In Vivo Identification of Adducts from the New Hypoxia-Activated Prodrug CP-506 Using DNA Adductomics

Many chemotherapeutic drugs exert their cytotoxicity through the formation of DNA modifications (adducts), which interfere with DNA replication, an overactive process in rapidly dividing cancer cells. Side effects from the therapy are common, however, because these drugs also affect rapidly dividing noncancerous cells. Hypoxia-activated prodrugs (HAPs) have been developed to reduce these side effects as they preferentially activate in hypoxic environments, a hallmark of solid tumors. CP-506 is a newly developed DNA-alkylating HAP designed to exert strong activity under hypoxia. The resulting CP-506-DNA adducts can be used to elucidate the cellular and molecular effects of CP-506 and its selectivity toward hypoxic conditions. In this study, we characterize the profile of adducts resulting from the reaction of CP-506 and its metabolites CP-506H and CP-506M with DNA. A total of 39 putative DNA adducts were detected in vitro using our high-resolution/accurate-mass (HRAM) liquid chromatography tandem mass spectrometry (LC-MS³) adductomics approach. Validation of these results was achieved using a novel strategy involving ¹⁵N-labeled DNA. A targeted MS/MS approach was then developed for the detection of the 39 DNA adducts in five cancer cell lines treated with CP-506 under normoxic and hypoxic conditions to evaluate the selectivity toward hypoxia. Out of the 39 DNA adducts initially identified, 15 were detected, with more adducts observed from the two reactive metabolites and in cancer cells treated under hypoxia. The presence of these adducts was then monitored in xenograft mouse models bearing MDA-MB-231, BT-474, or DMS114 tumors treated with CP-506, and a relative quantitation strategy was used to compare the adduct levels across samples. Eight adducts were detected in all xenograft models, and MDA-MB-231 showed the highest adduct levels. These results suggest that CP-506-DNA adducts can be used to better understand the mechanism of action and monitor the efficacy of CP-506 in vivo, as well as highlight a new role of DNA adductomics in supporting the clinical development of DNA-alkylating drugs.