- lapachone regulates mammalian inositol pyrophosphate levels in an NQO1-and oxygen- dependent manner

Inositol pyrophosphates (PP-InsPs) are energetic signaling molecules with important functions in mammals. As their biosynthesis depends on ATP concentration, PP- InsPs are tightly connected to cellular energy homeostasis. Consequently, an increasing num-ber of studies involve PP- InsPs in metabolic disorders, such as type 2 diabetes, aspects of tumorigenesis, and hyperphosphatemia. Research conducted in yeast suggests that the PP- InsP pathway is activated in response to reactive oxygen species (ROS). However, the precise modulation of PP- InsPs during cellular ROS signaling is unknown. Here, we report how mammalian PP- InsP levels are changing during exposure to exogenous (H2O2) and endogenous ROS. Using capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI- MS), we found that PP- InsP levels decrease upon expo -sure to oxidative stressors in HCT116 cells. Application of quinone drugs, particularly beta -lapachone (beta- lap), under normoxic and hypoxic conditions enabled us to produce ROS in cellulo and to show that beta- lap treatment caused PP- InsP changes that are oxygen-dependent. Experiments in MDA- MB- 231 breast cancer cells deficient of NAD(P)H:quinone oxidoreductase- 1 (NQO1) demonstrated that beta- lap requires NQO1 bioactivation to regulate the cellular metabolism of PP-InsPs. Critically, significant reductions in cellular ATP concentrations were not directly mirrored in reduced PP- InsP levels as shown in NQO1-deficient MDA- MB- 231 cells treated with beta- lap. The data presented here unveil unique aspects of beta- lap pharmacology and its impact on PP- InsP levels. The identification of different quinone drugs as modulators of PP- InsP synthesis will allow the overall impact on cellular function of such drugs to be better appreciated.