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Investigation of Activated Tyrosine Kinases in Myeloproliferative Neoplasms

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Investigation of Activated Tyrosine Kinases in Myeloproliferative Neoplasms Investigation of activated tyrosine kinases in myeloproliferative neoplasms by Michael Ross Marit A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Medical Biophysics University of Toronto Copyright © 2012 by Michael Ross Marit Abstract Investigation of activated tyrosine kinases in myeloproliferative neoplasms Michael Ross Marit Doctor of Philosophy Graduate Department of Medical Biophysics University of Toronto 2012 Myeloproliferative neoplasms (MPNs) are a group of disorders characterized by an excess production of a specific, fully functional blood cell type. Many cases involve deregulation of a protein tyrosine kinase. JAK2 is one such kinase, involved in a subset of MPNs. JAK2-selective inhibitors are currently being evaluated in clinical trials. In order to identify inhibitor-resistant JAK2 mutations before they appear in the clinic, we utilized TEL-JAK2 to conduct an in vitro random mutagenesis screen for JAK2 alleles resistant to JAK Inhibitor-I. Isolated mutations were evaluated for their ability to sustain cellular growth, stimulate downstream signalling pathways, and phosphorylate a novel JAK2 substrate in the presence of inhibitor. When testing the panel of mutations in the context of the Jak2 V617F allele, we observed that a subset of mutations conferred resistance to inhibitor. These results demonstrate that small-molecule inhibitors select for JAK2 inhibitor-resistant alleles. Chronic myeloid leukemia is an MPN characterized by the presence of the BCR-ABL fusion gene. We determined that a specific cohort bearing deletions near the ABL gene, which is associated with poor prognosis, do not suffer from genomic instability. We also examined the role of a putative tumour suppressor gene EXOSC2 as an explanation for the reduced survival time, and suggest it may have a role in disease progression. ii Contents Abstract ii List of Tables v List of Figures vi List of Abbreviations viii 1 Introduction 1 1.1 Hematopoiesis . .1 1.1.1 Epo signalling and erythropoiesis . .3 1.1.2 The tyrosine kinase domain and JAK2 . .3 1.1.3 JAK-STAT signalling pathway . .4 1.1.3.1 Stat activation . .5 1.1.3.2 Mitogen-activated protein kinase activation . .5 1.1.3.3 Phosphatidylinositol 3 kinase activation . .6 1.1.4 Tpo signalling and thrombopoiesis . .6 1.2 Myeloproliferative neoplasms . .8 1.3 Ph+ myeloproliferative neoplasm { CML . .9 1.3.1 CML history and the BCR-ABL translocation . 10 1.3.2 Functions of wild-type BCR and ABL . 12 1.3.3 Intracellular signalling downstream of BCR-ABL . 13 iii 1.3.4 The mouse bone marrow transplant model . 15 1.3.5 Deletions at chromosome 9q34 in CML . 18 1.3.5.1 The exosome and function of EXOSC2 . 19 1.3.5.2 Function of PRDM12 . 24 1.3.6 Historical therapies for CML . 24 1.3.7 Development and efficacy of imatinib mesylate . 25 1.3.8 Development of imatinib mesylate-insensitive relapse . 26 1.3.9 Next-generation ABL kinase inhibitors . 27 1.4 Ph{ myeloproliferative neoplasms { PV, ET, and MF . 29 1.4.1 MPN disease phenotypes . 30 1.4.2 Discovery of JAK2 V617F . 31 1.4.3 Genetic diversity among Ph{ MPN disease . 34 1.4.3.1 JAK2 exon 12 mutations . 34 1.4.3.2 MPL (Tpo-R) mutations . 35 1.4.3.3 LNK mutations . 35 1.4.3.4 TET2 mutations . 36 1.4.3.5 ASXL1 mutations . 37 1.4.3.6 EZH2 mutations . 37 1.4.3.7 JAK2 haplotype as a disease predictor . 38 1.4.3.8 Clonal diversity within disease and individual patients . 38 1.4.4 Inhibitors of JAK2 in the treatment of MPNs . 39 1.4.4.1 Ruxolitinib (INCB018424) . 40 1.4.4.2 SAR302503 (TG101348) . 41 1.4.4.3 Lestaurtinib (CEP-701) . 41 1.4.4.4 Momelotinib (CYT387) . 42 1.4.4.5 Pacritinib (SB1518) . 43 1.4.5 Mouse models for development and treatment of Ph{ MPNs . 43 iv 1.4.5.1 BMT models . 44 1.4.5.2 Transgenic models . 45 1.5 Rationale and hypothesis . 48 1.6 Thesis statement and study aims . 49 2 Random mutagenesis reveals residues of JAK2 critical in evading inhi- bition by a tyrosine kinase inhibitor 51 2.1 Abstract . 52 2.2 Introduction . 53 2.3 Materials and methods . 55 2.4 Results . 60 2.5 Discussion . 77 3 Examining Chromosome 9q34 Deletion as a Marker for Genomic Insta- bility in Chronic Myeloid Leukemia 85 3.1 Abstract . 86 3.2 Introduction . 87 3.3 Materials and methods . 89 3.4 Results . 96 3.5 Discussion . 117 4 Discussion 122 5 Concluding Remarks 136 References 138 v List of Tables 1.1 Exosome cross-kingdom protein sequence conservation . 22 2.1 Structural diagrams of selected JAK2 inhibitors . 62 2.2 JAK2 kinase domain mutations identified in an inhibitor-resistance screen 63 3.1 EXOSC2 and PRDM12 exon sequencing primers . 97 3.2 Quantitative PCR raw data from CML patient samples . 100 3.3 Quantitative PCR relative expression from CML patient samples . 101 3.4 9q34 and 22q11 status in CML samples as called by aCGH . 103 3.5 A genomic map of the 9q34 and 22q11 loci in CML patient samples . 106 3.6 CML patient copy number gains from related publications . 109 3.7 CML patient copy number losses from related publications . 110 3.8 EXOSC2 and PRDM12 exon sequencing . 116 vi List of Figures 1.1 Hematopoietic differentiation . .2 1.2 Epo signalling and JAK2 domain structure . .7 1.3 BCR-ABL structure and signalling . 16 1.4 The mammalian exosome . 23 2.1 Location of the putative JAK2 inhibitor-resistant mutations . 64 2.2 JAK2 mutations display resistance to JAK inhibitor-I . 65 2.3 TEL-JAK2 mutants are not resistant to TG101348 or CEP-701 . 66 2.4 TEL-JAK2 inhibitor-resistant mutants display enhanced phosphorylation of Stat5, Akt and Erk1/2 - I . 68 2.5 TEL-JAK2 inhibitor-resistant mutants display enhanced phosphorylation of Stat5, Akt and Erk1/2 - II . 69 2.6 TEL-JAK2 and Jak2 V617F phosphorylate JAK2 substrate activation loop sequences . 70 2.7 TEL-JAK2 mutants G935R and R975G display a strong degree of inhibitor resistance . 72 2.8 Jak2 V617F G935R is resistant to JAK Inhibitor-I . 74 2.9 Jak2 V617F G935R is not resistant to TG101348 or CEP-701 . 75 2.10 Jak2 V617F G935R displays enhanced Stat5 and Erk1/2 phosphorylation 76 2.11 Jak2 V617F G935R displays a strong degree of inhibitor resistance . 78 vii 2.12 JAK2 inhibitor-resistant residues mapped to the crystal structure bound to JAK Inhibitor-I . 82 2.13 Inhibitor-resistant mutations identified in mJak1 and hJAK2 mutagenesis screens . 84 3.1 EXOSC2 and PRDM12 quantitative PCR standard curves . 99 3.2 Nexus software visualization of the 9q34 locus in CML patient samples . 104 3.3 Nexus software visualization of the 22q11 locus in CML patient samples . 105 3.4 Nexus-generated virtual karyotype from CML patient samples . 108 3.5 Genomic loss at 12q13.32 in the RB1 tumour suppressor gene . 112 3.6 EXOSC2 antibody validation . 114 3.7 Exosc2 and L32 expression in hematopoietic progenitors . 115 viii List of Abbreviations 5-FU 5-fluorouracil BMT Bone marrow transplant 5mC 5-methylcytosine c-abl ABL1 tyrosine kinase, human orthologue ABL Abelson murine leukemia viral oncogene homo- c-mpl Thrombopoietin recep- logue tor, human orthologue CC Coiled coil aCGH Array comparative ge- nomic hybridization cDNA Complementary DNA AML Acute myeloid leukemia CFU-E Colony-forming unit, ery- throid ARE AU-rich element CLP Common lymphoid pro- ARED AU-rich element genitor database CML Chronic myeloid leu- ATP Adenosine tri-phosphate kemia CMML Chronic monomyelocytic aUPD Acquired uniparental dis- leukemia omy CMP Common myeloid pro- B-ALL B-cell acute lymphoblas- genitor tic leukemia der(9) Derivative chromosome 9 BAC Bacterial artificial chro- mosome DH DBL homology DNA Deoxyribonucleic acid BCR Breakpoint cluster region dNTP Dinucleotide triphos- BCR-ABL Fusion gene created with phate BCR and ABL1 ENU N-ethyl-N-nitrosourea BFU-E Burst-forming unit, ery- throid EPO Erythropoietin Epo Erythropoietin BID Bis in die (Latin: twice a day) EPO-R Erythropoietin receptor ix ET Essential thrombo- HSC Hematopoietic stem cell cythemia IFN-α Interferon alpha EXOSC Exosome sub-component IFN-γ Interferon gamma FCS Fetal calf serum IgG Immunoglobulin G FDA Food and drug adminis- IL-3 Interleukin 3 tration IL-3-R Interleukin 3 receptor FERM 4.1 / ezrin / radixin / moesin IL-6 Interleukin 6 FF1 Flip-flop1 IM Imatinib mesylate FISH Fluorescent in situ hy- ITD Internal tandem duplica- bridization tion G-CSF Granulocyte colony stim- JAK Janus kinase ulating factor JH1 JAK kinase domain GAS Gamma activated se- quence JH2 JAK dual-specificity kin- ase domain GDP Guanine diphosphate JI1 JAK Inhibitor-I GM-CSF Granulocyte macrophage colony stimulating factor LOH Loss of heterozygosity GMP Granulocyte macrophage LP Lymphoid progenitor progenitor LSK Lineage{, sca-1+, c-kit+ GST Glutathione-S-transferase MAP Mitogen activated pro- tein GST-J2s Fusion of glutathione-S- transferase and the 11 MAPK Mitogen activated pro- amino acid JAK2 sub- tein kinase strate Mast Mast cell GTP Guanine triphosphate MDR Minimal deleted region HCT Hematocrit MEP Megakaryocyte erythro- hmC 5-hydroxymethylcytosine cyte progenitor hopTum-1 HopscotchTumorous- MF Myelofibrosis lethal mJak Murine Jak orthologue HR Homologous repair MMEJ Microhomology-mediated HRP Horseradish peroxidase end joining x MPL Thrombopoietin receptor PRC2 Polycomb repressive complex 2 MPN Myeloproliferative neo- plasm / myeloprolifera- PRDM Positive regulatory do- tive disease main member NHEJ Non-homologous end PTK Protein tyrosine kinase joining PV Polycythemia vera NK Natural killer cell qPCR Quantitative real-time PCR p190 The 190 kDa isoform of BCR-ABL qRT-PCR Quantitative real-time PCR p210 The 210 kDa isoform of BCR-ABL RB1 Retinoblastoma
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