RESUMEN
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.
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Neoplasias del Colon/tratamiento farmacológico , ADN Glicosilasas/genética , ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Poli(ADP-Ribosa) Polimerasa-1/inmunología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/mortalidad , Daño del ADN , ADN Glicosilasas/antagonistas & inhibidores , ADN Glicosilasas/metabolismo , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , ADN de Neoplasias/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Guanina/análogos & derivados , Guanina/metabolismo , Células HCT116 , Humanos , Ratones , Ratones Desnudos , Terapia Molecular Dirigida , Estrés Oxidativo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Análisis de Supervivencia , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Enzimas Reparadoras del ADN/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Nucleótidos/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Animales , Dominio Catalítico , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cristalización , Daño del ADN , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/metabolismo , Nucleótidos de Desoxiguanina/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Masculino , Ratones , Modelos Moleculares , Conformación Molecular , Terapia Molecular Dirigida , Neoplasias/patología , Oxidación-Reducción/efectos de los fármacos , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/metabolismo , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Pirofosfatasas/antagonistas & inhibidores , Reproducibilidad de los Resultados , Ensayos Antitumor por Modelo de Xenoinjerto , Hidrolasas NudixRESUMEN
The mitotic MTH1 inhibitor TH1579 is a dual inhibitor that inhibits mitosis and incorporation of oxidative DNA damage and leads to cancer-specific cell death. The response to immune checkpoint inhibitor (ICI) treatment is often augmented by DNA damaging agents through the cGAS-STING pathway. This study investigates whether TH1579 can improve the efficacy of immune checkpoint blockades through its immunomodulatory properties. Various human and murine cancer cell lines were treated with mitotic MTH1i TH1579, and the expression of PD-L1 and T-cell infiltration-related chemokines was analysed by flow cytometry and real-time qPCR. Syngeneic mouse models were established to examine the combined effect of TH1579 and PD-L1 blockade. In our investigation, we found that TH1579 upregulates PD-L1 expression at both the protein and mRNA levels in human cancer cell lines. However, in murine cell lines, the increase was less pronounced. An in vivo experiment in a syngeneic mouse melanoma model showed that TH1579 treatment significantly increased the efficacy of atezolizumab, an anti-PD-L1 antibody, compared to vehicle or atezolizumab monotherapy. Furthermore, TH1579 exhibited immune-modulatory properties, elevating cytokines such as IFN-ß and chemokines including CCL5 and CXCL10, in a cGAS-STING pathway-dependent manner. In conclusion, TH1579 has the potential to improve ICI treatment by modulating immune checkpoint-related proteins and pathways.
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Brefeldin-A ADP-ribosylated substrate (BARS) and dynamin function in membrane fission in distinct intracellular transport pathways, but whether their functions are mechanistically similar is unclear. Here, we show that ARFGAP1, a GTPase-activating protein (GAP) for ADP-ribosylation factor 1 (ARF1), couples to either BARS or endophilin B for vesicle formation by the coat protein I (COPI) complex - a finding that reveals an unanticipated mechanistic flexibility in mammalian COPI transport. Because dynamin is coupled to endophilin A in vesicle formation by the clathrin-coat complex, our finding also predicts that dynamin and ARF GAPs are likely to be functional counterparts in membrane fission among different transport pathways that connect intracellular membrane compartments.
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Membranas Intracelulares/metabolismo , Aciltransferasas/metabolismo , Animales , Proteína Coat de Complejo I/metabolismo , Vesículas Cubiertas/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/citología , Aparato de Golgi/ultraestructura , Membranas Intracelulares/ultraestructura , Ratones , Factores de Transcripción/metabolismoRESUMEN
Epsin has been suggested to act as an alternate adaptor in several endocytic pathways. Its role in synaptic vesicle recycling remains, however, unclear. Here, we examined the role of epsin in this process by using the lamprey reticulospinal synapse as a model system. We characterized a lamprey ortholog of epsin 1 and showed that it is accumulated at release sites at rest and also at clathrin-coated pits in the periactive zone during synaptic activity. Disruption of epsin interactions, by presynaptic microinjection of antibodies to either the epsin-N-terminal homology domain (ENTH) or the clathrin/AP2 binding region (CLAP), caused profound loss of vesicles in stimulated synapses. CLAP antibody-injected synapses displayed a massive accumulation of distorted coated structures, including coated vacuoles, whereas in synapses perturbed with ENTH antibodies, very few coated structures were found. In both cases coated pits on the plasma membrane showed a shift to early intermediates (shallow coated pits) and an increase in size. Moreover, in CLAP antibody-injected synapses flat clathrin-coated patches occurred on the plasma membrane. We conclude that epsin is involved in clathrin-mediated synaptic vesicle endocytosis. Our results support a model, based on in vitro studies, suggesting that epsin coordinates curvature generation with coat assembly and further indicating that epsin limits clathrin coat assembly to the size of newly formed vesicles. We propose that these functions of epsin 1 provide an additional mechanism for generation of uniformly sized synaptic vesicles.
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Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Endocitosis , Lampreas/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Animales , Anticuerpos , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Microinyecciones , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Sinapsis/ultraestructura , Vesículas Sinápticas/ultraestructuraRESUMEN
Patients with hepatocellular carcinoma (HCC) suffer from few treatment options and poor survival rates. Here we report that endonuclease VIII-like protein 3 (NEIL3) is overexpressed in HCC and correlates with poor survival. All six HCC cell lines investigated were dependent on NEIL3 catalytic activity for survival and prevention of senescence, while NEIL3 was dispensable for nontransformed cells. NEIL3-depleted HCC cell lines accumulated oxidative DNA lesions specifically at telomeres, resulting in telomere dysfunctional foci and 53BP1 foci formation. Following oxidative DNA damage during mitosis, NEIL3 relocated to telomeres and recruited apurinic endonuclease 1 (APE1), indicating activation of base excision repair. META-FISH revealed that NEIL3, but not NEIL1 or NEIL2, is required to initiate APE1 and polymerase beta (POLB)-dependent base excision repair at oxidized telomeres. Repeated exposure of NEIL3-depleted cells to oxidizing damage induced chromatin bridges and damaged telomeres. These results demonstrate a novel function for NEIL3 in repair of oxidative DNA damage at telomeres in mitosis, which is important to prevent senescence of HCC cells. Furthermore, these data suggest that NEIL3 could be a target for therapeutic intervention for HCC. SIGNIFICANCE: This study describes compartmentalization of base excision repair during mitosis that is dependent on NEIL3, APE1, and POLB to repair oxidative damage accumulating at telomeres in hepatocellular carcinoma.
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Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Mitosis/genética , N-Glicosil Hidrolasas/metabolismo , Telómero/metabolismo , Humanos , Oxidación-Reducción , TransfecciónRESUMEN
The most common oxidative DNA lesion is 8-oxoguanine which is mainly recognized and excised by the 8-oxoG DNA glycosylase 1 (OGG1), initiating the base excision repair (BER) pathway. Telomeres are particularly sensitive to oxidative stress (OS) which disrupts telomere homeostasis triggering genome instability. In the present study, we have investigated the effects of inactivating BER in OS conditions, by using a specific inhibitor of OGG1 (TH5487). We have found that in OS conditions, TH5487 blocks BER initiation at telomeres causing an accumulation of oxidized bases, that is correlated with telomere losses, micronuclei formation and mild proliferation defects. Moreover, the antimetabolite methotrexate synergizes with TH5487 through induction of intracellular reactive oxygen species (ROS) formation, which potentiates TH5487-mediated telomere and genome instability. Our findings demonstrate that OGG1 is required to protect telomeres from OS and present OGG1 inhibitors as a tool to induce oxidative DNA damage at telomeres, with the potential for developing new combination therapies for cancer treatment.
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Antimetabolitos Antineoplásicos/farmacología , Bencimidazoles/farmacología , ADN Glicosilasas/antagonistas & inhibidores , Reparación del ADN/efectos de los fármacos , Metotrexato/farmacología , Estrés Oxidativo , Piperidinas/farmacología , Telómero/metabolismo , Ciclo Celular , Línea Celular Tumoral , ADN Glicosilasas/metabolismo , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Inestabilidad Genómica , Humanos , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
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Linfoma de Burkitt/tratamiento farmacológico , Enzimas Reparadoras del ADN/genética , Linfoma de Células B/tratamiento farmacológico , Monoéster Fosfórico Hidrolasas/genética , Pirimidinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Linfoma de Burkitt/genética , Linfoma de Burkitt/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN/biosíntesis , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Nucleótidos de Desoxiguanina/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patología , Ratones , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Crisis Blástica/tratamiento farmacológico , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Mitosis , Células Madre Neoplásicas/efectos de los fármacos , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Pirimidinas/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis , Crisis Blástica/genética , Crisis Blástica/metabolismo , Crisis Blástica/patología , Proliferación Celular , Citarabina/administración & dosificación , Doxorrubicina/administración & dosificación , Femenino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Pronóstico , Especies Reactivas de Oxígeno/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cutaneous malignant melanoma (CMM) is the deadliest form of skin cancer and clinically challenging due to its propensity to develop therapy resistance. Reactive oxygen species (ROS) can induce DNA damage and play a significant role in CMM. MTH1 protein protects from ROS damage and is often overexpressed in different cancer types including CMM. Herein, we report that MTH1 inhibitor TH1579 induced ROS levels, increased DNA damage responses, caused mitotic arrest and suppressed CMM proliferation leading to cell death both in vitro and in an in vivo xenograft CMM zebrafish disease model. TH1579 was more potent in abrogating cell proliferation and inducing cell death in a heterogeneous co-culture setting when compared with CMM standard treatments, vemurafenib or trametinib, showing its broad anticancer activity. Silencing MTH1 alone exhibited similar cytotoxic effects with concomitant induction of mitotic arrest and ROS induction culminating in cell death in most CMM cell lines tested, further emphasizing the importance of MTH1 in CMM cells. Furthermore, overexpression of receptor tyrosine kinase AXL, previously demonstrated to contribute to BRAF inhibitor resistance, sensitized BRAF mutant and BRAF/NRAS wildtype CMM cells to TH1579. AXL overexpression culminated in increased ROS levels in CMM cells. Moreover, silencing of a protein that has shown opposing effects on cell proliferation, CAV-1, decreased sensitivity to TH1579 in a BRAF inhibitor resistant cell line. AXL-MTH1 and CAV-1-MTH1 mRNA expressions were correlated as seen in CMM clinical samples. Finally, TH1579 in combination with BRAF inhibitor exhibited a more potent cell killing effect in BRAF mutant cells both in vitro and in vivo. In summary, we show that TH1579-mediated efficacy is independent of BRAF/NRAS mutational status but dependent on the expression of AXL and CAV-1.
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Caveolina 1/metabolismo , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Pirimidinas/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Daño del ADN , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , GTP Fosfohidrolasas/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Humanos , Melanoma/genética , Melanoma/patología , Proteínas de la Membrana/genética , Mitosis/efectos de los fármacos , Modelos Biológicos , Mutación/genética , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Pirimidinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Análisis de Supervivencia , Vemurafenib/farmacología , Pez Cebra , Tirosina Quinasa del Receptor Axl , Melanoma Cutáneo MalignoRESUMEN
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.
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8-Hidroxi-2'-Desoxicoguanosina , Monoéster Fosfórico Hidrolasas , Enzimas Reparadoras del ADN/genética , Genómica , Humanos , Mitosis/genética , Monoéster Fosfórico Hidrolasas/genética , Pirimidinas/farmacologíaRESUMEN
[This corrects the article DOI: 10.18632/oncotarget.19404.].
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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.
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Intersectin is a multidomain dynamin-binding protein implicated in numerous functions in the nervous system, including synapse formation and endocytosis. Here, we demonstrate that during neurotransmitter release in the central synapse, intersectin, like its binding partner dynamin, is redistributed from the synaptic vesicle pool to the periactive zone. Acute perturbation of the intersectin-dynamin interaction by microinjection of either intersectin antibodies or Src homology 3 (SH3) domains inhibited endocytosis at the fission step. Although the morphological effects induced by the different reagents were similar, antibody injections resulted in a dramatic increase in dynamin immunoreactivity around coated pits and at constricted necks, whereas synapses microinjected with the GST (glutathione S-transferase)-SH3C domain displayed reduced amounts of dynamin in the neck region. Our data suggest that intersectin controls the amount of dynamin released from the synaptic vesicle cluster to the periactive zone and that it may regulate fission of clathrin-coated intermediates.
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Proteínas Adaptadoras del Transporte Vesicular/fisiología , Dinaminas/metabolismo , Endocitosis/fisiología , Sinapsis/fisiología , Vesículas Sinápticas/fisiología , Secuencia de Aminoácidos , Animales , Dinaminas/antagonistas & inhibidores , Endosomas/fisiología , Endosomas/ultraestructura , Lampreas , Datos de Secuencia Molecular , Inhibición Neural/fisiología , Sinapsis/ultraestructura , Vesículas Sinápticas/ultraestructuraRESUMEN
Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor prognosis. Cancer cells are characterized by a specific redox environment that adjusts metabolism to its specific needs and allows the tumor to grow and metastasize. As a consequence, cancer cells and especially GBM cells suffer from elevated oxidative pressure which requires antioxidant-defense and other sanitation enzymes to be upregulated. MTH1, which degrades oxidized nucleotides, is one of these defense enzymes and represents a promising cancer target. We found MTH1 expression levels elevated and correlated with GBM aggressiveness and discovered that siRNA knock-down or inhibition of MTH1 with small molecules efficiently reduced viability of patient-derived GBM cultures. The effect of MTH1 loss on GBM viability was likely mediated through incorporation of oxidized nucleotides and subsequent DNA damage. We revealed that MTH1 inhibition targets GBM independent of aggressiveness as well as potently kills putative GBM stem cells in vitro. We used an orthotopic zebrafish model to confirm our results in vivo and light-sheet microscopy to follow the effect of MTH1 inhibition in GBM in real time. In conclusion, MTH1 represents a promising target for GBM therapy and MTH1 inhibitors may also be effective in patients that suffer from recurring disease.
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Cancer cells are commonly in a state of redox imbalance that drives their growth and survival. To compensate for oxidative stress induced by the tumor redox environment, cancer cells upregulate specific nononcogenic addiction enzymes, such as MTH1 (NUDT1), which detoxifies oxidized nucleotides. Here, we show that increasing oxidative stress in nonmalignant cells induced their sensitization to the effects of MTH1 inhibition, whereas decreasing oxidative pressure in cancer cells protected against inhibition. Furthermore, we purified zebrafish MTH1 and solved the crystal structure of MTH1 bound to its inhibitor, highlighting the zebrafish as a relevant tool to study MTH1 biology. Delivery of 8-oxo-dGTP and 2-OH-dATP to zebrafish embryos was highly toxic in the absence of MTH1 activity. Moreover, chemically or genetically mimicking activated hypoxia signaling in zebrafish revealed that pathologic upregulation of the HIF1α response, often observed in cancer and linked to poor prognosis, sensitized embryos to MTH1 inhibition. Using a transgenic zebrafish line, in which the cellular redox status can be monitored in vivo, we detected an increase in oxidative pressure upon activation of hypoxic signaling. Pretreatment with the antioxidant N-acetyl-L-cysteine protected embryos with activated hypoxia signaling against MTH1 inhibition, suggesting that the aberrant redox environment likely causes sensitization. In summary, MTH1 inhibition may offer a general approach to treat cancers characterized by deregulated hypoxia signaling or redox imbalance. Cancer Res; 76(8); 2366-75. ©2016 AACR.
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Hipoxia de la Célula , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Transducción de Señal , Microambiente Tumoral , Animales , Humanos , Oxidación-Reducción , Pez CebraRESUMEN
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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Enzimas Reparadoras del ADN/metabolismo , Nucleótidos de Desoxiguanina/metabolismo , Desoxirribonucleótidos/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Monoéster Fosfórico Hidrolasas/metabolismo , Pirofosfatasas/metabolismo , Western Blotting , Línea Celular Tumoral , Supervivencia Celular , Cristalización , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Pirofosfatasas/química , Especificidad por SustratoRESUMEN
During the past decade, many molecular components of clathrin-mediated endocytosis have been identified and proposed to play various hypothetical roles in the process [Nat. Rev. Neurosci. 1 (2000) 161; Nature 422 (2003) 37]. One limitation to the evaluation of these hypotheses is the efficiency and resolution of immunolocalization protocols currently in use. In order to facilitate the evaluation of these hypotheses and to understand more fully the molecular mechanisms of clathrin-mediated endocytosis, we have developed a protocol allowing enhanced and reliable subcellular immunolocalization of proteins in synaptic endocytic zones in situ. Synapses established by giant reticulospinal axons in lamprey are used as a model system for these experiments. These axons are unbranched and reach up to 80-100 microm in diameter. Synaptic active zones and surrounding endocytic zones are established on the surface of the axonal cylinder. To provide access for antibodies to the sites of synaptic vesicle recycling, axons are lightly fixed and cut along their longitudinal axis. To preserve the ultrastructure of the synaptic endocytic zone, antibodies are applied without the addition of detergents. Opened axons are incubated with primary antibodies, which are detected with secondary antibodies conjugated to gold particles. Specimens are then post-fixed and processed for electron microscopy. This approach allows preservation of the ultrastructure of the endocytic sites during immunolabeling procedures, while simultaneously achieving reliable immunogold detection of proteins on endocytic intermediates. To explore the utility of this approach, we have investigated the localization of a GTPase, dynamin, on clathrin-coated intermediates in the endocytic zone of the lamprey giant synapse. Using the present immunogold protocol, we confirm the presence of dynamin on late stage coated pits [Nature 422 (2003) 37] and also demonstrate that dynamin is recruited to the coat of endocytic intermediates from the very early stages of the clathrin coat formation. Thus, our experiments show that the current pre-embedding immunogold method is a useful experimental tool to study the molecular mechanisms of synaptic vesicle recycling.
Asunto(s)
Clatrina/fisiología , Endocitosis/fisiología , Inmunohistoquímica/métodos , Sinapsis/metabolismo , Animales , Vesículas Cubiertas por Clatrina/metabolismo , Dinamina I/análisis , Lampreas , Microscopía Electrónica/métodos , Ratas , Sinapsis/ultraestructura , Sinapsinas/análisis , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructuraRESUMEN
COPI (coat protein I) and the clathrin-AP-2 (adaptor protein 2) complex are well-characterized coat proteins, but a component that is common to these two coats has not been identified. The GTPase-activating protein (GAP) for ADP-ribosylation factor 1 (ARF1), ARFGAP1, is a known component of the COPI complex. Here, we show that distinct regions of ARFGAP1 interact with AP-2 and coatomer (components of the COPI complex). Selectively disrupting the interaction of ARFGAP1 with either of these two coat proteins leads to selective inhibition in the corresponding transport pathway. The role of ARFGAP1 in AP-2-regulated endocytosis has mechanistic parallels with its roles in COPI transport, as both its GAP activity and coat function contribute to promoting AP-2 transport.