Abstract A004: Targeting tumor hypoxia with prodrugs of DNA-dependent protein kinase inhibitors
Authors: Liew Lydia P. , Hong Cho R. , Dickson Benjamin D. , Wong Way W. , Jamieson Stephen M. , Wilson William R , Hay Michael P. 
Published: January 2024

Radiation therapy kills cancer cells through the generation of DNA double-strand breaks (DSB) which give rise to lethal chromosome aberrations. Intrinsic radiation resistance is mediated through several mechanisms including suppression of initial DSB formation by hypoxia in the tumor microenvironment and rapid repair of DSB by non-homologous end-joining (NHEJ). DNA-dependent protein kinase (DNA-PK) is a key element of NHEJ and is a well-validated target for tumor radiosensitization. However, DNA-PK inhibition has been demonstrated to radiosensitize normal tissues within the radiation field. Exploiting hypoxia to release DNA-PK inhibitors from hypoxia-activated prodrugs (HAPs) selectively in tumors is one strategy for enhancing tumor selectivity, with the advantage of sensitizing the most radioresistant (hypoxic) subpopulation and potentially sensitizing oxic tumor cells through a bystander effect. We recently discovered a new class of DNA-PK inhibitor based on an imidazo[4,5-c] pyridine-2-one scaffold. Our initial lead, SN39536, provided highly selective DNA-PK inhibition in biochemical and cell-based assays, potently radiosensitized parental HAP1 cells but not an isogenic knockout of PRKDC (encoding the DNA-PK catalytic subunit), and radiosensitized HCT116 cells both in vitro and as tumor xenografts. We explored three prodrug linker approaches (carbamate, ether, and quaternary ammonium salt linkers between nitroimidazole trigger units and DNA-PK inhibitors) to prepare HAPs of various DNA-PK inhibitors. The 2-nitroimidazole carbamate HAP, SN39884, was metabolized selectively in hypoxic cells to generate SN39536 in vitro, efficiently penetrated through hypoxic tumor tissue in multicellular layer cultures, and significantly radiosensitized HCT116 tumors in NIH-III mice at well-tolerated doses. However, the limited metabolic stability of SN39884 in mice led us to explore HAPs with an ether linker. Ether prodrug SN40458 released a phenolic DNA-PK inhibitor, SN39872, on reduction under hypoxic conditions. SN39872 provided robust sensitization of HCT116 cells under both oxic and hypoxic conditions in vitro, while the HAP, SN40458, only sensitized hypoxic cells. SN40458 provided significant radiosensitization of both HCT116 and UT-SCC-74B tumors in NIH-III mice, measured using an ex vivo clonogenic survival assay. Further optimization of prodrug metabolic stability is required before comparison of tumor and normal tissue radiosensitization by these second-generation HAPs and their corresponding DNA-PK inhibitors in mice. This comparison will provide a critical test of our hypothesis that hypoxia-dependent generation of the DNA-PK inhibitor enhances tumor selectivity.

Citation Format: Lydia P. Liew, Cho R. Hong, Benjamin D. Dickson, Way W. Wong, Stephen M. Jamieson, William R Wilson, Michael P. Hay. Targeting tumor hypoxia with prodrugs of DNA-dependent protein kinase inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A004.