Abstract 3836: Imbalanced nucleotide metabolism sensitizes breast cancer cells to anthracyclines

Abstract

Abstract Introduction. Triple negative breast cancer (TNBC) makes up 15% of breast cancers and is associated with poor prognosis. TNBC treatment remains hampered by early visceral metastasis and lymph node involvement at the time of diagnosis and limited effective therapeutic options. Advances in treatment that translate to significant improvements in outcome have been painstakingly incremental, despite advances in research technologies and TNBC subtyping. We have identified deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) as a critical gatekeeper that protects tumor DNA from the genotoxic misincorporation of uracil during treatment with anthracyclines that are commonly used in the FEC chemotherapy regimen. dUTPase catalyses the hydrolytic dephosphorylation of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate (dUMP). This reaction has the dual function of providing dUMP for thymidylate synthase (TS) as part of the thymidylate biosynthesis pathway and of maintaining low intracellular dUTP pools. This is crucial as DNA polymerase cannot distinguish between dUTP and deoxythymidylate triphosphate (dTTP), leading to dUTP misincorporated into DNA. dUTPase is being evaluated as a target in cancer therapy alongside TS-targeted therapies and uracil misincorporation is believed to be a potential mechanism of cytotoxicity. Hypothesis: Targeting dUTPase and inducing uracil misincorporation during the repair of DNA damage induced by anthracyclines represents a novel strategy to induce TNBC cell lethality. Methods. Inhibition of dUTPase (DUTi) was carried out using SMARTpool siRNA or small molecule inhibition. The effect of DUTi in combination with anthracyclines was determined by growth inhibition and clonogenics assays. DNA damage was assessed by Western blot, immune-fluorescent foci detection, and flow cytometry. Nucleotide metabolites were quantified by LC/MS. Results. DUTi significantly sensitised TNBC cell lines to doxorubicin and epirubicin. In the MDA-MB-231 cancer cells, loss of dUTPase expression resulted in a synergistic 80% reduction in survival compared to 0.075µM doxorubicin alone at 40%. This decrease in survival correlated with increased activation of proteins involved in DNA damage response, including γ-H2AX(Ser139), a marker for dsDNA breaks. We anticipate that quantification of nucleotides will show an imbalance of dUTP:dTTP, subsequently leading to uracil misincorporation and resulting cell death. Discussion. These results suggest that repair of anthracycline-induced DNA damage requires dUTPase to prevent uracil misincorporation and that inhibition of dUTPase is a potential novel strategy to enhance the efficacy of anthracyclines. This shows the potential advantage of a dUTPase inhibitor being added to current chemotherapy regimens for TNBC. Future work will elucidate the mechanism of sensitisation and in vivo translation of combination therapy for TNBC. Citation Format: Craig Davison, Olivier P. Chevallier, Catherine Knowlson, Melanie McKechnie, Robbie Carson, Jaime Esteve, Richard Wilson, Robert D. Ladner, Melissa J. LaBonte (Wilson). Imbalanced nucleotide metabolism sensitizes breast cancer cells to anthracyclines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3836.