Martina Anna Finetti

521 Pages·2015·16.73 MB·English

Next-Generation Sequencing Identifies
Mechanisms Of Tumourigenesis Caused By Loss
Of SMARCB1 In Malignant Rhabdoid Tumours

Martina Anna Finetti

Thesis submitted in partial fulfilment of the requirements
for the degree of Doctor of Philosophy
Newcastle University
Faculty of Medical Sciences
Northern Institute for Cancer Research
December 2014
Declaration
I certify that no part of the material documented in this thesis has previously been
submitted for a degree or other qualification in this or any other university. I declare that
this thesis represents my own unaided work, carried out by myself, except where it is
acknowledged otherwise in the thesis text.
Martina Finetti
December 2014

I
Acknowledgements
I would like to express my sincere gratitude to my supervisors, Dr. Daniel Williamson,
Professor Steven Clifford, and Professor Simon Bailey. Their continuous support and
their extraordinary knowledge of paediatric cancer and bioinformatics have helped me
immensely throughout my research.
I would also like to thank my assessors Professor John Lunec and Dr. Luke Gaughan
and Professor Craig Robson for their constant guidance and encouragement for the last
four years.
I would like to thank the members of the Paediatric Brain Tumour Research Group. In
particular I would like to thank Dr. Rebecca Hill, Matthew Selby and Dr Dolores
Hamilton for the support, the encouragement and the time spent together in good and
bad times.
Special thanks go to all people at Sir James Spence Institute for their technical help and
support. I would in particular thank Stefano and Alicia for our friendship: thank you for
the moral support and the all the laughs we shared in between experiments!
I would like to thank the NECCR, Children with cancer UK, Brain Trust, Love Oliver,
CCLG, Karen and Iain Wark, The Smiley Ridley Fund, whose financial support made
this project possible.
Finally, I would like to thank my family: my Mum Chiara, Lorenzo, Giulia and Al.
Thank you for supporting me especially in the bad times and for all your caring: without
your encouragement I would not have made it.
My final thanks are reserved for my Dad Roberto: your love for knowledge inspires me
to continue my studies and accomplish my dreams.

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Dedication
This thesis is dedicated to the memory of my Dad, Roberto Finetti.

How I wish, how I wish you were here.
We're just two lost souls swimming in a fish bowl, year after year,
Running over the same old ground.
What have we found?
The same old fears.
Wish you were here
-Pink Floyd-

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Abstract
Introduction: Malignant Rhabdoid Tumours (MRT) are unique malignancies caused by
biallelic inactivation of a single gene (SMARCB1). SMARCB1 encodes for a protein that
is part of the SWI/SNF chromatin remodelling complex, responsible for the regulation
of hundreds of downstream genes/pathways. Despite the simple biology of these
tumours, no studies have identified the critical pathways involved in tumourigenesis.
The understanding of downstream effects is essential to identifying therapeutic targets
that can improve the outcome of MRT patients.
Methods: RNA-seq and 450K-methylation analyses have been performed in MRT
human primary malignancies (n > 39) and in 4 MRT cell lines in which lentivirus was
used to re-express SMARCB1 (G401, A204, CHLA-266, and STA-WT1). The MRT cell
lines were treated with 5-aza-2 -deoxycytidine followed by global gene transcription
analysis (RNA-seq and 450K-methylation) to investigate how changes in methylation
lead to tumourigenesis.
Results: We show that primary Malignant Rhabdoid Tumours present a unique and
distinct expression/methylation profile which confirms that MRT broadly constitute a
single and different tumour type from other paediatric malignancies. However, despite
their common cause MRT can be can sub-group by location (i.e. CNS or kidney). We
observe that re-expression of SMARCB1 in MRT cell lines determines
activation/inactivation of specific downstream pathways such as IL-6/TGF beta. We
also observe a direct correlation between alterations in methylation and gene expression
in CD44, GLI2, GLI3, CDKN1A, CDKN2A and JARID after SMARB1 re-expression.
Loss of SMARCB1 also promotes expression of aberrant isoforms and novel transcripts
and causes genome-wide changes in SWI/SNF binding.
Conclusion: Next generation transcriptome and methylome analysis in primary MRT
and in functional models give us detailed downstream effects of SMARCB1 loss in
Malignant Rhabdoid Tumours. The integration of data from both primary and functional
models has provided, for the first time, a genome-wide catalogue of SMARCB1
tumourigenic changes (validated using systems biology). Here we show how a single
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deletion of SMARCB1 is responsible for deregulation of expression, methylation status
and binding at the promoter regions of potent tumour-suppressor genes. The genes,
pathways and biological mechanisms indicated as key in tumour development may
ultimately be targetable therapeutically and will lead to better treatments for what is
currently one of the most lethal paediatric cancers.

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List of abbreviation

H2A Histone 2A
µg Microgram
µl Microliter
3’ UTR Three Prime Untranslated Region
5-azaCdR 5-Aza-2’-Deoxycytidine
A Adenine
AATK Apoptosis-Associated Tyrosine Kinase
Ab Antibody
ABR Active BCR-Related
AKT V-Akt Murine Thymoma Viral Oncogene
AML Acute Myelogenous Leukaemia
APC Adenomatous Polyposis Coli
ARID1A/ BAF250a AT Rich Interactive Domain 1A (SWI-Like)
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ATPase Adenylpyrophosphatase
ATRT Atypical Teratoid Rhabdoid Tumours
AZA 5-Aza-2-Deoxycytidine
B2M Beta-2-Microglobulin
BAF155 BRG1-Associated Factor 155
BAF170 BRG1-Associated Factor 170
BASH Beadarray Subversion Of Harshlight
BCA Bicinchoninic Acid Assay
BCL2 B-Cell CLL / Lymphoma 2
B-FABP Brain Fatty Acid-Binding Protein
BHLHE40 Basic Helix-Loop-Helix Family, Member E40
BID BH3 Domain-Containing Proapoptotic Bcl2 Family
Member
BK PDZ-Binding Kinase
BLAST Basic Local Alignment Search Tool
VII
BM1 Polycomb Complex Protein
BMI1 BMI Polycomb Ring Finger Oncogene
BP Base Pair
BRAF V-Raf Murine Sarcoma Viral Oncogene
Homologue B1
BRCA1/2 Breast Cancer 1/2
BRG1 Brahma-Related Gene 1
BRM Brahma
BTG4 B-Cell Translocation Gene 4
C Cytosines
C2 Curated Gene Sets
C6 Oncogenic Signatures
CASP8 Caspase 8, Apoptosis-Related Cysteine Peptidase
CCDC46 Coiled-Coil Domain Containing 46
CCDC74B Coiled-Coil Domain Containing 74B
CCLG Children's Cancer And Leukaemia Group
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CCNB1 Cyclin B1
CD8 (Cluster Of Differentiation 8
CDH1 Cadherin 1, Type 1, E-Cadherin (Epithelial)
CDKN1A Cyclin Dependent Kinase Inhibitor 1A
CDKN2A Cyclin-Dependent Kinase Inhibitor 2A
cDNA Complementary DNA
CDS Similarly Coding Sequences
CHD5 Chromo domain Helicase DNA Binding Protein 5
ChIP Chromatin Immunoprecipitation
ChIP-seq Chromatin Immunoprecipitation Sequencing
CLAS Classic Type Medulloblastoma
CML Chronic Myelogenous Leukaemia
CNAs Copy-Number Aberrations
CNN Copy Number Neutral
CNS Central Nervous System
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