Exploiting nucleotide metabolism to improve cancer therapy : by targeting dUTPase, dCTPase and NUDT15

Sammanfattning: Synthetic nucleobase- and nucleoside-analogs have stood the test of time and remain a cornerstone in the treatment regimen against various forms of cancer. Due to their resemblance to endogenous nucleotides these antimetabolites interfere with cellular pathways, including nucleotide metabolism, as well as DNA and RNA synthesis. However, the treatment efficacy of nucleobase- and nucleoside-analogs can be hampered by “house-cleaning” enzymes involved in sanitation and balance of the nucleotide pool. In this work, we validated whether targeting nucleoside triphosphate hydrolases, involved in sanitation of the nucleotide pool, is a promising strategy to improve the efficacy of commonly used nucleobase- and nucleoside-analogs. These include: 1. dUTPase to potentiate 5-fluorouracil treatment 2. dCTPase to potentiate decitabine treatment 3. NUDT15 to potentiate 6-thioguanine treatment We characterized dUTPase, dCTPase and NUDT15 by various biochemical and biophysical techniques and assessed their role in intracellular nucleotide homeostasis using RNA interference. Through the development and use of small molecule inhibitors targeting these hydrolases, we highlighted the benefit of inhibiting nucleotide pool sanitization to improve nucleobase- and nucleoside-analog therapy. In Paper I we demonstrated that dUTPase inhibition reinforces 5-fluorouracil-induced replication defects and cytotoxicity. With this study, we contributed to the characterization of dUTPase inhibitors and increased our understanding of the mode of action of this combination treatment. In the second study (Paper II), we developed small molecule inhibitors against dCTPase to explore the biological function of this nucleoside triphosphate hydrolase in the context of endogenous nucleotide homeostasis and decitabine treatment. We showed that chronic inhibition of dCTPase has a cytostatic effect on cancer cells and potentiates cellular effects of decitabine therapy. Targeting enzymes involved in sanitation of the oxidized nucleotide pool is a novel treatment strategy that exploits the dysregulated reduction-oxidation environment of tumors. Based on the increasing attention to MTH1 as a prime example for this approach, we validated whether the sequence homolog NUDT15 (also known as MTH2) fulfills comparable enzymatic functions, making it a potential target for cancer therapy. With extensive biochemical and cellular experiments we demonstrated that NUDT15 possesses only minimal activity with oxidized nucleotides and is non-essential for cancer cell survival (Paper III). While assessing the cellular function of NUDT15, we discovered activity with the thiopurine effector metabolites, 6-thio-dGTP and 6-thio-GTP. In light of several pharmacogenetic studies, which link thiopurine hypersensitivity to the NUDT15 R139C variant, we further elucidated the role of NUDT15 (wild type and mutants) in thiopurine metabolism (Paper IV). We combined biochemical analyses with cellular experiments on genetically modified cell lines to demonstrate that NUDT15 has a strong preference for thiolated guanine substrates and that this activity counteracts thiopurine efficacy in cancer cells. Furthermore, we propose that the observed hypersensitivity of NUDT15 R139C positive patients is not caused by impaired enzymatic activity, but is a result ofuntenable protein stability. Inspired by these findings, we developed first-in-class NUDT15 inhibitors and validated whether pharmacological inhibition of NUDT15 is a promising strategy to sensitize leukemia cells to thiopurine treatment (Paper V). NUDT15 inhibition significantly increased the availability of thiopurine nucleoside triphosphates, leading to potentiation of DNA incorporation, DNA damage and cytotoxicity. Overall, these studies demonstrated that preventing the sanitation of nucleotide-analogs, by inhibiting nucleoside triphosphate hydrolases, is a promising strategy to improve the efficacy ofnucleobase- and nucleoside-analog treatments.