DOCSLIB.ORG

Efficacy of Cyclin-Dependent-Kinase 9 Inhibitors in a Murine Model Of

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Efficacy of Cyclin-Dependent-Kinase 9 Inhibitors in a Murine Model Of Leukemia (2014) 28, 1427–1435 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu ORIGINAL ARTICLE Efficacy of cyclin-dependent-kinase 9 inhibitors in a murine model of mixed-lineage leukemia M-P Garcia-Cuellar1,EFu¨ ller1,EMa¨thner1, C Breitinger1, K Hetzner1, L Zeitlmann2, A Borkhardt3 and RK Slany1 Mixed-lineage leukemia fusion proteins activate their target genes predominantly by stimulating transcriptional elongation. A core component necessary for this activity is cyclin-dependent kinase 9. Here we explored the effectiveness of small molecules targeting this enzyme as potential therapeutics. A screen of seven compounds with anti-CDK9 activity applied to a panel of leukemia cell lines identified flavopiridol and the experimental inhibitor PC585 as superior in efficacy with inhibitory concentrations in the submicromolar range. Both substances induced rapid dephosphorylation of the RNA polymerase II C-terminal domain, accompanied by downregulation of CDK9-dependent transcripts for MYC and HOXA9. Global gene expression analysis indicated the induction of a general stress response program, culminating in widespread apoptosis. Importantly, colony-forming activity in leukemia lines and primary patient samples could be completely inhibited under conditions that did not affect native precursors from bone marrow. In vivo application in a mouse transplant model significantly delayed disease with PC585 showing also oral activity. These results suggest CDK9 inhibition as novel treatment option for mixed-lineage leukemia. Leukemia (2014) 28, 1427–1435; doi:10.1038/leu.2014.40 Keywords: mixed-lineage leukemia; CDK9; inhibitor; preclinical study INTRODUCTION complex) including P-TEFb is responsible for elongation control,5 Despite considerable progress in leukemia treatment, mixed- DotCom (DOT1L complex) methylates histone H3 at lysine 79 lineage leukemia (MLL) remains a disease with a very dismal through the catalytic activity of the histone methyltransferase 12 prognosis. Five-year survival rates for affected patients hover well DOT1L, and the MLL fusion partner ENL forms a scaffold that below 50%.1,2 MLL occurs mostly either in infants or in adults as integrates these subcomplexes and in addition it neutralizes the 13 late sequelae after previous treatment for an unrelated neoplastic repressive activity of polycomb proteins. Importantly, all these condition. The hallmark of MLL is an aberration of chromosome functions are essential for the transforming activity of MLL fusion eleven, affecting the gene coding for the histone proteins. methyltransferase MLL. As a consequence, transforming fusion In an effort to develop new therapeutics with activity against proteins are created that join part of MLL with a variety of MLL fusions, small-molecule inhibitors of the DOT1L methyltrans- different fusion partners.3,4 We and others have shown that the ferase have been designed and tested in early preclinical trials 14,15 most frequent MLL partners are members of a complex that where they showed efficacy in mouse models of MLL. The specifically stimulates transcriptional elongation.5–8 By this strategies for pharmaceutical targeting of transcriptional mechanism, MLL fusions activate an oncogenic network that elongation are represented by recent publications that use the 16–19 includes the clustered HOX-homeobox genes as important experimental inhibitors JQ1 and/or iBET. These substances determinators of hematopoietic development.9 block the activity of the BET (bromodomain and extra Control of transcriptional elongation is the predominant bromodomain) protein family (BRD2/3/4 and BRDT) by regulatory checkpoint for many genes involved in proliferation, selectively disrupting their binding to chromatin. BET proteins differentiation, and for immediate response transcripts (for are normally instrumental for the proper recruitment of P-TEFb to reviews of this topic see Adelman and Lis10 and Peterlin and acetylated histones. This mark is particularly prevalent in the area Price11). On a molecular level, the rate of elongation is determined surrounding actively initiated transcription units. Because many by the phosphorylation status of the C-terminal repeat domain of growth-related transcripts, with MYC as a prime example, depend RNA polymerase II (CTD). During initiation, TFIIH-associated on P-TEFb, the effect of BET inhibitors is pleiotropic. Consequently, kinases CDK7 and CDK8 phosphorylate Ser5 of the CTD- these molecules have been variably proposed as general heptapeptide. Subsequently, Ser2 needs to be converted to anti-cancer agents targeting mainly MYC17,18 or as a tool for phosphoserine for efficient elongation. This reaction is catalyzed intervention in MLL-fusion leukemia because of the particular by P-TEFb (positive transcription elongation factor b) a dimer of importance of P-TEFb in this disease.16 Despite their promise, CDK9 and a cyclin T. P-TEFb purifies with MLL fusion partners in a these compounds are new, with partially difficult pharmacological large macromolecular complex called EAP (ENL-interacting properties, and ‘first in man’ studies have yet to be reported. proteins or elongation-assisting proteins).6 EAP contains at least In contrast, CDK9 inhibitors have been developed for many years. three separable functional modules: SEC (super elongation Although results are mostly outstanding, flavopiridol (alvocidib), 1Department of Genetics, University Erlangen, Erlangen, Germany; 2Ingenium Pharmaceuticals AG, Munich, Germany and 3Clinic of Paediatric Oncology, Haematology and Clinical Immunology, Centre for Child and Adolescent Health, Medical Faculty, Heinrich-Heine-University Du¨sseldorf, Du¨sseldorf, Germany. Correspondence: Professor RK Slany, Deptartment of Genetics, University Erlangen, Erwin Rommel Str. 3, Erlangen 91058, Germany. E-mail: [email protected] Received 11 November 2013; revised 10 January 2014; accepted 15 January 2014; accepted article preview online 21 January 2014; advance online publication, 11 February 2014 CDK9 inhibitors in mixed-lineage leukemia M-P Garcia-Cuellar et al 1428 the best investigated CDK9 inhibitor, was already included in a enriched with 10 ng/ml IL7 (Stemcell Technologies, Grenoble, France). number of mostly phase I and phase II studies testing safety and Primary precursor cells from mouse bone marrow were isolated by magnetic response in a wide variety of solid and hematological tumors selection of CD117 (c-kit)-positive cells according to the instructions (ClinicalTrials.gov). of the manufacturer (Miltenyi Biotech, BergischGladbach, Germany). Here we examined the potential of CDK9 inhibition as potential Transplantation studies were done in a syngeneic model. Three days before transplantation Balb/C mice were prophylactically treated with therapeutic intervention for mixed-lineage leukemia. We show 6 1.1 g/l neomycin and 1 Â 10 units/l polymixinB as addition to drinking efficacy of this approach in vitro and in vivo suggesting that MLL water. Twenty-four hours after sublethal total body irradiation (6 Gy), should be included as further indication for future CDK9 inhibitor 1 Â 106 MLL-ENL-transformed cells were transplanted by retroorbital trials. injection. Treatment commenced either 2 days or 14 days after transplant by subcutaneous injection or by oral gavage. Substances for injection were dissolved in 25% DMSO/75% PBS. Suspensions for oral gavage were MATERIALS AND METHODS prepared in 0.8% methocel in H2O with 300 ml administered per individual Cell lines dose. Animals with clear signs of disease (hunched posture, ruffled fur, labored breathing) were euthanized. Spleen weight was recorded as an Human leukemia lines with MLL rearrangement (HB11;19, SEM, KOPN-8, objective surrogate marker for leukemia burden. Cohorts were followed for MV4;11, RS4,11, THP1 and Karpas45) and cells without involvement of 11q23 (HL60, K562, U937, REH and Jurkat) were obtained from the German a maximum of 120 days and surviving animals without signs of collection of microorganisms and cell cultures (DSMZ, Braunschweig, leukemia (normal physiology, spleen weighto100 mg, no infiltration Germany) or they were laboratory stocks. All human lines were kept in of leukemia cells in lymph nodes or inner organs) were censored from RPMI1640 supplemented with 10% fetal calf serum (FCS), except Karpas45 the results. Engraftment rates were generally between 80% and 100%. Animal procedures have been approved by local and institutional animal that was grown in 20% FCS. Mouse MLL-ENL-transformed lines Me2 and welfare review boards under license numbers 54–2532.1–41/11 and Me3, as well as cells used for transplantation assays were obtained by 20 54–2532.2–5/12. retroviral transduction as described. MeerIV was derived from a mouse with a germ-line integration of an inducible MLL-ENL-ER construct.21 Murine cells were cultivated in RPMI, 10% FCS and recombinant murine cytokines: 50 ng/ml stem cell factor, 5 ng/ml each of IL3, IL6 and RESULTS GM-CSF. For MeerIV cells, 100 nM 4-hydroxytamoxifen was added to Leukemia cell lines are sensitive to CDK9 inhibition in vitro activate MLL-ENL. To select for the most efficient CDK9 inhibitors, we employed seven human leukemia cell lines with MLL rearrangement (Karpas, CDK9 inhibitors, proliferation assays, antibodies THP1, RS4;11, MV4;11, SEM, KOPN-8, HB11;19) and five human cell Alsterpaullone, CDK9 inhibitorII and CDC7_CDK9 inhibitor were bought lines derived from hematological malignancies without 11q23 from Merck/Calbiochem (Darmstadt, Germany).
Load more

Recommended publications
  • HEXIM1 (D5Y5K) Rabbit
    HEXIM1 (D5Y5K) Rabbit mAb Store at -20°C 3 n 100 µl Orders n 877-616-CELL (2355) (10 western blots) [email protected] Support n 877-678-TECH (8324) [email protected] Web n www.cellsignal.com rev. 01/05/15 #12604 For Research Use Only. Not For Use In Diagnostic Procedures. Entrez-Gene ID #10614 UniProt ID #O94992 Storage: Supplied in 10 mM sodium HEPES (pH 7.5), 150 Applications Species Cross-Reactivity* Molecular Wt. Isotype mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% W, IP, IF-IC H, Mk 60 kDa Rabbit IgG** sodium azide. Store at –20°C. Do not aliquot the antibody. Endogenous *Species cross-reactivity is determined by western blot. Background: Hexamethylene bis-acetamide-inducible ** Anti-rabbit secondary antibodies must be used to protein 1 (HEXIM1) was originally identified in vascular kDa HeLa A-431 Hep G2 COS-7 detect this antibody. smooth muscle cells as a protein that is upregulated upon 200 Recommended Antibody Dilutions: treatment with the differentiating agent hexamethylene bi- 140 Western blotting 1:1000 sacetamide (1). HEXIM1 binds 7SK RNA, a highly abundant 100 Immunoprecipitation 1:100 non-coding RNA, and together they act as a potent inhibitor 80 Immunofluorescence (IF-IC) 1:1200 of positive transcription elongation factor b (P-TEFb) (2,3). 60 HEXIM1 P-TEFb phosphorylates the C-terminal domain of the largest 50 For product specific protocols please see the web page subunit of RNA polymerase II and is an important regulator for this product at www.cellsignal.com. 40 of transcription elongation (4-8).
    [Show full text]
  • De Novo Mepcenonsense Variant Associated with a Neurodevelopmental Disorder Causes Disintegration of 7SK Snrnp and Enhanced Rna Polymerase Ii Activation
    www.nature.com/scientificreports OPEN de novo MEPCE nonsense variant associated with a neurodevelopmental disorder Received: 8 May 2019 Accepted: 19 August 2019 causes disintegration of 7SK snRNP Published: xx xx xxxx and enhanced RNA polymerase II activation Pauline E. Schneeberger1, Tatjana Bierhals1, Axel Neu2, Maja Hempel1 & Kerstin Kutsche1 In eukaryotes, the elongation phase of transcription by RNA polymerase II (RNAP II) is regulated by the transcription elongation factor b (P-TEFb), composed of Cyclin-T1 and cyclin-dependent kinase 9. The release of RNAP II is mediated by phosphorylation through P-TEFb that in turn is under control by the inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex. The 7SK snRNP consists of the 7SK non-coding RNA and the proteins MEPCE, LARP7, and HEXIM1/2. Biallelic LARP7 loss-of- function variants underlie Alazami syndrome characterized by growth retardation and intellectual disability. We report a boy with global developmental delay and seizures carrying the de novo MEPCE nonsense variant c.1552 C > T/p.(Arg518*). mRNA and protein analyses identifed nonsense-mediated mRNA decay to underlie the decreased amount of MEPCE in patient fbroblasts followed by LARP7 and 7SK snRNA downregulation and HEXIM1 upregulation. Reduced binding of HEXIM1 to Cyclin-T1, hyperphosphorylation of the RNAP II C-terminal domain, and upregulated expression of ID2, ID3, MRPL11 and snRNAs U1, U2 and U4 in patient cells are suggestive of enhanced activation of P-TEFb. Flavopiridol treatment and ectopic MEPCE protein expression in patient fbroblasts rescued increased expression of six RNAP II-sensitive genes and suggested a possible repressive efect of MEPCE on P-TEFb-dependent transcription of specifc genes.
    [Show full text]
  • A Structure-Based Mechanism for HEXIM Displacement from 7SK
    A structure-based mechanism for HEXIM displacement from 7SK Vincent Pham Harvard University Michael Gao Harvard University Jennifer Meagher University of Michigan Janet Smith University of Michigan https://orcid.org/0000-0002-0664-9228 Victoria D'Souza ( [email protected] ) Harvard University Article Keywords: Transcriptional Elongation, Conformational Plasticity, Remodeling Site, Local Destabilization, Competitive Regulation Posted Date: February 2nd, 2021 DOI: https://doi.org/10.21203/rs.3.rs-198962/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1 A structure-based mechanism for HEXIM displacement from 7SK 2 Vincent V. Pham1, Michael Gao1, Jennifer L. Meagher2, Janet L. Smith2,3 & Victoria M. 3 D’Souza1* 4 1Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, 5 02138, USA. 2Life Sciences Institute, University of Michigan, Ann Arbor, MI, 48109, USA. 6 3Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, 48109, 7 USA. *Correspondence and requests for materials should be addressed to V.M.D (email: 8 [email protected]). 9 10 Productive transcriptional elongation of many cellular and viral mRNAs requires 11 transcriptional factors to extract pTEFb from the 7SK snRNP by modulating the 12 association between the HEXIM protein and the 7SK snRNA. Here we report the 13 structure of the HEXIM arginine rich motif in complex with the apical stemloop-1 of 14 7SK (7SK-SL1apical) and detail how the HIV transcriptional regulator Tat from various 15 subtypes overcome the structural constraints required to displace HEXIM. While the 16 majority of interactions between 7SK and HEXIM and Tat are similar, critical 17 differences exist that guide function.
    [Show full text]
  • Datasheet: VPA00125KT Product Details
    Datasheet: VPA00125KT Description: HEXIM1 ANTIBODY WITH CONTROL LYSATE Specificity: HEXIM1 Format: Purified Product Type: PrecisionAb™ Polyclonal Isotype: Polyclonal IgG Quantity: 2 Westerns Product Details Applications This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit www.bio-rad-antibodies.com/protocols. Yes No Not Determined Suggested Dilution Western Blotting 1/1000 PrecisionAb antibodies have been extensively validated for the western blot application. The antibody has been validated at the suggested dilution. Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Further optimization may be required dependant on sample type. Target Species Human Species Cross Reacts with: Mouse, Rat Reactivity N.B. Antibody reactivity and working conditions may vary between species. Product Form Purified IgG - liquid Preparation 20μl Goat polyclonal antibody purified by affinity chromatography Buffer Solution TRIS buffered saline Preservative 0.02% Sodium Azide (NaN ) 0.5 % BSA Stabilisers 3 Immunogen Peptide with the sequence C-HRQQERAPLSKFGD, from the C Terminus of the protein sequence. External Database Links UniProt: O94992 Related reagents Entrez Gene: 10614 HEXIM1 Related reagents Synonyms CLP1, EDG1, HIS1, MAQ1 Page 1 of 2 Specificity Goat anti Human HEXIM1 antibody recognizes protein HEXIM1, also known as cardiac lineage protein 1, estrogen down-regulated gene 1 protein, hexamethylene bis-acetamide-inducible protein 1 or menage a quatre protein 1. Expression of HEXIM1 is induced by hexamethylene-bis-acetamide in vascular smooth muscle cells.
    [Show full text]
  • A Chromosome-Centric Human Proteome Project (C-HPP) To
    computational proteomics Laboratory for Computational Proteomics www.FenyoLab.org E-mail: [email protected] Facebook: NYUMC Computational Proteomics Laboratory Twitter: @CompProteomics Perspective pubs.acs.org/jpr A Chromosome-centric Human Proteome Project (C-HPP) to Characterize the Sets of Proteins Encoded in Chromosome 17 † ‡ § ∥ ‡ ⊥ Suli Liu, Hogune Im, Amos Bairoch, Massimo Cristofanilli, Rui Chen, Eric W. Deutsch, # ¶ △ ● § † Stephen Dalton, David Fenyo, Susan Fanayan,$ Chris Gates, , Pascale Gaudet, Marina Hincapie, ○ ■ △ ⬡ ‡ ⊥ ⬢ Samir Hanash, Hoguen Kim, Seul-Ki Jeong, Emma Lundberg, George Mias, Rajasree Menon, , ∥ □ △ # ⬡ ▲ † Zhaomei Mu, Edouard Nice, Young-Ki Paik, , Mathias Uhlen, Lance Wells, Shiaw-Lin Wu, † † † ‡ ⊥ ⬢ ⬡ Fangfei Yan, Fan Zhang, Yue Zhang, Michael Snyder, Gilbert S. Omenn, , Ronald C. Beavis, † # and William S. Hancock*, ,$, † Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States ‡ Stanford University, Palo Alto, California, United States § Swiss Institute of Bioinformatics (SIB) and University of Geneva, Geneva, Switzerland ∥ Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States ⊥ Institute for System Biology, Seattle, Washington, United States ¶ School of Medicine, New York University, New York, United States $Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia ○ MD Anderson Cancer Center, Houston, Texas, United States ■ Yonsei University College of Medicine, Yonsei University,
    [Show full text]
  • Anti-Cancer Drug HMBA Acts As an Adjuvant During Intracellular Bacterial Infections by Inducing Type I IFN Through STING
    Anti-Cancer Drug HMBA Acts as an Adjuvant during Intracellular Bacterial Infections by Inducing Type I IFN through STING This information is current as of September 26, 2021. Akshamal Mihiranga Gamage, Kok-Onn Lee and Yunn-Hwen Gan J Immunol 2017; 199:2491-2502; Prepublished online 21 August 2017; doi: 10.4049/jimmunol.1602162 Downloaded from http://www.jimmunol.org/content/199/7/2491 Supplementary http://www.jimmunol.org/content/suppl/2017/08/19/jimmunol.160216 http://www.jimmunol.org/ Material 2.DCSupplemental References This article cites 60 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/199/7/2491.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 26, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Anti-Cancer Drug HMBA Acts as an Adjuvant during Intracellular Bacterial Infections by Inducing Type I IFN through STING Akshamal Mihiranga Gamage,* Kok-Onn Lee,† and Yunn-Hwen Gan* The anti-proliferative agent hexamethylene bisacetamide (HMBA) belongs to a class of hybrid bipolar compounds developed more than 30 y ago for their ability to induce terminal differentiation of transformed cells.
    [Show full text]
  • Goat Anti-HEXIM1 Antibody Peptide-Affinity Purified Goat Antibody Catalog # Af1524a
    10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 Goat Anti-HEXIM1 Antibody Peptide-affinity purified goat antibody Catalog # AF1524a Specification Goat Anti-HEXIM1 Antibody - Product Information Application WB Primary Accession O94992 Other Accession NP_006451, 10614, 192231 (mouse) Reactivity Human Predicted Mouse Host Goat Clonality Polyclonal Concentration 100ug/200ul Isotype IgG Calculated MW 40623 Goat Anti-HEXIM1 Antibody - Additional Information AF1524a (0.1 µg/ml) staining of Human (A) Gene ID 10614 and Mouse (B) Heart lysates (35 µg protein in RIPA buffer). Primary incubation was 1 hour. Other Names Detected by chemiluminescence. Protein HEXIM1, Cardiac lineage protein 1, Estrogen down-regulated gene 1 protein, Hexamethylene bis-acetamide-inducible Goat Anti-HEXIM1 Antibody - Background protein 1, Menage a quatre protein 1, HEXIM1, CLP1, EDG1, HIS1, MAQ1 Expression of this gene is induced by hexamethylene-bis-acetamide in vascular Format smooth muscle cells. This gene has no introns. 0.5 mg IgG/ml in Tris saline (20mM Tris pH7.3, 150mM NaCl), 0.02% sodium azide, with 0.5% bovine serum albumin Goat Anti-HEXIM1 Antibody - References Storage HEXIM1 modulates vascular endothelial Maintain refrigerated at 2-8°C for up to 6 growth factor expression and function in breast months. For long term storage store at epithelial cells and mammary gland. Ogba N, -20°C in small aliquots to prevent et al. Oncogene, 2010 Jun 24. PMID 20453883. freeze-thaw cycles. A flexible bipartite coiled coil structure is required for the interaction of Hexim1 with the Precautions P-TEFB subunit cyclin T1.
    [Show full text]
  • Structure of the Cyclin T Binding Domain of Hexim1 and Molecular Basis for Its Recognition of P-Tefb
    Structure of the Cyclin T binding domain of Hexim1 and molecular basis for its recognition of P-TEFb Sonja A. Dames*†, Andre´ Scho¨ nichen‡, Antje Schulte‡, Matjaz Barboric§, B. Matija Peterlin§, Stephan Grzesiek*, and Matthias Geyer†‡ *Department of Structural Biology, Biozentrum Basel, University of Basel, 4003 Basel, Switzerland; ‡Abteilung Physikalische Biochemie, Max-Planck-Institut fu¨r molekulare Physiologie, 44227 Dortmund, Germany; and §Departments of Medicine, Microbiology, and Immunology, Rosalind Russell Medical Research Center, University of California, San Francisco, CA 94143 Edited by Ann E. McDermott, Columbia University, New York, NY, and approved July 11, 2007 (received for review March 1, 2007) Hexim1 is a cellular protein that associates with the positive tran- is suggested to have a self inhibitory function; a central nuclear scription elongation factor b (P-TEFb) to regulate RNA polymerase II localization signal (NLS, 150–177) that interacts with the nuclear elongation of nascent mRNA transcripts. It directly binds to Cyclin T1 transport machinery and directly binds to 7SK snRNA; a region of of P-TEFb and inhibits the kinase activity of Cdk9, leading to an arrest highest homology (185–220), including a negatively charged cluster of transcription elongation. Here, we report the solution structure of that might be involved in P-TEFb inhibition; and a C-terminal the Cyclin T binding domain (TBD) of Hexim1 that forms a parallel Cyclin T binding domain (TBD) (255–359) that leads to dimeriza- coiled-coil homodimer composed of two segments and a preceding tion of Hexim molecules (7–10, 15–18). The Cyclin T binding region alpha helix that folds back onto the first coiled-coil unit.
    [Show full text]
  • The Neurodegenerative Diseases ALS and SMA Are Linked at The
    Nucleic Acids Research, 2019 1 doi: 10.1093/nar/gky1093 The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex Downloaded from https://academic.oup.com/nar/advance-article-abstract/doi/10.1093/nar/gky1093/5162471 by [email protected] on 06 November 2018 Binkai Chi, Jeremy D. O’Connell, Alexander D. Iocolano, Jordan A. Coady, Yong Yu, Jaya Gangopadhyay, Steven P. Gygi and Robin Reed* Department of Cell Biology, Harvard Medical School, 240 Longwood Ave. Boston MA 02115, USA Received July 17, 2018; Revised October 16, 2018; Editorial Decision October 18, 2018; Accepted October 19, 2018 ABSTRACT Fused in Sarcoma (FUS) and TAR DNA Binding Protein (TARDBP) (9–13). FUS is one of the three members of Understanding the molecular pathways disrupted in the structurally related FET (FUS, EWSR1 and TAF15) motor neuron diseases is urgently needed. Here, we family of RNA/DNA binding proteins (14). In addition to employed CRISPR knockout (KO) to investigate the the RNA/DNA binding domains, the FET proteins also functions of four ALS-causative RNA/DNA binding contain low-complexity domains, and these domains are proteins (FUS, EWSR1, TAF15 and MATR3) within the thought to be involved in ALS pathogenesis (5,15). In light RNAP II/U1 snRNP machinery. We found that each of of the discovery that mutations in FUS are ALS-causative, these structurally related proteins has distinct roles several groups carried out studies to determine whether the with FUS KO resulting in loss of U1 snRNP and the other two members of the FET family, TATA-Box Bind- SMN complex, EWSR1 KO causing dissociation of ing Protein Associated Factor 15 (TAF15) and EWS RNA the tRNA ligase complex, and TAF15 KO resulting in Binding Protein 1 (EWSR1), have a role in ALS.
    [Show full text]
  • Structural Insights Into 7Sk Snrnp Complex and Its Implication for Hiv-1 Transcriptional Control
    STRUCTURAL INSIGHTS INTO 7SK SNRNP COMPLEX AND ITS IMPLICATION FOR HIV-1 TRANSCRIPTIONAL CONTROL by LE LUO Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor Blanton S. Tolbert, Ph.D. Department of Chemistry CASE WESTERN RESERVE UNIVERSITY January, 2019 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Le Luo candidate for the degree of Doctor of Philosophy. Committee Chair Mary Barkley, Ph.D. Committee Member Paul Carey, Ph.D. Committee Member Fu-Sen Liang, Ph.D. Committee Member Blanton S. Tolbert, Ph.D. Date of Defense November 26, 2018 *We also certify that written approval has been obtained for any proprietary material contained therein. Dedicated to my family and friends Table of Contents TITLE PAGE ........................................................................................................... COMMITTEE APPROVAL SHEET ......................................................................... DEDICATION .......................................................................................................... LIST OF FIGURES .............................................................................................. V ACKNOWLEDGEMENTS .................................................................................. VII LIST OF ABBREVIATIONS ................................................................................ IX ABSTRACT .........................................................................................................
    [Show full text]
  • Table S1. 103 Ferroptosis-Related Genes Retrieved from the Genecards
    Table S1. 103 ferroptosis-related genes retrieved from the GeneCards. Gene Symbol Description Category GPX4 Glutathione Peroxidase 4 Protein Coding AIFM2 Apoptosis Inducing Factor Mitochondria Associated 2 Protein Coding TP53 Tumor Protein P53 Protein Coding ACSL4 Acyl-CoA Synthetase Long Chain Family Member 4 Protein Coding SLC7A11 Solute Carrier Family 7 Member 11 Protein Coding VDAC2 Voltage Dependent Anion Channel 2 Protein Coding VDAC3 Voltage Dependent Anion Channel 3 Protein Coding ATG5 Autophagy Related 5 Protein Coding ATG7 Autophagy Related 7 Protein Coding NCOA4 Nuclear Receptor Coactivator 4 Protein Coding HMOX1 Heme Oxygenase 1 Protein Coding SLC3A2 Solute Carrier Family 3 Member 2 Protein Coding ALOX15 Arachidonate 15-Lipoxygenase Protein Coding BECN1 Beclin 1 Protein Coding PRKAA1 Protein Kinase AMP-Activated Catalytic Subunit Alpha 1 Protein Coding SAT1 Spermidine/Spermine N1-Acetyltransferase 1 Protein Coding NF2 Neurofibromin 2 Protein Coding YAP1 Yes1 Associated Transcriptional Regulator Protein Coding FTH1 Ferritin Heavy Chain 1 Protein Coding TF Transferrin Protein Coding TFRC Transferrin Receptor Protein Coding FTL Ferritin Light Chain Protein Coding CYBB Cytochrome B-245 Beta Chain Protein Coding GSS Glutathione Synthetase Protein Coding CP Ceruloplasmin Protein Coding PRNP Prion Protein Protein Coding SLC11A2 Solute Carrier Family 11 Member 2 Protein Coding SLC40A1 Solute Carrier Family 40 Member 1 Protein Coding STEAP3 STEAP3 Metalloreductase Protein Coding ACSL1 Acyl-CoA Synthetase Long Chain Family Member 1 Protein
    [Show full text]
  • Supplementary Data
    Supplementary information 1 Contents 1 Supplementary Methods 3 1.1 Prototype-based co-expression modules ...................... 3 1.2 Gene ontology and functional network analysis ................. 4 1.3 Survival meta-analysis ............................... 4 1.3.1 Univariate Cox regression ......................... 4 1.3.2 Variable selection for multivariate Cox regression ............ 5 2 Supplementary Figure 1 6 3 Supplementary Figure 2 7 4 Supplementary Figure 3 8 5 Supplementary Table 1 12 6 Supplementary Table 2 13 6.1 Funtional annotation of the gene expression modules .............. 13 7 Supplementary Table 3 14 8 Supplementary Table 4 16 9 Supplementary Table 5 17 10 Alternative computation of prognostic gene signatures 18 10.1 GENE70 in [van’t Veer et al., 2002] Dataset ................... 18 10.1.1 Score .................................... 18 10.1.2 Risk ..................................... 18 10.1.3 Stratified Survival Curves ......................... 19 10.2 GENE70 in [van de Vijver et al., 2002] Dataset ................. 19 10.2.1 Score .................................... 19 10.2.2 Risk ..................................... 19 10.2.3 Stratified Survival Curves ......................... 20 10.3 GENE76 in [Wang et al., 2005] .......................... 21 10.3.1 Score .................................... 21 10.3.2 Risk ..................................... 21 10.3.3 Stratified Survival Curves ......................... 21 10.4 GENE76 in [Foekens et al., 2006] Dataset .................... 21 10.4.1 Score ...................................
    [Show full text]