Abstract C059: Inhibition of thymidylate synthase by the ProTide NUC-3373: in vitro analysis and clinical validation
Authors: Blagden Sarah P , McKissock Fiona G , Graham Janet S , Ciombor Kristen K , Aroldi Francesca , Rodgers Lisa J , Myers Michelle , Berlin Jordan , Evans T.R. Jeffry , Harrison David J 
Published: March 2020

Background: Although 5-FU-based regimens such as FOLFOX and FOLFIRI remain the standard of care for treatment of patients with colorectal cancer (CRC), their clinical utility is limited by resistance mechanisms and the production of toxic by-products. Anti-cancer activity of 5-FU requires its conversion to the active metabolite, fluorodeoxyuridine-monophosphate (FUDR-MP), which binds to and inhibits thymidylate synthase (TS), depleting dTMP, resulting in DNA damage. Due to breakdown by dihydropyrimidine dehydrogenase (DPD), a reliance on enzymatic activation and a short plasma half-life; 5-FU is a sub-optimal inhibitor of TS. NUC-3373, a phosphoramidate transformation of FUDR-MP, was designed to bypass the key resistance mechanisms associated with the activation and breakdown of 5-FU. Herein, we characterize the impact of DPD on NUC-3373 and the role of NUC-3373 on TS biology in vitro. The NuTide:302 clinical study (NCT03428958) is investigating NUC-3373 pharmacokinetics and dTMP levels. Methods: Sensitivity of nine CRC cell lines was assessed by sulforhodamine B assay and IC50values established, with two (one sensitive and one less sensitive) selected for further evaluation. Pharmacological inhibition by gimeracil was used to investigate DPD-induced catabolism in cells treated with 5-FU and NUC-3373. Thymidine supplementation was used to indirectly assess the effect of NUC-3373 on the de novopathway of dTMP synthesis. Immunocytochemistry and Western blot analysis were used to assess the localization and expression of TS. NuTide:302 is a three-part, Phase Ib study in patients with advanced CRC who have relapsed after ≥2 prior lines of 5-FU-containing therapies. Pharmacokinetic analyses via LC-MS from patient PBMCs are intended to determine whether leucovorin (LV) augments NUC-3373 inhibition of TS and its depletion of the dTMP pool. Results: Unlike 5-FU-treated cells, inhibition of DPD by gimeracil had no significant effect on survival following NUC-3373 treatment. Supplementation with exogenous thymidine rescued cells from NUC-3373-induced death. No correlation was found between pre-treatment TS protein expression and the IC50for NUC-3373. In selected cell lines treated with NUC-3373, TS ternary complexes were detected for at least 72 hours. Treatment with NUC-3373 was also associated with increased cytoplasmic TS expression. In the clinical setting, 22 patients have received NUC-3373 in NuTide:302. The clinical impact of LV on TS inhibition and dTMP pool depletion in patient samples will be presented. Conclusions: Unlike 5-FU, NUC-3373 was not catabolised by DPD, highlighting the ability of NUC-3373 to bypass a key rate-limiting factor associated with 5-FU. NUC-3373 targets the de novo pathway of dTMP synthesis and its cytotoxicity is not dependent on basal TS expression. Formation of long-lasting ternary complexes and increased cytoplasmic expression suggest that NUC-3373 is a potent inhibitor of TS activity. Data describing whether LV potentiates the impact of NUC-3373 on TS in patients will be presented.

Citation Format: Sarah P Blagden, Fiona G McKissock, Janet S Graham, Kristen K Ciombor, Francesca Aroldi, Lisa J Rodgers, Michelle Myers, Jordan Berlin, T.R. Jeffry Evans, David J Harrison. Inhibition of thymidylate synthase by the ProTide NUC-3373: in vitro analysis and clinical validation [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C059. doi:10.1158/1535-7163.TARG-19-C059