Nuclear factor erythroid 2-related factor 1 (NFE2L1, NRF1) and nuclear factor erythroid 2-related factor 2 (NFE2L2, NRF2) are distinct oxidative stress response transcription factors, both of which have been shown to perform cytoprotective functions, modulating cell stress response and homeostasis. NAD(P)H:quinone oxidoreductase (NQO1) is a mutual downstream antioxidant gene target that catalyzes the two-electron reduction of an array of substrates, protecting against reactive oxygen species (ROS) generation. NQO1 is upregulated in non-small cell lung cancer (NSCLC) and is proposed as a predictive biomarker and therapeutic target. Antioxidant protein expression of immune cells within the NSCLC tumor microenvironment (TME) remains undetermined and may affect immune cell effector functions and survival outcomes. Multiplex immunofluorescence was performed to examine the co-localization of NQO1, NRF1 and NRF2 within the tumor and TME of 162 chemotherapy-naïve, early-stage NSCLC patients treated by primary surgical resection. This study demonstrates that NQO1 protein expression is high in normal, tumor-adjacent tissue and that NQO1 expression varies depending on the cell type. Inter and intra-patient heterogenous NQO1 expression was observed in lung cancer. Co-expression analysis showed NQO1 is independent of NRF1 and NRF2 in tumors. Density-based co-expression analysis demonstrated NRF1 and NRF2 double-positive expression in cancer cells is associated with improved overall survival.

Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates the cellular antioxidant response, allowing adaptation and survival under conditions of oxidative, electrophilic and inflammatory stress, and has a role in metabolism, inflammation and immunity. Activation of Nrf2 provides broad and long-lasting cytoprotection, and is often hijacked by cancer cells, allowing their survival under unfavorable conditions. Moreover, Nrf2 activation in established human tumors is associated with resistance to chemo-, radio-, and immunotherapies. In addition to cancer cells, Nrf2 activation can also occur in tumor-associated macrophages (TAMs) and facilitate an anti-inflammatory, immunosuppressive tumor immune microenvironment (TIME). Several cancer cell-derived metabolites, such as itaconate, L-kynurenine, lactic acid and hyaluronic acid, play an important role in modulating the TIME and tumor-TAMs crosstalk, and have been shown to activate Nrf2. The effects of Nrf2 in TIME are context-depended, and involve multiple mechanisms, including suppression of pro-inflammatory cytokines, increased expression of programmed cell death ligand 1 (PD-L1), macrophage colony-stimulating factor (M-CSF) and kynureninase, accelerated catabolism of cytotoxic labile heme, and facilitating the metabolic adaptation of TAMs. This understanding presents both challenges and opportunities for strategic targeting of Nrf2 in cancer.

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.

Introduction Fluoropyrimidines, principally 5-fluorouracil (5-FU), remain a key component of chemotherapy regimens for multiple cancer types, in particular colorectal and other gastrointestinal malignancies. To overcome key limitations and pharmacologic challenges that hinder the clinical utility of 5-FU, NUC-3373, a phosphoramidate transformation of 5-fluorodeoxyuridine, was designed to improve the efficacy and safety profile as well as the administration challenges associated with 5-FU. Methods Human colorectal cancer cell lines HCT116 and SW480 were treated with sub-IC50 doses of NUC-3373 or 5-FU. Intracellular activation was measured by LC–MS. Western blot was performed to determine binding of the active anti-cancer metabolite FdUMP to thymidylate synthase (TS) and DNA damage. Results We demonstrated that NUC-3373 generates more FdUMP than 5-FU, resulting in a more potent inhibition of TS, DNA misincorporation and subsequent cell cycle arrest and DNA damage in vitro. Unlike 5-FU, the thymineless death induced by NUC-3373 was rescued by the concurrent addition of exogenous thymidine. 5-FU cytotoxicity, however, was only reversed by supplementation with uridine, a treatment used to reduce 5-FU-induced toxicities in the clinic. This is in line with our findings that 5-FU generates FUTP which is incorporated into RNA, a mechanism known to underlie the myelosuppression and gastrointestinal inflammation associated with 5-FU. Conclusion Taken together, these results highlight key differences between NUC-3373 and 5-FU that are driven by the anti-cancer metabolites generated. NUC-3373 is a potent inhibitor of TS that also causes DNA-directed damage. These data support the preliminary clinical evidence that suggest NUC-3373 has a favorable safety profile in patients.