NAD+ Boosting Through NRH Supplementation Enhances Treatment Efficacy in EOC In Vitro

Dihydronicotinamide rioside (NRH), the reduced form of nicotinamide riboside (NR), is a recently identified, naturally occurring precursor of arguably the most crucial cofactor for cellular function, nicotinamide adenine dinucleotide (NAD+). Recent investigation suggests that NRH is more adept at increasing NAD+ stores than traditional NAD+ precursors, and such extreme NAD+ boosting via NRH supplementation induces cytotoxicity in certain cellular contexts. It has also been shown that the lack of functional BRCA protein in epithelial ovarian cancer (EOC) directly impacts intracellular NAD+ levels. Given that altered cellular metabolism and DNA repair mechanisms are central alterations in EOC, and these processes are functionally dependent on NAD+, we sought to assess whether NRH supplementation in EOC cell lines enhanced cellular cytotoxicity alone and in combination with standard therapeutic agents. Significant cytotoxicity was noted in NRH treated cells (~40%) with minimal cell death in the nicotinic acid (NA)-treated lines. Levels of NAD(P)H were confirmed to have increased with NRH supplementation, albeit at different levels among the different cell lines. Overall, the cytotoxicity associated with NRH supplementation appears to be independent of ROS generation. Strikingly, NRH supplementation enhanced cytotoxicity of carboplatin in OVCAR8, but not ES2 or SKOV3. Paclitaxel cytotoxicity was also enhanced by the addition of NRH in OVCAR8, but not ES2 or SKOV3 cell lines. NA supplementation had no effect on baseline treatment-induced cytotoxicity. PARP inhibition by olaparib requires NAD+. Interestingly, NRH supplementation enhanced olaparib cytotoxicity in SKOV3 and OVCAR8, but not ES2 cells. NRH in combination with olaparib completely altered mitochondrial respiration, thereby shutting down energy consumption, which would lead to cell death. Coupled together with expression data of key enzymes required for NRH/NAD metabolism, this could be key in understanding mechanisms of cell death with NRH supplementation. Here, we showed that in the context of EOC, exploitation of the NAD+ bioenergetic phenotype through NRH supplementation is a biologically feasible strategy to enhance the response of traditional therapy with potentially minimal toxicity. These data suggest several potential mechanisms by which cellular NAD+ availability impacts treatment efficacy and resistance and highlights the potential utility of NAD+ metabolomics as a biomarker to guide treatment decisions.