Characterization of molecular alterations in pediatric high- grade brain tumors

263 Pages·2014·39.95 MB·English

Characterization of molecular alterations in pediatric high-
grade brain tumors

Noha Bader Gerges
Department of Human Genetics
Faculty of Medicine
McGill University
Montreal, Quebec, Canada
August 2014

A thesis submitted to McGill University in partial fulfillment of the requirements of the
degree of Doctor of Philosophy
© Copyright Noha Bader Gerges, 2014
~ To you, I dedicate this thesis:

Bader and Nahed, my amazing parents
Nancy, my sister and friend
Fadi, my soulmate
ACKNOWLEDGMENTS

I am blessed to have had so much support during my PhD from different people
from across the world. Although I can’t name everyone here, you definitely know who
you are. Thank you.
I’d like to thank my supervisor Dr. Nada Jabado for her mentorship, patience, and
kindness throughout the past six years. Thank you for providing me with amazing
opportunities during my time here, for being supportive when things didn’t work out as
we expected them to, and for always being able to see (and teach me how to see) the
positive side of unexpected situations. I’ve learned valuable lessons from you that I will
definitely carry with me throughout my life. You’ve been a great supervisor and will be
dearly missed.
I would like to express my gratitude towards the Montreal Children’s Hospital
Research Institute, the Cedars Cancer Institute, and the Human Genetics department at
McGill University for providing me with generous fellowships and research/travel awards
during my time here.
To my dear committee members who have been instrumental to me during this
experience, and who were always very kind to me: Dr. Janusz Rak, Dr. Peter Siegel, Dr.
Jacek Majewski - thank you. It has been a pleasure discussing my research with such a
diverse group of scientists who each sees research through different colored lens.
You’ve truly made my experience more enjoyable.
My PhD would not have been possible without the friendships and collaborations
developed with many important people over the last years. This list is definitely not
exhaustive but highlights a few collaborators who have been a large part of my
experience here: Dr. Nahum Sonenberg – thank you very much for your generosity in
allowing me to spend time in your lab learning techniques essential to my research. Dr.
Maritza Jamarillo – working with you has undoubtedly been one of the most positive
experiences during my degree, and I thank you deeply for your support. Many thanks go
out to Dr. Steffen Albrecht, Genome Quebec’s many helpful residents (particularly Dr.
Jacek Majewski and his group, Dr. Tomi Pastinen and his group, and Francois
LeFebvre), Dr. Stephan Pfister, and Dr. Marcel Kool.
I’m very grateful towards a group of people who contributed to facilitating my time
here. Melanie Cotiangco, Thomas Leslie, Marlene Aardse and Eef Harmsen, thank you
for always lending a helping hand for all that was bureaucratic and for the conversations
we’ve had about unrelated subjects. A big thank you goes out to Ross Mackay and
Kandace Springer for all they have done, but most importantly, for alleviating any
concerns I had about the submission process as well as helping me get everything
ready to wrap up and answering my countless (and sometimes panicky) emails!

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To the amazing women who have become my sisters over the last six years,
you’re the best. Bahar, Cristiana, Gaby, and Olga: you ladies have been the most
phenomenal friends. I could write about all the awesome experiences we’ve all had
together but that would take me forever. I think you know what you all mean to me.
Thank you immensely for everything!
To all the Jabado lab members, you have all been great. Damien, thank you for
your patience and for always taking time out to discuss issues I was having, and for of
course never complaining when I asked you where something was many times in a
given period of time! Dong Anh, from the beginning, I had a feeling we were destined to
have a great friendship (even though we could barely have a full conversation!). I’ve
really enjoyed getting to know you, and am sincerely thankful for the countless hours we
spent discussing everything from work to life to randomness. Margaret, thank you for
always being willing to help out with anything and everything, and for making life at du
Fort much more interesting! Karine, thank you for making my transition to McGill easier,
for teaching me key lab-related things, for pushing me to always take proper notes
instead of trying to write what you were saying on my hand, and for being a great
person to talk to. Adam…where to start? Maybe at our chat sessions? I have enjoyed
getting to know you, and am glad that we could always find something to laugh about
despite whatever new disaster came our way on any given day. Thank you for teaching
me extremely important Italian phrases which I’m sure will be useful to me in the future
and for always lending an ear when needed. Xiaoyang, Takrima Tenzin, Denise,
Nisreen, Helene and Djihad, I have really enjoyed working with you and getting to know
you. Thanks for everything!
To my husband, my best friend, and my biggest fan, Fadi. Thank you for always
being there next to me, for always pushing me forward, and for making sure I smiled
throughout this entire process no matter what was going on. Each time you pushed me
when I thought I wouldn’t be able to continue, I got a newfound burst of energy that
drove me forward. This thesis wouldn’t have been possible without your calm and
grounded nature acting as a balance for me – for all this and so much more, thank you
forever.
To Nancy Gerges, my wonderful sister, thank you for always finding ridiculous
ways to make me laugh and for continuously leaving me funny notes around whenever
you came to visit.
Of course, none of this would have been possible without my extremely loving
and supportive parents, Bader Gerges and Nahed Meseiha. Without them being next to
me every step of the way from the beginning of my love for science at a very early age, I
wouldn’t have been able to accomplish all that I have, and for that I am eternally
grateful. Thank you for always encouraging me to move forward even when things
seemed like they would perpetually be at a standstill and for the countless hours of
conversations we had when things got tough. I couldn’t have asked for better parents.

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TABLE OF CONTENTS

ACKNOWLEDGMENTS II

TABLE OF CONTENTS IV

ABSTRACT VII

RÉSUMÉ IX

PREFACE AND CONTRIBUTION OF AUTHORS XI

LIST OF FIGURES XV

LIST OF TABLES XVII

LIST OF ABBREVIATIONS XIX

CHAPTER 1: INTRODUCTION 21

1.1 GLIOMAS 21

1.1.1
  CLASSIFICATION OF GLIOMAS 22

1.1.2 EPIDEMIOLOGY OF GLIOMAS 23

1.2 CHARACTERISTICS OF GLIOMAS 24

1.2.1 PILOCYTIC ASTROCYTOMAS (PA) 24

1.2.2 DIFFUSE ASTROCYTOMAS (DA) 25

1.2.3 OLIGODENDROGLIOMAS (OD) 27

1.2.4 OLIGOASTROCYTOMAS (OA) 28

1.2.5 ANAPLASTIC ASTROCYTOMAS (AA) 28

1.2.6 GLIOBLASTOMAS (GBM) 30

1.2.6.1 Adult glioblastoma 30

1.2.6.2 Pediatric glioblastoma 32

1.2.7 MOLECULAR STUDIES OF HIGH-GRADE GLIOMAS 35

1.2.7.1 Adult high-grade gliomas 36

1.2.7.2 Pediatric high-grade gliomas 38

1.3 CURRENT THERAPEUTIC AVENUES FOR HGA 40

1.4 Y-BOX BINDING PROTEIN 1 42

1.4.1 HISTORY 42

1.4.2 STRUCTURAL CHARACTERISTICS OF Y-BOX BINDING PROTEIN 1 43

1.4.3 POST-TRANSLATIONAL MODIFICATIONS OF YB1 43

1.4.4 Y-BOX BINDING PROTEIN 1 FUNCTIONS 44

1.4.5 MOUSE MODELS OF Y-BOX BINDING PROTEIN 1 46

1.4.6 Y-BOX BINDING PROTEIN 1 AND CANCER 47

1.5 EMBRYONAL TUMORS 49

1.5.1 CLASSIFICATION AND GRADING OF EMBRYONAL TUMORS 49

1.5.2 EMBRYONAL TUMORS WITH MULTILAYERED ROSETTES 50

1.5.2.1 Epidemiology 51

1.5.2.2 Molecular features 51

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1.5.2.3 Current clinical markers 53

1.6 TTYH1 AND DNMT3B 53

1.6.1 TTYH1 54

1.6.2 DNMT3B 55

1.7 INTRODUCTION TO HIGH THROUGHPUT TECHNOLOGIES 56

1.7.2 WHOLE EXOME SEQUENCING 58

1.7.3 RNA SEQUENCING 59

1.7.4 DNA METHYLATION ARRAYS 59

1.7.5 IMPACT ON CANCER RESEARCH 60

1.8 RATIONALE 62

1.9 HYPOTHESIS AND EXPERIMENTAL OBJECTIVES 63

1.10 FIGURES 66

1.11 TABLES 75

CHAPTER 2: UNRAVELING THE ROLE OF YB1 IN TRANSLATION: THE
ASTROCYTOMA EDITION 78

2.1 ABSTRACT 79

2.2 INTRODUCTION 80

2.3 MATERIALS AND METHODS 82

2.4 RESULTS 85

2.5 DISCUSSION 88

2.6 AUTHOR CONTRIBUTIONS 93

2.7 FIGURES 94

2.8 TABLES 98

2.9 SUPPLEMENTARY FIGURES 108

CONNECTING TEXT FROM CHAPTER 2 TO CHAPTER 3 111

CHAPTER 3: PEDIATRIC HIGH GRADE ASTROCYTOMAS – A LESSON ON
TUMOR SUBGROUPING 112

3.1 ABSTRACT 113

3.2 INTRODUCTION 114

3.3 MATERIALS AND METHODS 116

3.4 RESULTS 120

3.5 DISCUSSION 124

3.6 AUTHOR CONTRIBUTIONS 130

3.7 FIGURES 131

3.8 TABLES 133

3.9 SUPPLEMENTARY FIGURES 136

3.10 SUPPLEMENTARY TABLES 138

CONNECTING TEXT FROM CHAPTER 3 TO CHAPTER 4 151

CHAPTER 4: FUSION OF TTYH1 WITH THE C19MC MICRORNA CLUSTER DRIVES
EXPRESSION OF A BRAIN-SPECIFIC DNMT3B ISOFORM IN THE EMBRYONAL
BRAIN TUMOR ETMR 152

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4.1 ABSTRACT 154

4.2 INTRODUCTION 154

4.3 MATERIALS AND METHODS 155

4.4 RESULTS 165

4.5 DISCUSSION 172

4.6 AUTHOR CONTRIBUTIONS 173

4.7 FIGURES 174

4.8 SUPPLEMENTARY FIGURES 180

4.9 SUPPLEMENTARY TABLES 203

4.10 ACKNOWLEDGMENTS 221

CHAPTER 5: DISCUSSION 222

5.1 HOW DOES THE Y-BOX BINDING PROTEIN 1 AFFECT THE TRANSLATION OF ASTROCYTIC
CELLS? 223

5.2 ARE PEDIATRIC HGA REALLY TWO SEPARATE ENTITIES? 228

5.2.1 MICROARRAYS AND THEIR PERTINENT ISSUES 236

5.3 ETMR: IS THERE MORE TO THE STORY? 237

5.4 FINAL REMARKS AND ORIGINAL CONTRIBUTION TO KNOWLEDGE 244

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ABSTRACT
Primary brain tumors are the most common group of solid cancers in the
pediatric years and are the primary source of cancer related mortality and morbidity in
children. They are segregated into gliomas and embryonal tumors which are further
subdivided by the World Health Organization into pathological groups and graded
according to their degree of malignancy. To date, both gliomas and embryonal tumors
are diagnosed and treated based on this histopathological classification, yet several
molecular findings indicate this classification is obsolete and that this may partly
account for the dismal outcome seen for some of the high-grade tumours. To improve
molecular diagnosis and gain insight into novel targets, we investigated using genetic,
molecular and biochemical tools two groups of pediatric high-grade brain tumors,
namely from the glioma lineage anaplastic astrocytoma (AA) and glioblastoma (GBM),
and from the embryonal tumors lineage the embryonal tumors with multilayered rosettes
(ETMR). We initially investigated the effects of Y-Box Protein 1 (YB1) on translation of
astrocytic cells, a gene previously found by our lab to be overexpressed in pediatric
GBM. Overexpression of YB1 led to altered expression in astrocytomas. In pediatric
GBM cell lines, it led to the dysregulation of a core chromatin gene, HIST1H1A
highlighting a possible epigenetic role for YB1, and uncovered a set of new prospective
targets of YB1 with potential role in astrocytoma genesis. Our subsequent investigation
of AA and GBM using integrative high throughput analyses indicated that this grade
distinction is arbitrary as pediatric anaplastic astrocytoma (pAA, grade III) are
molecularly similar to pediatric glioblastoma (pGBM, grade IV) and that tumors

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segregate based on the underlying genetic mutations they carry. Hence, we propose
that future diagnoses and therapies be based on patho-molecular features rather than
the underlying tumor grade as per the current WHO classification. Our studies of a
newly described embryonal entity, ETMR, identify a novel fusion between TTYH1 and
the C19MC microRNA cluster in all samples. This characteristic TTYH1-C19MC fusion
in these tumors leads to extremely high expression levels of members of the C19MC
microRNA cluster in all ETMR investigated, and subsequently to de-repression of the
expression of an embryonic brain-specific isoform of DNMT3B, a de novo DNA
methyltransferase. Re-awakening or aberrant maintenance of an embryonic pathway in
these tumors affects the epigenome and is possibly at the origin of these intractable
tumors. This work as a whole has provided novel insight into three different pediatric
high-grade brain tumors, and has facilitated the discovery of novel markers and
underlying mechanisms which can now be used to improve the diagnoses and
treatment modalities of patients with these devastating cancers.

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RÉSUMÉ

Les tumeurs cérébrales primitives appartiennent au groupe le plus commun de tumeurs
solides rencontrées en pédiatrie. C’est la principale cause de mortalité et de morbidité
liée au cancer chez les enfants. Nous pouvons trouver deux subdivisions de ces entités
tumorales classifiées par l'Organisation Mondiale de la Santé (OMS). Cette
classification répertorie les tumeurs affectant le système nerveux central selon leur
histopathologie et leur degré de malignité. L'astrocytome anaplasique (AA), le
glioblastome (GBM) et les tumeurs embryonnaires avec les rosettes multicouches
(ETMR) sont des tumeurs cérébrales qui sont, actuellement, diagnostiquées et traitées
sur la base de cette classification. La découverte de marqueurs moléculaires
spécifiques à ces tumeurs cérébrales pédiatriques rend cette classification et les
traitements actuels non efficients en pédiatrie. Ainsi, afin d’améliorer le diagnostic
moléculaire et de mieux comprendre les nouvelles cibles potentielles, nous avons
étudié de manière approfondie deux groupes de tumeurs cérébrales pédiatrique de haut
grade : AA, GBM et ETMR en utilisant des outils génétiques, moléculaires et
biochimiques.
Dans un premier temps nous avons analysé les effets de la protéine YB1 (Y-Box
Protein 1) sur la conversion des cellules astrocytaires, un gène précédemment exploré
par notre laboratoire et surexprimé dans le cas des GBM pédiatriques. Dans les lignées
cellulaires de GBM pédiatriques, la surexpression de YB-1 conduit à une dérégulation
du gène HIST1H1A responsable de la compaction de la chromatine, mettant ainsi en
évidence un rôle épigénétique possible de YB1 et son rôle potentiel dans la
gliomagenèse. Dans un second temps, nous avons analysé par une méthode

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