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The Role of Deletions on Chromosomes 7P and 20Q in Myeloproliferative Disorders

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The Role of Deletions on Chromosomes 7P and 20Q in Myeloproliferative Disorders The role of deletions on chromosomes 7p and 20q in myeloproliferative disorders Doctoral thesis at the Medical University of Vienna for obtaining the academic degree Doctor of Philosophy Submitted by Roland Jäger Supervisor: Robert Kralovics, PhD CeMM – Center for Molecular Medicine of the Austrian Academy of Sciences Lazarettgasse 19/3, 1090 Vienna Vienna, 06/2010 TABLE OF CONTENTS ABSTRACT……………………………………………………………………………………………….. 4 ABSTRACT (GERMAN)………………………………………………………………………………… 5 1. INTRODUCTION ......................................................................................................................5 1.1. CLINICAL FEATURES OF MYELOPROLIFERATIVE NEOPLASMS...............................................................................5 1.2. GENETIC DEFECTS IN MPN PATHOGENESIS...........................................................................................................6 1.2.1. The V617F mutation of JAK2......................................................................................................................8 1.2.2. Other oncogenic mutations......................................................................................................................10 1.2.3. Loss of function of the tumor suppressor TET2........................................................................................11 1.2.4. CBL mutations – defects in a complex regulator of hematopoiesis..............................................13 1.2.5. Cytogenetic aberrations in MPN .............................................................................................................14 1.3. HEREDITARY FACTORS IN MPN............................................................................................................................15 2. HYPOTHESIS.........................................................................................................................18 3. METHODS...............................................................................................................................21 3.1. PATIENT COHORT, CELL SORTING, DNA ISOLATION, JAK2 AND MPL MUTATION ANALYSIS ............................21 3.2. MICROARRAY GENOTYPING, LOSS OF HETEROZYGOSITY AND COPY NUMBER ANALYSIS ...................................21 3.3. MICROSATELLITE PCR .........................................................................................................................................22 3.4. SEQUENCING .........................................................................................................................................................22 3.5. GENOTYPING OF HEMATOPOIETIC PROGENITORS ................................................................................................23 3.6. VECTORS, LENTIVIRAL PACKAGING ......................................................................................................................23 3.7. PRIMARY CELL ISOLATION AND VIRAL TRANSDUCTION ........................................................................................24 3.8. PROLIFERATION ASSAY– PRIMARY CELLS............................................................................................................24 3.9. INTRACELLULAR PSTAT5 AND STAT5 MRNA ASSAYS ........................................................................................25 3.10. GENE EXPRESSION PROFILING..............................................................................................................................25 3.11. STABLE TRANSFECTIONS – AMAXA DELIVERY OF SHRNA CONSTRUCTS TO CELL LINES ..................................27 3.12. TRANSIENT TRANSFECTIONS – DELIVERY OF SYNTHETIC SIRNAS TO UT7/TPO ..............................................27 3.13. MEASUREMENT KNOCK-DOWN EFFICIENCIES – REAL TIME PCR AND WESTERN BLOTTING.............................28 1 3.14. PROLIFERATION ASSAY – CELL LINES ..................................................................................................................28 3.15. SCREEN FOR DEL20Q TUMOR SUPRESSORS (POOLED BAR-CODED SHRNA SETUP) .........................................30 3.16. COMPETITIVE PROLIFERATION ASSAY (CANDIDATE VS. CONTROL BAR-CODED SHRNA SETUP) .......................30 3.17. LUMINEX QUANTIFICATION OF BAR-CODED SHRNAS ..........................................................................................31 4. RESULTS ................................................................................................................................32 4.1. DELETIONS ON CHROMOSOME 7P – A GENETIC DEFECT AT POST-MPN LEUKEMIC TRANSFORMATION.............32 4.1.1. High-resolution genomic analysis of patients with post-MPN leukemia ......................................................32 4.1.2. Frequency of del7p in MPN and mapping of the common deleted region....................................................33 4.1.3. Clinical phenotype of del7p patients...............................................................................................................37 4.1.4. Leukemic blasts carry del7p and JAK2-V617F..............................................................................................38 4.1.5. Absence of IKZF1 point mutations in del7p and MPN patients....................................................................38 4.1.6. Deletion of chromosome 7p is a late genetic event in MPN ..........................................................................39 4.1.7. Loss of IKZF1 increases cytokine sensitivity of primary progenitor cells...................................41 4.1.8. Ikaros haploinsufficiency increases Stat5 phosphorylation in primary progenitor cells ........43 4.2. DELETIONS ON CHROMOSOME 20Q – A FREQUENT DEFECT IN CHRONIC PHASE MPN......................................46 4.2.1. Screen for del20q events in three independent patient cohorts.....................................................46 4.2.2. Del20q acts independently and can occur before and after JAK2-V617F....................................46 4.2.3. The del20q common deleted region contains several non-expressed genes.............................49 4.2.4. Mybl2 is a putative tumor suppressor targeted by del20q...............................................................51 4.2.5. Top1 acts as a second putative tumor suppressor in a competitive RNAi setup ......................54 4.2.6. Top1 is outside of the del20q CDR assembled from two patient cohorts....................................60 5. DISCUSSION..........................................................................................................................62 5.1. IKAROS DEFECTS IN MPN AT TRANSFORMATION TO AML ..................................................................................62 5.2. PUTATIVE TUMOR SUPPRESSOR(S) IN THE DEL20Q CDR....................................................................................67 5.3. GENETIC COMPLEXITY IN MPN AND IMPLICATIONS FOR THERAPY ......................................................................72 REFERENCES…………………………………………………………………………………………. 76 ACKNOWLEDGEMENTS…………………………………………………………………………….. 87 Curriculum Vitae…………………………………………………………………………………….… 88 List of publications…………………………………………………………………………..…......... 91 2 ABSTRACT Somatic mutations play a central role in the pathogenesis of myeloproliferative neoplasms (MPNs), triggering clonal hematopoiesis and establishing the disease phenotype. After the discovery of JAK and MPL mutations, continual technological advances now allow the identification of increasing numbers of genetic defects in MPN patients, most of them chromosomal aberrations such as deletions and acquired uniparental disomies. Efforts to map the genetic lesions to single genes resulted in the discovery of defects in the TET2 and CBL genes, however, the target gene(s) comprised in the most frequent chromosomal lesion in MPN, deletions on chromosome 20q (del20q), still remain to be identified. Therefore, we determined the role of del20q in the evolution of the malignant clone, characterized three independent MPN patient cohorts (a total of 822 patients) for del20q and assembled a del20q common deleted region (CDR) using microarray karyotyping of nine patients. We further identified MYBL2 and TOP1 as two putative tumor suppressors on 20q by mimicking deletion-caused haploinsufficiency using different shRNA knock-down strategies in vitro. Comparing our mapping data in patient material with the functional in vitro studies, we could see MYBL2 within the del20q CDR, whereas TOP1 mapped outside of the CDR, questioning the exclusive relevance of CDRs in the search for tumor suppressors. Besides our work on del20q, a defect observed in the chronic phase of MPN, we were searching for cytogenetic aberrations associated with post-MPN transformation to acute leukemia. Leukemic transformation is a major complication of MPN, however, the genetic changes leading to transformation remain largely unknown. We screened nine patients with post-MPN leukemia for chromosomal aberrations using microarray karyotyping. Deletions on the short arm of chromosome 7 (del7p) emerged as a recurrent defect. We mapped the common deleted region to the IKZF1 gene, which encodes the transcription factor Ikaros. We further examined the frequency of IKZF1 deletions in a total of 29 post-MPN leukemia and 526 MPN patients without transformation and observed a strong association of IKZF1 deletions with post-MPN leukemia in two independent cohorts. Patients with IKZF1 loss exhibited complex karyotypes, and del7p was a late event in the genetic
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