DOCSLIB.ORG

RUNX1 Is an Oncogenic Transcription Factor That Regulates MYB and MYC Enhancer Activity in T-ALL

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
RUNX1 Is an Oncogenic Transcription Factor That Regulates MYB and MYC Enhancer Activity in T-ALL University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2018-02-13 RUNX1 Is an Oncogenic Transcription Factor that Regulates MYB and MYC Enhancer Activity in T-ALL AHyun Choi University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Cancer Biology Commons Repository Citation Choi A. (2018). RUNX1 Is an Oncogenic Transcription Factor that Regulates MYB and MYC Enhancer Activity in T-ALL. GSBS Dissertations and Theses. https://doi.org/10.13028/M20D6J. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/957 Creative Commons License This work is licensed under a Creative Commons Attribution 4.0 License. This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. RUNX1 IS AN ONCOGENIC TRANSCRIPTION FACTOR THAT REGULATES MYB AND MYC ENHANCER ACTIVITY IN T-ALL A Dissertation Presented By AHYUN CHOI Submitted to the Faculties of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY FEBRUARY 13, 2018 CANCER BIOLOGY ii RUNX1 IS AN ONCOGENIC TRANSCRIPTION FACTOR THAT REGULATES MYB AND MYC ENHANCER ACTIVITY IN T-ALL A Dissertation Presented By AHYUN CHOI This work was undertaken in the Graduate School of Biomedical Sciences Cancer Biology Under the mentorship of Michelle Kelliher, Ph.D., Thesis Advisor The signatures of the Dissertation Defense Committee signify completion and approval as to style and content of the Dissertation Lucio H. Castilla, Ph.D., Member of Committee Glen Raffel, MD., Ph.D., Member of Committee Thomas Fazzio, Ph.D., Member of Committee Alan B. Cantor, M.D., Ph.D., External Member of Committee Merav Socolovsky, MBBS., Ph.D., Chair of Committee Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences February 13, 2018 iii ACKNOWLEDGEMENTS These works could not be done without support and help from lots of people. First of all, I would like to thank my mentor, Dr. Michelle Kelliher, for her support and guidance. I have learned from her a way of thinking and handling the science. I also thank the lab member of the Kelliher lab, past and present, for their help and making the enjoyable lab life. Their advice and achievement have inspired me and kept me moving forward. I appreciate the advice and support from my committees, Dr. Lucio Castilla, Dr. Merav Socolovsky, Dr. Glen Raffel, and Dr. Thomas Fazzio. Additionally, I thank Dr. Alan Cantor for serving as an external examiner of my dissertation defense. I also appreciate Dr. John Pulikkan in the Castilla's lab for his friendship and guidance. I would like to thank my friends and family. Their support and advice help me going through this long journey. I also really enjoyed the adventures I have had with friends for years. Lastly, I cannot appreciate enough the understanding and support of my parents and husband, Hwanjong. They make me not to get lost, follow what I want, and smile every day. Thank you. iv ABSTRACT RUNX1, a transcription factor required for hematopoiesis and lymphocyte differentiation, is one of the most commonly targeted genes in hematopoietic malignancies. Mutations in the RUNX1 gene are associated with a poor prognosis in a subset of T cell acute lymphoblastic leukemia (T-ALL) and RUNX1 has been proposed as a tumor suppressor in TLX1/3-transformed human T-ALL. Recent ChIP-seq studies in human T-ALL cell lines demonstrated that a large portion of TAL1- and NOTCH1- bound regions contain RUNX binding sites in promoter or enhancer regions, suggesting oncogenic roles for RUNX1 in T-ALL. To interrogate RUNX1 functions in leukemogenesis, we depleted RUNX1 in a T-ALL mouse model and in human T-ALL cell lines. We found that RUNX1 is required for the maintenance of mouse T-ALL growth in vivo and the survival of human T-ALL cell lines in vitro. In addition, inhibition of the RUNX1 activity with a small molecule inhibitor impairs the growth of human T-ALL cell lines and primary patient samples. RUNX1 depletion reduces the expression of a subset of TAL1- and NOTCH1- regulated genes including the MYB and MYC oncogenes, respectively. We demonstrate that RUNX1 regulates transcription factor binding and acetylation of H3K27 at the Myb and Myc enhancer loci. These studies provide genetic and pharmacological evidences that RUNX1 supports T-ALL cell survival and suggest RUNX1 inhibitor as a therapeutic strategy in T-ALL treatment. v TABLE OF CONTENTS Signature Page ii Acknowledgements iii Abstract iv Table of Contents v List of Tables vii List of Figures viii Copyrighted Materials Produced by the Author xi Chapter I: Introduction 1 Chapter II: RUNX1/ and/or RUNX3 is required for T-ALL survival 50 Introduction 51 Results 52 Discussion 56 Methods and Materials 58 Figures 62 Chapter III: RUNX1 supports T-ALL survival by regulating Myb and Myc enhancer activity 76 Introduction 77 Results 78 Discussion 84 Methods and Materials 87 Figures 90 Chapter IV: Discussion 100 Appendix 122 vi Appendix I Evaluation of the efficacy of RUNX/CBFb inhibitor AI-12-126 on T-ALL progression in vivo 123 Appendix II RUNX1 regulates DNA accessibility in mouse T-ALL cells 128 Appendix III Repression of mTORC1 activity sensitizes T-ALL cells to ABT-263 treatment 139 Appendix IV Attempts to identify a compound that selectively kills ETP-ALL cells 148 Appendix V Identification of tumor suppressor(s) in chromosome 6q deleted region that cooperate with TAL1 to cause T-ALL 157 List of Primers 160 Reference 161 vii List of Tables Chapter II Table 2.1. Expression and mutation status in patient samples and human T- ALL cell lines. Table 2.2. Human T-ALL cell lines and primary patient samples are sensitive to AI-10-104 treatment. Table 2.3. shRNA clones used in the study Appendix Table A.II.1. Gene lists of functional annotations analyses Table A.IV.1. The list of compounds selected for the secondary screening Table A.IV.2. The list of 6 compounds identified in the secondary screening viii List of Figures Chapter I Figure 1.1. Thymocyte development Figure 1.2. The structure of RUNX proteins Chapter II Figure 2.1. RUNX1 is required for the maintenance of leukemic growth in vivo Figure 2.2. RUNX1 supports survival of mouse leukemic cells in vitro Figure 2.3. Cre activation has no significant effects on mouse T-ALL growth in vitro and RUNX3 protein expression in mouse leukemic cells Figure 2.4. RUNX1, but not RUNX3, is ubiquitously expressed in human T-ALL cell lines and primary patients T-ALL samples Figure 2.5. Knockdown of RUNX1, RUNX3, and CBFb results in apoptosis Figure 2.6. Treatment with a RUNX-CBFβ inhibitor impairs the growth of human T-ALL cell lines and primary pediatric T-ALL samples Figure 2.7. RUNX1/3-CBFb inhibitor is not detrimental to normal human hematopoietic stem and progenitor cells at low concentration Figure 2.8. Reduction of RUNX1 expression in TLX3-transformed T-ALL cell line induces cell apoptosis Chapter III Figure 3.1. RUNX1 regulates a subset of TAL1-, and NOTCH1-regulated genes ix Figure 3.2. RUNX1 knockdown in human T-ALL cell lines alters target the expression of target genes the expression of a subset of RUNX1-, TAL1-, and NOTCH1-regulated genes Figure 3.3. RUNX1 depletion derepresses CD4 cell surface expression on T- ALL cells Figure 3.4. RUNX1 is required for TAL1 binding to the Myb enhancers and for the retention of active chromatin marks Figure 3.5. RUNX1 is required for intracellular NOTCH1 binding and for chromatin accessibility at the N-Me Figure 3.6. Runx1 depletion has no effect on intracellular NOTCH1 binding to the Hes1 promoter or to gene desert regions Figure 3.7. RUNX1/3 binding to the oncogenic enhancers reflects their regulation on gene expression Figure 3.8. The reduced expression of RUNX1 or RUNX3 does not lead to increased expression of the other Figure 3.9. RUNX1 reduction interferes with the formation of loop between the MYB promoter and -94-kb and +14-kb enhancers Chapter VI Figure 4.1. Proposed model Appendix Figure A.I.1. AI-12-126 treatment does not suppress leukemia progression in mice transplanted with primary patient T-ALL sample x Figure A.I.2. Treatment of AI-12-126 does not inhibit leukemic growth in vivo Figure A.I.3. AI-12-126 treatment inhibits the growth of human T-ALL cell lines Figure A.II.1. Signals of ATAC-seq in Runx1 deleted mouse T-ALL cells and over-representative pathways Figure AII.2. The accessibility to the Myb and Myc enhancer regions is increased in Runx1-deleted cells. Figure A.III.1. Experimental strategy of used to determine the efficacy of combination treatment of AZD8055 and ATB-263 on leukemia progression in vivo Figure A.III.2. Treatment of AZD8055 and ABT-263 prolong survival of mice transplanted with primary T-ALL patient samples Figure A.III.3. Combination treatment of AZD8055 and ABT-263 inhibits leukemic burden in vivo Figure A.III.4. The in vivo growth of TALL-X-7 primary T-ALL patient sample is suppressed by treatment with ABT-263 Figure A.III.5. The expression of intracellular NOTCH1 and MCL-1 in primary T-ALL patient samples Figure A.IV.1. Secondary screening identifies compounds that are selectively effective against the Loucy ETP-ALL cell line Figure A.IV.2.
Load more

Recommended publications
  • Supplementary Data
    Figure 2S 4 7 A - C 080125 CSCs 080418 CSCs - + IFN-a 48 h + IFN-a 48 h + IFN-a 72 h 6 + IFN-a 72 h 3 5 MRFI 4 2 3 2 1 1 0 0 MHC I MHC II MICA MICB ULBP-1 ULBP-2 ULBP-3 ULBP-4 MHC I MHC II MICA MICB ULBP-1 ULBP-2 ULBP-3 ULBP-4 7 B 13 080125 FBS - D 080418 FBS - + IFN-a 48 h 12 + IFN-a 48 h + IFN-a 72 h + IFN-a 72 h 6 080125 FBS 11 10 5 9 8 4 7 6 3 MRFI 5 4 2 3 2 1 1 0 0 MHC I MHC II MICA MICB ULBP-1 ULBP-2 ULBP-3 ULBP-4 MHC I MHC II MICA MICB ULBP-1 ULBP-2 ULBP-3 ULBP-4 Molecule Molecule FIGURE 4S FIGURE 5S Panel A Panel B FIGURE 6S A B C D Supplemental Results Table 1S. Modulation by IFN-α of APM in GBM CSC and FBS tumor cell lines. Molecule * Cell line IFN-α‡ HLA β2-m# HLA LMP TAP1 TAP2 class II A A HC§ 2 7 10 080125 CSCs - 1∞ (1) 3 (65) 2 (91) 1 (2) 6 (47) 2 (61) 1 (3) 1 (2) 1 (3) + 2 (81) 11 (80) 13 (99) 1 (3) 8 (88) 4 (91) 1 (2) 1 (3) 2 (68) 080125 FBS - 2 (81) 4 (63) 4 (83) 1 (3) 6 (80) 3 (67) 2 (86) 1 (3) 2 (75) + 2 (99) 14 (90) 7 (97) 5 (75) 7 (100) 6 (98) 2 (90) 1 (4) 3 (87) 080418 CSCs - 2 (51) 1 (1) 1 (3) 2 (47) 2 (83) 2 (54) 1 (4) 1 (2) 1 (3) + 2 (81) 3 (76) 5 (75) 2 (50) 2 (83) 3 (71) 1 (3) 2 (87) 1 (2) 080418 FBS - 1 (3) 3 (70) 2 (88) 1 (4) 3 (87) 2 (76) 1 (3) 1 (3) 1 (2) + 2 (78) 7 (98) 5 (99) 2 (94) 5 (100) 3 (100) 1 (4) 2 (100) 1 (2) 070104 CSCs - 1 (2) 1 (3) 1 (3) 2 (78) 1 (3) 1 (2) 1 (3) 1 (3) 1 (2) + 2 (98) 8 (100) 10 (88) 4 (89) 3 (98) 3 (94) 1 (4) 2 (86) 2 (79) * expression of APM molecules was evaluated by intracellular staining and cytofluorimetric analysis; ‡ cells were treatead or not (+/-) for 72 h with 1000 IU/ml of IFN-α; # β-2 microglobulin; § β-2 microglobulin-free HLA-A heavy chain; ∞ values are indicated as ratio between the mean of fluorescence intensity of cells stained with the selected mAb and that of the negative control; bold values indicate significant MRFI (≥ 2).
    [Show full text]
  • Thesis Template
    Functional and Structural Characterization Reveals Novel FBXW7 Biology by Tonny Chao Huang A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Medical Biophysics University of Toronto © Copyright by Tonny Chao Huang 2018 Functional and Structural Characterization Reveals Novel FBXW7 Biology Tonny Chao Huang Master of Science Department of Medical Biophysics University of Toronto 2018 Abstract This thesis aims to examine aspects of FBXW7 biology, a protein that is frequently mutated in a variety of cancers. The first part of this thesis describes the characterization of FBXW7 isoform and mutant substrate profiles using a proximity-dependent biotinylation assay. Isoform-specific substrates were validated, revealing the involvement of FBXW7 in the regulation of several protein complexes. Characterization of FBXW7 mutants also revealed site- and residue-specific consequences on the binding of substrates and, surprisingly, possible neo-substrates. In the second part of this thesis, we utilize high-throughput peptide binding assays and statistical modelling to discover novel features of the FBXW7-binding phosphodegron. In contrast to the canonical motif, a possible preference of FBXW7 for arginine residues at the +4 position was discovered. I then attempted to validate this feature in vivo and in vitro on a novel substrate discovered through BioID. ii Acknowledgments The past three years in the Department of Medical Biophysics have defied expectations. I not only had the opportunity to conduct my own independent research, but also to work with distinguished collaborators and to explore exciting complementary fields. I experienced the freedom to guide my own academic development, as well as to pursue my extracurricular interests.
    [Show full text]
  • Functions of the Mineralocorticoid Receptor in the Hippocampus By
    Functions of the Mineralocorticoid Receptor in the Hippocampus by Aaron M. Rozeboom A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cellular and Molecular Biology) in The University of Michigan 2008 Doctoral Committee: Professor Audrey F. Seasholtz, Chair Professor Elizabeth A. Young Professor Ronald Jay Koenig Associate Professor Gary D. Hammer Assistant Professor Jorge A. Iniguez-Lluhi Acknowledgements There are more people than I can possibly name here that I need to thank who have helped me throughout the process of writing this thesis. The first and foremost person on this list is my mentor, Audrey Seasholtz. Between working in her laboratory as a research assistant and continuing my training as a graduate student, I spent 9 years in Audrey’s laboratory and it would be no exaggeration to say that almost everything I have learned regarding scientific research has come from her. Audrey’s boundless enthusiasm, great patience, and eager desire to teach students has made my time in her laboratory a richly rewarding experience. I cannot speak of Audrey’s laboratory without also including all the past and present members, many of whom were/are not just lab-mates but also good friends. I also need to thank all the members of my committee, an amazing group of people whose scientific prowess combined with their open-mindedness allowed me to explore a wide variety of interests while maintaining intense scientific rigor. Outside of Audrey’s laboratory, there have been many people in Ann Arbor without whom I would most assuredly have gone crazy.
    [Show full text]
  • Supplementary Materials: Evaluation of Cytotoxicity and Α-Glucosidase Inhibitory Activity of Amide and Polyamino-Derivatives of Lupane Triterpenoids
    Supplementary Materials: Evaluation of cytotoxicity and α-glucosidase inhibitory activity of amide and polyamino-derivatives of lupane triterpenoids Oxana B. Kazakova1*, Gul'nara V. Giniyatullina1, Akhat G. Mustafin1, Denis A. Babkov2, Elena V. Sokolova2, Alexander A. Spasov2* 1Ufa Institute of Chemistry of the Ufa Federal Research Centre of the Russian Academy of Sciences, 71, pr. Oktyabrya, 450054 Ufa, Russian Federation 2Scientific Center for Innovative Drugs, Volgograd State Medical University, Novorossiyskaya st. 39, Volgograd 400087, Russian Federation Correspondence Prof. Dr. Oxana B. Kazakova Ufa Institute of Chemistry of the Ufa Federal Research Centre of the Russian Academy of Sciences 71 Prospeсt Oktyabrya Ufa, 450054 Russian Federation E-mail: [email protected] Prof. Dr. Alexander A. Spasov Scientific Center for Innovative Drugs of the Volgograd State Medical University 39 Novorossiyskaya st. Volgograd, 400087 Russian Federation E-mail: [email protected] Figure S1. 1H and 13C of compound 2. H NH N H O H O H 2 2 Figure S2. 1H and 13C of compound 4. NH2 O H O H CH3 O O H H3C O H 4 3 Figure S3. Anticancer screening data of compound 2 at single dose assay 4 Figure S4. Anticancer screening data of compound 7 at single dose assay 5 Figure S5. Anticancer screening data of compound 8 at single dose assay 6 Figure S6. Anticancer screening data of compound 9 at single dose assay 7 Figure S7. Anticancer screening data of compound 12 at single dose assay 8 Figure S8. Anticancer screening data of compound 13 at single dose assay 9 Figure S9. Anticancer screening data of compound 14 at single dose assay 10 Figure S10.
    [Show full text]
  • Role of Mitochondrial Ribosomal Protein S18-2 in Cancerogenesis and in Regulation of Stemness and Differentiation
    From THE DEPARTMENT OF MICROBIOLOGY TUMOR AND CELL BIOLOGY (MTC) Karolinska Institutet, Stockholm, Sweden ROLE OF MITOCHONDRIAL RIBOSOMAL PROTEIN S18-2 IN CANCEROGENESIS AND IN REGULATION OF STEMNESS AND DIFFERENTIATION Muhammad Mushtaq Stockholm 2017 All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by E-Print AB 2017 © Muhammad Mushtaq, 2017 ISBN 978-91-7676-697-2 Role of Mitochondrial Ribosomal Protein S18-2 in Cancerogenesis and in Regulation of Stemness and Differentiation THESIS FOR DOCTORAL DEGREE (Ph.D.) By Muhammad Mushtaq Principal Supervisor: Faculty Opponent: Associate Professor Elena Kashuba Professor Pramod Kumar Srivastava Karolinska Institutet University of Connecticut Department of Microbiology Tumor and Cell Center for Immunotherapy of Cancer and Biology (MTC) Infectious Diseases Co-supervisor(s): Examination Board: Professor Sonia Lain Professor Ola Söderberg Karolinska Institutet Uppsala University Department of Microbiology Tumor and Cell Department of Immunology, Genetics and Biology (MTC) Pathology (IGP) Professor George Klein Professor Boris Zhivotovsky Karolinska Institutet Karolinska Institutet Department of Microbiology Tumor and Cell Institute of Environmental Medicine (IMM) Biology (MTC) Professor Lars-Gunnar Larsson Karolinska Institutet Department of Microbiology Tumor and Cell Biology (MTC) Dedicated to my parents ABSTRACT Mitochondria carry their own ribosomes (mitoribosomes) for the translation of mRNA encoded by mitochondrial DNA. The architecture of mitoribosomes is mainly composed of mitochondrial ribosomal proteins (MRPs), which are encoded by nuclear genomic DNA. Emerging experimental evidences reveal that several MRPs are multifunctional and they exhibit important extra-mitochondrial functions, such as involvement in apoptosis, protein biosynthesis and signal transduction. Dysregulations of the MRPs are associated with severe pathological conditions, including cancer.
    [Show full text]
  • Variations in Microrna-25 Expression Influence the Severity of Diabetic
    BASIC RESEARCH www.jasn.org Variations in MicroRNA-25 Expression Influence the Severity of Diabetic Kidney Disease † † † Yunshuang Liu,* Hongzhi Li,* Jieting Liu,* Pengfei Han, Xuefeng Li, He Bai,* Chunlei Zhang,* Xuelian Sun,* Yanjie Teng,* Yufei Zhang,* Xiaohuan Yuan,* Yanhui Chu,* and Binghai Zhao* *Heilongjiang Key Laboratory of Anti-Fibrosis Biotherapy, Medical Research Center, Heilongjiang, People’s Republic of China; and †Clinical Laboratory of Hong Qi Hospital, Mudanjiang Medical University, Heilongjiang, People’s Republic of China ABSTRACT Diabetic nephropathy is characterized by persistent albuminuria, progressive decline in GFR, and second- ary hypertension. MicroRNAs are dysregulated in diabetic nephropathy, but identification of the specific microRNAs involved remains incomplete. Here, we show that the peripheral blood from patients with diabetes and the kidneys of animals with type 1 or 2 diabetes have low levels of microRNA-25 (miR-25) compared with those of their nondiabetic counterparts. Furthermore, treatment with high glucose decreased the expression of miR-25 in cultured kidney cells. In db/db mice, systemic administration of an miR-25 agomir repressed glomerular fibrosis and reduced high BP. Notably, knockdown of miR-25 in normal mice by systemic administration of an miR-25 antagomir resulted in increased proteinuria, extracellular matrix accumulation, podocyte foot process effacement, and hypertension with renin-angiotensin system activation. However, excessive miR-25 did not cause kidney dysfunction in wild-type mice. RNA sequencing showed the alteration of miR-25 target genes in antagomir-treated mice, including the Ras-related gene CDC42. In vitro,cotrans- fection with the miR-25 antagomir repressed luciferase activity from a reporter construct containing the CDC42 39 untranslated region.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Inactivation of Fbxw7 Impairs Dsrna Sensing and Confers Resistance to PD-1 Blockade
    Published OnlineFirst May 5, 2020; DOI: 10.1158/2159-8290.CD-19-1416 RESEARCH ARTICLE Inactivation of Fbxw7 Impairs dsRNA Sensing and Confers Resistance to PD-1 Blockade Cécile Gstalder1,2, David Liu1,3, Diana Miao1, Bart Lutterbach1,2, Alexander L. DeVine1,2, Chenyu Lin4, Megha Shettigar1,2, Priya Pancholi1,2, Elizabeth I. Buchbinder1, Scott L. Carter5,6, Michael P. Manos1, Vanesa Rojas-Rudilla7, Ryan Brennick1, Evisa Gjini8, Pei-Hsuan Chen8, Ana Lako8, Scott Rodig8,9, Charles H. Yoon10, Gordon J. Freeman1, David A. Barbie1, F. Stephen Hodi1, Wayne Miles4, Eliezer M. Van Allen1, and Rizwan Haq1,2 Downloaded from cancerdiscovery.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst May 5, 2020; DOI: 10.1158/2159-8290.CD-19-1416 ABSTRACT The molecular mechanisms leading to resistance to PD-1 blockade are largely unknown. Here, we characterize tumor biopsies from a patient with melanoma who displayed heterogeneous responses to anti–PD-1 therapy. We observe that a resistant tumor exhibited a loss-of-function mutation in the tumor suppressor gene FBXW7, whereas a sensitive tumor from the same patient did not. Consistent with a functional role in immunotherapy response, inactivation of Fbxw7 in murine tumor cell lines caused resistance to anti–PD-1 in immunocompetent animals. Loss of Fbxw7 was associated with altered immune microenvironment, decreased tumor-intrinsic expression of the double-stranded RNA (dsRNA) sensors MDA5 and RIG1, and diminished induction of type I IFN and MHC-I expression. In contrast, restoration of dsRNA sensing in Fbxw7-deficient cells was suffi- cient to sensitize them to anti–PD-1.
    [Show full text]
  • Dimerization Specificity of Myogenic Helix-Loop-Helix DNA-Binding Factors Directed by Nonconserved Hydrophilic Residues
    Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Dimerization specificity of myogenic helix-loop-helix DNA-binding factors directed by nonconserved hydrophilic residues Masaki Shirakata, Fred K. Friedman, 1 Qin Wei, and Bruce M. Paterson Laboratory of Biochemistry, ~Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 USA The myogenic regulatory factor MyoD dimerizes with other positive and negative regulatory factors through a conserved region called the helix-loop-helix (HLH) domain. Using a non-DNA-binding MyoD mutant with a normal HLH domain as a dimerization competitor in gel mobility shift assays in conjunction with various MyoD HLH mutants, nonhydrophobic amino acids were identified in the HLH domain that contribute to dimerization specificity with El2. The assay detected subtle differences in dimerization activity among the mutant MyoD proteins that correlated with their ability to activate transcription in vivo, but this correlation was not apparent in the absence of competitor. The identification of such nonhydrophobic residues enabled us to predict the differences in dimerization affinity among the four vertebrate myogenic factors with El2. The experiments confirmed the prediction. Furthermore, a high-affinity homodimerizing analog of MyoD was designed by a single substitution at one of these residue positions. These experimental results were strengthened when they were analyzed in terms of the crystal structure for the Max bHLHZip domain homodimer. This analysis has allowed us to identify those residues that form charged residue pairs between the two HLH domains of MyoD and El2 and determine the dimerization specificity of the bHLH proteins.
    [Show full text]
  • RING1 Antibody (N-Term) Affinity Purified Rabbit Polyclonal Antibody (Pab) Catalog # AP14560A
    10320 Camino Santa Fe, Suite G San Diego, CA 92121 Tel: 858.875.1900 Fax: 858.622.0609 RING1 Antibody (N-term) Affinity Purified Rabbit Polyclonal Antibody (Pab) Catalog # AP14560A Specification RING1 Antibody (N-term) - Product Information Application WB,E Primary Accession Q06587 Other Accession Q6MGB6, O35730, NP_002922.2 Reactivity Human Predicted Mouse, Rat Host Rabbit Clonality Polyclonal Isotype Rabbit Ig Antigen Region 95-123 RING1 Antibody (N-term) - Additional Information RING1 Antibody (N-term) (Cat. #AP14560a) Gene ID 6015 western blot analysis in MDA-MB453 cell line lysates (35ug/lane).This demonstrates the Other Names RING1 antibody detected the RING1 protein E3 ubiquitin-protein ligase RING1, 632-, (arrow). Polycomb complex protein RING1, RING finger protein 1, Really interesting new gene 1 protein, RING1, RNF1 RING1 Antibody (N-term) - Background Target/Specificity This gene belongs to the RING finger family, This RING1 antibody is generated from members of rabbits immunized with a KLH conjugated synthetic peptide between 95-123 amino which encode proteins characterized by a RING acids from the N-terminal region of human domain, a RING1. zinc-binding motif related to the zinc finger domain. The gene Dilution product can bind DNA and can act as a WB~~1:1000 transcriptional repressor. It is associated with the multimeric polycomb Format group protein complex. Purified polyclonal antibody supplied in PBS The gene product interacts with the polycomb with 0.09% (W/V) sodium azide. This group proteins BMI1, antibody is purified through a protein A EDR1, and CBX4, and colocalizes with these column, followed by peptide affinity proteins in large purification.
    [Show full text]
  • Clinical Utility of Recently Identified Diagnostic, Prognostic, And
    Modern Pathology (2017) 30, 1338–1366 1338 © 2017 USCAP, Inc All rights reserved 0893-3952/17 $32.00 Clinical utility of recently identified diagnostic, prognostic, and predictive molecular biomarkers in mature B-cell neoplasms Arantza Onaindia1, L Jeffrey Medeiros2 and Keyur P Patel2 1Instituto de Investigacion Marques de Valdecilla (IDIVAL)/Hospital Universitario Marques de Valdecilla, Santander, Spain and 2Department of Hematopathology, MD Anderson Cancer Center, Houston, TX, USA Genomic profiling studies have provided new insights into the pathogenesis of mature B-cell neoplasms and have identified markers with prognostic impact. Recurrent mutations in tumor-suppressor genes (TP53, BIRC3, ATM), and common signaling pathways, such as the B-cell receptor (CD79A, CD79B, CARD11, TCF3, ID3), Toll- like receptor (MYD88), NOTCH (NOTCH1/2), nuclear factor-κB, and mitogen activated kinase signaling, have been identified in B-cell neoplasms. Chronic lymphocytic leukemia/small lymphocytic lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, Burkitt lymphoma, Waldenström macroglobulinemia, hairy cell leukemia, and marginal zone lymphomas of splenic, nodal, and extranodal types represent examples of B-cell neoplasms in which novel molecular biomarkers have been discovered in recent years. In addition, ongoing retrospective correlative and prospective outcome studies have resulted in an enhanced understanding of the clinical utility of novel biomarkers. This progress is reflected in the 2016 update of the World Health Organization classification of lymphoid neoplasms, which lists as many as 41 mature B-cell neoplasms (including provisional categories). Consequently, molecular genetic studies are increasingly being applied for the clinical workup of many of these neoplasms. In this review, we focus on the diagnostic, prognostic, and/or therapeutic utility of molecular biomarkers in mature B-cell neoplasms.
    [Show full text]
  • UNIVERSITY of CALIFORNIA, IRVINE Combinatorial Regulation By
    UNIVERSITY OF CALIFORNIA, IRVINE Combinatorial regulation by maternal transcription factors during activation of the endoderm gene regulatory network DISSERTATION submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biological Sciences by Kitt D. Paraiso Dissertation Committee: Professor Ken W.Y. Cho, Chair Associate Professor Olivier Cinquin Professor Thomas Schilling 2018 Chapter 4 © 2017 Elsevier Ltd. © 2018 Kitt D. Paraiso DEDICATION To the incredibly intelligent and talented people, who in one way or another, helped complete this thesis. ii TABLE OF CONTENTS Page LIST OF FIGURES vii LIST OF TABLES ix LIST OF ABBREVIATIONS X ACKNOWLEDGEMENTS xi CURRICULUM VITAE xii ABSTRACT OF THE DISSERTATION xiv CHAPTER 1: Maternal transcription factors during early endoderm formation in 1 Xenopus Transcription factors co-regulate in a cell type-specific manner 2 Otx1 is expressed in a variety of cell lineages 4 Maternal otx1 in the endodermal conteXt 5 Establishment of enhancers by maternal transcription factors 9 Uncovering the endodermal gene regulatory network 12 Zygotic genome activation and temporal control of gene eXpression 14 The role of maternal transcription factors in early development 18 References 19 CHAPTER 2: Assembly of maternal transcription factors initiates the emergence 26 of tissue-specific zygotic cis-regulatory regions Introduction 28 Identification of maternal vegetally-localized transcription factors 31 Vegt and OtX1 combinatorially regulate the endodermal 33 transcriptome iii
    [Show full text]