Abstract 1003: NUC-1031 causes incorporation of fluorinated deoxycytidine into DNA, inducing persistent damage in biliary tract cancer cells
Authors: Patel Dillum , Dickson Alison L. , Read Oliver J. , Czekster Clarissa M. , Ghazaly Essam A. , Harrison David J. , Bré Jennifer 
Published: July 2021

Abstract
Background: NUC-1031 is a ProTide transformation of the nucleoside analog gemcitabine, designed to overcome key cancer resistance mechanisms and generate significantly higher levels of the active metabolite, dFdCTP. Its incorporation into DNA interrupts cell replication and induces DNA damage, leading to cell death. NUC-1031 has shown broad clinical activity across a range of solid tumors as both a single agent and in combination with platinum agents. Increasing evidence in the clinical setting in both ovarian and biliary tract cancer populations suggests potential synergy between NUC-1031 and platinum agents, which may lead to increased anti-tumor activity. The aim of this study was to investigate the dose and time relationships between the incorporation of dFdCTP into DNA, the effect on the cell cycle and DNA damage response in biliary tract cancer cells treated with NUC-1031.
Methods: Human intrahepatic cholangiocarcinoma HuCCT1 cells were treated with 500 nM (half-IC50 dose) or 1 µM (IC50 dose) NUC-1031 for 24h and samples were collected every 24h up to 96h. The intracellular conversion of NUC-1031 to its active metabolite dFdCTP was assessed by liquid chromatography mass spectrometry (LC-Q-TOF). This technique was also used to determine the incorporation of dFdCTP into DNA, using the dFdC signature as a surrogate and deoxyguanosine (2dG) for normalization. Flow cytometry was used to measure cell cycle populations and to analyse histone variant H2AX phosphorylation (γH2AX) as a marker for response to DNA damage, where increased signal is related to increase in response.
Results: NUC-1031 generated dFdCTP in HuCCT1 cells. The incorporation of this fluorinated deoxycytidine into DNA was dose-dependent over time. At 48h post-treatment, the ratio of dFdC:2dG was 0.70 at half-IC50 dose, with comparable levels at IC50 dose. After 48h, the ratio continued to rise for the 1 µM dose but decreased with 500 nM. The incorporation of the active metabolite was accompanied by an increase in cells in S phase, up to 46% for 500 nM and 64% for 1 µM. This dose-dependent increase coincides with H2AX phosphorylation over time up to 48h. Active metabolite incorporation, S phase population and yH2AX signals reduced towards the 96h time point.
Conclusion: NUC-1031 is a potent cytotoxic agent that causes DNA damage through the incorporation of its active metabolite into DNA, in a cell cycle dependant manner. This incorporation induces S phase arrest and evokes the DNA damage response via generation of double-strand breaks. The cytotoxic effect of NUC-1031 is prolonged post-treatment. Ongoing studies are investigating NUC-1031 in combination with cisplatin, where relative levels of dFdCTP incorporated in tumor DNA may act as a pharmacodynamic biomarker to determine synergy in patients who receive the combination treatment.
Citation Format: Dillum Patel, Alison L. Dickson, Oliver J. Read, Clarissa M. Czekster, Essam A. Ghazaly, David J. Harrison, Jennifer Bré. NUC-1031 causes incorporation of fluorinated deoxycytidine into DNA, inducing persistent damage in biliary tract cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1003.