Many chemotherapeutic drugs exert their cytotoxic activity through the formation of mono- or bifunctional DNA modifications (adducts), which can interfere with replication, an over-active process in rapidly dividing cancer cells. Hypoxia is a distinct characteristic of solid tumors, resulting from an imbalance between the cellular oxygen consumption rate and the tumor-oxygen supply. To selectively target tumor cells, the pro-drug CP-506 was designed to convert into strong alkylating agents under hypoxic environments. The goal is to demonstrate proof of concept, verifying CP-506 selectivity to hypoxic tumor cells. The initial work focused on adduct discovery, involving exposure of purified calf thymus DNA with CP-506 or its metabolites, followed by enzymatic digestion of the DNA into its nucleosides, adduct enrichment, and identification using high-resolution/accurate-mass (HR/AM) LC-MS3 targeted and untargeted adductomic analyses. This strategy is based on data-dependent MS2 fragmentation of the most abundant ions in the full scan. The neutral loss of the deoxyribose moiety or any of the four DNA bases in the MS2 spectra triggers MS3 fragmentation, revealing adduct identity. Validation of the identified adducts was conducted using internal standards using similarly exposed15N-labeled DNA. A targeted adductomics approach was then developed for detection of previously identified adducts in CP-506-treated cancer cells, under normoxic and hypoxic conditions to ascertain specificity. Cell exposure to CP-506 resulted to formation of various adducts with the pro-drug’s active metabolites including cross-links, only on hypoxic cancer cells. We are currently looking into off-target specimens by conducting analyses on blood samples from treated animal models. Additionally, further efficacy and specificity analyses are being conducted on tumor biopsies obtained from animal models treated with CP-506. Future work will be focused on exploring these adducts’ potential as biomarkers of therapeutic potency, to be used to select the ideal target population for a precision, patient-oriented, and personalized chemotherapeutic approach.

High-resolution/accurate-mass DNA adductomics to identify adducts formed by the hypoxia-activated alkylating agent, CP-506 and its metabolites

2019 Fall National Meeting

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