Development of a molecular biomarker screen to detect oxidoreductase activation of hypoxia-selective prodrugs in cancer therapy

Abstract

Hypoxia is a common feature of solid tumours and is associated with poor prognosis and therapeutic resistance. The absence of severe hypoxia in normal tissues has led to the development of a number of hypoxia-selective prodrugs, several of which have advanced to clinical trial but to date none have been approved for the treatment of cancer. Two examples of hypoxia-selective prodrugs that have advanced to the clinical trial are PR-104 and TH-302, both masked alkylating agents which undergo metabolic activation by cellular oxidoreductases in the absence of oxygen to their cytotoxic species. The successful clinical application of these prodrugs, in part, depends on the complement of cellular oxidoreductases which may also aid prodrug development. This project sought to develop a novel flow cytometry-based screen to identify potential prodrug-activating oxidoreductases, through activation of DNA damage response (DDR) and phosphorylation of 53bp1 (p-53bp1) DNA repair protein. The extent of TH-302- induced DDR in wild type cells was compared to that of cells expressing candidate oxidoreductases to identify those capable of bioreductive drug activation. Initial optimisation under oxygenated conditions was performed using cells engineered to express oxygen-insensitive bacterial nitroreductases (NTRs). NTR expression resulted in marked increases in p-53bp1 levels in response to aerobic TH-302 exposure, thus validating the potential of the screen to identify prodrug-activating enzymes. Analysis of p-53bp1 levels in cells treated with TH-302 under anoxia showed large responses in WT cells with no further increases in 13 candidate oxidoreductase-overexpressing or NTR-expressing cells. In contrast, SN29176 (an analogue of PR-104) was demonstrated to be highly dependent on additional reductive activity in order to provoke a robust p-53bp1 response. The findings presented here suggests that TH-302 is efficiently activated under anoxia by endogenous levels of oxidoreductases in three cell lines tested and that overexpression of additional reductive capacity does not alter the p-53bp1 response. SN29176, however, shows great potential as a reporter molecule that could aid in the identification of oxidoreductases involved in activation of hypoxia-selective prodrugs. These observations are of importance for clinical application of TH-302 which has shown promising activity in preclinical and various clinical studies.