Function of BCL6 in pre-B cells and Philadelphia chromosome-positive acute lymphoblastic leukemia [Elektronische Ressource] / Cihangir Duy. Gutachter: Markus Müschen ; Dieter Willbold ; Ari Melnick

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Sujets

• Biologie

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2011
• Nombre de lectures: 123
• Langue: Deutsch
• Poids de l'ouvrage: 30 Mo

Extrait

Function of BCL6 in pre-B cells and Philadelphia
chromosome-positive acute lymphoblastic
leukemia





Inaugural-Dissertation



zur Erlangung des Doktorgrades

der Mathematisch-Naturwissenschaftlichen Fakultät

der Heinrich-Heine-Universität Düsseldorf




vorgelegt von

Cihangir Duy

aus Dinslaken



Januar 2011
Die vorliegende Arbeit wurde unter Anleitung von Herrn Prof. Dr. Markus
Müschen zum Großteil in der Abteilung für Hämatologie und Onkologie am
Children’s Hospital Los Angeles der University of Southern California (CA, USA)
durchgeführt. Die Arbeit wurde fakultätsübergreifend mitbetreut von Herrn Prof.
Dr. Dieter Willbold seitens der Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf.















Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf

Referent: Prof. Dr. Markus Müschen
Koreferent: Prof. Dr. Dieter Willbold
Externer Referent: Prof. Dr. Ari Melnick

Tag der mündlichen Prüfung: 11.07.2011











To those who never give up and are still able to see the world with open and
imaginative eyes
























Table of Contents
Related Articles .............................................................................................. i
List of Figures................................................................................................ ii
List of Tables vi
1 Introduction 1
1.1 B cells........................................................................................................................ 1
1.1.1 V(D)J recombination ......................................................................................... 1
1.1.2 B cell development ............................................................................................ 5
1.1.3 B cell activation ............................................................................................... 11
1.1.4 Malignant transformation................................................................................. 13
1.2 Philadelphia (Ph) chromosome............................................................................... 17
1.2.1 BCR-ABL1-positive leukemia......................................................................... 19
1.2.2 Drug treatment and resistance.......................................................................... 20
1.2.3 Quiescent leukemia cells.................................................................................. 23
1.3 BCL6....................................................................................................................... 26
1.3.1 Structure and regulation................................................................................... 27
1.3.2 Biological relevance......................................................................................... 30
1.3.3 Aberrant expression 32
1.4 Aims. 34
2 Materials and Methods............................................................................ 36
2.1 Patient samples, cell lines and human bone marrow cells...................................... 36
2.2 Mouse models ......................................................................................................... 37
2.3 Extraction and culturing of bone marrow cells from mice ..................................... 37
2.4 In vivo model for BCR-ABL1-transformed ALL and bioluminescence imaging.. 38
2.5 Retroviral transduction............................................................................................ 39
2.6 BCR-ABL1 Tyrosine Kinase Inhibitors (TKI)....................................................... 40
2.7 Flow cytometry ....................................................................................................... 40
2.8 In vitro pre-B cell differentiation assays................................................................. 41
2.9 V(D)J recombination reporter assay ....................................................................... 41
2.10 Clonality analysis and spectratyping of B cell populations.................................. 42
2.11 Retro-Inverso BCL6 Peptide Inhibitor (RI-BPI) .................................................. 42
2.12 Quantitative RT-PCR............................................................................................ 43
2.13 Western blotting.................................................................................................... 43
2.14 Affymetrix GeneChip analysis ............................................................................. 44
2.15 Chromatin immunoprecipitation, ChIP-on-chip ................................................... 45
2.16 Quantitative chromatin immunoprecipitation (QChIP) ........................................ 45
+2.17 Target validation of RI-BPI in human Ph ALL cells .......................................... 46
2.18 ChIP-on-chip analysis........................................................................................... 47
2.19 Data analysis of ChIP-on-chip experiments ......................................................... 48
2.20 Comparative Genomic Hybridization (CGH)....................................................... 49
2.21 Cell viability assay................................................................................................ 49
2.22 Colony forming assay 50
2.23 Cell cycle analysis................................................................................................. 50
2.24 Senescence-associated -galactosidase assay 50
2.25 MicroRNA-155 expression in mouse and human CML-LBC.............................. 51
2.26 Mutation analysis and ligation-mediated PCR ..................................................... 51
2.27 Single-cell RT-PCR analysis ................................................................................ 52
2.28 Single nucleotide polymorphism mapping assay and comparative genomic
hybridization ................................................................................................................. 53
3 Results ....................................................................................................... 54
3.1 BCL6 is critical for the development of a diverse primary B cell repertoire ......... 54
3.2 BCL6 enables leukemia cells to survive inhibition of oncogenic tyrosine kinases 84
3.3 The B cell mutator AID promotes B lymphoid blast crisis and drug-resistance in
chronic myeloid leukemia........................................................................................... 124
4 Discussion................................................................................................ 152
5 Appendix................................................................................................. 162
5.1 Abbreviations........................................................................................................ 162
5.2 Supplementary Information .................................................................................. 165
5.3 Contribution to Publication................................................................................... 172
6 References............................................................................................... 177
7 Summary 219
8 Zusammenfassung ................................................................................. 221
9 Danksagung ............................................................................................ 224
10 Curriculum vitae.................................................................................. 226
11 List of Publications .............................................................................. 228
12 Erklärung.............................................................................................. 229 Related Articles
Related Articles

This thesis is based on the following papers, which will be referred to in the results
section by their numbers:

1. Duy C, Yu JJ, Nahar R, Swaminathan S, Kweon SM, Polo JM, Valls E, Klemm
L, Shojaee S, Cerchietti L, Schuh W, Jack HM, Hurtz C, Ramezani-Rad P, Jäck
HM, Herzog S, Jumaa H, Koeffler HP, de Alborán IM, Melnick AM, Ye BH &
Müschen M. BCL6 is critical for the development of a diverse primary B cell
repertoire. J Exp Med. 2010; 207:1209-1221

2. Duy C, Hurtz C, Shojaee S, Cerchietti L, Geng H, Swaminathan S, Klemm L,
Kweon SM, Nahar R, Braig M, Park E, Kim YM, Hofmann W-K, Herzog S,
Jumaa H, Koeffler PH, Yu JJ, Heisterkamp N, Graeber TG, Wu H, Ye BH,
+Melnick A & Müschen M. BCL6 enables Ph acute lymphoblastic leukaemia cells
to survive BCR–ABL1 kinase inhibition. Nature. 2011; in press

3. Klemm L, Duy C, Iacobucci I, Kuchen S, von Levetzow