenovirus on transforming growth factor- β-induced epithelial-mesenchymal transition in human cancer cells

Abstract Background: Epithelial-mesenchymal transition (EMT) is a tumor progression-related process by which epithelial cells lose their epithelial characteristics and acquire mesenchymal properties. EMT promotes the malignant phenotypes including invasion, metastasis and drug resistance, resulting in tumor recurrence and poor prognosis. Therefore, to prevent tumor progression, EMT targeted therapy is required. Oncolytic virotherapy is a promising strategy for anticancer therapy. Although a variety of oncolytic adenoviruses have been developed to induce tumor-selective cell death, the effect of oncolytic adenovirus on the EMT-induced tumor progression remains unclear. In this study, we investigated the biological effect of oncolytic adenovirus on EMT in human cancer cells. Methods: We previously generated a telomerase-specific replication-selective oncolytic adenovirus (OBP-301; Telomelysin), in which the human telomerase reverse transcriptase promoter drives the expression of E1A and E1B for virus replication. Transforming growth factor-β (TGF-β) was used to induce EMT in human lung (A549) and pancreatic (Panc-1) cancer cells. To investigate whether OBP-301 infection affects TGF-β-induced EMT in these cells, western blot and real-time PCR analysis for EMT-related markers were performed. We also examined cell mobility using transwell migration and invasion assays. In vitro antitumor effect of OBP-301 and chemotherapeutic agents in TGF-β-treated cancer cells were assessed by trypan blue exclusion assay. Results: Administration of TGF-β induced mesenchymal characteristics, including spindle-like cell morphology, down-regulation of E-cadherin, up-regulation of vimentin and high motility, in A549 and Panc-1 cells. When TGF-β-treated cancer cells were infected with OBP-301, suppression of E-cadherin expression was attenuated. Conversely, the expression levels of mesenchymal markers, vimentin and N-cadherin, and EMT-inducing transcription factors, Snail, Slug and ZEB1, were reduced. OBP-301 infection also inhibited TGF-β-induced enhancement of migration and invasion in cancer cells. Moreover, OBP-301 efficiently killed TGF-β-treated cancer cells, whereas TGF-β-treated cancer cells were resistant to cisplatin and docetaxel. Conclusion: Our results suggest that telomerase-specific oncolytic adenovirus OBP-301 inhibits TGF-β-induced EMT in human cancer cells. OBP-301 has therapeutic potential against cancer cells that have undergone EMT. Therefore, OBP-301 might be a promising anticancer agent to suppress EMT-mediated cancer progression and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 343. doi:1538-7445.AM2012-343

Abstract 5415: Preclinical evaluation of telomerase-specific oncolytic virotherapy for human bone and soft tissue sarcomas

Abstract Purpose: Bone and soft tissue sarcomas frequently occur in young children and show aggressive progression, resistance to conventional chemotherapy and poor prognosis, indicating a requirement for novel antitumor therapy to improve the clinical outcome. Tumor-specific replication-selective oncolytic virotherapy is a promising antitumor therapy for induction of cell death in tumor, but not of normal, cells. We previously developed an oncolytic adenovirus, OBP-301, that kills human epithelial malignant cells in a telomerase-dependent manner. Recent evidence suggests that non-epithelial malignant cells, which have low telomerase activity, maintain telomere length through alternative lengthening of telomeres (ALT). However, it remains unclear if OBP-301 is cytopathic for non-epithelial malignant cells. Here, we evaluated the antitumor effect of OBP-301 on human bone and soft tissue sarcoma cells. Experimental Design: The cytopathic activity of OBP-301, coxsackie and adenovirus receptor (CAR) expression, and telomerase activity were examined in 10 bones (OST, U2OS, HOS, HuO9, MNNG/HOS, SaOS-2, NOS-2, NOS-10, NDCS-1 and OUMS-27) and in 4 soft tissue (CCS, NMS-2, SYO-1 and NMFH-1) sarcoma cell lines. OBP-301 antitumor effects were assessed using orthotopic tumor xenograft models. The fiber-modified-OBP-301, (OBP-405), was used to confirm an antitumor effect on OBP-301-resistant sarcomas. Results: OBP-301 was cytopathic for 12 sarcoma cell lines but not for the non-CAR-expressing OUMS-27 and NMFH-1 cells. Sensitivity to OBP-301 was dependent on CAR expression, not on telomerase activity. ALT-type sarcomas were also sensitive to OBP-301 because of upregulation of human telomerase reverse transcriptase (hTERT) mRNA following virus infection. Intratumoral injection of OBP-301 significantly suppressed the growth of OST and SYO-1 tumors. Furthermore, fiber-modified OBP-405 showed antitumor effects on OBP-301-resistant OUMS-27 and NMFH-1 cells. Conclusions: We showed that OBP-301 has antitumor effects on both non-ALT-type and ALT-type sarcoma cells through upregulation of human telomerase reverse transcriptase mRNA. Furthermore, coxsackie and adenovirus receptor-negative sarcoma cells were efficiently killed by fiber-modified OBP-301-derived-OBP-405 through virus-integrin binding. A telomerase-specific oncolytic adenovirus is a promising antitumor reagent for the treatment of bone and soft tissue sarcomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5415. doi:10.1158/1538-7445.AM2011-5415