www.nature.com/npjbcancer ARTICLE OPEN Targeting nucleotide metabolism enhances the efficacy of anthracyclines and anti-metabolites in triple-negative breast cancer Craig Davison1, Roisin Morelli1, Catherine Knowlson1, Melanie McKechnie1, Robbie Carson1, Xanthi Stachtea1, Kylie A. McLaughlin2, Vivien E. Prise2, Kienan Savage 1, Richard H. Wilson3, Karl A. Mulligan2, Peter M. Wilson2, Robert D. Ladner1,5 and ✉ Melissa J. LaBonte 1,4,5 Triple-negative breast cancer (TNBC) remains the most lethal breast cancer subtype with poor response rates to the current chemotherapies and a lack of additional effective treatment options. We have identified deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) as a critical gatekeeper that protects tumour DNA from the genotoxic misincorporation of uracil during treatment with standard chemotherapeutic agents commonly used in the FEC regimen. dUTPase catalyses the hydrolytic dephosphorylation of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP), providing dUMP for thymidylate synthase as part of the thymidylate biosynthesis pathway and maintaining low intracellular dUTP concentrations. This is crucial as DNA polymerase cannot distinguish between dUTP and deoxythymidylate triphosphate (dTTP), leading to dUTP misincorporation into DNA. Targeting dUTPase and inducing uracil misincorporation during the repair of DNA damage induced by fluoropyrimidines or anthracyclines represents an effective strategy to induce cell lethality. dUTPase inhibition significantly sensitised TNBC cell lines 1234567890():,; to fluoropyrimidines and anthracyclines through imbalanced nucleotide pools and increased DNA damage leading to decreased proliferation and increased cell death. These results suggest that repair of treatment-mediated DNA damage requires dUTPase to prevent uracil misincorporation and that inhibition of dUTPase is a promising strategy to enhance the efficacy of TNBC chemotherapy. npj Breast Cancer (2021) 7:38 ; https://doi.org/10.1038/s41523-021-00245-5 INTRODUCTION reprogramming has been characterized in TNBC following Triple-negative breast cancer (TNBC) is an aggressive breast cancer genotoxic chemotherapy with doxorubicin and identified de novo 6 subtype that lacks oestrogen receptor, progesterone receptor, and pyrimidine synthesis as a promoter of therapy resistance . human epidermal receptor 21 and is associated with an overall In this study, we sought to identify and characterize new poor prognosis2,3. At present, the treatment of TNBC lacks any therapeutic opportunities to enhance current SoC chemotherapies effective targeted therapies, leaving chemotherapy regimens that that incorporate anthracyclines and the anti-metabolite 5-Fluor- incorporate anthracycline and taxane-based therapeutics as the ouracil (5-FU) in TNBC through further modulation of pyrimidine standard of care (SoC). While 30% of patients respond well to and uracil nucleotide metabolism pathways. We hypothesized that this could be achieved through inhibition of the gatekeeper chemotherapy, the remaining patients have limited improvements enzyme, deoxyuridine 5′-triphosphate nucleotidohydrolase (dUT- in clinical outcomes, highlighting the critical need for effective 4 Pase), as this enzyme functions to prevent uracil misincorporation therapeutic strategies to treat TNBC . into DNA9. While clinical trials for immunotherapy, checkpoint inhibition, 5-FU exerts its anticancer activity via the inhibition of the antibody-drug conjugates, and other promising agents are under pyrimidine biosynthetic enzyme thymidylate synthase (TS), indu- investigation in the evolving treatment landscape, this study cing a metabolic blockade that results in the acute depletion of fi focuses on the identi cation of metabolic vulnerabilities in TNBC thymidylate and subsequently dTTP which is required for DNA that represent potential new therapeutic strategies to improve synthesis and repair. This results in rapid growth arrest, and in SoC anthracycline/taxane-based chemotherapy for early TNBC some instances of prolonged dTTP depletion, and tumour cell and anthracycline or anti-metabolite-based treatments for lethality. Inhibition of TS also results in the accumulation of the TS metastatic TNBC. enzyme-substrate dUMP, which, upon further phosphorylation by The metabolic landscape of TNBC is typified by significant ubiquitous pyrimidine monophosphate and diphosphate kinases, intrinsic heterogeneity in cellular metabolism that drives pro- can lead to rapid and abnormal expansion of the deoxyuridine 1,5,6 liferation, survival, and metastasis . The targeting of aberrant triphosphate (dUTP) pool. DNA polymerases cannot distinguish metabolic pathways may identify vulnerabilities that can be between dUTP and dTTP and will incorporate either substrate into therapeutically targeted to disrupt the biosynthesis and proces- DNA. Therefore, as TS inhibition has depleted cells of dTTP, the sing of key macromolecules including proteins, lipids, or nucleo- abnormal and unusually high availability of dUTP leads to its tides7,8. Beyond intrinsic metabolic alterations, adaptive metabolic misincorporation into newly synthesized DNA as uracil10. As uracil 1Medicine, Dentistry and Biomedical Sciences: Patrick G Johnston Centre for Cancer Research, Queen’s University Belfast, Belfast, UK. 2CV6 Therapeutics (NI) Ltd, Belfast, UK. 3Translational Research Centre, University of Glasgow, Glasgow, UK. 4Present address: Medicine, Dentistry and Biomedical Sciences: Patrick G Johnston Centre for Cancer ✉ Research, Queen’s University Belfast, Belfast, UK. 5These authors contributed equally: Robert D. Ladner, Melissa J. LaBonte. email: [email protected] Published in partnership with the Breast Cancer Research Foundation C. Davison et al. 2 is not a native component of DNA, the base excision repair (BER) effects from the anthracycline-induced DNA damage, but perhaps pathway attempts to remove the misincorporated uracil. However, impairs DNA repair and cell recovery (Supplementary Fig. 3). as dTTP levels remain low due to TS inhibition and dUTP pools remain high, futile cycles of uracil misincorporation, failed repair Inhibition of dUTPase enhances DNA damage and cell death and further misincorporation ensue resulting in catastrophic DNA induced by fluoropyrimidine and anthracycline damage and cancer cell death. DNA damage due to misincorpora- chemotherapies tion of dUTP is highly dependent on the levels of the enzyme FUdR induced inhibition of de novo pyrimidine synthesis dUTPase, which rapidly degrades dUTP to dUMP (and PPi) and, efficiently blocks DNA synthesis and repair which results in DNA therefore prevents dUTP pool accumulation, protecting tumour damage10,17. Anthracyclines-induced DNA double-strand breaks DNA from the lethal effects of uracil misincorporation during TS (DSBs) also require increasing de novo nucleotide production for inhibition. Our group and others have demonstrated conclusively efficient repair via new DNA synthesis6. We, therefore, investigated that targeting dUTPase sensitises cancer cells to TS-targeting therapies10–14. In addition, recent research has also demonstrated the impact of DUTi in combination with FUdR or epirubicin on the induction of DNA damage and subsequent repair. For FUdR, a low- that causing uracil misincorporation at sites of DNA damage repair fi can sensitise cancer cells to anthracyclines15,16. Based on these dose (0.1 µM) was selected that alone showed no signi cant alteration of proliferation or survival, but in combination with CV6- observations, this study sought to explore the potential applica- fi tion of promising combination strategies in TNBC. 530 signi cantly reduced survival both in MDA-MB-231 and MDA- MB-468 cells (Fig. 1). The combination in both cell lines resulted in a significant increase in DNA damage compared with single RESULTS agents (as determined by the γH2AX foci) after 4 h (P = 0.001; P < Inhibition of dUTPase sensitises TNBC cells to 0.0001, respectively) that further increased at 24 h and remained fluoropyrimidine chemotherapy elevated to 24 h post-drug removal (Fig. 2A, B). Epirubicin at 0.075 µM increased the number of DSB positive cells to 82.7% ± To examine the potential of inducing uracil DNA misincorporation 3.0 (P < 0.0001) after a 4 h treatment, which was subsequently as an effective therapeutic strategy to enhance TNBC response to repaired and reduced to 47.4% (±4.3%) at 16 h. DUTi or siDUT in TS inhibitors and anthracyclines, the catalytic activity of dUTPase combination with epirubicin did not significantly alter the total was blocked, either by small interfering RNA (siRNA) or by small- number of DSB positive cells following 4 h epirubicin treatment molecule inhibition and the impact on cancer cell proliferation (82.7% vs. 84.2%; P = 0.74), however, the combination resulted in 1234567890():,; and survival in combination with anthracyclines or TS inhibitors a decrease in the repair of the DSB breaks out to 16 h (Fig. 2C; was evaluated. Supplementary Fig. 4) suggesting that the sensitisation caused by Silencing of dUTPase was achieved by pre-treating cells with targeting dUTPase with anthracyclines is not due to changes in 10 nM siDUT which resulted in a reduction in dUTPase mRNA of the initial DNA damage, but rather interferes with the subsequent 89% and 85% in MDA-MB-231 and MDA-MB-468 cells, respec- DSB repair leading to the persistence of DSBs. tively, and a decrease in dUTPase protein of >80% in both cell The ability of the DUTi combinations to enhance apoptosis in lines. Small