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Isoprene pyrophosphates play a crucial role in the synthesis of a diverse array of essential nonsterol and sterol biomolecules and serve as substrates for posttranslational isoprenylation of proteins, enabling specific anchoring to cellular membranes. Hydrolysis of isoprene pyrophosphates would be a means to modulate their levels, downstream products, and protein isoprenylation. While NUDIX hydrolases from plants have been described to catalyze the hydrolysis of isoprene pyrophosphates, homologous enzymes with this function in animals have not yet been reported. In this study, we screened an extensive panel of human NUDIX hydrolases for activity in hydrolyzing isoprene pyrophosphates. We found that human nucleotide triphosphate diphosphatase NUDT15 and 8-oxo-dGDP phosphatase NUDT18 efficiently catalyze the hydrolysis of several physiologically relevant isoprene pyrophosphates. Notably, we demonstrate that geranyl pyrophosphate is an excellent substrate for NUDT18, with a catalytic efficiency of 2.1 × 105 m-1·s-1, thus making it the best substrate identified for NUDT18 to date. Similarly, geranyl pyrophosphate proved to be the best isoprene pyrophosphate substrate for NUDT15, with a catalytic efficiency of 4.0 × 104 M-1·s-1. LC-MS analysis of NUDT15 and NUDT18 catalyzed isoprene pyrophosphate hydrolysis revealed the generation of the corresponding monophosphates and inorganic phosphate. Furthermore, we solved the crystal structure of NUDT15 in complex with the hydrolysis product geranyl phosphate at a resolution of 1.70 Å. This structure revealed that the active site nicely accommodates the hydrophobic isoprenoid moiety and helped identify key binding residues. Our findings imply that isoprene pyrophosphates are endogenous substrates of NUDT15 and NUDT18, suggesting they are involved in animal isoprene pyrophosphate metabolism.
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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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Somatic copy number alterations (SCNAs) are pervasive in advanced human cancers, but their prevalence and spatial distribution in early-stage, localized tumors and their surrounding normal tissues are poorly characterized. Here, we perform multi-region, single-cell DNA sequencing to characterize the SCNA landscape across tumor-rich and normal tissue in two male patients with localized prostate cancer. We identify two distinct karyotypes: 'pseudo-diploid' cells harboring few SCNAs and highly aneuploid cells. Pseudo-diploid cells form numerous small-sized subclones ranging from highly spatially localized to broadly spread subclones. In contrast, aneuploid cells do not form subclones and are detected throughout the prostate, including normal tissue regions. Highly localized pseudo-diploid subclones are confined within tumor-rich regions and carry deletions in multiple tumor-suppressor genes. Our study reveals that SCNAs are widespread in normal and tumor regions across the prostate in localized prostate cancer patients and suggests that a subset of pseudo-diploid cells drive tumorigenesis in the aging prostate.
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Variaciones en el Número de Copia de ADN , Neoplasias de la Próstata , Análisis de la Célula Individual , Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Aneuploidia , Próstata/patología , Próstata/metabolismo , Células Clonales , Diploidia , AncianoRESUMEN
INTRODUCTION: Immunotherapies have revolutionized cancer treatment, but often fail to produce desirable therapeutic outcomes in all patients. Due to the inter-patient heterogeneity and complexity of the tumor microenvironment, personalized treatment approaches are gaining demand. Researchers have long been using a range of in-vitro assays including 2D models, organoid co-cultures, and cancer-on-a-chip platforms for cancer drug screening. A comparative analysis of these assays with their suitability, high-throughput capacity, and clinical translatability is required for optimal translational use. AREAS COVERED: The review summarized in-vitro platforms with their comparative advantages and limitations including construction strategies, and translational potential for immuno-oncology drug efficacy assessment. We also discussed end-point analysis strategies so that researchers can contextualize their usefulness and optimally design experiments for personalized immunotherapy efficacy prediction. EXPERT OPINION: Researchers developed several in-vitro platforms that can provide information on personalized immunotherapy efficacy from different angles. Image-based assays are undoubtedly more suitable to gather a wide range of information including cellular morphology and phenotypical behaviors but need significant improvement to overcome issues including background noise, sample preparation difficulty, and long duration of experiment. More studies and clinical trials are needed to resolve these issues and validate the assays before they can be used in real-life scenarios.
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Inmunoterapia , Neoplasias , Medicina de Precisión , Humanos , Medicina de Precisión/métodos , Neoplasias/inmunología , Neoplasias/terapia , Neoplasias/tratamiento farmacológico , Inmunoterapia/métodos , Microambiente Tumoral/inmunología , Animales , Ensayos de Selección de Medicamentos Antitumorales/métodosRESUMEN
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.
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SAMHD1 is a dNTP triphosphohydrolase governing nucleotide pool homeostasis and can detoxify chemotherapy metabolites controlling their clinical responses. To understand SAMHD1 biology and investigate the potential of targeting SAMHD1 as neoadjuvant to current chemotherapies, we set out to discover selective small-molecule inhibitors. Here, we report a discovery pipeline encompassing a biochemical screening campaign and a set of complementary biochemical, biophysical, and cell-based readouts for rigorous characterization of the screen output. The identified small molecules, TH6342 and analogs, accompanied by inactive control TH7126, demonstrated specific, low µM potency against both physiological and oncology-drug-derived substrates. By coupling kinetic studies with thermal shift assays, we reveal the inhibitory mechanism of TH6342 and analogs, which engage pre-tetrameric SAMHD1 and deter oligomerization and allosteric activation without occupying nucleotide-binding pockets. Altogether, our study diversifies inhibitory modes against SAMHD1, and the discovery pipeline reported herein represents a thorough framework for future SAMHD1 inhibitor development.
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Complex DNA damage (CDD), containing two or more DNA lesions within one or two DNA helical turns, is a signature of ionising radiation (IR) and contributes significantly to the therapeutic effect through cell killing. The levels and complexity of CDD increases with linear energy transfer (LET), however, the specific cellular response to this type of DNA damage and the critical proteins essential for repair of CDD is currently unclear. We performed an siRNA screen of ~240 DNA damage response proteins to identify those specifically involved in controlling cell survival in response to high-LET protons at the Bragg peak, compared to low-LET entrance dose protons which differ in the amount of CDD produced. From this, we subsequently validated that depletion of 8-oxoguanine DNA glycosylase (OGG1) and poly(ADP-ribose) glycohydrolase (PARG) in HeLa and head and neck cancer cells leads to significantly increased cellular radiosensitivity specifically following high-LET protons, whilst no effect was observed after low-LET protons and X-rays. We subsequently confirmed that OGG1 and PARG are both required for efficient CDD repair post-irradiation with high-LET protons. Importantly, these results were also recapitulated using specific inhibitors for OGG1 (TH5487) and PARG (PDD00017273). Our results suggest OGG1 and PARG play a fundamental role in the cellular response to CDD and indicate that targeting these enzymes could represent a promising therapeutic strategy for the treatment of head and neck cancers following high-LET radiation.
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ADN Glicosilasas , Neoplasias de Cabeza y Cuello , Humanos , Protones , Transferencia Lineal de Energía , Daño del ADN , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/radioterapia , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismoRESUMEN
The one-carbon folate enzyme methylenetetrahydrofolate dehydrogenase/cyclohydrolase 2 (MTHFD2) is a promising therapeutic target in cancer. MTHFD2 is upregulated across numerous cancer types, promotes growth and metastasis of cancer, and correlates with poorer survival. Recent studies have developed small-molecule inhibitors to the isozymes MTHFD2 and MTHFD1 that show promise as anticancer agents through different mechanisms. This review discusses the current understanding of the function of MTHFD2 in cancer and the status of inhibitors for treating MTHFD2-overexpressing cancers.
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Antineoplásicos , Neoplasias , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Ácido Fólico , Reparación del ADNRESUMEN
The DNA damage response (DDR) protein MTH1 is sanitising the oxidized dNTP pool and preventing incorporation of oxidative damage into DNA and has an emerging role in mitosis. It is a stress-induced protein and often found to be overexpressed in cancer. Mitotic MTH1 inhibitors arrest cells in mitosis and result in incorporation of oxidative damage into DNA and selective killing of cancer cells. Here, I discuss the leading mitotic MTH1 inhibitor TH1579 (OXC-101, karonudib), now being evaluated in clinical trials, and describe its dual effect on mitosis and incorporation of oxidative DNA damage in cancer cells. I describe why MTH1 inhibitors that solely inhibits the enzyme activity fail to kill cancer cells and discuss if MTH1 is a valid target for cancer treatment. I discuss emerging roles of MTH1 in regulating tubulin polymerisation and mitosis and the necessity of developing the basic science insights along with translational efforts. I also give a perspective on how edgetic perturbation is making target validation difficult in the DDR field.
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Enzimas Reparadoras del ADN , Neoplasias , Humanos , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , ADN/genética , ADN/uso terapéutico , Daño del ADNRESUMEN
With diminishing returns and high clinical failure rates from traditional preclinical and animal-based drug discovery strategies, more emphasis is being placed on alternative drug discovery platforms. Ex vivo approaches represent a departure from both more traditional preclinical animal-based models and clinical-based strategies and aim to address intra-tumoural and inter-patient variability at an earlier stage of drug discovery. Additionally, these approaches could also offer precise treatment stratification for patients within a week of tumour resection in order to direct tailored therapy. One tumour group that could significantly benefit from such ex vivo approaches are high-grade gliomas, which exhibit extensive heterogeneity, cellular plasticity and therapy-resistant glioma stem cell (GSC) niches. Historic use of murine-based preclinical models for these tumours has largely failed to generate new therapies, resulting in relatively stagnant and unacceptable survival rates of around 12-15 months post-diagnosis over the last 50 years. The near universal use of DNA damaging chemoradiotherapy after surgical resection within standard-of-care (SoC) therapy regimens provides an opportunity to improve current treatments if we can identify efficient drug combinations in preclinical models that better reflect the complex inter-/intra-tumour heterogeneity, GSC plasticity and inherent DNA damage resistance mechanisms. We have therefore developed and optimised a high-throughput ex vivo drug screening platform; GliExP, which maintains GSC populations using immediately dissociated fresh surgical tissue. As a proof-of-concept for GliExP, we have optimised SoC therapy responses and screened 30+ small molecule therapeutics and preclinical compounds against tumours from 18 different patients, including multi-region spatial heterogeneity sampling from several individual tumours. Our data therefore provides a strong basis to build upon GliExP to incorporate combination-based oncology therapeutics in tandem with SoC therapies as an important preclinical alternative to murine models (reduction and replacement) to triage experimental therapeutics for clinical translation and deliver rapid identification of effective treatment strategies for individual gliomas.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Animales , Ratones , Glioblastoma/tratamiento farmacológico , Evaluación Preclínica de Medicamentos , Avatar , Neoplasias Encefálicas/tratamiento farmacológico , Detección Precoz del Cáncer , Células Madre NeoplásicasRESUMEN
Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase-cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a 'folate trap'. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.
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Metilenotetrahidrofolato Deshidrogenasa (NADP) , Neoplasias , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Ácido Fólico/metabolismo , Formiatos , Purinas , TetrahidrofolatosRESUMEN
Dysregulation of messenger RNA (mRNA) translation, including preferential translation of mRNA with complex 5' untranslated regions such as the MYC oncogene, is recognized as an important mechanism in cancer. Here, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which is inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis performed in samples from patients with CLL and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibiting translation induced a proliferation arrest and a rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the eukaryotic initiation factor (eIF)4F translation complex and are targeted by FL3. Knockdown of PHBs resembled FL3 treatment. Importantly, inhibition of translation controlled CLL development in vivo, either alone or combined with immunotherapy. Finally, high expression of translation initiation-related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in patients with CLL. Overall, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for patients with CLL.
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Leucemia Linfocítica Crónica de Células B , Humanos , Ratones , Animales , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Factor 4F Eucariótico de Iniciación/genética , Prohibitinas , Genes myc , ARN Mensajero/genéticaRESUMEN
OBJECTIVES: Androgen deprivation therapy (ADT) has long been a cornerstone in treatment of advanced prostate cancer (PCa), and is known to improve the results of radiotherapy (RT) for high-risk disease. The purpose of our study was to use a multiplexed immunohistochemical (mIHC) approach to investigate the infiltration of immune cells in PCa tissue after eight weeks of ADT and/or RT with 10 Gy. METHODS: From a cohort of 48 patients divided into two treatment arms, we obtained biopsies before and after treatment and used a mIHC method with multispectral imaging to analyze the infiltration of immune cells in tumor stroma and tumor epithelium, focusing on areas with high infiltration. RESULTS: Tumor stroma showed a significantly higher infiltration of immune cells compared to tumor epithelium. The most prominent immune cells were CD20+ B-lymphocytes, followed by CD68+ macrophages, CD8+ cytotoxic T-cells, FOXP3+ regulatory T-cells (Tregs), and T-bet+ Th1-cells. Neoadjuvant ADT followed by RT significantly increased the infiltration of all five immune cells. Numbers of Th1-cells and Tregs significantly increased after single treatment with ADT or RT. In addition, ADT alone increased the number of cytotoxic T-cells and RT increased the number of B-cells. CONCLUSIONS: Neoadjuvant ADT in combination with RT results in a higher inflammatory response compared to RT or ADT alone. The mIHC method may be a useful tool for investigating infiltrating immune cells in PCa biopsies to understand how immunotherapeutic approaches can be combined with current PCa therapies.
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Neoplasias de la Próstata , Masculino , Humanos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/radioterapia , Antagonistas de Andrógenos/uso terapéutico , Andrógenos , Biopsia , Linfocitos BRESUMEN
BACKGROUND: Chemotherapy (CT) is central to the treatment of triple negative breast cancer (TNBC), but drug toxicity and resistance place strong restrictions on treatment regimes. Fasting sensitizes cancer cells to a range of chemotherapeutic agents and also ameliorates CT-associated adverse effects. However, the molecular mechanism(s) by which fasting, or short-term starvation (STS), improves the efficacy of CT is poorly characterized. METHODS: The differential responses of breast cancer or near normal cell lines to combined STS and CT were assessed by cellular viability and integrity assays (Hoechst and PI staining, MTT or H2DCFDA staining, immunofluorescence), metabolic profiling (Seahorse analysis, metabolomics), gene expression (quantitative real-time PCR) and iRNA-mediated silencing. The clinical significance of the in vitro data was evaluated by bioinformatical integration of transcriptomic data from patient data bases: The Cancer Genome Atlas (TCGA), European Genome-phenome Archive (EGA), Gene Expression Omnibus (GEO) and a TNBC cohort. We further examined the translatability of our findings in vivo by establishing a murine syngeneic orthotopic mammary tumor-bearing model. RESULTS: We provide mechanistic insights into how preconditioning with STS enhances the susceptibility of breast cancer cells to CT. We showed that combined STS and CT enhanced cell death and increased reactive oxygen species (ROS) levels, in association with higher levels of DNA damage and decreased mRNA levels for the NRF2 targets genes NQO1 and TXNRD1 in TNBC cells compared to near normal cells. ROS enhancement was associated with compromised mitochondrial respiration and changes in the metabolic profile, which have a significant clinical prognostic and predictive value. Furthermore, we validate the safety and efficacy of combined periodic hypocaloric diet and CT in a TNBC mouse model. CONCLUSIONS: Our in vitro, in vivo and clinical findings provide a robust rationale for clinical trials on the therapeutic benefit of short-term caloric restriction as an adjuvant to CT in triple breast cancer treatment.
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Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Neoplasias de la Mama Triple Negativas , Animales , Ratones , Humanos , Dieta Reductora , Especies Reactivas de Oxígeno , ObesidadRESUMEN
The NUDIX hydrolase NUDT22 converts UDP-glucose into glucose-1-phosphate and the pyrimidine nucleotide uridine monophosphate but a biological significance for this biochemical reaction has not yet been established. Glucose-1-phosphate is an important metabolite for energy and biomass production through glycolysis and nucleotides required for DNA replication are produced through energetically expensive de novo or energy-efficient salvage pathways. Here, we describe p53-regulated pyrimidine salvage through NUDT22-dependent hydrolysis of UDP-glucose to maintain cancer cell growth and to prevent replication stress. NUDT22 expression is consistently elevated in cancer tissues and high NUDT22 expression correlates with worse survival outcomes in patients indicating an increased dependency of cancer cells to NUDT22. Furthermore, we show that NUDT22 transcription is induced after inhibition of glycolysis, MYC-mediated oncogenic stress, and DNA damage directly through p53. NUDT22-deficient cancer cells suffer from growth retardation, S-phase delay, and slower DNA replication fork speed. Uridine supplementation rescues replication fork progression and alleviates replication stress and DNA damage. Conversely, NUDT22 deficiency sensitizes cells to de novo pyrimidine synthesis inhibition in vitro and reduces cancer growth in vivo. In conclusion, NUDT22 maintains pyrimidine supply in cancer cells and depletion of NUDT22 leads to genome instability. Targeting NUDT22 therefore has high potential for therapeutic applications in cancer therapy.
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Neoplasias , Proteína p53 Supresora de Tumor , Humanos , Glucosa , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Pirimidinas/farmacología , Uridina/metabolismo , Uridina DifosfatoRESUMEN
8-oxo Guanine DNA Glycosylase 1 is the initiating enzyme within base excision repair and removes oxidized guanines from damaged DNA. Since unrepaired 8-oxoG could lead to G : CâT : A transversion, base removal is of utmost importance for cells to ensure genomic integrity. For cells with elevated levels of reactive oxygen species this dependency is further increased. In the past we and others have validated OGG1 as a target for inhibitors to treat cancer and inflammation. Here, we present the optimization campaign that led to the broadly used tool compound TH5487. Based on results from a small molecule screening campaign, we performed hit to lead expansion and arrived at potent and selective substituted N-piperidinyl-benzimidazolones. Using X-ray crystallography data, we describe the surprising binding mode of the most potent member of the class, TH8535. Here, the N-Piperidinyl-linker adopts a chair instead of a boat conformation which was found for weaker analogues. We further demonstrate cellular target engagement and efficacy of TH8535 against a number of cancer cell lines.
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ADN Glicosilasas , Neoplasias , Humanos , ADN Glicosilasas/química , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Guanina/química , Guanina/metabolismo , Reparación del ADN , Bencimidazoles/farmacología , Daño del ADNRESUMEN
Previously, we reported that MTH1 inhibitors TH588 and TH1579 selectively induce oxidative damage and kill Ras-expressing or -transforming cancer cells, as compared to non-transforming immortalized or primary cells. While this explains the impressive anti-cancer properties of the compounds, the molecular mechanism remains elusive. Several oncogenes induce replication stress, resulting in under replicated DNA and replication continuing into mitosis, where TH588 and TH1579 treatment causes toxicity and incorporation of oxidative damage. Hence, we hypothesized that oncogene-induced replication stress explains the cancer selectivity. To test this, we overexpressed c-Myc in human epithelial kidney cells (HA1EB), resulting in increased proliferation, polyploidy and replication stress. TH588 and TH1579 selectively kill c-Myc overexpressing clones, enforcing the cancer cell selective killing of these compounds. Moreover, the toxicity of TH588 and TH1579 in c-Myc overexpressing cells is rescued by transcription, proteasome or CDK1 inhibitors, but not by nucleoside supplementation. We conclude that the molecular toxicological mechanisms of how TH588 and TH1579 kill c-Myc overexpressing cells have several components and involve MTH1-independent proteasomal degradation of c-Myc itself, c-Myc-driven transcription and CDK activation.
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Enzimas Reparadoras del ADN , Estrés Oxidativo , Humanos , Enzimas Reparadoras del ADN/metabolismo , Línea Celular Tumoral , Pirimidinas/farmacología , Daño del ADNRESUMEN
To understand complex diseases, high-throughput data are generated at large and multiple levels. However, extracting meaningful information from large datasets for comprehensive understanding of cell phenotypes and disease pathophysiology remains a major challenge. Despite tremendous advances in understanding molecular mechanisms of cancer and its progression, current knowledge appears discrete and fragmented. In order to render this wealth of data more integrated and thus informative, we have developed a GECIP toolbox to investigate the crosstalk and the responsible genes'/proteins' connectivity of enriched pathways from gene expression data. To implement this toolbox, we used mainly gene expression datasets of prostate cancer, and the three datasets were GSE17951, GSE8218, and GSE1431. The raw samples were processed for normalization, prediction of differentially expressed genes, and the prediction of enriched pathways for the differentially expressed genes. The enriched pathways have been processed for crosstalk degree calculations for which number connections per gene, the frequency of genes in the pathways, sharing frequency, and the connectivity have been used. For network prediction, protein-protein interaction network database FunCoup2.0 was used, and cytoscape software was used for the network visualization. In our results, we found that there were enriched pathways 27, 45, and 22 for GSE17951, GSE8218, and GSE1431, respectively, and 11 pathways in common between all of them. From the crosstalk results, we observe that focal adhesion and PI3K pathways, both experimentally proven central for cellular output upon perturbation of numerous individual/distinct signaling pathways, displayed highest crosstalk degree. Moreover, we also observe that there were more critical pathways which appear to be highly significant, and these pathways are HIF1a, hippo, AMPK, and Ras. In terms of the pathways' components, GSK3B, YWHAE, HIF1A, ATP1A3, and PRKCA are shared between the aforementioned pathways and have higher connectivity with the pathways and the other pathway components. Finally, we conclude that the focal adhesion and PI3K pathways are the most critical pathways, and since for many other pathways, high-rank enrichment did not translate to high crosstalk degree, the global impact of one pathway on others appears distinct from enrichment.
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Fosfatidilinositol 3-Quinasas , Neoplasias de la Próstata , Humanos , Masculino , Fosfatidilinositol 3-Quinasas/genética , Perfilación de la Expresión Génica/métodos , Neoplasias de la Próstata/genética , Mapas de Interacción de Proteínas/genética , Simulación por Computador , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
Background and aim: Allergic asthma is a complex inflammatory disease involving type 2 innate lymphoid cells, type 2 T helper cells, macrophages, and eosinophils. The disease is characterized by wheezing, dyspnea, coughing, chest tightness and variable airflow limitation for which there is no cure and is symptomatically treated with inhaled corticosteroids and ß2-agonists. Molecular mechanisms underlying its complex pathogenesis are not fully understood. However, 8-oxoguanine DNA glycosylase-1 (OGG1), a DNA repair protein may play a central role, as OGG1 deficiency decreases both innate and allergic inflammation. Methods: Using a murine ovalbumin (OVA) model of allergic airway inflammation we assessed the utility of an inhibitor of OGG1 (TH5487) in this disease context. Cytokines and chemokines, promoting immune cell recruitment were measured using a 23-multiplex assay and Western blotting. Additionally, immune cell recruitment to bronchi was measured using flow cytometry. Histological analyses and immunofluorescent staining were used to confirm immune cell influx and goblet cell hyperplasia of the airways. A PCR array was used to assess asthma-related genes in murine lung tissue following TH5487 treatment. Finally, airway hyperresponsiveness was determined using in vivo lung function measurement. Results: In this study, administration of TH5487 to mice with OVA-induced allergic airway inflammation significantly decreased goblet cell hyperplasia and mucus production. TH5487 treatment also decreased levels of activated NF-κB and expression of proinflammatory cytokines and chemokines resulting in significantly lower recruitment of eosinophils and other immune cells to the lungs. Gene expression profiling of asthma and allergy-related proteins after TH5487 treatment revealed differences in several important regulators, including down regulation of Tnfrsf4, Arg1, Ccl12 and Ccl11, and upregulation of the negative regulator of type 2 inflammation, Bcl6. Furthermore, the gene Clca1 was upregulated following TH5487 treatment, which should be explored further due to its ambiguous role in allergic asthma. In addition, the OVA-induced airway hyperresponsiveness was significantly reduced by TH5487 treatment. Conclusion: Taken together, the data presented in this study suggest OGG1 as a clinically relevant pharmacological target for the treatment of allergic inflammation.
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Despite advances in cancer genomics and the increased use of genomic medicine, metastatic cancer is still mostly an incurable and fatal disease. With diminishing returns from traditional drug discovery strategies, and high clinical failure rates, more emphasis is being placed on alternative drug discovery platforms, such as ex vivo approaches. Ex vivo approaches aim to embed biological relevance and inter-patient variability at an earlier stage of drug discovery, and to offer more precise treatment stratification for patients. However, these techniques also have a high potential to offer personalised therapies to patients, complementing and enhancing genomic medicine. Although an array of approaches are available to researchers, only a minority of techniques have made it through to direct patient treatment within robust clinical trials. Within this review, we discuss the current challenges to ex vivo approaches within clinical practice and summarise the contemporary literature which has directed patient treatment. Finally, we map out how ex vivo approaches could transition from a small-scale, predominantly research based technology to a robust and validated predictive tool. In future, these pre-clinical approaches may be integrated into clinical cancer pathways to assist in the personalisation of therapy choices and to hopefully improve patient experiences and outcomes.