Novel Molecular Insights And Targeted Therapies In T-cell .
Novel molecular insights and targeted therapiesin T-cell acute lymphoblastic leukemiaSofie PeirsPromotors: Prof. Dr. Bruce Poppe, Prof. Dr. Pieter Van VlierbergheSubmitted to the Faculty of Medicine and Health Sciences of Ghent University, in fulfilment of therequirements for the degree of Doctor in Health SciencesAcademic year: 2016-2017
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Thesis submitted to fulfill the requirements for the degree of Doctor in Health SciencesPromotors:prof. dr. Bruce PoppeGhent University, Belgiumprof. dr. Pieter Van VlierbergheGhent University, BelgiumMembers of the examination committee:prof. dr. Triona Ni ChonghaileRoyal College of Surgeons in Ireland, Irelanddr. Charles de BockKULeuven, Belgiumprof. dr. Nadine Van RoyGhent University, Belgiumprof. dr. Katleen De PreterGhent University, Belgiumprof. dr. Jan PhilippéGhent University, Belgiumdr. Bram De WildeGhent University, Belgiumiii
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De auteur en de promotoren geven de toelating deze scriptie voor consultatie beschikbaar testellen en delen ervan te kopiëren voor persoonlijk gebruik. Elk ander gebruik valt onder debeperkingen van het auteursrecht, in het bijzonder met betrekking tot de verplichtinguitdrukkelijk de bron te vermelden bij het aanhalen van resultaten uit deze scriptie.The author and the promotors give the permission to use this thesis for consultation and tocopy parts of it for personal use. Every other use is subject to the copyright law, morespecifically the source must be extensively specified when using results from this thesis.The research described in this thesis was conducted at the Center for Medical Genetics,Ghent University, Ghent, Belgium.This work was supported by the Fund for Scientific Research (FWO) Flanders (PhD grant toSofie Peirs), Stand Up To Cancer (KOTK; PhD grant to Sofie Peirs) and project fundingobtained from the Foundation Against Cancer (STK), the Children Cancer Fund Ghent(Kinderkankerfonds VZW), the Swiss Bridge Foundation and Worldwide Cancer Research.v
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Table of ContentsList of abbreviations . ixCHAPTER 1 Introduction . 1Outline of the introduction . 3PART I Description and contemporary treatment of T-cell acute lymphoblastic leukemia . 41.What is T-cell acute lymphoblastic leukemia (T-ALL)? . 42.Incidence. 63.T-ALL subgroups . 64.Treatment. 85.Prognosis .11PART II The genetic and epigenetic landscape of T-ALL .121.T-ALL development is a multistep process .122.Genetic alterations and opportunities for targeted therapies in T-ALL .123.2.1.Overview of genetic alterations in T-ALL .122.2.Opportunities to target genetic alterations in T-ALL .142.3.The transcription factor ZEB2 as an oncogene in early immature T-ALL .15Epigenetic alterations and opportunities for targeted therapies in T-ALL .173.1.Overview of epigenetic alterations in T-ALL .173.2.Opportunities to target epigenetic alterations in T-ALL .223.3.Inhibition of BRD4 to repress the transcription of oncogenes .223.4. Targeting the lysine demethylase KDM1A as a therapeutic strategy in manycancers .25PART III Targeting apoptosis in cancer .301.Apoptosis: programmed cell death .302.Intrinsic pathway of apoptosis .312.1.BCL-2 family of proteins governs the intrinsic pathway of apoptosis.312.2.BH3 profiling .332.3.Chemotherapy and apoptosis.342.4.Pathways that contribute to the evasion of apoptosis in T-ALL .343.Extrinsic pathway of apoptosis .394.Strategies to target apoptosis pathways in cancer .404.1.Targeting the anti-apoptotic BCL-2 family.404.2.SMAC mimetics.444.3.Recombinant TRAIL and TRAIL receptor agonists .45References .46vii
CHAPTER 2 Research objectives .61CHAPTER 3 Results .65PART I The BCL-2 inhibitor ABT-199 as monotherapy and part of combination therapiesfor the treatment of T-ALL .67Paper 1: ABT-199 mediated inhibition of BCL-2 as a novel therapeutic strategy in T-cellacute lymphoblastic leukemia .69Paper 2: Targeting BET proteins improves the therapeutic efficacy of BCL-2 inhibition inT-cell acute lymphoblastic leukemia .96PART II Pharmacological inhibition of LSD1/KDM1A for the treatment of T-ALL .131Paper 3: Oncogenic ZEB2 activation drives sensitivity towards KDM1A inhibition in T-cellacute lymphoblastic leukemia .133CHAPTER 4 Discussion and Future Perspectives .1591. The BCL-2 inhibitor ABT-199 as monotherapy and part of combination therapies for thetreatment of T-ALL .1611.1. How to select T-ALL patients that may benefit from ABT-199 treatment .1611.2. Impact of ABT-199 on healthy blood cells .1621.3. Acquired resistance to ABT-199 and how to overcome it .1621.4. Combination therapies with ABT-199 in T-ALL .1652. Pharmacological inhibition of LSD1/KDM1A for the treatment of T-ALL .1662.1. Limited in vivo activity of GSK2879552 in T-ALL xenograft models .1662.2. Combinations with KDM1A inhibitor that are worthwhile to test in the context of TALL.1672.3. Understanding sensitivity towards KDM1A inhibition in ZEB2-negative T-ALL .1673. General conclusions .169References .170CHAPTER 5 Summary .173Summary .175Samenvatting .177CHAPTER 6 Curriculum Vitae & Word of thanks .179Curriculum Vitae Sofie Peirs .181Dankwoord (word of thanks) .185viii
List of CREBCREBBPCSCCtBPcTECCyt cAbsence of biallelic TCRγ locus deletionAcute lymphoblastic leukemiaAcute myeloid leukemiaAmine oxidase domainApoptotic protease-activating factor 1Androgen receptorAntisense oligonucleotideAll-trans-retinoic acidArea under the curveAdolescents and young adultsBrain and acute leukemia, cytoplasmicBCL-2 associated agonist of cell deathBCL-2 homologous antagonist/killerBCL-2 associated XB-cell CLL/lymphoma 11BB-cell lymphoma 2BCL2 related protein A1BCL2-like 1B cell precursorBromodomain and extra-terminal family of proteinsBlocks of sequence homologyBone marrowBromodomainBreakthrough therapy designationBruton tyrosine kinasecyclin D3Cluster of differentiationcyclin dependent kinaseCyclin-dependent kinase inhibitor 2AChromatin immunoprecipitationCombination indexCellular inhibitor of apoptosisCtBP interacting domainChronic lymphocytic leukemiaCorticomedullary junctionChronic myeloid leukemiaCommon myeloid progenitorCCR4-NOT transcription complex subunit 3Calibrated normalized relative quantitiescAMP response element-binding proteinCREB binding proteinCancer stem cellC-terminal binding proteinCortical thymic epithelial cellCytochrome cix
HAThCD45HDHDACHES1HOXADendritic cellDiffuse large B-cell lymphomaDimethylsulfoxideDouble negative (CD4- CD8-)DNA interferenceDNA methyltransferase 1DOT1-like histone lysine methyltransferaseDouble positive (CD4 CD8 )Death receptorEffective doseEmbryonic ectoderm developmentEnhanced green fluorescent proteinEuropean Group for the Immunological Characterization of LeukemiasEukaryotic initiating factorElongation factor for RNA polymerase IIMaximal efficacyEpithelial-mesenchymal transitionEMT transcription factorsE1A binding protein p300Estrogen receptorExtracellular signal-regulated kinaseEmbryonic stem cellEarly thymic progenitorEarly T-cell precursor ALLETS variant 6Enhancer of zeste 2 PRC2 subunitFluorescence-activated cell sorterFlavin adenine dinucleotideFAS-associated protein with death domainFetal bovine serumU.S. Food and Drug AdministrationFollicular lymphomaFms related tyrosine kinase 3Forkhead box OGATA binding protein 3Gene expression omnibusGrowth factor independent 1Green fluorescent proteinGranulocyte-monocyte progenitorGene set enrichment analysisγ-secretase inhibitorGlycogen synthase kinase-3 betaHistone acetyltransferaseHuman CD45HomeodomainHistone deacetylaseHes family bHLH transcription factor 1Homeobox A clusterx
TGAMJAKJARID1AJMJD3JNK1/2KDM1A TECmTORMYBBP1AMYPT1HarakiriHematopoietic stem cellHematopoietic stem cell transplantationHeat shock protein 90Inhibitor of apoptosisIntracellular BH3 profilingHalf maximal inhibitory concentrationIntracellular NOTCH1Isocitrate dehydrogenaseInsulin like growth factor binding protein 7Interleukin-7Interleukin-7 receptorImmature single positiveIntegrin subunit alpha MJanus kinaseJumonji/ARID domain-containing protein 1AJumonji domain containing 3c-Jun N-terminal kinase 1 or 2Lysine demethylase 1A (Lysine-specific demethylase 1)Lysine-specific demethylase 5ALiquid chromatography tandem mass spectrometryLeukemic stem cellLymphoid enhancer binding factor 1Library of integrated cellular signaturesLIM domain onlyMagnetic activated cell sortingMonoamine oxidaseMitogen-activated protein kinase 1BCL-2 family apoptosis regulatorMyelodysplastic syndromeMediator complex subunit 1Myocyte enhancer factor 2Mitogen-extracellular signal-regulated kinaseMegakaryocyte-erythroid progenitorMixed lineage leukemiaMLL-rearrangedMultilymphoid progenitormultiple myelomaMeningioma 1Mitochondrial outer membraneMitochondrial outer membrane permeabilizationMultipotent progenitorMinimal residual diseaseMesenchymal stromal cellsMedullary thymic epithelial cellMechanistic target of rapamycinMYB binding protein 1AMyosin phosphatase target subunit 1xi
eurofibromin 1Non-Hodgkin lymphomaNK2 homeobox 1NK2 homeobox 2Non-small cell lung cancerNuclear receptor binding SET domain proteinNonobese diabetic/severe combined immunodeficient gammaNuclear mitotic apparatus protein 1Nucleoporin 214Nucleosome remodeling/histone deacetylatingPolymerase chain reactionPatient-derived xenograftPropidium iodidePlant homeodomain finger 6Phosphoinositide-3-kinasePhosphatidylinositol 4,5-bisphosphatePhosphatidylinositol 3,4,5-trisphosphatePolycomb repressive complex 2Positive transcription elongation factor bPhosphatase and tensin homologProtein tyrosine phosphatase non-receptor type 11p53 upregulated modulator of apoptosisQuantitative real-time PCRQuantitative real-time reverse transcription PCRRetinoblastoma-binding protein 2Radioimmunoprecipitation assayRunt related transcription factor 1SMAD binding domainSmall cell lung cancerSubcapsular zoneSarco/endoplasmic reticulum calcium ATPaseSET domain containing 2Small lymphocytic lymphomaSecond mitochondria-derived activator of caspaseSnail family transcriptional repressor 2Single positive (CD4 or CD8 )Signal transducer and activator of transcriptionT-cell acute lymphoblastic leukemiaTruncated BIDT cell receptorTen-Eleven Translocation protein familyT-cell lymphoblastic leukemia/lymphomaTumor lysis syndromeT-cell leukemia homeobox 1T-cell leukemia homeobox 3Tumor necrosis factorType 1 tumor necrosis factor receptorTNFR1-accociated death domain proteinxii
lated apoptosis-inducing ligand receptorThymus seeding progenitorTwist family bHLH transcription factor 1Tyrosine kinase 2Untranslated regionUbiquitously transcribed tetratricopeptide repeat, X chromosomeVimentinVariance stabilization and calibrationWilms tumor 1X-linked inhibitor of apoptosis proteinZinc finger E-box binding homeobox 2Zinc fingerxiii
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Chapter 1: IntroductionCHAPTER 1Introduction1
Chapter 1: Introduction2
Chapter 1: IntroductionOutline of the introductionThe research performed as part of this doctoral thesis aimed to gain novel insights into themolecular mechanisms driving T-cell acute lymphoblastic leukemia (T-ALL) and to identifynew therapeutic strategies for patients with T-ALL. In this first introductory chapter, the topicscovered in the thesis are introduced. The state-of-the-art knowledge and broader contextprovided in Chapter 1 allow a critical reading of the results and discussion.In part I, the disease T-ALL and its current treatment are described. The short-comings andside effects of the current treatment strategies indicate that there is still room forimprovement. Within T-ALL, several subgroups can be distinguished. The main focus in thisdoctoral thesis was on the early immature subgroup, a subgroup associated with a poorprognosis. Searching new treatment strategies for these patients by investigating themolecular mechanisms that play a role in this subgroup was the goal of this work.One way to find more effective and targeted therapies is increasing our knowledge on thegenetic and epigenetic alterations present in T-ALL. In that way, druggable targets can beidentified. Part II of the introduction discusses therefore the genetic and epigenetic factorsthat play a role in the pathogenesis of T-ALL and the opportunities for targeted therapies. Adetailed description of ZEB2 (zinc finger E-box binding homeobox 2), KDM1A (lysinedemethylase 1A) and BET (bromodomain and extraterminal) proteins is given since thesewere central to the research performed in this thesis. The transcription factor ZEB2 is anoncogenic driver of early immature T-ALL and the determination of its interaction partnerswas part of our objectives. One of the newly identified interaction partners, KDM1A, turnedout to be druggable. Consequently, KDM1A inhibition was one of the treatment strategiesevaluated in this thesis. BET bromodomain inhibitors were also studied in this work and arethus also discussed in detail in this part of the introduction.Part III deals with the role of apoptosis, a form of programmed cell death, in cancer. Thepathways of apoptosis are described and ways to exploit apoptosis in cancer therapy aregiven. The emphasis is on the intrinsic pathway of apoptosis, a pathway that is governed bythe B-cell lymphoma 2 (BCL-2) family of proteins, since inhibition of the anti-apoptotic factorBCL-2 was the subject of two publications that are part of this PhD thesis.3
Chapter 1: IntroductionPART I Description and contemporary treatment of T-cell acutelymphoblastic leukemia1. What is T-cell acute lymphoblastic leukemia (T-ALL)?Cancer is a genetic disease because the transformation of a healthy cell to a cancer cell iscaused by changes in genetic material. This transformation is a multistep process duringwhich the cells acquire capabilities that enable them to become malignant. The hallmarkcapabilities that allow cancer cells to survive, proliferate and disseminate are sustainingproliferative signaling, evading growth suppressors, resisting cell death, enabling replicativeimmortality, inducing angiogenesis and activating invasion and metastasis1. Leukemias arecancers of the white blood cells and depending on the type of cell that becomes malignant,several types of leukemias can be distinguished.Blood cells are lifelong produced from hematopoietic stem cells (HSCs) residing in the bonemarrow. These HSCs have the capacity of self-renewal and to differentiate in any type ofblood cell. During this differentiation process, progenitor cell intermediates are formed(Figure 1)2.Figure 1. Schematic representation of human hematopoiesis. HSCs in the bone marrow (left) candifferentiate via several progenitor cell intermediates to terminally differentiated blood cells (right). Via the clusterof differentiation (CD) cell surface markers, a distinction between the different progenitors can be made.Multilymphoid progenitors (MLPs) also possess myeloid, but not erythroid and megakaryocytic potential.Accumulation of mutations in a T-cell progenitor can cause T-cell acute lymphoblastic leukemia (T-ALL). HSC:hematopoietic stem cell; MPP: multipotent progenitor; MLP: multilymphoid progenitor; ETP: early thymicprogenitor, CMP: common myeloid progenitor; GMP: granulocyte-monocyte progenitor; MEP: megakaryocyteerythroid progenitor; Lin: cocktail containing cell surface markers for all terminally differentiated populations.2Adap
IDH Isocitrate dehydrogenase IGFBP7 Insulin like growth factor binding protein 7 IL7 Interleukin-7 IL7R Interleukin-7 receptor ISP Immature single positive ITGAM Integrin subunit alpha M JAK Janus kinase JARID1A Jumonji/ARID domain-containing protein 1A JMJD3 Jumonji domain containing 3 JNK1/2 c-Jun N-terminal kinase 1 or 2
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