Abstract:
This thesis examines novel inhibitors of the enzyme DNA-dependent protein kinase (DNAPK), and hypoxia-activated prodrugs of these inhibitors, in the context of their use as radiation sensitisers. Radiation therapy is a widely used treatment modality for many cancer types and is commonly used as a primary definitive treatment for head and neck squamous cell carcinoma (HNSCC). The efficacy of radiation therapy is often limited by normal tissue toxicity with oral mucositis being a common dose limiting toxicity for head and neck cancer patients. DNA-PK is an important component of the DNA damage response, and is involved in the pathway for repair of radiation-induced DNA double strand breaks. DNA-PK inhibitors have been explored as radiosensitisers, but as a drug class have yet to be proven in the clinical setting. The in vitro aspect of this thesis focused on the DNA-PK inhibitor IC87361, and its hypoxiaactivated prodrug SN38023, with their activity being explored in human and murine HNSCC cell lines through the use of western blotting, γH2AX immunofluorescence and the clonogenic survival assay. IC87361 radiosensitised cells under both oxic and anoxic conditions and SN38023 was a modest radiosensitiser of murine cells under anoxic conditions, indicating release of IC87361. The activity of IC87361 and SN38023 in murine cells had previously not been explored. Radiosensitisation of murine HNSCC cells by the DNA-PK inhibitor AZD7648 was also explored through clonogenic assay with AZD7648 acting as a potent radiosensitiser. The in vivo aspect of this study focused on the use of an immunocompetent mouse model of HNSCC to compare the radiosensitisation of normal tissues and SCCVII tumours by the recently described DNA-PK inhibitor AZD7648. The crypt assay was used to quantify radiosensitisation of the small intestine by AZD7648. Development of an analogous assay for the oral mucosa was also carried out. AZD7648 was an effective radiosensitiser of SCCVII tumours but was shown to radiosensitise the small intestine and oral mucosa even more effectively than the tumours. The crypt assay is a well validated assay for studying repair of the small intestine following irradiation and the assay was modified in this study to use EdU labelling of S-phase cells. Implementation of an algorithm for counting EdU+ cells is a novel aspect of this thesis which increases the information obtained from the crypt assay. The longer term intention is to use the immunocompetent mouse model of HNSCC, with the crypt assay and oral mucosa assay, to test if hypoxia-activated prodrugs of DNA-PK inhibitors cause less normal tissue radiosensitisation than systemically administered DNA-PK inhibitors.