Abstract A020: NAD+ Boosting through NRH supplementation enhances treatment efficacy in EOC

Abstract

Abstract Background: Dihydronicotinamide riboside (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. Methods: We supplemented 3 ovarian cancer cell lines (SKOV3, OVCAR8 and ES2) with NRH and traditional NAD precursor, NA (nicotinic acid) alone and in combination with carboplatin, paclitaxel, combination, and olaparib. Cytotoxicity was measured via CellTiter-Glo. RNA sequencing of EOC cell lines was performed to determine the differential expression of NAD+ anabolic and catabolic enzymes. Of note, all three cell lines are known to be BRCA1 &2 wildtype while OVCAR8 cells are hypermethylated and harbor loss of heterozygosity (LOH high) at the BRCA1 loci. Results: Significant cytotoxicity was noted in NRH treated cells (~40 %) with minimal cell death in the NA treated lines. Strikingly, NRH supplementation enhanced carboplatin cytotoxicity by 25-50 % depending on cell line, paclitaxel cytotoxicity by 10-25 %, and the combination by 10-35 % whereas NA supplementation had no effect on the baseline treatment induced cytotoxicity. Interestingly, the effect of NRH supplementation with olaparib differed between the ES2 and OVCAR8 cell lines, with a significant 20 % increase in cell death in ES2 vs OVCAR8 cells with a negligible increase in cell death. PARP inhibition by olaparib requires NAD+. RNA sequencing revealed an increased expression in NAMPT, an enzyme involved in the NAD+ salvage pathway in the ES2 cell line relative to the OVCAR8, suggesting that the ES2 cells harbor a baseline NAD+ deficit rendering them more vulnerable to PARP inhibition following NAD+ boosting. This is also consistent with the OVCAR8 data suggesting that hypermethylated BRCA1 results in a relative increase in NAD+ to the extent that NRH supplementation does not enhance the efficacy of PARP inhibition. Conclusions: Here we show 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. This data suggests several potential mechanisms by which cellular NAD+ availability belies treatment efficacy and resistance and highlights the potential utility of NAD+ metabolomics as a biomarker to guide treatment decisions. Citation Format: Kevin Lee, Tanvi Joshi, Catherine Lyons, Mary Howard Singleton, Elizabeth Catranis, Sagar Chokshi, Luciana Madiera da Silva, Marie Migaud, Jennifer Scalici. NAD+ Boosting through NRH supplementation enhances treatment efficacy in EOC [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A020.