Investigating β‐Lapachone Mediated Metabolic Disruption Using Stable Isotope Tracers

Abstract

β‐lapachone has been found to be a highly potent anti‐cancer agent in phase 1 clinical trials, but has dose limiting toxicity in red blood cells [Gerber et. al Br J Cancer. 2018 Oct;119(8):928‐936]. Identifying a safe and quantitative method of monitoring treatment efficacy will be highly beneficial to establishing an adaptive dosing platform. Adaptative dosing can allow personalized treatments that significantly reduce off target effects while maintaining an effective therapeutic regimen. In this study we present the use of a safe and noninvasive tracer strategy utilizing both [2H7]glucose and [U‐13C]glucose tracers to assess the metabolic effects caused by β‐lapachone treatment in vitro. Treating cancers with β‐lapachone bioactivates NAD(P)H: quinone oxidoreductase 1 (NQO1) to produce a highly unstable hydroquinone intermediate that reestablishes itself through an aggressive futile cycle, generating reactive oxygen species (ROS) in the form of hydrogen peroxide. Accelerated accumulation of ROS causes large amounts of DNA damage, triggers poly‐ADP‐ribose polymerase‐I hyperactivation, induces global depletion of NAD+ and ATP, and hinders overall glycolytic flux [Moore et. al Cell Death Dis. 2015 Jan; 6(1): e159]. Cancer cells that overexpress NQO1 subsequently die through NAD+‐keresis. Measuring changes in glycolysis and downstream metabolism caused by the bioactivation of NQO1 would establish a platform for assessing the efficacy of treatment, potentially allowing the reduction of chemotherapeutic dosage thereby diminishing off‐target toxicities. Changes in central carbon metabolism caused by NQO1 bioactivation were detected and assessed in various cancer cells using both tracer strategies. Deuterated lactate and HDO (deuterated water) were easily quantified, and the production of these metabolic products were linearly correlated with [2H7]glucose consumption. Mass isotopologue distribution analysis by gas chromatography‐mass spectrometry demonstrated downregulated energy metabolism with a reduction in 2H and 13C labeling in NQO1+ cancer cells treated with β‐lapachone. These methods are highly beneficial for measuring rates of glycolytic metabolism, identifying potential therapeutic synergies, the efficacy of chemotherapeutic agents that hamper central carbon metabolism, and can be potentially translated to in vivo models.