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1.
Nat Chem Biol ; 16(10): 1120-1128, 2020 10.
Article in English | MEDLINE | ID: mdl-32690945

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.


Subject(s)
Pyrophosphatases/antagonists & inhibitors , Pyrophosphatases/metabolism , Binding Sites , Cell Line , Drug Design , Drug Development , Escherichia coli , Humans , Inorganic Pyrophosphatase/antagonists & inhibitors , Inorganic Pyrophosphatase/genetics , Inorganic Pyrophosphatase/metabolism , Models, Molecular , Protein Binding , Protein Conformation , Pyrophosphatases/chemistry , Pyrophosphatases/genetics , Structure-Activity Relationship
2.
Cancer Res ; 80(17): 3530-3541, 2020 09 01.
Article in English | MEDLINE | ID: mdl-32312836

ABSTRACT

Reactive oxygen species (ROS) oxidize nucleotide triphosphate pools (e.g., 8-oxodGTP), which may kill cells if incorporated into DNA. Whether cancers avoid poisoning from oxidized nucleotides by preventing incorporation via the oxidized purine diphosphatase MTH1 remains under debate. Also, little is known about DNA polymerases incorporating oxidized nucleotides in cells or how oxidized nucleotides in DNA become toxic. Here we show that replacement of one of the main DNA replicases in human cells, DNA polymerase delta (Pol δ), with an error-prone variant allows increased 8-oxodG accumulation into DNA following treatment with TH588, a dual MTH1 inhibitor and microtubule targeting agent. The resulting elevated genomic 8-oxodG correlated with increased cytotoxicity of TH588. Interestingly, no substantial perturbation of replication fork progression was observed, but rather mitotic progression was impaired and mitotic DNA synthesis triggered. Reducing mitotic arrest by reversin treatment prevented accumulation of genomic 8-oxodG and reduced cytotoxicity of TH588, in line with the notion that mitotic arrest is required for ROS buildup and oxidation of the nucleotide pool. Furthermore, delayed mitosis and increased mitotic cell death was observed following TH588 treatment in cells expressing the error-prone but not wild-type Pol δ variant, which is not observed following treatments with antimitotic agents. Collectively, these results link accumulation of genomic oxidized nucleotides with disturbed mitotic progression. SIGNIFICANCE: These findings uncover a novel link between accumulation of genomic 8-oxodG and perturbed mitotic progression in cancer cells, which can be exploited therapeutically using MTH1 inhibitors.See related commentary by Alnajjar and Sweasy, p. 3459.


Subject(s)
8-Hydroxy-2'-Deoxyguanosine , Phosphoric Monoester Hydrolases , DNA Repair Enzymes/genetics , Genomics , Humans , Mitosis/genetics , Phosphoric Monoester Hydrolases/genetics , Pyrimidines/pharmacology
4.
Oncotarget ; 8(14): 23713-23726, 2017 Apr 04.
Article in English | MEDLINE | ID: mdl-28423595

ABSTRACT

The antimetabolite 5-Fluorouracil (5-FU) is used in the treatment of various forms of cancer and has a complex mode of action. Despite 6 decades in clinical application the contribution of 5-FdUTP and dUTP [(5-F)dUTP] and 5-FUTP misincorporation into DNA and RNA respectively, for 5-FU-induced toxicity is still under debate.This study investigates DNA replication defects induced by 5-FU treatment and how (5-F)dUTP accumulation contributes to this effect. We reveal that 5-FU treatment leads to extensive problems in DNA replication fork progression, causing accumulation of cells in S-phase, DNA damage and ultimately cell death. Interestingly, these effects can be reinforced by either depletion or inhibition of the deoxyuridine triphosphatase (dUTPase, also known as DUT), highlighting the importance of (5-F)dUTP accumulation for cytotoxicity.With this study, we not only extend the current understanding of the mechanism of action of 5-FU, but also contribute to the characterization of dUTPase inhibitors. We demonstrate that pharmacological inhibition of dUTPase is a promising approach that may improve the efficacy of 5-FU treatment in the clinic.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , DNA Replication/drug effects , Enzyme Inhibitors/pharmacology , Fluorouracil/pharmacology , Neoplasms/drug therapy , Pyrophosphatases/antagonists & inhibitors , Antimetabolites, Antineoplastic/administration & dosage , Antimetabolites, Antineoplastic/pharmacology , Cell Line, Tumor , Drug Synergism , Enzyme Inhibitors/administration & dosage , Fluorouracil/administration & dosage , HeLa Cells , Humans , Neoplasms/enzymology , Neoplasms/genetics
5.
Nat Med ; 23(2): 256-263, 2017 Feb.
Article in English | MEDLINE | ID: mdl-28067901

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), which causes DNA damage through perturbation of DNA synthesis. 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. 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.


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Cytarabine/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Monomeric GTP-Binding Proteins/drug effects , Viral Regulatory and Accessory Proteins/pharmacology , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Antimetabolites, Antineoplastic/therapeutic use , Arabinofuranosylcytosine Triphosphate/metabolism , Child , Child, Preschool , Cytarabine/therapeutic use , Disease Models, Animal , Female , Humans , In Vitro Techniques , Infant , Leukemia, Myeloid, Acute/metabolism , Male , Mice , Molecular Targeted Therapy , Monomeric GTP-Binding Proteins/metabolism , Prognosis , SAM Domain and HD Domain-Containing Protein 1
7.
PLoS One ; 11(12): e0165214, 2016.
Article in English | MEDLINE | ID: mdl-27959931

ABSTRACT

BACKGROUND: There is great interest in repurposing the commonly prescribed anti-diabetic drug metformin for cancer therapy. Intracellular uptake and retention of metformin is affected by the expression of organic cation transporters (OCT) 1-3 and by multidrug and toxic compound extrusion (MATE) 1-2. Inside cells, metformin inhibits mitochondrial function, which leads to reduced oxygen consumption and inhibition of proliferation. Reduced oxygen consumption can lead to improved tumor oxygenation and radiation response. PURPOSE: Here we sought to determine if there is an association between the effects of metformin on inhibiting oxygen consumption, proliferation and expression of OCTs and MATEs in a panel of 19 cancer cell lines. RESULTS: There was relatively large variability in the anti-proliferative response of different cell lines to metformin, with a subset of cell lines being very resistant. In contrast, all cell lines demonstrated sensitivity to the inhibition of oxygen consumption by metformin, with relatively small variation. The expression of OCT1 correlated with expression of both OCT2 and OCT3. OCT1 and OCT2 were relatively uniformly expressed, whereas expression of OCT3, MATE1 and MATE2 showed substantial variation across lines. There were statistically significant associations between resistance to inhibition of proliferation and MATE2 expression, as well as between sensitivity to inhibition of oxygen consumption and OCT3 expression. One cell line (LNCaP) with high OCT3 and low MATE2 expression in concert, had substantially higher intracellular metformin concentration than other cell lines, and was exquisitely sensitive to both anti-proliferative and anti-respiratory effects. In all other cell lines, the concentration of metformin required to inhibit oxygen consumption acutely in vitro was substantially higher than that achieved in the plasma of diabetic patients. However, administering anti-diabetic doses of metformin to tumor-bearing mice resulted in intratumoral accumulation of metformin and reduced hypoxic tumor fractions. CONCLUSIONS: All cancer cells are susceptible to inhibition of oxygen consumption by metformin, which results in reduced hypoxic tumor fractions beneficial for the response to radiotherapy. High MATE2 expression may result in resistance to the anti-proliferative effect of metformin and should be considered as a negative predictive biomarker in clinical trials.


Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor/metabolism , Drug Resistance, Neoplasm , Hypoglycemic Agents/pharmacology , Metformin/pharmacology , Organic Cation Transport Proteins/metabolism , Animals , Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/genetics , Cell Proliferation/drug effects , Cells, Cultured , HCT116 Cells , HeLa Cells , Humans , Hypoglycemic Agents/therapeutic use , Metformin/therapeutic use , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Octamer Transcription Factors/genetics , Octamer Transcription Factors/metabolism , Organic Cation Transport Proteins/genetics , Oxygen Consumption/drug effects
8.
Cancer Res ; 76(18): 5501-11, 2016 09 15.
Article in English | MEDLINE | ID: mdl-27530327

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. Cancer Res; 76(18); 5501-11. ©2016 AACR.


Subject(s)
Antimetabolites, Antineoplastic/metabolism , Antimetabolites, Antineoplastic/pharmacology , Pyrophosphatases/metabolism , Thioguanine/metabolism , Thioguanine/pharmacology , Blotting, Western , Cell Line, Tumor , Chromatography, High Pressure Liquid , Crystallography, X-Ray , Humans , Mutation, Missense , Protein Stability , Pyrophosphatases/chemistry , Pyrophosphatases/genetics
9.
Cell Rep ; 14(2): 298-309, 2016 Jan 12.
Article in English | MEDLINE | ID: mdl-26748709

ABSTRACT

ATR and CHK1 maintain cancer cell survival under replication stress and inhibitors of both kinases are currently undergoing clinical trials. As ATR activity is increased after CHK1 inhibition, we hypothesized that this may indicate an increased reliance on ATR for survival. Indeed, we observe that replication stress induced by the CHK1 inhibitor AZD7762 results in replication catastrophe and apoptosis, when combined with the ATR inhibitor VE-821 specifically in cancer cells. Combined treatment with ATR and CHK1 inhibitors leads to replication fork arrest, ssDNA accumulation, replication collapse, and synergistic cell death in cancer cells in vitro and in vivo. Inhibition of CDK reversed replication stress and synthetic lethality, demonstrating that regulation of origin firing by ATR and CHK1 explains the synthetic lethality. In conclusion, this study exemplifies cancer-specific synthetic lethality between two proteins in the same pathway and raises the prospect of combining ATR and CHK1 inhibitors as promising cancer therapy.


Subject(s)
Protein Kinases/genetics , Apoptosis , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Line, Tumor , Checkpoint Kinase 1 , DNA Damage , Humans , Protein Kinases/metabolism
10.
Nat Commun ; 6: 7871, 2015 Aug 04.
Article in English | MEDLINE | ID: mdl-26238318

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.


Subject(s)
DNA Repair Enzymes/metabolism , Deoxyguanine Nucleotides/metabolism , Deoxyribonucleotides/metabolism , Oxidation-Reduction , Oxidative Stress , Phosphoric Monoester Hydrolases/metabolism , Pyrophosphatases/metabolism , Blotting, Western , Cell Line, Tumor , Cell Survival , Crystallization , HCT116 Cells , HeLa Cells , Humans , MCF-7 Cells , Pyrophosphatases/chemistry , Substrate Specificity
11.
Nature ; 508(7495): 215-21, 2014 Apr 10.
Article in English | MEDLINE | ID: mdl-24695224

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 bind in 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.


Subject(s)
DNA Repair Enzymes/antagonists & inhibitors , Neoplasms/drug therapy , Neoplasms/metabolism , Nucleotides/metabolism , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Animals , Catalytic Domain , Cell Death/drug effects , Cell Survival/drug effects , Crystallization , DNA Damage , DNA Repair Enzymes/chemistry , DNA Repair Enzymes/metabolism , Deoxyguanine Nucleotides/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Female , Humans , Male , Mice , Models, Molecular , Molecular Conformation , Molecular Targeted Therapy , Neoplasms/pathology , Oxidation-Reduction/drug effects , Phosphoric Monoester Hydrolases/chemistry , Phosphoric Monoester Hydrolases/metabolism , Pyrimidines/chemistry , Pyrimidines/pharmacokinetics , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Pyrophosphatases/antagonists & inhibitors , Reproducibility of Results , Xenograft Model Antitumor Assays , Nudix Hydrolases
12.
Nucleic Acids Res ; 40(17): 8440-8, 2012 Sep 01.
Article in English | MEDLINE | ID: mdl-22753029

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 η (Polη), 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.


Subject(s)
DNA Damage , DNA Replication , Protein Kinases/metabolism , Ultraviolet Rays , Caffeine/pharmacology , Cell Cycle Checkpoints/drug effects , Cell Line, Transformed , Cell Survival , Checkpoint Kinase 1 , DNA Breaks, Double-Stranded , DNA Replication/drug effects , DNA Replication/radiation effects , DNA-Directed DNA Polymerase/deficiency , Humans , Protein Kinase Inhibitors/pharmacology , Signal Transduction/radiation effects
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