368 results on '"Michael Boutros"'
Search Results
52. Supplementary Figures from CAMK1D Triggers Immune Resistance of Human Tumor Cells Refractory to Anti–PD-L1 Treatment
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Philipp Beckhove, Nisit Khandelwal, Gyorgy Vereb, Arpad Szoor, Mathias Witzens-Harig, Martin Ehrenschwender, Hartmut Goldschmidt, Dirk Hose, Anja Seckinger, Christian H. Wetzel, Mark Berneburg, Sebastian Haferkamp, Michael Boutros, Klaus Griewank, Anchana Rathinasamy, Chih-Yeh Chen, Vladimir M. Milenkovic, Madlen Ditz, Gertrud Knoll, Ayse N. Menevse, Antonio Sorrentino, Tillmann Michels, and Valentina Volpin
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Supplementary Figures 1-7
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- 2023
53. Supplementary movie S1 from Pharmacological Inhibition of Centrosome Clustering by Slingshot-Mediated Cofilin Activation and Actin Cortex Destabilization
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Marc Steffen Raab, Alwin Krämer, Kensaku Mizuno, Michael Boutros, Axel Benner, Natalia Becker, Andrey Turchinovich, Tomoaki Nagai, Elena Bausch, and Gleb Konotop
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Representative fluorescence time-lapse microscopy comparing mitotic events of H2B-mCherry-α-tubulin-EGFP- HeLa cells treated with DMSO.
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- 2023
54. Supplementary Table S1 from Pharmacological Inhibition of Centrosome Clustering by Slingshot-Mediated Cofilin Activation and Actin Cortex Destabilization
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Marc Steffen Raab, Alwin Krämer, Kensaku Mizuno, Michael Boutros, Axel Benner, Natalia Becker, Andrey Turchinovich, Tomoaki Nagai, Elena Bausch, and Gleb Konotop
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Screen result. For each library and concentration, compounds are divided into two groups, according to readout significance (black, P < 0.05; grey P > 0.05) and sorted from highest to lowest CCI index. Compounds highlighted in bold represent a CCI index higher than 0.3, indicating that viability of cells with CA was significantly affected in comparison to cells with normal centrosome content (by approx. 20%). Relative overall viability of each drug-treated cell population in comparison to the corresponding DMSO-treated population (maximum viability = 1) is indicated by rel.Control or rel.Tet. Compounds that eradicated both cell populations, giving a false positive result are highlighted in red (i.e. Crizotinib).
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- 2023
55. Supplementary data from Pharmacological Inhibition of Centrosome Clustering by Slingshot-Mediated Cofilin Activation and Actin Cortex Destabilization
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Marc Steffen Raab, Alwin Krämer, Kensaku Mizuno, Michael Boutros, Axel Benner, Natalia Becker, Andrey Turchinovich, Tomoaki Nagai, Elena Bausch, and Gleb Konotop
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Extended experimental procedures Supplementary Fig. S1. Effect of CP-673451 and Crenolanib on centrosome amplification in U2OS cells. Supplementary Fig. S2. Crenolanib prolongs of mitotic duration. Supplementary Fig. S3. CP-673451 activates cofilin in a dose-dependent manner in a variety of cancer cell lines. Supplementary Fig. S4. Inhibition of cofilin phosphorylation by LIMKi3 and Damnacanthal. Supplementary Fig. S5. Immunoblot analysis showing transient overexpression of different cofilin constructs and representative images of transfection efficiencies in U2OS-EGFP-PLK4 cells. Supplementary Fig. S6. Validation of SSH knock-down by real-time PCR. Supplementary Fig. S7. Effect of CP-673451 on SSH1 phosphatase activity. Supplementary Fig. S8. Immunoblot analysis of different cell lines showing that CP-673451 increases Akt and MEK1/2 phosphorylation under normal cultivation conditions. Supplementary Table S2. Average percentages of multipolar telophases in various cell lines after 24 hours of exposure to CP-673451 or crenolanib.
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- 2023
56. The role of Evi/Wntless in exporting Wnt proteins
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Lucie Wolf and Michael Boutros
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Molecular Biology ,Developmental Biology - Abstract
Intercellular communication by Wnt proteins governs many essential processes during development, tissue homeostasis and disease in all metazoans. Many context-dependent effects are initiated in the Wnt-producing cells and depend on the export of lipidated Wnt proteins. Although much focus has been on understanding intracellular Wnt signal transduction, the cellular machinery responsible for Wnt secretion became better understood only recently. After lipid modification by the acyl-transferase Porcupine, Wnt proteins bind their dedicated cargo protein Evi/Wntless for transport and secretion. Evi/Wntless and Porcupine are conserved transmembrane proteins, and their 3D structures were recently determined. In this Review, we summarise studies and structural data highlighting how Wnts are transported from the ER to the plasma membrane, and the role of SNX3-retromer during the recycling of its cargo receptor Evi/Wntless. We also describe the regulation of Wnt export through a post-translational mechanism and review the importance of Wnt secretion for organ development and cancer, and as a future biomarker.
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- 2023
57. USP47 deubiquitylates Groucho/TLE to promote Wnt–β-catenin signaling
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Sara Kassel, Alison J. Hanson, Hassina Benchabane, Kenyi Saito-Diaz, Carly R. Cabel, Lily Goldsmith, Muhammad Taha, Aksheta Kanuganti, Victoria H. Ng, George Xu, Fei Ye, Julia Picker, Fillip Port, Michael Boutros, Vivian L. Weiss, David J. Robbins, Curtis A. Thorne, Yashi Ahmed, and Ethan Lee
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Cell Biology ,Molecular Biology ,Biochemistry - Abstract
The Wnt–β-catenin signal transduction pathway is essential for embryonic development and adult tissue homeostasis. Wnt signaling converts TCF from a transcriptional repressor to an activator in a process facilitated by the E3 ligase XIAP. XIAP-mediated monoubiquitylation of the transcriptional corepressor Groucho (also known as TLE) decreases its affinity for TCF, thereby allowing the transcriptional coactivator β-catenin to displace it on TCF. Through a genome-scale screen in cultured Drosophila melanogaster cells, we identified the deubiquitylase USP47 as a positive regulator of Wnt signaling. We found that USP47 was required for Wnt signaling during Drosophila and Xenopus laevis development, as well as in human cells, indicating evolutionary conservation. In human cells, knockdown of USP47 inhibited Wnt reporter activity, and USP47 acted downstream of the β-catenin destruction complex. USP47 interacted with TLE3 and XIAP but did not alter their amounts; however, knockdown of USP47 enhanced XIAP-mediated ubiquitylation of TLE3. USP47 inhibited ubiquitylation of TLE3 by XIAP in vitro in a dose-dependent manner, suggesting that USP47 is the deubiquitylase that counteracts the E3 ligase activity of XIAP on TLE. Our data suggest a mechanism by which regulated ubiquitylation and deubiquitylation of TLE enhance the ability of β-catenin to cycle on and off TCF, thereby helping to ensure that the expression of Wnt target genes continues only as long as the upstream signal is present.
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- 2023
58. HTSvis: a web app for exploratory data analysis and visualization of arrayed high-throughput screens.
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Christian Scheeder, Florian Heigwer, and Michael Boutros
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- 2017
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59. Analyse von Zellfunktionen mit Hochdurchsatz-Mikroskopie und KI
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Michael Boutros, Christian Scheeder, and Florian Heigwer
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Pharmacology toxicology ,Cellular pathways ,Computational biology ,Allele ,Biology ,Molecular Biology ,Gene ,Phenotype ,Human genetics ,Biotechnology ,Genetic screen - Abstract
Genes that share a distinct phenotype often share biological functions. A principle that is used in genetic screens and that provides the basis for our understanding of key biological processes. Traditionally, individual phenotypes were used to group mutant alleles into cellular pathways. Today, high-throughput technologies allow the screening of thousands of perturbations. Using computational methods and machine learning, millions of images are profiled to assign biological effects to genes and drugs.
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- 2021
60. caRpools: an R package for exploratory data analysis and documentation of pooled CRISPR/Cas9 screens.
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Jan Winter, Marco Breinig, Florian Heigwer, Dirk Brügemann, Svenja Leible, Oliver Pelz, Tianzuo Zhan, and Michael Boutros
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- 2016
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61. Tissue-Specific CRISPR-Cas9 Screening in Drosophila
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Fillip, Port and Michael, Boutros
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Mutagenesis ,Animals ,Drosophila ,Genetic Testing ,Genomics ,CRISPR-Cas Systems ,RNA, Guide, Kinetoplastida - Abstract
Over the last century research in Drosophila has resulted in many fundamental contributions to our understanding of the biology of multicellular organisms. Many of these breakthroughs have been based on the identification of novel gene functions in large-scale genetic screens. However, conventional forward-genetic screens have been limited by the random nature of mutagenesis and difficulties in mapping causal mutations, while reverse-genetic RNAi screens suffer from incomplete knockdown of gene expression. Recently developed large-scale CRISPR-Cas9 libraries promise to address these limitations by allowing the induction of targeted mutations in genes with spatial and temporal control. Here, we provide a guide for tissue-specific CRISPR screening in Drosophila, including the characterization of Gal4 UAS-Cas9 lines, selection of sgRNA libraries, and various quality control measures. We also discuss confounding factors that can give rise to false-positive and false-negative results in such experiments and suggest strategies on how to detect and avoid them. Conditional CRISPR screening represents an exciting new approach for functional genomics in vivo and is set to further expand our knowledge of the molecular underpinning of development, homeostasis, and disease.
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- 2022
62. A global genetic interaction network by single-cell imaging and machine learning
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Florian Heigwer, Christian Scheeder, Josephine Bageritz, Schayan Yousefian, Benedikt Rauscher, Christina Laufer, Sergi Beneyto-Calabuig, Maja Christina Funk, Vera Peters, Maria Boulougouri, Jana Bilanovic, Thilo Miersch, Barbara Schmitt, Claudia Blass, Fillip Port, and Michael Boutros
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Histology ,Cell Biology ,Pathology and Forensic Medicine - Published
- 2023
63. Superresolution microscopy localizes endogenous Dvl2 to Wnt signaling-responsive biomolecular condensates
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Antonia Schubert, Oksana Voloshanenko, Franziska Ragaller, Philipp Gmach, Dominique Kranz, Christian Scheeder, Thilo Miersch, Matthias Schulz, Lorenz Trümper, Claudia Binder, Marko Lampe, Ulrike Engel, and Michael Boutros
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Biomolecular Condensates ,Wnt Proteins ,Multidisciplinary ,Microscopy, Fluorescence ,Protein Domains ,Dishevelled Proteins ,Humans ,Wnt Signaling Pathway ,beta Catenin - Abstract
During organismal development, homeostasis, and disease, Dishevelled (Dvl) proteins act as key signaling factors in beta-catenin–dependent and beta-catenin–independent Wnt pathways. While their importance for signal transmission has been genetically demonstrated in many organisms, our mechanistic understanding is still limited. Previous studies using overexpressed proteins showed Dvl localization to large, punctate-like cytoplasmic structures that are dependent on its DIX domain. To study Dvl’s role in Wnt signaling, we genome engineered an endogenously expressed Dvl2 protein tagged with an mEos3.2 fluorescent protein for superresolution imaging. First, we demonstrate the functionality and specificity of the fusion protein in beta-catenin–dependent and beta-catenin–independent signaling using multiple independent assays. We performed live-cell imaging of Dvl2 to analyze the dynamic formation of the supramolecular cytoplasmic Dvl2_mEos3.2 condensates. While overexpression of Dvl2_mEos3.2 mimics the previously reported formation of abundant large “puncta,” supramolecular condensate formation at physiological protein levels is only observed in a subset of cells with approximately one per cell. We show that, in these condensates, Dvl2 colocalizes with Wnt pathway components at gamma-tubulin and CEP164-positive centrosomal structures and that the localization of Dvl2 to these condensates is Wnt dependent. Single-molecule localization microscopy using photoactivated localization microscopy (PALM) of mEos3.2 in combination with DNA-PAINT demonstrates the organization and repetitive patterns of these condensates in a cell cycle–dependent manner. Our results indicate that the localization of Dvl2 in supramolecular condensates is coordinated dynamically and dependent on cell state and Wnt signaling levels. Our study highlights the formation of endogenous and physiologically regulated biomolecular condensates in the Wnt pathways at single-molecule resolution.
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- 2022
64. Targeting euchromatic histone lysine methyltransferases sensitizes colorectal cancer to histone deacetylase inhibitors
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Leonhard Valentin Bamberg, Florian Heigwer, Anna Maxi Wandmacher, Ambika Singh, Johannes Betge, Niklas Rindtorff, Johannes Werner, Julia Josten, Olga Valerievna Skabkina, Isabel Hinsenkamp, Gerrit Erdmann, Christoph Röcken, Matthias P Ebert, Elke Burgermeister, Tianzuo Zhan, and Michael Boutros
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Histone Deacetylase Inhibitors ,Cancer Research ,Oncology ,Cell Line, Tumor ,Histocompatibility Antigens ,Humans ,Acetylation ,Antineoplastic Agents ,Histone-Lysine N-Methyltransferase ,Colorectal Neoplasms ,Histone Deacetylases - Abstract
Epigenetic dysregulation is an important feature of colorectal cancer (CRC). Combining epigenetic drugs with other antineoplastic agents is a promising treatment strategy for advanced cancers. Here, we exploited the concept of synthetic lethality to identify epigenetic targets that act synergistically with histone deacetylase (HDAC) inhibitors to reduce the growth of CRC. We applied a pooled CRISPR-Cas9 screen using a custom sgRNA library directed against 614 epigenetic regulators and discovered that knockout of the euchromatic histone-lysine N-methyltransferases 1 and 2 (EHMT1/2) strongly enhanced the antiproliferative effect of clinically used HDAC inhibitors. Using tissue microarrays from 1066 CRC samples with different tumor stages, we showed that low EHMT2 protein expression is predominantly found in advanced CRC and associated with poor clinical outcome. Cotargeting of HDAC and EHMT1/2 with specific small molecule inhibitors synergistically reduced proliferation of CRC cell lines. Mechanistically, we used a high-throughput Western blot assay to demonstrate that both inhibitors elicited distinct cellular mechanisms to reduce tumor growth, including cell cycle arrest and modulation of autophagy. On the epigenetic level, the compounds increased H3K9 acetylation and reduced H3K9 dimethylation. Finally, we used a panel of patient-derived CRC organoids to show that HDAC and EHMT1/2 inhibition synergistically reduced tumor viability in advanced models of CRC.
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- 2022
65. GenomeRNAi: a database for cell-based and in vivo RNAi phenotypes, 2013 update.
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Esther E. Schmidt, Oliver Pelz, Svetlana Buhlmann, Grainne Kerr, Thomas Horn, and Michael Boutros
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- 2013
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66. Extracellular vesicles and oncogenic signaling
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Michael Boutros and Antonia Schubert
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0301 basic medicine ,Cancer Research ,Carcinogenesis ,Cancer therapy ,tumor progression ,Review Article ,exosomes ,Biology ,Models, Biological ,Extracellular vesicles ,03 medical and health sciences ,0302 clinical medicine ,Neoplasms ,Oncogenic signaling ,Genetics ,Animals ,Humans ,metastasis ,RC254-282 ,Wnt signaling pathway ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,General Medicine ,Microvesicles ,3. Good health ,Cell biology ,030104 developmental biology ,Oncology ,Tumor progression ,030220 oncology & carcinogenesis ,Molecular Medicine ,Signal transduction ,extracellular vesicles ,signaling ,microvesicles ,Biogenesis ,Signal Transduction - Abstract
Extracellular vesicles (EVs) emerged as potential diagnostic and prognostic markers for cancer therapy. This review summarizes mechanisms of signaling specificity and cargo transfer by EVs in the oncogenic and cancer‐associated signaling cascades Wnt, TGF‐β, ErbB, VEGF, and PD1–PD‐L1. Translatable EV functions and existing knowledge gaps are discussed to possibly further exploit EVs for diagnostics and therapeutic approaches in the future., In recent years, extracellular vesicles (EVs) emerged as potential diagnostic and prognostic markers for cancer therapy. While the field of EV research is rapidly developing and their application as vehicles for therapeutic cargo is being tested, little is still known about the exact mechanisms of signaling specificity and cargo transfer by EVs, especially in vivo. Several signaling cascades have been found to use EVs for signaling in the tumor–stroma interaction. These include potentially oncogenic, verbatim transforming, signaling cascades such as Wnt and TGF‐β signaling, and other signaling cascades that have been tightly associated with tumor progression and metastasis, such as PD‐L1 and VEGF signaling. Multiple mechanisms of how these signaling cascades and EVs interplay to mediate these complex processes have been described, such as direct signal activation through pathway components on or in EVs or indirectly by influencing vesicle biogenesis, cargo sorting, or uptake dynamics. In this review, we summarize the current knowledge of EVs, their biogenesis, and our understanding of EV interactions with recipient cells with a focus on selected oncogenic and cancer‐associated signaling pathways. After an in‐depth look at how EVs mediate and influence signaling, we discuss potentially translatable EV functions and existing knowledge gaps.
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- 2021
67. Multi‐omics integration identifies a selective vulnerability of colorectal cancer subtypes to <scp>YM155</scp>
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Michael Boutros, Tianzuo Zhan, Faehling Verena, Matthias P. Ebert, Johannes Betge, and Benedikt Rauscher
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Drug ,Cancer Research ,Cell Survival ,Colorectal cancer ,media_common.quotation_subject ,Antineoplastic Agents ,Apoptosis ,Computational biology ,Biology ,Polymorphism, Single Nucleotide ,law.invention ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,law ,Cell Line, Tumor ,medicine ,Humans ,CRISPR ,Gene ,media_common ,Cas9 ,Gene Expression Profiling ,Imidazoles ,HCT116 Cells ,medicine.disease ,digestive system diseases ,Gene Expression Regulation, Neoplastic ,Oncology ,030220 oncology & carcinogenesis ,Suppressor ,Multi omics ,RNA Interference ,Caco-2 Cells ,Colorectal Neoplasms ,Genome-Wide Association Study ,Naphthoquinones - Abstract
Tumor heterogeneity is a major challenge to the treatment of colorectal cancer (CRC). Recently, a transcriptome-based classification was developed, segregating CRC into four consensus molecular subtypes (CMS) with distinct biological and clinical characteristics. Here, we applied the CMS classification on CRC cell lines to identify novel subtype-specific drug vulnerabilities. We combined publicly available transcriptome data from multiple resources to assign 157 CRC cell lines to CMS. By integrating results from large-scale drug screens, we discovered that the CMS1 subtype is highly vulnerable to the BIRC5 suppressor YM155. We confirmed our results using an independent panel of CRC cell lines and demonstrated a 100-fold higher sensitivity of CMS1. This vulnerability was specific to YM155 and not observed for commonly used chemotherapeutic agents. In CMS1 CRC, low concentrations of YM155 induced apoptosis and expression signatures associated with ER stress-mediated apoptosis signaling. Using a genome-wide CRISPR/Cas9 screen, we further discovered a novel role of genes involved in LDL-receptor trafficking as modulators of YM155 sensitivity in the CRC cell line HCT116. Our work shows that combining drug response data with CMS classification in cell lines can reveal selective vulnerabilities and proposes YM155 as a novel subtype-specific drug.
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- 2020
68. E-RNAi: a web application for the multi-species design of RNAi reagents - 2010 update.
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Thomas Horn and Michael Boutros
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- 2010
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69. GenomeRNAi: a database for cell-based RNAi phenotypes. 2009 update.
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Moritz Gilsdorf, Thomas Horn, Zeynep Arziman, Oliver Pelz, Evgeny Kiner, and Michael Boutros
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- 2010
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70. CAMK1D Triggers Immune Resistance of Human Tumor Cells Refractory to Anti–PD-L1 Treatment
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Christian H. Wetzel, Hartmut Goldschmidt, Vladimir M. Milenkovic, Mark Berneburg, Mathias Witzens-Harig, György Vereb, Ayse Nur Menevse, Arpad Szoor, Michael Boutros, Antonio Sorrentino, Chih-Yeh Chen, Anchana Rathinasamy, Philipp Beckhove, Sebastian Haferkamp, Klaus G. Griewank, Martin Ehrenschwender, Gertrud Knoll, Tillmann Michels, Madlen Ditz, Valentina Volpin, Anja Seckinger, Dirk Hose, Nisit Khandelwal, Hematology, and Basic (bio-) Medical Sciences
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0301 basic medicine ,endocrine system ,Cancer Research ,Calcium-Calmodulin-Dependent Protein Kinase Type 1/biosynthesis ,medicine.medical_treatment ,Immunology ,B7-H1 Antigen/antagonists & inhibitors ,Medizin ,Dermatology ,Drug resistance ,B7-H1 Antigen ,Mice ,03 medical and health sciences ,Multiple Myeloma/immunology ,0302 clinical medicine ,Cancer immunotherapy ,Neoplasms ,Neoplasms/immunology ,medicine ,Animals ,Humans ,Multiple myeloma ,hematology ,business.industry ,Melanoma ,Cancer ,T-Lymphocytes, Cytotoxic/immunology ,medicine.disease ,Immune checkpoint ,CTL ,030104 developmental biology ,Calcium-Calmodulin-Dependent Protein Kinase Type 1 ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,oncology ,Immunotherapy/methods ,Cancer research ,Immunotherapy ,Multiple Myeloma ,business ,T-Lymphocytes, Cytotoxic ,Genetic screen - Abstract
The success of cancer immunotherapy is limited by resistance to immune checkpoint blockade. We therefore conducted a genetic screen to identify genes that mediated resistance against CTLs in anti–PD-L1 treatment–refractory human tumors. Using PD-L1–positive multiple myeloma cells cocultured with tumor-reactive bone marrow–infiltrating CTL as a model, we identified calcium/calmodulin-dependent protein kinase 1D (CAMK1D) as a key modulator of tumor-intrinsic immune resistance. CAMK1D was coexpressed with PD-L1 in anti–PD-L1/PD-1 treatment–refractory cancer types and correlated with poor prognosis in these tumors. CAMK1D was activated by CTL through Fas-receptor stimulation, which led to CAMK1D binding to and phosphorylating caspase-3, -6, and -7, inhibiting their activation and function. Consistently, CAMK1D mediated immune resistance of murine colorectal cancer cells in vivo. The pharmacologic inhibition of CAMK1D, on the other hand, restored the sensitivity toward Fas-ligand treatment in multiple myeloma and uveal melanoma cells in vitro. Thus, rapid inhibition of the terminal apoptotic cascade by CAMK1D expressed in anti–PD-L1–refractory tumors via T-cell recognition may have contributed to tumor immune resistance.
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- 2020
71. Evolutionary conserved NSL complex/BRD4 axis controls transcription activation via histone acetylation
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Julien Thevenon, M. Felicia Basilicata, Aline Gaub, Bilal N. Sheikh, Marie Vincent, Asifa Akhtar, Cindy Colson, Mathilde Nizon, James E. Bradner, Michael Boutros, Matthew Bird, Max Planck Institute of Immunobiology and Epigenetics (MPI-IE), Max-Planck-Gesellschaft, Service de Génétique Médicale [Nantes], Centre hospitalier universitaire de Nantes (CHU Nantes), Service de Génétique Clinique [CHU Caen], Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Tumorothèque de Caen Basse-Normandie (TCBN), Biologie, génétique et thérapies ostéoarticulaires et respiratoires (BIOTARGEN), Normandie Université (NU)-Normandie Université (NU), Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven), Novartis Institutes for Biomedical Research [Cambridge, MA, États-Unis], Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), German Cancer Research Center - Deutsches Krebsforschungszentrum [Heidelberg] (DKFZ), Heidelberg University, This work was supported by DFG funded CRC992, CRC1140 awarded to A.A. and DFG (DRIC infrastructure grant) awarded to MB. This study was supported by the German Research Foundation under Germany’s Excellence Strategy (CIBSS—EXC-2189—Project ID 390939984)., and Bodescot, Myriam
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Epigenomics ,Male ,Transcriptional Activation ,0301 basic medicine ,Science ,General Physics and Astronomy ,Cellular homeostasis ,Article ,General Biochemistry, Genetics and Molecular Biology ,Histones ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Pregnancy ,Transcription (biology) ,RNA interference ,Animals ,Drosophila Proteins ,Epigenetics ,Promoter Regions, Genetic ,lcsh:Science ,Cells, Cultured ,Multidisciplinary ,biology ,Gene Expression Profiling ,Nuclear Proteins ,Acetylation ,[SDV.BBM.MN]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN] ,General Chemistry ,Chromatin ,Cell biology ,030104 developmental biology ,Histone ,[SDV.BBM.MN] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular Networks [q-bio.MN] ,RNAi ,biology.protein ,Drosophila ,Female ,RNA Interference ,lcsh:Q ,Transcription ,030217 neurology & neurosurgery ,NSL complex - Abstract
Cells rely on a diverse repertoire of genes for maintaining homeostasis, but the transcriptional networks underlying their expression remain poorly understood. The MOF acetyltransferase-containing Non-Specific Lethal (NSL) complex is a broad transcription regulator. It is essential in Drosophila, and haploinsufficiency of the human KANSL1 subunit results in the Koolen-de Vries syndrome. Here, we perform a genome-wide RNAi screen and identify the BET protein BRD4 as an evolutionary conserved co-factor of the NSL complex. Using Drosophila and mouse embryonic stem cells, we characterise a recruitment hierarchy, where NSL-deposited histone acetylation enables BRD4 recruitment for transcription of constitutively active genes. Transcriptome analyses in Koolen-de Vries patient-derived fibroblasts reveals perturbations with a cellular homeostasis signature that are evoked by the NSL complex/BRD4 axis. We propose that BRD4 represents a conserved bridge between the NSL complex and transcription activation, and provide a new perspective in the understanding of their functions in healthy and diseased states., The MOF acetyltransferase-containing Non-Specific Lethal (NSL) complex is a broad transcription regulator and haploinsufficiency of its KANSL1 subunit results in the Koolen-de Vries syndrome in humans. Here, the authors identify the BET protein BRD4 as evolutionary conserved co-factor of the NSL complex and provide evidence that NSL-deposited histone acetylation induces BRD4 recruitment for transcription of constitutively active genes.
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- 2020
72. JNK-dependent intestinal barrier failure disrupts host–microbe homeostasis during tumorigenesis
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Jun Zhou and Michael Boutros
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MAP Kinase Kinase 4 ,Biology ,medicine.disease_cause ,Immune system ,stem cells ,medicine ,Animals ,Homeostasis ,Tumor growth ,Microbiome ,Intestinal Mucosa ,Barrier function ,Multidisciplinary ,epithelial barrier defects ,Biological Sciences ,Intestinal epithelium ,Epithelium ,Gastrointestinal Microbiome ,Cell biology ,Cell Transformation, Neoplastic ,Drosophila melanogaster ,medicine.anatomical_structure ,Host-Pathogen Interactions ,Drosophila ,JNK ,Stem cell ,Carcinogenesis ,host–microbe homeostasis ,Signal Transduction ,Developmental Biology - Abstract
Significance The intestinal epithelium forms a tight barrier to the environment and is constantly regenerated. Precise control of barrier function and tissue renewal is important to maintain homeostasis. Using an inducible tumor model in the Drosophila intestine, this study shows that tumor progression disrupts the intestinal barrier and leads to commensal dysbiosis, thereby further fueling tumor growth. This reenforcing feedback loop can be interrupted by treatments with JNK inhibitor or antibiotics., In all animals, the intestinal epithelium forms a tight barrier to the environment. The epithelium regulates the absorption of nutrients, mounts immune responses, and prevents systemic infections. Here, we investigate the consequences of tumorigenesis on the microbiome using a Drosophila intestinal tumor model. We show that upon loss of BMP signaling, tumors lead to aberrant activation of JNK/Mmp2 signaling, followed by intestinal barrier dysfunction and commensal imbalance. In turn, the dysbiotic microbiome triggers a regenerative response and stimulates tumor growth. We find that inhibiting JNK signaling or depletion of the microbiome restores barrier function of the intestinal epithelium, leading to a reestablishment of host–microbe homeostasis, and organismic lifespan extension. Our experiments identify a JNK-dependent feedback amplification loop between intestinal tumors and the microbiome. They also highlight the importance of controlling the activity level of JNK signaling to maintain epithelial barrier function and host–microbe homeostasis.
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- 2020
73. Gut Microbiota-Derived Propionate Regulates the Expression of Reg3 Mucosal Lectins and Ameliorates Experimental Colitis in Mice
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Raquel Mejias-Luque, Markus Gerhard, Adrian Niemann, Dirk H. Busch, Roland M. Schmid, Michael Boutros, Elena Tonin, Tsuyoshi Miki, Bernd Schnabl, Maja C. Funk, Christoph K. Stein-Thoeringer, Melissa D. Docampo, Anna-Katharina Hillmer, Danica Bajic, Marcel R.M. van den Brink, and Sena Bluemel
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Antimicrobial peptides ,Pancreatitis-Associated Proteins ,Inflammation ,Gut flora ,Microbiology ,Mice ,Lectins ,medicine ,Organoid ,Animals ,Intestinal Mucosa ,Colitis ,Letters to the Editor ,Receptor ,Cell Proliferation ,AcademicSubjects/MED00260 ,chemistry.chemical_classification ,biology ,business.industry ,Interleukins ,Toll-Like Receptors ,Gastroenterology ,LGR5 ,General Medicine ,Protective Factors ,medicine.disease ,biology.organism_classification ,Gastrointestinal Microbiome ,Disease Models, Animal ,chemistry ,Propionate ,Propionates ,medicine.symptom ,business ,Signal Transduction - Abstract
Background and Aims Regenerating islet-derived protein type 3 [Reg3] lectins are antimicrobial peptides at mucosal surfaces of the gut, whose expression is regulated by pathogenic gut microbes via interleukin-22- or Toll-like receptor signalling. In addition to antimicrobial effects, tissue protection is hypothesized, but has been poorly investigated in the gut. Methods We applied antibiotic-induced microbiota perturbations, gnotobiotic approaches and a dextran-sodium sulfate [DSS] colitis model to assess microbial Reg3 regulation in the intestines and its role in colitis. We also used an intestinal organoid model to investigate this axis in vitro. Results First, we studied whether gut commensals are involved in Reg3 expression in mice, and found that antibiotic-mediated reduction of Clostridia downregulated intestinal Reg3B. A loss in Clostridia was accompanied by a significant reduction of short-chain fatty acids [SCFAs], and knock-out [KO] mice for SCFA receptors GPR43 and GPR109 expressed less intestinal Reg3B/-G. Propionate was found to induce Reg3 in intestinal organoids and in gnotobiotic mice colonized with a defined, SCFA-producing microbiota. Investigating the role of Reg3B as a protective factor in colitis, we found that Reg3B-KO mice display increased inflammation and less crypt proliferation in the DSS colitis model. Propionate decreased colitis and increased proliferation. Treatment of organoids exposed to DSS with Reg3B or propionate reversed the chemical injury with a loss of expression of the stem-cell marker Lgr5 and Olfm4. Conclusions Our results suggest that Clostridia can regulate Reg3-associated epithelial homeostasis through propionate signalling. We also provide evidence that the Reg3–propionate axis may be an important mediator of gut epithelial regeneration in colitis.
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- 2020
74. Allele-specific endogenous tagging and quantitative analysis of ��-catenin in colorectal cancer cells
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Giulia Ambrosi, Oksana Voloshanenko, Antonia F Eckert, Dominique Kranz, G Ulrich Nienhaus, and Michael Boutros
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Carcinoma, Hepatocellular ,QH301-705.5 ,Carcinogenesis ,Science ,fluorescence correlation spectroscopy ,Oncogenic signaling ,General Biochemistry, Genetics and Molecular Biology ,Humans ,CTNNB1 ,ddc:530 ,Biology (General) ,Wnt Signaling Pathway ,Alleles ,beta Catenin ,Cancer ,Genome ,General Immunology and Microbiology ,General Neuroscience ,Physics ,Liver Neoplasms ,Genetic Variation ,Genetics and Genomics ,FCS ,Cell Biology ,General Medicine ,β-catenin ,HCT116 Cells ,Wnt signaling ,Tools and Resources ,CRISPR ,Mutation ,endogenous tagging ,Medicine ,Colorectal Neoplasms ,Genetic Engineering ,Human - Abstract
Wnt signaling plays important roles in development, homeostasis, and tumorigenesis. Mutations in β-catenin that activate Wnt signaling have been found in colorectal and hepatocellular carcinomas. However, the dynamics of wild-type and mutant forms of β-catenin are not fully understood. Here, we genome-engineered fluorescently tagged alleles of endogenous β-catenin in a colorectal cancer cell line. Wild-type and oncogenic mutant alleles were tagged with different fluorescent proteins, enabling the analysis of both variants in the same cell. We analyzed the properties of both β-catenin alleles using immunoprecipitation, immunofluorescence, and fluorescence correlation spectroscopy approaches, revealing distinctly different biophysical properties. In addition, activation of Wnt signaling by treatment with a GSK3β inhibitor or a truncating APC mutation modulated the wild-type allele to mimic the properties of the mutant β-catenin allele. The one-step tagging strategy demonstrates how genome engineering can be employed for the parallel functional analysis of different genetic variants.
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- 2022
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75. Borrow Now, Pay Even Later: A Quantitative Analysis of Student Debt Payment Plans
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Michael Boutros, Nuno Clara, and Francisco Gomes
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History ,Polymers and Plastics ,Business and International Management ,Industrial and Manufacturing Engineering - Published
- 2022
76. Epithelial cells of the intestine acquire cell-intrinsic inflammation signatures during ageing
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Maja C. Funk, Jan G. Gleixner, Florian Heigwer, Erica Valentini, Zeynep Aydin, Elena Tonin, Jenny Hetzer, Danijela Heide, Oliver Stegle, Mathias Heikenwalder, and Michael Boutros
- Abstract
During ageing, cell-intrinsic and extrinsic factors lead to the decline of tissue function and organismal health. Disentangling these factors is important for developing effective strategies to prolong organismal healthspan. Here, we addressed this question in the mouse intestinal epithelium, which forms a dynamic interface with its microenvironment and receives extrinsic signals affecting its homeostasis and tissue ageing. We systematically compared transcriptional profiles of young and aged epithelial cells in vivo and ex vivo in cultured intestinal organoids. We found that all cell types of the aged epithelium exhibit an inflammation phenotype, which is marked by MHC class II upregulation and most pronounced in enterocytes. This was accompanied by elevated levels of the immune tolerance markers PD-1 and PD-L1 in the aged tissue microenvironment, indicating dysregulation of immunological homeostasis. Intestinal organoids from aged mice still showed an inflammation signature after weeks in culture, which was concurrent with increased chromatin accessibility of inflammation-associated loci. Our results reveal a cell-intrinsic, persistent inflammation phenotype in aged epithelial cells, which might contribute to systemic inflammation observed during ageing.
- Published
- 2021
77. The drug-induced phenotypic landscape of colorectal cancer organoids
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Johannes Betge, Niklas Rindtorff, Jan Sauer, Benedikt Rauscher, Clara Dingert, Haristi Gaitantzi, Frank Herweck, Kauthar Srour-Mhanna, Thilo Miersch, Erica Valentini, Kim E. Boonekamp, Veronika Hauber, Tobias Gutting, Larissa Frank, Sebastian Belle, Timo Gaiser, Inga Buchholz, Ralf Jesenofsky, Nicolai Härtel, Tianzuo Zhan, Bernd Fischer, Katja Breitkopf-Heinlein, Elke Burgermeister, Matthias P. Ebert, and Michael Boutros
- Subjects
Organoids ,Multidisciplinary ,Phenotype ,General Physics and Astronomy ,Humans ,General Chemistry ,Colorectal Neoplasms ,General Biochemistry, Genetics and Molecular Biology ,Signal Transduction - Abstract
Patient-derived organoids resemble the biology of tissues and tumors, enabling ex vivo modeling of human diseases. They have heterogeneous morphologies with unclear biological causes and relationship to treatment response. Here, we use high-throughput, image-based profiling to quantify phenotypes of over 5 million individual colorectal cancer organoids after treatment with >500 small molecules. Integration of data using multi-omics modeling identifies axes of morphological variation across organoids: Organoid size is linked to IGF1 receptor signaling, and cystic vs. solid organoid architecture is associated with LGR5 + stemness. Treatment-induced organoid morphology reflects organoid viability, drug mechanism of action, and is biologically interpretable. Inhibition of MEK leads to cystic reorganization of organoids and increases expression ofLGR5, while inhibition of mTOR induces IGF1 receptor signaling. In conclusion, we identify shared axes of variation for colorectal cancer organoid morphology, their underlying biological mechanisms, and pharmacological interventions with the ability to move organoids along them.
- Published
- 2021
78. Conditional CRISPR-Cas Genome Editing in Drosophila to Generate Intestinal Tumors
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Tianyu Wang, Shivohum Bahuguna, Siamak Redhai, Michael Boutros, Fillip Port, and Jun Zhou
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tumors ,Time Factors ,Notch ,QH301-705.5 ,Mutagenesis (molecular biology technique) ,Mitosis ,Article ,Genome editing ,CRISPR-Associated Protein 9 ,Intestinal Neoplasms ,CRISPR ,Animals ,BMP ,Genes, Tumor Suppressor ,Guide RNA ,Biology (General) ,Cas9 ,Gene knockout ,intestinal stem cells ,Gene Editing ,biology ,Stem Cells ,aging ,General Medicine ,biology.organism_classification ,Cell biology ,Drosophila melanogaster ,Mutation ,JNK ,Stem cell ,CRISPR-Cas Systems ,Digestive System - Abstract
CRISPR-Cas has revolutionized genetics and extensive efforts have been made to enhance its editing efficiency by developing increasingly more elaborate tools. Here, we evaluate the CRISPR-Cas9 system in Drosophila melanogaster to assess its ability to induce stem cell-derived tumors in the intestine. We generated conditional tissue-specific CRISPR knockouts using different Cas9 expression vectors with guide RNAs targeting the BMP, Notch, and JNK pathways in intestinal progenitors such as stem cells (ISCs) and enteroblasts (EBs). Perturbing Notch and BMP signaling increased the proliferation of ISCs/EBs and resulted in the formation of intestinal tumors, albeit with different efficiencies. By assessing both the anterior and posterior regions of the midgut, we observed regional differences in ISC/EB proliferation and tumor formation upon mutagenesis. Surprisingly, high continuous expression of Cas9 in ISCs/EBs blocked age-dependent increase in ISCs/EBs proliferation and when combined with gRNAs targeting tumor suppressors, it prevented tumorigenesis. However, no such effects were seen when temporal parameters of Cas9 were adjusted to regulate its expression levels or with a genetically modified version, which expresses Cas9 at lower levels, suggesting that fine-tuning Cas9 expression is essential to avoid deleterious effects. Our findings suggest that modifications to Cas9 expression results in differences in editing efficiency and careful considerations are required when choosing reagents for CRISPR-Cas9 mutagenesis studies. In summary, Drosophila can serve as a powerful model for context-dependent CRISPR-Cas based perturbations and to test genome-editing systems in vivo.
- Published
- 2021
79. GenomeRNAi: a database for cell-based RNAi phenotypes.
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Thomas Horn, Zeynep Arziman, Juerg Berger, and Michael Boutros
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- 2007
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80. FlyRNAi: the Drosophila RNAi screening center database.
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Ian Flockhart, Matthew Booker, Amy Kiger, Michael Boutros, Susan Armknecht, Nadire Ramadan, Kris Richardson, Andrew Xu, Norbert Perrimon, and Bernard Mathey-Prevot
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- 2006
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81. E-RNAi: a web application to design optimized RNAi constructs.
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Zeynep Arziman, Thomas Horn, and Michael Boutros
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- 2005
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- View/download PDF
82. EVI/WLS function is regulated by ubiquitylation and is linked to ER-associated degradation by ERLIN2
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Lucie M. Wolf, Julie Haenlin, Annika M. Lambert, and Michael Boutros
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Ubiquitylation ,Ubiquitin-Protein Ligases ,Proteolysis ,Endogeny ,Biology ,Endoplasmic-reticulum-associated protein degradation ,Endoplasmic Reticulum ,Ubiquitin ,medicine ,Animals ,Humans ,Wnt signalling ,Secretory pathway ,EVI/WLS ,medicine.diagnostic_test ,Endoplasmic reticulum ,Ubiquitination ,Wnt signaling pathway ,Membrane Proteins ,Endoplasmic Reticulum-Associated Degradation ,Cell Biology ,ERAD ,Cell biology ,Ubiquitin-Conjugating Enzymes ,biology.protein ,Function (biology) ,Research Article - Abstract
WNT signalling is important for development in all metazoans and is associated with various human diseases. The ubiquitin–proteasome system (UPS) and regulatory endoplasmic reticulum-associated degradation (ERAD) have been implicated in the production of WNT proteins. Here, we investigated how the WNT secretory factor EVI (also known as WLS) is ubiquitylated, recognised by ERAD components and subsequently removed from the secretory pathway. We performed a focused immunoblot-based RNAi screen for factors that influence EVI/WLS protein stability. We identified the VCP-binding proteins FAF2 and UBXN4 as novel interaction partners of EVI/WLS and showed that ERLIN2 links EVI/WLS to the ubiquitylation machinery. Interestingly, we also found that EVI/WLS is ubiquitylated and degraded in cells irrespective of their level of WNT production. This K11, K48 and K63-linked ubiquitylation is mediated by the E2 ubiquitin-conjugating enzymes UBE2J2, UBE2K and UBE2N, but is independent of the E3 ubiquitin ligases HRD1 (also known as SYVN1) and GP78 (also known as AMFR). Taken together, our study identifies factors that link the UPS to the WNT secretory pathway and provides mechanistic details of the fate of an endogenous substrate of regulatory ERAD in mammalian cells. This article has an associated First Person interview with the first author of the paper., Summary: The WNT secretory factor EVI/WLS is ubiquitylated and linked to ER-associated degradation by multiple proteins, providing insight into the link between WNT signalling and the ubiquitin–proteasome system.
- Published
- 2021
83. EBImage - an R package for image processing with applications to cellular phenotypes.
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Grégoire Pau, Florian Fuchs 0003, Oleg Sklyar, Michael Boutros, and Wolfgang Huber
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- 2010
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84. Author response: Allele-specific endogenous tagging and quantitative analysis of β-catenin in colorectal cancer cells
- Author
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Antonia F Eckert, Oksana Voloshanenko, Giulia Ambrosi, Dominique Kranz, G Ulrich Nienhaus, and Michael Boutros
- Published
- 2021
85. Glyoxal as alternative fixative for single cell RNA sequencing
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Josephine Bageritz, Krausse N, Yousefian S, Michael Boutros, Erica Valentini, and S Leible
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Transcriptome ,Cell type ,medicine.anatomical_structure ,Cellular differentiation ,Gene expression ,Cell ,medicine ,Sample collection ,Biology ,Gene ,Fixative ,Cell biology - Abstract
Single cell RNA sequencing (scRNA-seq) has become an important method to identify cell types, delineate the trajectories of cell differentiation in whole organisms and understand the heterogeneity in cellular responses. Nevertheless, sample collection and processing remain a severe bottleneck for scRNA-seq experiments. Cell isolation protocols often lead to significant changes in the transcriptomes of cells, requiring novel methods to preserve cell states. Here, we developed and benchmarked protocols using glyoxal as a fixative for scRNA-seq application. Using Drop-seq methodology, we detected high numbers of transcripts and genes from glyoxal-fixed Drosophila cells after scRNA-seq. The effective glyoxal fixation of transcriptomes in Drosophila and human cells was further supported by a high correlation of gene expression data between glyoxal-fixed and unfixed samples. Accordingly, we also found highly expressed genes overlapping to a large extent between experimental conditions. These results indicated that our fixation protocol did not induce considerable changes in gene expression and conserved the transcriptome for subsequent single cell isolation procedures. In conclusion, we present glyoxal as a suitable fixative for Drosophila cells and potentially cells of other species that allows high-quality scRNA-seq applications.
- Published
- 2021
86. The extracellular matrix proteins type I collagen, type III collagen, fibronectin, and laminin 421 stimulate migration of cancer cells
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Patrick Horn, Michael Boutros, Christian Maercker, Anthony D. Ho, and Fabian Graf
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0301 basic medicine ,Integrin ,Apoptosis ,Biochemistry ,Collagen Type I ,Extracellular matrix ,03 medical and health sciences ,0302 clinical medicine ,Cell surface receptor ,Laminin ,Cell Movement ,Neoplasms ,Genetics ,Biomarkers, Tumor ,Tumor Cells, Cultured ,Humans ,Molecular Biology ,Cell Proliferation ,biology ,Chemistry ,Mesenchymal stem cell ,Mesenchymal Stem Cells ,Cell biology ,Fibronectins ,Fibronectin ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,Collagen Type III ,Cancer cell ,biology.protein ,030217 neurology & neurosurgery ,Type I collagen ,Biotechnology - Abstract
For metastasis formation, individual cells from a primary tumor must migrate toward other tissues. The aim of this study was to determine if mesenchymal stromal cells (MSCs) from human bone marrow are able to emit signals that induce this migratory activity in cancer cells. We separated the supernatant of MSCs derived from human bone marrow by size-exclusion and ion-exchange chromatography and have subsequently studied the migratory behavior of the prostate cancer cell line PC3 and the breast cancer cell line MDA-MB-231 toward the respective fractions in a transwell migration assay. We identified the extracellular matrix (ECM) proteins type I collagen, type III collagen, fibronectin, and laminin 421 as potential drivers of cancer cell migration. These results could be reproduced using the corresponding isolated or recombinant ECM proteins. Knockdown of the gene encoding beta 1 integrin, an important cell surface receptor for fibronectin, has led to inhibition of cancer cell migration. This supports the hypothesis that beta 1 integrin signaling represents an initial event that leads to metastasis, and that signaling is triggered by binding of integrin heterodimers to ECM molecules. Further characterization of signaling factors and their respective receptors will have implications for anticancer drug development.
- Published
- 2021
87. Detection of mutational patterns in cell‐free DNA of colorectal cancer by custom amplicon sequencing
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Tianzuo Zhan, Nicolai Härtel, Nadine Schulte, Simon Herrmann, Benedikt Rauscher, Ralf Jesenofsky, Sebastian Belle, Matthias P. Ebert, Michael Boutros, Timo Gaiser, Tobias Gutting, Johannes Betge, and Ralf Hofheinz
- Subjects
0301 basic medicine ,Cancer Research ,Colorectal cancer ,next‐generation sequencing ,colorectal cancer ,Computational biology ,Drug resistance ,Disease ,Biology ,lcsh:RC254-282 ,DNA sequencing ,03 medical and health sciences ,0302 clinical medicine ,Multiplex polymerase chain reaction ,Genetics ,medicine ,Humans ,Liquid biopsy ,cfDNA ,Research Articles ,liquid biopsy ,High-Throughput Nucleotide Sequencing ,Reproducibility of Results ,General Medicine ,Amplicon ,medicine.disease ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,030104 developmental biology ,Oncology ,Drug Resistance, Neoplasm ,030220 oncology & carcinogenesis ,Mutation ,Amplicon sequencing ,Molecular Medicine ,Colorectal Neoplasms ,Cell-Free Nucleic Acids ,Research Article - Abstract
Monitoring the mutational patterns of solid tumors during cancer therapy is a major challenge in oncology. Analysis of mutations in cell-free (cf) DNA offers a noninvasive approach to detect mutations that may be prognostic for disease survival or predictive for primary or secondary drug resistance. A main challenge for the application of cfDNA as a diagnostic tool is the diverse mutational landscape of cancer. Here, we developed a flexible end-to-end experimental and bioinformatic workflow to analyze mutations in cfDNA using custom amplicon sequencing. Our approach relies on open-software tools to select primers suitable for multiplex PCR using minimal cfDNA as input. In addition, we developed a robust linear model to identify specific genetic alterations from sequencing data of cfDNA. We used our workflow to design a custom amplicon panel suitable for detection of hotspot mutations relevant for colorectal cancer and analyzed mutations in serial cfDNA samples from a pilot cohort of 34 patients with advanced colorectal cancer. Using our method, we could detect recurrent and patient-specific mutational patterns in the majority of patients. Furthermore, we show that dynamic changes of mutant allele frequencies in cfDNA correlate well with disease progression. Finally, we demonstrate that sequencing of cfDNA can reveal mechanisms of resistance to anti-Epidermal Growth Factor Receptor(EGFR) antibody treatment. Thus, our approach offers a simple and highly customizable method to explore genetic alterations in cfDNA.
- Published
- 2019
88. CRISPR/Cas9 for cancer research and therapy
- Author
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Johannes Betge, Tianzuo Zhan, Matthias P. Ebert, Michael Boutros, and N Rindtorff
- Subjects
Gene Editing ,0301 basic medicine ,Cancer Research ,Genome, Human ,Cas9 ,Research ,Cancer ,Synthetic lethality ,Biology ,medicine.disease ,Genome ,Genome engineering ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Drug development ,Neoplasms ,030220 oncology & carcinogenesis ,Cancer research ,medicine ,Humans ,CRISPR ,CRISPR-Cas Systems ,Functional genomics - Abstract
CRISPR/Cas9 has become a powerful method for making changes to the genome of many organisms. First discovered in bacteria as part of an adaptive immune system, CRISPR/Cas9 and modified versions have found a widespread use to engineer genomes and to activate or to repress the expression of genes. As such, CRISPR/Cas9 promises to accelerate cancer research by providing an efficient technology to dissect mechanisms of tumorigenesis, identify targets for drug development, and possibly arm cells for cell-based therapies. Here, we review current applications of the CRISPR/Cas9 technology for cancer research and therapy. We describe novel Cas9 variants and how they are used in functional genomics to discover novel cancer-specific vulnerabilities. Furthermore, we highlight the impact of CRISPR/Cas9 in generating organoid and mouse models of cancer. Finally, we provide an overview of the first clinical trials that apply CRISPR/Cas9 as a therapeutic approach against cancer.
- Published
- 2019
89. Monetary Policy Implementation in a Negative Rate Environment
- Author
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Jonathan Witmer and Michael Boutros
- Subjects
Bank rate ,Economics and Econometrics ,050208 finance ,Overnight rate ,Inflation targeting ,media_common.quotation_subject ,05 social sciences ,Monetary policy ,Monetary economics ,Interest rate ,Credit channel ,Accounting ,Overnight market ,0502 economics and business ,Economics ,050207 economics ,Cash management ,Finance ,media_common - Abstract
Monetary policy implementation could, in theory, be constrained by deeply negative rates since overnight market participants may have an incentive to invest in cash rather than lend to other participants.
- Published
- 2019
90. Clinical relevance of gene expression in localized and metastatic prostate cancer exemplified by FABP5
- Author
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Philipp Nuhn, J. von Hardenberg, Thomas Stefan Worst, Philipp Erben, Frank Waldbillig, Michael Boutros, Katja Nitschke, Cleo-Aron Weis, Maria Gottschalt, Maurice-Stephan Michel, Abdallah Abdelhadi, and Sarah Wahby
- Subjects
Hepatocyte Nuclear Factor 3-alpha ,Male ,Oncology ,medicine.medical_specialty ,Oncogene Proteins, Fusion ,Microarray ,Urology ,medicine.medical_treatment ,Peroxisome Proliferator-Activated Receptors ,Prostatic Hyperplasia ,030232 urology & nephrology ,Gene Expression ,SPOP ,Fatty Acid-Binding Proteins ,TMPRSS2 ,03 medical and health sciences ,Prostate cancer ,0302 clinical medicine ,Internal medicine ,medicine ,Humans ,RNA, Messenger ,Neoplasm Metastasis ,Aged ,Neoplasm Staging ,Transurethral resection of the prostate ,Aged, 80 and over ,Prostatectomy ,Reverse Transcriptase Polymerase Chain Reaction ,business.industry ,Carcinoma ,Palliative Care ,Transurethral Resection of Prostate ,Nuclear Proteins ,Prostatic Neoplasms ,Cancer ,Middle Aged ,medicine.disease ,Repressor Proteins ,Case-Control Studies ,030220 oncology & carcinogenesis ,Mutation ,Neoplasm Grading ,FOXA1 ,business ,Signal Transduction - Abstract
Fatty acid-binding protein 5 (FABP5), a transport protein for lipophilic molecules, has been proposed as protein marker in prostate cancer (PCa). The role of FABP5 gene expression is merely unknown. In two cohorts of PCa patients who underwent radical prostatectomy (n = 40 and n = 57) and one cohort of patients treated with palliative transurethral resection of the prostate (pTUR-P; n = 50) FABP5 mRNA expression was analyzed with qRT-PCR. Expression was correlated with clinical parameters. BPH tissue samples served as control. To independently validate findings on FABP5 expression, three microarray and sequencing datasets were reanalyzed (MSKCC 2010 n = 216; TCGA 2015 n = 333; mCRPC, Nature Medicine 2016 n = 114). FABP5 expression was correlated with ERG-fusion status, TCGA subtypes, cancer driver mutations and the expression of druggable downstream pathway components. FABP5 was overexpressed in PCa compared to BPH in the cohorts analyzed by qRT-PCR (radical prostatectomy p = 0.003, p = 0.010; pTUR-P p = 0.002). FABP5 expression was independent of T stage, Gleason Score, nodal status and PSA level. FABP5 overexpression was associated with the absence of TMPRSS2:ERG fusion (p
- Published
- 2019
91. Salt-inducible kinase 3 protects tumor cells from cytotoxic T-cell attack by promoting TNF-induced NF-κB activation
- Author
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Antonio Sorrentino, Ayse Nur Menevse, Tillmann Michels, Valentina Volpin, Franziska Christine Durst, Julian Sax, Maria Xydia, Abir Hussein, Slava Stamova, Steffen Spoerl, Nicole Heuschneider, Jasmin Muehlbauer, Katharina Marlene Jeltsch, Anchana Rathinasamy, Melanie Werner-Klein, Marco Breinig, Damian Mikietyn, Christian Kohler, Isabel Poschke, Sabrina Purr, Olivia Reidell, Catarina Martins Freire, Rienk Offringa, Claudia Gebhard, Rainer Spang, Michael Rehli, Michael Boutros, Christian Schmidl, Nisit Khandelwal, and Philipp Beckhove
- Subjects
Tumor Necrosis Factor-alpha/metabolism ,ddc:004 ,Pharmacology ,ddc:610 ,Cancer Research ,Tumor Necrosis Factor-alpha ,T-Lymphocytes ,Immunology ,NF-kappa B ,610 Medizin ,tumor escape, immunotherapy, cytokines, immunomodulation, CD8-positive T-lymphocytes ,Apoptosis ,T-Lymphocytes/metabolism ,004 Informatik ,Oncology ,NF-kappa B/metabolism ,Humans ,Molecular Medicine ,Immunology and Allergy ,Phosphorylation - Abstract
BackgroundCancer immunotherapeutic strategies showed unprecedented results in the clinic. However, many patients do not respond to immuno-oncological treatments due to the occurrence of a plethora of immunological obstacles, including tumor intrinsic mechanisms of resistance to cytotoxic T-cell (TC) attack. Thus, a deeper understanding of these mechanisms is needed to develop successful immunotherapies.MethodsTo identify novel genes that protect tumor cells from effective TC-mediated cytotoxicity, we performed a genetic screening in pancreatic cancer cells challenged with tumor-infiltrating lymphocytes and antigen-specific TCs.ResultsThe screening revealed 108 potential genes that protected tumor cells from TC attack. Among them, salt-inducible kinase 3 (SIK3) was one of the strongest hits identified in the screening. Both genetic and pharmacological inhibitions of SIK3 in tumor cells dramatically increased TC-mediated cytotoxicity in several in vitro coculture models, using different sources of tumor and TCs. Consistently, adoptive TC transfer of TILs led to tumor growth inhibition of SIK3-depleted cancer cells in vivo. Mechanistic analysis revealed that SIK3 rendered tumor cells susceptible to tumor necrosis factor (TNF) secreted by tumor-activated TCs. SIK3 promoted nuclear factor kappa B (NF-κB)nuclear translocation and inhibited caspase-8 and caspase-9 after TNF stimulation. Chromatin accessibility and transcriptome analyses showed that SIK3 knockdown profoundly impaired the expression of prosurvival genes under the TNF–NF-κBaxis. TNF stimulation led to SIK3-dependent phosphorylation of the NF-κB upstream regulators inhibitory-κB kinase and NF-kappa-B inhibitor alpha on the one side, and to inhibition of histone deacetylase 4 on the other side, thus sustaining NF-κB activation and nuclear stabilization. A SIK3-dependent gene signature of TNF-mediated NF-κB activation was found in a majority of pancreatic cancers where it correlated with increased cytotoxic TC activity and poor prognosis.ConclusionOur data reveal an abundant molecular mechanism that protects tumor cells from cytotoxic TC attack and demonstrate that pharmacological inhibition of this pathway is feasible.
- Published
- 2022
92. Local emergence and decline of a SARS-CoV-2 variant with mutations L452R and N501Y in the spike protein
- Author
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Niklas Weidner, Paul Schnitzler, Nayara Trevisan Doimo de Azevedo, Katharina Bauer, Jan-Philipp Mallm, Simon Steiger, Kathleen Börner, Vladimir Benes, Karsten Rippe, Daniel Hübschmann, Ralf Bartenschlager, Michael Boutros, Christian Bundschuh, Anja Telzerow, Tobias Rausch, Barbara Müller, Heeyoung Kim, Katharina Laurence Jost, Isabelle Lander, Hans-Georg Kräusslich, and Sylvia Parthé
- Subjects
Whole genome sequencing ,Vaccination ,Phylogenetic tree ,biology.protein ,Biology ,Antibody ,Vaccine efficacy ,Clade ,Gene ,Virology ,Virus - Abstract
SummaryVariants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are replacing the initial wild-type strain, jeopardizing current efforts to contain the pandemic. Amino acid exchanges in the spike protein are of particular concern as they can render the virus more transmissible or reduce vaccine efficacy. Here, we conducted whole genome sequencing of SARS-CoV-2 positive samples from the Rhine-Neckar district in Germany during January-March 2021. We detected a total of 166 samples positive for a variant with a distinct mutational pattern in the spike gene comprising L18F, L452R, N501Y, A653V, H655Y, D796Y and G1219V with a later gain of A222V. This variant was designated A.27.RN according to its phylogenetic clade classification. It emerged in parallel with the B.1.1.7 variant, increased to >50% of all SARS-CoV-2 variants by week five. Subsequently it decreased to
- Published
- 2021
93. Systematic functional analysis of rab GTPases reveals limits of neuronal robustness to environmental challenges in flies
- Author
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Thomas F. Mathejczyk, Chih-Chiang Chan, Bojana Pavlovic, Michael Boutros, Friederike Elisabeth Kohrs, P. Robin Hiesinger, Heike Wolfenberg, Gerit A. Linneweber, Eugene Jennifer Jin, F Ridvan Kiral, Shih-Ching Lin, Ilsa-Maria Daumann, and Filip Port
- Subjects
Nervous system ,QH301-705.5 ,Science ,Mutant ,GTPase ,Biology ,General Biochemistry, Genetics and Molecular Biology ,flies ,medicine ,Animals ,Drosophila Proteins ,neuronal robustness ,Gene Knock-In Techniques ,Biology (General) ,Gene ,Neurons ,mutant collection ,D. melanogaster ,General Immunology and Microbiology ,Functional analysis ,General Neuroscience ,fungi ,Imidazoles ,Temperature ,Robustness (evolution) ,Cell Biology ,500 Naturwissenschaften und Mathematik::570 Biowissenschaften ,Biologie::570 Biowissenschaften ,Biologie ,General Medicine ,rab GTPases ,Tools and Resources ,Cell biology ,Drosophila melanogaster ,medicine.anatomical_structure ,rab GTP-Binding Proteins ,Medicine ,Drosophila ,Rab ,Rab GTPase ,Function (biology) ,Neuroscience - Abstract
Rab GTPases are molecular switches that regulate membrane trafficking in all cells. Neurons have particular demands on membrane trafficking and express numerous Rab GTPases of unknown function. Here, we report the generation and characterization of molecularly defined null mutants for all 26 rab genes in Drosophila. In flies, all rab genes are expressed in the nervous system where at least half exhibit particularly high levels compared to other tissues. Surprisingly, loss of any of these 13 nervous system-enriched Rabs yielded viable and fertile flies without obvious morphological defects. However, all 13 mutants differentially affected development when challenged with different temperatures, or neuronal function when challenged with continuous stimulation. We identified a synaptic maintenance defect following continuous stimulation for six mutants, including an autophagy-independent role of rab26. The complete mutant collection generated in this study provides a basis for further comprehensive studies of Rab GTPases during development and function in vivo.
- Published
- 2021
94. Author response: Systematic functional analysis of rab GTPases reveals limits of neuronal robustness to environmental challenges in flies
- Author
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Friederike Elisabeth Kohrs, F Ridvan Kiral, Heike Wolfenberg, Thomas F. Mathejczyk, Ilsa-Maria Daumann, Eugene Jennifer Jin, Shih-Ching Lin, Filip Port, P. Robin Hiesinger, Gerit A. Linneweber, Chih-Chiang Chan, Michael Boutros, and Bojana Pavlovic
- Subjects
Functional analysis ,Computer science ,Robustness (evolution) ,Rab ,GTPase ,Computational biology - Published
- 2021
95. PPARγ induces PD-L1 expression in MSS+ colorectal cancer cells
- Author
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Maximilian Eckardt, Kay Klapproth, Veronika Hauber, Philip Weidner, Michael Boutros, Tianzuo Zhan, Matthias P. Ebert, Beifang Li, Timo Gaiser, Wenyue Wu, Jens Pahl, Sebastian Belle, Frank Herweck, Elke Burgermeister, Adelheid Cerwenka, Torsten Schroeder, Juliane Reichling, Laura Helm, Ioana Dobrota, Carsten Sticht, Tobias Gutting, and Johannes Betge
- Subjects
0301 basic medicine ,mss ,medicine.medical_treatment ,Immunology ,Peroxisome proliferator-activated receptor ,B7-H1 Antigen ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,pd-l1 ,PD-L1 ,Tumor Microenvironment ,medicine ,Humans ,cancer ,Immunology and Allergy ,RC254-282 ,Original Research ,colorectal ,chemistry.chemical_classification ,Tumor microenvironment ,biology ,business.industry ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Cancer ,Microsatellite instability ,Immunotherapy ,RC581-607 ,medicine.disease ,Immune checkpoint ,PPAR gamma ,030104 developmental biology ,Oncology ,chemistry ,030220 oncology & carcinogenesis ,biology.protein ,Cancer research ,Microsatellite Instability ,immunotherapy ,Immunologic diseases. Allergy ,Colorectal Neoplasms ,ppar ,business ,Research Article - Abstract
Only a small subset of colorectal cancer (CRC) patients benefits from immunotherapies, comprising blocking antibodies (Abs) against checkpoint receptor “programmed-cell-death-1” (PD1) and its ligand (PD-L1), because most cases lack the required mutational burden and neo-antigen load caused by microsatellite instability (MSI) and/or an inflamed, immune cell-infiltrated PD-L1+ tumor microenvironment. Peroxisome proliferator-activated-receptor-gamma (PPARγ), a metabolic transcription factor stimulated by anti-diabetic drugs, has been previously implicated in pre/clinical responses to immunotherapy. We therefore raised the hypothesis that PPARγ induces PD-L1 on microsatellite stable (MSS) tumor cells to enhance Ab-target engagement and responsiveness to PD-L1 blockage. We found that PPARγ-agonists upregulate PD-L1 mRNA/protein expression in human gastrointestinal cancer cell lines and MSS+ patient-derived tumor organoids (PDOs). Mechanistically, PPARγ bound to and activated DNA-motifs similar to cognate PPARγ-responsive-elements (PPREs) in the proximal −2 kb promoter of the human PD-L1 gene. PPARγ-agonist reduced proliferation and viability of tumor cells in co-cultures with PD-L1 blocking Ab and lymphokine-activated killer cells (LAK) derived from the peripheral blood of CRC patients or healthy donors. Thus, metabolic modifiers improved the antitumoral response of immune checkpoint Ab, proposing novel therapeutic strategies for CRC.
- Published
- 2021
96. EVI/WLS function is regulated by ubiquitination and linked to ER-associated degradation by ERLIN2
- Author
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Michael Boutros, Julie Haenlin, Lucie Wolf, and Annika Lambert
- Subjects
Ubiquitin ,biology ,biology.protein ,Wnt signaling pathway ,Endogeny ,Secretion ,Ubiquitin-conjugating enzyme ,Endoplasmic-reticulum-associated protein degradation ,Secretory pathway ,Function (biology) ,Cell biology - Abstract
Wnt signalling is important for development in all metazoan animals and associated with various human diseases. The Ubiquitin-Proteasome System (UPS) and regulatory ER-associated degradation (ERAD) has been implicated in the secretion of WNT proteins. Here, we investigated how the WNT secretory factor EVI/WLS is ubiquitinated, recognised by ERAD components, and subsequently removed from the secretory pathway. We performed a focused, immunoblot-based RNAi screen for factors that influence EVI/WLS protein stability. We identified the VCP-binding proteins FAF2 and UBXN4 as novel interaction partners of EVI/WLS and showed that ERLIN2 links EVI/WLS to the ubiquitination machinery. Interestingly, we found in addition that EVI/WLS is ubiquitinated and degraded in cells irrespective of their level of WNT production. K11, K48, and K63-linked ubiquitination is mediated by the E2 ubiquitin conjugating enzymes UBE2J2, UBE2N, and UBE2K, but independent of the E3 ligases HRD1/SYVN. Taken together, our study identified factors that link UPS to the WNT secretory pathway and provides mechanistic details on the fate of an endogenous substrate of regulatory ERAD in mammalian cells.
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- 2020
97. Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library
- Author
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Benedikt Rauscher, Jan Winter, Luisa Henkel, Barbara Schmitt, and Michael Boutros
- Subjects
Physiology ,Genome scale ,Plant Science ,Computational biology ,Biology ,Genome ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,0302 clinical medicine ,Structural Biology ,Humans ,CRISPR ,CRISPR/Cas9 ,Gene ,Gene essentiality ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,Subgenomic mRNA ,0303 health sciences ,Genes, Essential ,Genome, Human ,Cas9 ,Methodology Article ,Functional genomics ,Genetic screens ,Cell Biology ,sgRNA design ,CRISPR-Cas Systems ,Single-Cell Analysis ,General Agricultural and Biological Sciences ,030217 neurology & neurosurgery ,Developmental Biology ,Biotechnology ,Genetic screen - Abstract
Background In recent years, large-scale genetic screens using the CRISPR/Cas9 system have emerged as scalable approaches able to interrogate gene function with unprecedented efficiency and specificity in various biological contexts. By this means, functional dependencies on both the protein-coding and noncoding genome of numerous cell types in different organisms have been interrogated. However, screening designs vary greatly and criteria for optimal experimental implementation and library composition are still emerging. Given their broad utility in functionally annotating genomes, the application and interpretation of genome-scale CRISPR screens would greatly benefit from consistent and optimal design criteria. Results We report advantages of conducting viability screens in selected Cas9 single-cell clones in contrast to Cas9 bulk populations. We further systematically analyzed published CRISPR screens in human cells to identify single-guide (sg) RNAs with consistent high on-target and low off-target activity. Selected guides were collected in a novel genome-scale sgRNA library, which efficiently identifies core and context-dependent essential genes. Conclusion We show how empirically designed libraries in combination with an optimized experimental design increase the dynamic range in gene essentiality screens at reduced library coverage.
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- 2020
98. The Role of Organelles in Intestinal Function, Physiology, and Disease
- Author
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Michael Boutros and Siamak Redhai
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Organelles ,0303 health sciences ,Cell type ,Regeneration (biology) ,Stem Cells ,Cell Differentiation ,Cell Biology ,Biology ,Intestinal epithelium ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Organelle ,Stem cell ,Signal transduction ,Intestinal Mucosa ,030217 neurology & neurosurgery ,Homeostasis ,030304 developmental biology ,Cell Proliferation - Abstract
The intestine maintains homeostasis by coordinating internal biological processes to adjust to fluctuating external conditions. The intestinal epithelium is continuously renewed and comprises multiple cell types, including absorptive cells, secretory cells, and resident stem cells. An important feature of this organ is its ability to coordinate many processes including cell proliferation, differentiation, regeneration, damage/stress response, immune activity, feeding behavior, and age-related changes by using conserved signaling pathways. However, the subcellular spatial organization of these signaling events and the organelles involved has only recently been studied in detail. Here we discuss how organelles of intestinal cells serve to initiate, mediate, and terminate signals, that are vital for homeostasis.
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- 2020
99. Establishment of a simplified preparation method for single-nucleus RNA-sequencing and its application to long-term frozen tumor tissues
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Konstantin Okonechnikov, Marc Zuckermann, Josephine Bageritz, Dtw Jones, Andrea Wittmann, Jan-Philipp Mallm, Kendra K. Maass, Michael Boutros, Kati Ernst, Stefan M. Pfister, and S Leible
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education.field_of_study ,Pilocytic astrocytoma ,Population ,RNA ,Genomics ,Computational biology ,Biology ,medicine.disease ,Tumor tissue ,Preparation method ,Transcriptome ,medicine.anatomical_structure ,medicine ,education ,Nucleus - Abstract
Recent advances allowing the genomic analysis of individual cells from a bulk population have provided intriguing new insights into areas such as developmental processes and tumor heterogeneity. Most approaches to date, however, rely on the availability of fresh surgical specimens, thereby dramatically reducing the ability to profile particularly rare tissue types. Pediatric central nervous system tumors – the leading cause of childhood cancer deaths – represent one such example, where often only frozen rather than native material is available. Due to an increasing need for advanced techniques to understand the heterogeneity of these tumors, we optimized a method to isolate intact nuclei from long-term frozen pediatric glioma tissues. We performed a technical comparison between different single nucleus RNA-sequencing (snRNA-seq) systems using a patient-derived xenograft model as a test sample. Further, we applied the established nucleus isolation method to analyze frozen primary tissue from two pediatric central nervous system tumors – one pilocytic astrocytoma and one glioblastoma – allowing the identification of distinct tumor cell populations and infiltrating microglia. The results show that our fast, simple and low-cost nuclear isolation protocol provides intact nuclei, which can be used in both droplet-based 3’ transcriptome amplification (10X Genomics) and plate-based whole transcriptome amplification (Fluidigm C1) single-cell sequencing platforms, thereby dramatically increasing the potential for application of such methods to rare entities.
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- 2020
100. The Persistence of Miscalibration
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John R. Graham, Michael Boutros, Itzhak Ben-David, John W. Payne, and Campbell R. Harvey
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Persistence (psychology) ,Download ,Economics ,Econometrics ,Conviction ,Developing country ,Bayesian inference ,Behavioral economics ,Confidence interval - Abstract
Using 14,800 forecasts of one-year S&P 500 returns made by Chief Financial Officers over a 12-year period, we track the individual executives who provide multiple forecasts to study how their beliefs evolve dynamically. While CFOs’ return forecasts are systematically unbiased, their confidence intervals are far too narrow, implying significant miscalibration. We find that when return realizations fall outside of ex-ante confidence intervals, CFOs’ subsequent confidence intervals widen considerably. These results are consistent with a model of Bayesian learning which suggests that the evolution of beliefs should be impacted by return realizations. However, the magnitude of the updating is dampened by the strong conviction in beliefs inherent in the initial miscalibration and, as a result, miscalibration persists. Institutional subscribers to the NBER working paper series, and residents of developing countries may download this paper without additional charge at www.nber.org.
- Published
- 2020
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