| 1. |
- Gad, Helge, et al.
(author)
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MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool
- 2014
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In: Nature. - : Nature Publishing Group. - 0028-0836 .- 1476-4687. ; 508:7495, s. 215-221
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Journal article (peer-reviewed)abstract
- Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bindin the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.
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| 2. |
- Carter, Megan, et al.
(author)
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Crystal structure, biochemical and cellular activities demonstrate separate functions of MTH1 and MTH2
- 2015
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 6
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Journal article (peer-reviewed)abstract
- Deregulated redox metabolism in cancer leads to oxidative damage to cellular components including deoxyribonucleoside triphosphates (dNTPs). Targeting dNTP pool sanitizing enzymes, such as MTH1, is a highly promising anticancer strategy. The MTH2 protein, known as NUDT15, is described as the second human homologue of bacterial MutT with 8-oxo-dGTPase activity. We present the first NUDT15 crystal structure and demonstrate that NUDT15 prefers other nucleotide substrates over 8-oxo-dGTP. Key structural features are identified that explain different substrate preferences for NUDT15 and MTH1. We find that depletion of NUDT15 has no effect on incorporation of 8-oxo-dGTP into DNA and does not impact cancer cell survival in cell lines tested. NUDT17 and NUDT18 were also profiled and found to have far less activity than MTH1 against oxidized nucleotides. We show that NUDT15 is not a biologically relevant 8-oxo-dGTPase, and that MTH1 is the most prominent sanitizer of the cellular dNTP pool known to date.
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| 3. |
- Elvers, Ingegerd, et al.
(author)
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CHK1 activity is required for continuous replication fork elongation but not stabilization of post-replicative gaps after UV irradiation
- 2012
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 40:17, s. 8440-8448
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Journal article (peer-reviewed)abstract
- Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine-inhibiting ATM and ATR amongst other kinases-is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase eta (Pol eta), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation.
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| 4. |
- Hagenkort, Anna
(author)
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Exploiting nucleotide metabolism to improve cancer therapy : by targeting dUTPase, dCTPase and NUDT15
- 2016
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Doctoral thesis (other academic/artistic)abstract
- 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 of untenable 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.
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| 5. |
- Herold, Nikolas, et al.
(author)
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Targeting SAMHD1 with the Vpx protein to improve cytarabine therapy for hematological malignancies
- 2017
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In: Nature Medicine. - : Springer Science and Business Media LLC. - 1078-8956 .- 1546-170X. ; 23:2, s. 256-263
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Journal article (peer-reviewed)abstract
- The cytostatic deoxycytidine analog cytarabine (ara-C) is the most active agent available against acute myelogenous leukemia (AML). Together with anthracyclines, ara-C forms the backbone of AML treatment for children and adults'. In AML, both the cytotoxicity of ara-C in vitro and the clinical response to ara-C therapy are correlated with the ability of AML blasts to accumulate the active metabolite ara-C triphosphate (ara-CTP)(2-5), which causes DNA damage through perturbation of DNA synthesis(6). Differences in expression levels of known transporters or metabolic enzymes relevant to ara-C only partially account for patient-specific differential ara-CTP accumulation in AML blasts and response to ara-C treatment(7-9). Here we demonstrate that the deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) promotes the detoxification of intracellular ara-CTP pools. Recombinant SAMHD1 exhibited ara-CTPase activity in vitro, and cells in which SAMHD1 expression was transiently reduced by treatment with the simian immunodeficiency virus (SIV) protein Vpx were dramatically more sensitive to ara-C-induced cytotoxicity. CRISPR-Cas9-mediated disruption of the gene encoding SAMHD1 sensitized cells to ara-C, and this sensitivity could be abrogated by ectopic expression of wild-type (WT), but not dNTPase-deficient, SAMHD1. Mouse models of AML lacking SAMHD1 were hypersensitive to ara-C, and treatment ex vivo with Vpx sensitized primary patient derived AML blasts to ara-C. Finally, we identified SAMHD1 as a risk factor in cohorts of both pediatric and adult patients with de novo AML who received ara-C treatment. Thus, SAMHD1 expression levels dictate patient sensitivity to ara-C, providing proof-of-concept that the targeting of SAMHD1 by Vpx could be an attractive therapeutic strategy for potentiating ara-C efficacy in hematological malignancies.
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| 6. |
- Valerie, Nicholas C. K., et al.
(author)
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NUDT15 Hydrolyzes 6-Thio-DeoxyGTP to Mediate the Anticancer Efficacy of 6-Thioguanine
- 2016
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In: Cancer Research. - 0008-5472 .- 1538-7445. ; 76:18, s. 5501-5511
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Journal article (peer-reviewed)abstract
- Thiopurines are a standard treatment for childhood leukemia, but like all chemotherapeutics, their use is limited by inherent or acquired resistance in patients. Recently, the nucleoside diphosphate hydrolase NUDT15 has received attention on the basis of its ability to hydrolyze the thiopurine effector metabolites 6-thio-deoxyGTP (6-thio-dGTP) and 6-thio-GTP, thereby limiting the efficacy of thiopurines. In particular, increasing evidence suggests an association between the NUDT15 missense variant, R139C, and thiopurine sensitivity. In this study, we elucidated the role of NUDT15 and NUDT15 R139C in thiopurine metabolism. In vitro and cellular results argued that 6-thio-dGTP and 6-thio-GTP are favored substrates for NUDT15, a finding supported by a crystallographic determination of NUDT15 in complex with 6-thio-GMP. We found that NUDT15 R139C mutation did not affect enzymatic activity but instead negatively influenced protein stability, likely due to a loss of supportive intramolecular bonds that caused rapid proteasomal degradation in cells. Mechanistic investigations in cells indicated that NUDT15 ablation potentiated induction of the DNA damage checkpoint and cancer cell death by 6-thioguanine. Taken together, our results defined how NUDT15 limits thiopurine efficacy and how genetic ablation via the R139C missense mutation confers sensitivity to thiopurine treatment in patients.
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| 7. |
- Zhang, Si Min, et al.
(author)
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Development of a chemical probe against NUDT15
- 2020
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In: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 16:10, s. 1120-1128
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Journal article (peer-reviewed)abstract
- The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15-dependent chemotherapeutics. Lead compound TH1760 demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.
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