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Antibodies Targeted to Radiation Inducible Neoantigens in Cancer
Proceedings of the 53rd Annual ASTRO Meeting
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WITHDRAWN
TORC1/2 Inhibition with Concurrent Radiation Controls Inflammatory Breast Cancer in a Preclinical Animal Model Through Selective Blockade of Translation
E. P. Connolly, D. Silvera, T. Venuto, A. Sawai, R. J. Schneider New York University Medical Center, New York, NY Purpose/Objective(s): The PI3K/AKT/mTOR pathway is frequently deregulated in human cancers, including inflammatory breast cancer (IBC). mTOR (mammalian target of rapamycin) is a key regulator of protein synthesis; it links mRNA translation to the metabolic state of the cell and plays a key role in the signaling of malignant cell growth, proliferation, differentiation, migration, and survival. We have shown that mTOR activation following treatment with DNA damaging agents such as ionizing radiation (IR) is a primary protector of advanced breast cancer cells, including IBC cells. In these cells IR selectively increases translation of mRNAs for survival and DNA repair genes, including survivin, PARP, and DNA repair enzymes. We hypothesized that treatment with an mTOR inhibitor coupled with IR would provide a synergistic ability to control IBC. We examined the cytotoxic effects of combining mTOR inhibition and radiation in an IBC xenograft model, using either the catalytic mTORC1/2 inhibitor pp242, or allosteric mTORC1 inhibitor RAD001 (Everlimus). Materials/Methods: Experiments were conducted in SUM149 IBC cells. Cells were treated with RAD001 or pp242 alone or in combination with increasing IR from 0 - 8 Gy. In vitro studies preformed included: cell survival assay, immunoblot analysis, 35Smethionine labeling, cell cycle analysis by FACs, and caspase-3 activity assay. In vivo studies were performed in a SUM149 xenograft nude mouse model. Animals were treated with RAD001 and pp242 individually or in combination with IR and tumor growth was monitored. IHC, immune-fluorescence and TUNEL assays were also performed. Results: pp242 inhibited both mTORC1/2 signaling in IBC cells and blocked feedback upregulation of AKT and ERK, while RAD001 inhibited only mTORC1 and activated AKT through up-regulation of mTORC2. pp242 when combined with IR showed significantly greater tumor control in IBC xenografts and strongly enhanced radio-sensitivity in vitro, in contrast to RAD001, which had no added effect. Combining pp242 with IR promoted apoptosis, inhibition of both endogenous and radiation-induced AKT activation, and greater inhibition of mTOR signaling to its downstream effectors. This combination more efficiently inhibited overall translation, and likely blocks translation of specific mRNAs involved in DNA repair and survival functions. Conclusions: These studies demonstrate that targeted inhibition of mTORC1/2 synergizes with IR in a model system of IBC. Mechanistically, mTORC1/2 inhibition combined with IR increases apoptosis, prevents radiation induced pro-survival signals mediated through constitutively active AKT and selectively blocks translation of survival and DNA response mRNAs. Author Disclosure: E.P. Connolly: None. D. Silvera: None. T. Venuto: None. A. Sawai: None. R.J. Schneider: None.
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Radiation-Guided 64Cu-radiolabeled Anti-GRP78 To Glioblastoma
A. J. Chang, J. Jaboin, D. E. Hallahan Washington University in St. Louis, St. Louis, MO Purpose/Objective(s): Radiation-guided delivery of systemic agents may improve tumor response, while reducing normal tissue toxicity. We have found that a member of the heat-shock protein-70 family, GRP78, is induced in tumor microvasculature after exposure to low doses of ionizing radiation. We subsequently utilized high throughput screening of an antibody library in irradiated cancer cells to generate a highly specific and avid monoclonal antibody to GRP78. The purpose of this study was to image radiation-induced GRP78 expression in vivo using optical and PET imaging in a heterotopic glioblastoma model with our antibody engineered against GRP78. Materials/Methods: For optical imaging, anti-GRP78 was conjugated to Alexa Fluor 750 (AF750). For microPET/CT imaging, anti-GRP78 was conjugated with the chelator, p-SCN-Bn-NOTA, and labeled with 64Cu. 64Cu, a radionuclide with a half-life of 12.7 h, has both imaging and radiotherapeutic properties with its positron and beta decay. The radiolabeled antibody was checked for purity by TLC and HPLC analysis. Athymic nude mice were inoculated into the bilateral hindlimbs with GL261 glioblastoma cells and tumors were allowed to grow to approximately 0.7 cm. The left hindlimb was exposed to 3 Gy of ionizing radiation while special attention was made to block the right hindlimb from ionizing radiation. Optical imaging and microPET/CT studies were performed at various time points with AF750-anti-GRP78 and 64Cu-NOTA-anti-GRP78, respectively. Tumors were harvested and GRP78 expression was evaluated by immunohistochemical staining. Results: The anti-GRP78 antibody was successfully conjugated to AF750 and also efficiently radiolabeled with 64Cu. After 3 Gy of irradiation of GL261 tumors, significant binding of anti-GRP78 was observed within 24 hours as demonstrated by optical and microPET/CT imaging. In contrast, the unirradiated tumor on the contralateral hindlimb did not demonstrate any significant antiGRP78 binding. Upregulated GRP78 expression was confirmed in irradiated tumors in comparison to non-irradiated tumors by immunohistochemistry. Conclusions: Optical imaging and microPET/CT imaging are effective methods for visualizing in vivo targeting of radiation-induced GRP78 in GL261 glioblastoma tumors. This study suggests that fluorescent-labeled and radiolabeled antiGRP78 can be used to target radiation-induced GRP78 for tumor-specific delivery of therapeutic drugs and radionuclides in vivo. Author Disclosure: A.J. Chang: None. J. Jaboin: None. D.E. Hallahan: None.
3133
Albendazole Sensitizes Melanoma and Small Cell Lung Cancer Cells to Ionizing Radiation
S751
3130
3131
WITHDRAWN
TORC1/2 Inhibition with Concurrent Radiation Controls Inflammatory Breast Cancer in a Preclinical Animal Model Through Selective Blockade of Translation
E. P. Connolly, D. Silvera, T. Venuto, A. Sawai, R. J. Schneider New York University Medical Center, New York, NY Purpose/Objective(s): The PI3K/AKT/mTOR pathway is frequently deregulated in human cancers, including inflammatory breast cancer (IBC). mTOR (mammalian target of rapamycin) is a key regulator of protein synthesis; it links mRNA translation to the metabolic state of the cell and plays a key role in the signaling of malignant cell growth, proliferation, differentiation, migration, and survival. We have shown that mTOR activation following treatment with DNA damaging agents such as ionizing radiation (IR) is a primary protector of advanced breast cancer cells, including IBC cells. In these cells IR selectively increases translation of mRNAs for survival and DNA repair genes, including survivin, PARP, and DNA repair enzymes. We hypothesized that treatment with an mTOR inhibitor coupled with IR would provide a synergistic ability to control IBC. We examined the cytotoxic effects of combining mTOR inhibition and radiation in an IBC xenograft model, using either the catalytic mTORC1/2 inhibitor pp242, or allosteric mTORC1 inhibitor RAD001 (Everlimus). Materials/Methods: Experiments were conducted in SUM149 IBC cells. Cells were treated with RAD001 or pp242 alone or in combination with increasing IR from 0 - 8 Gy. In vitro studies preformed included: cell survival assay, immunoblot analysis, 35Smethionine labeling, cell cycle analysis by FACs, and caspase-3 activity assay. In vivo studies were performed in a SUM149 xenograft nude mouse model. Animals were treated with RAD001 and pp242 individually or in combination with IR and tumor growth was monitored. IHC, immune-fluorescence and TUNEL assays were also performed. Results: pp242 inhibited both mTORC1/2 signaling in IBC cells and blocked feedback upregulation of AKT and ERK, while RAD001 inhibited only mTORC1 and activated AKT through up-regulation of mTORC2. pp242 when combined with IR showed significantly greater tumor control in IBC xenografts and strongly enhanced radio-sensitivity in vitro, in contrast to RAD001, which had no added effect. Combining pp242 with IR promoted apoptosis, inhibition of both endogenous and radiation-induced AKT activation, and greater inhibition of mTOR signaling to its downstream effectors. This combination more efficiently inhibited overall translation, and likely blocks translation of specific mRNAs involved in DNA repair and survival functions. Conclusions: These studies demonstrate that targeted inhibition of mTORC1/2 synergizes with IR in a model system of IBC. Mechanistically, mTORC1/2 inhibition combined with IR increases apoptosis, prevents radiation induced pro-survival signals mediated through constitutively active AKT and selectively blocks translation of survival and DNA response mRNAs. Author Disclosure: E.P. Connolly: None. D. Silvera: None. T. Venuto: None. A. Sawai: None. R.J. Schneider: None.
3132
Radiation-Guided 64Cu-radiolabeled Anti-GRP78 To Glioblastoma
A. J. Chang, J. Jaboin, D. E. Hallahan Washington University in St. Louis, St. Louis, MO Purpose/Objective(s): Radiation-guided delivery of systemic agents may improve tumor response, while reducing normal tissue toxicity. We have found that a member of the heat-shock protein-70 family, GRP78, is induced in tumor microvasculature after exposure to low doses of ionizing radiation. We subsequently utilized high throughput screening of an antibody library in irradiated cancer cells to generate a highly specific and avid monoclonal antibody to GRP78. The purpose of this study was to image radiation-induced GRP78 expression in vivo using optical and PET imaging in a heterotopic glioblastoma model with our antibody engineered against GRP78. Materials/Methods: For optical imaging, anti-GRP78 was conjugated to Alexa Fluor 750 (AF750). For microPET/CT imaging, anti-GRP78 was conjugated with the chelator, p-SCN-Bn-NOTA, and labeled with 64Cu. 64Cu, a radionuclide with a half-life of 12.7 h, has both imaging and radiotherapeutic properties with its positron and beta decay. The radiolabeled antibody was checked for purity by TLC and HPLC analysis. Athymic nude mice were inoculated into the bilateral hindlimbs with GL261 glioblastoma cells and tumors were allowed to grow to approximately 0.7 cm. The left hindlimb was exposed to 3 Gy of ionizing radiation while special attention was made to block the right hindlimb from ionizing radiation. Optical imaging and microPET/CT studies were performed at various time points with AF750-anti-GRP78 and 64Cu-NOTA-anti-GRP78, respectively. Tumors were harvested and GRP78 expression was evaluated by immunohistochemical staining. Results: The anti-GRP78 antibody was successfully conjugated to AF750 and also efficiently radiolabeled with 64Cu. After 3 Gy of irradiation of GL261 tumors, significant binding of anti-GRP78 was observed within 24 hours as demonstrated by optical and microPET/CT imaging. In contrast, the unirradiated tumor on the contralateral hindlimb did not demonstrate any significant antiGRP78 binding. Upregulated GRP78 expression was confirmed in irradiated tumors in comparison to non-irradiated tumors by immunohistochemistry. Conclusions: Optical imaging and microPET/CT imaging are effective methods for visualizing in vivo targeting of radiation-induced GRP78 in GL261 glioblastoma tumors. This study suggests that fluorescent-labeled and radiolabeled antiGRP78 can be used to target radiation-induced GRP78 for tumor-specific delivery of therapeutic drugs and radionuclides in vivo. Author Disclosure: A.J. Chang: None. J. Jaboin: None. D.E. Hallahan: None.
3133
Albendazole Sensitizes Melanoma and Small Cell Lung Cancer Cells to Ionizing Radiation
S751