ABSTRACT
INTRODUCTION AND IMPORTANCE: Pituitary apoplexy is a clinical syndrome that occurs secondary to abrupt haemorrhage or infarction and can cause a presentation with headache, visual disturbances, and various neurological deficits. Pituitary apoplexy is a rare clinical syndrome that is often misdiagnosed or discovered late in its clinical course due to its relatively nonspecific clinical presentation. CASE PRESENTATION: The case describes patient with a giant prolactinoma which developed haemorrhagic progression in the context of previously undiagnosed coagulopathy from idiopathic thrombocytopaenia. Transcranial (rather than transnasal) surgery was performed for evacuation and debulking. CONCLUSION: The case highlights the management of an exceedingly large adenoma in the context of coagulopathy contributing to rapid clinical progression, as well as a positive clinical outcome in a patient with restored vision and pituitary function.
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 , ThymidineABSTRACT
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/metabolismABSTRACT
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 AssaysABSTRACT
Inflammatory diseases, including psoriasis, are characterized by changes in redox regulation. The MTH1 prevents the incorporation of oxidized nucleotides during DNA replication. Using MTH1 small-molecule inhibitors, we found induced apoptosis through 8-oxodeoxyguanosine triphosphate accumulation and DNA double-strand breaks after oxidative stress in normal and malignant keratinocytes. In psoriasis, we detected increased MTH1 expression in lesional skin and PBMCs compared with that in the controls. Using the imiquimod psoriasis mouse model, we found that MTH1 inhibition diminished psoriatic histological characteristics and normalized the levels of neutrophils and T cells in the skin and skin-draining lymph nodes. The inhibition abolished the expression of T helper type 17âassociated cytokines in the skin, which was in line with decreased levels of IL-17-producing γδ T cells in lymph nodes. In human keratinocytes, MTH1 inhibition prevented the upregulation of IL-17âdownstream genes, which was independent of ROS-induced apoptosis. In conclusion, our data support MTH1 inhibition using small molecules suitable for topical application as a promising therapeutic approach to psoriasis.
Subject(s)
DNA Repair Enzymes/antagonists & inhibitors , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Psoriasis/drug therapy , Skin/pathology , Administration, Cutaneous , Animals , Apoptosis/drug effects , Apoptosis/immunology , Biopsy , Cell Line, Tumor , DNA Repair Enzymes/metabolism , Disease Models, Animal , Female , Humans , Imiquimod/administration & dosage , Keratinocytes/drug effects , Keratinocytes/immunology , Keratinocytes/metabolism , Mice , Oxidative Stress/drug effects , Oxidative Stress/immunology , Phosphoric Monoester Hydrolases/metabolism , Primary Cell Culture , Psoriasis/immunology , Psoriasis/pathology , Skin/drug effects , Skin/immunologyABSTRACT
Chemo-immunotherapy has improved survival in B-cell lymphoma patients, but refractory/relapsed diseases still represent a major challenge, urging for development of new therapeutics. Karonudib (TH1579) was developed to inhibit MTH1, an enzyme preventing oxidized dNTP-incorporation in DNA. MTH1 is highly upregulated in tumor biopsies from patients with diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma, hence confirming a rationale for targeting MTH1. Here, we tested the efficacy of karonudib in vitro and in preclinical B-cell lymphoma models. Using a range of B-cell lymphoma cell lines, karonudib strongly reduced viability at concentrations well tolerated by activated normal B cells. In B-cell lymphoma cells, karonudib increased incorporation of 8-oxo-dGTP into DNA, and prominently induced prometaphase arrest and apoptosis due to failure in spindle assembly. MTH1 knockout cell lines were less sensitive to karonudib-induced apoptosis, but were displaying cell cycle arrest phenotype similar to the wild type cells, indicating a dual inhibitory role of the drug. Karonudib was highly potent as single agent in two different lymphoma xenograft models, including an ABC DLBCL patient derived xenograft, leading to prolonged survival and fully controlled tumor growth. Together, our preclinical findings provide a rationale for further clinical testing of karonudib in B-cell lymphoma.
Subject(s)
Burkitt Lymphoma/drug therapy , DNA Repair Enzymes/genetics , Lymphoma, B-Cell/drug therapy , Phosphoric Monoester Hydrolases/genetics , Pyrimidines/pharmacology , Animals , Apoptosis/drug effects , Burkitt Lymphoma/genetics , Burkitt Lymphoma/pathology , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , DNA/biosynthesis , DNA Repair Enzymes/antagonists & inhibitors , Deoxyguanine Nucleotides/antagonists & inhibitors , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lymphoma, B-Cell/genetics , Lymphoma, B-Cell/pathology , Mice , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Xenograft Model Antitumor AssaysABSTRACT
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 AssaysABSTRACT
BACKGROUND: Osteosarcoma (OS) is the most common primary malignant bone tumour. Unfortunately, no new treatments are approved and over the last 30 years the survival rate remains only 30% at 5 years for poor responders justifying an urgent need of new therapies. The Mutt homolog 1 (MTH1) enzyme prevents incorporation of oxidized nucleotides into DNA and recently developed MTH1 inhibitors may offer therapeutic potential as MTH1 is overexpressed in various cancers. METHODS: The aim of this study was to evaluate the therapeutic benefits of targeting MTH1 with two chemical inhibitors, TH588 and TH1579 on human osteosarcoma cells. Preclinical efficacy of TH1579 was assessed in human osteosarcoma xenograft model on tumour growth and development of pulmonary metastases. FINDINGS: MTH1 is overexpressed in OS patients and tumour cell lines, compared to mesenchymal stem cells. In vitro, chemical inhibition of MTH1 by TH588 and TH1579 decreases OS cells viability, impairs their cell cycle and increases apoptosis in OS cells. TH1579 was confirmed to bind MTH1 by CETSA in OS model. Moreover, 90â¯mg/kg of TH1579 reduces in vivo tumour growth by 80.5% compared to non-treated group at day 48. This result was associated with the increase in 8-oxo-dG integration into tumour cells DNA and the increase of apoptosis. Additionally, TH1579 also reduces the number of pulmonary metastases. INTERPRETATION: All these results strongly provide a pre-clinical proof-of-principle that TH1579 could be a therapeutic option for patients with osteosarcoma. FUNDING: This study was supported by La Ligue Contre le Cancer, la SFCE and Enfants Cancers Santé.
Subject(s)
Antineoplastic Agents/therapeutic use , Bone Neoplasms/drug therapy , DNA Repair Enzymes/antagonists & inhibitors , Lung Neoplasms/drug therapy , Osteosarcoma/drug therapy , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Pyrimidines/therapeutic use , 8-Hydroxy-2'-Deoxyguanosine/metabolism , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Bone Neoplasms/pathology , Cell Line, Tumor , DNA/genetics , DNA Repair Enzymes/metabolism , Humans , Lung Neoplasms/secondary , Mice , Osteosarcoma/pathology , Phosphoric Monoester Hydrolases/metabolism , Pyrimidines/pharmacology , Tumor Cells, CulturedABSTRACT
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.
ABSTRACT
BACKGROUND AND PURPOSE: Small cell lung cancer (SCLC) is an aggressive disease with median survival of <2 years. Tumour biopsies for research are scarce, especially from extensive-stage patients, with repeat sampling at disease progression rarely performed. We overcame this limitation for relevant preclinical models by developing SCLC circulating tumour cell derived explants (CDX), which mimic the donor tumour pathology and chemotherapy response. To facilitate compound screening and identification of clinically relevant biomarkers, we developed short-term ex vivo cultures of CDX tumour cells. EXPERIMENTAL APPROACH: CDX tumours were disaggregated, and the human tumour cells derived were cultured for a maximum of 5 weeks. Phenotypic, transcriptomic and pharmacological characterization of these cells was performed. KEY RESULTS: CDX cultures maintained a neuroendocrine phenotype, and most changes in the expression of protein-coding genes observed in cultures, for up to 4 weeks, were reversible when the cells were re-implanted in vivo. Moreover, the CDX cultures exhibited a similar sensitivity to chemotherapy compared to the corresponding CDX tumour in vivo and were able to predict in vivo responses to therapeutic candidates. CONCLUSIONS AND IMPLICATIONS: Short-term cultures of CDX provide a tractable platform to screen new treatments, identify predictive and pharmacodynamic biomarkers and investigate mechanisms of resistance to better understand the progression of this recalcitrant tumour.
Subject(s)
Antineoplastic Agents/pharmacology , Indazoles/pharmacology , Lung Neoplasms/drug therapy , Neoplastic Cells, Circulating/drug effects , Small Cell Lung Carcinoma/drug therapy , Sulfonamides/pharmacology , Animals , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Drug Screening Assays, Antitumor , Humans , Indazoles/chemistry , Lung Neoplasms/pathology , Mice , Mice, Inbred Strains , Mice, SCID , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Neoplastic Cells, Circulating/pathology , Small Cell Lung Carcinoma/pathology , Structure-Activity Relationship , Sulfonamides/chemistry , Tumor Cells, CulturedABSTRACT
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/pharmacologyABSTRACT
In spite of the fact that cholesterol does not pass the blood-brain barrier, treatment of mice with dietary cholesterol causes significant effects on a number of genes in the brain and in addition a memory impairment. We have suggested that these effects are mediated by 27-hydroxycholesterol, which is able to pass the blood-brain barrier. To test this hypothesis we utilized Cyp27-/- mice lacking 27-hydroxycholesterol. The negative effect on memory observed after treatment of wildtype mice with dietary cholesterol was not observed in these mice. The cholesterol diet reduced the levels of the "memory protein" Arc (Activity Regulated Cytoskeleton associated protein) in the hippocampus of the wildtype mice but not in the hippocampus of the Cyp27-/- mice. The results are consistent with 27-hydroxycholesterol as the mediator of the negative effects of cholesterol on cognition.
Subject(s)
Cholesterol/adverse effects , Cognition Disorders/etiology , Cognition Disorders/genetics , Hydroxycholesterols/therapeutic use , AIDS-Related Complex/genetics , AIDS-Related Complex/metabolism , Animals , Body Weight/drug effects , Body Weight/genetics , Cholestanetriol 26-Monooxygenase/deficiency , Cholestanetriol 26-Monooxygenase/genetics , Cholic Acid/administration & dosage , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Male , Maze Learning/drug effects , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Messenger/metabolism , Spatial Learning/drug effectsABSTRACT
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 HydrolasesABSTRACT
Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents.
Subject(s)
Antineoplastic Agents/pharmacology , DNA Repair Enzymes/antagonists & inhibitors , DNA Repair Enzymes/metabolism , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Phosphoric Monoester Hydrolases/metabolism , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyridines/pharmacology , Aminoquinolines/pharmacology , Animals , Antineoplastic Agents/chemistry , Colonic Neoplasms/drug therapy , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Crizotinib , Crystallization , DNA Breaks, Single-Stranded/drug effects , DNA Repair , DNA Repair Enzymes/biosynthesis , DNA Repair Enzymes/chemistry , Disease Models, Animal , Female , Homeostasis/drug effects , Humans , Mice , Mice, SCID , Models, Molecular , Nucleotides/metabolism , Phosphoric Monoester Hydrolases/biosynthesis , Phosphoric Monoester Hydrolases/chemistry , Protein Conformation , Protein Kinase Inhibitors/chemistry , Proteomics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Pyrazoles/chemistry , Pyridines/chemistry , Substrate Specificity , Xenograft Model Antitumor Assays , ras Proteins/geneticsABSTRACT
Apolipoprotein E (apoE) has an intricate biological function in modulating immune responses and apoE isoforms exhibit diverse effects on neurodegenerative and neuroinflammatory disorders. In the present study, we investigated the individual roles of apoE isoforms in the kainic acid (KA)-induced hippocampal neurodegeneration with focus on immune response and microglia functions. ApoE2, 3 and 4 transgenic mice as well as wild-type (WT) mice were treated with KA by intranasal route. ApoE4 overexpressing mice revealed several peculiarities as compared with other transgenic mice and WT mice, i.e. (1) they had more severe KA-induced seizures than apoE2 and 3 mice, (2) they exhibited neuron loss in hippocampus that was higher than in apoE2, 3 and WT mice, (3) KA administration resulted in higher counts of their head drops in the cross-area of elevated plus-maze, (4) they showed lower KA-induced rearing activity than apoE2 mice in the open-field test, (5) their KA-induced microglial expression of MHC-II and CD86 was elevated compared to apoE3 mice, (6) the KA-induced increase of microglial iNOS was higher than that in the other groups of mice, and (7) the TNF-α and IL-6 expression was decreased 7 days after KA application compared to untreated mice and mice treated 1 day with KA. However, the signaling pathway of NFκB or Akt seemed not to be involved in apoE-isoform dependent susceptibility to KA-induced neurotoxicity. In conclusion, over-expression of apoE4 deteriorated KA-induced hippocampal neurodegeneration in C57BL/6 mice, which might result from a higher up-regulation of microglia activation compared to apoE2 and 3 transgenic mice and WT mice.
Subject(s)
Apolipoprotein E4/metabolism , Gene Expression Regulation/physiology , Hippocampus/metabolism , Neurodegenerative Diseases/pathology , Neurodegenerative Diseases/physiopathology , Analysis of Variance , Animals , Apolipoprotein E2 , Apolipoprotein E3 , Apolipoprotein E4/genetics , Astrocytes/drug effects , Astrocytes/metabolism , CD11b Antigen/metabolism , Cytokines , Enzyme-Linked Immunosorbent Assay , Excitatory Amino Acid Agonists/toxicity , Exploratory Behavior/drug effects , Flow Cytometry , Gene Expression Regulation/drug effects , Glial Fibrillary Acidic Protein/metabolism , Hippocampus/pathology , Hippocampus/physiopathology , Kainic Acid/toxicity , Male , Maze Learning/drug effects , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neurodegenerative Diseases/chemically induced , Seizures/chemically induced , Statistics, NonparametricABSTRACT
N-formylpeptide receptor 1 (FPR1) is a G protein-coupled receptor that mediates pro-inflammatory chemotactic responses by phagocytic leukocytes to N-formylpeptides produced by bacteria or mitochondria. Mice lacking Fpr1 (Fpr1 (-/-) mice) have increased susceptibility to challenge with certain bacteria. FPR1 is also a receptor for annexin-1, which mediates the anti-inflammatory effects of glucocorticoids as well as negative feedback by glucocorticoids of the hypothalamic-pituitary-adrenocortical axis. However, homeostatic functions of FPR1 in the neuroendocrine system have not previously been defined. Here we show that in systematic behavioral testing Fpr1 (-/-) mice exhibited increased exploratory activity, reduced anxiety-like behavior, and impaired fear memory, but normal spatial memory and learning capacity. Consistent with this, the homeostatic serum level of corticosterone in Fpr1 (-/-) mice was significantly lower compared with wild-type mice. The data implicate Fpr1 in modulation of anxiety-like behavior and fear memory by regulating glucocorticoid production.
Subject(s)
Anxiety/genetics , Fear , Memory , Receptors, Formyl Peptide/genetics , Adrenal Cortex Hormones/metabolism , Animals , Anxiety/metabolism , Behavior, Animal , Disease Models, Animal , Glucocorticoids/metabolism , Homeostasis , Inflammation , Male , Maze Learning , Mice , Mice, Knockout , Mitochondria/metabolism , N-Formylmethionine Leucyl-Phenylalanine/metabolism , Phagocytes/metabolismABSTRACT
Recently, interest in the rat as an animal model of Alzheimer's disease (AD) has been growing. We have previously described the Tg6590 transgenic rat line expressing the amyloid precursor protein containing the Swedish AD mutation (K670M/N671L) that shows early stages of Abeta deposition, predominantly in cerebrovascular blood vessels, after 15 months of age. Here we show that by the age of 9 months, that is long before the appearance of Abeta deposits, the Tg6590 rats exhibit deficits in the Morris water maze spatial navigation task and altered spontaneous behaviour in the open-field test. The levels of soluble Abeta were elevated both in the hippocampus and cortex of transgenic animals. Magnetic resonance imaging showed no major changes in the brains of transgenic animals, although they tended to have enlarged lateral ventricles when compared to control animals. The Tg6590 transgenic rat line should prove a suitable model of early AD for advanced studies including serial cerebrospinal fluid sampling, electrophysiology, neuroimaging or complex behavioural testing.
Subject(s)
Alzheimer Disease/complications , Amyloid beta-Protein Precursor/metabolism , Cognition Disorders/complications , Disease Models, Animal , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Alzheimer Disease/physiopathology , Animals , Behavior, Animal , Biomarkers/metabolism , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Cognition Disorders/metabolism , Cognition Disorders/pathology , Cognition Disorders/physiopathology , Hippocampus/metabolism , Hippocampus/pathology , Humans , Magnetic Resonance Imaging , Male , Maze Learning , Neurons/metabolism , Neurons/pathology , Phosphorylation , Rats , Rats, Transgenic , Synapses/metabolism , Synapses/pathology , tau Proteins/metabolismABSTRACT
Formation of lasting memories is believed to rely on structural alterations at the synaptic level. We had found that increased neuronal activity down-regulates Nogo receptor-1 (NgR1) in brain regions linked to memory formation and storage, and postulated this to be required for formation of lasting memories. We now show that mice with inducible overexpression of NgR1 in forebrain neurons have normal long-term potentiation and normal 24-h memory, but severely impaired month-long memory in both passive avoidance and swim maze tests. Blocking transgene expression normalizes these memory impairments. Nogo, Lingo-1, Troy, endogenous NgR1, and BDNF mRNA expression levels were not altered by transgene expression, suggesting that the impaired ability to form lasting memories is directly coupled to inability to down-regulate NgR1. Regulation of NgR1 may therefore serve as a key regulator of memory consolidation. Understanding the molecular underpinnings of synaptic rearrangements that carry lasting memories may facilitate development of treatments for memory dysfunction.
Subject(s)
Gene Expression Regulation/physiology , Memory/physiology , Myelin Proteins/physiology , Prosencephalon/metabolism , Animals , Brain-Derived Neurotrophic Factor/metabolism , Chromatography, High Pressure Liquid , Electrophysiology , Immunoblotting , Immunohistochemistry , In Situ Hybridization , Maze Learning/physiology , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Myelin Proteins/metabolism , Nerve Tissue Proteins/metabolism , Nogo Proteins , Receptors, Tumor Necrosis Factor/metabolism , Rotarod Performance Test , Transgenes/geneticsABSTRACT
Social housing is the optimal way of housing female laboratory mice. However, individual housing may be required in experimental designs, for example after surgery. We therefore investigated whether housing two female mice in a cage, separated by a grid partition ('living apart together', LAT), counters the adverse effects of individual housing on postoperative recovery. Ten individually housed (IND) mice, nine socially housed (SOC) mice and nine mice, housed LAT, were surgically implanted with a telemetry transmitter. From one week prior to surgery until three weeks thereafter, several physiological and behavioural parameters were measured in the mice subjected to surgery. The telemetry transmitter measured heart rate (HR), body temperature and activity continuously. Body weight, food and water intake were scored regularly, as were wound healing, ease of handling, nest building and behaviour. Results indicated that SOC mice appear to be less affected by abdominal surgery than IND mice, as indicated by HR and behaviour. LAT, however, did not appear to be beneficiary to the mice. Increased HR levels and differences in behaviour as compared with both SOC and IND animals indicate that LAT may even be the most stressful of the three housing conditions. We therefore conclude that mice benefit most from social housing after surgery. If, however, social housing is not possible, individual housing appears to be a better option than separating mice by a grid partition.