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1.
Mol Oncol ; 2024 Mar 27.
Article in English | MEDLINE | ID: mdl-38533616

ABSTRACT

The one-carbon metabolism enzyme bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase 2 (MTHFD2) is among the most overexpressed proteins across tumors and is widely recognized as a promising anticancer target. While MTHFD2 is mainly described as a mitochondrial protein, a new nuclear function is emerging. Here, we observe that nuclear MTHFD2 protein levels and association with chromatin increase following ionizing radiation (IR) in an ataxia telangiectasia mutated (ATM)- and DNA-dependent protein kinase (DNA-PK)-dependent manner. Furthermore, repair of IR-induced DNA double-strand breaks (DSBs) is delayed upon MTHFD2 knockdown, suggesting a role for MTHFD2 in DSB repair. In support of this, we observe impaired recruitment of replication protein A (RPA), reduced resection, decreased IR-induced DNA repair protein RAD51 homolog 1 (RAD51) levels and impaired homologous recombination (HR) activity in MTHFD2-depleted cells following IR. In conclusion, we identify a key role for MTHFD2 in HR repair and describe an interdependency between MTHFD2 and HR proficiency that could potentially be exploited for cancer therapy.

2.
Int J Mol Sci ; 24(13)2023 Jul 06.
Article in English | MEDLINE | ID: mdl-37446337

ABSTRACT

The introduction of anti-amyloid monoclonal antibodies against Alzheimer's disease (AD) is of high importance. However, even though treated patients show very little amyloid pathology, there is only a modest effect on the rate of cognitive decline. Although this effect can possibly increase over time, there is still a need for alternative treatments that will improve cognitive function in patients with AD. Therefore, the purpose of this study was to characterize the triazinetrione ACD856, a novel pan-Trk positive allosteric modulator, in multiple models to address its neuroprotective and potential disease-modifying effects. The pharmacological effect of ACD856 was tested in recombinant cell lines, primary cortical neurons, or animals. We demonstrate that ACD856 enhanced NGF-induced neurite outgrowth, increased the levels of the pre-synaptic protein SNAP25 in PC12 cells, and increased the degree of phosphorylated TrkB in SH-SY5Y cells. In primary cortical neurons, ACD856 led to increased levels of phospho-ERK1/2, showed a neuroprotective effect against amyloid-beta or energy-deprivation-induced neurotoxicity, and increased the levels of brain-derived neurotrophic factor (BDNF). Consequently, administration of ACD856 resulted in a significant increase in BDNF in the brains of 21 months old mice. Furthermore, repeated administration of ACD856 resulted in a sustained anti-depressant effect, which lasted up to seven days, suggesting effects that go beyond merely symptomatic effects. In conclusion, the results confirm ACD856 as a cognitive enhancer, but more importantly, they provide substantial in vitro and in vivo evidence of neuroprotective and long-term effects that contribute to neurotrophic support and increased neuroplasticity. Presumably, the described effects of ACD856 may improve cognition, increase resilience, and promote neurorestorative processes, thereby leading to a healthier brain in patients with AD.


Subject(s)
Alzheimer Disease , Cognitive Dysfunction , Neuroblastoma , Neuroprotective Agents , Rats , Mice , Humans , Animals , Alzheimer Disease/metabolism , Brain-Derived Neurotrophic Factor/metabolism , Neuroblastoma/drug therapy , Amyloid beta-Peptides/metabolism , PC12 Cells , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , Cognitive Dysfunction/drug therapy , Cognitive Dysfunction/etiology
3.
Science ; 376(6600): 1471-1476, 2022 06 24.
Article in English | MEDLINE | ID: mdl-35737787

ABSTRACT

Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed ß,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.


Subject(s)
DNA Damage , DNA Glycosylases , DNA Repair , Oxidative Stress , Biocatalysis/drug effects , DNA Damage/drug effects , DNA Glycosylases/chemistry , DNA Glycosylases/drug effects , DNA Repair/drug effects , Enzyme Activation , Glycine/chemistry , Humans , Ligands , Oxidative Stress/genetics , Phenylalanine/chemistry , Substrate Specificity
4.
Nat Cancer ; 3(2): 156-172, 2022 02.
Article in English | MEDLINE | ID: mdl-35228749

ABSTRACT

The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors.


Subject(s)
Aminohydrolases , Leukemia, Myeloid, Acute , Aminohydrolases/genetics , Humans , Hydrolases , Leukemia, Myeloid, Acute/drug therapy , Methylenetetrahydrofolate Dehydrogenase (NADP)/genetics , Multifunctional Enzymes/genetics , Thymidine
5.
Cell Death Differ ; 29(1): 246-261, 2022 01.
Article in English | MEDLINE | ID: mdl-34453118

ABSTRACT

T cell-driven diseases account for considerable morbidity and disability globally and there is an urgent need for new targeted therapies. Both cancer cells and activated T cells have an altered redox balance, and up-regulate the DNA repair protein MTH1 that sanitizes the oxidized nucleotide pool to avoid DNA damage and cell death. Herein we suggest that the up-regulation of MTH1 in activated T cells correlates with their redox status, but occurs before the ROS levels increase, challenging the established conception of MTH1 increasing as a direct response to an increased ROS status. We also propose a heterogeneity in MTH1 levels among activated T cells, where a smaller subset of activated T cells does not up-regulate MTH1 despite activation and proliferation. The study suggests that the vast majority of activated T cells have high MTH1 levels and are sensitive to the MTH1 inhibitor TH1579 (Karonudib) via induction of DNA damage and cell cycle arrest. TH1579 further drives the surviving cells to the MTH1low phenotype with altered redox status. TH1579 does not affect resting T cells, as opposed to the established immunosuppressor Azathioprine, and no sensitivity among other major immune cell types regarding their function can be observed. Finally, we demonstrate a therapeutic effect in a murine model of experimental autoimmune encephalomyelitis. In conclusion, we show proof of concept of the existence of MTH1high and MTH1low activated T cells, and that MTH1 inhibition by TH1579 selectively suppresses pro-inflammatory activated T cells. Thus, MTH1 inhibition by TH1579 may serve as a novel treatment option against autoreactive T cells in autoimmune diseases, such as multiple sclerosis.


Subject(s)
DNA Repair Enzymes , Phosphoric Monoester Hydrolases , Animals , DNA Damage , DNA Repair Enzymes/metabolism , Lymphocyte Count , Mice , Phosphoric Monoester Hydrolases/genetics , T-Lymphocytes/metabolism
6.
Cancer Res ; 81(22): 5733-5744, 2021 11 15.
Article in English | MEDLINE | ID: mdl-34593524

ABSTRACT

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, exhibiting high levels of reactive oxygen species (ROS). ROS levels have been suggested to drive leukemogenesis and is thus a potential novel target for treating AML. MTH1 prevents incorporation of oxidized nucleotides into the DNA to maintain genome integrity and is upregulated in many cancers. Here we demonstrate that hematologic cancers are highly sensitive to MTH1 inhibitor TH1579 (karonudib). A functional precision medicine ex vivo screen in primary AML bone marrow samples demonstrated a broad response profile of TH1579, independent of the genomic alteration of AML, resembling the response profile of the standard-of-care treatments cytarabine and doxorubicin. Furthermore, TH1579 killed primary human AML blast cells (CD45+) as well as chemotherapy resistance leukemic stem cells (CD45+Lin-CD34+CD38-), which are often responsible for AML progression. TH1579 killed AML cells by causing mitotic arrest, elevating intracellular ROS levels, and enhancing oxidative DNA damage. TH1579 showed a significant therapeutic window, was well tolerated in animals, and could be combined with standard-of-care treatments to further improve efficacy. TH1579 significantly improved survival in two different AML disease models in vivo. In conclusion, the preclinical data presented here support that TH1579 is a promising novel anticancer agent for AML, providing a rationale to investigate the clinical usefulness of TH1579 in AML in an ongoing clinical phase I trial. SIGNIFICANCE: The MTH1 inhibitor TH1579 is a potential novel AML treatment, targeting both blasts and the pivotal leukemic stem cells while sparing normal bone marrow cells.


Subject(s)
Blast Crisis/drug therapy , DNA Repair Enzymes/antagonists & inhibitors , Gene Expression Regulation, Leukemic/drug effects , Leukemia, Myeloid, Acute/drug therapy , Mitosis , Neoplastic Stem Cells/drug effects , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Pyrimidines/pharmacology , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis , Blast Crisis/genetics , Blast Crisis/metabolism , Blast Crisis/pathology , Cell Proliferation , Cytarabine/administration & dosage , Doxorubicin/administration & dosage , Female , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Prognosis , Reactive Oxygen Species/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
7.
Nucleic Acids Res ; 48(21): 12234-12251, 2020 12 02.
Article in English | MEDLINE | ID: mdl-33211885

ABSTRACT

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.


Subject(s)
Colonic Neoplasms/drug therapy , DNA Glycosylases/genetics , DNA, Neoplasm/genetics , Gene Expression Regulation, Neoplastic , Poly (ADP-Ribose) Polymerase-1/immunology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/mortality , DNA Damage , DNA Glycosylases/antagonists & inhibitors , DNA Glycosylases/metabolism , DNA Repair/drug effects , DNA Replication/drug effects , DNA, Neoplasm/metabolism , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Guanine/analogs & derivatives , Guanine/metabolism , HCT116 Cells , Humans , Mice , Mice, Nude , Molecular Targeted Therapy , Oxidative Stress , Poly (ADP-Ribose) Polymerase-1/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Reactive Oxygen Species/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Signal Transduction , Survival Analysis , Tumor Burden/drug effects , Xenograft Model Antitumor Assays
8.
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
9.
EMBO Mol Med ; 12(3): e10419, 2020 03 06.
Article in English | MEDLINE | ID: mdl-31950591

ABSTRACT

The deoxycytidine analogue cytarabine (ara-C) remains the backbone treatment of acute myeloid leukaemia (AML) as well as other haematological and lymphoid malignancies, but must be combined with other chemotherapeutics to achieve cure. Yet, the underlying mechanism dictating synergistic efficacy of combination chemotherapy remains largely unknown. The dNTPase SAMHD1, which regulates dNTP homoeostasis antagonistically to ribonucleotide reductase (RNR), limits ara-C efficacy by hydrolysing the active triphosphate metabolite ara-CTP. Here, we report that clinically used inhibitors of RNR, such as gemcitabine and hydroxyurea, overcome the SAMHD1-mediated barrier to ara-C efficacy in primary blasts and mouse models of AML, displaying SAMHD1-dependent synergy with ara-C. We present evidence that this is mediated by dNTP pool imbalances leading to allosteric reduction of SAMHD1 ara-CTPase activity. Thus, SAMHD1 constitutes a novel biomarker for combination therapies of ara-C and RNR inhibitors with immediate consequences for clinical practice to improve treatment of AML.


Subject(s)
Cytarabine/pharmacology , Leukemia, Myeloid, Acute , Pyrophosphatases/metabolism , Ribonucleotide Reductases/antagonists & inhibitors , SAM Domain and HD Domain-Containing Protein 1/metabolism , Animals , Arabinofuranosylcytosine Triphosphate/metabolism , Mice
10.
Ther Adv Med Oncol ; 11: 1758835919866960, 2019.
Article in English | MEDLINE | ID: mdl-31489034

ABSTRACT

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common form of liver cancer and is generally caused by viral infections or consumption of mutagens, such as alcohol. While liver transplantation and hepatectomy is curative for some patients, many relapse into disease with few treatment options such as tyrosine kinase inhibitors, for example, sorafenib or lenvatinib. The need for novel systemic treatment approaches is urgent. METHODS: MTH1 expression profile was first analyzed in a HCC database and MTH1 mRNA/protein level was determined in resected HCC and paired paracancerous tissues with polymerase chain reaction (PCR) and immunohistochemistry. HCC cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA. 8-oxoG was measured by the modified comet assay. The effect of MTH1 inhibition on tumor growth was explored in HCC xenograft in vivo models. RESULTS: MTH1 protein level is elevated in HCC tissue compared with paracancerous liver tissue and indicates poor prognosis. The MTH1 inhibitor Karonudib (TH1579) and siRNA effectively introduce toxic oxidized nucleotides into DNA, 8-oxoG, and kill HCC cell lines in vitro. Furthermore, we demonstrate that HCC growth in a xenograft mouse model in vivo is efficiently suppressed by Karonudib. CONCLUSION: Altogether, these data suggest HCC relies on MTH1 for survival, which can be targeted and may open up a novel treatment option for HCC in the future.

11.
Science ; 362(6416): 834-839, 2018 11 16.
Article in English | MEDLINE | ID: mdl-30442810

ABSTRACT

The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor-α-induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Benzimidazoles/pharmacology , DNA Glycosylases/antagonists & inhibitors , Enzyme Inhibitors/therapeutic use , Gene Expression/drug effects , Inflammation/drug therapy , Piperidines/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Benzimidazoles/therapeutic use , DNA Glycosylases/metabolism , DNA Repair/drug effects , DNA Repair/genetics , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Gene Knockout Techniques , Guanine/analogs & derivatives , Guanine/antagonists & inhibitors , Guanine/metabolism , HEK293 Cells , Humans , Inflammation/genetics , Jurkat Cells , Mice , Mice, Mutant Strains , NF-kappa B/genetics , NF-kappa B/metabolism , Piperidines/therapeutic use , Promoter Regions, Genetic , Tumor Necrosis Factor-alpha/pharmacology
12.
Nucleic Acids Res ; 46(20): 10888-10904, 2018 11 16.
Article in English | MEDLINE | ID: mdl-30304478

ABSTRACT

Nucleotides in the free pool are more susceptible to nonenzymatic methylation than those protected in the DNA double helix. Methylated nucleotides like O6-methyl-dGTP can be mutagenic and toxic if incorporated into DNA. Removal of methylated nucleotides from the nucleotide pool may therefore be important to maintain genome integrity. We show that MutT homologue 1 (MTH1) efficiently catalyzes the hydrolysis of O6-methyl-dGTP with a catalytic efficiency similar to that for 8-oxo-dGTP. O6-methyl-dGTP activity is exclusive to MTH1 among human NUDIX proteins and conserved through evolution but not found in bacterial MutT. We present a high resolution crystal structure of human and zebrafish MTH1 in complex with O6-methyl-dGMP. By microinjecting fertilized zebrafish eggs with O6-methyl-dGTP and inhibiting MTH1 we demonstrate that survival is dependent on active MTH1 in vivo. O6-methyl-dG levels are higher in DNA extracted from zebrafish embryos microinjected with O6-methyl-dGTP and inhibition of O6-methylguanine-DNA methyl transferase (MGMT) increases the toxicity of O6-methyl-dGTP demonstrating that O6-methyl-dGTP is incorporated into DNA. MTH1 deficiency sensitizes human cells to the alkylating agent Temozolomide, a sensitization that is more pronounced upon MGMT inhibition. These results expand the cellular MTH1 function and suggests MTH1 also is important for removal of methylated nucleotides from the nucleotide pool.


Subject(s)
DNA Repair Enzymes/physiology , Deoxyguanine Nucleotides/chemistry , Phosphoric Monoester Hydrolases/physiology , Animals , Catalytic Domain , Crystallography, X-Ray , DNA Modification Methylases/chemistry , DNA Repair Enzymes/chemistry , Dogs , Escherichia coli/genetics , HL-60 Cells , Humans , Hydrolysis , Kinetics , Mice , Nucleotides , Phosphoric Monoester Hydrolases/chemistry , Pyrophosphatases/chemistry , Species Specificity , Swine , Temozolomide/pharmacology , Tumor Suppressor Proteins/chemistry , Zebrafish
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