Abstract 1835: NUC-3373 targets the DNA-directed pathway more effectively than 5-FU

Abstract

Abstract Background: Over the past 60 years, numerous strategies have been employed to enhance the generation of the anticancer metabolite FUDR-MP (FdUMP) by 5-FU and to reduce the generation of toxic metabolites. FUDR-MP, the primary anticancer metabolite of 5-FU, exerts its activity via TS inhibition which results in increased dUMP, depletion of dTMP and DNA damage. 5-FU can also generate FdUTP, which is incorporated into DNA, resulting in DNA damage. 5-FU can also be metabolized to FUTP, which results in the RNA mediated dose-limiting toxicities of myelosuppression and GI tract inflammation. NUC-3373 is a phosphoramidate transformation of FUDR designed specifically to inhibit TS. It generates higher intracellular levels of FUDR-MP, lower levels of toxic metabolites and through its favorable PK profile can be administered via a short infusion. NUC-3373 is a more potent inhibitor of TS than 5-FU and we tested the hypothesis that it more effectively targets DNA than 5-FU. Methods: CRC cells (HCT116 & SW480) were exposed to sub-IC50 doses of NUC-3373 or 5-FU for 6 or 24h. Cells were harvested and analyzed as follows: metabolite levels and incorporation of FdUTP into DNA and FUTP into RNA (using FdUr and FUr as surrogates) by LC-MS and LC-MS/MS. Gene expression of dUTPase was knocked down by siRNA and protein assessed by western blot. Cytotoxicity of NUC-3373 and 5-FU was determined by IC50, measured with sulforhodamine B. Results: NUC-3373 generated significantly higher levels of FUDR-MP compared to 5-FU (AUC >45x greater) resulting in a pronounced increase in dUMP levels (AUC >162x compared to 5-FU). NUC-3373 treated cells incorporated FdUTP into DNA, while incorporation by 5-FU treated cells was below the LLOQ (0.1 nM). NUC-3373 was effective in the presence of dUTPase, but its potency increased after dUTPase knockdown demonstrating that FdUTP incorporation contributes to NUC-3373’s cytotoxicity. At equimolar doses, 42x more FUTP was incorporated into RNA in 5-FU treated cells compared to NUC-3373. Conclusion: NUC-3373 generates higher intracellular levels of the anticancer metabolite FUDR-MP and is a more potent inhibitor of TS than 5-FU resulting in markedly greater accumulation of intracellular dUMP. Additionally, in contrast to 5-FU, NUC-3373 treatment results in incorporation of FdUTP into DNA which indicates that NUC-3373 is a more efficient DNA damaging agent. Through bypassing the FUTP-generation pathway, NUC-3373 avoids the RNA damage that is known to be associated with dose-limiting toxicities such as myelosuppression and GI tract inflammation. This is consistent with the very low rates of FUTP-related toxicities observed in patients treated with NUC-3373 (NCT03428958). By exploiting a more DNA-directed approach, NUC-3373 provides a potentially more effective, safer and convenient therapeutic option than 5-FU for patients with cancer. Citation Format: Jennifer Bré, Alison L. Dickson, Oliver J. Read, Ying Zhang, Clarissa M. Czekster, David J. Harrison. NUC-3373 targets the DNA-directed pathway more effectively than 5-FU [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1835.