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THE EXTENSIVE PROLIFERATION OF HUMAN

CANCER CELLS WITH EVER-SHORTER

Rebecca Dagg

A thesis submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy

Faculty of Medicine

The University of Sydney

2017

1

Statement of Originality

This is to certify that to the best of my knowledge, the content of this thesis is my own work. This thesis has not been submitted for any degree or other purposes.

I certify that the intellectual content of this thesis is the product of my own work and that all the assistance received in preparing this thesis and sources have been acknowledged.

Rebecca Dagg

i

Abstract

Cellular immortalisation is currently regarded as an essential step in malignant transformation and is consequently considered a hallmark of . Acquisition of replicative immortality is achieved by activation of a lengthening mechanism (TLM), either or the alternative lengthening of telomeres (ALT), to counter normal telomere attrition. However, a substantial proportion of , liposarcomas, retinoblastomas, and osteosarcomas are reported to be

TLM-negative. The lack of serial untreated malignant human tumour samples over time has made it impossible to examine telomere length over time and therefore determine whether they are truly

TLM-deficient, or whether this is the result of false-negative assays. Here we describe a subset (11%) of high-risk neuroblastomas (NB; MYCN-amplified or metastatic disease) that lack evidence of any significant TLM activity despite a 51% 5-year mortality rate. NB cell lines (COG-N-291 and LA-N-6) derived from such tumours proliferated for 500 population doublings (PDs) with ever-shorter telomeres (EST). COG-N-291 and LA-N-6 cells had exceptionally long and heterogeneous telomere lengths as measured by terminal restriction fragment (TRF) analysis (mean TRF of 31 and 37.8 kb, respectively) and telomere fluorescence in situ hybridisation. Both cell lines were telomerase negative during culturing and did not have elevated markers of ALT or associated . The telomeres of these cells shortened by 80 and 55 bases/PD, consistent with telomere attrition due to normal cell division, but did not reach after 500 PDs in culture. This is conclusive evidence that cells from highly malignant, lethal tumours are able to undergo continuous proliferation in spite of an EST phenotype. The EST phenotype was rescued by activation of telomerase (via transduction with hTERT expression constructs) or ALT (spontaneous occurrence of a nonsense TP53 , followed by spontaneous activation of ALT after 100 PDs). We also found that NB EST cells are very sensitive to I inhibitors indicating the potential to target the EST phenotype with topoisomerase I inhibitors in high-risk NB.

ii

Acknowledgements

The first person that I need to thank is my supervisor Dr Loretta Lau. I cannot express how grateful I am that you took a chance and employed me eight years ago. I am the scientist that I am today because of you. Thank you for your guidance and support and putting up with short deadlines with my writing.

You have been a great teacher, friend and mentor and I couldn’t have asked for a better supervisor.

I would also like to thank my co-supervisor Prof. Roger Reddel. I am constantly astounded by your replies to emails at all times of the day and I am so grateful for your guidance and insights. Thank you for always having an open door, because of you I have learnt to be a better scientist and critical thinker. The success of my PhD is in large part due to the support that I have received from both of my supervisors.

To all of the members of the CCRU past and present you have made the long hours in the lab far more enjoyable and become some of my dearest friends. I would especially like to thank Vanita for always answering my questions (no matter how many times I had to ask about the same form), Belinda for always offering me advice with a smile, and lunches and dinners spent laughing with Radikha, Jess,

Cuc, Kylie, Michael, Greg, Nick, Amanda, Oksana, Natalie, Namratha, Camilla, and Yu Wooi. Yuyan your support has helped me more than I can express and I will miss being able to chat with you at the bench.

To Peta, Sarah, Naz, Kaitlin and Amy I can’t believe we decided to start Stress whilst doing our PhD but

I’m so proud of what we achieved! Ladies you have become some of my dearest friends and I can’t imagine having survived the last few years without you. I also need to thank the past and present members of the cancer research and telomere length regulation units at CMRI for all of the advice and assistance I’ve received which have made this a better thesis.

During my PhD I have spent more time in the lab than anywhere else and I would like to thank all of my friends and family for being so understanding about the events that I’ve missed, left early or been late to, or had to reschedule around experiments. You have all been so understanding and I

iii cannot say how much I love you and appreciate your support. I would especially like to thank Chris and my grandparents for being an unending source of support, you have helped me become who I am today and I love you more than I can say. To Mum and Katherine, I don’t think that there are words to say what you mean to me, but to start with I love you. You have seen me at my best and worst during the PhD and done everything you can to make sure that I’m still sane at the end of it. Thank you for always listening, being there when I need you, and doing everything from eating cake with me to getting posters printed when I was on the other side of the world. I never doubted that I would get to the end of this because of your unending love, support and encouragement. In many ways, I think of this as something that we have achieved together, so thank you for getting onto this rollercoaster with me and helping me become the person that I am today.

iv

Publications

Dagg RA, Pickett HA, Neumann AA, Napier CE, Henson JD, Teber ET, Arthur JW, Reynolds CP, Murray

J, Haber M, Lau L MS, Reddel RR (2017). Extensive proliferation of human cancer cells with ever-shorter telomeres. Cell Reports. Vol 20;19(12): 2544-2556.

Hayward NK, Wilmott JS, Waddell N, Johansson PA, Field MA, Nones K, Patch A, Kakavand H,

Alexandrov LB, Burke H, Jakrot V, Kazakoff S, Holmes O, Leonard C, Sabarinathan R, Mularoni L, Wood

S, Xu Q, Waddell N, Tembe V, Pupo GM, Paoli-Iseppi RD, Vilain RE, Shang P, Lau L MS, Dagg RA,

Schramm S, Pritchard A, Dutton-Regester K, Newll F, Fitzgerald A, Shang CA, Grimmond SM, Pickett

HA, Jean Y. Yang JY, Stretch JR, Behren A, Kefford RF, Hersey P, Long GV, Cebon J, Shackleton M,

Spillane AJ, Saw R PM, Lopez-Bigas N, Pearson JV, Thompson JF, Scolyer RA, Mann GJ (2017). Whole landscapes of major subtypes. Nature. Vol 11;545(7653): 175-180.

Bradbury P, Turner K, Mitchell C, Griffin K, Lau L, Dagg RA, Taran E, Cooper-White J, Fabry B, O’Neill GM

(2017). The focal adhesion targeting (FAT) of p130 Crk associated substrate (p130Cas) confers mechanosensing function. Journal of Cell Science. Vol 1;130(7): 1263-1273.

Scarpa A, Chang DK, Nones k, Corbo V, Patch A, Bailey P, Lawlor RT, Johns AL, Miller DK, Mafficini A,

Rusev B, Scardoni M, Antonello D, Barbi S, Sikora KO, Cingarlini S, Vicentini C, Simpson S, Quinn MCJ,

Bruxner TJC, Christ AN, Harliwong I, Idrisoglu S, Manning S, Nourse C, Nourbakhsh E, Wilson PJ,

Anderson MJ, Fink JL, Newell F, Waddell N, Holmes O, Kazakoff SH, Leonard C, Wood S, Xu Q, Nagaraj

SH, Amato E, Dalai I, Bersani S, Cataldo I, Capelli P, Davì MV, Landoni L, Malpaga A, Miotto M, Whitehall

V, Leggett B, Khanna KK, Harris J, Jones MD, Humphris J, Chantrill LA, Chin V, Nagril A, Pajic M, Scarlett

v

CJ, Pinho A, Rooman I, Toon C, Wu J, Pinese M, Cowley M, Giry-Laterriere M, Mawson A, Humphrey

ES, Colvin EK, Chou A, Lovell JA, Jamieson NB, Duthie F, Gingras M, Muzny D, Dagg RA, Lau LMS, Lee

M, Pickett HA, Reddel RR, Samra JS, Kench JG, Merrett ND, Epari K, Nguyen NQ, Zeps N, Falconi M,

Simbolo M, Butturini G, Fassan M, Australian Pancreatic Cancer Genome Initiative, Gill AJ, Wheeler

DA, Gibbs RA, Musgrove EA, Bassi C, Tortora G, Pederzoli P, Pearson JV, Waddell N, Biankin AV and

Grimmond SM (2017). Whole-genome landscape of pancreatic neuroendocrine tumours. Nature. Vol

543: 65–71.

Henson JD, Lau L MS, Koch S, Martin La Rotta N, Dagg RA, Reddel RR (2016). The C-circle assay for

Alternative-Lengthening-of-Telomeres activity. Methods. http://dx.doi.org/10.1016/j.ymeth.2016.

08.01

Farooqi AS, Dagg RA, Choi LM R, Shay JW, Reynolds P, Lau L MS (2014). Alternative Lengthening of

Telomeres in neuroblastoma cell lines is associated with a lack of MYCN genomic amplification and with pathway aberrations. Journal of Neuro-Oncology. Vol 119(1): DOI:10.1007/s11060-014-

1456-8.

Lee M, Hills M, Conomos D, Stutz M, Dagg RA, Lau L MS, Reddel RR, Pickett HA (2014). Telomere extension by telomerase and ALT generates variant repeats by mechanistically distinct processes.

Nucleic Acids Research. Vol 42 (3): 1733-1746.

Lau L MS, Dagg RA, Henson J, Au A, Royds J, Reddel RR (2013). Detection of Alternative Lengthening of Telomeres (ALT) by telomere quantitative PCR. Nucleic Acids Research. Vol 41 (2): e34.

vi

List of Abbreviations

ɸ29 phi 29 β-actin beta-actin 32P phosphorus-32 123I-MIBG meta-iodobenzylguanidine 6-thio-dG 6-thio-2’-deoxyguanosine 36B4 acidic ribosomal phosphoprotein P0 53BP1 p53-binding 1 γ-H2AX H2AX phosphorylated at serine 139 ALT alternative lengthening of telomeres ABDIL antibody dilution buffer ACRF Australian Cancer Research Foundation ALU Short stretch of DNA, transposable element ANOVA analysis of variance APB ALT‐associated PML body ALK anaplastic lymphoma ASF1 anti‐silencing factor 1 ATM ataxia telangiectasia mutated ATO arsenic trioxide ATP adenosine triphosphate ATR ataxia telangiectasia and Rad3‐related ATRA all trans retinoic acid ATRX alpha thalassemia/mental retardation syndrome X‐linked AU arbitrary units Alt-NHEJ alternative non-homologous end joining BCA Bicinchoninic acid BLM Bloom syndrome bp BRCA1 type 1 susceptibility protein BSA bovine serum albumin C Cytosine CAF1 chromatin assembly factor 1

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CC/C-circle C‐rich telomere circle cDNA complementary DNA CDKN1B cyclin dependent kinase inhibitor 1B cells/mL Cells/millilitre ChIP chromatin immunoprecipitation CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate c- V-Myc Avian Myelocytomatosis Viral Oncogene Homolog

CO2 carbon dioxide COG Children’s Oncology Group CpG 5’-C-phosphate-G-3’ CST CTC1-STN1-TEN1 complex Ct cycle threshold DABCO 1,4 diazabicyclo(2.2. 2)octane DAPI 4',6‐diamidino‐2‐phenylindole dihydrochloride dATP deoxyadenosine triphosphate DAXX death domain‐associated protein 6 DC dyskeratosis congenital DDR DNA damage response dGTP deoxyguanosine triphosphate DKC1 gene D‐loop displacement loop DMEM Dulbecco’s modified Eagle’s medium DMs double-minute chromatin bodies DMSO Dimethylsulphoxide DNA deoxyribonucleic acid DNAse Deoxyribonuclease DNMT DNA methyltransferase: 1, 3a or 3b dNTP deoxyribonucleotide triphosphate DOX Doxorubicin dsDNA double-stranded DNA DTT Dithiothreitol dTTP deoxythymidine triphosphate EDTA ethylenediaminetetraacetic acid EGTA ethyleneglycoltetraacetic acid

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EFS event-free survival EST ever-shorter telomeres ExoI I F Forward FANCA Fanconi anaemia group A FAND2 Fanconi anaemia group D2 FBS fetal bovine serum FEN1 1 FFPE formalin-fixed paraffin-embedded FISH fluorescence in situ hybridisation g Gravity G Guanine GAPDH glyceraldehyde‐3‐phosphate dehydrogenase G‐circle G‐rich telomere circle GD2 ganglioside 2 G-MCSF granulocyte macrophage colony-stimulating factor G-quadruplex secondary structures formed in guanine rich sequences GWAS genome-wide association study cell cycle phase gap 1 G2 phase cell cycle phase gap 2 H2AX variant H2AX H3 histone H3 H3.3 histone variant H3.3 H3F3A H3 Histone Family Member 3A H3K9me3 H3 trimethylated at lysine 9 H4 histone H4 H4K20me3 H4 trimethylated at lysine 20 HBG Haemoglobin HCl hydrochloric acid HEPES 4‐(2‐hydroxyethyl)piperazine‐1‐ethanesulphonic acid HIRA histone regulator A HJ Holliday junction HMTase histone methyltransferase hnRNPs heterogeneous nuclear ribonucleoproteins

ix

HP1 heterochromatin protein-1 HPV human papillomavirus hr(s) hour(s) HRP horseradish peroxidase HSRs homogeneously staining regions hTERT human telomerase HSP70 heat shock protein 70 HSP90 heat shock protein 90 hTR human telomerase RNA IARC International Agency for Research on Cancer IC50 inhibitor concentration required for 50% growth inhibition IF Immunofluorescence IgG immunoglobulin G IL-2 interleukin 2 IMDM Iscove's Modified Dulbecco's Media Indel insertion or deletion INPC International neuroblastoma pathology classification system IP Immunoprecipitation INRGSS International neuroblastoma risk group staging system INSS International neuroblastoma staging system ITS insulin-transferrin-selenium-sodium pyruvate IVA Ingenuity Variant Analysis kb Kilobase KCl potassium chloride kDa Kilodalton kg Kilogram KOH potassium hydroxide LgT large T antigen LHC-MM Laboratory of Human mesothelial medium LOH Loss of heterozygosity M Molar M/MUT Mutant MAPK mitogen-activated MEM minimum essential medium

x meta‐TIF metaphase‐TIF

MgCl2 magnesium chloride min(s) minute(s) mL Millilitre mM Millimolar MMS21 methyl methanesulphonate-sensitivity 21 MOPS 3-(N-morpholino)propanesulphonic acid MRN MRE11/RAD50/NBS1 mRNA messenger RNA MYCN V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog n number of replicates NaCl sodium chloride NaF sodium fluoride

NaHCO3 sodium bicarbonate

Na2HPO4 disodium hydrogen phosphate NaOH sodium hydroxide

Na3V04 sodium orthovanadate NB Neuroblastoma NBS1 Nijmegen breakage syndrome 1 (Nibrin) gene NCBI National Center for Biotechnology Information NEAA non-essential amino acids ng Nanogram ng/mL nanogram/millilitre ng/µg nanogram/microgram ng/µL nanogram/microliter nM Nanomolar nm Nanometre NHEJ non-homologous end joining NP-40 Nonidet P-40 NuRD nucleosome remodelling and histone deacetylation ORC origin replication complex p16INK4A cyclin-dependent kinase inhibitor 2A cyclin-dependent kinase inhibitor 1

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PAGE polyacrylamide gel electrophoresis PanNET pancreatic neuroendocrine tumour PBS phosphate-buffered saline PCNA proliferating cell nuclear antigen PCR polymerase chain reaction PCT pressure cycling technology PD(s) population doubling(s) PFA Paraformaldehyde pH potential of hydrogen PML promyelocytic leukaemia PML-NB promyelocytic leukaemia nuclear body PMSF phenylmethanesulphonyl fluoride PNA peptide nucleic acid POT1 protection of telomeres protein 1 PPTP Pediatric Preclinical Testing Program pRb PTEN phosphatase and tensin homolog PVDF polyvinylidene fluoride qPCR quantitative polymerase chain reaction R Reverse RAP1 repressor-activator protein 1 RFC RNA ribonucleic acid RNase Ribonuclease RNaseH1 ribonuclease H1 RPMI-1640 Roswell Park Memorial Institute 1640 Medium RPA RTEL1 regulator of telomere elongation 1 RT-PCR reverse transcriptase polymerase chain reaction SCG single copy gene SD standard deviation SDS sodium dodecyl sulphate sec Second SIOPEN International Society of Paediatric Oncology Europe Neuroblastoma

xii shelterin protein complex that binds the telomere SKP2 S-phase kinase-associated protein 2 SLX4 SLX4 Structure-Specific Endonuclease Subunit gene SNP single polymorphism SNV single nucleotide variation S-phase cell cycle phase- DNA synthesis SSC saline-sodium citrate ssDNA single-stranded DNA STR short tandem repeat SV structural variation SV40 Simian 40 SWATH sequential window acquisition of theoretical fragment ion spectra TBE tris-borate-EDTA TBS tris-buffered saline TC telomere content T-circle telomere circle TE tris-EDTA TEP telomerase-associated protein 1 TERC telomerase RNA component gene TERT telomerase reverse transcriptase gene TERRA telomeric repeat containing RNA TIF telomere dysfunction induced focus TIN2 TRF1- and TRF2-interacting nuclear factor 2 T-loop telomere loop TLM telomere lengthening mechanism TOPO IIIα topoisomerase III alpha TP53/p53 tumour protein 53 TPE telomere position effect TPP1 Shelterin protein encoded by the ACD gene TRAP telomere repeat amplification protocol TRF terminal restriction fragment TRF1 telomeric repeat-binding factor 1 TRF2 telomeric repeat-binding factor 2 Tris tris (hydroxymethyl) aminomethane

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T-SCE telomere-sister chromatid exchange TTBS Tween-20, tris buffered saline TZAP and BTB domain containing 48 (ZBTB48) U/µg Unit/microgram µg Microgram µg/mL microgram/millilitre µg/well microgram/well µL Microliter µm Micrometre µM Micromolar USA United States of America UTR untranslated region UV VAV2 Vav Guanine Nucleotide Exchange Factor 2 V/cm volts/centimetre V/sec volts/second v/v volume/volume WGS whole genome sequence WRN RecQ like helicase w/v weight/volume W/WT wild type XRCC3 X-Ray Repair Cross Complementing 3 gene YFP yellow fluorescent protein yr Year

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Table of Contents

Statement of Originality ...... i

Abstract ...... ii

Acknowledgements ...... iii

Publications ...... v

List of Abbreviations ...... vii

Table of Contents ...... xv

List of Figures ...... xxi

List of Tables ...... xxiii

Chapter 1: Introduction ...... 2

1.1. Telomere biology ...... 2

1.1.1. Telomeres and end-protection ...... 2

1.1.2. Telomeric chromatin ...... 5

1.1.3. Telomere replication ...... 7

1.1.4. Telomere ...... 8

1.2. Telomeres and replicative aging ...... 9

1.2.1. Senescence ...... 9

1.2.2. Immortalisation ...... 11

1.3. Telomere length regulation ...... 14

1.4. Telomere lengthening mechanisms ...... 14

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1.4.1. Telomerase ...... 14

1.4.2. Alternative Lengthening of Telomeres (ALT) ...... 19

1.4.3. Telomere lengthening maintenance negative samples ...... 35

1.5. Neuroblastoma ...... 36

1.5.1. Disease stages ...... 36

1.5.2. Risk stratification...... 37

1.5.3. Treatment ...... 41

1.5.4. Predisposition ...... 43

1.5.5. Genetic features of sporadic neuroblastoma ...... 43

1.5.6. Prognostic relevance of telomerase activity ...... 46

1.5.7. ALT in neuroblastoma ...... 48

1.6. Aims of the project...... 49

Chapter 2: Materials and Methods ...... 52

2.1. Buffers and solutions ...... 52

2.2. Patient samples ...... 52

2.3. Cell culture ...... 53

2.4. Vectors and viral transfections ...... 53

2.5. Cell proliferation assay ...... 55

2.6. Immunostaining and fluorescence in situ hybridisation (FISH) ...... 55

2.6.1. ATRX and DAXX immunofluorescence (IF) ...... 55

2.6.2. APB detection...... 58

2.6.3. MetaTIF assay ...... 59

xvi

2.6.4. Telomere FISH on metaphase spreads ...... 61

2.6.5. Telomere sister chromatid exchange (T-SCE) analysis ...... 61

2.7. DNA extraction and quantitation ...... 62

2.8. C-circle assay ...... 62

2.8.1. Slot-blot detection ...... 62

2.8.2. Exonuclease treatment of DNA ...... 63

2.8.3. Telomere quantitative polymerase chain reaction (qPCR) detection ...... 64

2.9. Telomere length analysis ...... 65

2.9.1. Telomere restriction fragment (TRF) analysis ...... 65

2.9.2. Telomere qPCR ...... 65

2.10. T-circle detection ...... 67

2.11. Telomere chromatin immunoprecipitation (ChIP)...... 67

2.11.1. Cell lysis and chromatin isolation...... 67

2.11.2. Immunoprecipitation (IP) ...... 68

2.11.3. DNA isolation ...... 68

2.11.4. Quantitation ...... 68

2.12. Labelling probes with 32P ...... 69

2.13. Whole genome sequencing (WGS) and bioinformatics ...... 69

2.13.1. WGS and read alignment ...... 69

2.13.2. Variant annotations ...... 71

2.14. Sanger sequencing ...... 72

2.15. RNA extraction ...... 73

xvii

2.16. qPCR analysis ...... 73

2.16.1. Reverse transcriptase qPCR (RT-qPCR) ...... 73

2.16.2. Genomic qPCR ...... 73

2.17. Telomeric repeat-containing transcript (TERRA) ...... 74

2.18. Telomerase activity ...... 74

2.18.1. Protein Lysis ...... 74

2.18.2. Immunoprecipitation ...... 75

2.18.3. Telomeric repeat amplification protocol (TRAP) ...... 75

2.19. Immunoblotting ...... 75

2.20. Statistical analyses ...... 77

Chapter 3: A subgroup of high-risk MYCN non-amplified neuroblastomas lacks a telomere lengthening mechanism ...... 79

3.1. Introduction ...... 79

3.2. Results ...... 80

3.2.1. Unique subgroup of high-risk MYCN non-amplified neuroblastoma ...... 80

3.2.2. ALT-negative and telomerase-negative cancer cell lines ...... 84

3.3. Discussion ...... 97

Chapter 4: Long-term proliferation occurs in cancer cells with ever-shorter telomeres ...... 101

4.1. Introduction ...... 101

4.2. Results ...... 102

4.2.1. Cells with ever-shorter telomeres are capable of long-term proliferation ...... 102

4.2.2. Characteristics of EST cells ...... 102

xviii

4.2.3. Genetics of EST cells ...... 109

4.3. Discussion ...... 111

Chapter 5: Activation of a telomere lengthening mechanism rescues the ever-shorter telomere phenotype ...... 115

5.1. Introduction ...... 115

5.2. Results ...... 115

5.2.1. Activation of telomerase rescues the EST phenotype ...... 115

5.2.2. Activation of ALT rescues the EST phenotype ...... 120

5.2.3. EST neuroblastoma cell lines are unable to generate t-circles ...... 124

5.3. Discussion ...... 127

Chapter 6: Neuroblastoma EST cells are sensitive to topoisomerase inhibitors ...... 131

6.1. Introduction ...... 131

6.2. Results ...... 132

6.2.1. EST cells are sensitive to topoisomerase inhibitors ...... 132

6.2.2. EST cells do not exhibit increased cell death with potential ALT inhibitors or after induction

of replication stress ...... 136

6.2.3. EST cells have alterations to genes involved in DNA damage repair and replication ...... 137

6.3. Discussion ...... 137

Chapter 7: Discussion ...... 143

7.1. The ever-shorter telomeres phenotype in human cancer ...... 143

7.2. Targeting telomere biology in the clinic ...... 145

7.3. Conclusion ...... 146

xix

Chapter 8: References ...... 149

Appendix I: Information Relating to Chapter 2 ...... 193

Appendix II: Data Pertaining to Chapter 4 ...... 199

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List of Figures

Figure 1.1. Structure of the human telomere...... 4

Figure 1.2. Telomere length during cell division...... 13

Figure 1.3. Potential templates of mediated telomere lengthening...... 27

Figure 1.4. Prevalence of ALT in human cancer based on tumour tissue type...... 33

Figure 2.1. Schematic of neuroblastoma (NB) tumours used in this study based on MYCN amplification status...... 53

Figure 3.1. A subgroup of high-risk MYCN non-amplified neuroblastoma with long telomeres are ALT- negative...... 81

Figure 3.2. A unique subgroup of high-risk ALT-negative/MYCN non-amplified/TC≥15 NBs have poor survival...... 83

Figure 3.3. Two neuroblastoma cell lines (COG-N-291 and LA-N-6) have long and heterogeneous telomere lengths similar to the ALT-negative/TC≥15 tumour group...... 86

Figure 3.4. The COG-N-291 and LA-N-6 cell lines are phenotypically similar to the ALT-negative/TC≥15 tumour group...... 89

Figure 3.5. The cell lines COG-N-291 and LA-N-6 lack characteristic of ALT...... 90

Figure 3.6. COG-N-291 and LA-N-6 lack DNA damage at the telomere and characteristics of ALT. .... 92

Figure 3.7. The level of telomere bound does not differ between ALT neuroblastoma cell lines and COG-N-291 and LA-N-6...... 93

Figure 3.8. Some melanoma tumours are ALT-negative with TC≥15...... 95

Figure 3.9. ALT-negative/long telomere melanoma cell lines are telomerase positive...... 96

Figure 4.1. Two neuroblastoma cell lines (COG-N-291 and LA-N-6) are capable of long term proliferation despite the ever-shorter telomere (EST) phenotype...... 103

Figure 4.2. EST cells are capable of proliferation for hundreds of population doublings...... 104

xxi

Figure 4.3. Characteristics of EST cells during continuous proliferation for 600 population doublings.

...... 105

Figure 4.4. A subpopulation of COG-N-291 cells are responsible for sporadic increases in C-circles in the first 300 PDs of culture...... 107

Figure 4.5. Periodically a population of COG-N-291 cells activate an ALT mechanism but a slower proliferation rate prevents them outgrowing the EST cells...... 108

Figure 4.6. Characteristics of COG-N-291 sublines during continuous long-term proliferation...... 110

Figure 5.1. Retroviral transduction of hTERT results in telomerase activity in LA-N-6 cells...... 116

Figure 5.2. Ectopic expression of telomerase rescues the EST phenotype...... 118

Figure 5.3. No change to ALT characteristics following ectopic expression of hTERT...... 119

Figure 5.4. A LA-N-6 subline spontaneously acquired a premature stop codon in TP53...... 120

Figure 5.5. Activation of ALT results in rescue of the EST phenotype...... 122

Figure 5.6. Loss of p53 results in activation of ALT in an EST culture (LA-N-6)...... 123

Figure 5.7. LA-N-6 MUT p53 cells did not activate ALT due to aberrations to ATRX or DAXX...... 125

Figure 5.8. EST cells do not form t-circles after activation of ALT or extreme telomere lengthening by telomerase activity...... 127

Figure 5.9. EST cells express genes involved in t-circle formation...... 128

Figure 6.1. EST cells are sensitive to topoisomerase inhibitors...... 133

Figure 6.2. Telomere biology does not influence sensitivity of NB cells to common NB therapies. .. 135

Figure 6.3. Telomere biology does not predict the response of cell lines to inhibitors that cause replication stress or reduce factors critical to the ALT mechanism...... 139

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List of Tables

Table 1.2. Neuroblastoma risk group classification for stages L1/L2, L1 and MS...... 39

Table 1.3. Neuroblastoma risk group classification of stage L2...... 39

Table 1.4. Neuroblastoma risk group classification of stage M...... 39

Table 2.1. Composition of common buffers and solutions...... 52

Table 2.2. Characteristics of NB cell lines ...... 54

Table 2.3. Telomere lengthening mechanism of control cell lines ...... 54

Table 2.4. Cell culture medium...... 56

Table 2.5. Seeding density of cell lines in the cell proliferation assay...... 58

Table 2.6. List of drugs and stock concentrations...... 60

Table 2.7. List of primers...... 66

Table 2.8. Antibodies for telomere ChIP...... 70

Table 2.8. TRAP PCR conditions...... 76

Table 2.9. Antibodies and conditions used for Western blot analysis...... 76

Table 3.1. Characteristics of 35 neuroblastoma cell lines...... 85

Table 6.1. IC50 values for common NB chemotherapeutics...... 136

Table 6.2. IC50 values for inhibitors that cause replication stress and inhibit factors associated with

ALT...... 138

Table A1.1. Primers used to Sanger sequence ATRX...... 193

Table A1.2. Primers used to Sanger sequence DAXX...... 195

Table A1.3. PCR cycling conditions for program 1...... 196

Table A1.4. PCR cycling conditions for program 2...... 196

Table A1.5. PCR cycling conditions for program 3...... 197

Table A1.6. PCR cycling conditions for program 4...... 197

Table A1.7. PCR cycling conditions for program 5...... 198

xxiii

Table A1.8. PCR cycling conditions for program 6...... 198

Table A2.1. Characteristics of high-risk NB ALT tumours (n=36)...... 199

Table A2.2. Characteristics of high-risk MYCN amplified NB tumours (n=55)...... 201

Table A2.3. Characteristics of high-risk ALT-negative/long telomere NB tumours (n=17)...... 204

Table A2.4. Characteristics of high-risk MYCN non-amplified/short telomere NB tumours (n=41). . 205

Table A2.5. Genes examined by whole genome sequencing...... 208

Table A2.6. Genetic events identified by whole genome sequencing...... 249

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Chapter 1: Introduction

1

Chapter 1: Introduction

1.1. Telomere biology

1.1.1. Telomeres and chromosome end-protection

Chromosomes contain genetic material that must be conserved for correct cellular function. The linear of mammalian cells terminate with 3-12 kilobases (kb) of tandem arrays of TTAGGG repeats (Moyzis et al., 1988), known as the telomere. The proximal 2 kb region of human telomeres consist of non-random arrays of variant (TCAGGG, TGAGGG, and TTGGGG) and canonical (TTAGGG) repeats (Allshire et al., 1989; Baird et al., 1995; Coleman et al., 1999). The telomere sequence is predominantly double-stranded with the terminal 100-200 bases on the G-rich strand ending in a 3’ single-stranded overhang (Makarov et al., 1997) (Figure 1.1.A). To prevent recognition of the telomeric

3’ overhang as a double-stranded DNA (dsDNA) break which would lead to activation of a DNA damage response (DDR) at chromosome ends or chromosome end-to-end fusion, the telomere forms a protein-

DNA complex that can induce a unique chromatin structure at the telomere known as the T-loop (Griffith et al., 1999) (Figure 1.1.B). A T-loop is formed when the 3’ overhang invades the upstream duplex telomeric DNA forming a Holliday junction (HJ) (Liu and West, 2004) before annealing to the complementary C-rich strand inducing a displacement or D-loop (Figure 1.1.B). This sequesters the single-stranded telomeric DNA and provides a protective cap that defines the end of the chromosome, ultimately preventing it initiating a DDR (de Lange, 2004).

Telomeres provide specific binding sites for a number of proteins, of which the six-subunit protein complex known as shelterin is the best characterised. The shelterin complex is involved in the formation and stabilisation of the T-loop structure and is critical to preventing a DDR at the telomere

(Liu et al., 2004a; Ye et al., 2004c) (Figure 1.1.C, D). The shelterin complex binds to telomeric DNA through three sequence-specific DNA binding proteins: telomeric repeat binding factor 1 (TRF1) and

2

3

Figure 1.1. Structure of the human telomere.

(A) The human telomere is a predominantly double-stranded sequence of (TTAGGG)n repeats that

ends in a 3’ single-stranded overhang of 100-200 bp on the G-rich strand. (B) The single-stranded 3’

overhang can invade the duplex sequence to form a T-loop. (C & D) The shelterin complex is able to

bind telomeric DNA through TRF1 and TRF2 interaction with the dsDNA of the telomere and through

POT1 binding to the ssDNA of the 3' overhang or the D-loop. TIN2 and TPP1 connect POT1 to TRF1

and TRF2 and RAP1 binds directly to TRF2.

telomeric repeat binding factor 2 (TRF2) which bind directly to double-stranded telomeric DNA

(Benarroch-Popivker et al., 2016; Bilaud et al., 1997; Broccoli et al., 1997; Chong et al., 1995; van

Steensel et al., 1998), and protection of telomeres 1 (POT1) which specifically binds to the single- stranded telomeric DNA (Baumann and Cech, 2001; Loayza et al., 2004). TRF1 mediates T-loop formation by bending the dsDNA, and this allows the single-stranded overhang to invade the duplex telomere sequence and form a HJ, a process mediated by TRF2 (Griffith et al., 1999; Stansel et al., 2001).

Telomeres represent a challenge to the DNA replication machinery and cells require TRF1 to ensure efficient telomere replication (Chastain et al., 2016; Martinez et al., 2009; Sfeir et al., 2009; Stewart et al., 2012). TRF2 prevents an ataxia telangiectasia mutated (ATM) dependent DDR (Celli and de Lange,

2005; Denchi and de Lange, 2007; Karlseder et al., 2004) through two mechanisms: firstly the formation and stabilisation of the T-loop structure (Doksani et al., 2013; Griffith et al., 1999; Poulet et al., 2009) which prevents classical non-homologous end joining (NHEJ) at telomeres (Smogorzewska et al., 2002; van Steensel et al., 1998) or cleavage of the structure by resolvases (Poulet et al., 2009) and secondly, by direct inhibition of ATM (Okamoto et al., 2013). TRF2 recruits and binds the shelterin component repressor-activator protein 1 (RAP1) to the telomere, which aids in suppressing illegitimate recombination events at telomeres (Li et al., 2000; Martinez et al., 2010; Sfeir et al., 2010; Ye and de

Lange, 2004). The fifth component of the shelterin complex, TRF1- and TRF2-interacting nuclear factor

2 (TIN2), can bind both TRF1 and TRF2 and acts to stabilise the binding of these two proteins to the

4 telomere (Houghtaling et al., 2004; Kim et al., 2004; Kim et al., 1999; Liu et al., 2004a; Ye et al., 2004c).

TPP1, the final component of the shelterin complex, directly binds to POT1 and with TIN2 mediates POT1 interaction with TRF1 and TRF2 (Frescas and de Lange, 2014a; Frescas and de Lange, 2014b; Liu et al.,

2004b; Takai et al., 2011; Ye et al., 2004b). The TPP1/POT1 complex suppresses ataxia telangiectasia and Rad3 related (ATR) dependent DNA repair (Denchi and de Lange, 2007; Wu et al., 2006), likely through exclusion of the DDR protein replication protein A (RPA) from the single-stranded overhang

(Gong and de Lange, 2010).

In addition to the T-loop, telomeres can form G-quadruplex secondary structures. G- quadruplexes are formed by G-rich DNA sequences which are able to associate with each other by

Hoogsteen base pairing (Gellert et al., 1962; Gilbert and Feigon, 1999). G-quadruplexes have been detected in telomere sequence in vitro (Sen and Gilbert, 1988; Sundquist and Klug, 1989) and in vivo in organisms including ciliates (Paeschke et al., 2008; Paeschke et al., 2005) and humans (Biffi et al., 2013).

Such structures can induce DNA damage at the telomere and hence must be unfolded by including Bloom syndrome helicase (BLM) for correct cellular functions including DNA replication (Wu et al., 2015).

1.1.2. Telomeric chromatin

DNA is condensed into chromatin, a structure comprised of DNA bound to histone proteins, to allow all genetic material to fit into the nucleus of a cell. Chromatin also provides a mechanism to regulate DNA processes such as transcription. There are two types of chromatin; heterochromatin and euchromatin.

Heterochromatin is a compact structure that renders the DNA transcriptionally inactive and is consequently associated with gene sparse regions of the chromosome (Pontecorvo, 1944). In contrast, euchromatin is less condensed and therefore found in regions of the genome that are transcriptionally active (Pontecorvo, 1944). The nucleosome is the basic subunit of chromatin and is comprised of 147 base pairs (bp) of DNA wrapped tightly around a disc-shaped histone octamer (Kornberg, 1974;

Kornberg and Thomas, 1974). A histone octamer contains two molecules each of H2A, H2B, H3 and H4

5 (Felsenfeld and Groudine, 2003; Kornberg, 1974; Kornberg and Thomas, 1974). Histones sterically prevent factors such as RNA polymerase from binding to the DNA, and chromatin remodelling is therefore required to allow transcription to occur. There are two methods of chromatin remodelling: nucleosomes can slide or be temporarily disassembled by adenosine triphosphate (ATP) dependent molecules (Cote et al., 1994; Lia et al., 2006; Saha et al., 2002; Saha et al., 2005), or the post-translational modification of histones (acetylation or ) can modulate DNA-histone binding or recruit other proteins to the nucleosome (Pogo et al., 1966; Schubeler et al., 2004).

Telomeric chromatin has features consistent with heterochromatin, including histone H3 tri- methylation at lysine 9 (H3K9me3) and histone H4 tri-methylation at lysine 20 (H4K20me3) (Garcia-Cao et al., 2004). The histone methyltransferase (HMTase) Suv39h mediates trimethylation of H3K9 (Peters et al., 2001) which recruits heterochromatin protein-1 (HP1), necessary for chromatin compaction, to the telomere and subtelomere regions (Cheutin et al., 2003). HP1 then recruits the HMTase Suv4-20h resulting in trimethylation of H4K20 (Benetti et al., 2007b). Telomeric heterochromatin can also influence the chromatin structure of reporter genes in the subtelomere resulting in gene silencing through a process known as the telomere position effect (TPE) (Baur et al., 2001; Gottschling et al.,

1990). There is a low level of H3 and H4 acetylation at sub- and telomeric regions in mortal cells (Benetti et al., 2007a). Mammalian telomeres lack CpG sequences and hence are not methylated, compared to the sub-telomeric regions which may be highly methylated by the action of the DNA methyltransferases

DNMT1, DNMT3a and DNMT3b (Brock et al., 1999; Gonzalo et al., 2006). Nucleosomes have been detected in human telomeres although they have an altered nucleosome structure compared to non- telomeric regions of the genome (Ichikawa et al., 2014; Lejnine et al., 1995; Makarov et al., 1993;

Tommerup et al., 1994). Repetitive sequences are predicted to be a difficult substrate to maintain packaged as a nucleosome, suggesting telomeres may also have rapid nucleosome turnover

(Schneiderman et al., 2009).

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1.1.3. Telomere replication

Mammalian telomeres are replicated throughout (Arnoult et al., 2010; Verdun and Karlseder,

2006; Wright et al., 1999) from replication start sites largely originating in the subtelomere (Drosopoulos et al., 2012). However, there is emerging evidence that replication can also initiate in the telomere itself

(Drosopoulos et al., 2012; Kurth and Gautier, 2010; Sfeir et al., 2009). Telomeric structures such as the

T-loop, G-quadruplexes and heterochromatin present a barrier to replication forks (Paeschke et al.,

2011). Therefore, proteins that unwind structures within the telomere or restart stalled replication forks associate with the telomere during S or G2 phase (Chawla et al., 2011; Crabbe et al., 2004; Gu et al.,

2012; Sfeir et al., 2009; Uringa et al., 2012; Vannier et al., 2013; Ye et al., 2010). To facilitate replication of the telomere, telomeric structures must unfold either in response to the DNA polymerase or by active unwinding by helicases (Vannier et al., 2012). The helicases Werner syndrome ATP-dependent helicase

(WRN), regulator of telomere elongation helicase 1 (RTEL1), and BLM are recruited to the mammalian telomere by TRF1, TRF2 and POT1 which subsequently remove T-loop and G-quadruplex structures to allow telomeric replication (Crabbe et al., 2004; Drosopoulos et al., 2015; Opresko et al., 2002; Sarek et al., 2015; Sfeir et al., 2009; Vannier et al., 2012; Wu et al., 2015; Zaug et al., 2005). There is evidence that during telomere replication single-stranded DNA (ssDNA) becomes exposed and induces the formation of a structure similar to the stalled replication fork (Verdun and Karlseder, 2006). This is detected by MRE11 and RPA which trigger an ATR/ATM-dependent response that leads to the completion of replication (Verdun and Karlseder, 2006).

Upon completion of replication, there is a small 3’ overhang caused by the inability of the replicating DNA to fill the terminal RNA primer during lagging strand synthesis (Levy et al., 1992). This generates an ATM-dependent DNA damage response, indicated by the localisation of MRE11 and ATM to the telomere during G2/M (Verdun and Karlseder, 2006). Consequently, the end of the telomere is processed by Apollo, Exo1, the CST (CTC1-STN1-TEN1) complex and POT1 (Chow et al., 2012; Huang et al., 2012; Lam et al., 2010; Stewart et al., 2012; Wu et al., 2012b; Wu et al., 2010) to generate a long 3’ overhang before the shelterin proteins and recombination proteins including RAD51, RAD52 and XRCC3

7

(X-Ray Repair Cross Complementing 3 protein) reform the T-loop at the end of the telomere (Amiard et al., 2007; Doksani et al., 2013; Stansel et al., 2001; Verdun and Karlseder, 2006).

1.1.4. Telomere transcription

Telomeres are transcribed into long non-coding RNA termed TERRA (telomere repeat-containing RNA) by RNA polymerase II from transcription initiation sites in the subtelomere (Azzalin et al., 2007;

Nergadze et al., 2009; Schoeftner and Blasco, 2008). TERRA transcripts consist of UUAGGG repeats ranging in size from 100 bases to 9 kb (Azzalin et al., 2007) and about 7% of human TERRA transcripts have a poly-A tail (Azzalin and Lingner, 2008; Porro et al., 2010). Initial reports suggested approximately

25% of telomeres in human cells transcribe TERRA from specific transcription start sites defined by CpG

DNA islands (Nergadze et al., 2009; Porro et al., 2010), although, controversially, it has recently been proposed that only one or two subtelomeres are responsible for TERRA transcription (Lopez de Silanes et al., 2014; Montero et al., 2016). Cohesin, involved in regulating sister-chromatid cohesion, and CTCF, a chromatin organising factor, are components of human subtelomeres which are involved in the regulation of TERRA expression (Deng et al., 2012). DNMT3B, a DNA methyltransferase which regulates the methylation of repetitive sequences (Hansen et al., 1999), has also been implicated in the control of

TERRA transcription (Yehezkel et al., 2008). Telomere length is involved in the regulation of TERRA levels via telomeric H3K9me3 density (Arnoult et al., 2012). Interaction between TRF1 and RNA polymerase II is also involved in regulating TERRA levels (Schoeftner and Blasco, 2008). TERRA expression is associated with the cell cycle, with levels peaking during early S phase before decreasing towards the end of S phase/G2 (Arnoult et al., 2012; Flynn et al., 2011; Porro et al., 2010). The low TERRA levels in late S phase facilitate DNA replication of the telomeres and as cells pass through mitosis and into the following G1 phase TERRA expression increases again (Porro et al., 2010).

TERRA transcripts are retained at telomeres post-transcription (Azzalin et al., 2007; Ho et al.,

2008; Schoeftner and Blasco, 2008) and can form DNA-RNA hybrids due to the GC-rich nature of the sequences (Arora et al., 2014; Balk et al., 2013; Pfeiffer et al., 2013). Telomere DNA-RNA hybrids are

8 regulated by the RNA endonuclease RNaseH1 (Arora et al., 2014). TERRA is also able to form G- quadruplex structures and can bind to telomere interacting proteins (Biffi et al., 2014; Xu et al., 2010).

Heterogeneous nuclear ribonucleoproteins (hnRNPs) bind to telomeric ssDNA (Lopez de Silanes et al.,

2010), an interaction regulated by TERRA (Yamada et al., 2016). TERRA assists in T-loop formation by promoting POT1 binding to the telomeric ssDNA after DNA replication through its interaction with hnRNPA1 which will lead to RPA displacement from the ssDNA to allow POT1 binding (Flynn et al., 2011).

Telomeric transcripts can bind to TRF1 and TRF2 (Azzalin et al., 2007; Deng et al., 2009; Schoeftner and

Blasco, 2008) and telomeric heterochromatin formation is in part mediated by the interaction of TRF2 and TERRA (Deng et al., 2009). This interaction also recruits the origin replication complex (ORC) to the telomere for the initiation of replication and formation of heterochromatin (Deng et al., 2009).

1.2. Telomeres and replicative aging

1.2.1. Senescence

Normal somatic cells have a limited proliferative capacity in vitro (Hayflick, 1965; Hayflick and

Moorhead, 1961). As proliferation ceases the cells undergo numerous biochemical and morphological changes as they enter a state of permanent growth arrest known as senescence. It was postulated by

Olovnikov that the end replication problem was responsible for the initiation of senescence (Olovnikov,

1971). The end replication problem refers to the inability of the DNA replication machinery to fully replicate the end of linear DNA molecules. DNA are unidirectional and require a primer to initiate synthesis therefore, the gap caused by the terminal RNA primer cannot be filled which ultimately results in the loss of telomeric sequence during each round of cell division (Levy et al., 1992). However, this process generates only a small overhang, which is converted to a 100-200 bp overhang by an exonuclease that acts in the 5’ to 3’ direction, shortening the C-rich telomere strand and thereby increasing the length of the G-rich single-stranded telomeric tail (Makarov et al., 1997). In fibroblasts telomere lengths decrease by 31-85 bp/population doubling (PD) (Harley et al., 1990) and mean telomere lengths decrease with age in the human population (Hastie et al., 1990; Lindsey et al., 1991).

9

Thus, the terminal telomere sequence is lost at the end of each cell division until the cell is prompted to exit the cell cycle.

Cells require functional telomeres to form structures such as the T-loop to prevent chromosome end fusions or recognition of the end of the chromosome as a dsDNA break. However, as normal cell division erodes the telomere sequence the telomeres become critically short and dysfunctional.

Depletion of shelterin proteins results in a dissociation between telomere length and the induction of senescence (Karlseder et al., 2002; van Steensel et al., 1998) implying that alterations to the telomere structure, and not just shorter telomere lengths, result in activation of senescence due to the DDR that forms at a subset of telomeres (d'Adda di Fagagna et al., 2003). This DDR is visualised by the localisation of DDR components such as p53-binding protein 1 (53BP1) or phosphorylated histone H2AX (γ-H2AX) to the telomere where they form a telomere dysfunction induced focus (TIF) (Takai et al., 2003).

Visualisation of telomeres and γ-H2AX in metaphase spreads allow an accurate quantification of TIFs

(Cesare et al., 2009; Thanasoula et al., 2010). In young primary human cells a very low frequency of TIFs is observed however, they accumulate in presenescent cells until at least five foci are present in senescent nuclei (Kaul et al., 2012). Consequently, it has been postulated that telomeres exist in three states (Cesare et al., 2013). The first state is a ‘closed-state’ where telomeres form structures that prevent a DDR at the end of the chromosome. If the structure fails to form, the cells have an

‘intermediate-state’ telomere which induces a DDR but is still able to prevent repair by end-joining; if a sufficient number of intermediate-state telomeres is present, then senescence is induced, associated with changes in the level of histones and consequently the chromatin architecture (O'Sullivan et al.,

2010). The ‘uncapped-state’ allows chromosome end fusions to occur when cells continue to proliferate beyond the normal induction of senescence.

Dysfunctional telomeres are only one of the stimuli that can induce senescence. Indeed, chronic activation of a DDR regardless of the genomic site will induce senescence (Nakamura et al., 2008).

Double-stranded DNA breaks, potent activators of senescence, can be the result of ionising radiation,

10 topoisomerase inhibitors, and other types of cytotoxic chemotherapies (Chang et al., 2002; Novakova et al., 2010; Robles and Adami, 1998; Schmitt et al., 2002; Sedelnikova et al., 2004). Oxidative stress results in DNA damage to a nucleotide or a ssDNA break which gets converted to a dsDNA break during or DNA replication (Kuzminov, 2001; Wilstermann and Osheroff, 2001). The resulting dsDNA break can ultimately initiate senescence. Oxidative stress has also been implicated in telomere shortening by inducing damage at the G-rich sites of the telomere sequence (Kawanishi and

Oikawa, 2004). Therefore, senescence is frequently induced as a result of directly or indirectly generated dsDNA damage.

Senescence can also be induced by upregulated or unbalanced mitogenic and proliferation- associated signals. Chronic activation of the mitogen-activated protein kinase (MAPK) signalling pathway will induce senescence in normal cells (Lin et al., 1998). Mutations to the Ras pathway including the H-RASV12 mutant (Serrano et al., 1997) and oncogenic mutations to BRAF (Michaloglou et al., 2005) also result in senescence when the p53 and p16INK4A pathways are intact. Subsequent studies determined that senescence is mediated by activation of the DNA damage signalling pathway in response to oncogenic mutations (Bartkova et al., 2006; Di Micco et al., 2006; Mallette et al., 2007).

There is emerging evidence that genes frequently altered in cancer, including c-Myc (Wu et al., 2007),

CDKN1B (Lin et al., 2010), SKP-2 (Lin et al., 2010) and PTEN (Chen et al., 2005), are involved in mediating senescence in normal cells.

1.2.2. Immortalisation

Senescence is primarily established or maintained by activation of the p53/p21 and p16INK4A/pRB pathways (Beausejour et al., 2003). Therefore, the p53, pRb and p16INK4A proteins are considered tumour suppressor proteins and inactivation of their normal function is associated with an increased proliferative capacity in cells (Beausejour et al., 2003; Gire and Wynford-Thomas, 1998). The normal function of p53 can be abrogated as the result of spontaneous loss of the wild type TP53 allele of Li-

Fraumeni cells (a syndrome where one TP53 allele contains a null-mutation) (Rogan et al., 1995),

11 transduction with mutated TP53 (Bond et al., 1994), or infection with human papillomavirus (HPV).

Regardless of the mechanism, loss of p53 function results in proliferation beyond the PDs that generally induce senescence (Bond et al., 1999). Adenovirus, HPV and Simian virus 40 (SV40) are DNA that express proteins capable of binding to and inactivating p53 and pRB tumour suppressor proteins. When the SV40 T-antigen (binds p53 and pRB), E1A/E1B (binds pRB/p53 respectively) adenovirus genes, or the

E6/E7 (binds p53/pRB respectively) HPV genes are artificially expressed in human fibroblasts the cells have an increased proliferative capacity (Shay et al., 1991). Loss of up- or down-stream genes in the p53 or pRB pathways also result in the bypass of senescence (Brown et al., 1997; Dimri et al., 2000;

Huschtscha et al., 1998; O'Loghlen et al., 2015; Okamoto et al., 1994; Vogt et al., 1998). Therefore, a critical step in the development of cells with indefinite replicative potential, or cellular immortality, is inactivation of the p53 or pRB pathways (Duncan et al., 1993; Lehman et al., 1993; Rogan et al., 1995;

Shay et al., 1995).

With bypass of senescence by inactivation of the p53 or pRB pathways the telomere sequence continues to be eroded during each round of cell division. The short, unprotected telomere ends are able to undergo classic NHEJ or alternative end joining (alt-NHEJ) (Rai et al., 2010; Sfeir and de Lange,

2012) when there are <13 pure TTAGGG repeats distal to the final telomere variant repeat (Capper et al., 2007). The resultant di-centric chromosomes lead to chromatin bridges between the daughter cells during cell division and the resolution of these bridges causes genomic instability (Artandi et al., 2000;

Soler et al., 2005). The telomeres will eventually become critically short and induce the cells to enter a period of crisis where most cells will undergo (Girardi et al., 1965). In the case of human fibroblasts, approximately 1 in 107 cells escape crisis (Huschtscha and Holliday, 1983) by activation of a telomere lengthening mechanism (TLM) which stabilises the telomere lengths and allows unlimited proliferation of the cells hence the immortal nature of post-crisis cells (Counter et al., 1992; Meyerson et al., 1997; Rogan et al., 1995) (Figure 1.2). Cellular immortalisation is a hallmark of cancer (Hanahan and Weinberg, 2000; Hanahan and Weinberg, 2011) and to date two TLMs have been described:

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Figure 1.2. Telomere length during cell division. Cells that have constitutively active telomerase activity such as embryonic stem cells may

completely maintain telomere length (blue line). Stem cells that express tightly regulated levels of

telomerase partially maintain telomere length (green line). In normal somatic cells telomere

erosion occurs as a result of normal cell division (grey line) until cells reach senescence. A

proportion of cells may acquire an abrogation to the p53 or pRb pathway that allows the cells to

overcome senescence. Cell division continues until cells enter crisis. A small proportion of cells may

escape crisis through activation of a telomere lengthening mechanism which allows continued cell

division without telomere length erosion (red line).

telomerase (Greider and Blackburn, 1989) and alternative lengthening of telomeres (ALT) (detailed in section 1.4.).

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1.3. Telomere length regulation

The telomere length of normal human cells is determined by a balance of lengthening and shortening events (Gilson and Londono-Vallejo, 2007). Overlengthening of telomeres by activation of a TLM results in telomere trimming and the generation of t-circles (Pickett et al., 2009). A t-circle refers to double- stranded circular telomeric DNA that has a nick in both the C- and G-rich strands preventing the structure from becoming supercoiled (Cesare and Griffith, 2004; Wang et al., 2004). They are generated when telomere lengths are extended past a threshold length, likely due to resolution of the HJ formed by the

T-loop (Pickett et al., 2009). A number of genes are involved in the regulation of t-circles (XRCC3

(Compton et al., 2007; Wang et al., 2004), NBS1 (Compton et al., 2007; Wang et al., 2004), SLX4 (Sarkar et al., 2015), ZBTB48 (encodes TZAP) (Li et al., 2017) and RTEL1 (Deng et al., 2013)) and their involvement in homologous recombination and DNA replication supports this hypothesis. Recently, TZAP has been implicated in the initiation of telomere trimming as long telomeres have a reduced concentration of shelterin proteins which allows TZAP to bind to the telomere, and it is this switch from shelterin to TZAP that is proposed to initiate telomere trimming (Li et al., 2017). T-circles are conserved in

(Pickett et al., 2011; Rivera et al., 2017), yeast (Lustig, 2003), and plants (Watson and Shippen, 2007) and observed in normal human and mouse cells (Pickett et al., 2011) indicating that telomere trimming is a conserved process with a role in normal telomere biology. This process is tightly controlled and does not result in activation of a DNA damage response or telomere fusions (Pickett et al., 2009).

1.4. Telomere lengthening mechanisms

1.4.1. Telomerase

1.4.1.1. Structure and function

In 1985 Greider and Blackburn discovered an in Tetrahymena capable of synthesising telomeric sequence (Greider and Blackburn, 1985) by reverse transcribing the template region of its RNA subunit

(Greider and Blackburn, 1989) and in 1989 Morin described the human equivalent, telomerase (Morin,

14

1989). Telomerase, a ribonucleoprotein, is comprised of the catalytic reverse transcriptase domain hTERT (human telomerase reverse transcriptase) (Nakamura et al., 1997), the human telomerase RNA hTR (Feng et al., 1995), and the associated protein dyskerin (Cohen et al., 2007). In vitro studies indicate that telomerase is a dimer comprised of two molecules each of hTERT, hTR and dyskerin (Cohen et al.,

2007; Sauerwald et al., 2013; Wenz et al., 2001) although there is debate about whether a higher order structure is strictly necessary for telomerase function as several studies indicate that telomerase is functional as a monomer (Alves et al., 2008; Errington et al., 2008). Experiments to isolate subunits of the enzyme responsible for telomere elongation in yeast utilised mutants defective for telomere addition (Lundblad and Szostak, 1989). This led to a continual loss of telomere length and ultimately senescence, a phenotype known as ever shorter telomeres (EST) with the gene subsequently termed EST1. Further studies identified other EST genes including EST2 which was subsequently identified as the catalytic component of telomerase in S.cerevisiae (Counter et al., 1997;

Lendvay et al., 1996).

The RNA subunit of telomerase permits de novo addition of six nucleotide repeats of telomere sequence, 5’-GGTTAG-3’, to the 3’ single-stranded overhang of telomeres. The hTR transcript is comprised of 451 with an eleven nucleotide (5’-CUAACCCUAAC-3’) template region (Feng et al., 1995), encoded from the TERC gene. The hTR RNA is composed of four domains; a pseudoknot domain (CR2/CR3), a H/ACA box domain (CR6/CR8), CR4/CR5 domain, and C7 domain (Chen et al., 2000).

The H/ACA box domain is a characteristic of small nucleolar RNA and the region also contains a targeting sequence (CAB box) which facilitates localisation and retention of telomerase to Cajal bodies (Ganot et al., 1997). Cajal bodies are subnuclear structures that contain several proteins such as coilin and TCAB1. They provide a site for RNA processing and ribonucleoprotein assembly (Bauer and

Gall, 1997; Jady et al., 2003) and facilitate the recruitment of telomerase to telomeres.

The reverse transcriptase hTERT features: a reverse transcriptase motif located in the C-terminal end of the protein, a telomerase specific region (T motif), dissociates activities of telomerase (DAT)

15 domain, the telomerase essential N-terminal (TEN) domain and the N-terminal end of the protein

(Armbruster et al., 2001; Moriarty et al., 2002; Nakamura et al., 1997; Xia et al., 2000). hTERT N-terminal regions are essential for hTR binding (Moriarty et al., 2002; Xia et al., 2000), whilst the C-terminus of hTERT and the TEN domain are responsible for the of the telomerase enzyme (Akiyama et al., 2015; Bryan et al., 2000; Hossain et al., 2002; Peng et al., 2001), and the DAT domain is essential for correct telomerase localisation to the telomere (Armbruster et al., 2004).

Telomerase requires accessory proteins for in vivo synthesis, subcellular localisation and function. The proteins dyskerin, NHP2, NOP10, GAR1, Nopp140, NAF1, pontin/reptin and TCAB1 associate with the hTR/hTERT complex (Venteicher et al., 2009; Venteicher et al., 2008). The H/ACA ribonucleoproteins dyskerin, GAR1, NOP10 and NHP2 form a complex that can bind to H/ACA such as hTR (Ganot et al., 1997; Henras et al., 1998; Watkins et al., 1998). This complex is required to stabilise hTR and facilitate the synthesis of telomerase. Pontin and reptin act as a complex that mediates telomerase synthesis through an interaction with hTERT and dyskerin (Venteicher et al., 2008). These proteins are hypothesised to accumulate and stabilise hTR, potentially by pontin’s ATPase activity, and are present at a pre-telomerase intermediate complex with hTERT. TCAB1 binds the CAB box region of hTR, accumulates telomerase at Cajal bodies, and traffics it to the telomere (Chen et al., 2015; Stern et al., 2012). Recently, additional proteins including ATR and ATM were identified as contributing to telomerase recruitment to the telomere (Lee et al., 2015; Tong et al., 2015). Therefore, accurate telomerase function in vivo requires an array of telomerase-associated proteins in addition to the enzyme components hTR, hTERT and dyskerin.

1.4.1.2. Role in cancer and normal cells

Telomerase activity in normal human and cancer cells has been extensively characterised by telomerase activity assays, most commonly the telomerase repeat amplification assay (TRAP) (Kim et al., 1994).

Telomerase activity is tightly controlled in normal human tissue, with activity detected during embryogenesis in tissues such as the liver, skin, lung and muscle which is subsequently downregulated

16 prior to birth (Ulaner and Giudice, 1997; Ulaner et al., 2001; Wright et al., 1996). However, highly proliferative tissues such as male germ cells, haematopoietic stem cells, epithelial cells including the gastrointestinal tract and hair follicle, and activated lymphocytes require tightly regulated telomerase activity to maintain their cell population (Bachor et al., 1999; Broccoli et al., 1995; Counter et al., 1995;

Harle-Bachor and Boukamp, 1996; Hiyama et al., 1996; Hiyama et al., 1995b; Ramirez et al., 1997;

Tanaka et al., 1998; Yashima et al., 1998; Yui et al., 1998).

Telomere dysfunction can result in short telomere syndromes. was the first short telomere syndrome identified and is generally caused by mutations in the genes for hTR

(Mitchell et al., 1999; Vulliamy et al., 2001a), hTERT (Armanios et al., 2005) or dyskerin (Heiss et al.,

1998) that lead to defective telomerase, excessively short telomeres and reduced proliferation of highly regenerative cells such as the bone marrow and skin (Drachtman and Alter, 1992; Tummala et al., 2015).

Mutations in telomerase genes, RTEL1, TCAB1, TIN2, TPP1 and PARN (Calado et al., 2011; Guo et al.,

2014; Kocak et al., 2014; Stuart et al., 2015; Tummala et al., 2015) have been identified in aplastic anaemia (Vulliamy et al., 2002), Hoyeraal-Hreidarsson syndrome (Knight et al., 1999; Marrone et al.,

2007), Coats plus syndrome (Anderson et al., 2012; Polvi et al., 2012), Revesz syndrome (Savage et al.,

2008), and idiopathic pulmonary fibrosis (Armanios et al., 2007; Tsang et al., 2012). Patients who have germ-line mutations in a telomerase gene also have significantly shorter telomeres than healthy age matched individuals (Alder et al., 2008; Marrone et al., 2007; Vulliamy et al., 2002; Vulliamy et al.,

2001b).

To ensure that telomerase activity only occurs in specific normal cell types there is tight regulation over the expression of hTERT and hTR. hTERT is a limiting factor for telomerase expression as it has either low expression or is absent in telomerase-negative cells and ectopic expression of hTERT in these cells is sufficient to reactivate telomerase (Bodnar et al., 1998; Counter et al., 1998b; Hahn et al.,

1999; Meyerson et al., 1997; Wen et al., 1998). Expression of hTERT can be regulated at several levels; including transcription, , and post-translational modifications (Cong et al., 1999; Cong

17 et al., 2002; Horikawa et al., 1999; Liu et al., 1999; Takakura et al., 1999). hTR expression is present at limiting levels in all cell types and can become upregulated once hTERT is expressed (Avilion et al., 1996;

Cao et al., 2008a; Cao et al., 2008b; Feng et al., 1995). Immortalisation is considered a critical step to oncogenesis, achieved when cells in crisis activate a TLM. Telomerase is responsible for the immortalisation of up to 90% of human (Kim et al., 1994; Shay and Bacchetti, 1997) by preferentially extending the shortest telomeres in the cell (Britt-Compton et al., 2009; Hemann et al.,

2001; Teixeira et al., 2004). Initially two models were proposed to explain the presence of telomerase activity in cancer cells; the expansion of a stem cell population that failed to undergo differentiation and subsequently formed cancers (Greaves, 1996), or the dysregulation of endogenously expressed telomerase to overcome crisis (Shay and Wright, 1996). Subsequent studies identified upregulation of endogenously expressed hTERT or hTR as frequent events in cancer cells suggesting telomerase activity is upregulated during tumourigenesis to bypass crisis (Bell et al., 2015; Cao et al., 2008b; Fredriksson et al., 2014; Horn et al., 2013; Huang et al., 2013; Horikawa et al., 1999; Peifer et al., 2015; Wang et al.,

1998; Wu et al., 1999; Zhang et al., 2000). Endogenous expression of hTERT can be upregulated in cancer cells through a number of mechanisms including abnormally high levels of Myc activity (Horikawa et al.,

1999; Wang et al., 1998; Wu et al., 1999), gene amplification (Cao et al., 2008b; Zhang et al., 2000),

C228T and C250T mutations in the region leading to the introduction of binding sites (Bell et al., 2015; Fredriksson et al., 2014; Horn et al., 2013; Huang et al., 2013), and structural rearrangements upstream of the promoter region that result in super- elements

(Peifer et al., 2015).

1.4.1.3. Telomerase inhibitors as anti-cancer therapeutics

Telomerase is an attractive target for anti-cancer therapies because its expression is crucial for the majority of cancers and a minority of normal cells. There are a number of strategies for telomerase anti- cancer therapeutics: the direct inhibition of telomerase, stabilisation of the telomere structure to prevent telomerase binding, or inducing cell death specifically in telomerase expressing cells (Shay and

Keith, 2008). A number of potential anti-cancer therapies have progressed to clinical trials including the

18 antisense oligonucleotide GRN163L (targeting hTR) (Chanan-Khan et al., 2008; Roth et al., 2010; Ruden and Puri, 2013), immunotherapy GV1001 (hTERT peptide based vaccine) (Bernhardt et al., 2006;

Brunsvig et al., 2011; Buanes et al., 2009; Kyte, 2009; Ruden and Puri, 2013; Shaw et al., 2010a; Shaw et al., 2010b), and immunotherapy GRNVAC1 (dendritic cell vaccine to stimulate TERT-specific CD4+ &

CD8+ cells) (DiPersio et al., 2009; Khoury et al., 2010; Ruden and Puri, 2013). However, the results of these trials are still pending and there is currently no data available suggesting that these drugs improve patient outcome by inhibiting telomerase activity (Jafri et al., 2016). There are also concerns about the length of time required for telomerase inhibitors to induce telomere shortening and hence cell death or senescence. This has led to the development of novel drugs such as 6-thio-2'-deoxyguanosine (6-thio- dG), a nucleoside analogue which can be incorporated into telomerase mediated de novo synthesised telomeric DNA, resulting in telomere dysfunction which leads to rapid cell death (Mender et al., 2015).

There is also evidence that treating cells with a combination of PARP and telomerase inhibitors reduces the time required to observe telomere shortening (Lu et al., 2014; Seimiya et al., 2005). So, whilst there are currently no telomerase targeted therapies as a standard cancer treatment, telomerase remains a potential target for the development of novel therapies.

1.4.2. Alternative Lengthening of Telomeres (ALT)

The most common TLM used by human cancers is telomerase, however, a subset of cancer cells and immortalised cell lines utilise the alternative lengthening of telomeres (ALT) to confer immortalisation.

ALT is defined as any telomerase-independent method of telomere length maintenance, which generally utilises a DNA template to synthesise de novo telomeric sequence (Dilley et al., 2016; Dunham et al.,

2000). The existence of ALT in human cells was first demonstrated by the observation that some immortalised cell lines maintain their telomere length without any detectable telomerase activity (Bryan et al., 1997; Bryan et al., 1995) and it has subsequently been estimated 10-15% of human cancers utilise the ALT mechanism to achieve unlimited proliferation (Henson et al., 2002). The molecular details of the ALT mechanism are not yet fully understood but studies have ascertained a number of characteristics that are hallmarks of the ALT mechanism.

19

1.4.2.1. Phenotypic characteristics of ALT

For over a decade since the discovery of ALT there was no biochemical assay to determine ALT activity and the only definitive way to confirm cells utilise the ALT mechanism is to examine telomere length in telomerase-negative cells over time. However, this requires serial untreated samples from the same patient which are generally impossible to procure. Hence, a combination of ALT markers have historically been used to determine ALT activity in human cells.

One of the most notable phenotypes of ALT is the presence of long and heterogeneous telomere lengths within cells. Telomere lengths, in a mass population of cells, are observable by Southern blot analysis of pulse field gel electrophoresed terminal restriction fragment (TRF) lengths, considered the gold-standard technique to measure telomere lengths from a population of cells. From this technique, the range of telomere lengths in ALT cells varies from <3 kb to >50 kb with an average length of 20 kb

(Bryan, 1997; Bryan et al., 1995; Grobelny et al., 2000; Murnane et al., 1994; Yeager et al., 1999) compared to telomerase-positive cells which have relatively homogeneous telomere lengths with an average length usually less than 10 kb (Bryan et al., 1995; Park et al., 1998). However, a large amount of high quality genomic DNA (at least 2 g) is required to perform the analysis, often a limitation when examining tumour samples. Telomere lengths within a single cell can be observed by telomere fluorescent in situ hybridisation (FISH) on metaphase spreads. The telomere probe is visible at a range of intensities within an individual ALT cell, with some chromosomes lacking a detectable signal altogether, so called signal-free ends. Comparisons of FISH and TRF analysis confirm that TRFs reflect the heterogeneous nature of telomere lengths within a single ALT cell (Lansdorp et al., 1998; Perrem et al., 2001). Alternatively, telomere FISH can be performed on interphase cells allowing the examination of telomere lengths in a range of sample types including formalin-fixed paraffin-embedded (FFPE) tissues. Although long and heterogeneous telomere lengths are a marker of ALT in most situations, ectopic overexpression of hTR and/or hTERT can result in extreme telomere lengthening and an average length comparable to ALT cells (Cao et al., 2008b; Pickett et al., 2009). Therefore, telomere length alone is not sufficient to indicate ALT activity. The ability to examine individual telomeres has also shown that

20 telomeres in ALT are very dynamic with rapid unsynchronised elongation and deletion events (Murnane and Sabatier, 2004).

Promyelocytic leukaemia nuclear bodies (PML-NBs) are present in the nuclei of the majority of mammalian cells (Bernardi and Pandolfi, 2007) and have been implicated in a number of cellular processes including cell cycle regulation and DDR (Bernardi and Pandolfi, 2003; Dellaire and Bazett-

Jones, 2004; Dellaire et al., 2006), tumour suppression (He et al., 1997), induction of senescence and apoptosis (Bernardi and Pandolfi, 2003; Ferbeyre et al., 2000; Jiang and Ringertz, 1997), transcriptional regulation (Luciani et al., 2006; Zhong et al., 2000), protein refolding and degradation (Mattsson et al.,

2001), the immune system (Zheng et al., 1998), and viral replication (Chung and Tsai, 2009). PML-NBs contain a number of proteins, including Sp100 and isoforms of PML, and in ALT cells a subset also contain telomeric DNA and shelterin proteins, in addition to DNA repair, recombination and replication factors

(Henson et al., 2002; Jiang et al., 2005; Jiang et al., 2007; Yeager et al., 1999; Zhong et al., 2007). Hence, such foci are termed ALT-associated promyelocytic leukemia nuclear bodies (APBs) (Yeager et al., 1999).

APBs associate with the termini of chromosomal telomeric DNA implying that this is a transient structure and the PML-NBs can dissociate from the telomere to allow normal cellular processes to occur

(Molenaar et al., 2003). Methionine restriction in cells leads to an increase in the number of discernible

APBs and has shown that TRF1, TRF2, TIN2, RAP1, PML, HP1 and the MRN (comprised of MRE11, RAD50 and NBS1) complex are necessary for APB formation (Henson et al., 2002; Jiang et al., 2005; Jiang et al.,

2007; Zhong et al., 2007). APB formation also requires proteins of the SMC5/6 complex (Potts and Yu,

2007). It has been hypothesised that APBs are the site of ALT activity, however, to date there is no conclusive proof that this is the case. Evidence in support of this hypothesis includes a reduction in the number of APBs following inhibition of ALT (Jiang et al., 2005; Perrem et al., 2001) by sequestration of the MRN complex (a complex involved in homologous recombination), and the increased frequency of

APBs when cells approach senescence (Fasching et al., 2007; Jiang et al., 2007; Jiang et al., 2009). There is also evidence that the intensity of telomeric FISH signals increase in a subset of APBs compared to other telomeres (Yeager et al., 1999). Additionally, APB levels increase during the G2 phase of the cell

21 cycle which is when homologous recombination occurs most frequently and is likely when the ALT mechanism is active (Grobelny et al., 2000; Wu et al., 2000). The APB assay requires interphase cells and can be performed on a range of fixed cells including FFPA tissues and has often been used, in conjunction with telomere length, to determine the frequency of ALT in tumour cohorts.

ALT cells have a high abundance of extrachromosomal telomeric repeats which can be either linear or circular in form (Cesare and Griffith, 2004; Nabetani and Ishikawa, 2009; Ogino et al., 1998;

Pickett et al., 2009; Tokutake et al., 1998; Wang et al., 2004). Extrachromosomal telomeric DNA likely results from rapid deletion events, and is a feature of ALT cells. T-circles are the product of telomere trimming (see section 1.3.) and are more abundant in ALT cells than normal cells or endogenously expressing telomerase cells (Cesare and Griffith, 2004; Fasching et al., 2005; Henson et al., 2009; Wang et al., 2004). This greater proportion of t-circles is likely due to resolution of the T-loop during telomere trimming of over-lengthened telomeres as a result of the rapid elongation events caused by the ALT mechanism (Pickett and Reddel, 2012). When telomeres are over-lengthened by telomerase, generally via overexpression of hTERT and/or hTR, abundant t-circles are also generated at levels comparable to

ALT-positive cell lines indicating it is telomere length and not a specific aspect of the ALT mechanism that results in t-circle formation (Pickett et al., 2009).

C-circles are a type of circular telomeric DNA that appear to be closely related to the ALT mechanism. C-circles are a C-rich circular telomeric sequence that is only partially double-stranded and therefore able to self-prime a rolling circle amplification assay (Henson et al., 2009). G-circles are also specific to ALT cells however, they occur in far lower frequency than C-circles (Henson et al., 2009). C- circles are a specific and quantitative marker of ALT activity, not found in mortal or telomerase-positive cells but detectable in all ALT cell lines assayed to date (Henson et al., 2009). Most importantly, treatment of ALT cells with high levels of YFP-Sp100 fusion protein to sequester the MRN complex and inhibit ALT, produced a rapid decrease in C-circle levels (Henson et al., 2009). Patients with ALT-positive osteosarcomas had detectable C-circles in their blood samples (Henson et al., 2009), signifying the

22 potential the C-circle assay has as a routine clinical test to diagnose ALT tumours. The discovery of C- circles and a simple, rapid detection method for ALT is an important development for identifying TLMs in cancer patients and facilitates the development of anti-cancer therapeutics to target TLMs.

Telomere variant repeats have also been examined in the context of ALT. Minisatellites are tandem variant repeat sequences 6-100 bp in length (Vergnaud and Denoeud, 2000). Notably, there is increased instability at the MS32 minisatellite in ALT cells although the frequency of mutation varies between cell lines (Jeyapalan et al., 2005). Further structural differences have been noted between the telomeres of ALT cells and non-ALT cells. In mortal or telomerase-positive cells, variant repeats are confined to the 2 kb region proximal to the end of the telomere (Baird et al., 1995; Coleman et al., 1999). However, homologous recombination in ALT cells has led to the dispersal of variant repeats throughout their telomeres, most frequently the TCAGGG variant but TTCGGG and GTAGGG have also been observed (Conomos et al., 2012; Varley et al., 2002). Orphan nuclear receptors have been identified at ALT telomeres through binding to the variant telomeric sequence TCAGGG, which in turn recruits the zinc finger protein ZNF827 to the telomere (Conomos et al., 2014; Conomos et al., 2012;

Dejardin and Kingston, 2009). Subsequently, the histone deacetylation and nucleosome remodelling complex NuRD is recruited to the telomere through its binding with ZNF827 which leads to telomeric chromatin remodelling and facilitates the telomere-telomere interaction required for homologous recombination and ALT activity (Conomos et al., 2014).

The telomeric transcript TERRA can form DNA-RNA hybrids with telomeres and has been found to localise to APBs (Arora et al., 2014). TERRA levels are higher in ALT-positive cells than telomerase- positive cells and these levels are associated with telomere length (Arora et al., 2014; Episkopou et al.,

2014; Kreilmeier et al., 2016; Ng et al., 2009). The association between ALT activity and TERRA level may be the result of reduced sub-telomeric methylation in ALT cells (Ng et al., 2009) or the reduced chromatin compaction at telomeres which promotes not only the ALT mechanism but telomere transcription (Episkopou et al., 2014). Similarly, depletion of the chromatin remodeller α-thalassemia

23 mental retardation X-linked (ATRX) protein, an event observed in a subset of ALT cells and tumours

(discussed in section 1.4.2.3) (Heaphy et al., 2011a; Lovejoy et al., 2012), results in decreased TERRA expression in telomerase-positive cells due to increased telomeric chromatin compaction, reduced RNA polymerase II recruitment, and decreased cohesion abundance at telomeres (Eid et al., 2015; Episkopou et al., 2014). A study in S. cerevisiae suggests that TERRA expression is sufficient to induce recombination

(Yu et al., 2014). The evidence thus suggests that TERRA may be involved in telomere maintenance, although we do not currently understand the role of TERRA in the human ALT mechanism.

Telomere-sister chromatid exchange (T-SCE) is a reflection of recombination at the telomere and has been used as a marker for ALT (Bechter et al., 2004; Londono-Vallejo et al., 2004). Whilst some studies reported that post-replicative telomere exchange events were detected at a greater frequency in ALT-positive cells compared to telomerase-positive cells (Bechter et al., 2004; Londono-Vallejo et al.,

2004), there is also evidence that some telomerase-positive cell lines have comparable T-SCE levels to

ALT cells (Vera et al., 2008). T-SCEs may be more frequent in ALT cells because of the increased number of short, more recombinogenic telomeres resulting from the ALT mechanism. However, T-SCEs can also result from dsDNA break repair in the telomere and hence are not universally indicative of ALT activity

(Doksani and de Lange, 2016).

As identification of the ALT mechanism relies on the presence of phenotypic characteristics, some more specific for ALT than others, it is important to confirm the classification via as many techniques as possible. Given the specificity, and comparative ease of detection, the C-circle assay in conjunction with telomere length testing can be rapidly used to identify ALT in tumour samples, even with limited quantities of DNA (Lau et al., 2013).

1.4.2.2. Mechanistic details of ALT

The exact details of the ALT mechanism are currently unknown. The present model proposes that the 3’ single-stranded overhang of a telomere invades double-stranded telomeric DNA and anneals to the complementary telomere strand which can act as a primer to synthesise new telomeric DNA, which is

24 then converted to dsDNA. As outlined below, there are a number of potential sources of template telomeric DNA, including: sister chromatid exchange, replication within the t-loop structure, homologous recombination from linear extrachromosomal DNA, and DNA rolling circle replication

(summarised in Figure 1.3.A, B, C, D).

Homologous recombination was first hypothesised to be involved in ALT telomere length maintenance because of the dynamic nature of the telomeres observed in ALT cells. Homologous recombination at the telomere can be detected in vivo in mammalian cells by inserting a DNA tag into one telomere and demonstrating that it is copied to other non-homologous telomeres (Dunham et al.,

2000; Neumann et al., 2013; O'Sullivan et al., 2014) or duplicated in the original location (Muntoni et al., 2009). This was observed in ALT but not telomerase-positive cells. These observations indicate that

ALT cells can use a telomere on a sister chromatid, another telomere, or even itself as a template to copy telomeric sequence (Figure 1.3.A, B, C). This process appears to rely on break-induced replication

(Dilley et al., 2016; Roumelioti et al., 2016).

Break-induced replication is a process to repair DNA damage at the termini of the chromosome

(McEachern and Haber, 2006). A recent study indicates replication factor C (RFC) loads proliferating cell nuclear antigen (PCNA) onto the exposed end of the chromosome, which is recognised as a site of DNA damage, leading to polymerase δ recruitment to the telomere (Dilley et al., 2016). This leads to one end of the break invading a template which is used for -directed telomere synthesis. In addition to linear chromosomes, circular extrachromosomal telomeric DNA can act as a template for telomere elongation via rolling circle amplification machinery (Henson et al., 2009; Natarajan and McEachern,

2002)(Figure 1.3.D). Introduction of exogenous t-circles to the telomerase negative Kluyveromyces lactis

25

26

Figure 1.3. Potential templates of homologous recombination mediated telomere lengthening. Homologous recombination can use a number of templates to mediate telomere lengthening as

proposed in the ALT mechanism. These include (A) another telomere, (B) the T-loop of the same

telomere, and possibly (C) linear extrachromosomal DNA or (D) circular extrachromosomal DNA.

resulted in incorporation of the exogenous sequence into the chromosomal telomere and subsequent spreading of the sequence by homologous recombination (Natarajan et al., 2003; Natarajan and

McEachern, 2002; Topcu et al., 2005).

The telomere templates used for homology-directed synthesis may be spatially close to the termini of the telomere for example a sister chromatid, or spatially separated, such as a distant telomere. To allow telomeres to find templates over long distances the cells require a mechanism to move telomeres within the nuclei. Loss of the shelterin protein TRF2 results in a DDR at the telomere and an increased mobility within the nuclei (Dimitrova et al., 2008). A spontaneous DDR is commonly detected at the telomere of ALT cells (Cesare et al., 2009) and a subset of ALT telomeres have directional movement which allows transient binding with each other and APBs (Molenaar et al., 2003). Of late, evidence has emerged of a HOP2-MND1- and RAD51 dependent homology search mechanism that drives rapid, directional, long-range movement of telomeres leading to telomere clustering, ultimately initiating homology based telomere synthesis (Cho et al., 2014; Zhao and Sung, 2015). It is currently unknown what proportion of the telomere ends utilise long-range movement to find template DNA for the ALT mechanism.

1.4.2.3. Genetic changes and telomeric chromatin in ALT

It has been presumed that for the ALT mechanism to occur in cells, p53 would need to be inhibited to allow recombination events to occur. There is some indirect evidence to suggest this is the case: in , 78% of ALT tumours were p53 deficient but only 21% of telomerase-positive tumours

27 lacked p53 (Chen et al., 2006). However, this finding may be specific to glioblastoma, as ALT has also been detected in other tumour types, such as primary neuroblastoma (NB) tumours which have a very low incidence of p53 deficiency (Vogan et al., 1993; Kurihara et al., 2014; Lundberg et al., 2011; Onitake et al., 2009; Valentijn et al., 2015). Of the ALT cell lines characterised to date only four have a wild type

TP53 allele and only one of these is known to have normal p53 protein levels (Henson and Reddel, 2010).

These data suggest that inhibition of p53 may be required in the majority of circumstances to permit the ALT mechanism to activate, however, it is clearly not an absolute requirement for ALT to occur.

Genes that are involved in chromatin remodelling have recently been implicated in activation of the ALT mechanism. An association between ALT-positive tumours and mutations to ATRX or Death-

Associated protein 6 (DAXX) was established in pancreatic neuroendocrine tumours (PanNETs) (Heaphy et al., 2011a; Jiao et al., 2011; Scarpa et al., 2017). Studies in other tumour types including glioblastoma

(Schwartzentruber et al., 2012), liposarcoma (Lee et al., 2015) and NB (Kurihara et al., 2014) have confirmed there is a correlation between ALT-positive tumours and ATRX mutations, although DAXX mutations are rarely observed. Mutations in the histone variant H3.3, encoded by the gene H3F3A, have also been associated with ALT (Schwartzentruber et al., 2012). ALT-positive cell lines frequently lack

ATRX expression (Bower et al., 2012; Lovejoy et al., 2012) although this is not necessarily due to loss-of- function mutations in the gene (Lovejoy et al., 2012). A study of 22 ALT cell lines indicated that loss of

ATRX expression is not necessarily the result of somatic mutations, 19/22 cell lines had loss of ATRX and/or DAXX protein but only 7/19 cell lines contained a loss-of-function mutation in ATRX and 0/19 had mutations in DAXX or H3F3A (Lovejoy et al., 2012). It is clear that loss of ATRX, DAXX and/or H3.3 expression is not solely responsible for initiation of the ALT mechanism as 14% of ALT cell lines exist with normal levels of ATRX and DAXX and knockdown of either protein did not result in ALT immortalisation of mortal cells (Lovejoy et al., 2012; Napier et al., 2015).

ATRX is a member of the ATP dependent SWI2/SNF2 family (Picketts et al., 1996) and mutations to the ATRX gene cause alpha thalassemia in patients with a normal alpha globin gene which led to the

28 hypothesis that ATRX is involved in the regulation of (Gibbons et al., 1995). ATRX is localised to PML bodies, pericentric heterochromatin, and sub-telomeric and telomeric DNA where the protein is involved in regulating DNA methylation and the chromatin structure (De La Fuente et al., 2004;

Gibbons et al., 2000; Ritchie et al., 2008). To perform its chromatin remodelling function, ATRX forms a complex with DAXX (Xue et al., 2003). DAXX can bind to H3.3 and the ATRX/DAXX complex can then deposit H3.3 into ribosomal, telomeric, and pericentric repeat sequences (Drane et al., 2010; Goldberg et al., 2010; Lewis et al., 2010). H3.3 is a histone variant associated with nucleosomes undergoing rapid turnover at transcriptionally active sites (Ahmad and Henikoff, 2002). Therefore, H3.3 may be stabilising the telomere chromatin structure as it does at other sites of rapid nucleosome turnover. Loss of either

ATRX or H3.3 in mouse embryonic stem cells resulted in an increased number of cells with five or more

TIFs (Wong et al., 2010) demonstrating that both proteins are important to maintaining telomere function. ATRX and H3.3 are involved in maintaining the heterochromatin state across the genome and mediate silencing of genetic regions including endogenous retroviral elements (Elsasser et al., 2015;

Sadic et al., 2015) and imprinted loci (Voon et al., 2015). Defective function of ATRX or H3.3 results in hypo-methylation (Voon et al., 2015) and ALT cells have reduced compaction of telomeric chromatin compared to telomerase-positive cells (Episkopou et al., 2014). However, knockdown of ATRX in isogenic telomerase and ALT-positive cells led to increased telomeric chromatin compaction suggesting other factors mediate the reduced telomeric chromatin compaction observed in ALT cells (Episkopou et al.,

2014). Furthermore, depletion of ASF1, a histone chaperone that interacts with histone regulator A

(HIRA) and the chromatin assembly factor 1 (CAF1) complex, led to the activation of ALT phenotypes in a mortal and telomerase-positive cell line providing further evidence that normal chromatin structure is critical in repressing ALT in normal cells (O'Sullivan et al., 2014).

1.4.2.4. ALT proteins

The hypothesis that ALT exploits normal homologous recombination pathways is supported by the involvement of proteins utilised in DDR, homologous recombination, and DNA replication in the ALT mechanism. Notably, a large proportion of these proteins are present in APBs lending support to the

29 hypothesis that they are the active site of the ALT mechanism (Henson and Reddel, 2010). Proteins required for homologous recombination including RAD51, RAD52, RPA, NBS1, SLX4, BLM, MRN, BRCA1 and BRIT1, have been identified in the APBs of ALT-positive cells (Bhattacharyya et al., 2009; Cesare and

Reddel, 2010; Conomos et al., 2014; Wan et al., 2013; Wilson et al., 2013; Yeager et al., 1999). The members of the MRN complex, involved in the early steps of homologous recombination (van den Bosch et al., 2003), were some of the first proteins identified as critical for telomere lengthening by ALT. The

MRN complex recruits ATM to dsDNA breaks and facilitates resection of the chromosome end to create the 3’ overhang for strand invasion and subsequent homologous recombination (Lee and Paull, 2007).

Therefore, the MRN complex may be involved in the initiation of ALT activity by recruiting ATM to the telomere and creating a larger 3’ overhang at the telomere end to be used for strand invasion of template telomeric DNA. Sp100 binds to and sequesters the MRN complex via its interaction with NBS1, and overexpression of Sp100 led to the repression of ALT, where normal cell division based telomere length attrition and a reduction in APBs was observed (Jiang et al., 2005). In support of the hypothesis that the MRN complex is critical for the ALT mechanism, depletion of any individual component of the complex resulted in loss of ALT mediated telomere maintenance (Zhong et al., 2007). The SMC5/6 recombination complex is comprised of 8 subunits, three of which (methy methanesulphonate- sensitivity 21 (MMS21), SMC5, and SMC6) are required for ALT-mediated telomere maintenance (Potts and Yu, 2007). The MMS21 SUMO ligase is required to sumoylate the telomere binding proteins TRF1,

TRF2, TIN2, and RAP1 (Potts and Yu, 2007; Wu et al., 2012a). Whilst the exact role of the SMC5/6 complex in ALT remains to be elucidated, the complex may recruit the telomere to APBs via sumoylation of shelterin proteins.

Other components of dsDNA break repair machinery and DNA replication including Fanconi anaemia group A (FANCA) (Fan et al., 2009), Fanconi anaemia group D2 (FANCD2) (Fan et al., 2009), flap enonuclease 1 (FEN1) (Saharia and Stewart, 2009), and MUS81 endonuclease (Zeng et al., 2009), have been implicated in the ALT mechanism although depletion of these proteins does not result in overall loss of telomere length due to ALT inhibition. RecQ family DNA helicases are important for the resolution

30 of atypical DNA structures and mutations to the proteins result in chromosomal abnormalities and disease in patients (Croteau et al., 2014). RecQ like proteins in humans include WRN, RECQL4, and BLM

(Ellis et al., 1995; Killoran and Keck, 2006; Yu et al., 1996), mutations in which are responsible for

Werner, Rothmund-Thomson, and Bloom syndrome, respectively. The binding partners of the BLM protein, as identified by mass spectrometry, include heat shock protein 90 (HPSP90), telomerase- associated protein 1 (TEP1), topoisomerase III alpha (TOPO IIIα), and the shelterin proteins TRF1 and

TRF2 (Bhattacharyya et al., 2009). Depletion of TOPO IIIα in ALT but not telomerase-positive cells resulted in increased cell death and DNA damage at the telomere but a concurrent reduction in the level of TRF2 protein could also account for these observations (Bhattacharyya et al., 2009; Temime-Smaali et al., 2008). Interestingly, the WRN protein is not essential to the ALT mechanism (Laud et al., 2005) despite its role in the separation of dsDNA, likely due to redundancy in the function of other RecQ helicases. Indeed, recent studies have suggested that many DDR and homologous recombination proteins maintain normal telomere function rather than, or perhaps in addition to, being required for the ALT mechanism (Fan et al., 2009; Saharia and Stewart, 2009; Saharia et al., 2010; Verdun and

Karlseder, 2006; Zeng and Yang, 2009).

1.4.2.5. ALT in cancer and normal cells

The role of ALT has not been established in normal human cells and currently the best evidence for ALT function in embryogenesis and normal somatic cells comes from murine models. Telomere length analysis of oocytes and early embryos revealed that telomeres were short in oocytes but then lengthened until blastocysts were formed (Liu et al., 2007). Telomerase activity remained consistently low until blastocyst formation, demonstrating that telomerase activity alone was not responsible for the increase in telomere length and indeed, the early cleavage embryos contained evidence of telomere sister chromatid exchange and recombination suggestive of the ALT mechanism (Liu et al., 2007).

However, there is not complete concordance with these results, as a subsequent study incorporating a tag into a single telomere only observed copying of the telomere via recombination in somatic cells and not germ-line cells (Neumann et al., 2013). This discrepancy may be due to the different detection

31 methods used by each group and the inability of the telomere tag to detect intra-telomeric recombination arising from the ALT mechanism (Neumann et al., 2013). The ALT mechanism has also been detected in a range of including mosquitoes (Mason and Biessmann, 1995), the plant

Arabidopsis thaliana (Riha et al., 2001), the worm Caenorhabditis elegans (Cheng et al., 2012; Lackner et al., 2012) and the yeasts C. cerevisiae (Lundblad and Blackburn, 1993) and K. lactis (McEachern and

Blackburn, 1996). These studies provide evidence that ALT activity is present in a range of cells and the

ALT mechanism observed in human cancers may be the result of a dysregulated normal TLM.

The prevalence of ALT in human cancers has not been as widely studied as telomerase due to the difficulty in obtaining enough tumour material for assays and the need to perform multiple assays for the identification of the ALT mechanism. However, this has changed with the recent development of a telomere quantitative polymerase chain reaction (qPCR) based assay to detect C-circles which allows ALT to be detected from as little as 30 ng of DNA (Lau et al., 2013). Lau et al. (2013) described a cut-off for C-circle levels indicative of an active ALT mechanism, detected by qPCR, which gives 100% concordance with APB assay results and the conventional C-circle assay detected by a P32-radiolabelled telomere probe, validated in a cohort of 23 cell lines and 43 tumours including glioblastoma, melanoma, and soft tissue sarcoma samples. The prevalence of ALT has been studied in cancers including osteosarcoma, gastric carcinoma, soft tissue sarcoma, astrocytoma, mesothelioma, adrenocortical carcinoma, ovarian carcinoma, melanoma, NB, medulloblastoma, breast carcinoma, colon carcinoma,

Ewings sarcoma, PanNETs and lung carcinoma (Figure 1.4. indicates prevalence of ALT according to tumour tissue type) (Bryan et al., 1997; Costa et al., 2006; Else et al., 2008; Guilleret et al., 2002; Hakin-

Smith et al., 2003; Heaphy et al., 2011b; Henson et al., 2005; Jiao et al., 2011; Johnson et al., 2005;

Matsuo et al., 2009; Montgomery et al., 2004; Omori et al., 2009; Onitake et al., 2009; Sanders et al.,

2004; Scarpa et al., 2017; Subhawong et al., 2009; Ulaner et al., 2004; Ulaner et al., 2003; Villa et al.,

2008; Yan et al., 2002).

32

Figure 1.4. Prevalence of ALT in human cancer based on tumour tissue type. The prevalence of ALT varies depending on the tumour tissue type with ALT yet to be identified in

any hematopoietic, pancreas or prostate tumours. Adapted from Dilley and Greenberg 2015.

The association between upregulation of ALT and prognosis is dependent on the tumour type.

ALT is utilised for immortalisation by approximately 50% of osteosarcomas but there is no difference in survival between ALT-positive and ALT-negative patients (Henson et al., 2005). Interestingly, 29% of liposarcomas are ALT-positive and these patients have a worse survival than telomerase-positive patients (Pricolo et al., 1996). In contrast, glioblastoma patients with ALT tumours have a longer median survival compared to non-ALT tumours (Hakin-Smith et al., 2003). The prevalence of ALT is also highly variable in different tumour types, up to 60% of astrocytomas and sarcomas (neuroepithelial and mesenchymal origin respectively) are ALT-positive compared to carcinomas (epithelial in origin) where

5-15% of tumours utilise ALT. This difference may be due to the regulation of ALT in specific cell types

(Henson and Reddel, 2010).

33

Normal cells appear to contain repressors of the ALT mechanism since the fusion of the ALT cell line GM847 and normal fibroblasts resulted in a rapid loss of telomere sequence and onset of senescence (Perrem et al., 1999). Hybridisation of ALT and telomerase-positive immortalised cell lines created cells that utilised only one telomere maintenance mechanism (Bower et al., 2012; Ishii et al.,

1999; Perrem et al., 2001). This implies that telomerase-positive cells contain a repressor of ALT and

ALT-positive cells contain repressors of telomerase (Bower et al., 2012). Hence, the tumour types with low or no ALT activity may contain repressors of ALT and tumour types with higher ALT prevalence may more tightly regulate telomerase expression.

1.4.2.6. Anti-cancer therapeutics and ALT

To date, there are no ALT targeted anti-cancer therapeutics available, although there is some debate regarding the potential to use ATR inhibitors as an anti-ALT therapy (Deeg et al., 2016; Flynn et al., 2015).

A better understanding of the mechanism and the prevalence of ALT is necessary to develop treatments and determine patient groups eligible for targeted therapies. There is also evidence that cells within a single tumour can utilise both telomerase and ALT (Bryan et al., 1997), although it is unclear if both exist in a single cell or it is the result of heterogeneity within the tumour. It is possible to artificially generate telomerase expression in ALT immortalised cell lines, and this has been used to demonstrate that both mechanisms can co-exist in a single cell (Cerone et al., 2001; Grobelny et al., 2001; Perrem et al., 2001).

Consequently, anti-cancer therapies targeted to a TLM must take into consideration whether one or both TLMs are present in the tumour.

Another concern with telomere maintenance mechanism targeted therapies is the possibility that cells can change their active mechanism if one becomes non-functional. Inhibition of hTERT in a telomerase positive cell line resulted in cells that could maintain telomere length and grow for over 200

PDs once telomerase became inactive, although the cells possessed no typical features of ALT such as

APBs or long and heterogeneous telomere lengths (Kumakura et al., 2005). This indicates, in principle, that cells have the ability to activate alternative pathways for telomere length maintenance if their TLM

34 becomes compromised. Whilst, there is no evidence to suggest that human cancers treated with a telomerase targeted anti-cancer therapeutic activate the ALT mechanism to continue telomere length maintenance, telomerase inhibition has resulted in the emergence of ALT cells in mouse cells (Hu et al.,

2012). Therefore, targeting telomerase as an anti-cancer therapeutic has the potential to select for ALT- positive cells which would be resistant to such therapies supporting the need to develop anti-cancer therapeutics against both TLMs.

1.4.3. Telomere lengthening maintenance negative samples

Immortalisation via activation of a TLM is currently regarded as a hallmark of cancer (Hanahan and

Weinberg, 2000; Hanahan and Weinberg, 2011) although, it has been postulated that immortalisation may not be required in cancers with a low level of tumour cell death or genetically simple cancers that do not require extensive clonal evolution (Kipling, 1995; Parkinson, 1996; Reddel, 2000). A human fibroblast isolated from a middle-aged person is capable of an additional 20-40 PDs (Hayflick, 1965). If every cell survives, 40 PDs can generate 240 cells which would form a tumour mass of 1 kg. However, cell death occurs in most tumours due to factors like an inadequate blood supply resulting in hypoxia and necrosis, or due to non-viable cells created during the clonal evolution of the tumour. Consequently, the majority of tumours activate a TLM to obtain the proliferative capacity to form a tumour mass. This may not be required for tumours that have long telomere lengths in their progenitor cells, in cancers such as leukaemias which do not undergo cell death due to limited blood supply, and in genetically simple cancers. In keeping with the hypothesis that immortalisation is not an absolute requirement of carcinogenesis, a substantial proportion of liposarcomas (Costa et al., 2006; Jeyapalan et al., 2008), glioblastoma (Hakin-Smith et al., 2003), retinoblastomas (Gupta et al., 1996), and osteosarcomas

(Ulaner et al., 2003) have been reported as TLM-negative. While these tumours may be truly TLM- negative, this could also be the result of false negative assays or an as-yet unidentified ALT mechanism lacking the usual phenotypic characteristics. To date, it has been impossible to test which of these scenarios is true, as the definitive test for telomere length maintenance is to examine telomere length over time which would require serial samples of untreated human tumours, samples impossible to

35 obtain. It is increasingly important to define what is happening in this group of tumours as TLM- inhibitors are moving toward clinical use and TLM-negative tumours would be resistant to such treatment. Large-scale studies are also moving towards genetic markers such as telomere length and hTERT and ATRX/DAXX mutations to define TLMs (Barthel et al., 2017; Pekmezci et al., 2017; Scarpa et al., 2017; Valentijn et al., 2015) and the molecular details of TLM-negative tumours would ensure that tumours are not misclassified.

1.5. Neuroblastoma

Neuroblastoma (NB) is an embryonal cancer that arises in the sympathetic nervous system. The disease predominantly affects infants or young children, with an annual incidence of 29 patients/million infants

<1-year-old, which is reduced to one patient/million in children 10-14 years old (Lau and Irwin, 2017).

NB is the most common extracranial solid tumour in children, responsible for 7-10% of cancers in children under 15 years, yet it accounts for 15% of deaths in paediatric oncology patients (Maris et al.,

2007).

1.5.1. Disease stages

The majority of NB patients (65%) present with a primary tumour in the abdomen, most commonly adjacent to the adrenal gland. Other sites of primary disease include the pelvis, chest and neck (Maris et al., 2007). Metastatic disease is present in nearly half of patients at diagnosis, either in lymph nodes adjacent to the primary tumour, or at distant sites such as the bone marrow, cortical bones, the liver and non-adjacent lymph nodes (Maris et al., 2007). Diagnosis can be made by the histology of the tumour or the presence of tumour cells in the bone marrow combined with elevated catecholamine levels in the urine. The tumour size and invasiveness and metastatic sites can be determined with CT or

MRI scan in conjunction with radiolabelled meta-iodobenzylguanidine (123I-MIBG).

Established in 1993, The International Neuroblastoma Staging System (INSS) (Brodeur et al.,

1993) is a classification system based on clinical and biological prognostic markers. However, a more

36 recent system of classification, the International Neuroblastoma Risk Group Staging System (INRGSS)

(Cohn et al., 2009), has been developed to ensure uniform risk stratification and comparison of clinical trials across large international bodies such as the Children’s Oncology Group (COG) and the

International Society of Paediatric Oncology Europe Neuroblastoma (SIOPEN). The INRGSS classifies NB into stages L1, L2, M, and MS. Stage L1 and L2 represent locoregional disease in the absence and presence of image-defined risk factors, respectively. Stage M (equivalent to INSS stage 4) includes all metastatic disease unless classified stage MS. Stage MS is similar to INSS Stage 4S with metastases limited to the liver, skin and bone marrow, except that it includes patients up to age 18 months and large primary tumours (L1 or L2) (Monclair et al., 2009). Stage MS, which will be referred to as stage 4S in remainder of this thesis, is a unique group of NB that has the propensity to spontaneously regress in the absence of treatment (D'Angio et al., 1971). The staging of NB reflects the heterogeneity of the disease, ranging from tumours that can spontaneously regress, to metastatic disease that conveys a 2- year event free survival of only 66% (Yu et al., 2010).

1.5.2. Risk stratification

There are a number of biological and clinical factors that need to be considered when stratifying NB patients into risk groups, including: stage, age at diagnosis, histology of the tumour, differentiation within the tumour, MYCN amplification, 11q aberration, and ploidy of the tumour. Based on these features the INRGSS classifies NB patients into 16 pre-treatment risk categories (A to R) which are further classified into risk subsets based on the 5-year event-free survival (EFS) of each category (Table

1.2); very low risk (5-year EFS >85%), low-risk (5-year EFS >75 to ≤85%), intermediate-risk (5-year EFS

≥50% to ≤75%) and high-risk (5-year EFS <50%) (Cohn et al., 2009; Monclair et al., 2009).

1.5.2.1. Histology and degree of differentiation

NB tumours are comprised of small, round blue cells with reduced cytoplasm and darkly staining nuclei.

A panel of antibodies is used to distinguish NB from other solid tumour types and the International

Neuroblastoma Pathology Classification System (INPC) uses a modified Shimada system to determine

37 the degree of Schwannian stroma (NB, stroma-poor; ganglioblastoma intermixed, stroma-rich; ganglioneuroma, stroma-dominant; or ganglioneuroblastoma, composite rich/dominant and stroma- poor), neuroblast differentiation (differentiating, poorly differentiated, or undifferentiated) and mitosis- karyorrhexis index (high, intermediate or low) of the tumour (Shimada et al., 1999). The tumours are classified into favourable or unfavourable histology groups based on the combination of these features and age at diagnosis (Shimada et al., 2001).

1.5.2.2. Age at diagnosis

The median age at diagnosis of NB is 19 months, and 90% of patients are younger than 5 years of age at diagnosis (London et al., 2005). Age at diagnosis correlates to the EFS of patients with Stage 4 MYCN non-amplified NB, with children >24 months at diagnosis (6-year EFS of 31%) having a significantly poorer prognosis than patients 12 to 18 months old at diagnosis (6-year EFS of 74%) (Schmidt et al.,

2005).

1.5.2.3. MYCN amplification

The proto-oncogene MYCN is located at chromosome 2p24 and amplification of the gene occurs in approximately 22% of primary tumours and is associated with high-risk NB across all stages of disease

(Ambros et al., 2009; Bagatell et al., 2009; Brodeur et al., 1984; Cohn et al., 2009; Seeger et al., 1985).

Amplified MYCN DNA can be found in double-minute chromatin bodies (DMs) or homogeneously staining regions (HSRs) which can be detected by MYCN FISH in the nuclei of NB cells (Corvi et al., 1994;

Schwab et al., 1984). MYCN amplification is defined as ≥10 copies of MYCN in a diploid cell or a >4-fold increase in signal compared to . MYCN oncoprotein is a transcription factor that binds to the promoter region of target genes involved in differentiation, cellular proliferation, and apoptosis, through binding to E-box recognition sites (Murphy et al., 2009). Transgenic mouse models of MYCN overexpression result in spontaneous NB, indicating MYCN expression has a role in NB oncogenesis

(Weiss et al., 1997), however, it remains to be elucidated by which mechanisms MYCN amplification results in aggressive NB tumours. Currently, there is no successful therapeutic to target MYCN but as

38

Table 1.2. Neuroblastoma risk group classification for stages L1/L2, L1 and MS. Stage L1/L2 L1 MS Age (months) <18 Any except Ganglioneuroma ganglioneuroma maturing; Histology maturing or ganglineuroblastoma ganglineuroblastoma intermixed intermixed Differentiation Non- MYCN Amplified Non-amplified Amplified amplified 11q loss of No Yes heterozygosity Ploidy INRG category A B K C Q R Risk group Very low Very low High Very low High High

Table 1.3. Neuroblastoma risk group classification of stage L2. Stage L2 Age (months) <18 ≥18 Any except ganglioneuroma maturing Histology Ganglineuroblastoma nodular or neuroblastoma or ganglineuroblastoma intermixed Poorly or un- Differentiation Differentiating differentiated

MYCN Non-amplified Non-amplified Non-amplified Amplified 11q loss of No Yes No Yes heterozygosity Ploidy INRG category D G E H H N Risk group Low Intermediate Low Intermediate Intermediate High

Table 1.4. Neuroblastoma risk group classification of stage M. Stage M Age (months) <18 <12 Dec-18 <18 ≥18 Histology Differentiation

MYCN Non-amplified Non-amplified Non-amplified Amplified 11q loss of

heterozygosity Ploidy Hyperdiploid Diploid Diploid INRG category F I J O P Risk group Low Intermediate Intermediate High High

39

Aurora kinase A is required to stabilise MYCN protein, the inhibitor MLN8237 is currently in clinical trial in combination with chemotherapy (Mosse et al., 2012) (clinical trial: NCT01601535). Inhibition of the

MYCN downstream target ornithine decarboxylase by difluoromethylorthinine is also in clinical trial

(clinical trial: NCT02030964).

1.5.2.4. Tumour ploidy and chromosome aberrations

Tumour ploidy is an independent prognostic factor, predominantly in patients less than 18 months of age (George et al., 2005; Schleiermacher et al., 2010). The ploidy of a NB tumour refers to the DNA content of the cells and is indicative of the type of genetic rearrangement that the tumour has undergone. Hyperdiploid tumours have whole chromosome gains and therefore, little structural rearrangement of chromosomes, which confers a more favourable outcome for patients (Kaneko et al.,

1987; Look et al., 1991). In contrast, diploid tumours are associated with advanced-stage disease and poor prognostic markers including MYCN amplification and loss of 1p or 11q (Attiyeh et al., 2005; Cohn et al., 2009; Kaneko et al., 1987; Look et al., 1991).

Loss of heterozygosity (LOH) of 11q has been identified in 34-45% of primary NBs (Attiyeh et al.,

2005; Guo et al., 1999; Plantaz et al., 1997; Spitz et al., 2003). 11q LOH does not correlate with MYCN amplification but is associated with high-risk features such as unfavourable histology and poor survival.

Consequently, LOH of 11q has been identified as a prognostic factor in MYCN non-amplified tumours and is used in the INRGSS to predict outcome in the stage L2 MYCN non-amplified tumours. The short arm of (1p) is deleted in 25-35% of NBs and correlates with MYCN amplification (Attiyeh et al., 2005; Brodeur et al., 1981; Christiansen et al., 2000; Kaneko et al., 1987; Plantaz et al., 1997).

Allelic loss at 1p36 is associated with an increased risk of relapse, although it is only an independent prognostic factor in low or intermediate risk tumours. Gain of additional copies of 17q, often occurs through unbalanced translocation with chromosome 1 or 11, and is associated with aggressive disease

(Bown et al., 1999; Van Roy et al., 1997). It has been suggested that changes to ploidy are required for the gain of tumour driver genes on 17q and a loss of tumour suppressor genes on 1p or 11q, however,

40 a recent study identified loss of 1p and 11q in relapse but not matched primary tumours (Eleveld et al.,

2015), suggesting these events are not tumour initiating but rather a step in aggressive tumour evolution.

1.5.3. Treatment

NB treatment may vary depending on clinical trials group. The following therapies reflect COG protocols for NB treatment. Low-risk NB has a good prognosis and a 5-year EFS of 75-85% (Monclair et al., 2009).

As such, minimal treatment is offered to patients who have tumours with favourable biology. If surgery is performed, partial resection with residual disease is not a risk for relapse in these patients (Strother et al., 2012). Chemotherapy (with or without surgery) consisting of low doses of carboplatin, cyclophosphamide, doxorubicin and etoposide is given to patients who have life-threatening symptoms, progressive disease after surgery, or unfavourable tumour biology in the resected tumour. In contrast, stage 4S NB with no high-risk markers can be observed and given supportive care. Treatment with chemotherapy is only given if the patient becomes symptomatic or the tumour has unfavourable biology as most tumours spontaneously regress without any intervention (Nickerson et al., 2000).

Intermediate risk NB have a poorer survival (5-year EFS 50-75%) than low risk patients, and are treated with chemotherapy and surgery (Baker et al., 2010). Chemotherapy can consist of 4 or 8 cycles of carboplatin, cyclophosphamide, doxorubicin and etoposide. Four cycles are given for tumours with favourable biology whilst 8 cycles are used when the biology is unfavourable.

The standard regimen for high-risk NB treatment consists of three phases: induction, consolidation, and post-consolidation (Matthay et al., 1999; Park et al., 2011). The current COG trial protocol for induction therapy involves 6 cycles of dose-intensive chemotherapy. The first two cycles include a combination of topotecan and cyclophosphamide, then a combination of etoposide and cisplatin for cycles 3 and 5, and for cycles 4 and 6 a combination of doxorubicin, cyclophosphamide and vincristine (Park et al., 2011). The response to induction therapy is a prognostic indicator for high-risk patients, and correlates with patient survival after the completion of therapy (Cheung et al., 1991).

41

Consequently, tumours are monitored with MIBG scoring systems at diagnosis and at the end of induction therapy (Yanik et al., 2013). Local resection of the tumour is recommended after 4-6 cycles of chemotherapy, although it is debatable that complete resection improves the outcome of patients

(Alonso et al., 2006; Castel et al., 2002; Du et al., 2014; Simon et al., 2013). Patients then receive consolidation therapy consisting of myeloablative chemotherapy and autologous stem cell transplantation. Myeloablative chemotherapy comprises of treatment with melphalan, followed by carboplatin and etoposide. After completion of consolidation therapy, patients may also receive radiation treatment. Though patients receive multi-modal therapy, relapse remains common in high- risk NB, suggesting that minimal residual disease is an important factor in recurrence. To treat minimal residual disease, it is standard to use non-cytotoxic differentiation therapy (treatment with 13-cis retinoic acid), as post-consolidation therapy for high-risk NB (Matthay et al., 2009b) in addition to immunotherapy with anti-ganglioside 2 (GD2) chimeric antibody (Simon et al., 2011) and

(interleukin-2 [IL-2] and granulocyte macrophage colony-stimulating factor [GMCSF]) (Yu et al., 2010).

Despite intensive treatment many high-risk patients have refractory disease or experience recurrence, and there is no curative therapy for the majority of these patients (London et al., 2011).

Indeed, stage 4 patients with relapsed disease only have a 5-year overall survival of 8% (London et al.,

2011). Relapsed patients generally present with metastatic disease, that has acquired mutations as a result of primary therapy and are consequently drug resistant (Carr-Wilkinson et al., 2010; Keshelava et al., 2001; Tweddle et al., 2001). Therefore, patients that are treated with further chemotherapy, receive agents with a mode of action distinct from therapies used in the initial treatment. Examples of relapse therapy include temozolomide with irinotecan, topotecan in combination with etoposide or cyclophosphamide, high dose 131I-MIBG therapy and a second autologous stem cell transplantation

(Kushner et al., 2006; Matthay et al., 2009a; Simon et al., 2007). Patients can also be enrolled in early phase clinical trials.

42

1.5.4. Predisposition genes

Familial NB is rare, accounting for only 1-2% of NB cases, and is generally autosomal dominant with incomplete (Kushner et al., 1986; Maris et al., 2002). Correlation between mutations and cases of familial NB led to candidate predisposition regions being located at 16p, 12p and 2p23-26 (Maris et al., 1997), ultimately resulting in the identification of the anaplastic lymphoma kinase (ALK) gene. A proportion of the ALK mutations identified in patients are the result of germline missense mutations

(Chen et al., 2008; George et al., 2008; Janoueix-Lerosey et al., 2008; Mosse et al., 2008). Patients with

NB associated with Hirschsprung disease or congenital central hypoventilation have germline PHOX2B mutations (Mosse et al., 2004; Trochet et al., 2004). Consequently, the two most common predisposition genes associated with NB tumour formation are ALK and PHOX2B. Single nucleotide polymorphisms (SNPs) that have been reported to increase the risk of NB include HACE1 (Diskin et al.,

2012), LIN28B (Diskin et al., 2012), BARD1 (Capasso et al., 2009; Pugh et al., 2013), FGFR4 (Whittle et al., 2016), CHEK2 (Pugh et al., 2013), PINK1 (Pugh et al., 2013), TP53 (Pugh et al., 2013) and LM01 (Wang et al., 2011).

1.5.5. Genetic features of sporadic neuroblastoma

Despite the ability to stratify patients there is very little understood about how these factors drive the

NB tumour. Recently, studies utilising next generation sequencing techniques have been undertaken to determine the genetic mutations that are responsible for driving NB pathogenesis in an effort to design targeted therapies. Genome-wide association studies (GWAS) have identified several SNPs that are associated with sporadic NB. Low-risk NB is associated with SNPs in DUSP12, HSD17B12, and

DDX4/IL31RA at chromosome 5q11.2 (Nguyen le et al., 2011), whereas high-risk tumours are associated with SNPs within or upstream of 6p22 (Maris et al., 2008), HACE1 (Diskin et al., 2012), LIN28B (Diskin et al., 2012), BARD1 (Capasso et al., 2009), FGFR4 (Whittle et al., 2016), and LM01 (Wang et al., 2011). A common copy number variation in chromosome 1q21 that effects NBPF23 expression was also identified in NB tumours (Diskin et al., 2009). In addition to familial NB, ALK mutations are present in

43 sporadic cases: 6-12% carry somatic mutations, 15-22% have gained copies of ALK and 3-4% have ALK amplification (Chen et al., 2008; George et al., 2008; Janoueix-Lerosey et al., 2008; Mosse et al., 2008;

Pugh et al., 2013). Recent evidence suggests that there is epigenetic regulation of ALK expression in samples with wild type ALK sequence (Gomez et al., 2015). Due to the prevalence of upregulated ALK expression in unfavourable NB, ALK has become a target of novel NB therapies. Currently the ALK inhibitor, crizotinib, is in clinical trial (Mosse et al., 2013) (clinical trial NCT01606878) and newer inhibitors are under development.

With the increased ability to perform whole exome and whole genome sequencing several genome wide studies (Cheung et al., 2012; Molenaar et al., 2012; Peifer et al., 2015; Pugh et al., 2013;

Sausen et al., 2013) have been conducted to determine the somatic alterations present in NB tumours.

As expected, genetic events previously linked to NB prognosis were identified including MYCN amplification, 1p and 11q deletion and 17q gains. A very low level of recurrent gene mutations was detected, only 12-14 amino acid affecting mutations were present per tumour (Molenaar et al., 2012;

Peifer et al., 2015; Pugh et al., 2013; Sausen et al., 2013). This is consistent with evidence that cancers in paediatric patients carry fewer genetic alterations than adult cancers (Alexandrov et al., 2013; Jones et al., 2008; Parsons et al., 2011; Vogelstein et al., 2013). The most frequently mutated genes in high- risk NB are ALK (9.2%), ATRX (9.6%), PTPN11 (2.9%), MYCN (1.7%), and NRAS (0.83%) (Pugh et al., 2013).

Of interest, alterations to the ATRX gene were seen at rates comparable to ALK (Molenaar et al., 2012;

Peifer et al., 2015; Pugh et al., 2013) and predominantly in older patients without MYCN amplification

(Cheung et al., 2012; Peifer et al., 2015; Pugh et al., 2013). This suggests that ALT may be a significant factor in NB pathogenesis, particularly for older patients. One study also identified the chromatin remodelling genes ARID1A and ARID1B as altered in 11% of tumours and associated with poor survival

(Sausen et al., 2013). The Rac/Rho and neuritogenesis pathways were identified as frequently defective in NB tumours in one study (Molenaar et al., 2012) however, this was not evident in the high-risk tumour cohort of Pugh and colleagues (Pugh et al., 2013). Whilst mutations to TP53 are one of the most common

44 events in cancer overall, the gene is rarely mutated at diagnosis in NB (Vogan et al., 1993), although 15% of recurrent tumours have acquired a TP53 mutation (Carr-Wilkinson et al., 2010).

Chromothripsis is a form of chromosomal rearrangement caused by the shattering of a chromosomal region which results in the random reassembly of most of the fragments by DNA repair mechanisms (Stephens et al., 2011). Analysis of structural rearrangements in NB identified chromothripsis in 18% of high-stage tumours and absent in low-stage tumours (Molenaar et al., 2012).

Consequently, chromothripsis correlated with poor outcome although, subsequent studies found chromothripsis in only 0-4% of NB (Peifer et al., 2015; Sausen et al., 2013). Another structural event that has been identified in 24-31% of high-risk NB is a structural rearrangement 30-50 kb upstream of the hTERT promoter on (Peifer et al., 2015; Valentijn et al., 2015). This rearrangement results in an upregulation of hTERT mRNA expression due to super-enhancer elements now adjacent to the hTERT transcription start site (Peifer et al., 2015; Valentijn et al., 2015). The implication of these studies is that NB is not a disease driven by a single mutation but rather by rare mutations, copy number variation, structural rearrangements, and possibly epigenetic changes.

Mutations to the p53 pathway have been identified as events that contribute to drug resistance and ultimately relapse in NB (Keshelava et al., 2001). Recently, next generation sequencing approaches have been used to identify novel mutations acquired by relapse tumours. Relapse tumours have a 1.5-

2-fold increase in single nucleotide variations (SNVs) compared to primary tumours (Eleveld et al., 2015;

Schramm et al., 2015). Like primary tumours, relapse is not driven by one causative genetic aberration.

Schramm and colleagues identified genes involved in the epithelial-mesenchymal transition, a process involved in cell migration, invasion and metastasis, as the most common pathway with aberrations in relapse tumours (Schramm et al., 2015) however, this has yet to be validated in another tumour cohort.

The increased allelic frequency of CHD5, a putative tumour suppressor gene, from the primary to relapse tumour suggests that clonal selection of the primary tumour occurs, likely due to therapy (Schramm et al., 2015). A similar clonal evolution was observed in relapse tumours with activating ALK mutations in

45 which both increased allelic fraction of the ALK mutation, and an increase in the overall number of tumours effected by ALK mutations (7-9% of primary tumours to 20-26% of relapse tumours) was observed (Padovan-Merhar et al., 2016; Schleiermacher et al., 2014). Activating mutations to the

RAS/MAPK pathway have been identified in primary tumours and several studies have found this is enriched in relapse tumours due to novel mutations and clonal selection (Eleveld et al., 2015; Padovan-

Merhar et al., 2016). Hence, the genome of NB tumours continually evolve under the selective pressure of treatment and biopsies of relapse tumours are required for the use of targeted therapy in these patients.

1.5.6. Prognostic relevance of telomerase activity

Telomerase expression in NB was first described over two decades ago. The initial studies utilised the

TRAP assay to determine telomerase activity. In 1995 Hiyama and colleagues detected telomerase activity in 94% of tumours although, the level of activity ranged from high (20% of tumours) to low (76% of tumours) (Hiyama et al., 1995a). Low telomerase activity was detected in the majority of stage 1 and

2 tumours whereas high telomerase activity was observed in stage 3 and 4 tumours. This correlated with a poor patient outcome in 75% of tumours with high telomerase activity but only 3.3% of tumours which exhibited low telomerase activity. Another study reported a similar number of tumours (96%) had detectable telomerase activity and that the level of activity correlated with patient outcome (Hiyama et al., 1997). It was therefore, concluded that telomerase activity could be a prognostic factor for NB.

Further studies confirmed that telomerase activity correlated with NB prognostic risk group although, the total percentage of NB tumours reported as telomerase-positive decreased compared to the initial two studies. These studies described 20% (Poremba et al., 1999), 24% (Krams et al., 2001),

29% (Poremba et al., 2000), 35% (Choi et al., 2000), and 37% (Onitake et al., 2009) of NBs as telomerase- positive. The discrepancy between studies may be the result of false-positive TRAP results due to the sensitivity of the PCR. Approximately 10% of localised NB tumours maintain telomere length via telomerase compared to nearly 60% of metastatic disease (Poremba et al., 1999). Despite the varying

46 percentages of telomerase-positive NBs there is a correlation between high telomerase activity and poor prognosis (30-45% 5-year overall survival depending on tumour cohort) (Ohali et al., 2006; Onitake et al., 2009; Poremba et al., 2000).

Telomerase activity has also been identified in some, but not all, stage 4S tumours that exhibit progressive disease (Brinkschmidt et al., 1998) and has been associated with fatal 4S tumours (Choi et al., 2000). It has been proposed that the spontaneous regression of stage 4S tumours is the result of telomere shortening and subsequent cellular senescence that occurs due to the low telomerase activity in these tumours (Brinkschmidt et al., 1998). Indeed, examination of the telomere length of progressive liver metastases biopsied from a single patient at different time points showed telomere shortening in spite of low levels of telomerase (Hiyama et al., 1995a).

There are several ways that NB cells can upregulate telomerase activity. The amplification of

MYCN has been associated with upregulated telomerase activity, in one study 65% of tumours with high telomerase activity were also MYCN amplified (Hiyama et al., 1997). This correlation is likely due to

MYCN driven telomerase activity as the hTERT promoter contains binding sequences for the MYCN transcription factor (Cong et al., 1999; Mac et al., 2000; Wang et al., 1998). Expression of hTERT can also be upregulated as a result of two mutations upstream of the promoter. However, this appears to be a rare event in NB with 0/131 primary tumours harbouring either a C228T or a C250T mutation and only

3/19 cell lines (all derived from the same tumour) having a C228T mutation (Lindner et al., 2015).

Instead, up to 31% of NB tumours activate telomerase via chromosome 5 structural rearrangements

(Peifer et al., 2015; Valentijn et al., 2015). A breakpoint 30-50 kb upstream or 6-40 kb downstream of the hTERT promoter results in the introduction of super-enhancer elements and the consequent upregulation of hTERT mRNA expression. This event is mutually exclusive with MYCN amplification and also with ATRX aberrations (Peifer et al., 2015; Valentijn et al., 2015). Telomerase activity has also been identified in all but four NB cell lines screened to date (4/40 cell lines; Farooqi et al., 2014).

47

Given the correlation between telomerase activity and poor prognosis in NB, the mechanism has become a target for the development of novel NB therapy. Long term treatment (4-6 weeks) with the telomerase inhibitor telomestatin, at non-toxic concentrations in four NB cell lines, results in a reduction in the growth rate of cells, a decrease in telomere length, and an increase in apoptosis (Binz et al., 2005). This suggests that therapies targeting TLMs may have therapeutic relevance in NB.

1.5.7. ALT in neuroblastoma

A subgroup of NB tumours upregulates telomerase activity to maintain their telomere length however, between 20 and 60% of high-risk tumours do not have detectable telomerase activity (Krams et al.,

2001; Onitake et al., 2009; Poremba et al., 2000; Poremba et al., 1999). The first evidence of ALT in NB relied on telomere length to identify the mechanism. Long and heterogeneous telomere lengths, a phenotypic characteristic of ALT, were observed in 5/9 telomerase-negative patients who had protracted but fatal disease (Onitake et al., 2009). Another study utilising telomere length as a marker of ALT indicated the mechanism may be used by up to 59% of NB tumours (Pezzolo et al., 2015). A subsequent study that utilised telomere length and APBs to determine ALT activity confirmed that ALT tumours have a poor outcome, approximately 45% 5-year overall survival, despite lacking poor prognostic markers such as MYCN amplification (Lundberg et al., 2011).

Aberrations of the ATRX gene have been reported in NB tumours (Kurihara et al., 2014;

Lundberg et al., 2011; Onitake et al., 2009; Valentijn et al., 2015) and correlate with an upregulated ALT mechanism in other tumour types (Chen et al., 2014; Heaphy et al., 2011a; Jiao et al., 2012; Jiao et al.,

2011). In NB, alterations to the ATRX gene associate with long telomeres, suggestive of ALT, and patients with these tumours are older at diagnosis (ATRX mutations in 44% of patients aged ≥12 years, 17% of patients aged 18 months to <12 years and 0% patients aged <18 months) (Cheung et al., 2012). A retrospective examination of the tumours by Onitake and colleagues found all ALT tumours had an aberration to ATRX or DAXX and that ATRX alterations led to a 5-year overall survival of approximately

40% (Kurihara et al., 2014). Mutations in ATRX or DAXX have been identified in 10-22% of high-risk NB

48 tumours (Cheung et al., 2012; Peifer et al., 2015; Valentijn et al., 2015). In most studies alterations to the ATRX, hTERT, and MYCN genes are mutually exclusive (Cheung et al., 2012; Peifer et al., 2015;

Valentijn et al., 2015) and lead to a similarly poor overall survival rate of approximately 25% (Valentijn et al., 2015).

A caveat of using large genome studies to identify ALT is that ATRX and DAXX mutations are not a universal feature of the mechanism and relying on these genetic events would underestimate ALT in

NB. To date four telomerase-negative NB cell lines derived from metastatic disease have been identified

(Choi et al., 2000; Farooqi et al., 2014). Only one of these cell lines (CHLA-90) exhibited classical ALT phenotypes including abundant C-circles and APBs, was MYCN non-amplified and ATRX and TP53 mutated, whilst a second cell line (SK-N-FI) retained wild type ATRX/DAXX expression but otherwise exhibited classical features of ALT (Farooqi et al., 2014). Therefore, it appears that ALT is present and clinically relevant in NB, although no study to date has utilised C-circles, the quantifiable ALT marker, to identify ALT in NB tumours.

1.6. Aims of the project

High-risk NB confers a very poor prognosis and the identification of novel biomarkers is required for the development of better therapy for these patients. Recent studies have identified mutations to genes involved in TLMs as relatively frequent events in NB (Cheung et al., 2012; Peifer et al., 2015; Valentijn et al., 2015). Telomerase activity is associated with poor outcome (Ohali et al., 2006; Onitake et al.,

2009; Poremba et al., 2000), whilst the identification of ALT has relied on genetic markers or telomere length to identify the presence of this mechanism rather than C-circles, the active marker of ALT (Cheung et al., 2012; Lundberg et al., 2011; Onitake et al., 2009; Peifer et al., 2015; Valentijn et al., 2015).

Consequently, the initial aim of this study was to determine the prognostic relevance of ALT in high-risk

NB utilising telomere length and abundant C-circles (Lau et al., 2013). Unexpectedly, we identified a subgroup of high-risk tumours that lacked C-circles but had a telomere content indistinguishable from

49

ALT tumours and no telomerase activity. Therefore, the aim of this study became to investigate the telomere biology of ALT-negative/long telomere NB. Specifically we sought to:

1. Determine if a subgroup of fatal malignant NB tumours can develop in the absence of a TLM

and the consequence of extensive proliferation on the telomere length of these cells.

2. Examine the effect of activation of a TLM on ALT-negative/long telomere NB cells.

3. Study the association between telomere biology and sensitivity to chemotherapeutics in NB.

4. Investigate similar phenotype in other tumour types.

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Chapter 2:

Materials and Methods

51

Chapter 2: Materials and Methods

2.1. Buffers and solutions

The buffers and solutions used throughout the methods are listed in Table 2.1.

Table 2.1. Composition of common buffers and solutions. Solution Composition 6X DNA loading buffer 0.0001% (v/v) SDS 15% (w/v) Ficoll 400 0.25% (w/v) Bromophenol blue 6X TBE (Tris, borate and EDTA) 10X phosphate buffered saline (PBS) 1.37 M NaCl 0.1 M Na2HPO4 0.018 M KH2PO4 20X saline-sodium citrate (SSC) 3 M NaCl 0.3 M Tri-sodium citrate Tris, borate and EDTA (TBE) 0.9 M Tris pH 8 0.9 M Boric acid 0.02 M ethylenediaminetetraacetic acid (EDTA) Tris-EDTA (TE) 10 mM Tris pH 7.6 0.1 mM EDTA

2.2. Patient samples

The study cohort contained 149 high-risk NB samples: 19 frozen tumour specimens from The Children’s

Hospital at Westmead Tumour Bank (Australia), 46 DNA samples from the Children’s Cancer Institute

Tumour Bank (Australia), and 84 samples (35 frozen and 49 DNA) from the Children’s Oncology Group

Neuroblastoma Biobank (USA). High-risk NB was defined as tumours with MYCN amplification or metastatic disease in patients aged ≥12 months. The samples were obtained at original diagnosis and data on sex, age at diagnosis, tumour stage, MYCN status (Figure 2.1), and survival were retrieved from the clinical database from the source of the sample. The Sydney Children’s Hospital Network and

Northern Hospital Network Human Research Ethics Committees provided ethics approval for this study.

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Figure 2.1. Schematic of neuroblastoma (NB) tumours used in this study based on MYCN amplification status. One hundred and forty-nine high-risk NB tumours were examined in this study, 55 were MYCN

amplified and 94 were MYCN non-amplified tumours.

2.3. Cell culture

NB cell lines (characteristics of 6 cell lines predominantly used in the thesis in Table 2.2), and ALT and telomerase lines used as controls (Table 2.3), were cultured with medium supplemented with fetal bovine serum (FBS) and additional additives, as listed in Table 2.4. All cell lines were grown in a humidified incubator at 37°C with atmospheric oxygen and 5% CO2, except COG-N-328h which was grown under normoxic conditions (5% oxygen balanced with nitrogen). Cell lines were authenticated by

16-locus short tandem repeat (STR) profiling and were found to be free of Mycoplasma species by

CellBank Australia (Children’s Medical Research Institute, Westmead, NSW, Australia).

2.4. Vectors and viral transfections

Plasmid contructs pBABE-hTERT (Counter et al., 1998a), pBABE-hTR (Wong and Collins, 2006), pBABE hTR/hTERT (Wong and Collins, 2006), and pBABE empty vector (Morgenstern and Land, 1990) were obtained from Addgene (Plasmid #1771, 27666, 27665, 1764, respectively). Retrovirus was generated by Lipofectamine 2000 (Life Technologies) transfection of Phoenix-AMPHO cells (ATCC CRL-3213) with the plasmids, followed by harvest of the retrovirus 24 hours (hrs) later. LA-N-6 cells were retrovirally transduced by two rounds of viral infection with 4 µg/mL polybrene (Sigma) and retrovirus. Cells that stably expressed the plasmid were selected with culture medium containing 0.3 µg/mL puromycin (Life

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Table 2.2. Characteristics of NB cell lines Cell Line Patient Tissue sample Tumour References Characteristics of origin Formation in Mice SH-SY5Y Stage 4, Progressive Yes (Ross et al., 1983) Female, disease, post (Nevo et al., 2008) 48 months old at chemotherapy, diagnosis bone marrow SK-N-BE2c Stage 4, Progressive Yes (Biedler et al., 1978) Male, disease, post (Ciccarone et al. 1989) 24 months old at chemotherapy, (Reynolds et al., 1988) diagnosis bone marrow Stage 4, Progressive Yes (Reynolds et al., 1988) Male, disease, post (Tsutsumimoto et al., SK-N-FI 132 months old chemotherapy, 2014) at diagnosis bone marrow (Farooqi et al., 2014) Stage 4, Progressive (Keshelava et al., 1998) Male, disease, bone CHLA-90 102 months old marrow at diagnosis transplant Stage 4, Progressive Yes (Reynolds et al., 1988) Male, disease, post (Wada et al., 1993) LA-N-6 60 months old at chemotherapy, diagnosis bone marrow Progressive (Farooqi et al., 2014) COG-N-291 disease, post chemotherapy

Table 2.3. Telomere lengthening mechanism of control cell lines Cell Line Telomere Lengthening Reference Mechanism A2182 Telomerase + (Ng et al., 2009) DOS16 ALT + (Henson et al., 2009) G292 ALT + (Bryan et al., 1997) GM639 Telomerase + (Bryan et al., 1995) GM847 ALT + (Bryan et al., 1995) HeLa 1.2.11 Telomerase + (Whitaker et al., 1995) HFF5 Mortal (Bryan et al., 1995) HT1080 Telomerase + (Whitaker et al., 1995) HT1080 hTR Telomerase + (Pickett et al., 2009) IIICF/c ALT + (Bryan et al., 1995) MeT-4A ALT + (Bryan et al., 1995) MG-63 Telomerase + (Bryan et al., 1997) MRC-5 Mortal (Huschtscha and Holliday, 1983) SAOS-2 ALT + (Bryan et al., 1997) U-2 OS ALT + (Bryan et al., 1997)

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Technologies) for 7 days. Sub-clones were isolated by plating cells, post puromycin selection, at a density of 100 cells/mL with conditioning medium.

2.5. Cell proliferation assay

Cells were seeded in 96-well plates according to the density in Table 2.5 and treated with serial dilutions of drugs (6 wells were treated with every drug concentration; Table 2.6) at 37°C with 5% CO2 for 6 PDs.

Drugs were replenished every 72 hrs and experiments were performed in triplicate. At the end of treatment 10 µL of AlamarBlue (Thermo Fischer Scientific) was added to each well containing 100 µL of medium/drug and incubated at 37°C with 5% CO2 for 4 hrs. AlamarBlue is a fluormetric indicator, where the change in fluorescence correlates with the number of metabolically active cells. The fluorescence was measured in a fluorescence plate reader (EnSpire, Perkin Elmer) with an excitation wavelength 570 nm and an emission wavelength at 585 nm. The results were expressed as a percentage of the untreated control and GraphPad Prism6 was used to determine the IC50 dose (the dose that inhibited 50% of untreated control cells growth).

2.6. Immunostaining and fluorescence in situ hybridisation (FISH)

2.6.1. ATRX and DAXX immunofluorescence (IF)

Cells grown in two-well chamber slides were washed once with 1X PBS and then fixed with 4% PFA for

10 minutes (mins) at room temperature. The cells were rinsed with 2X PBS and permeabilised in 0.1%

Triton-X for 10 mins, and then washed again with 2X PBS. Cells were blocked, at room temperature, with

5% goat serum/bovine serum albumin (BSA) for 20 mins before the cells were incubated for 1 hr at 37°C with either anti-ATRX or DAXX rabbit polyclonal antibody (Sigma) diluted 1:500 in 3% BSA/4X SSC. To remove excess antibody, the cells were washed three times with 0.1% Tween-20/4X SSC and were then incubated with Alexa Fluor 594 goat anti-rabbit secondary antibody (Invitrogen) diluted 1:1000 in 3%

BSA/4X SSC. After incubation at room temperature for 1 hr, the cells were washed three times with 0.1%

Tween-20/4X SSC and the DNA was stained with 20 µg/mL 4, 6 diamidino-2-phenylindole (DAPI) in 1X

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Table 2.4. Cell culture medium.

Cell Type Medium FBS (%) Additives Cell Line

A2182 Lung Carcinoma DMEM- with L-glutamine 10 2% L-glutamine CHLA-42 Neuroblastoma IMDM 15 1X ITS 2% L-glutamine CHLA-90 Neuroblastoma IMDM 15 1X ITS 2% L-glutamine CHLA-95 Neuroblastoma IMDM 15 1X ITS 2% L-glutamine CHLA-119 Neuroblastoma IMDM 15 1X ITS 2% L-glutamine CHLA-122 Neuroblastoma IMDM 15 1X ITS 2% L-glutamine CHLA-136 Neuroblastoma IMDM 15 1X ITS 2% L-glutamine CHLA-140 Neuroblastoma IMDM 15 1X ITS 2% L-glutamine CHLA-171 Neuroblastoma IMDM 15 1X ITS 2% L-glutamine CHLA-172 Neuroblastoma IMDM 15 1X ITS 2% L-glutamine CHLA-225 Neuroblastoma IMDM 15 1X ITS CHP-134 Neuroblastoma RPMI-1640 10 1% L-glutamine 2% L-glutamine COG-N-291 Neuroblastoma IMDM 15 1X ITS 2% L-glutamine COG-N-328h Neuroblastoma IMDM 20 1X ITS 2% L-glutamine COG-N-347 Neuroblastoma IMDM 20 1X ITS DOS16 Leiomyosarcoma RPMI-1640 10 2% L-glutamine FU-NB-2006 Neuroblastoma IMDM 15 1X ITS McCoy's 5a Medium G292 Osteosarcoma 10 Modified GM639 Fibroblast, skin DMEM- with L-glutamine 10 GM847 Fibroblast, skin DMEM- with L-glutamine 10 HeLa 1.2.11 Cervical carcinoma RPMI-1640 10 1% L-glutamine HFF5 Fibroblast, foreskin DMEM- with L-glutamine 10 HT1080 Fibrosarcoma DMEM- with L-glutamine 10

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HT1080 hTR Fibrosarcoma DMEM- with L-glutamine 10 Fibroblast, breast, IIICF/c Li-Fraumeni DMEM- with L-glutamine 10 syndrome IMR32 Neuroblastoma DMEM- with L-glutamine 10 KELLY Neuroblastoma RPMI-1640 10 1% L-glutamine LA-N-1 Neuroblastoma MEM-F12 with L-glutamine 10 1% NEAA LA-N-2 Neuroblastoma MEM-F12 with L-glutamine 10 1% NEAA LA-N-5 Neuroblastoma RPMI-1640 10 1% L-glutamine LA-N-6 Neuroblastoma RPMI-1640 10 1% L-glutamine MeT-4A Mesothelial LHC-MM 3 MG-63 Osteosarcoma DMEM- with L-glutamine 10 MRC-5 Fibroblast, lung DMEM- with L-glutamine 10 NB69 Neuroblastoma RPMI-1640 10 1% L-glutamine SAOS-2 Osteosarcoma DMEM- with L-glutamine 10 SH-EP Neuroblastoma DMEM- with L-glutamine 10 SH-SY5Y Neuroblastoma DMEM- with L-glutamine 10 1% NEAA SK-LU-1 Adenocarcinoma DMEM- with L-glutamine 10 SK-N-AS Neuroblastoma DMEM- with L-glutamine 10 SK-N-BE1 Neuroblastoma RPMI-1640 10 1% L-glutamine SK-N-BE2c Neuroblastoma DMEM- with L-glutamine 10 SK-N-DZ Neuroblastoma DMEM- with L-glutamine 10 SK-N-FI Neuroblastoma RPMI-1640 10 1% L-glutamine SK-N-SH Neuroblastoma DMEM- with L-glutamine 10 SMS-KANR Neuroblastoma RPMI-1640 10 1% L-glutamine SMS-KCN Neuroblastoma RPMI-1640 10 1% L-glutamine SMS-KCNR Neuroblastoma RPMI-1640 10 1% L-glutamine SMS-LHN Neuroblastoma RPMI-1640 10 1% L-glutamine SMS-SAN Neuroblastoma RPMI-1640 10 1% L-glutamine U-2 OS Osteosarcoma DMEM- with L-glutamine 10

Abbreviations: DMEM, Dulbecco’s Modified Eagle’s Medium; IMDM, Iscove's Modified Dulbecco's Medium; LHC-MM, Laboratory of Human Carcinogenesis Mesothelial Medium; MEM, minimum essential medium; ITS, insulin-transferrin-selenium-sodium pyruvate; NEAA, non-essential amino acids; RPMI-1640, Roswell Park Memorial Institute 1640.

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Table 2.5. Seeding density of cell lines in the cell proliferation assay. Cell Line Seeding Density (X104 cells/well) COG-N-291 1.6 CHLA-90 0.25 GM639 0.125 GM847 0.1 HeLa 1.2.11 0.38 HT1080 0.1 HT1080 hTR 0.2 LA-N-6 2.5 SK-N-BE2c 0.15 SK-N-FI 1 SH-SY5Y 0.375 U-2 OS 0.125

PBS for 1 min. The slides were rinsed with water and mounted in DABCO (2.33% antifade compound

1,4-Diazabicyclo[2.2.2]octane [Sigma], 90% [v/v] glycerol, 20 mM Tris-HCl pH 8.0). Cells were considered ATRX or DAXX positive when foci were present in the nucleus.

2.6.2. APB detection

Cell cultures were harvested by trypsinisation and resuspended in buffered hypotonic solution (United

Biosciences). The cells (110,000 cells/mL) were cytocentrifuged onto SuperFrost Plus glass slides

(Menzel-Glaser) at 110 x g for 10 min in a Shandon Cytospin 4 with medium acceleration. Slides were permeabilised at room temperature for 10 mins with KCM buffer (120 mM KCl, 20 mM NaCl, 10 mM Tris pH 7.5, 0.1% Triton X-100), rinsed in 1X PBS, and fixed in 4% (v/v) formaldehyde in 1X PBS at room temperature for 10 mins. After several rinses with 1X PBS, the slides were treated with 100 µg/mL

DNase-free RNase A (Sigma) in antibody dilution buffer (ABDIL; 20 mM Tris pH 7.5, 2% BSA, 0.2% fish gelatin, 150 mM NaCl, 0.1% Triton X-100, 0.1% sodium azide) at 37°C for 30 mins. The slides were then incubated at 37°C for 1 hr with mouse anti-PML antibody (PG-M3; Santa Cruz), diluted 1:250 in ABDIL.

Slides were washed three times with 0.1% Tween-20/1X PBS and then incubated with Alexa Fluor 594 goat anti-mouse secondary antibody (Invitrogen; 1:1000). Following the incubation at 37°C for 30 mins, the cells were washed three times with 0.1% Tween-20/1X PBS, and proteins were cross-linked with 4%

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PFA for 10 mins at room temperature. The cells were dehydrated with an ethanol series (70%, 90%, and

100%) and then telomeric DNA was detected using the 5’ labelled telomere-specific peptide nucleic acid

(PNA) probe, Alexa 488-(CCCTAA)3 PNA probe (PNA Bio) (Chen et al., 1999; Perrem et al., 2001). After the probe was placed on the slide, the DNA was denatured at 80°C for 3 mins and the probe was hybridised for 3 hrs at room temperature. Unhybridised probe was removed by washing with Buffer A

(70% formamide, 0.01 M Tris pH 7.5, 0.1% BSA) for 15 mins, and then the nuclei were stained with DAPI

(20 µg/mL) in wash Buffer B (0.1 M Tris pH 7.5, 0.15 M NaCl, 0.08% Tween-20) for 3 mins. The slides were washed with 1X PBS and rinsed in water before mounting in DABCO antifade mounting medium.

A Zeiss Axio Imager Z2 with ApoTome was used to capture Z-stacked images (ACRF Telomere Analysis

Centre). Quantitation was completed on three independent experiments, on at least 100 cells/experiment, using Zeiss ZEN software. An APB was defined as the colocalisation of telomeric DNA and PML protein of any size (Yeager et al., 1999).

2.6.3. MetaTIF assay

Cell cultures were treated with 50 µg/mL vinblastine for 1 hr at 37°C in 5% CO2 to obtain metaphase chromosomes. Cells were harvested by trypsinisation and resuspended in buffered hypotonic solution at 37°C. NB cultures were incubated for 20 mins whereas control cell lines were incubated for 5 mins.

To obtain metaphase spreads 110,000 cells/mL were cytocentrifuged onto SuperFrost Plus glass slides at 450 x g (high acceleration) for 10 min in a Shandon Cytospin 4. Slides were then permeabilised with

KCM buffer for 10 mins, rinsed in 1X PBS three times, and fixed in 4% (v/v) formaldehyde in 1X PBS for

10 mins at room temperature. The slides were treated with 100 µg/mL RNase A in ABDIL at 37°C for 30 mins, and then incubated for 1 hr at 37°C with mouse anti-γH2AX antibody (Ser139 Clone JBW301;

Merck Millipore) diluted 1:300 with ABDIL. To remove excess antibody, the slides were washed three times and then incubated with Alexa Fluor 594 anti-mouse secondary antibody (1:1000 in ABDIL) at 37°C for 30 mins. Slides were washed three more times with 0.1% Tween-20/1X PBS, cross-linked with 4%

PFA for 10 mins, and dehydrated with increasing concentrations of ethanol (70%, 90% and 100%).

Telomeric DNA was detected using the Alexa 488-(CCCTAA)3 PNA probe, applied to the slide prior to

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Table 2.6. List of drugs and stock concentrations. Dilution Highest Drug Factor Drug Company Target Diluent Concentration for Serial Dilution All trans AbCam Differentiation induction Ethanol 45 µM 3 retinoic acid Aphidicolin AbCam DNA replication inhibitor DMSO 30 µM 6 Sigma- Complex mode of action, 1 M Arsenic Trioxide 10 µM 3 Aldrich causes apoptosis NaOH 0.9% Cayman Cisplatin Cross-links DNA Saline 30 µM 4 Chemicals (w/v) Interferes with Cayman Doxorubicin involved in DNA DMSO 1.5 µM 5 Chemicals replication Cayman Topoisomerase II Etoposide DMSO 400 µM 10 Chemicals inhibitor Ribonucleotide reductase Hydroxyurea AbCam DMSO 1600 µM 2 inhibitor Cayman Irinotecan Topoisomerase I inhibitor DMSO 600 µM 6 Chemicals -60019 Selleckchem ATM inhibitor DMSO 24 µM 2 Interferes with enzymes Mitomycin C Selleckchem involved in DNA Water 3 µg/mL 4 replication Sigma- ML216 Bloom helicase inhibitor DMSO 225 µM 3 Aldrich Cayman Alkylating agent, induce Temozolomide DMSO 400 µM 6 Chemicals DNA damage Cayman Topotecan Topoisomerase I inhibitor DMSO 90 µM 12 Chemicals VE-822 Selleckchem ATR inhibitor DMSO 7 µM 5 Vincristine AbCam Mitotic inhibitor DMSO 0.05 µM 6

denature at 80°C for 3 mins. After hybridisation of the probe at room temperature for 3 hrs, the slides were washed with Buffer A for 15 mins. The DNA was stained with 20 µg/mL DAPI/Buffer B for 3 mins and rinsed with water several times before mounting in DABCO. Images were obtained on the Metafer

60 slide scanning system (ACRF Telomere Analysis Centre) and a meta-TIF was defined as the localisation of γ-H2AX protein to the end of the chromosome (with or without the presence of telomere signal)

(Cesare et al., 2009). Quantitation was performed, in triplicate, on at least 1000 chromosome ends.

2.6.4. Telomere FISH on metaphase spreads

Metaphase chromosomes were obtained by treating cells with vinblastine (50 µg/mL) overnight for 16 hrs. Cell cultures were harvested by trypsinisation, resuspended in buffered hypotonic solution at 37°C for 20 mins, fixed (3:1 methanol/glacial acetic mix) and dropped onto SuperFrost Plus glass slides. The slides were baked at room temperature overnight, then treated with 100 µg/mL RNase A at 37°C for 30 mins, and then dehydrated with increasing concentrations of ethanol. FISH was performed with an Alexa

Fluor 488 telomere specific PNA probe applied to the slide prior to denaturing the DNA for 3 mins at

80°C. After a 3 hr hybridisation, the slides were washed for 15 mins with Buffer A to remove excess probe. A second wash (with Buffer B) was performed for 5 mins at room temperature, and then the slides were stained with DAPI (20 µg/mL in PBS) for 3 mins to visualise the DNA. The slides were washed with 1X PBS, rinsed with water and mounted in DABCO. Fluorescence microscopy images were obtained on the Metafer slide scanning system (ACRF Telomere Analysis Centre) and the intensity of the telomere signal was quantitated using TFL-Telo software (Zijlmans et al., 1997) in fifteen metaphase spreads for each sample.

2.6.5. Telomere sister chromatid exchange (T-SCE) analysis

T-SCE analysis was performed as previously described (Bailey et al., 2004). Briefly, cells were cultured in medium containing a 3:1 ratio of BrdU:BrdC (Sigma) with vinblastine (50 µg/mL) added for the final 16 hrs. Metaphases were harvested and resuspended in buffered hypotonic solution at 37°C for 20 mins.

Chromosomes were then fixed with 3:1 ice-cold methanol:acetic acid and dropped onto SuperFrost Plus glass slides. The slides were incubated at 37°C for 30 mins with 100 µg/mL DNase-free RNase A (Sigma) in 2X SSC. Slides were postfixed with 4% formaldehyde in PBS for 10 mins and then dehydrated in an ice-cold ethanol series (75, 85, 100%) for 2 mins each. Metaphase spreads were stained with 0.5 µg/mL

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Hoechst 33258 at room temperature for 15 mins and exposed to long wave 365 nm UV light for 45 mins.

An exonuclease III (NEB) digest was then performed for 30 mins at 37°C. The chromosomes were denatured for 5 mins at 80°C in 70% formamide/30% 2X SSC before the slides were dehydrated in an ice-cold ethanol series as previously described. The slides were incubated overnight with 0.3 µg/ml

TAMRA–OO-(TTAGGG)3 PNA probe (PNA Bio) and washed with Buffer A for 15 mins followed by a wash with Buffer B for 5 mins at room temperature. Slides were incubated with 0.3 µg/ml Alexa 488–OO-

(CCCTAA)3 PNA probe overnight and washed with buffer A and B as previously described prior to staining with DAPI (20 µg/mL in PBS) for 3 mins to visualise the DNA. Lastly, the slides were washed with 1X PBS, rinsed with water and mounted in DABCO. Imaging was performed on the Metafer slide scanning system

(ACRF Telomere Analysis Centre).

2.7. DNA extraction and quantitation

Genomic DNA was extracted by lysis at 37°C overnight with 2% sodium dodecyl sulphate (SDS) buffer containing 50 mM Tris, 20 mM EDTA, and 200 µg/mL Pronase (Sigma). Lysis was followed by 2 hrs salt precipitation at 4°C with 1.4 M NaCl, followed by serial centrifugation (15 mins at 3900 x g) until the white precipitant was removed. The DNA was precipitated at 4°C overnight with 100% ethanol (2.5

X volume of lysis buffer with NaCl), before centrifugation at 3900 x g for 30 mins. The DNA pellet was washed with 70% ethanol, centrifuged for 15 mins, and then allowed to air-dry. The DNA was resuspended in TE and quantitated with a Qubit Fluorometer (Invitrogen).

2.8. C-circle assay

2.8.1. Slot-blot detection

Rolling circle amplification of C-circles was performed on 10 ng of genomic DNA. The assay was performed with and without Φ29 polymerase for each sample. The DNA (10 µL) was added to 10 µL of the amplification mix, for a 20 µL reaction containing 4 µM DL-Dithiothreitol (DTT), 200 µg/mL BSA, 0.1%

Tween-20, 1X Φ29 buffer (New England BioLabs), 7.5 Units Φ29 polymerase (New England BioLabs), and

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1 mM each of dATP, dGTP and dTTP (Roche), which was incubated at 30°C for 8 hrs and then at 65°C for

20 mins to heat inactivate the enzyme (Henson et al., 2009). The C-circle assay product was diluted with

100 µL of 2X SSC, and using slot-blot apparatus, blotted onto a Biodyne B 0.45 µM nylon membrane

(Pall). The membrane was dried and then the DNA was UV-cross-linked onto the membrane in a

Stratagene UV Stratalinker 1800. PerfectHyb Plus buffer (Sigma) was used to pre-hybridise the

32 membrane for 1 hr at 37°C, and a P-ATP-labelled (CCCTAA)3 telomeric probe was hybridised to the

DNA overnight, under native conditions. The membrane was washed four times at 37°C with 0.5X

SSC/0.1% SDS for 15 mins and exposed to a storage phosphor screen, which was scanned on a Typhoon imager (GE Healthcare) the following day. Signals were quantitated using ImageQuant (GE Healthcare) and the level of C-circles was calculated by subtracting the 32P-probe signal of the C-circle assay without

Φ29 from that of the C-circle assay with Φ29.

2.8.2. Exonuclease treatment of DNA

Genomic DNA was pretreated with 6 U/ug of the restriction enzymes HinfI and RsaI (New England

BioLabs) and 25 ng/µg RNase (Roche) at 37°C for 12 hrs, followed by heat inactivation of the enzymes at 80°C for 20 mins. The digested DNA was ethanol precipitated (0.3 volume of 10 M ammonium acetate and 3.3 volumes of 100% ethanol with 1 µL glycogen) and resuspended in TE, prior to quantitation by the Qubit Fluorometer. To remove linear DNA from the C-circle reaction, 1 µg of pretreated DNA was digested with lambda exonuclease (12.5 U/µg; New England BioLabs), exonuclease I (100 U/µg; New

England BioLabs), and 1X lambda buffer (New England BioLabs) at 37°C for 20 mins followed by 80°C for

20 mins (Henson et al., 2009). The DNA was quantitated, and additional rounds of exonuclease digestion were performed, until there was <100 ng of DNA remaining. As a control, a mock reaction without was also carried out for each sample. The exonuclease digested DNA, and control sample, were precipitated by ethanol precipitation as described above and resuspended in 20 µL Tris. To perform the C-circle amplification reaction, 5 µL of the DNA was diluted with 5 µL of Tris and added to the reaction. The rolling circle amplification product was quantitated by slot-blot analysis as described in section 2.8.1.

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2.8.3. Telomere quantitative polymerase chain reaction (qPCR) detection

Rolling circle amplification of C-circles was performed on 16 ng (in 5 µL) of genomic DNA, with and without Φ29 polymerase for each sample. The total reaction volume was 10 µL (including DNA), and the components and conditions were the same as section 2.8.1, with a modification to the amount of Φ29 polymerase (Lau et al., 2013), which was halved. The 10 µL C-circle assay product was diluted with 30

µL of TE, and 5 µL of the diluted product was used for each PCR. For every sample, the following PCRs were performed in triplicate: telomere PCR of C-circle assay/Φ29+, telomere PCR of C-circle assay/

Φ29-, single copy gene (SCG) PCR of C-circle assay/Φ29+, and SCG PCR of C-circle assay/Φ29-. The SCG for NB cell lines and tumours was VAV2 (Lau et al., 2013), whilst the SCG for oesophageal and PanNETs was HBG, and the SCG for melanoma tumours and cell lines was 36B4 (Lau et al., 2013). Primer sequences are listed in Table 2.7. A standard curve of DNA was included for each PCR, to allow for subsequent analysis. Concentrations in the telomere PCR were 2.5, 1, 0.2, 0.04, 0.02, 0.01 ng/µL and concentrations of the SCG genes were 2.5, 1.25, 0.625, 0.3125, 0.156, 0.078 ng/µL. Each qPCR reaction consisted of 1X Rotor-Gene SYBR Green (Qiagen), 5 µL C-circle assay product or standard DNA, and the primer sets. The final primer concentrations were, telomere: forward 500 nM and reverse 500 nM,

VAV2: forward 700 nM and reverse 400 nM, 36B4: forward 300 nM and 500 nM reverse, and HBG: forward 500 nM and reverse 500 nM. PCR conditions were, telomere: 95°C for 15 mins, 30 cycles of 95°C for 7 sec and 58°C for 10 sec, VAV2: 95°C for 5 mins, 43 cycles of 95°C for 15 sec, 61°C for 30 sec, and

72°C for 20 sec, and 36B4/HBG: 95°C for 5 mins, 40 cycles of 95°C for 15 sec and 58°C for 30 sec. All PCR conditions concluded with a melt curve analysis. Analysis was conducted using the two standard curves method (Cawthon, 2002), and the telomere product was corrected for loading with the SCG product. All

PCR results were expressed as the mean of triplicate reactions. The relative telomeric DNA content (TC) of a sample was obtained from the mean normalised C-circle assay/Φ29- reaction, relative to the TC of

SK-N-FI or IIICF/c, ALT-positive cell lines with arbitrary TC values of 14.0 and 30.0, respectively. The relative C-circle level of a sample was the normalised C-circle assay/Φ 29+ subtracted by the normalised

C-circle assay/Φ29-, relative to the C-circle level of SK-N-FI and IIICF/c, with arbitrary values of 196 and

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100, respectively. Long telomeres were indicated by a relative TC≥15, and a sample was considered ALT+ when the relative C-circle level was ≥7.5 (Lau et al., 2013).

2.9. Telomere length analysis

2.9.1. Telomere restriction fragment (TRF) analysis

Genomic DNA was digested at 37°C for 12 hrs with 6 U/µg of the restriction enzymes HinfI and RsaI (New

England BioLabs) and 25 ng/µg RNase (Roche) (Perrem et al., 2001), followed by heat inactivation of the enzymes at 80°C for 20 mins. Prior to gel electrophoresis, the DNA was ethanol precipitated (0.3 volume of 10 M ammonium acetate and 3.3 volumes of 100% ethanol with 1 µL glycogen) and then resuspended in TE. The digested DNA was quantitated by the Qubit Fluorometer and 1.5 µg/well was loaded on a 1%

(w/v) agarose gel in 0.5X TBE. To resolve bands in COG-N-291 and LA-N-6, the DNA was separated using pulsed-field gel electrophoresis (BioRad) in 14°C recirculating 0.5X TBE buffer at 6 V/sec for 16 hrs, with an initial switch time of 1 and final switch time of 6. Gels containing other samples were run for 14 hrs.

The gels were dried for 2 hrs at 65°C, denatured in 0.82 M NaOH/2.5 M NaCl for 1 hr, and neutralised in

0.7 M Tris pH 8/1.5 M NaCl for 1 hr at room temperature. Gels were pre-hybridised for 1 hr at 50°C in

32 Church buffer (0.5 M Na2HPO4 pH 7.2, 1 mM EDTA, 7% SDS, and 1% BSA) and then hybridised with a P-

ATP-labelled (CCCTAA)4 telomeric probe overnight. The gels were washed for 30 mins in 4X SSC, three times at room temperature and once at 50°C, and then exposed to a storage phosphor screen. The screen was scanned the following day with a Typhoon imager and the signals were quantitated using

Las4000-Multigauge software. The unweighted mean TRF length was calculated using Telorun software

(Baur et al., 2004), which utilised linear regression analysis of molecular weight marker positions to calculate telomere length.

2.9.2. Telomere qPCR

The TC of cells, independent of C-circles, can be determined via the protocol described in section 2.8.3. with modification to the input sample. Rather than rolling circle amplification product, 5 µL of 1 ng/µL

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Table 2.7. List of primers. Methods Gene Primer sequence section F: 5’-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT- 3’ Telomere 2.8.3. R: 5’-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT- 3’ F: 5’ -TGGGCATGACTGAAG ATGAC- 3’ VAV2 2.8.3. R: 5’ -ATCTGCCCTCACCTTCTCAA- 3’ F: 5’ -CAGCAAGTGGGAAGGTGTAATCC- 3’ 36B4 2.8.3. R: 5’ -CCCATTCTATCATCAACGGGTACAA- 3’ F: 5’ -GCTTCTGACACAACTGTGTTCACTAGC- 3’ HBG 2.8.3. R: 5’ - CACCAACTTCATCCACGTTCACC- 3’ TP53 F: 5’ -CCAGGTGACCCAGGGTTGGA- 3’ 2.14. 2-3 R: 5’ -AGCATCAAATCATCCATTGC- 3’ TP53 exon F: 5’ -TGCTCTTTTCACCCATCTAC- 3’ 2.14. 4 R: 5’ -ATACGGCCAGGCATTGAAGT- 3’ TP53 exon F: 5’ -TGTTCACTTGTGCCCTGACT- 3’ 2.14. 5-6 R: 5’ -TTAACCCCTCCTCCCAGAGA- 3’ TP53 exon F: 5’ -CTTGCCACAGGTCTCCCCAA- 3’ 2.14. 7 R: 5’ -AGGGGTCAGCGGCAAGCAGA- 3’ TP53 exon F: 5’ -TTGGGAGTAGATGGAGCCT- 3’ 2.14. 8-9 R: 5’ -AGTGTTAGACTGGAAACTTT- 3’ TP53 exon F: 5’ -CAATTGTAACTTGAACCATC- 3’ 2.14. 10 R: 5’ -GGATGAGAATGGAATCCTAT- 3’ TP53 exon F: 5’ -AGACCCTCTCACTCATGTGA- 3’ 2.14. 11 R: 5’ -TGACGCACACCTATTGCAAG- 3’ H3F3A F: 5’ -GATTTTGGGTAGACGTAATCTTCA- 3’ 2.14. exon 1 R: 5’ -AACAAAAAGATGGATAACAGGAAA- 3’ H3F3A F: 5’ -TTTCTTTGAAGCTGCCCACT- 3’ 2.14. exon 2 R: 5’ -TCCAAATTGTTGCATGTGCT- 3’ H3F3A F: 5’ -AGCATCTTGCCCAGTCATTTT- 3’ 2.14. exon 3 R: 5’ -CAGGTATTGGCAGTTTTTCCA- 3’ F: 5’-CGGAAGAGTGTCTGGAGCAA- 3’ TERT 2.16.1. R: 5’-TCGTAGTTGAGCACGCTGAACAG- 3’ F: 5’-CTAACCCTAACTGAGAAGGGCGT- 3’ TERC 2.16.1. R: 5’-GGCGAACGGGCCAGCAGCTGAC- 3’ F: 5’-GCCGAAATACAACACGCTGAAG- 3’ Dyskerin 2.16.1. R: 5’-CAGAGGATTTGAACCACATGCA- 3’ F: 5‘-ACCCACTCCTCCACCTTTG- 3’ GAPDH 2.16.1. R: 5’-CTCTTGTGCTCTTGCTGGG- 3’ F: 5’-CTGTGCCATCAAAGCAGAAA-3’ SLX4 2.16.1. R: 5’-CGGAGATTTTTCTGGGAACA- 3’ F: 5’-CCATTCCGCTGTGAATTTG-3’ XRCC3 2.16.1. R: 5’-GCCTCTGTCACCTGGTTGAT- 3’ F: 5’-CACTCGCAACTCACACAGGT-3’ RTEL1 2.16.1. R: 5’-TTACGGCACAAGTGGATCTG- 3’ F: 5‘-GGGTGAGGAAGTTTGAGTGC- 3’ ZBTB48 2.16.1. R: 5’-ACCACCACCATCTTCTCGTC- 3’ F: 5‘-CCCACCTCTTGATGAACCAT- 3’ NBS1 2.16.1. R: 5’-CCCACCTCCAAAGACAACTG- 3’

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F: 5’-GCCGGAAGAGACAGATAAGC-3’ MYCN 2.16.2. R: 5’-TTCTCCACAGTGACCACGTC-3’ M2 5’-AAT CCG TCG AGC AGA GTT-3’ 2.18.3. ACX 5’-GCGC GGC TTA CCC TTA CCC TTA CCC TAA CC-3’ 2.18.3. Abbreviations: F, forward; R, reverse. Primer sequences for section 2.8.3. were described in Henson et al., 2017 & Lau et al., 2013. TP53 primer sequences were obtained from the International Agency for Research on Cancer (IARC) website (http://p53.iarc.fr/Download/TP53_DirectSequencing_IARC.pdf). H3F3A primers were obtained from (Bower et al., 2012) and RT-qPCR primers from section 1.15.1 were obtained from (Cao et al., 2008b). The M2 and ACX primers were described in (Kim and Wu, 1997; Piatyszek et al., 1995).

genomic DNA was added to the PCR. The relative TC of a sample was obtained from the mean of triplicate telomere reactions normalised to the SCG reactions.

2.10. T-circle detection

T-circles were detected using two-dimensional TRF analysis. Genomic DNA was restriction digested with

HinfI and RsaI, using the TRF protocol. Digested DNA was separated in a 0.6% agarose gel at 1 V/cm for

13.5 hr, in the first dimension. Lanes were excised and run in a 1.1% agarose gel containing 300 ng/mL ethidium bromide at 6 V/cm for 4 hrs, in the second dimension. Following the TRF protocol in section

32 2.9.1, gels were dried, denatured and hybridised overnight to a P-ATP-labelled (CCCTAA)4 telomeric probe in Church buffer. Gels were washed, exposed to a storage phosphor screen and scanned on a

Typhoon imager.

2.11. Telomere chromatin immunoprecipitation (ChIP)

2.11.1. Cell lysis and chromatin isolation

Cells were harvested by trypsinisation, washed in 1X PBS and collected as 1X107 cell pellets. Pellets were resuspended in 1 mL of cell lysis buffer (5 mM HEPES-KOH pH 8, 85 mM KCl, 0.4% [v/v] NP-40, and, added fresh each time, 1X complete protease inhibitor (Roche), and 1 mM phenylmethylsulphonyl fluoride [PMSF]) and incubated on ice for 10 mins. To pellet the nuclei, samples were centrifuged at

6000 x g for 15 sec at 4°C, and the supernatant discarded. The chromatin bound proteins were cross- linked at room temperature with 1% (v/v) formaldehyde in cell lysis buffer, lacking NP-40, for 10 mins.

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To end the fixation, 75 µL of 1.5 M glycine was incubated with the nuclei for 5 mins at room temperature.

The nuclei were centrifuged at 4°C for 15 sec at 6000 x g, and the supernatant was discarded. Nuclei were lysed with 50 µL nuclei lysis buffer (50 mM Tris-HCl pH 8.1, 1 mM EDTA, 1% [v/v] SDS, added fresh each time: 1X complete protease inhibitor, and 1 mM PMSF) on ice for 10 mins, and then diluted with

750 µL buffer A, containing 20 mM HEPES-KOH pH 8, 2 mM MgCl2, 300 mM KCl, 1 mM EDTA, 10% (v/v) glycerol, and 0.1% (v/v) Triton X-100. The DNA was fragmented by sonication on a Branson sonifier in an ice/ethanol bath. Sonication was performed with a duty cycle of 10, and 60 second pulse/pause cycles at outputs 3, 4, and 5. Solubilised chromatin was isolated by retaining the supernatant after centrifugation at 17,000 x g for 10 mins at 4°C.

2.11.2. Immunoprecipitation (IP)

IP was conducted in a buffer of 20 mM HEPES-KOH pH 7.9, 150 mM KCl, 1.5 mM EDTA, 1% (v/v) Triton

X-100, and, added fresh each time, 1X complete protease inhibitor, and 1 mM PMSF. The chromatin

(112 µL) was rotated at 4°C for 1 hr with 225 µL of the IP buffer, and the antibody as indicated in Table

2.8. A pre-blocked suspension of Protein G agarose beads (Roche), were bound to the antibody by rotating 60 µL/sample at 4°C overnight. The next day, the chromatin/antibody/bead complexes were collected by centrifugation at room temperature for 30 sec at 17,000 x g. Unbound chromatin and antibodies were removed by washes: two washes with buffer A and once with TE, with the centrifuge step repeated between each wash.

2.11.3. DNA isolation

Immunoprecipitated DNA was eluted from the beads by digestion of the antibody with 200 µg of proteinase K (Sigma) in elution buffer (50 mM NaHCO3, 1% [v/v] SDS) at 45°C for 2 hrs. The DNA was purified with the Qiagen PCR purification kit and eluted in 140 µL of TE.

2.11.4. Quantitation

The amount of telomeric chromatin immunopurified by each antibody was quantitated using slot-blot analysis. The purified DNA was denatured with 980 µL of 0.46 M NaOH and heated at 95°C for 5 mins.

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The cooled samples were then halved, and 560 µL loaded into a slot-blot well and blotted onto

Biodyne B 0.45 µM nylon membrane (Pall). The remainder of the sample was used to make a duplicate membrane. For the purpose of quantitation, a standard was included for every sample; 1%, 5%, 10% and 25% purified input (lacking IP) was also blotted. The membranes were air-dried, cross-linked in a

Stratagene UV Stratalinker 1800, and pre-hybridised with Church buffer at 50°C for 1 hr. One membrane

32 was hybridised overnight with a P-ATP-labelled (CCCTAA)3 telomere probe, and the other was hybridised with 32P-dATP-labelled ALU sequence. The following day, both membranes were washed four times: telomere membranes with 2X SSC at room temperature for 15 mins, and ALU membranes with

0.1% SDS/0.2X SSC at 37°C for 15 mins, and then exposed to a phosphor screen overnight. The phosphor screen was imaged with a Typhoon imager and then signals were quantitated using ImageQuant software. Standard curves were generated for each sample and used to normalise the telomeric DNA immunoprecipitated. All experiments were performed on two biological replicates.

2.12. Labelling probes with 32P

Experiments used two types of C-rich telomere probes, [CCCTAA]3 or [CCCTAA]4. Both probes were end labelled with ATP-γ32-P radiation (Perkin Elmer) by a T4 polynucleotide kinase (New England BioLabs) at

37°C for 30 mins. Unlabelled probe was removed using a Quick Spin Mini Oligo Column (Roche). The same method was used to end-label the GAPDH probe in section 2.17. The ALU probe in section 2.11.4, was labelled with α-32P-dATP (Perkin Elmer) using the DECAprime II Random Primed DNA Labeling Kit

(Ambion). Unlabelled DNA was removed using a Quick Spin Mini DNA Column (Roche).

2.13. Whole genome sequencing (WGS) and bioinformatics

2.13.1. WGS and read alignment

Whole genome sequencing was performed on the Illumina HiSeq X Ten platform, by Macrogen Inc.

(Korea) and the Kinghorn Centre for Clinical Genomics/Garvan Medical Research Institute, to generate paired-end (2 × 150 bp) sequence reads. Subsequent analysis was performed by Dr Erdahl Teber. The

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Table 2.8. Antibodies for telomere ChIP.

Antibody Company Isolated from Dilution Novus Biologicals TERF21P/ RAP1 Rabbit 1:40 (NB100-292) Novus Biologicals TRF2 Rabbit 1:70 (NB110-57130) Merck Millipore Normal Mouse IgG Mouse 1:30 (12-371) Cell Signaling Technology Normal Rabbit IgG Rabbit 1:30 (2729) Cell Signaling Technology Histone H3 Rabbit 1:40 (4620) Abcam H3K9me3 Rabbit 1:30 (ab8898)

Cell Signaling Technology H3K4me2 Rabbit 1:50 (9725)

reads were trimmed, and poor quality reads were removed using Trimmomatic (v0.32; parameters:

ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:75). The remaining paired-end reads were aligned to NCBI hg19 reference genome using BWA (v0.7.10; main parameters: mem -M). Using Picard (v1.119), the generated BAM file was sorted; duplicates were removed, and the reads placed into read groups and indexed. Picard (v1.1.19) modules,

CollectAlignmentSummaryMetrics and CollectMultipleMetrics, determined the mapping quality metrics. Insertion or deletion (indel) realignment was carried out with RealignerTargetCreator and

IndelRealigner, and known indel sites were based on Mills_and_1000G_gold_standard.indels.hg19.vcf and 1000G_phase1.indels.hg19.vcf (GATK v3.2.2 resource bundle). This was followed by

BaseRecalibrator (parameters for KnownSites are: Mills_ and_1000G_gold_standard.indels.hg19.vcf,

1000G_phase1.indels.hg19.vcf,dbsnp_138.hg19.vcf) and PrintReads (GATK v3.2.2). MuTect v1.1.4

(Cibulskis et al., 2013) (parameters: --dbsnp dbsnp_138.hg19.vcf --cosmic cosmic_v71.vcf) was used to generate single nucleotide variants (SNVs), only a ‘PASS’ entry in the FILTER column was accepted. Indels were generated using GATK v3.2.2 HaplotypeCaller (paremeters: --genotyping_mode DISCOVERY - stand_call_conf 30 -stand_emit_conf 10), filtered by GATK VariantFiltration (parameters: -- filterExpression "QD < 2.0 || FS > 200.0 || ReadPosRankSum < -20.0"), and were decomposed and left

70 normalised (Vt normalisation reference, http://genome.sph.umich.edu/wiki/Vt) (Tan et al., 2015). Short indel calls were generated by PINDEL v0.2.4w (Ye et al., 2009a) and were left normalised. For each cell line, a final vcf file was generated, and used in all downstream analysis. This file contained the intersecting variant calls between normalised Haplotypecaller and PINDEL, all unique HaplotypeCaller indels sized 1 to 9 bp, unique PINDEL calls ≥100 bp, and all SNV ‘PASS’ Mutect calls.

Delly v0.6.7 (Rausch et al., 2012) identified structural variant (SV) calls to detect the four SV types (deletion, DEL; duplication, DUP; inversion, INV; translocation, TRA). The results were concatenated and sorted using vcftools (v0.1.13) and annotated into Class A and Class B categories, whilst TxDb.Hsapiens.UCSC.hg19.knownGene (v3.2.2) R package was used to map the transcript and gene structures. A Class A annotation indicated the gene(s) overlapping at a breakpoint, and the Class B annotation, indicated the gene(s) between two breakpoints or within the SV.

2.13.2. Variant annotations

To determine potentially pathogenic genetic alterations Ingenuity Variant Analysis (IVA, v4.2.20160927) was used to filter and annotate SNV and indel results. To remove variants present in a healthy population, the analysis parameters included: a read depth of at least 10, variants had to be outside of the top 1% most exonically variable genes in healthy (1000 genomes), and variants observed in at least 0.001% of the 1000 genomes project, ExAc and NHLBI ESP exomes were excluded. The next step was to determine potentially deleterious events using the predicted deleterious filter. From the settings used we obtained a gene list including mutations: pathogenic or likely pathogenic, established in literature, gene fusion, inferred activating mutation by Ingenuity, predicted gain of function by BSIFT, frameshift/in-frame indel or start/stop codon change, missense unless predicted tolerated by SIFT or

PolyPhen-2, splice site loss up to 2 bases into or as predicted by MaxEntScan, or a loss of function structural variant.

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2.14. Sanger sequencing

All ATRX and DAXX were amplified using the primers, PCR buffer and PCR conditions previously reported (Jiao et al., 2011), and summarised in Appendix I. The protocol for TP53 sequencing was described on the International Agency for Research on Cancer (IARC) website

(http://p53.iarc.fr/Download/TP53_DirectSequencing_IARC.pdf) and the primers are listed in Table 2.7.

Briefly, the Qiagen HotStar Taq (0.8 Units) was used to amplify the TP53 exons 2-3 and 7 in a reaction mix containing 1X PCR buffer, 1X Q-solution, 0.2 mM each dNTP, 0.4 µM forward primer and 0.4 µM reverse primer. The PCR conditions for exon 2-3 were: 94°C for 2 mins, followed by 50 cycles of 94°C for

30 sec, 61°C for 45 sec and 72°C for 45 sec, and the final cycle of 72°C for 10 mins, and for exon 7 were:

95°C for 15 mins, followed by 50 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec, and the final cycle of 72°C for 10 mins. Whereas, 0.8 Units of Taq Platinum (Invitrogen) were used to amplify

TP53 exons 4, 5-6, 8-9 and 11 in a reaction containing 1X PCR buffer, 1.5 mM MgCl2, 0.2 mM each dNTP,

0.4 µM forward primer and 0.4 µM reverse primer. Exons 4, 5-6, 8-9, and 11 were amplified with the conditions: 94°C for 2 mins, followed by 20 cycles of 94°C for 30 sec, 63°C for 45 sec (-0.5°C every 3 cycles) and 72°C for 60 sec, then 30 cycles of 94°C for 30 sec, 60°C for 45 sec and 72°C for 60 sec, and the final cycle of 72°C for 10 mins. Finally, TP53 exon 10 was amplified with the PCR conditions: 94°C for

2 mins, followed by 20 cycles of 94°C for 30 sec, 58.5°C for 45 sec (-0.5°C every 3 cycles) and 72°C for 60 sec, then 30 cycles of 94°C for 30 sec, 55°C for 45 sec and 72°C for 60 sec, and the final cycle of 72°C for

10 mins. The gene H3F3A was sequenced using primers previously reported to amplify exons 1, 2 and 3 of the gene (Bower et al., 2012). Invitrogen’s Taq Platinum was used to amplify the exons, using the primer sequences listed in Table 2.7, with the PCR conditions: 95°C for 5 mins, followed by 30 cycles of

94°C for 30 sec, 60°C for 1 min and 72°C for 2 mins, and the final cycle of 72°C for 10 mins. Prior to sequencing, all PCR products were purified with the QIAquick PCR Purification Kit (Qiagen), and subsequent Sanger sequencing was conducted at the Australian Genome Research Facility.

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2.15. RNA extraction

RNA was extracted from cell pellets using the RNeasy Mini Kit (Qiagen) with on column DNA digestion

(Qiagen DNase). The RNA was resuspended in RNase-free water and quantitated using a NanoDrop spectrophotometer.

2.16. qPCR analysis

2.16.1. Reverse transcriptase qPCR (RT-qPCR)

Complementary DNA (cDNA) was synthesised from 1 µg of RNA using the Superscript III First Strand

Synthesis Kit (Invitrogen) with random primers. Transcripts of TERT, TERC, DKC1, and GAPDH were detected as previously described (Cao et al., 2008b), using the primers in Table 2.7. Expression of NBS1,

XRCC3, RTEL1, ZBTB48, and SLX4 mRNA was detected using the primers described in Table 2.7. PCR reactions contained 1X QuantiFAST SYBR Green PCR buffer (Qiagen), and 0.4 µM forward and reverse primers for TERC, DKC1, NBS1, XRCC3, RTEL1, ZBTB48, SLX4 and GAPDH transcripts or 0.5 µM forward and reverse primers for TERT transcripts. The cDNA reaction was diluted 1:2.5 with TE, and 2 µL was loaded into each PCR reaction. The PCR was performed in triplicate with the following conditions: 1 cycle at 95°C for 15 mins, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec for TERT, TERC, DKC1, and GAPDH or 40 cycles of 95°C for 15 sec, 60°C for 20 sec and 72°C for 30 sec for NBS1, XRCC3, RTEL1, and SLX4 or 40 cycles of 95°C for 15 sec and 60°C for 60 sec for ZBTB48 and then melt curve analysis.

The level of expression was calculated using a delta delta cycle threshold (Ct) method, with GAPDH as a reference gene, and expressed as RNA level relative to the appropriate control sample.

2.16.2. Genomic qPCR

MYCN copy number was determined by qPCR of 5 ng of genomic DNA. PCR reactions were performed in triplicate using 1X QuantiFAST SYBR Green PCR buffer (Qiagen) containing 0.5 µM of forward and reverse primers (Table 2.7) with the PCR conditions: 95°C for 5 mins then 43 cycles of 95°C for 15 sec,

61°C for 30 sec and 72°C for 20 sec, and melt curve analysis. Samples were quantitated using a standard

73 curve method, MYCN was normalised to the VAV2 gene, and copy number was expressed relative to the normal DNA control (Applied Biosystems).

2.17. Telomeric repeat-containing transcript (TERRA)

The quantitation of TERRA was performed by slot-blot analysis of 1 µg of DNase treated RNA (Ng et al.,

2009). In addition to a DNase treated RNA sample for each cell line, 1 µg of RNA was also treated with

25 ng/µg RNase A (Sigma) to confirm that there was no DNA contamination. All samples were denatured at 65°C for 15 mins in a solution of 66% (v/v) formamide, 2.6 M formaldehyde, and 13% (v/v) 3-(N- morpholino)propanesulphonic acid (MOPS) solution (0.5 M sodium acetate, 0.01 M EDTA, 0.2 M MOPS).

The denatured RNA was diluted with 100 µL of 2X SSC and slot-blotted onto a Biodyne B nylon membrane. Duplicates of all membranes were prepared. Membranes were dried and the RNA was crossed-linked to the membrane using the Stratagene UV Stratalinker 1800. After UV-cross-linking, both membranes were pre-hybridised with Church buffer at 55°C for 1 hr, then one membrane was

32 32 hybridized with a P-ATP-labelled (CCCTAA)3 telomeric probe and the other a P-labelled GAPDH probe

(ACCCACTCCTCCACCTTTG), overnight at 55°C. The following day, the membranes were washed three times for 15 mins at room temperature with 2X SSC/0.1% SDS and then at 50°C with 0.2X SSC/0.1% SDS for 15 mins. Membranes were exposed to a storage phosphor screen and scanned on a Typhoon imager.

Images were quantitated with ImageQuant software and the amount of TERRA was corrected for loading by dividing by the intensity of the corresponding GAPDH transcript. Three biological replicates were analysed.

2.18. Telomerase activity

2.18.1. Protein Lysis

Cells were trypsinised, washed with 1X PBS and pelleted. An equal number of cells were lysed at 4°C for

30 mins with 3-([3-cholamidopropyl] dimethylammonio)-1-propanesulphonate (CHAPS) lysis buffer (10 mM Tris-HCl pH 7.5, 1 mM MgCl2, 1 mM ethylene glycol-bis[2-aminoethylether]-N,N,N',N'-tetraacetic

74 acid [EGTA], 10% glycerol, 10% CHAPS), supplemented with 1mM PMSF and 5 mM 2-mercaptoethanol.

Following lysis, the samples were centrifuged at 15,500 x g for 20 mins at 4°C, and then the supernatant was transferred to a new tube to remove cellular debris.

2.18.2. Immunoprecipitation

To purify the lysate and remove potential PCR inhibitors, anti-hTERT antibody (10 µg; obtained from Dr

Scott Cohen) was bound to telomerase by rotating samples at 4°C for 30 mins (Cohen and Reddel, 2008).

A 50% (v/v) suspension of Protein G beads (Roche), pre-blocked in BSA (New England BioLabs), was then bound to the antibody at 4°C for 1 hr. The telomerase/antibody/bead complex was collected in an

Illustra micro-spin column (GE Healthcare) with vacuum suction, and the supernatant discarded. The beads were washed with ice cold lysis buffer to remove any unbound proteins. To release the telomerase enzyme from the bead/antibody complex, 1.5 mM antigenic peptide solution was combined with the complex in the column and incubated at room temperature for 1 hr before collection of the supernatant.

2.18.3. Telomeric repeat amplification protocol (TRAP)

Immunoprecipitated telomerase was added to 0.09 µg of the M2 primer (Table 2.7) and allowed to extend via telomerase mediated base addition in a buffer containing 45 mM Tris pH 8.8, 11 mM ammonium sulphate, 4.5 mM MgCl2, 10 mM EDTA pH 8, 76 mM 2-mercaptoethanol, 113 µg/mL BSA

(Ambion), 1 mM each of dNTPs (Roche), 0.045 µg of the reverse primer ACX (Table 2.7), and 2 Units (Roche). The TRAP reaction was performed under the conditions in Table 2.8. PCR products were separated on a 10% denaturing polyacrylamide gel in 0.5X TBE and detected with SYBR Green I

(Molecular Probes), visualised on a Typhoon imager.

2.19. Immunoblotting

Cells were harvested, pelleted and resuspended in cold lysis buffer supplemented with complete protease inhibitor (Roche), 100 mM NaF, and 200 µM Na3VO4 immediately before use. For analysis of

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Table 2.8. TRAP PCR conditions. Number of cycles Step Temperature Time Telomerase extension of 1 cycle 25°C 30 mins primer 1 cycle Taq Hot start 95°C 2 mins 35 cycles Denaturation 95°C 10 sec Annealing 50°C 25 sec Extension 72°C 30 sec 1 cycle Denaturation 95°C 15 sec Annealing 50°C 25 sec Extension 72°C 60 sec

Table 2.9. Antibodies and conditions used for Western blot analysis. Name Manufacturer Isolated Blocking agent Diluent Dilution Incubation from conditions Room Abcam anti-ATRX Rabbit 5% (w/v) skim milk 1X TBS 1:500 temperature, (ab72124) 2.5 hrs Sigma Room anti-DAXX (HPA008736- Rabbit 5% (w/v) skim milk 1X TBS 1:250 temperature, 100UL) 2 hrs Merck Same as Room anti-p53 (DO- 5% (w/v) skim Millipore Mouse blocking 1:1000 temperature, 1) milk/1X TTBS (OP43L) agent 1 hr Same as Room anti-p21 Cell Signalling 5% (w/v) BSA/1X Rabbit blocking 1:1000 temperature, (Waf1/Cip1) (2947) TTBS agent 1 hr ThermoFisher Same as 5% (w/v) skim Overnight, anti-GAPDH Scientific Mouse blocking 1:5000 milk/1X TTBS 40C (MA5-15738) agent Sigma Overnight, anti-β-actin Mouse 5% (w/v) skim milk 1X TBS 1:3000 (A2228-100UL) 40C Sigma Overnight, anti-HSP70 (H5147- Mouse 5% (w/v) skim milk 1X TBS 1:3000 40C 100UL)

ATRX and DAXX proteins the cells were lysed in EBC buffer (50 mM Tris pH 8, 120 mM NaCl, and 0.5%

(v/v) NP-40), and for p53 and p21 immunoblots the cells were lysed in RIPA buffer (50 mM Tris pH 8,

150 mM NaCl, 0.1% (v/v) SDS, 1% (v/v) NP-40, and 0.5% (v/v) sodium deoxycholate). Cells were lysed for 30 mins at 4°C and centrifuged at 15,500 x g for 20 mins to remove cell debris. The protein concentration of the whole cell extract was determined against known concentrations of BSA standards, using the bicinchoninic acid (BCA) protein assay kit.

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Equal concentrations of protein lysates were mixed with NuPAGE LDS Sample Buffer (Life

Technologies) and denatured by boiling at 95°C for 10 mins. Proteins were resolved on a 3–8% NuPAGE

Tris-acetate polyacrylamide SDS gel (Life Technologies) (running buffer: 2.5 mM tricine, 2.5 mM Tris,

0.005% (v/v) SDS, pH 8.24) for ATRX, on a 10% Tris-glycine SDS polyacrylamide gel for DAXX, and on a

12% Tris-glycine SDS polyacrylamide gel for p53 and p21 (running buffer: 0.02 M Tris, 0.2 M glycine,

0.1% SDS (v/v), pH 8.3). Separated proteins were transferred to a methanol-activated 0.45 mm PVDF membrane (Millipore), in chilled transfer buffer (22 mM Tris, 171 mM glycine, 0.01% (v/v) SDS, 20% (v/v) methanol), and blocked for 1 hr at room temperature and then probed according to the conditions in

Table 2.9. The membranes were washed with a solution of 0.1% Tween-20, 50 mM Tris, and 150 mM

NaCl (TTBS) three times for 15 mins each, then probed at room temperature for 1 hr with HRP- conjugated secondary antibodies (DakoCytomation) diluted 1:5000 in TTBS, and followed by another wash series with TTBS. Chemiluminescence detection was performed using SuperSignal West Pico chemiluminescent substrate (ThermoFisher Scientific) at room temperature for 5 mins, exposed to X- ray film (Fujifilm), and developed on an X-ray developer (Konica).

2.20. Statistical analyses

GraphPad Prism 6 was used to generate graphs and perform statistical analysis. The Kaplan-Meier product-limit method was used to estimate survival, comparisons of survival curves were performed by the log-rank test, and medians and proportions were compared by the Mann-Whitney test and Chi- square test, respectively. Two data sets were compared with unpaired Student’s two-tailed t tests or two-tailed Mann-Whitney tests, and multiple data sets were compared using one-way ANOVA.

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Chapter 3:

A subgroup of high-risk MYCN non- amplified neuroblastomas lacks a telomere lengthening mechanism

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Chapter 3: A subgroup of high‐risk MYCN non‐amplified neuroblastomas lacks a telomere lengthening mechanism

3.1. Introduction

The acquisition of replicative immortality is a hallmark of human cancer, achieved by activation of a TLM to counter the normal telomere attrition that accompanies cellular proliferation (Hanahan and

Weinberg, 2000; Hanahan and Weinberg, 2011). However, a substantial proportion of glioblastomas

(Hakin-Smith et al., 2003), liposarcomas (Costa et al., 2006; Jeyapalan et al., 2008), osteosarcomas

(Ulaner et al., 2003), and retinoblastomas (Gupta et al., 1996) have been reported as telomerase- negative with no evidence of ALT activity. The consensus view in the telomere research field has been that these results are due to false-negative assays; however, there can be other plausible explanations.

Firstly, telomerase-negative tumours may be able to maintain their telomere length by a different ALT mechanism which lacks the known phenotypic characteristics of ALT. A second potential explanation is that it is theoretically possible some tumours can achieve long term proliferation without an active TLM

(Reddel, 2000). To date, it has been impossible to demonstrate the latter scenario as serial sampling of human tumours which are required to show continuous telomere shortening over extended periods of time cannot usually be obtained. Therefore, suitable cell line models are required to determine if an active TLM is a requirement for the long-term proliferation of malignant cells.

NB is an embryonal tumour of the sympathetic nervous system (Maris, 2010). Telomerase activity has been identified as a poor prognostic factor in NB (Ohali et al., 2006; Onitake et al., 2009;

Poremba et al., 2000; Poremba et al., 1999) although it is absent in 20-60% of metastatic NB (Krams et al., 2001; Onitake et al., 2009; Poremba et al., 2000; Poremba et al., 1999), supporting the presence of

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ALT in NB. Thus far, ALT has either been diagnosed in NB tumours by long telomeres (a feature of ALT) in the absence of telomerase (Onitake et al., 2009) or by the presence of mutation/deletion in ATRX

(Cheung et al., 2012; Kurihara et al., 2014; Molenaar et al., 2012; Peifer et al., 2015; Valentijn et al.,

2015), a chromatin remodelling factor where loss of function is associated with ALT activity (Bower et al., 2012; Chen et al., 2014; Heaphy et al., 2011a; Lovejoy et al., 2012; Schwartzentruber et al., 2012).

However, C-circles, a quantifiable marker of ALT activity (Henson et al., 2009), have not been used to detect ALT in any of these studies. Relying solely on long telomeres and the absence of telomerase can potentially misclassify tumours as ALT-positive, especially in embryonal tumours that tend to have longer telomeres, while using only ATRX mutations will likely underestimate the prevalence of ALT as

ATRX mutations are not a universal feature of ALT (Lovejoy et al., 2012).

We hypothesised that (1) a proportion of high-risk NB with long telomeres were ALT-negative and (2) a subset of ALT-positive high-risk NB tumours were ATRX wild-type. Utilising quantitative PCR to measure telomere content (TC) and C-circle levels (Lau et al., 2013) in a cohort of high-risk NB, we detected ALT in a significantly higher frequency than previously reported on the basis of ATRX mutation frequency, supporting the hypothesis that some ALT-positive NB are ATRX wild-type. Furthermore, we found that a subset of MYCN non-amplified tumours with long telomeres lacked ALT activity. We also went on to screen a large panel of NB cell lines and identified two NB cultures with exceptionally long telomeres which were negative for both telomerase and C-circles.

3.2. Results

3.2.1. Unique subgroup of high-risk MYCN non-amplified neuroblastoma

Here we describe a cohort of 149 high-risk NB screened for C-circles and TC using telomere quantitative

PCR where ALT-positive tumours were defined as those with a relative C-circle level of ≥7.5 compared to the ALT+ IIICF/c cell line with an arbitrary value of 100 (Lau et al., 2013) (Appendix II Table A2.1-4).

Twenty-four percent (36/149) of tumours were found to be C-circle/ALT-positive. None (0/36) of the

ALT-positive tumours had MYCN amplification, consistent with other studies reporting MYCN

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Figure 3.1. A subgroup of high-risk MYCN non-amplified neuroblastoma with long telomeres are ALT-negative. (A) Relative telomere content (TC) in arbitrary units (AU), measured by telomere qPCR for individual patient tumours (n=149), are plotted according to ALT status (ALT+ n=36, ALT- n=113). ALT+ was defined by a relative C-circle (CC) level ≥7.5 compared to the ALT+ IIICF/c cell line with an arbitrary value of 100. (B) Age at diagnosis for individual patient tumours is plotted according to ALT status

(n=149). (C) Relative TC of MYCN non-amplified tumours (n=94) is plotted according to ALT status

(ALT+ n=36, ALT- n=58). A subgroup of ALT- tumours (n=17) had long telomeres (TC≥15, indicated by the bracket). (D) Age at diagnosis for individual MYCN non-amplified ALT-negative patient tumours (n=58) is plotted according to TC.

Data points represent the average of triplicate TC measurements by qPCR. Horizontal red bar indicates group median. * P<0.01

81 amplification and ATRX aberrations are mutually exclusive (Cheung et al., 2012; Kurihara et al., 2014;

Peifer et al., 2015; Pugh et al., 2013; Valentijn et al., 2015). This is contrast with ALT-negative tumours where 49% (55/113) had MYCN amplification. The relative TC of ALT-positive tumours was significantly higher than that of ALT-negative tumours (median 19.7 vs. 6.2; P<0.0001; Figure 3.1.A); this was not due to younger patients in the ALT-positive group as patients with ALT NB were significantly older at diagnosis than patients with ALT-negative NB (median age 4.4 vs. 2.3 years; P<0.0001; Figure 3.1.B). Of note, within the MYCN non-amplified group, 29% (17/58) of tumours with long telomeres (TC≥15) were

ALT-negative (Figure 3.1.C). In addition, patients with ALT-negative/TC≥15 tumours were older at diagnosis than patients with ALT-negative/TC<15 tumours (median age 4.67 vs. 2.72 years; P<0.01;

Figure 3.1.D). There was sufficient DNA to assess telomere length by Southern blot terminal restriction fragment (TRF) analysis for 45 samples, including four of the 17 ALT-negative/TC≥15 tumours. The TRF telomere length profile of the four ALT-negative/TC≥15 tumours was long and heterogeneous (relative

TC≥15 corresponds to mean TRF length 20 kb; Figure 3.2.A), with a mean length indistinguishable from

ALT-positive tumours (P>0.05). Frozen tumour samples were available for the four ALT-negative/TC≥15 tumours which allowed us to perform TRAP assays; all four tumours were telomerase-negative (Figure

3.2.B). This suggests that a subgroup of metastatic NB with long telomeres may not require a TLM to allow long term proliferation as a result of their extensive telomere reserves.

The molecular details of ALT upregulation are not well defined but there is a correlation between ALT activity and the loss of expression of ATRX or its binding partner DAXX (Bower et al., 2012;

Heaphy et al., 2011a; Lovejoy et al., 2012). Inactivation of p53 is also strongly correlated with ALT in cell lines although this is less apparent in tumours (Henson and Reddel, 2010). Next-generation sequencing results were available for 32 tumour samples; 13 ALT-positive and 19 ALT-negative. This includes previously reported whole exome sequence data for 26 samples (Pugh et al., 2013) and whole genome sequence data for nine tumours obtained as part of this study (of which 3 had existing whole exome data from Pugh et al., 2013). Of the 13 ALT-positive tumours, eight had an ATRX mutation, one had a

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Figure 3.2. A unique subgroup of high-risk ALT-negative/MYCN non-amplified/TC≥15 NBs have poor survival. (A) Terminal restriction fragment (TRF) analysis of 13 selected tumour samples. Telomere content

(TC) and C-circle (CC) status measured by telomere qPCR is indicated at the bottom with mean TRF lengths. Black boxes represent the four ALT-negative/TC≥15 samples, green boxes represent the

ALT-positive samples, and ALT-negative/TC<15 are indicated by white boxes. (B) Telomere repeat amplification protocol (TRAP) to detect telomerase activity in six selected tumour samples. Cell lysates were immunopurified with anti-hTERT antibody to remove potential inhibitors. The ALT sample U-2 OS was negative for telomerase and SH-SY5Y was included as a telomerase positive control. (C, D) Event-free and overall survival of ALT-negative/TC≥15 (ALT-/ TC≥15) (n=17), ALT- positive (n=36), and MYCN amplified (amp) (n=55) tumours.

TRF and IP-TRAP were performed by Dr Loretta Lau.

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DAXX mutation, and one had a TP53 mutation. In contrast, abnormalities of ATRX, DAXX or TP53 were observed in 0/19 ALT-negative tumours.

It is well established that MYCN amplification results in a worse outcome for patients than MYCN non-amplified NB tumours (Ambros et al., 2009; Seeger et al., 1985). In our cohort, the outcome for patients with ALT NB was as poor as the outcome for MYCN amplified patients (5-year event-free survival 28% vs. 24%; 5-year overall survival 36% vs 28%; Figure 3.2.C, D), despite all ALT-positive tumours being MYCN non-amplified. Like ALT NB, ALT-negative/TC≥15 NB also had later events or deaths and a poor survival that was not significantly different from the ALT tumours, although this may be due to the number of cases in each subgroup (5-year overall survival 49% vs. 36%; P=0.1908; Figure

3.2.C, D), ultimately leading to death in 51% of patients with this subgroup of disease.

3.2.2. ALT-negative and telomerase-negative cancer cell lines

3.2.2.1. Neuroblastoma

To determine whether ALT-negative/TC≥15 NB cells lack a TLM, or have an as-yet unidentified telomerase-independent mechanism, the telomere length of these cells must be examined over time.

To perform this study, we screened 35 cell lines derived from metastatic NB for a phenotype corresponding to the ALT-negative/TC≥15 tumours (TC≥15 and C-circle negative via quantitative PCR and MYCN non-amplified). We identified three cell lines that have a TC≥15 (CHLA-90, COG-N-291, LA-N-

6) and one cell line with a TC of 14 (SK-N-FI); two cell lines (SK-N-FI and CHLA-90) were C-circle positive, as previously reported (Farooqi et al., 2014; Lau et al., 2013), and two were C-circle negative (COG-N-

291 and LA-N-6; Table 3.1) and also had the highest TC of the 35 cell lines (25 and 38, respectively). TRF analysis confirmed long and heterogeneous telomere lengths in COG-N-291 and LA-N-6 cells (mean 31.0 and 37.8 kb, respectively), comparable to U-2 OS, an ALT cell line with very long telomeres (mean TRF

38.8 kb; Figure 3.3.A). The TRF blots of LA-N-6 and COG-N-291 showed a banding pattern that has only been observed when ALT activity was suppressed in ALT-positive cells by overexpression of Sp100 a component of PML bodies (Jiang et al., 2005), and when telomerase-mediated telomere extension was

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Table 3.1. Characteristics of 35 neuroblastoma cell lines.

Telomere C- SD SD hTERT SD hTR SD Dyskerin SD MYCN SD content circle LA-N-6 38.0 0.047 -0.4 0.036 0.0 0.01 1.1 0.07 0.5 0.05 3.9 0.4 COG-N-291 25.0 0.006 -3.3 0.022 0.0 0.00 0.0 0.00 0.9 0.13 2.7 0.2 CHLA-90 15.6 0.005 139 0.285 0.1 0.06 2.8 0.44 0.8 0.04 18.9 0.8 SK-N-FI 14.0 0.013 196 0.695 0.0 0.00 0.3 0.04 1.4 0.24 4.2 0.5 CHLA-119 2.0 0.002 2.1 0.014 0.1 0.02 0.7 0.10 0.9 0.14 262 16.0 CHLA-122 0.8 0.001 -0.1 0.000 3.0 0.40 4.2 0.29 1.2 0.08 154 14.5 CHLA-136 0.8 0.003 -0.1 0.000 0.5 0.07 1.7 0.07 1.3 0.13 141 24.0 CHLA-140 1.2 0.009 -0.2 0.001 4.6 0.56 2.2 0.34 0.3 0.01 6.9 0.7 CHLA-171 1.3 0.001 0.3 0.001 3.3 0.28 1.1 0.07 0.6 0.11 2.7 0.1 CHLA-225 2.5 0.001 0.0 0.006 2.2 0.29 2.6 0.29 1.5 0.18 127 14.2 CHLA-42 0.4 0.001 0.3 0.001 0.6 0.09 2.5 0.08 1.0 0.13 4.8 0.6 CHLA-95 1.0 0.001 0.1 0.002 0.6 0.04 0.7 0.07 0.8 0.10 495 41.9 CHP-134 1.3 0.002 -0.1 0.001 2.1 0.07 1.0 0.14 1.6 0.07 485 60.5 COG-N-328h 0.5 0.001 -0.1 0.000 0.9 0.13 1.9 0.15 2.0 0.22 252 26.7 COG-N-347 0.4 0.002 -0.1 0.000 0.6 0.08 1.5 0.11 0.3 0.02 1066 77.0 FU-NB-2006 0.6 0.000 0.3 0.004 1.0 0.25 5.5 0.53 1.2 0.18 5.7 0.7 IMR32 0.5 0.002 0.0 0.002 1.0 0.10 1.0 0.11 1.0 0.10 120 25.4 KELLY 0.6 0.001 0.0 0.002 2.5 0.26 0.8 0.08 0.7 0.07 676 56.5 LA-N-1 1.3 0.001 0.0 0.002 0.3 0.05 0.8 0.13 1.0 0.09 392 85.5 LA-N-5 0.5 0.000 0.0 0.001 2.7 0.25 1.2 0.14 1.4 0.17 256 10.7 NB69 1.1 0.000 0.1 0.002 2.7 0.36 0.7 0.07 0.6 0.09 4.5 0.7 SH-EP 2.5 0.002 0.0 0.004 8.1 0.50 1.1 0.12 1.3 0.11 1.0 0.1 SH-SY5Y 1.2 0.003 -0.2 0.001 2.9 0.31 1.2 0.06 0.8 0.05 3.7 0.3 SK-N-AS 1.8 0.004 0.2 0.001 2.3 0.29 0.3 0.02 1.0 0.14 2.8 0.7 SK-N-BE1 1.8 0.005 0.3 0.005 1.0 0.14 1.6 0.14 0.4 0.05 475 41.6 SK-N-BE2c 0.6 0.001 0.0 0.002 0.4 0.05 0.9 0.11 1.0 0.14 115 8.4 SK-N-DZ 2.1 0.036 -0.3 0.001 0.8 0.11 0.9 0.08 1.0 0.08 220 36.3 SK-N-SH 1.2 0.001 -0.1 0.002 1.8 0.19 0.9 0.15 0.6 0.07 3.1 0.3 SMS-KANR 0.4 0.000 0.0 0.002 0.8 0.11 1.6 0.08 1.1 0.12 164 17.7 SMS-KCN 0.7 0.001 0.1 0.000 0.4 0.06 0.7 0.07 0.5 0.04 909 55.7 SMS-KCNR 0.4 0.000 0.0 0.002 0.5 0.07 1.2 0.09 0.6 0.02 537 32.5 SMS-LHN 1.3 0.002 -0.3 0.001 0.2 0.02 2.3 0.33 1.3 0.17 6.2 0.7 SMS-SAN 0.5 0.001 0.0 0.001 0.2 0.03 1.8 0.10 0.9 0.10 835 64.1

Note: Relative hTERT, hTR, and dyskerin mRNA levels (samples normalised to IMR32 set to 1) and MYCN copy number were measured by qPCR and expressed as the mean of triplicates.

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Figure 3.3. Two neuroblastoma cell lines (COG-N-291 and LA-N-6) have long and heterogeneous telomere lengths similar to the ALT-negative/TC≥15 tumour group. (A) TRF analysis of a panel of seven cell lines. ALT-positive (ALT+) and telomerase-positive (TEL+) cell lines are indicated. The HT1080 hTR line is a clone of HT1080 with telomeres over-lengthened due to overexpression of hTR and upregulation of telomerase. (B) Representative images of telomere fluorescent in situ hybridisation (FISH) including the ALT cell line U-2 OS and telomerase-positive cell line SH-SY5Y. Telomeres are represented by green signals at the end of the chromosomes. (C)

Quantitation of telomere FISH in a panel of eleven cell lines. Dots in graph represent individual telomere signals with mean telomere fluorescence indicated by red horizontal bar. 15 metaphases were analysed per cell line. (D) Percentage of chromosome ends without telomere signal (images from Figure 3.3.B). 15 metaphase spreads were analysed per cell line.

86 inhibited by deletion of the essential DNA helicase RTEL1 (Uringa et al., 2012). Telomere fluorescence in situ hybridisation (FISH) analysis showed COG-N-291 and LA-N-6 have a greater range of fluorescent intensities and fewer short telomeres (signal-free ends) than ALT cell lines (Figure 3.3.B, C, D).

Although COG-N-291 and LA-N-6 had very long telomeres, they did not have elevated markers of ALT or telomerase activity. Both cell lines were telomerase-negative by TRAP (Figure 3.4.A). To confirm this was not the result of PCR inhibitors present in the sample lysate resulting in a false-negative

TRAP result, the samples were spiked with a telomerase-positive lysate (Figure 3.4.B). The presence of telomerase activity in the spiked samples confirmed these two cell lines were telomerase-negative. The cells also lacked detectable mRNA expression for hTERT, the catalytic subunit of telomerase (Table 3.1,

Figure 3.4.C). Although a low level of APBs was identified in COG-N-291 and LA-N-6, the level was similar to telomerase-positive samples and significantly lower than ALT-positive cell lines (P<0.001; Figure

3.4.D). C-circle levels were measured using a second method where a 32P-labelled telomeric probe was used to detect products of the C-circle rolling circle amplification catalysed by Φ29 polymerase (Henson et al., 2009). This detection method identified very low levels of signal above the no Φ29 polymerase control (Figure 3.4.E). To determine whether this low level of signal was due to extension of genomic linear telomeric DNA or amplification of C-circles, exonuclease digestion of linear telomeric DNA was performed prior to the C-circle assay. The presence of radioactive signals following exonuclease digestion confirmed that the cells contained very low levels of C-circles (Figure 3.4.F).

Both COG-N-291 and LA-N-6 lacked genetic variations associated with ALT activation, and like the ALT-negative/TC≥15 tumours were MYCN non-amplified (Table 3.1). Whole genome sequencing indicated both cell lines were wild-type for H3F3A, TP53, ATRX and DAXX genes (Appendix II Table A2.5,

6) and was confirmed by Sanger sequencing, although it has been previously reported that LA-N-6 possesses a homozygous deletion of CDKN2A (Garcia et al., 2010) and COG-N-291 possesses a TP53 polymorphism (Farooqi et al., 2014). Consistent with wild type TP53, the downstream target p21 was induced when cells were treated with the DNA damaging agent doxorubicin (Figure 3.5.A). Full- length

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Figure 3.4. The COG-N-291 and LA-N-6 cell lines are phenotypically similar to the ALT- negative/TC≥15 tumour group. (A) TRAP assay to detect telomerase activity in a panel of NB cell lines. Cell lysates were

immunopurified (IP) with anti-hTERT antibody to remove potential PCR inhibitors. (B) Telomerase

activity measured by TRAP. All telomerase-negative samples were spiked with lysate from the

telomerase-positive sample, SH-SY5Y, to confirm that no PCR inhibitors were present. (C) Melt curves

from SYBR Green hTERT RT-qPCR. Arrows indicate that primer dimers formed when no template was

present in CHLA-90 (ALT+) and LA-N-6. Second peak represents product formed by qPCR

amplification of template in telomerase-positive samples (SK-N-BE2c and SH-SY5Y). (D) ALT-

associated promyelocytic leukaemia bodies (APBs) in a panel of cell lines (co-localisation of telomeric

foci (green) and PML nuclear body (red), indicated by arrows). ALT-positive (ALT+) and telomerase-

positive (TEL+) cell lines are indicated. Data represent mean (+SD) of 3 independent experiments.

Scale bar represents 3 µM. (E) Representative C-circle (CC) assay with and without Φ29 polymerase,

followed by detection using a 32P labelled telomeric probe in slot-blot analysis. CC level was

calculated relative to that of the ALT-positive U-2 OS cell line (designated to be 100 AU). ALT-positive

(ALT+) and telomerase-positive (TEL+) cell lines are indicated. (F) Confirmation of C-circle assay

products in COG-N-291 and LA-N-6 at three different population doublings (PD). To remove linear

telomeric DNA prior to the C-circle assay, restriction enzyme-treated DNA was subjected to

exonuclease digestion. MeT-4A and DOS16 are ALT-positive cell lines with low CC levels (Henson et

al., 2009). SK-N-BE2c, a telomerase-positive cell line, was used as negative control.

ATRX and DAXX proteins were also expressed in both cell lines (Figure 3.5.B) and localised to the nucleus

(Figure 3.5.C). As expected for TP53 wild-type cultures (Cesare et al., 2009), the cells do not accumulate

DNA damage at the telomere (Figure 3.6.A). The level of the telomeric transcript TERRA has been associated with telomere length and accordingly, levels are higher in ALT cells than telomerase-positive cells (Arora et al., 2014; Episkopou et al., 2014; Ng et al., 2009). However, TERRA levels in COG-N-291 and LA-N-6 were well below those of any other sample with long telomeres (Figure 3.6.B). Taken

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Figure 3.5. The cell lines COG-N-291 and LA-N-6 lack characteristic of ALT. (A) p53 and p21 immunoblot with GAPDH loading control. Cultures were treated for 72 hrs with a doxorubicin (DOX) dose equivalent to their IC50. p53 status is indicated for each cell line (W, wild type; M, mutant). (B) ATRX and DAXX immunoblot with HSP70 loading control. CHLA-90 and U-2 OS

(ATRX-negative cell line with partial deletion of ATRX gene) were used as negative controls. (C)

Representative images of ATRX and DAXX foci (red) in the nuclei (blue) of NB cell lines.

90 together these data suggest that COG-N-291 and LA-N-6 cells lack the genetic and/or epigenetic

(Episkopou et al., 2014) changes that allow upregulation of ALT in some cancer cells.

To determine whether differences in the chromatin structure of COG-N-291 and LA-N-6 were responsible for preventing upregulation of ALT in these cells, we performed telomere chromatin immunoprecipitation (ChIP) for shelterin proteins and histone 3 (H3) marks. There was no difference in the level of TRF2 or RAP1 shelterin proteins bound to the telomeres of ALT or COG-N-291 and LA-N-6 cells (Figure 3.7.A). Similarly, there was no increase in repressive histone marks at H3 compared to ALT

NB cell lines (Figure 3.7.B).

From the work described in this section, we conclude that the cell line screen has identified two cell lines (COG-N-291 and LA-N-6) that are phenotypically and genetically similar to the ALT- negative/TC≥15 tumours.

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Figure 3.6. COG-N-291 and LA-N-6 lack DNA damage at the telomere and characteristics of ALT. (A) Representative images of meta-TIF assay (telomere foci green and γ-H2AX red, arrows indicate damage at telomere). Bottom panel is percentage of chromosome ends with or without telomere signal that have DNA damage. p53 status is indicated below each cell line (WT wild type; M, mutant;

LgT, SV40 large T antigen). Mean +SD from 3 independent experiments. (B) Telomeric repeat- containing RNA (TERRA) was quantitated from DNase treated RNA, followed by detection using a 32P labelled telomeric or GAPDH (loading control) probe in slot-blot analysis. The TERRA levels of ALT samples (CHLA-90, SK-N-FI, U-2 OS, SK-LU-1, SAOS-2 and G292), telomerase-positive cells (SH-SY5Y,

SK-N-BE2c, A2182 and MG63) and mortal cell lines (HFF5 and MRC-5) were compared to COG-N-291 and LA-N-6. Bar graph shows mean (+SD) TERRA levels from 3 independent experiments.

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Figure 3.7. The level of telomere bound proteins does not differ between ALT neuroblastoma cell lines and COG-N-291 and LA-N-6. (A) Telomere-ChIP against the shelterin complex components RAP1 and TRF2. The telomerase- positive cell lines SK-N-BE2c and SH-SY5Y, and ALT-positive cell lines SK-N-FI and CHLA-90 are included for comparison. Quantitation of the percentage of telomeric pulled down is shown in the graph. Mean +SD from 2 independent experiments. (B) Telomere-ChIP against histone H3 and histone marks. The telomerase-positive cell line SH-SY5Y and ALT-positive cell lines SK-N-FI and

CHLA-90 are included for comparison. Quantitation of the percentage of telomeric pulled down and normalised to H3 is shown in the panel below. Mean +SD from 2 independent experiments.

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3.2.2.2. Other tumour types

In addition to NB, we screened a cohort of 94 PanNET tumours, 48 oesophageal tumours, and 2 cohorts of melanoma tumours (n=82 and n=62) for the ALT-negative/TC≥15 phenotype. Four melanoma tumours met the criteria, C-circle negative and TC≥15, whilst one oesophageal tumour had a TC just below the cut-off for long telomeres (Figure 3.8.A, B, C, D). As with the NB tumours, to determine if these samples are utilising an as-yet unidentified ALT mechanism, or if they proliferate despite lack of a

TLM, we require cell line models to examine telomere length over time. Subsequently, 146 melanoma cell lines were examined for C-circle level and TC. There were four melanoma cell lines with longer telomere lengths (TC>10; Figure 3.9.A) and no C-circles, however, they had detectable mRNA expression for hTERT and hTR RNA (Figure 3.9.B), components of telomerase, and had telomerase activity (Figure

3.9.C). Therefore, the ALT-negative/TC≥15 phenotype is present in melanoma tumours but the current study did not identify any corresponding cell line model.

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Figure 3.8. Some melanoma tumours are ALT-negative with TC≥15. (A) Relative TC in arbitrary units (AU) of pancreatic neuroendocrine tumours (PanNET), measured by telomere qPCR for individual patient tumours (n=94), plotted according to ALT status (ALT+ n=33,

ALT- n=61). ALT+ was defined by a relative C-circle level ≥7.5 compared to the ALT+ IIICF/c cell line with an arbitrary value of 100. (B) Relative TC of oesophageal tumours (n=48) is plotted according to

ALT status (ALT+ n=0, ALT- n=48). (C) Relative TC of melanoma tumours cohort 1 (n=82) is plotted according to ALT status (ALT+ n=12, ALT- n=70). ALT-negative samples with TC≥15 compared to ALT+

IIICF/c cell line with arbitrary value of 30 are indicated by the bracket. (D) Relative TC of melanoma tumours cohort 2 (n=62) is plotted according to ALT status (ALT+ n=1, ALT- n=61), ALT-negative samples with TC≥15 compared to ALT+ IIICF/c cell line with arbitrary value of 30 are indicated by the bracket.

Data points represent the average of triplicate measurements by qPCR. Horizontal red bar indicates mean.

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Figure 3.9. ALT-negative/long telomere melanoma cell lines are telomerase positive. (A) Relative telomere content, in arbitrary units (AU), of ALT-negative cell lines. Data points represent the average of triplicate TC measurements by qPCR. A cohort of 146 melanoma cell lines are represented. The box indicates 4 melanoma cell lines with long telomeres (TC>10). (B) mRNA expression of the telomerase components hTR and hTERT, measured by RT-qPCR, for the TC>10 melanoma cell lines in Figure 3.8.A. (C) TRAP to detect telomerase activity in the long telomere TC>10 melanoma cell lines from Figure 3.8.A, B. The cell lines LA-N-6 and U-2 OS (ALT) are included as negative controls whilst SH-SY5Y is a telomerase positive NB cell line.

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3.3. Discussion

In the past two decades, numerous reports have identified telomerase activity as a poor prognostic factor in NB (Ohali et al., 2006; Onitake et al., 2009; Poremba et al., 2000; Poremba et al., 1999).

However, reports also identified a subgroup of metastatic disease that does not utilise telomerase for continued proliferation (Choi et al., 2000; Krams et al., 2001; Poremba et al., 2000; Poremba et al., 1999;

Reynolds et al., 1997). Studies of ALT in NB tumours thus far have relied on telomere length (Onitake et al., 2009), or ATRX/DAXX mutations (Cheung et al., 2012; Kurihara et al., 2014; Molenaar et al., 2012;

Peifer et al., 2015; Valentijn et al., 2015) to identify the mechanism. This is the first study of ALT in NB tumours to utilise telomere length and C-circles, a quantifiable marker of ALT activity, to determine the prevalence of ALT in metastatic NB.

We found 24% of high-risk NB were ALT-positive and that MYCN amplification and ALT were mutually exclusive, in agreement with previous studies (Cheung et al., 2012; Farooqi et al., 2014;

Molenaar et al., 2012; Peifer et al., 2015; Pugh et al., 2013). Interestingly, ALT-positive patients had more late events than MYCN amplified patients. The presence of ALT correlated with poor outcome in high-risk NB as previously reported in ATRX mutated NB tumours (Kurihara et al., 2014; Lundberg et al.,

2011; Onitake et al., 2009; Valentijn et al., 2015), liposarcoma, and malignant fibrous histiocytoma tumours (Henson and Reddel, 2010). Thirteen ALT tumours had whole exome or whole genome sequence data available and four of these tumours were wild type for ATRX and DAXX. Mutations in

ATRX/DAXX, and consequently the prevalence of the ALT mechanism, have been reported in 10-22% of high-risk NB tumours (Cheung et al., 2012; Peifer et al., 2015; Valentijn et al., 2015). The broad range of

ALT prevalence reported via ATRX/DAXX sequencing indicates inter-study variables impact significantly on the final prevalence estimates obtained from sequencing. These studies under-report the prevalence of ALT by 2-12% compared to ALT in high-risk NB detected by the presence of C-circles. Therefore, relying on ATRX and DAXX mutations to identify ALT-positive NB tumours would underestimate the prevalence of this mechanism.

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Of note, we identified a subgroup of high-risk NB that had telomere lengths similar to the length of ALT cells but with very low levels of C-circles and no telomerase activity. In addition, 51% of the patients in this subgroup died from the disease. This is distinct from Stage 4S NB, a group of unique tumours that generally spontaneously regress and has a survival of 70-90% (Nickerson et al., 2000; van

Noesel et al., 1997). Like ALT-negative/telomerase-negative tumours reported in liposarcoma, glioblastoma, osteosarcoma, and retinoblastoma (Costa et al., 2006; Gupta et al., 1996; Hakin-Smith et al., 2003; Jeyapalan et al., 2008; Ulaner et al., 2003), serial tumour samples are required to determine if these tumours represent an unidentified ALT mechanism lacking phenotypic characteristics of ALT, or if these tumours do not require a TLM for long term proliferation. The only definitive way to determine which hypothesis is correct, is to determine what happens to telomere length over time. In the first scenario telomere lengths would be maintained, whilst in the second telomeres would shorten over time. To determine if telomere length shortened during tumour growth we had to identify corresponding cell line models, as it is not possible to obtain serial tumours samples from the same patient over an extended period of time when the high-risk NB is left untreated. Two NB cell lines, COG-

N-291 and LA-N-6, derived from patients who died of metastatic disease, were identified with characteristics that correspond to the ALT-negative/TC≥15 tumours. Interestingly, these cells have a very low level of C-circle activity (at least 2 times lower than MeT-4A which has one of the lowest C- circle levels reported in ALT-positive cells (Henson et al., 2009)) which indicates a very low level of ALT activity in these cells. The only definitive way to determine if this is sufficient for telomere maintenance is to examine telomere length over time. Subsequent chapters of this thesis describe the consequences of continued proliferation on telomere length in these cells.

We were unable to identify potential TLM-negative cell lines in other tumour types. The criteria used to identify ALT-negative/TC≥15 NB may not reflect the phenotype of ALT-negative/telomerase- negative cells in other tumour types. NB is a childhood cancer and consequently the tumour initiating cells have not undergone as many cycles of replication induced telomere shortening as in adult cancers.

The NB ALT-negative/TC≥15 cells have extreme telomere lengths, hence cell lines derived from these

98 tumours still have very long telomeres despite the PDs, and consequent telomere shortening, undergone by the cells during the establishment of the cell line. Whilst adult tumour types, such as melanoma, have ALT-negative/TC≥15 tumours, they do not have the extreme telomere lengths seen in

NB. Hence cell lines derived from these tumours may not have lengths above the normal range after the

PDs required to derive a cell line from the tumour. Indeed, this hypothesis is supported by the observation, by the Decottignies laboratory, that melanoma cells with telomeres which are at the upper end of the normal length do not have a TLM (Viceconte et al., 2017). Consequently, when screening adult cancer types for ALT-negative/telomerase-negative cell lines the telomere length cut-off may need to be adjusted.

In conclusion, telomere biology plays an important role in NB pathogenesis as activation of either TLM confers poor outcome, although the disease course differs between ALT and telomerase- positive tumours. Currently, TLM inhibitors are being developed as anti-cancer therapies and may be effective in improving the outcome in high-risk NB with TLM. However, we require a greater understanding of the ALT-negative/TC≥15 phenotype to determine whether such inhibitors would be effective in these tumours. Success of targeted therapies requires appropriate biomarkers to predict response. Establishing the TLM status of a tumour is therefore required prior to treatment with TLM inhibitors or other TLM-targeted therapies. Care must be taken to correctly define ALT tumours, because neither high telomere content nor ATRX/DAXX mutation is sufficient to classify ALT tumours, and are best complemented with C-circles as an ALT activity assay.

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Chapter 4: Long-term proliferation occurs in cancer cells with ever-shorter

telomeres

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Chapter 4: Long‐term proliferation occurs in cancer cells with ever‐shorter telomeres

4.1. Introduction

A subgroup of high-risk NB tumours lack telomerase activity and upregulate the ALT mechanism to maintain telomere lengths; however, previous identification of ALT in NB has been limited to examination of telomere length (Onitake et al., 2009) or ATRX/DAXX mutations (Cheung et al., 2012;

Kurihara et al., 2014; Molenaar et al., 2012; Peifer et al., 2015; Valentijn et al., 2015). In the previous chapter we examined ALT in high-risk NB by simultaneous detection of long telomeres and abundant C- circles. This method identified ALT in 24% of high-risk NB, as well as a subgroup of TLM-negative tumours that had extremely long telomeres. In previous studies, TLM-negative tumours were assumed to result from false-negative assays. Yet, it is possible that the cells utilise a yet to be identified mechanism to lengthen telomeres. It has also been hypothesised that extreme telomere length in tumour progenitor cells would be sufficient for the tumour to achieve long term proliferation, without the need to activate a TLM (Reddel, 2000). The aim of this chapter was to determine the consequence of continued proliferation on the telomere length of two NB cell lines, COG-N-291 and LA-N-6. These cells are characteristic of ALT-negative/TC≥15 tumours when screened by qPCR. However, detection by 32P labelled telomere probe revealed the cells have a very low level of C-circles (compared to MeT-4A, an

ALT cell line with low C-circle levels (Henson et al., 2009)) indicative of low levels of ALT activity. To determine if this was sufficient to maintain telomeres in these cells, telomere length was examined during four years of continuous cell culture. We found that the COG-N-291 and LA-N-6 telomeres undergo continuous shortening during continuous proliferation for 500 PDs, a phenotype we termed ever-shorter telomeres (EST) in accordance with terminology from S. cerevisiae mutants (Lundblad and

Szostak, 1989).

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4.2. Results

4.2.1. Cells with ever-shorter telomeres are capable of long-term proliferation

To determine whether the NB cell lines COG-N-291 and LA-N-6 utilise an alternative non-telomerase method to maintain telomeres, or if they are truly TLM-negative, we examined the telomere length of the cells during continual proliferation for 500 PDs. If the first scenario was true, telomere lengths would be maintained, whilst truly TLM-negative cells would have continuous telomere length shortening.

Southern blot TRF analysis showed the telomeres of both cell lines shortened continuously during culture (Figure 4.1.A, B), at a rate of 80 and 55 bp/PD for COG-N-291 and LA-N-6, respectively (Figure

4.2.A). Normal somatic cell division results in a 31-85 bp/PD loss of telomere sequence (Harley et al.,

1990). Hence, the shortening exhibited by COG-N-291 and LA-N-6 is the result of cell division related telomere erosion, indicating the cells lack a TLM. A reduction in telomere content by qPCR confirmed the telomere shortening demonstrated by TRF analysis (Figure 4.2.B). Despite shortening telomeres and a very slow proliferation rate (3.4 days/PD for COG-N-291 and 3 days/PD for LA-N-6), the cells have yet to reach senescence after 500 PDs and over four years in culture (Figure 4.2.C). Therefore, NB cells derived from lethal high-risk tumours continue to proliferate long-term in spite of their EST phenotype.

4.2.2. Characteristics of EST cells

Telomerase activity and levels of ALT characteristics were monitored during continuous proliferation of the EST cells. The cells remained telomerase negative throughout the culture period (Figure 4.3.A) and the APB levels did not change (Figure 4.3.B). The C-circle levels were compared to the ALT cell line MeT-

4A set at 10 arbitrary units. This ALT cell line has one of the lowest levels of C-circles previously detected

(Henson et al., 2009). The C-circles in COG-N-291 and LA-N-6 persisted at very low levels with sporadic increases for the first 300 PDs in culture, but for the next 200 PDs of culture there was a stabilisation of

C-circle levels, at an amount similar to MeT-4A (Figure 4.3.C). ATRX and DAXX were expressed across the period of culture (Figure 4.3.D).

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Figure 4.1. Two neuroblastoma cell lines (COG-N-291 and LA-N-6) are capable of long term proliferation despite the ever-shorter telomere (EST) phenotype. (A, B) TRF analysis of COG-N-291 and LA-N-6 telomere lengths at progressive population doublings

(PD). The white gaps between lanes and different well heights indicate individual TRF analysis performed at progressive time points throughout the culture period. Individual blots are aligned with each other by molecular weight marker.

Initial half of TRFs were completed by Dr Loretta Lau.

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Figure 4.2. EST cells are capable of proliferation for hundreds of population doublings. (A) The average rate of telomere shortening was calculated by plotting the average TRF length of four individual TRF bands (indicated by asterisks in Figure 4.1) against the number of population doublings (PD). (B) Telomere content measured by telomere qPCR. Data represent mean + SD, n=3.

(C) Growth curves of COG-N-291 and LA-N-6 demonstrate long-term proliferation.

Calculations in panel (A) completed by Dr Loretta Lau.

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Figure 4.3. Characteristics of EST cells during continuous proliferation for 600 population doublings. (A) Telomerase activity was monitored by TRAP at the specified PDs in COG-N-291 and LA-N-6 cells.

SH-SY5Y and SK-N-BE2c were used as positive controls, and SK-N-FI and CHLA-90 (ALT+) as negative controls. (B) Frequency of APBs in EST cells at early and late population doublings (PD). Data

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represent mean +SD of 3 independent experiments. (C) C-circle levels detected using 32P labelled

telomeric DNA probe on slot blot. Levels were calculated relative to those of the ALT+ MeT-4A cell

line (designated to be 10 AU). SK-N-BE2c (telomerase-positive) was included as a negative control.

(D) ATRX and DAXX immunoblot at specified population doublings (PD) with β-actin as loading

control.

IP-TRAP was, in part, performed by Dr Christine Napier.

4.2.2.1. ALT and the EST phenotype

C-circles were present at low levels in early populations of the EST cell lines. The cells undergo continuous telomere shortening during this time, indicating that a small amount of ALT activity may be present in these cells but at levels insufficient to confer telomere maintenance. This is consistent with the observation that telomere shortening can occur in telomerase-positive normal cells (Broccoli et al.,

1995; Marion et al., 2009), and that ALT activity has been detected in somatic cells of mice (Neumann et al., 2013). However, within the first 300 PDs of culture the cells had sporadic increases in C-circle levels despite continuous telomere shortening ultimately resulting in a level of C-circles similar to the

ALT cell line MeT-4A after PD400 and telomere length stabilisation. To determine if a subpopulation of the EST mass culture was responsible for the sporadic increases in C-circle levels at early PDs, COG-N-

291 cells at PD247 (the first timepoint where COG-N-291 had increased C-circle levels, Figure 4.3.C) were sub-cloned by plating cells at very low density until colonies from a single cell arose. The TRF analysis of subclones indicated clone 4 has long and heterogeneous telomere lengths, typical of ALT cells, whilst the remaining cell lines have the banding pattern seen in the EST mass culture (Figure 4.4.A). The level of C-circles in clone 4 was at least five-fold higher than the other COG-N-291 subclones (Figure 4.4.B), which indicates that a subpopulation of cells is responsible for the increased C-circle level in the mass culture.

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Figure 4.4. A subpopulation of COG-N-291 cells are responsible for sporadic increases in C-circles in the first 300 PDs of culture. (A) TRF of the telomere length of COG-N-291 clones isolated at PD247. (B) C-circle slot blot detected

with 32P-labelled telomeric DNA probe. SK-N-FI was used as an ALT-positive, C-circle positive control

and SK-N-BE2c a telomerase-positive, C-circle negative control.

The TRF pattern and C-circle level in COG-N-291 clone 4 suggests that these cells have upregulated the ALT mechanism whilst the other subclones isolated were EST cells. Examination of telomere length over time by both TRF and qPCR analysis showed telomere maintenance in clone 4, whereas clones 3 and 11 exhibited the EST phenotype (Figure 4.5.A, B). As expected, none of the subclones exhibited telomerase activity during 100 PDs of culture (Figure 4.5.C), confirming that, by definition, the clone 4 cells must be utilising some form of an ALT mechanism to maintain telomere

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Figure 4.5. Periodically a population of COG-N-291 cells activate an ALT mechanism but a slower proliferation rate prevents them outgrowing the EST cells. (A) TRF analysis of the COG-N-291 subclones telomere lengths at progressive population doublings

(PD). (B) Telomere content measured by telomere qPCR in arbitrary units (AU). Data represent mean + SD, n=3. (C) Telomerase activity was monitored by TRAP at the specified PDs. SH-SY5Y was

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included as a positive control and U-2 OS as a negative control. (D) Growth curves of COG-N-291

subclones over 50 population doublings (PD).

lengths. The EST subclones had faster growth rates than clone 4; 3.3 days/PD for clone 3, 2 days/PD for clone 11 vs. 3.8 days/PD for clone 4 but this did not result in senescence in the cultures after 100 PDs of continuous proliferation (Figure 4.5.E). The slower growth rate of clone 4 implies that activation of the

ALT mechanism has not provided a survival advantage for these cells, and consequently they do not become the predominant population in a mixed culture with the EST cells in the first 300 PDs of growth

(Figure 4.3C). Presumably, further molecular changes, caused by long term culture, were required for these ALT-like cells to become the predominant population of cells observed after PD400 leading to the increased C-circle levels (Figure 4.3C).

Subclone 4 was able to upregulate the ALT mechanism to a sufficient level to confer telomere length maintenance; however, these cells do not possess any of the other characteristic features of ALT.

As expected, clone 4 cells maintained a high level of C-circles during culture whilst the EST subclones had very low levels although, like the mass culture, clone 3 had a sporadic increase in C-circles (Figure

4.6.A). There was no increase in the level of APBs observed in clone 4 compared to the EST controls

(Figure 4.6.B). Gene mutations associated with ALT, such as alterations to ATRX, DAXX and TP53, were absent in the clone 4 culture and ATRX and DAXX full length proteins were expressed (Figure 4.6.C).

There was also no increase in TERRA levels with activation of ALT (Figure 4.6.D) and as expected for TP53 wild type cells meta-TIFs did not accumulate (Figure 4.6.E).

4.2.3. Genetics of EST cells

To determine whether EST cells had genetic events that prevented sufficient upregulation of a TLM to counteract telomere attrition, we used whole genome sequencing to examine 656 genes related to telomere biology, ALT, and processes relating to RNA or DNA replication, repair and damage response

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Figure 4.6. Characteristics of COG-N-291 sublines during continuous long-term proliferation. (A) C-circle levels of three sub-clones of COG-N-291 (clone 4, 3, 11) detected using 32P-labelled telomeric DNA probe on slot blot. Levels were calculated (as arbitrary units [AU]) relative to those of the ALT cell line MeT-4A set to 10 AU. SK-N-BE2c (telomerase-positive) was included as a negative control. (B) Frequency of APBs in COG-N-291 sublines compared to the COG-N-291 mass culture. Graph shows the mean +SD of three independent experiments. (C) ATRX and DAXX immunoblot with GAPDH as loading control. (D) Graph of meta-TIF assay results, percentage of chromosome ends with or without telomere signal that have DNA damage. Mean +SD from 3

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independent experiments. (E) TERRA detected by slot-blot analysis with GAPDH loading control.

Quantitation in bottom panel from 3 independent experiments (mean +SD).

(Appendix II Table A2.5). To represent each of the three telomere-biology groups (EST, ALT and telomerase-positive), a total of 6 cell lines and 9 tumours were analysed (two NB cell lines and three tumours per group). As matched normal DNA was not available for the samples, we filtered the data using Ingenuity Variant Analysis to exclude genetic events previously reported in publicly available data sets of normal genomes. Relevant alterations were those predicted to have unknown or deleterious consequences and were not present in the ALT or telomerase-positive samples. There were no specific genetic events identified for the EST phenotype (Appendix II Table A2.6).

4.3. Discussion

Cellular immortalisation, via activation of a TLM to counteract normal telomere attrition, is considered to be an essential step in malignant transformation and therefore a hallmark of cancer (Hanahan and

Weinberg, 2000; Hanahan and Weinberg, 2011). To form a mass of approximately 1 kg, a single cell needs to go through 40 cell divisions (240 cells), which many types of normal cells are able to achieve

(Parkinson, 1996; Reddel, 2000). However, this would require all cells to survive, which is not possible in a tumour environment where there is an inadequate supply of oxygen and nutrients. Oncogenesis also requires multistep oncogenic mutation and associated clonal evolution, resulting in an increase in the number of cell divisions required for a tumour mass to form, and consequently the cells may require immortalisation in order to form a clinically significant tumour (Parkinson, 1996; Reddel, 2000).

However, it has been postulated that immortalisation is not required if the progenitor cells have long telomeres, the tumours are genetically simple such as paediatric cancers (Alexandrov et al., 2013; Jones et al., 2008; Parsons et al., 2011; Vogelstein et al., 2013) and don’t require multiple rounds of clonal evolution, or the tumour environment supports a high surviving fraction such as leukaemias (Reddel,

2000). The presence of the EST phenotype in malignant NB cells provides direct evidence to support the

111 hypothesis that immortalisation is not an absolute requirement for oncogenic development. The NB EST cells have very long telomeres that can act as a substitute for an active TLM, permitting the cell divisions required for additional oncogenic changes which are essential to the formation of malignant tumours.

The discovery of malignant and lethal TLM-negative cancer cells has implications for the development of TLM inhibitors for clinical use as anti-cancer therapies. Tumours that rely on extreme telomere length, and not a TLM, for continual proliferation will be resistant to these inhibitors. The existence of EST cells illustrates the need to assay for TLMs prior to therapy with such drugs. The similarity of the telomere lengths in EST and ALT cells indicate that it is not appropriate to define ALT by telomere length alone: measurement by telomere qPCR, TRF or telomere FISH is not sufficient to differentiate the two phenotypes. Furthermore, care must be taken when utilising C-circles to define

ALT; presence of low levels of C-circles does not indicate sufficient ALT activity to counter the EST phenotype. This is further complicated by the evidence that EST cultures have periodic increases in C- circles. Although misclassification of such a phenotype as ALT could result in the use of an ALT inhibitor that targets a subpopulation of the tumour cells, the bulk of the tumour would be resistant to such treatment.

The majority of ALT cell lines report inactivation of p53 function, either by viral oncoproteins or

TP53 mutations (Henson and Reddel, 2010). This implies that p53 has a role in supressing ALT, likely due to p53 mediated senescence or apoptosis which may be triggered by the high levels of chromosomal instability (Montgomery et al., 2004; Scheel et al., 2001; Ulaner et al., 2004) and telomeric DNA damage signals present in ALT cells (Cesare et al., 2009; Stagno d'Alcontres et al., 2007). COG-N-291 subclone 4 is an example of a cell line that can maintain telomeres by a telomerase-independent mechanism while retaining functional p53. As expected, these cells do not accumulate DNA damage signals at the telomere, although they have reduced proliferation rates compared to the EST cells. In a mixed culture of ALT and EST COG-N-291 cells, clone 4 is unable to outgrow the EST cells due to this reduced rate of proliferation, resulting in their removal from the population and the fluctuating C-circle levels we

112 observed. Previous studies indicate ATRX can supress the ALT mechanism (Clynes et al., 2015; Napier et al., 2015). Clone 4 cells lack abundant APBs but have wild type ATRX and DAXX, which may act with p53 to suppress some of the characteristics of ALT. However, a small proportion of ALT cell lines retain ATRX and DAXX protein, whilst exhibiting classic ALT phenotypes (Lovejoy et al., 2012), indicating that retention of p53, ATRX and DAXX protein is not soley responsible for the repressed ALT phenotype in

COG-N-291 clone 4 cells.

In conclusion, this chapter has provided evidence that lethal, malignant human cancers may, in some cases, contain cells that can proliferate long term with continuously shortening telomeres. The extreme telomere length which was presumably present in the progenitor cells gave rise to EST cells that do not require immortalisation for proliferation in excess of 500 PDs. The overlap in some features of the EST and ALT phenotypes indicates that appropriate assaying for TLMs is required before the use of TLM inhibitors as anti-cancer therapeutics.

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Chapter 5: Activation of a telomere lengthening mechanism rescues the

ever-shorter telomere phenotype

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Chapter 5: Activation of a telomere lengthening mechanism rescues the ever‐shorter telomere phenotype

5.1. Introduction

The previous chapter describes two NB EST cell lines that were capable of extensive proliferation in spite of their continually shortening telomeres. Examination of C-circle levels in these cells identified a subpopulation of cells that were able to activate a telomerase-independent TLM, although these cells did not possess many of the characteristics of ALT. In principle, these cells show that activation of a TLM would rescue the EST phenotype. The aim of this chapter is to provide evidence that ectopic expression of hTERT in EST cells results in telomerase activity, and that TP53 mutated EST cells activate a typical

ALT mechanism, and ultimately, that activation of either TLM rescues the EST phenotype. In addition to proving that activation of a TLM abolishes the EST phenotype, we determined that the EST lines were t- circle deficient, suggesting that the EST phenotype may arise due to a failure of t-circle formation and telomere trimming.

5.2. Results

5.2.1. Activation of telomerase rescues the EST phenotype

To confirm that activation of a TLM would abolish the EST phenotype we activated telomerase in LA-N-

6 cells at PD83. As established in Chapter 3, LA-N-6 cells lack expression of the telomerase catalytic subunit, hTERT. Retroviral transduction of hTERT, or of hTERT plus hTR (telomerase RNA subunit; Figure

5.1.A), resulted in telomerase activity (Figure 5.1.B). To confirm that the telomerase activity detected

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Figure 5.1. Retroviral transduction of hTERT results in telomerase activity in LA-N-6 cells. (A) mRNA expression of the three telomerase components (hTR, hTERT and dyskerin; measured by

RT-qPCR) in LA-N-6 infected with plasmids for either hTR (clones, hTR C3 & C4), hTERT (clones, hTERT C4 & C23) or hTR/hTERT (clones, hTR/hTERT C19 & C23). (B) Telomerase activity detected by TRAP. U-2 OS and SH-SY5Y cells are negative and positive controls, respectively, for telomerase activity. (C) TRAP assay performed on heat-inactivated lysates (55°C for 20 mins) from Figure 5.1.B shows that there is no PCR product contamination. (D) Growth curves of LA-N-6 derivatives.

*P<0.0003

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Figure 5.2. Ectopic expression of telomerase rescues the EST phenotype. (A) TRF analysis of the telomere length of LA-N-6 clones at the indicated PDs. (B) Verification of TRF

results by telomere qPCR at the indicated PDs. qPCR data represent mean of triplicates +SD. (C)

Relative telomere fluorescent signals measured from telomere FISH (15 metaphases analysed per

cell line). Dots represent individual telomere signals and red horizontal bar indicates mean. (D) Graph

indicates the percentage of chromosome ends without telomere signal in telomere FISH from Figure

5.2.C.

was not due to PCR contamination a proportion of all samples had the telomerase enzyme heat inactivated prior to the TRAP assay. These samples were negative on a TRAP gel (Figure 5.1.C) confirming that the banding pattern in Figure 5.1.B was due to the presence of the telomerase enzyme in these cells. As expected, expression of hTR alone did not result in telomerase activity. Activation of telomerase led to a significant increase in the growth rate of these cells compared to LA-N-6 transduced with hTR alone (Figure 5.1.D). Telomere length was examined by both TRF and qPCR, which showed that the EST phenotype was abolished in all clones expressing telomerase (Figure 5.2.A, B). Two clones expressing hTERT (C4 and C23) and one clone expressing hTR/hTERT (C19) maintained telomere length, whereas one hTR/hTERT expressing clone (C23) underwent telomere lengthening over the 50 PDs of culture

(Figure 5.2.A, B). In the telomerase-positive cells, the shortest telomeres were preferentially elongated

(Figure 5.2.C, D). In contrast, two independent clones with overexpression of hTR did not abolish the

EST phenotype (Figure 5.2.A, B). The activation of telomerase resulted in elongated telomere lengths in

LA-N-6 but as expected this did not result in any changes to ALT characteristics. The level of APBs, C- circles and TERRA did not differ between the hTR, hTERT, and hTR/hTERT clones (Figure 5.3.A, B, C, D).

Consequently, we have demonstrated that activation of telomerase in an EST cell line rescued the EST phenotype.

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Figure 5.3. No change to ALT characteristics following ectopic expression of hTERT. (A) APB (localisation of telomere FISH signal to PML body) analysis of LA-N-6 derived cell lines. Mean

+SD from 3 independent experiments. (B) C-circle levels were detected by slot blot analysis. MeT-4A and SK-N-BE2c were included as the ALT-positive control and the telomerase-positive negative control, respectively. (C) Quantitation of C-circle results in Figure 5.3.B. (D) TERRA detected by slot- blot analysis. Quantitation in bottom panel from 3 independent experiments (mean +SD).

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Figure 5.4. A LA-N-6 subline spontaneously acquired a premature stop codon in TP53. (A) Chromatograph from Sanger sequencing of LA-N-6 TP53 wild-type cells (LA-N-6 WT p53) and

the LA-N-6 subline that spontaneously acquired a stop codon (Y236*) (LA-N-6 MUT p53). (B) p53

immunoblot of LA-N-6 cells with wild-type and mutant TP53. GAPDH was used as loading control.

Western blot performed by Dr Loretta Lau.

5.2.2. Activation of ALT rescues the EST phenotype

Activation of telomerase in telomerase-negative cells only requires ectopic expression of the telomerase components hTR and hTERT (Weinrich et al., 1997). By comparison activating ALT is far more challenging. Currently, no one gene has been identified that is responsible for activating the ALT mechanism. In the previous chapter, we provided evidence that COG-N-291 cells can spontaneously activate ALT. In contrast to COG-N-291, LA-N-6 cells have a single copy of TP53 and consequently there is a high likelihood that these cells may develop a spontaneous TP53 mutation during long-term culture.

Therefore, to determine if activation of ALT can rescue the EST phenotype, we developed a strategy to identify a spontaneously activated ALT mechanism in LA-N-6 TP53 mutated cells.

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Figure 5.5. Activation of ALT results in rescue of the EST phenotype. (A) TRF analysis of the LA-N-6 culture from acquisition of the p53 mutation (PD90) to PD654.

(B) Telomere content of LA-N-6 MUT p53 cells measured by telomere qPCR. Mean +SD, n=3.

(C) Percentage of chromosome ends free of telomere signal (obtained from telomere FISH) in LA-

N-6 parental (wild-type TP53) and LA-N-6 MUT p53 cells. Quantitation from 15 metaphase spreads.

(D) Growth curves of LA-N-6 parental (wild-type TP53) and LA-N-6 MUT p53 culture. (E) Telomerase

activity was monitored by TRAP at the specified PDs in LA-N-6 MUT p53 cells. Lysates were

immunopurified (IP) with anti-hTERT antibody to remove potential inhibitors which could lead to

false negative results. SH-SY5Y and SK-N-BE2c were used as positive controls.

We first identified a subculture of LA-N-6 that spontaneously acquired a TP53 mutation at PD90. The subclone had spontaneously acquired a base change in exon 7 of TP53 (Figure 5.4.A), resulting in a premature stop codon (Y236*) and loss of p53 protein expression (hereafter referred to as LA-N-6 MUT p53; Figure 5.4.B). As expected, the loss-of-function mutation was insufficient to activate ALT, and the telomeres of this LA-N-6 MUT p53 subculture continued to shorten until PD209, i.e., for another 119 PD

(Figure 5.5.A). By PD222, the telomere length profile of the cells had changed to have the typical appearance of ALT (Figure 5.5.A). Over the next 99 PD, the mean telomere length fluctuated slightly, but after PD240 the continuous telomere shortening previously exhibited by these cells had ceased, demonstrating that a TLM had been activated (Figure 5.5.A). Telomere qPCR confirmed telomere length maintenance in LA-N-6 MUT p53 cells after PD209 (Figure 5.5.B). From PD222 onwards, the shortest telomeres in the TRF were elongated, an observation supported by a decrease in the percentage of chromosomes with telomere signal-free ends (Figure 5.5.C). The growth rate of the culture was unchanged by the loss of p53 at PD90 and by activation of a TLM at PD209 (Figure 5.5.D). The LA-N-6

MUT p53 cells exhibited telomere maintenance in the absence of telomerase (Figure 5.5.E), therefore the TLM that was activated was, by definition, an ALT mechanism. Consistent with this conclusion, we observed a 2 to 4-fold increase in C-circle levels from PD222 until PD503, after which a further 2 to

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Figure 5.6. Loss of p53 results in activation of ALT in an EST culture (LA-N-6). (A) C-circle assay product was detected using slot-blot analysis. Levels were calculated relative to the ALT-positive MeT-4A cell line with an arbitrary unit of 10 and graphed on the right. SK-N-BE2c was included as a negative control. (B) Percentage of APB positive cells in TP53 wild type LA-N-6 at

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PD95 compared to LA-N-6 mutant cells at PD704 (mean +SD, n=3 independent experiments). (C)

TERRA level in the ALT-positive LA-N-6 MUT p53 PD618 compared to TP53 wild-type LA-N-6 PD93

cells. Quantitation shows mean +SD (n=3 independent experiments). (D) Telomere sister chromatid

exchange (T-SCE) events indicated by colocalisation of red and green FISH labelled telomeres

(shown by arrows) and quantitated in the graph (mean +SD, n=2 independent experiments).

4-fold increase was observed (Figure 5.6.A), there was a 4-fold increase in APBs (Figure 5.6.B), a 2-fold increase in TERRA (Figure 5.6.C), and a 4-fold increase in T-SCE events (Figure 5.6.D). The mechanism of

ALT activation in LA-N-6 MUT p53 cells is currently unknown; although down-regulated, ATRX and DAXX expression was not abolished, and the proteins continued to localise to the nucleus (Figure 5.7.A, B).

Although loss of p53 removes a barrier to the accumulation of DNA damage responses at the telomere

(Cesare et al., 2009), the number of meta-TIFs remained unchanged (Figure 5.7.C). Irrespective of the mechanism utilised to activate ALT, our results demonstrate that ALT is able to rescue the EST phenotype.

5.2.3. EST neuroblastoma cell lines are unable to generate t-circles

During the investigation of spontaneous ALT activation in COG-N-291 clone 4 and LA-N-6 MUT p53 cells, we examined the level of t-circles, a type of double-stranded circular telomeric DNA. T-circles are abundant in ALT cells (Cesare and Griffith, 2004), and it has been proposed that this is the result of telomere trimming following telomere over-lengthening by ALT (Pickett and Reddel, 2012). T-circles have also been shown to form as a result of telomere trimming in cells that have had telomeres over- lengthened by telomerase (Pickett et al., 2009). The two EST cell lines lack t-circles, and as expected, t- circles are present in the two ALT NB cell lines (Figure 5.8.A). Contrary to what was expected, no t-circles were generated in the LA-N-6 clones after activation of telomerase (Figure 5.8.B), even though all hTERT or hTERT/hTR expressing clones had extremely long telomeres after activation of telomerase. The

LA-N-6 MUT p53 culture also failed to generate t-circles, even after hundreds of PDs utilising the ALT mechanism, and perhaps less surprisingly the COG-N-291 clone 4 cells also lacked t-circles (Figure 5.8.C).

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Figure 5.7. LA-N-6 MUT p53 cells did not activate ALT due to aberrations to ATRX or DAXX. (A) ATRX and DAXX immunoblot with HSP70 loading control. (B) Immunofluorescence indicating

the presence of ATRX or DAXX foci (red) in the nucleus (blue). CHLA-90 is included as the ATRX

negative control and SH-SY5Y as the positive control for ATRX and DAXX expression. (C) Graph of

meta-TIF assay results demonstrating percentage of chromosome ends which have both DNA

damage and telomere signal (-H2AX/TTAGGG+) and chromosome ends which have DNA damage

in the absence of telomere signal (-H2AX/TTAGGG-) in LA-N-6 TP53 wild type PD96 and LA-N-6

MUT p53 PD707. Mean +SD from 3 independent experiments.

These results suggest that both of the EST cell lines are unable to undergo telomere trimming and generate t-circles. A number of proteins are known to be involved in t-circle formation including: XRCC3

(Compton et al., 2007; Wang et al., 2004), NBS1 (Wang et al., 2004), SLX4 (Sarkar et al., 2015), TZAP (Li et al., 2017), and RTEL1 (Deng et al., 2013), but whole genome sequencing of the EST cells did not reveal any mutations in these genes (Appendix II Table A2.6) and all NB cell lines have detectable transcript for the five genes involved in t-circle formation (Figure 5.9).

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Figure 5.8. EST cells do not form t-circles after activation of ALT or extreme telomere lengthening by telomerase activity. (A) Two-dimensional gel electrophoresis of restriction-digested genomic DNA. Denatured gels were

hybridised with a 32P-labelled C-rich telomeric DNA probe. T-circle arcs are indicated by the arrow.

(B) hTR and hTERT expressing LA-N-6 clones do not have t-circles at early or later PDs. (C) T-circles

(indicated by arrow) are present in the ALT control but not in LA-N-6 MUT p53 or COG-N-291

clone 4.

The investigation of t-circles was conducted by A/Prof Hilda Pickett.

5.3. Discussion

This chapter describes the features of EST cultures that have activated a TLM, and thereby abolished the EST phenotype. Spontaneous activation of a telomerase independent TLM, which is by definition

ALT, in a p53-mutant LA-N-6 clone (presented in this chapter) resulted in a different phenotype than a

COG-N-291 clone (described in Chapter 4). The COG-N-291 clone had a reduced proliferation rate with no increase in APBs and TERRA, compared to COG-N-291 EST cells. In contrast, the LA-N-6 clone had a similar proliferation rate, and increased APB and TERRA levels compared to LA-N-6 EST cells.

Interestingly, both cultures have a typical ALT TRF profile, an increase in C-circles, and are wild type for

ATRX and DAXX. The most notable difference between the ALT COG-N-291 and LA-N-6 cultures relates to TP53 status. The COG-N-291 clone (clone 4) is the first cell line reported to retain expression of wild type TP53 and full length ATRX protein whilst utilising a telomerase-independent mechanism to prevent telomere shortening (Henson and Reddel, 2010; Lovejoy et al., 2012). The presence of functional p53 may account for the reduced proliferation rate in the COG-N-291 ALT clone that was not observed in

LA-N-6. Currently, we do not understand the mechanism that these cells have used to activate telomere lengthening, but as we know that a proportion of ALT NB tumours are wild type for both TP53 and ATRX

(Chapter 3), this cell line will provide a valuable model for understanding this disease.

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Figure 5.9. EST cells express genes involved in t-circle formation. (A) Transcript levels of NBS1, TZAP, RTEL1, SLX4, and XRCC3, genes involved in t-circle formation.

The cell lines GM847, SK-N-FI and CHLA-90 are ALT cell lines that form t-circles.

The EST cells that we have identified indicate that a TLM may not be required for the formation of metastatic and lethal tumours, presumably when the progenitor cells have telomeres long enough to sustain extensive proliferation, though we do not know how this initial lengthening occurred in EST NB cells. Telomerase is active during human embryogenesis, prior to downregulation during the late stages of gestation, leaving somatic cells telomerase-negative (Ulaner and Giudice, 1997; Wright et al., 1996).

Telomere trimming, a process that generates t-circles, regulates telomere length in cells that have been over-lengthened by telomerase (Pickett et al., 2009). We propose that a failure of telomere trimming, and subsequently lack of t-circle formation, in EST NB cells resulted in in excessive telomere lengths during embryogenesis. The EST NB cells lacked detectable t-circles, even in circumstances where their long telomeres were lengthened further by telomerase. Abundant t-circles are also a feature of lengthening by ALT (Cesare and Griffith, 2004), but t-circles were not detected in either of the COG-N-

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291 or LA-N-6 clones that spontaneously activated an ALT mechanism. A lack of telomere trimming may cause inhibition to the full expression of the ALT phenotype as observed in the ALT COG-N-291 or LA-N-

6 clones. Presumably, the EST cells downregulated telomerase activity as normal during embryogenesis, leaving excessively lengthened telomeres, which have allowed very extensive proliferation to occur without requiring significant upregulation of a TLM. As with tumours that activate a TLM, additional oncogenic changes would need to have occurred to result in the development of malignant EST tumours, and the excessively long telomeres presumably provide sufficient reserves of telomere content to obviate the need for an activated TLM.

In summary, we have shown that activation of a TLM is sufficient to rescue the EST phenotype.

EST cells lack t-circles implying a failure of telomere trimming, regardless of the mechanism (either activation of telomerase or ALT) used to overlengthen the telomeres. This is the first example of a disease that may result from a failure of telomere trimming. Despite treatment in these patients, EST tumours are lethal in approximately 50% of patients and the telomere trimming mechanism may provide a unique way to target therapies to these cells.

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Chapter 6: Neuroblastoma EST cells are

sensitive to topoisomerase inhibitors

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Chapter 6: Neuroblastoma EST cells are sensitive to topoisomerase inhibitors

6.1. Introduction

The aggressive behaviour of high-risk NB requires extensive cellular proliferation made possible via activation of a TLM, either telomerase or ALT, or by an EST phenotype which is characterised by extremely long telomeres that allow proliferation without significant upregulation of a TLM. In Chapter

3 we show that the prognosis of each of these groups of tumours is poor: the 5-year overall survival is

28% for patients with MYCN amplified tumours (which are generally telomerase positive (Hiyama et al.,

1997)), 36% for ALT-positive patients, and 49% for EST NB. In addition, telomere biology has an impact on disease course, with ALT and EST NB patients having late events compared to MYCN amplified tumours. Recently, several large scale genomic studies have been conducted to elucidate driver mutations in NB (Molenaar et al., 2012; Pugh et al., 2013). Whilst these studies were unable to identify a driver mutation, approximately 10% of tumours had mutations to the ATRX gene which is associated with ALT activity (Cheung et al., 2012; Kurihara et al., 2014; Molenaar et al., 2012; Pugh et al., 2013), or rearrangements upstream of hTERT which results in reactivation of telomerase (Peifer et al., 2015;

Valentijn et al., 2015). Therefore, telomere biology has become an attractive target for the development of novel NB therapies.

Over the last two decades telomerase inhibitors have gone through various stages of development, resulting in a number of clinical trials in diseases such as breast and lung cancer, glioblastoma, and lympho-proliferative disorders (Arndt and MacKenzie, 2016). In contrast, ALT inhibitors have yet to enter clinical trials and are still being developed in preclinical laboratory models

(Deeg et al., 2016; Flynn et al., 2015).

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The EST NB cell lines have very low levels of C-circles which likely represent a very low level of

ALT activity that is insufficient to confer telomere maintenance in these cells. In yeast, ALT acts preferentially on the shortest telomeres (Fu et al., 2014) although, Murnane and colleagues suggest that this is not necessarily the case in human cells (Murnane et al., 1994). In the experiments described in this chapter, we tested the hypothesis that the low level of ALT in EST cells may preferentially lengthen the shortest telomeres as critically short telomeres will eventually trigger p53-mediated senescence or apoptosis. If this was the case, even though ALT activity is clearly insufficient to prevent telomere shortening, EST cells might rely on low levels of ALT to survive, and consequently inhibition of ALT would result in increased cell death. The ALT mechanism relies on homologous recombination. APBs potentially play a role in this process as proteins associated with homologous recombination are found at APBs

(Pickett and Reddel, 2015). One such protein, RPA, is associated with replication stress via its interaction with ATR (Zou and Elledge, 2003). It has been suggested that ALT cells are acutely sensitive to ATR inhibitors (Flynn et al., 2015), implying that replication stress has a role in ALT activity that can be targeted by anti-cancer therapeutics. Therefore, the aim of this chapter was to determine the sensitivity of EST cells to common NB chemotherapeutics, and to determine if treatment with drugs that target the ALT mechanism led to an increased rate of cell death.

6.2. Results

6.2.1. EST cells are sensitive to topoisomerase inhibitors

EST cells represent a novel prognostic group of NB, with an approximately 50% 5-year mortality in high- risk patients. To develop better treatments for these patients we first need to understand the role telomere biology plays in the response of high-risk NB to chemotherapy. We asked whether cells of different telomere biology groups (ALT-positive, telomerase-positive, or EST) had different sensitivity to common NB chemotherapeutics. Using AlamarBlue, a redox indicator that provides a quantitative measure of cell viability, we determined the effect of chemotherapeutics on the proliferation of six telomerase-positive (SH-SY5Y and SK-N-BE2c NB cell lines; HT1080, GM639, HT1080 hTR and

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Figure 6.1. EST cells are sensitive to topoisomerase inhibitors. Cell lines were treated with increasing concentrations of (A) doxorubicin, (B) etoposide, (C, D) irinotecan, and (E, F) topotecan for 6 PDs before proliferation was quantified using AlamarBlue. U-

2 OS and GM847 ALT cell lines, and HT1080, GM639, HT1080 hTR and HeLa 1.2.11 telomerase- positive cell lines were included as non-NB controls. Results in panel (A), (B), (C), (E) are expressed as percentages of growth compared to untreated controls and as means of triplicate independent experiments +SD. Panel (D) displays the percentage of cell growth, compared to control cells, when

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cells were treated with 2.8 µM irinotecan. COG-N-291 had significantly reduced growth compared

to all cell lines except SH-SY5Y and HT1080 hTR whilst LA-N-6 had significantly reduced growth

compared to U-2 OS, SK-N-BE2c and HeLa 1.2.11 cells (when significant P<0.05). Panel (F) displays

the percentage of cell growth, compared to control cells, when cells were treated with 0.0004 µM

topotecan. COG-N-291 and LA-N-6 cells had significantly reduced growth compared to all other cell

lines (P<0.001).

HeLa 1.2.11 non-NB cell line), four ALT-positive (SK-N-FI and CHLA-90 NB cell lines; U-2 OS and GM847 non-NB cell lines), and the two EST (COG-N-291 and LA-N-6 NB cells) cell lines. As the cell lines in this panel had a range of growth rates, of which COG-N-291 and LA-N-6 were the slowest, it was not possible to treat for a fixed length of time, instead cells were treated for a duration of 6 PDs. The DNA damaging agent doxorubicin reduced proliferation of all cell lines, although there was no differential response to doxorubicin based on telomere biology (Figure 6.1.A; Table 6.1). Treatment with etoposide, a topoisomerase II inhibitor, resulted in the most significant growth inhibition of COG-N-291 cells (IC50

0.08 µM; the dose to inhibit 50% of control growth) (Figure 6.1.B), although sensitivity to topoisomerase

II inhibitors is not a general feature of EST cells as LA-N-6 had an IC50 of 0.23 µM, similar to telomerase- positive cell lines (0.17 to 0.56 µM). In contrast, EST cells show a striking sensitivity to the topoisomerase

I inhibitors, irinotecan and topotecan (Figure 6.1.C, D, E, F). At a dose of 2.8 µM irinotecan COG-N-291 had significantly reduced cell growth compared to all cell lines except SH-SY5Y and HT1080 hTR whilst

LA-N-6 cells had a similar trend of sensitivity with significantly reduced growth compared to U-2 OS, SK-

N-BE2c and HeLa.1.2.11 cells (where significant P<0.05, Figure 6.1D). The EST cells were particularly sensitive to topotecan treatment, 0.0004 µM topotecan led to significantly reduced EST cell growth compared to all other cell lines tested (P<0.001, Figure 6.1F). The IC50 of irinotecan and topotecan for non-EST cell lines with long telomeres (U-2 OS, HT1080 hTR, and HeLa 1.2.11), was generally higher than the EST cells (Figure 6.1.C, D; Table 6.1), indicating that the telomere length of EST cells is not entirely

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Figure 6.2. Telomere biology does not influence sensitivity of NB cells to common NB therapies. Cell lines were treated with increasing concentrations of (A) vincristine, (B) cisplatin, (C)

temozolomide, and (D) all-trans retinoic acid (ATRA) for 6 PDs before proliferation was quantified

using AlamarBlue. U-2 OS and GM847 ALT cell lines, and HT1080, GM639, HT1080 hTR and HeLa

1.2.11 telomerase-positive cell lines were included as controls. Results are expressed as

percentages of growth compared to untreated controls and as means of triplicate independent

experiments +SD.

responsible for their sensitivity to topoisomerase I inhibitors. The response of cell lines to vincristine, cisplatin and temozolomide (Figure 6.2.A, B, C) varied depending on the cell line and was not associated with telomere length maintenance phenotype. All-trans retinoic acid (ATRA), a differentiating agent, did not reduce the proliferation of cells in a treatment period of 6 PDs (Figure 6.2.D).

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Table 6.1. IC50 values for common NB chemotherapeutics. IC50 (µM) All Trans Retinoic Doxorubicin Etoposide Cisplatin Irinotecan Topotecan Temozolomide Vincristineb Acid LA-N-6 0.40 0.23 4.23 1.04 <0.0004 324.33 8.07 >45 COG-N-291 0.01 0.08 0.77 0.31 <0.0004 58.87 12.64 >45 SK-N-FI 0.69 3.65 2.62 5.64 0.07 >400 >25 >45 CHLA-90 0.20 8.11 6.60 30.44 0.42 >400 2.21 >45 GM847 0.01 0.41 1.57 6.14 0.02 191.54 0.59 >45 U-2 OS 0.02 0.74 5.93 11.64 0.16 >400 6.02 >45 SK-N-BE2c 0.03 0.29 16.19 8.59 0.09 >400 1.61 >45 SH-SY5Y 0.03 0.56 2.20 2.50 0.01 98.96 19.35 >45 HT1080 0.01 0.19 1.88 5.00 0.01 389.18 0.18 >45 GM639 0.01 0.17 1.75 3.59 0.01 126.93 1.02 >45 HT1080 hTRa 0.12 0.66 8.39 2.08 0.11 365.30 2.79 HeLa 1.2.11a 0.12 0.51 0.98 17.21 <0.0004 >400 22.67 aLong telomeres/telomerase-positive bIC50 (nM)

6.2.2. EST cells do not exhibit increased cell death with potential ALT inhibitors or after

induction of replication stress

The EST cells have a low level of ALT activity which we hypothesise is preferentially lengthening the

shortest telomeres, consequently these cells could be sensitive to agents that target the ALT

mechanism. Arsenic trioxide (ATO; results in PML degradation) and ML216 (BLM helicase inhibitor) have

been shown to downregulate C-circles in ALT cells (unpublished data). However, neither ALT nor EST

cells showed greater sensitivity for these agents than telomerase-positive cells (Figure 6.3.A, B; Table

6.2). Previous work by Flynn and colleagues found that ALT cells are hypersensitive to ATR inhibition

(Flynn et al., 2015). In this panel of cell lines, the IC50 values for the ATR inhibitor VE-822 ranged from

0.02-0.53 µM for telomerase-positive cells, 0.02-6.22 µM for ALT cells, and 0.39-3.52 µM for EST cells

(Figure 6.3.C; Table 6.2), which is consistent with a later report that shows ALT cells are not more

sensitive to ATR inhibition than telomerase-positive cells (Deeg et al., 2016). There was a slight increase

in the IC50 doses for EST and ALT cells in response to the ATM inhibitor Ku60019 (Figure 6.3.D; Table

6.2). Agents that induce replication stress, including mitomycin C, hydroxyurea, and aphidicolin reduce

136 proliferation in all cells; however, the extent of reduction was not related to the telomere biology of the cells (Figure 6.3.E, F, G; Table 6.2). Whilst COG-N-291 cells were most sensitive to all three inhibitors, the IC50s for LA-N-6 were similar to ALT and telomerase-positive cells, and we found no evidence that

ALT cells are more sensitive to replication stress than other cell types (Table 6.2). In summary, the current study failed to identify any drug that specifically inhibits either ALT or telomerase-positive cells.

However, we were able to identify the significant sensitivity of EST cells to topoisomerase I inhibitors.

6.2.3. EST cells have alterations to genes involved in DNA damage repair and replication

We have previously shown (Chapter 3) that the EST NB cell lines have functional p53 capable of activating downstream targets such as p21 and triggering p53-mediated apoptosis in response to DNA damage. Mutations to pathways involved in DNA damage repair and replication would prevent the EST cells from repairing damage caused by chemotherapeutics leading to apoptosis through the p53 pathway. Whole genome sequencing data revealed heterozygous alterations of a number of genes involved in DNA damage repair and replication in the EST cell lines (Appendix II A2.6). The COG-N-291 cells have mutations in GEN1 a resolvase that cleaves Holliday junctions (Ip et al., 2008), HLTF which is involved in DNA damage repair and replication fork reversal (Achar et al., 2011; Blastyak et al., 2010;

Kile et al., 2015), and MCM4, a gene involved in the initiation and progression of replication forks (Sheu et al., 2014; Sheu et al., 2016). Moreover, LA-N-6 cells have altered EME2 which is involved in the cleavage and restart of replication forks (Pepe and West, 2014), and POLM, a polymerase used for gap filling in DNA damage repair by NHEJ (Pryor et al., 2015). The effect of heterozygous mutations of these genes on DNA damage repair and replication remains to be elucidated.

6.3. Discussion

The poor survival rates of NB patients with high-risk disease indicate the need to develop better therapies for these patients. Our results, in conjunction with a number of genomic studies (Cheung et al., 2012; Kurihara et al., 2014; Molenaar et al., 2012; Peifer et al., 2015; Pugh et al., 2013; Valentijn et

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Table 6.2. IC50 values for inhibitors that cause replication stress and inhibit factors associated with ALT. IC50 (µM) Arsenic ML216 Trioxide VE-822 Ku60019 Mitomycin Ca Hydroxyurea Aphidicolin LA-N-6 >225 3.24 3.52 >24 0.94 429.24 0.62

COG-N-291 >225 3.51 0.39 >24 0.05 84.33 0.08

SK-N-FI >225 2.55 6.22 >24 >3 1199.17 2.38

CHLA-90 5.04 7.00 0.33 20.91 1.22 257.94 4.45

GM847 110.53 0.77 0.02 4.97 0.18 193.23 0.51

U-2 OS 95.98 2.19 0.09 13.37 1.32 731.70 16.13

SK-N-BE2c >225 4.88 0.07 12.91 0.51 348.27 0.19

SH-SY5Y >225 3.94 0.53 10.77 0.43 >1600 0.50

HT1080 26.98 1.26 0.04 3.52 0.52 229.45 0.97

GM639 95.11 1.26 0.02 11.79 0.49 262.79 0.57 aIC50 (µg/mL)

al., 2015), suggest that telomere biology may be a target for novel NB therapies. The EST cells are highly sensitive to the topoisomerase I inhibitors topotecan and irinotecan. Topotecan and irinotecan are derivatives of camptothecin, a topoisomerase I poison that stabilises the topoisomerase I-DNA cleavage complex during DNA replication (Hsiang et al., 1985). This results in a dsDNA break when the advancing

DNA replication fork collides with the topoisomerase I-DNA complex leading to cell cycle arrest and apoptosis (Hsiang et al., 1989). The differential response of EST cells to this form of DNA damage compared with ALT or telomerase-positive cells was not due to therapy-acquired resistance in the ALT or telomerase-positive NB cell lines as neither topotecan nor irinotecan were used to treat NB patients from which these cells were derived more than two decades ago (Biedler et al., 1973; Keshelava et al.,

1998; Wada et al., 1993). Whilst COG-N-291 and LA-N-6 have obvious sensitivity to topoisomerase I inhibitors the analysis is limited by the number of EST cell lines. The Pediatric Preclinical Testing Program

(PPTP) identified NB-1643 as acutely sensitive to topotecan in their cell line panel with an IC50 of 0.71 nM (Carol et al., 2010) and the Reynold’s lab has identified a further four cell lines that are telomerase-

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Figure 6.3. Telomere biology does not predict the response of cell lines to inhibitors that cause replication stress or reduce factors critical to the ALT mechanism. Cell lines were treated with increasing concentrations of (A) arsenic trioxide (ATO) which degrades

PML, (B) the BLM inhibitor ML216, (C) the ATR inhibitor VE-822, (D) the ATM inhibitor KU60019, (E)

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mitomycin C, (F) hydroxyurea, and (G) aphidicolin for 6 PDs before proliferation was quantified

using AlamarBlue. U-2 OS and GM847 ALT cell lines, and HT1080 and GM639 telomerase-positive

cell lines were included as controls. Results are expressed as percentages of growth compared to

untreated controls and as means of triplicate independent experiments +SD.

negative and lack C-circles (unpublished data). Therefore, further testing is required to establish if any of these cell lines have the EST phenotype, with the potential to expand the EST NB cohort for further drug screening. Genz-644282 is a recently developed new generation of topoisomerase I inhibitor, not derived from camptothecin (Li et al., 2003). According to the PPTP screen, NB cells are generally more sensitive to Genz-644282 than topotecan (Houghton et al., 2012), therefore, establishing the acute sensitivity of EST cells to Genz-644282 will confirm EST cells are particularly sensitive to topoisomerase

I inhibitors.

One possible explanation for the EST cell sensitivity to topoisomerase inhibitors are the very long telomeres of these cells. Telomeres are known to form G-quadruplex structures (Sen and Gilbert,

1988; Sundquist and Klug, 1989) which need to be removed prior to telomere replication in S. cerevisiae

(Paeschke et al., 2011). Long telomeres have a greater potential to form such structures which could reduce the efficiency of telomeric replication and make complete DNA replication more difficult.

Furthermore, the replication fork must travel a further distance to fully replicate long telomeres. In this study, we found no evidence that telomere length as a single factor was responsible for the EST sensitivity to topoisomerase I inhibitors. The cell line U-2 OS has wild type TP53 which can induce p21

(Bensaad et al., 2006; Cesare et al., 2009) and has similar telomere lengths to EST cell lines however, it is not sensitive to treatment with topotecan or irinotecan. Whole genome sequencing identified aberrations in DNA damage repair and replication genes in the EST cell lines but further studies are required to determine the effect of these heterozygous mutations. The EST cells may be particularly sensitive to DNA damage by topoisomerase I inhibitors due to a combination of very long telomeres,

140 wild-type TP53 and impaired DNA repair/replication machinery, which means that the additional DNA damage caused by topoisomerase I inhibition during replication, cannot be repaired by the cells leading to p53-mediated apoptosis.

Topoisomerase levels may predict the response of cells to topoisomerase inhibitors (Burgess et al., 2008). We are currently using pressure cycling technology (PCT) and sequential window acquisition of all theoretical fragment ion spectra (SWATH) mass spectrometry (Guo et al., 2015) to measure the level of proteins in EST, telomerase and ALT NB which will allow us to determine if the level of topoisomerase I and II correlates with sensitivity to topoisomerase inhibitors. This study will also allow us to determine the effect of heterozygous mutations on the protein expression of GEN1, HLTF, MCM4,

EME2, and POLM in the EST cells. We will also use the mass spectrometry results to determine a proteomic profile of the EST cells compared to ALT and telomerase-positive NB to aid the development of targeted therapies based on the telomere biology of NB cells.

The use of topoisomerase I inhibitors is a recent development in NB treatment. Topotecan has only recently been incorporated into COG clinical trials as a part of induction therapy (Park et al., 2011) or to treat relapsed/refractory disease (Garaventa et al., 2003) whilst irinotecan is only used to treat relapsed or refractory NB (Kushner et al., 2006). Our results indicate that 11% of high-risk NB, i.e. the

EST tumours, could be acutely sensitive to upfront treatment with topotecan and irinotecan. Further preclinical studies are required to confirm whether topoisomerase I inhibition results in better outcome for EST tumours but we have shown that targeting telomere biology may lead to novel treatment regimens for high-risk NB.

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Chapter 7: Discussion

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Chapter 7: Discussion

The acquisition of cellular immortality, via activation of a TLM, is currently considered a hallmark of cancer (Hanahan and Weinberg, 2000; Hanahan and Weinberg, 2011). However, studies of some cancers including glioblastoma (Hakin-Smith et al., 2003), liposarcoma (Costa et al., 2006; Jeyapalan et al., 2008), osteosarcoma (Ulaner et al., 2003), and retinoblastoma (Gupta et al., 1996) have reported that a substantial proportion of tumours are TLM-negative, and the widely-held explanation for this has been that these are false negatives caused by deficiencies in the standard TLM assays. Our study has identified a subset of NB tumours that lack telomerase activity, have very long telomeres, and have such low levels of ALT that they are unable to prevent telomere shortening. Cells derived from such tumours are capable of long-term proliferation despite ever-shorter telomeres. Subsequent investigations showed NB EST cells were acutely sensitive to topoisomerase I inhibitors, indicating TLMs are potential targets for novel NB therapies.

7.1. The ever-shorter telomeres phenotype in human cancer

Telomerase activity was established as a poor prognostic factor in NB over two decades ago (Poremba et al., 2000; Poremba et al., 1999) and recent studies have utilised telomere length (Onitake et al., 2009), or ATRX/DAXX mutations (Cheung et al., 2012; Kurihara et al., 2014; Molenaar et al., 2012; Peifer et al.,

2015; Valentijn et al., 2015) to identify ALT tumours. Ours is the first study to utilise C-circles as a quantifiable marker of ALT activity to determine the prevalence of ALT in metastatic NB. Using this method, we identified 11% of high-risk NB tumours as ALT-negative/TC≥15. These tumours lacked telomerase activity, indicating that a subgroup of high-risk NBs lack evidence of a TLM, consistent with previous studies (Choi et al., 2000; Krams et al., 2001; Poremba et al., 2000; Poremba et al., 1999;

Reynolds et al., 1997). These studies were unable to distinguish among the possibilities that the apparent lack of significant ALT or telomerase activity was due to: (1) false-negative assays, (2) an as- yet unidentified ALT mechanism lacking the common phenotypic characteristics of the "classic" ALT

143 mechanism, or (3) a true lack of significant TLM activity in the cells. The only conclusive test to determine if cells are TLM-negative is to observe telomere length over time, which cannot be done ethically in human tumour samples as it would require serial samples from the same patient over time in the absence of treatment. Consequently, cell line models were required to determine which of these scenarios was true. We present evidence that human cell lines derived from fatal metastatic disease are capable of proliferating for 500 PDs, despite ever-shorter telomeres.

The observation of the EST phenotype in high-risk NB provides direct evidence that, as previously postulated (Reddel, 2000), immortalisation is not an absolute requirement of oncogenesis.

Telomerase activity is detectable during the early stages of human embryogenesis however, it is downregulated in late embryogenesis resulting in telomerase-negative somatic cells (Ulaner and

Giudice, 1997; Wright et al., 1996). NB is an embryonal cancer, and we propose that excessive telomerase activity during embryogenesis resulted in very long telomeres in the EST progenitor cells before telomerase activity was downregulated at the end of embryogenesis. The EST progenitor cells would not require significant upregulation of a TLM to allow extensive proliferation to occur in these cells although further oncogenic events would be required to form a malignant tumour. Telomere trimming is a process that regulates telomere length in cells that have been over-lengthened by telomerase (Pickett et al., 2009). The lack of t-circles, a product of telomere trimming, in LA-N-6 cells with over-lengthened telomeres due to ectopic telomerase expression suggests that EST cells have a failure of telomere trimming. T-circles are also detected in cells that have upregulated ALT (Cesare and

Griffith, 2004), however we were unable to detect t-circles either in COG-N-291 clone 4, or in the LA-N-

6 MUT p53 clone that spontaneously activated ALT, further confirming that these cells have a failure of telomere trimming. This is the first report of a disease that may result from a failure of telomere trimming, although the mechanism leading to failure of telomere trimming remains to be elucidated.

Whole genome sequencing did not identify genetic events unique to the EST cells and further studies are required to examine their epigenetic and proteomic profile compared to ALT and telomerase- positive cells and consequently identify events responsible for the failure of telomere trimming. The

144 failure of telomere trimming in EST cells may be responsible for their sensitivity to topoisomerase I inhibitors. If this is the case, knockdown of genes associated with telomere trimming would lead to the increased sensitivity of non-EST cells to topoisomerase I inhibitors, providing further evidence that the

EST cells are a disease resulting from the failure of telomere trimming.

Our study did not identify potential EST cell lines in other tumour types although this is likely due to the criteria used to identify EST cell lines. The criteria for NB EST may not reflect the phenotype of EST cells in other tumour types, as NB is a childhood cancer and consequently the tumour progenitor cells have not undergone many cycles of replication induced telomere shortening. In contrast, the progenitor cells of adult cancers may have undergone many rounds of cell division, and associated telomere shortening, before oncogenesis occurs. Cell lines derived from these tumours may not have very long telomere lengths after the PDs required to derive a cell line from the tumour. Indeed, data from the Decottignies laboratory, found melanoma cells lacking evidence of a TLM had telomere lengths at the upper end of the normal range (Viceconte et al., 2017). Another explanation for the lack of EST cell lines in other tumour types may be the difficulty in deriving cell lines from such tumours. The NB

EST cell lines grow very slowly compared to cell lines with an upregulated TLM and consequently other

EST cell lines may never have been characterised or grown long term in laboratories due to the bias towards with faster growing cell lines for in vitro experiments. Therefore, it is likely that cell line studies will underestimate the occurrence of the EST phenotype.

7.2. Targeting telomere biology in the clinic

Since the discovery of telomerase, inhibitors have been developed to target the enzyme translating to clinical trials in cancers including breast and lung cancer, glioblastoma, and lympho-proliferative disorders, although to date there is no evidence of their effectiveness in treating malignancies (Arndt and MacKenzie, 2016). In contrast, ALT inhibitors have yet to enter clinical trials and are currently being developed in preclinical laboratory models (Deeg et al., 2016; Flynn et al., 2015). High-risk NB patients have very poor survival rates and novel targets for therapy need to be identified. Our study, in addition

145 to a number of large scale genomic studies (Cheung et al., 2012; Kurihara et al., 2014; Molenaar et al.,

2012; Peifer et al., 2015; Pugh et al., 2013; Valentijn et al., 2015), indicate that telomere biology plays a significant role in NB pathogenesis and may be a target for novel NB therapies. We have shown that NB

EST cells are highly sensitive to the topoisomerase I inhibitors topotecan and irinotecan and therefore

11% of high-risk NB could be acutely sensitive to upfront treatment with these agents. The identification of further EST NB cell lines, currently underway through expansion of the cell line screen, will aid in the identification of other agents that uniquely target these cells. The identification of EST cell lines in other cancer types will facilitate further studies to determine if sensitivity to topoisomerase I inhibition is a universal feature of EST cells.

EST cells provide the first evidence of a disease that may result from a deficiency in telomere trimming. This provides a novel area for drug development as there is currently no therapy to target the telomere trimming mechanism. The molecular details of the trimming mechanism are still under investigation which may lead to the identification of novel therapeutic targets.

The development of drugs which target features of telomere biology require appropriate biomarkers to predict response to therapy. The discovery of malignant and lethal EST cancer cells illustrates the need to assay for TLMs prior to treatment with drugs that target these mechanisms. The phenotypic similarity of EST and ALT cells illustrates that care must be taken to correctly define ALT tumours: ATRX/DAXX mutations are not a universal feature of ALT cells (Lovejoy et al., 2012), telomere length (as measured by telomere qPCR, TRF analysis or telomere FISH) is insufficient to differentiate ALT and EST cells, and the presence of low levels of C-circles does not necessarily indicate sufficient ALT activity to counter the EST phenotype. Therefore, studies that define a molecular profile for the EST cells will be critical in developing accurate clinical assays to differentiate between EST and ALT cells.

7.3. Conclusion

In conclusion, this study has provided evidence of lethal, malignant human cancers that can proliferate continuously for hundreds of PDs with ever-shorter telomeres. This indicates that the upregulation of a

146

TLM to confer immortalisation is not an absolute requirement for oncogenesis. Patients with high-risk

NB have a poor survival rate and we need to develop novel therapies to improve the outcome of these patients. Our results, indicate that telomere biology is a relevant target for novel NB therapies and EST tumours are potentially very susceptible to treatment with topoisomerase I inhibitors. The similarity of the EST and ALT phenotypes indicates that rapid and specific assays for TLMs are required to facilitate the use of TLM targeting inhibitors as anti-cancer therapeutics.

147

Chapter 8: References

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Appendices

192

Appendix I: Information Relating to Chapter 2

ATRX and DAXX sequencing was performed with the primers listed in Table A1.1. and A1.2., respectively.

The PCR conditions are listed in the subsequent tables.

Table A1.1. Primers used to Sanger sequence ATRX. PCR PCR Taq used in Exon product M13 primer Other primer Program PCR size Taq Platinum 35 552 ATGGTCCTGTGAATGCCATC GGCATTTAAGGGGACCAAAC 1 (Invitrogen) Taq Platinum 34 339 ACCTTGGGAAATCCCGAATA CAATGACTATCCATCCCTCCA 1 (Invitrogen) Taq Platinum 33 237 GTTGGCAAATGGAAGGATTC AGTAGGGGGTGGAGGGTACA 2 (Invitrogen) GGGTGAAAAGGGTGTTTTGT HotStar Taq 32 353 GCATAGGGAACCCTCAACAA 3 T (Qiagen) GGTTTTAGTTTCTAGTACAGT Taq Platinum 31 372 GGGGAATGTGTTCCTAAAACC 1 TGACCA (Invitrogen) GCAAAATTGCTGATGAGTTTT CATTTTATTATCCTTGAAAAAT Taq Platinum 30 500 1 T TCTGA (Invitrogen) AAGAAATGAATTCTCTGAACT Taq Platinum 29 405 CCAACTTTGTTTCCCTCTCTG 1 CTTGA (Invitrogen) TGATGAGCAAGGTGGAAAAT Taq Platinum 28 329 TGACACTGTTTTGCAACCTGA 1 C (Invitrogen) GGGTAGTTTTGTTTCTTTTGTT Taq Platinum 27 313 AAATCCTGCTGGGATTTTTG 1 GC (Invitrogen) Taq Platinum 26 392 CCCCATGGGTAGGTCTTTTT TTGCTTGTATTGGCCTAGCA 1 (Invitrogen) HotStar Taq 24-25 554 TTCCTCAGTCCTTCCTCAGC CCCCAAATACCAGAGAGCAC 3 (Qiagen) GCTTCTCTACACTGCCAAAAG Taq Platinum 23 289 TTCTGCTTCCAATAGATGCTTT 1 TG (Invitrogen) TTGTGGGTTTAGAAAGGGTA TGCAAAACTGAAAAAGAACA Taq Platinum 22 387 1 AA ACA (Invitrogen) Taq Platinum 21 367 TGAGCATTTCATTGGGGAAT TGAAAGAGCGGGAAAGAAAA 1 (Invitrogen) TTAACCAAATACGGGAGCAG TTTCACAGCAGACTAAGATGA Taq Platinum 20 449 1 A ACC (Invitrogen) GGCAAGAGGGATTAAAAGAT Taq Platinum 19 576 TGGCGACATTAAGGGTGATT 1 GA (Invitrogen) TTCCCACTGAAATATGCATCA Taq Platinum 18 387 TTGGAAATTCTGGCCGTTTA 1 C (Invitrogen) Taq Platinum 17 418 TCTTCAGCCCCTACGACTGT GAAGGAAAGTCCCCCTGTTC 1 (Invitrogen) Taq Platinum 16 379 CAATTGGATTTGTGGTGTGG CCACCCCACTCACCAATTTA 1 (Invitrogen)

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HotStar Taq 15 503 TCTTCCACCTTTTCCTGCTG AGGCTGGGTGTGTTGACTC 3 (Qiagen) TGGAACAGAGAGGTAACAGC Taq Platinum 14 398 AACAAACCTCCCCTCAGGAT 1 A (Invitrogen) TGCTCTGTTTTAATGTCGAGT Taq Platinum 13 245 TGAAGGCATGGTCATTCAGA 1 CA (Invitrogen) HotStar Taq 12 314 CAGCTTCCCAAAGTGCTAGG CGAGGCATTTTAAAGGCTGA 3 (Qiagen) TTGGCCTCCCAAAGTCCTGAG Taq Platinum 11 391 TCTATTGGCACATTTATTTCT 1 ATT (Invitrogen) TTTGGAGTCCAGAGTTTAGA AACTTGCAGGAAGACTGTGA Taq Platinum 10 343 1 CC GCGA (Invitrogen) CATCTGATGCTGAGGAAAGT GAGATCCCTGATACTGAATAC Taq Platinum 9(g) 493 1 TCTG TAGC (Invitrogen) TGAATCTTCATCTGATGGCAC TTTCTGTTCATCGCTGCTTCCC Taq Platinum 9(f) 649 2 TGA TC (Invitrogen) AGGAATGGATAATCAAGGGC TCCTTTCCCTGTTGACTTCTCA Taq Platinum 9(e) 640 1 ACA GC (Invitrogen) GGATAAGCGTAATTCTTCTGA AGCACTTGCTTGCTGCTTCTTA Taq Platinum 9(d) 655 1 CAGTGC GG (Invitrogen) CCGGTGGTGAACATAAGAAA AACTGTGACTCATCCTGCTCA Taq Platinum 9(c) 777 4 TCTG CCT (Invitrogen) CTTGTTCAGTTCCACTGCTGC CCTGTTCTGGCTCTGTAACCT Taq Platinum 9(b) 663 1 CAT ACT (Invitrogen) GAGTAAGCAGATGACCTAAA Taq Platinum 9(a) 742 CCAATGCAAGATGAGCCTTC 1 TTACCAC (Invitrogen) CACACCAGTGTCCTGGAGAT AGGAAACACTGAATGTTAGCT Taq Platinum 8 226 1 TT CATCT (Invitrogen) TGCCAAGGTTGTCATGTGCTT GAAGTCTTCCAAGGGCAGAT Taq Platinum 7 415 1 AG ACCA (Invitrogen) CCAGCAATGTTGGCTTTATCT Taq Platinum 6 276 AAGCACATCCGATTTTCCAA 1 GAACTG (Invitrogen) TTCCTTGTTGAGACCCACTGC GCCATGTTTGGTCGTTTGTAC Taq Platinum 5 348 1 TCA ATAGT (Invitrogen) GCTAATTGTAGGGATGCCGT CTCAGAATAGTGGTTGACATG Taq Platinum 4 219 1 TTCG AGTTCAG (Invitrogen) AGTGTGAGAATGGGTTTGTG TGGGTATCAGTAGCCTTCGAC HotStar Taq 3 259 1 GAGT ACA (Qiagen) GGGCTTCTATAAAGCTTGCTA ACACCCACAACTGTAACATTT Taq Platinum 2 424 1 ATCTGTC CCC (Invitrogen) TGTGCTTTGGAGGAGGTAGC TAAGCAACACACAGGCCTAAC Taq Platinum 1 418 1 CAAT CCA (Invitrogen) M13 primer: refers to the addition of the M13 sequence (5’ -GTAAAACGACGGCCAGT- 3’) at the 5’ end of the listed primer.

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Table A1.2. Primers used to Sanger sequence DAXX. PCR PCR Exon product M13 primers Other primers PCR Master Mix Program size Taq Platinum 8 409 GTGCCACATCCTGTCTCTTCC GGGACAGCTAATGCCAATCTG 5 (Invitrogen) Taq Platinum 7 404 AAGAGACAGGATGTGGCACG GTCTGCTGGGAGAGACTGGAC 2 (Invitrogen) Taq Platinum 6(b) 466 TCTCCCAGCAGACTCAGTTCC CAAAGGACGCATAGTGTCACC 2 (Invitrogen) Taq Platinum 6(a) 534 CAAGGGAACATTCTCCTCACC CGCCTCCATTGAAGGAAGTAG 1 (Invitrogen) Taq Platinum 5 383 GAGTCCAGGTTGACTGATGGG GAAAGGTTTCAAACAGGTGGC 2 (Invitrogen) Taq Platinum 4 397 ATGTCAGGTATGAGGCGGATG CATCAGTCAACCTGGACTCCC 2 (Invitrogen) Taq Platinum 3(d) 576 GTCAGAGCACTCAGCCCTTG GTAAGCTGATCCGCCTCTTTG 2 (Invitrogen) Taq Platinum 3(c) 543 TAACCCTCCCACACACCTCTC TGGCAGCCAAAGTTGTAGATG 1 (Invitrogen) Taq Platinum 3(b) 553 CGCCTGTTAACCTCTGGGTAG CATTCCTCTATAACCGGCAGC 1 (Invitrogen) Taq Platinum 3(a) 502 CAGAGGAAGCAGTAGTTCGGG AGGTGTGTGGGAGGGTTATTC 6 (Invitrogen) GGGTTAGTGGGAAAGAAAGG Taq Platinum 2 443 GGGCTGGATGTTACTGAAACC 6 AC (Invitrogen)

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Table A1.3. PCR cycling conditions for program 1. Number of cycles Step Temperature Time

1 cycle Taq Hot start 94°C 2 mins

3 cycles Denaturation 94°C 30 sec

Annealing 64°C 45 sec

Extension 70°C 1 min

41 cycles Denaturation 94°C 30 sec

Annealing 57°C 45 sec

Extension 70°C 1 min

1 cycle Extension 72°C 10 mins

Table A1.4. PCR cycling conditions for program 2. Number of cycles Step Temperature Time

1 cycle Taq Hot start 94°C 2 mins

3 cycles Denaturation 94°C 30 sec

Annealing 65°C 45 sec

Extension 70°C 1 min

41 cycles Denaturation 94°C 30 sec

Annealing 59°C 45 sec

Extension 70°C 1 min

1 cycle Extension 72°C 10 mins

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Table A1.5. PCR cycling conditions for program 3. Number of cycles Step Temperature Time

1 cycle Taq Hot start 94°C 2 mins

20 cycles Denaturation 94°C 30 sec

58.5°C

Annealing -0.5°C from 45 sec annealing temp every 3 cycles Extension 72°C 1 min

30 cycles Denaturation 94°C 30 sec

Annealing 55°C 45 sec

Extension 72°C 1 min

1 cycle Extension 72°C 10 mins

Table A1.6. PCR cycling conditions for program 4. Number of cycles Step Temperature Time

1 cycle Taq Hot start 94°C 2 mins

3 cycles Denaturation 94°C 30 sec

Annealing 67°C 45 sec

Extension 70°C 1 min

41 cycles Denaturation 94°C 30 sec

Annealing 60°C 45 sec

Extension 70°C 1 min

1 cycle Extension 72°C 10 mins

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Table A1.7. PCR cycling conditions for program 5. Number of cycles Step Temperature Time

1 cycle Taq Hot start 94°C 2 mins

3 cycles Denaturation 94°C 30 sec

Annealing 63°C 45 sec

Extension 70°C 1 min

41 cycles Denaturation 94°C 30 sec

Annealing 55°C 45 sec

Extension 70°C 1 min

1 cycle Extension 72°C 10 mins

Table A1.8. PCR cycling conditions for program 6. Number of cycles Step Temperature Time

1 cycle Taq Hot start 94°C 2 mins

3 cycles Denaturation 94°C 30 sec

Annealing 62°C 45 sec

Extension 70°C 1 min

41 cycles Denaturation 94°C 30 sec

Annealing 55°C 45 sec

Extension 70°C 1 min

1 cycle Extension 72°C 10 mins

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Appendix II: Data Pertaining to Chapter 4

Table A2.1. Characteristics of high-risk NB ALT tumours (n=36).

C-circle

Telomere Assay Age Content Telomere Product C-circle

Sample Gender Dx Outcome (Arbitrary Content (Arbitrary Assay TRF Telomerase ID MYCN (Yr) (Yr) Unit) Group Unit) Classification (kb) Activity DOD CHW 21 F NA 4.49 (2.96yr) 68.7 long 106 + ND -

DOD CHW 27 F NA 4.04 (2.1yr) 19.1 long 43.0 + ND +

DOD CHW 3 F NA 3.65 (2.62yr) 16.7 long 41.0 + 28.3 ND

DOD NB 157 M NA 4.24 (3.6yr) 72.1 long 134 + ND ND

DOD NB 31 F NA 2.78 (4.24yr) 60.5 long 1311 + ND ND

DOD NB 71 M NA 4.08 (1.32yr) 43.5 long 38.7 + ND ND

OS NB 74 F NA 3.44 (2.77yr) 27.9 long 666 + ND ND

DOD NB 8 F NA 5.41 (1.63yr) 20.2 long 249 + ND ND

DOD NB 92 F NA 4.49 (2.96yr) 75.7 long 54.7 + ND ND

DOD PAHSVL M NA 6.7 (7.62yr) 16.02 long 111 + 11.0 -

DOD PAIHWT F NA 4.2 (4.27yr) 28.27 long 129 + 11.1 +

OS PAISZV M NA 5.1 (5.35yr) 65.89 long 125 + 22.0 -

DOD PAIUTE M NA 5.5 (4.01yr) 75.32 long 14.8 + 22.0 +

OS PAPKXS M NA 6.98 (5.4yr) 17.57 long 15.1 + 18.4 +

OS PAPNHR M NA 3.6 (5.08yr) 15.92 long 36.0 + 1.9 -

OS PAPTAN F NA 4.0 (5.61yr) 26.45 long 141 + 19.9 -

DOD PAPUWY M NA 5.4 (2.98yr) 23.85 long 16.0 + 19.3 +

OS PARACS F NA 3.2 (3.61yr) 15.03 long 341 + ND ND

199

DOD PARASL M NA 4.4 (0.53yr) 34.04 long 25.0 + 24.1 +

DOD PARBGT F NA 2.6 (2.74yr) 29.58 long 14.0 + ND ND

DOD PARKNP M NA 8.8 (1.75yr) 20.87 long 1149 + 20.2 -

PARYM DOD N M NA 4.4 (2.95yr) 42.07 long 462 + ND ND

OS PASAAN F NA 4.2 (3.76yr) 16.39 long 348 + 24.8 +

DOD PASJRH F NA 7.04 (3.33yr) 21.82 long 26.3 + 23.1 +

OS PASPIJ F NA 8.2 (2.81yr) 18.86 long 11.2 + 29.2 -

OS PASRTR F NA 4.4 (2.6yr) 18.08 long 7.6 + ND ND

OS PASVYG M NA 5.1 (2.3yr) 23.94 long 45.8 + ND ND

DOD CHW 24 F NA 2.69 (5.03yr) 14.1 short 249 + ND +

OS CHW 25 F NA 3.59 (11.72yr) 12.7 short 111 + ND -

DOD PALRGK M NA 5.4 (2.52yr) 13.84 short 104 + 17.0 +

PAMHM DOD K M NA 5.7 (3.18yr) 13.61 short 47.5 + 18.3 -

DOD PANLET M NA 5.4 (3.32yr) 8.07 short 29.5 + 9.0 +

OS PANRVJ F NA 13.1 (6.19yr) 5.30 short 41.7 + 9.8 -

OS PANUIF M NA 6.1 (6.22yr) 7.21 short 14.5 + ND ND

DOD PARVLK M NA 16.5 (2.36yr) 7.91 short 219 + 12.7 -

OS PASWLY F NA 2.7 (2.72yr) 12.17 short 166 + 14.6 +

F = female NA = Not Amplified DOD = Died of Disease M = male Yr = year OS = Overall survival

A= Amplified ND = not determined DX = Diagnosis

200

Table A2.2. Characteristics of high-risk MYCN amplified NB tumours (n=55).

C-circle

Telomere Assay Age Content Telomere Product C-circle Sample Dx Outcome (Arbitrary Content (Arbitrary Assay TRF Telomerase ID Gender MYCN (yr) (yr) Unit) Group Unit) Classification (kb) Activity OS CHW 7 F A 1.71 (7.33yr) 18.2 long 1.4 - ND ND

DOD NB 114 M A 1.73 (1.28yr) 18.1 long 0.0 - ND ND

DOD NB 195 M A 2.27 (1.28yr) 23.6 long 0.0 - ND ND

DOD NB 45 F A 3.75 (1.56yr) 20.4 long 0.0 - ND ND

OS PAKPHB M A 0.4 (4.25yr) 35.25 long 0.0 - ND ND

DOD PAPWFY F A 2.1 (2.75yr) 25.55 long 0.0 - ND ND

DOD PARYXW F A 6.0 (1.17yr) 43.20 long 1.5 - ND ND

DOD CHW 12 M A 1.78 (1.64yr) 10.8 short 0.0 - 14.9 ND

DOD CHW 13 M A 0.78 (0.44yr) 6.5 short 0.0 - 10.4 ND

DOD CHW 15 F A 1.96 (0.87yr) 4.7 short 0.0 - 8.6 ND

OS CHW 16 F A 2.19 (5.54yr) 7.3 short 0.1 - 11.4 ND

OS CHW 2 F A 1.59 (7.41yr) 9.1 short 1.3 - 11.9 ND

OS CHW 20 F A 2.51 (4.98yr) 11.1 short 0.0 - ND ND

OS CHW 8 F A 1.32 (5.62yr) 3.4 short 0.0 - 11.7 ND

OS NB 149 F A 1.11 (8.33yr) 2.5 short 0.6 - ND ND

DOD NB 166 F A 5.43 (1.09yr) 13.4 short 0.0 - ND ND

DOD NB 169 F A 0.44 (0.66yr) 13.2 short 0.0 - ND ND

DOD NB 171 M A 2.93 (0.61yr) 7.6 short 0.0 - ND ND

DOD NB 172 F A 2.39 (0.65yr) 3.4 short 0.0 - ND ND

OS NB 23 F A 2.01 (9.33yr) 7.3 short 0.3 - ND ND

201

DOD NB 40 F A 0.64 (0.05yr) 2.9 short 1.6 - ND ND

DOD NB 43 F A 5.81 (0.86yr) 12.3 short 0.5 - ND ND

OS NB 44 F A 1.55 (6.76yr) 3.3 short 0.0 - ND ND

DOD NB 48 F A 0.73 (0.69yr) 9.7 short 0.0 - ND ND

DOD NB 51 M A 1.22 (0.95yr) 2.5 short 0.0 - ND ND

DOD NB 53 F A 1.51 (2.15yr) 3.0 short 0.3 - ND ND

DOD NB 56 M A 1.38 (1.26yr) 8.0 short 0.0 - ND ND

DOD NB 6 F A 1.41 (0.5yr) 14.0 short 0.0 - ND ND

DOD NB 98 M A 0.91 (0.63yr) 10.0 short 0.0 - ND ND

DOD PAKNYZ F A 1.7 (0.41yr) 4.44 short 0.0 - ND ND

DOD PAKPXS M A 3.0 (0.07yr) 2.53 short 0.12 - ND ND

OS PALHHT F A 1.6 (9.97yr) 2.09 short 0.27 - ND ND

DOD PALUVT F A 2.4 (3.18yr) 4.17 short 0.72 - ND ND

DOD PALZMB F A 0.8 (0.53yr) 2.29 short 0.18 - ND ND

DOD PAMRAA F A 0.4 (0.5yr) 5.04 short 0.0 - ND ND

OS PANHRZ 1 A 4.1 (6.86yr) 1.82 short 0.42 - ND ND

DOD PANLTR F A 1.2 (0.41yr) 9.62 short 1.5 - ND ND

DOD PAPRHX F A 10.1 (1.01yr) 5.30 short 0.82 - ND ND

DOD PAPRPR M A 1.7 (4.32yr) 2.31 short 0.19 - ND ND

OS PAPRXW F A 2.4 (5.62yr) 2.03 short 0.0 - ND ND

OS PARELJ F A 0.3 (4.3yr) 6.75 short 0.26 - ND ND

DOD PARETE F A 1.9 (0.7yr) 4.47 short 0.02 - ND ND

202

DOD PARHDE F A 2.3 (1.3yr) 5.79 short 0.41 - ND ND

DOD PARSCY M A 1.4 (0.64yr) 9.62 short 1.1 - ND ND

DOD PARSUF F A 1.3 (1.02yr) 1.51 short 0.07 - ND ND

DOD PARVVM F A 0.9 (0.83yr) 4.89 short 0.0 - ND ND

DOD PARXBV M A 3.6 (1.32yr) 8.25 short 0.0 - ND ND

OS PARYRE M A 3.2 (4.11yr) 4.38 short 0.29 - ND ND

DOD PASBTL F A 1.3 (0.61yr) 3.80 short 0.42 - ND ND

OS PASFKX F A 0.7 (3.01yr) 2.10 short 0.23 - ND ND

DOD PASLXS M A 2.4 (0.33yr) 2.38 short 0.24 - ND ND

DOD PASTGD F A 2.5 (1.5yr) 4.12 short 0.43 - ND ND

DOD PASWFB M A 2.0 (0.2yr) 2.10 short 0.54 - ND ND

DOD PASYIP F A 2.3 (0.6yr) 3.07 short 0.86 - ND ND

OS PASZPI F A 1.8 (2.3yr) 9.61 short 0.32 - ND ND

F = female NA = Not Amplified DOD = Died of Disease M = male Yr = year OS = Overall survival

A= Amplified ND = not determined DX = Diagnosis

203

Table A2.3. Characteristics of high-risk ALT-negative/long telomere NB tumours (n=17).

C-circle

Telomere Assay Age Content Telomere Product C-circle Sample Dx Outcome (Arbitrary Content (Arbitrary Assay TRF Telomerase ID Gender MYCN (yr) (yr) Unit) Group Unit) Classification (kb) Activity OS CHW 11 F NA 3.81 (14.54yr) 22.3 long 3.8 - 24.3 -

DOD CHW 9 F NA 4.67 (2.78yr) 21.1 long 2.3 - 36.3 -

DOD NB 128 F NA 4.45 (1.74yr) 27.3 long 0.0 - ND ND

OS NB 19 F NA 3.29 (11.98yr) 16.1 long 0.0 - ND ND

DOD NB 3 F NA 3.05 (1.27yr) 19.4 long 0.0 - ND ND

OS NB 36 F NA 5.65 (9.49yr) 15.2 long 0.0 - ND ND

OS NB 4 F NA 3.83 (10.63yr) 73.5 long 0.0 - ND ND

OS NB 60 F NA 1.26 (7.38yr) 18.3 long 0.5 - ND ND

DOD NB 70 F NA 2.43 (3.71yr) 36.8 long 0.0 - ND ND

OS NB 95 M NA 5.30 (12.5yr) 17.8 long 1.4 - ND ND

DOD PAIDDG F NA 9.4 (2.5yr) 31.64 long 5.7 - ND ND

DOD PAIFXV M NA 5.5 (3.04yr) 20.68 long 1.5 - 18.7 -

OS PALPYT F NA 5.8 (9.75yr) 50.67 long 0.0 - ND ND

DOD PAMFUX M NA 3.6 (4.52yr) 24.22 long 2.7 - ND ND

DOD PAMTPS F NA 11.2 (5.23yr) 18.08 long 4.0 - ND ND

OS PARXFT F NA 5.1 (2.05yr) 69.95 long 6.6 - 24.5 -

OS PATHVK M NA 5.8 (2.0yr) 52.55 long 0.0 - ND ND

F = female NA = Not Amplified DOD = Died of Disease M = male Yr = year OS = Overall survival

A= Amplified ND = not determined DX = Diagnosis

204

Table A2.4. Characteristics of high-risk MYCN non-amplified/short telomere NB tumours (n=41).

C-circle

Telomere Assay Age Content Telomere Product C-circle Sample Dx Outcome (Arbitrary Content (Arbitrary Assay TRF Telomerase ID Gender MYCN (yr) (yr) Unit) Group Unit) Classification (kb) Activity

OS CHW 22 M NA 2.00 (15.93yr) 2.9 short 1.5 - ND +

OS CHW 23 F NA 1.47 (11.06yr) 2.5 short 0.2 - ND +

OS CHW 26 F NA 1.25 (9.7yr) 4.2 short 0.0 - ND +

DOD CHW 4 F NA 3.69 (2.47yr) 7.1 short 1.0 - 13.2 ND

OS NB 112 M NA 1.99 (5.48yr) 7.4 short 5.0 - ND ND

OS NB 12 F NA 2.12 (3.67yr) 9.4 short 0.5 - ND ND

DOD NB 140 F NA 3.46 (0.78yr) 6.6 short 1.4 - ND ND

DOD NB 17 M NA 3.59 (0.6yr) 5.7 short 1.4 - ND ND

DOD NB 174 F NA 3.44 (1.07yr) 13.8 short 0.8 - ND ND

DOD NB 198 F NA 4.18 (0.37yr) 2.7 short 0.0 - ND ND

OS NB 216 F NA 1.97 (4.65yr) 6.1 short 0.0 - ND ND

DOD NB 217 F NA 2.88 (0.97yr) 9.4 short 0.0 - ND ND

DOD NB 226 M NA 3.99 (3.2yr) 5.9 short 1.3 - ND ND

DOD NB 24 M NA 2.41 (1.68yr) 9.1 short 1.5 - ND ND

DOD NB 25 M NA 6.63 (0.67yr) 5.4 short 0.0 - ND ND

DOD NB 65 M NA 2.72 (5.71yr) 6.2 short 0.8 - ND ND

DOD NB 77 F NA 1.55 (2.42yr) 6.3 short 1.8 - ND ND

OS NB 78 F NA 1.78 (3.17yr) 4.6 short 0.0 - ND ND

OS PAHVCS M NA 1.2 (5.52yr) 7.17 short 1.1 - 8.5 +

205

DOD PAHXUD M NA 1.4 (3.24yr) 5.10 short 0.0 - 6.6 +

OS PAILRI M NA 1.2 (8.25yr) 3.70 short 0.0 - 9.5 +

OS PALKNC M NA 1.0 (1.77yr) 3.26 short 0.0 - 9.1 +

OS PALSRF F NA 1.0 (8.96yr) 3.44 short 0.0 - 10.2 -

OS PAMHFI F NA 1.1 (5.91yr) 6.20 short 0.0 - 8.7 -

OS PAMPXA F NA 1.9 (7.39yr) 3.64 short 0.20 - ND ND

OS PAMRTG F NA 1.4 (7.65yr) 8.86 short 1.5 - ND ND

DOD PAMYBA F NA 12.6 (1.48yr) 3.63 short 0.34 - ND ND

DOD PANHVF F NA 6.6 (3.56yr) 0.93 short 0.0 - 4.1 +

DOD PAPFSL M NA 6.0 (0.61yr) 2.87 short 0.0 - 9.4 +

OS PAPMUT F NA 2.7 (5.25yr) 5.30 short 1.1 - ND ND

OS PAPRGH M NA 1.2 (5.14yr) 4.60 short 0.0 - 6.5 -

OS PAPZFW F NA 3.7 (4.61yr) 2.13 short 0.80 - ND ND

DOD PARADX F NA 4.4 (0.5yr) 5.39 short 0.32 - ND ND

OS PARGIW F NA 3.2 (4.69yr) 2.37 short 0.56 - 9.1 +

OS PARNNG F NA 3.7 (4.65yr) 3.69 short 0.0 - ND ND

DOD PARRLH M NA 3.6 (1.48yr) 4.27 short 0.06 - ND ND

OS PASHVB M NA 5.1 (3.17yr) 7.28 short 0.0 - 12.1 +

DOD PASJNY M NA 3.4 (1.22yr) 5.41 short 0.0 - 7.9 +

DOD PASLUP M NA 4.5 (2.53yr) 2.28 short 0.01 - 5.5 +

DOD PASRII M NA 2.3 (1.8yr) 5.04 short 0.59 - ND ND

OS PASUCB F NA 2.8 (2.45yr) 2.98 short 0.0 - 13.8 -

206

F = female NA = Not Amplified DOD = Died of Disease M = male Yr = year OS = Overall survival

A= Amplified ND = not determined DX = Diagnosis

207

Table A2.5. Genes examined by whole genome sequencing.

Name ID Summary Synonym

Protein involved in a variety of cellular processes, including cell division, DNA damage response, adhesion, differentiation, and response ABL1 25 to stress, SWI/SNF complex JTK7, P150

ACD 65057 Shelterin protein TINT1, TPP1

Required for SMARCA4/BRG1/BAF190A containing remodelling complex BAF ACTL6A 86 with chromatin/nuclear matrix. BAF53A, INO80K

ACTL6B 51412 Role in remodelling mononucleosomes BAF53B

Recognises cytosolic dsDNA, putative role in tumorigenic reversion and may AIM2 9447 control cell proliferation. PYHIN4

Regulates many processes including metabolism, proliferation, cell survival, AKT1 207 growth and angiogenesis.

ALKBH 8846 DNA repair gene

ALKBH2 121642 DNA repair gene

Role in the cellular response to APEX1 328 oxidative stress.

Involved in DNA damage and repair APITD1 378708 (involved in Fanconi anaemia)

APTX 54840 Editing and processing nucleases aprataxin

ARID1A 8289 SWI/SNF complex SMARCF, C1orf4

ARID1B 57492 Involved in chromatin remodelling

ARID3A 1820 Transcription factor

208

ASF1A 25842 Histone chaperone HAsf1, HCIA, HSPC146

Member of the H3/H4 family of histone ASF1B 55723 chaperone proteins HAsf1b

ASH1, KMT2H, ASH1L 55870 Histone methyltransferase KIAA1420

Component of the Set1/Ash2 histone ASH2L 9070 methyltransferase (HMT) complex ASH2L1, Bre2

Transcription coactivator activity, ASXL1 171023 chromatin modifier KIAA0978, MDS, BOPS

ATM 472 DNA damage response ATA, ATDC, ATC, ATD

ATP5C1 509 Subunit of mitochondrial ATP synthase. ATP5CL1, ATP5C

ATR 545 Conserved DNA damage response gene MEC1, FRP1

ATRIP 84126 Conserved DNA damage response gene AGS1

Chromatin Modifier/epigenetic ATRX 546 remodeller RAD54, JMS, MRX52

DNA damage repair, ubiquitination and BARD1 580 transcriptional regulation

Chromatin Modifier/epigenetic BAZ1A 11177 remodeller WCRF18, HACF10

Chromatin Modifier/epigenetic WSTF, WBSCR10, BAZ1B 9031 remodeller WBSCR9

BCCIP 56647 DNA repair and cell cycle TOK1

BCL2 596 Apoptosis Bcl-2, PPP1R50

BLM 641 DNA helicase RECQL3, RECQ2

Chromatin Modifier/epigenetic BMI1 648 remodeller PCGF4, RNF51

209

Role in maintaining genomic stability, BRCA1 672 and it also acts as a tumour suppressor. FANCS

Involved in double-strand break repair BRCA2 675 and/or homologous recombination. FANCD1

Chromatin Modifier, transcriptional BRD2 6046 regulator KIAA9001, RING3

BRD4 23476 Chromatin/epigenetic remodeller HUNKI, MCAP

Involved in cell cycle and cell death DBC1, DBCCR1, BRINP1 1620 pathways FAM5A

BRIP1 83990 DNA-dependent ATPase FANCJ

BTAF1 9044 Regulates transcription MOT1

Involved in replication-independent CABIN1 23523 chromatin assembly. PPP3IN, KIAA0330

CARM1 10498 Chromatin regulation PRMT4

CBX1 10951 Component of heterochromatin. CBX, M31, HP1Hsbeta

CBX2 84733 Involved in chromatin regulation CDCA6, SRXY5

Binds DNA and is a component of CBX3 11335 heterochromatin.

CBX5 23468 Component of heterochromatin HP1A

Protein is enriched in the heterochromatin and associated with CCAR2 23468 centromeres. HEL25

CCNH 902 Transcription P34, P37, CycH, CAK

CCNO 10309 Cell cycle CCNU, UDG2, CILD29

CCNT1 904 Transcription HIVE1, CCNT, CYCT1

CDC25A 993 Cell cycle CDC25A2

210

CDC25B 994 Cell cycle CDC25HU2

CDK2 1017 Cell cycle P33(CDK2)

Cell cycle, transcription initiating, DNA CDK7 1022 repair gene CAK, STK1

CDKN1, P21, WAF1, CDKN1A 1026 Cell cycle, DNA repair gene MDA-6, CAP20

CENPA 1058 Chromatin CENP-A

CENPT 80152 Chromatin C16orf56, ICEN22

Involved in microtubule organization, DNA damage response, and Cep164, CEP164 22897 . NPHP15, KIAA1052

Involved in DNA replication and DNA CHAF1A 10036 repair. CAF1

Chromatin Modifier/epigenetic CHD1 1105 remodeller CHD-1

Chromatin Modifier/epigenetic CHD2 1106 remodeller CHD-2, EEOC

NuRD (nucleosome remodelling and Mi2-ALPHA, ZFH, CHD- CHD3 1107 histone deacetylation) 3

NuRD (nucleosome remodelling and CHD4 1108 histone deacetylation) Mi2-BETA, CHD-4

Chromatin regulator, transcription CHD7 55636 regulator CRG, KAL5

CHD8 57680 Chromatin remodeller HELSNF1, AUTS18

CHEK1 1111 Conserved DNA damage response gene CHK1

CHEK2 11200 Conserved DNA damage response gene RAD53, CHK2

CHFR 55743 Cell Cycle RNF116, RNF196

211

C16orf41, RUVBL, CHTF18 63922 Cell replication CHL12

CLK2 1196 Transcription

CLOCK 9575 Transcription KAT13D, KIAA0334

CLSPN 63967 Cell cycle, DNA damage

Chromatin regulator, transcription CREBBP 1387 factor RSTS, KAT3A

CTBP1 1487 Transcription regulator BARS

CTCF 10664 Chromatin regulator MRD21

CTCFL 140690 DNA binding protein CT27, HMGB1L1

CTDP1 9150 Involved in initiation of translation CCFDN, FCP1

CTR9 9646 Chromatin regulation SH2BP1, TSBP, P150

DAXX 1616 Chromatin remodeller DAP6, EAP1, BING2

ASKL1, DRF1, CHIFB, DBF4B 80174 Roll in DNA replication and proliferation ZDBF1B

Required for DNA inter-strand cross-link SNM1, PSO2, HSNM1, DCLRE1A 9937 repair and cell cycle arrest HSNM1A, KIAA0086

Involved in t-loop formation, telomere SNM1B, APOLLO, DCLRE1B 64858 replication and DNA damage HSNM1B

SCIDA, ARTEMIS, DCLRE1C 64421 Involved in DNA damage repair HSNM1C, DCLREC1C

DDBA, XPCE, XAP1, DDB1 1642 Required for DNA repair UV-DDB1

DDB2 1643 Required for DNA repair DDBB, XPE, UV-DDB2

RNA helicase involved in translation DHX29 54505 initiation. DDX29

212

Role in transcriptional regulation and DDX36, MLEL1, G4R1, DHX36 170506 mRNA stability. RHAU, KIAA1488

DHX58 79132 Recognises ssRNA, dsRNA LGP2, RLR

DDX60 55601 Binds ssRNA, dsRNA and dsDNA

DHX9 1660 Functions as a transcriptional activator. DDX9, LKP, RHA, NDH2

DKC, NOLA4, CBF5, DKC1 1736 Component of telomerase NAP57, Dyskerin

Chromatin regulator, methylates CpG DNMT, MCMT, AIM, DNMT1 1786 residues. ADCADN, HSN1E

DNA methyltransferase, NuRD DNMT3A 1788 associated TBRS

DNA methyltransferase, NuRD DNMT3B 1789 associated M.HsaIIIB, ICF1

DNTT 1791 DNA polymerase TDT

DR1 1810 Chromatin regulator NC2B

E2F1 1869 Transcription activator RBBP3, RBAP1, -1

E2F2 1870 Transcription factor, cell cycle

Transcription factor, also involved in cell 1871 cycle and DNA repair KIAA0075

Transcription factor, also involved in cell P107/P130-Binding 1874 cycle and DNA repair Protein

ECT2 1894 Involved in cell cycle ARHGEF31

EGF 1950 Involved in cell growth and proliferation HOMG4, URG

Methyltransferase that methylates EHMT2 10919 lysine residues of histone H3. C6orf30, BAT8

Involved in repair of DNA double-strand EID3 493861 breaks by homologous recombination NSE4B

213

ELP3 55140 Chromatin remodelling/regulation HELP3, KAT9

Homologous recombination, interacts with MUS81 to form a DNA structure- EME1 146956 specific endonuclease SLX2A, MMS4

Homologous recombination, interacts with MUS81 to form a DNA structure- EME2 197342 specific endonuclease SLX2B, Gs125

ENDOV 284131 Editing and processing RNAs

EP300 2033 histone acetyltransferase, remodeller P300, RSTS2, KAT3B

ERCC1 2067 Involved in DNA repair RAD10, UV20, COFS4

Transcription factor also involved in XPD, TTD1, COFS2, ERCC2 2068 DNA repair EM9

DNA helicase and transcription factor RAD25, GTF2H, BTF2, ERCC3 2071 also involved in DNA repair TTD2, XPB

XPF, RAD1, FANCQ, ERCC4 2072 Involved in DNA repair XFEPS, XPF, ERCC11

ERCM2, XPGC, COFS3, Involved in DNA repair ERCC5 2073 UVDR, XPG

CKN2, RAD26, ARMD5, Involved in DNA repair ERCC6 2074 UVSS1, COFS

ERCC8 1161 Involved in DNA repair CKN1, UVSS2

RNA exonuclease, involved in histone ERI1 90459 RNA degradation THEX1, HEXO

EWSR1 2130 Transcription factor- repressor

EXO1 9156 DNA exonuclease HEX1, EXOI

Chromatin remodeller and transcription EZH2 2146 repressor

214

FAAP20 199990 Involved in DNA damage repair C1orf86, FP7162

FAAP24 91442 Involved in DNA damage repair C19orf40

Involved in Fanconi anaemia DNA FAAP100 80233 damage response C17orf70

FAN1 22909 Editing and processing nucleases MTMR15

FANCA 2175 Involved in DNA repair FACA, FANCH

FANCB 2187 Involved in DNA repair

FANCC 2176 Involved in DNA repair FACC

FANCD2 2177 Involved in DNA repair FACD, FANCD

FANCE 2178 Involved in DNA repair FACE

FANCF 2188 Involved in DNA repair

FANCG 2189 Involved in DNA repair XRCC9

FANCI 55215 Involved in DNA repair KIAA1794

FANCL 55120 Involved in DNA repair PHF9, POG

FANCM 57697 Involved in DNA repair KIAA1596, FAAP250

Methyltransferase that has the ability to FBL 2091 methylate both RNAs and proteins. RNU3IP1

FBXO18 84893 Involved in homologous recombination HFBH1

FEN1 2237 Involved in DNA repair DNase IV, RAD2

HSPC215, Foxp1 27086 NuRD associated, transcription QRF1, 12CC4, HFKH1B

TNRC10, Foxp2 93986 NuRD associated, transcription SPCH1, CAGH44

NuRD associated, transcriptional Foxp4 116113 repressor HFKHLA, FKHLA

215

FSBP 100861412 Transcriptional repressor

ALS6, ETM4, FUS1, FUS 2521 Chromatin regulator POMP75, TLS

GAR1 54433 Involved in telomerase NOLA1

Homologous recombination, Endonuclease which resolves Holliday GEN1 48654 junctions

GET4 51608 Post translational trafficking C7orf20

GMNN 51053 Involved in cell cycle regulation MGORS6

E2IG3, NNP47, NS, GNL3 26354 Involved in cell proliferation C77032

GTF2B 2959 Transcription factor S300-II, TFIIB, TF2B

Transcription initiation factor that binds RAP74, TF2F1, TFIIF, GTF2F1 2962 to RNA polymerase II BTF4

GTF2H1 2965 Transcription factor BTF2, TFIIH, TFB1, P62

T-BTF2P44, TFIIH, GTF2H2 2966 Transcription factor BTF2, P44

GTF2H3 2967 Transcription factor TFB4, TFIIH, BTF2, P34

GTF2H4 2968 Transcription factor TFB2, TFIIH, P52

H2AFX 3014 Chromatin Structure H2AX

H2AFY 9555 Chromatin Structure H2AF12M, MH2A1

H2AFZ 3015 Chromatin Structure H2AZ

HAT1 8520 Involved in nucleosome assembly KAT1

Responsible for the deacetylation of lysine residues on the N-terminal part HDAC1 3065 of the core histones RPD3L1, GON-10, HD1

216

Responsible for the deacetylation of lysine residues on the N-terminal part HDAC10 83933 of the core histones

Responsible for the deacetylation of lysine residues on the N-terminal part YAF1, HD2, RPD3, YY1- HDAC2 3066 of the core histones Associated Factor 1

Responsible for the deacetylation of lysine residues on the N-terminal part HDAC3 8841 of the core histones

Responsible for the deacetylation of lysine residues on the N-terminal part HDAC4 9759 of the core histones

Responsible for the deacetylation of lysine residues on the N-terminal part HD5, KIAA0600, NY- HDAC5 10014 of the core histones CO-9

Responsible for the deacetylation of lysine residues on the N-terminal part HDAC6 10013 of the core histones

Responsible for the deacetylation of lysine residues on the N-terminal part HDAC7 51564 of the core histones

HELLS 3070 Chromatin regulation LSH, ICF4

DNA-dependent ATPase and 5 to 3 DNA HELQ 113510 helicase. HEL308

Helicase that plays a role in RNA KIAA0054, HELZ 9931 metabolism DHRC, HUMORF5

HES1 3280 Transcriptional repressor HRY, HL, HES-1

217

Transcriptional regulator which functions as a general RNA polymerase HEXIM1 10614 II transcription inhibitor. HIS1, EDG1, CLP1

HIC1 3090 Transcriptional repressor ZNF901, ZBTB29

Chromatin Modifier/epigenetic HIRA 7290 remodeller TUPLE1, DGCR1, HIR

HIST1H1A 3024 Replication Dependent Histone H1F1, H1.1, H1a

Replication Dependent Histone H1F5, H1.5, H1b, H1s- HIST1H1B 3009 3

HIST1H1C 3006 Replication Dependent Histone H1F2, H1.2, H1s-1, H1c

Replication Dependent Histone H1F3, H1.3, H1d, H1s- HIST1H1D 3007 2

Replication Dependent Histone H1F4, H1.4, H1e, H1s- HIST1H1E 3008 4

HIST1H1PS1 387325 Replication Dependent Histone DJ34B20.16, H1F6P

HIST1H1PS2 10338 Replication Dependent Histone H3FEP, PH3/e

HIST1H1T 3010 Replication Dependent Histone H1FT, H1t

Replication Dependent Histone BA317E16.2, H2AFR, HIST1H2AA 221613 H2AA, TH2A

HIST1H2AB 8335 Replication Dependent Histone H2AFM, H2A/m

HIST1H2AC 8334 Replication Dependent Histone H2AFL

HIST1H2AD 3013 Replication Dependent Histone H2AFG, H2A/g, H2A.3

HIST1H2AE 3012 Replication Dependent Histone H2AFA, H2A/a, H2A.1

Replication Dependent Histone H2AFP, pH2A/f, HIST1H2AG 8969 H2A/p, H2A.1b

Replication Dependent Histone H2AFALii, dJ86C11.1, HIST1H2AH 85235 H2A/S

218

HIST1H2AI 8329 Replication Dependent Histone H2AFC, H2A/c

HIST1H2AJ 8331 Replication Dependent Histone H2AFE, H2A/E

HIST1H2AK 8330 Replication Dependent Histone H2AFD, H2A/d

Replication Dependent Histone H2AFI, H2A/I, HIST1H2AL 8332 dJ193B12.9

HIST1H2AM 8336 Replication Dependent Histone H2AFN, H2A/n, H2A.1

Replication Dependent Histone bA317E16.3, STBP, HIST1H2BA 255626 TSH2B, H2BFU

HIST1H2BB 3018 Replication Dependent Histone H2BFF, H2B/f

HIST1H2BC 8347 Replication Dependent Histone H2BFL, H2B/l, H2B.1

HIST1H2BD 8347 Replication Dependent Histone H2BFB, H2B/b

HIST1H2BE 8344 Replication Dependent Histone H2BFH, H2B/h, H2B.h

HIST1H2BF 8343 Replication Dependent Histone H2BFG, H2B/g

HIST1H2BG 8339 Replication Dependent Histone H2BFA, H2B/a, H2B.1A

HIST1H2BH 8345 Replication Dependent Histone H2BFJ, H2B/j

HIST1H2BI 8346 Replication Dependent Histone H2BFK, H2B/k

HIST1H2BJ 8970 Replication Dependent Histone H2BFR, H2B/r

HIST1H2BK 85236 Replication Dependent Histone H2BFT, H2BFAiii

Replication Dependent Histone H2BFC, H2B/c, HIST1H2BL 8340 dJ97D16.4

Replication Dependent Histone H2BFE, H2B/e, HIST1H2BM 8342 dJ160A22.3

HIST1H2BN 8341 Replication Dependent Histone H2BFD, H2B/d

HIST1H2BO 8348 Replication Dependent Histone H2BFN, H2B/n, H2B.2

HIST1H3A 8350 Replication Dependent Histone H3FA, H3/A

219

HIST1H3B 8358 Replication Dependent Histone H3FL, H3/l

HIST1H3C 8352 Replication Dependent Histone H3FC, H3/c, H3.1

HIST1H3D 8351 Replication Dependent Histone H3FB, H3/b,

HIST1H3E 8353 Replication Dependent Histone H3FD, H3/d, H3.1

HIST1H3F 8968 Replication Dependent Histone H3FI, H3/i

HIST1H3G 8355 Replication Dependent Histone H3FH, H3/h

HIST1H3H 8357 Replication Dependent Histone H3FK, H3/k, H3F1K

HIST1H3I 8354 Replication Dependent Histone H3FF, H3/f, H3.f

HIST1H3J 8356 Replication Dependent Histone H3FJ, H3/j

HIST1H4A 8359 Replication Dependent Histone H4FA

HIST1H4B 8366 Replication Dependent Histone H4FI, H4/I

Replication Dependent Histone H4FG, H4/g, HIST1H4C 8364 dJ221C16.1

HIST1H4D 8360 Replication Dependent Histone H4FB, H4/b

HIST1H4E 8367 Replication Dependent Histone H4FJ, H4/j

HIST1H4F 8361 Replication Dependent Histone H4FC, H4/c, H4

HIST1H4G 8369 Replication Dependent Histone H4FL, H4/l

HIST1H4H 8365 Replication Dependent Histone H4FH, H4/h

HIST1H4I 8294 Replication Dependent Histone H4FM, H4/m

HIST1H4J 8363 Replication Dependent Histone H4FE, H4/e, H4F2iv

Replication Dependent Histone H4FD, H4/d, H4F2iii, HIST1H4K 8362 dJ160A22.1

HIST1H4L 8368 Replication Dependent Histone H4FK, H4.k, H4/k

Replication Dependent Histone H2AFO, HIST2H2AA, HIST2H2AA3 8337 H2A.2, H2A/q

220

Replication Dependent Histone H2A/r, H2BFQ, H2B/q, HIST2H2AA4 723790 H2B.1

HIST2H2AB 317772 Replication Dependent Histone H2AB

HIST2H2AC 8338 Replication Dependent Histone H2A, H2AFQ, H2A/q

HIST2H2BA 337875 Replication Dependent Histone

HIST2H2BB 338391 Replication Dependent Histone

HIST2H2BC 337873 Replication Dependent Histone H2B/t, HIST2H2BD

HIST2H2BD 337874 Replication Dependent Histone H2BFO, H2B/s, H2B/o

Replication Dependent Histone H2B, H2BQ, H2BFQ, HIST2H2BE 8349 H2BGL105

HIST2H2BF 440689 Replication Dependent Histone

HIST2H3A 333932 Replication Dependent Histone H3/N, H3/O

HIST2H3C 126961 Replication Dependent Histone H3/M, H3, H3.2

HIST2H3D 653604 Replication Dependent Histone

Replication Dependent Histone H4F2, H4FN, HIST2H4, HIST2H4A 8370 H4/n

HIST2H4B 554313 Replication Dependent Histone H4/o

HIST3H2A 92815 Replication Dependent Histone MGC3165

HIST3H2BA 337872 Replication Dependent Histone

HIST3H2BB 128312 Replication Dependent Histone

HIST3H3 8290 Replication Dependent Histone H3FT, H3t, H3/g, H3.4

HIST4H4 121504 Replication Dependent Histone MGC24116

SWI/SNF family, helicase and HLTF 6596 ligase functions SMARCA3

221

HMGIY, HMGA1A, HMGA1 3159 Transcription HMG-R

HMGIC, LIPO, BABL, HMGA2 8091 Transcription STQTL9

Involved in transcription and chromatin HMGB1 3146 remodelling HMG1, SBP-1, HMG1

Involved in transcription and chromatin HMGB2 3148 remodelling HMG2

Involved in transcription and chromatin HMGN1 3150 remodelling HMG14

Involved in transcription and chromatin HMGN2 3151 remodelling HMG17

HNRNPA0 10949 RNA processing HNRPA0

HNRPA1, IBMPFD3, HNRNPA1 3178 RNA processing ALS19, ALS20, UP 1

HNRPA2B1, IBMPFD2, HNRPA2, HNRPB1, HNRNPA2B1 3181 RNA processing RNPA2

Involved in RNA processing and DNA AUF1, HNRPD, P37, HNRNPD 3184 replication HnRNPD0, HNRPD

Involved in cell cycle and signal HSPC1, HSPCA, LAP2, HSP90AA1 3320 transduction EL52, Hsp90

HSPA1L 3305 Protein stabilization HSP70T

HUS1 3364 Role in DNA repair

IDPC, IDCD, HEL-216, IDH1 3417 Chromatin regulation PICD

IDPM, IDHM, D2HGA2, IDH2 3418 Chromatin regulation IDH

222

MDA5, IDDM19, IFIH1 64135 RNA helicase IDDM19, RH116, AGS7

IGF1 3479 Growth factor and replication IGFI, IBP1, IGF-I, MGF

Involved in transcription and chromatin DYS, ELP1, IKAP, P150, IKBKAP 8518 remodelling IKI3, DYS, FD

DNA helicase and transcription factor INO80 54617 also involved in DNA repair INOC1, KIAA1259

Involved in transcriptional regulation, INO80C 125476 DNA replication and DNA repair. C18orf37

Involved in transcriptional regulation, INO80E 283899 DNA replication and DNA repair. CCDC95

Exoribonuclease that acts on single- ISG20 3669 stranded RNA CD25, HEM45

Histone acetyltransferase that KAT2A 2648 promotes transcriptional activation. GCN5L2, STAF97

Histone acetyltransferase that PCAF, CAF, Lysine KAT2B 8850 promotes transcriptional activation. Acetyltransferase 2B

HTATIP, TIP60, HTATIP, ESA1, PLIP, KAT5 10524 Transcriptional activation ZC2HC5

lysine (K)-specific demethylase KDM1A 23028 Histone demethylase, NuRD associated 1A (LSD1)

C5orf7, JMJD1B, KDM3B 51780 Demethylates Lys-9 of histone H3 KIAA1082, NET22

Demethylates Lys-9 and Lys-36 residues JMJD2A, JHDM3A, KDM4A 9682 of histone H3 KIAA0677, TDRD14A

223

JMJD2B, KIAA0876, KDM4B 23030 Demethylates Lys-9 of histone H3 TDRD14B, JHDM3B

JMJD2C, TDRD14C, Demethylates Lys-9 and Lys-36 residues KIAA0780, GASC1, KDM4C 23081 of histone H3 JHDM3C

RBBP2, JARID1A, KDM5A 5927 Demethylates Lys-4 of histone H3 RBBP2

JARID1B, RBP2-H1, KDM5B 10765 PLU-1, CT31, PUT1, Demethylates Lys-4 of histone H3 RBBP2H1A, PPP1R98

SMCX, JARID1C, MRX13, MRXJ, MRXSCJ, XE169, KDM5C 8242 Demethylates Lys-4 of histone H3 DXS1272E

Histone demethylase, demethylates KDM6B 23135 Lys-27 of histone H3

MLL4, MLL1B, CXXC10, 9757 KIAA0304, TRX2, KMT2B Methylates Lys-4 of histone H3 WBP7

MLL2, TNRC21, KMT2D 8085 Methylates Lys-4 of histone H3 AAD10, KABUK1, (H3K4me). CAGL114, ALR

Mono- and di-methylates Lys-4 of MLL5, NKp44L, 55904 KMT2E histone H3 (H3K4me1 and H3K4me2) HDCMC04P

Monomethylates both histones and SETD8, H4-K20- non-histone proteins, involved in Specific Histone KMT5A 387893 mitosis Methyltransferase

224

SUV420H1, Lysine- Specific KMT5B 51111 Trimethylates Lys-20 of histone H4. Methyltransferase 5B

SUV420H2, Lysine (K)- Specific KTM5C 84787 Histone methyltransferase Methyltransferase 5C

KRAS 3845 Cell proliferation

KRIT1 889 Cell proliferation CCM1, CaM

LIG1 3978 DNA repair

LIG3 3980 DNA repair LIG2

LIG4 3981 DNA repair LIG4S

MAD2L2 10459 Cell cycle and DNA repair REV7, POLZ2, MAD2B

VISA, IPS1, KIAA1271, MAVS 57506 Detect intracellular dsRNA CARDIF

Transcriptional corepressor and MBD1 4152 coactivator, Chromatin Modifier/epigenetic remodeller

Transcriptional corepressor and DMTase, NY-CO-41, MBD2 8932 coactivator, NuRD complex demethylase

Transcriptional corepressor and MBD3 53615 coactivator, NuRD complex

Transcriptional corepressor and MBD4 8930 coactivator

CCNL1, CDCL1, MITOTIN, D3S3194, MCM2 4171 DNA replication KIAA0030

RLFB, HCC5, P1- MCM3 4172 DNA replication MCM3, P102

225

MCM4 4173 Initiation of DNA replication CDC21

MCM5 4174 Cell cycle CDC46

MCM6 4175 Initiation of DNA replication

MCM2, CDC47, P1CDC47, PPP1R104, MCM7 4176 DNA replication PNAS146, P85MCM

MCPH1 79648 DNA damage response BRIT1, MCT

MSP58, P78, ICP22BP, MCRS1 10445 Transcription regulator INO80Q

MDC1 9656 Cell cycle and DNA damage response NFBD1, KIAA0170

RTT, MRX16, MRX79, MECP2 4204 Mediates transcriptional expression AUTSX3, RTS, MRXSL

TNRC11, FGS1, ARC240, CAGH45, MED12 9968 Transcription HOPA, MED12S

MEN1 4221 Transcriptional regulator SCG2, Menin

Protein catalyses transfer of methyl groups from O(6)-alkylguanine and other methylated moieties of the DNA O6-Methylguanine- to its own molecule, which repairs the DNA MGMT 4255 toxic lesions. Methyltransferase

MKI67 4288 Cell proliferation MIB-1, KIA, PPP1R105

FCC2, HMLH1, HNPCC, DNA repair MLH1 4292 COCA2

YEATS1, LTG19, ENL, MLLT1 4298 Transcription regulator CTC-503J8.6

CAP35, RNF66, MAT1, MNAT1 4331 Transcription regulator P36, TFB3

226

ADPG, AAG, MDG, APNG, PIG16, PIG11, MPG 4350 DNA repair CRA36.1

MPLKIP 136647 Cell cycle TTDN1, C7orf11

Component of the MRN complex, role in double-strand break repair, DNA recombination, maintenance of MRE11A 4361 telomere integrity and meiosis. MRE11

Post-replicative DNA mismatch repair MSH2 4436 system

MRP1, DUP, DUC1, MSH3 4437 DNA repair DUG

MSH4 4438 Involved in meiotic recombination.

MSH5 4439 Involved in meiotic recombination. MUTSH5, NG23, G7

GTBP, HNPCC5, HMSH6, HSAP, P160, MSH6 2956 DNA repair GTMBP

Transcriptional corepressor and p70, MTA1L1, PID, MTA1 9112 coactivator, NuRD complex KIAA1264

Transcriptional corepressor and p70, MTA1L1, PID, MTA2 9219 coactivator, NuRD complex KIAA1265

Transcriptional corepressor and p70, MTA1L1, PID, MTA3 57504 coactivator, NuRD complex KIAA1266

M96, TDRD19A, MTF2 22823 Transcriptional activator DJ976O13.2, PCL2

DNA repair and homologous MUS81 80198 recombination SLX3

MUTYH 4595 DNA repair HMYH

227

C-Myc, MYCC, MRTL, MYC 4609 Transcription factor BHLHe39

ARD1, ARD1A, TE2, Catalytic subunit of the N-terminal NATD, OGDNS, NAA10 8260 acetyltransferase A (NatA) complex DXS707, MCOPS1

SSB2, OBFC2A, SOSS- NABP1 64859 DNA repair and cell cycle B2

SSB1, OBFC2B, NABP2 79035 DNA repair and cell cycle LP3587, SOSS-B1

Component of MRN complex, DNA NBS1 4683 repair NBS, NBN, ATV, P95

YCG1, CHCG, HCAP-G, NYMEL3, NY-MEL-3, NCAPG 64151 Involved in meiosis CAPG

Chromatin decondensation by binding NCL 4691 H1

F-SRC-1, BHLHe42, KAT13A, NCoA-1, SRC- NCOA1 8648 Involved in transcription 1, RIP160

NCOA3 8202 Chromatin Modifier

NCOR1 9611 Chromatin Modifier

transcriptional regulator, promoting NCOR2 9612 chromatin condensation

KNTC2, HEC, KNTC2, Ndc80 10403 Involved in cell cycle TID3

NELFCD 51497 Transcription TH1L, HSPC130, NELFD

Involved in transcriptional regulation NFRKB 4798 and DNA repair INO80G

228

NHEJ1 79840 DNA repair XLF, XRCC4-Like Factor

NHP2 55651 Involved in trafficking hTR NOLA2, DKCB2, NHP2P

NOP10 55505 Involved in trafficking hTR NOLA3, DKCB1

Component of the SMC5-SMC6 complex, a complex involved in DNA double-strand break repair by NSMCE2 286053 homologous recombination. C8orf36

NDNL2, MAGEG1, NSMCE3 56160 DNA repair HCA4, NSE3

NSMCE4A 54780 DNA repair C10orf86, NSE4A

NTHL1 4913 DNA repair NTH1, FAP3, OCTS3

C15orf55, FAM22H, NUTM1 256646 Chromatin regulation NUT

OBFC1 79991 DNA replication RPA-32, STN1, AAF44

MMH, HOGG1, OGH1, OGG1 4968 DNA repair genes MUTM

ORC1L, PARC1, ORC1 4998 DNA replication HSORC1

ORC2 4999 DNA replication ORC2L

ORC3 23595 DNA replication ORC3L, LATHEO, LAT

ORC4 5000 DNA replication ORC4L

ORC5L, ORC5P, ORC5 5001 DNA replication ORC5T, PPP1R117

ORC6 23594 DNA replication ORC6L

PAB1, PABPC2, PABPC1 26986 RNA processing PABPL1

PAF1 54623 Transcription PD2

229

PALB2 79728 DNA repair FANCN, PNCA3

Editing and processing nucleases, 3' PAPOLG 64895 mRNA PAP2, PAPG

ADPRT1, PARP, PPOL, PARP1 142 DNA damage response ARTD1

ADPRTL2, ADPRTL3, ARTD2, PARP-2, PARP2 10038 DNA damage response PADPRT-2

ADPRTL1, KIAA0177, VAULT3, VWA5C, PARP4 143 DNA damage response P193, PH5P, PARP-4

PAX8 7849 Transcription factor

PAXIP1L, TNRC2, Involved in DNA damage response and PACIP1, CAGF29, PAXIP1 22976 transcriptional regulation CAGF28, PAXIP1L

PCNA 5111 DNA polymerase

PER1 5187 Transcriptional repressor PER, KIAA0482, RIGUI

Involved in cell proliferation and RNA PES1 23481 processing PES

PGBD3 267004 Transcription

HPCl1, PCL1, PHF1 5252 Transcriptional repressor TDRD19C, MTF2L2

PHF2 5253 Lysine demethylase KIAA0662, JHDM1E

C20orf104, GLEA2, PHF20 51230 Transcriptional regulator HCA58, NZF, TZP

URLC1, CGI-72, PHF20L1 51105 Involved in chromatin regulation TDRD20B

230

PIF1 80119 DNA helicase C15orf20, PIF

LPTL, LPTL, Involved in chromosome segregation PIN2/TERF1 and TRF1 and TERT accumulation in Interacting, PINX1 54984 nucleus Telomerase Inhibitor 1

PKMYT1 9088 Cell cycle PPP1R126, MyT1

PLK1 5347 Cell cycle PLK, STPK13, EC 2.7.11

Involved in tumour suppression, transcriptional regulation, apoptosis, senescence, DNA damage response, and PP8675, TRIM19, PML 5371 viral defence mechanisms. RNF71, MYL

PMSL1, HNPCC3, PMS1 5378 DNA repair HPMS1, MLH2

PMSL2, HNPCC4, PMS2 5395 DNA repair PMS2CL, MLH4

POLA1 5422 DNA polymerase POLA, NSX, P180

POLA2 23649 DNA replication

POLB 5423 DNA polymerase

POLD, CDC2, CRCS10, POLD1 5424 DNA polymerase MDPL

POLD2 5425 Subunit of DNA polymerase

p66, p68, KIAA0039, POLD3 10714 Subunit of DNA polymerase PPP1R128

POLD4 57804 Subunit of DNA polymerase P12, POLDS

POLE 5426 Subunit of DNA polymerase POLE1, FILS, CRCS12

POLE2 5427 Subunit of DNA polymerase DPE2

231

Role in allowing polymerase epsilon to carry out its replication and/or repair POLE3 54107 function- chromatin remodelling P17, YBL1, CHARAC17

Role in allowing polymerase epsilon to carry out its replication and/or repair POLE4 56655 function- chromatin remodelling P12, YHHQ1

POLG1, POLGA, MDP1, SANDO, MIRAS, SCAE, POLG 5428 Mitochondrial DNA polymerase PEO

POLH 5429 DNA polymerase RAD30A, XPV

POLI 11201 DNA polymerase RAD30B, RAD3OB

DINB1, POLQ, DINB1, POLK 51426 DNA polymerase DINP

POLL 27343 DNA polymerase BETAN, POLKAPPA

DNA polymerase, involved in repair of DNA double-strand breaks by non- POLM 27434 homologous end joining

POLN 353497 DNA polymerase POL4P

DNA polymerase that promotes POLQ 10721 microhomology-mediated end-joining

POLR2A 5430 RNA polymerase II

POL2RB, HRPB140, POLR2B 5431 RNA polymerase subunit RPB2

POLR2C 5432 RNA polymerase subunit RPB3, HRPB33

POLR2D 5433 RNA polymerase subunit HSRBP4, RPB16, RBP4

POLRF, RPABC2, RPB6, POLR2F 5435 RNA polymerase subunit RPC15

232

RPB19, RPB7, HRPB19, POLR2G 5436 RNA polymerase subunit HsRPB7

POLR2H 5437 RNA polymerase subunit RPB17, RPABC3

POLR2I 5438 RNA polymerase subunit RPB9, RPB14.5

RPB11, HRPB14, POLR2J 5439 RNA polymerase subunit POLR2J1

POLR2K 5440 Subunit of RNA polymerase II RPB12, RPABC4

POLR2L 5441 RNA polymerase subunit RPB10, RPABC5

Catalytic component of RNA RPC1, HLD7, RPC155, POLR3A 11128 polymerase III ADDH

POT1 25913 Shelterin complex GLM9, CMM10

PPARG 5468 Transcription factor NR1C3, GLM1, CIMT1

PPP1CA 5499 Involved in cell division PPP1A

PPP1CB 5500 Involved in cell division PP1B

PPP1CC 5501 Involved in cell division PP1C

Involved in cell cycle progression, DNA CAT53, PP1R10, PPP1R10 5514 repair and apoptosis PNUTS, FB19, P99

Involved in the negative control of cell PPP2R1A 5518 growth and division. MRD36, PR65A

Involved in the negative control of cell PPP2R1B 5519 growth and division. PR65B

PPP2R2A 5520 Cell division and growth

PFM13, MEL1, PRDM16 63976 KIAA1675, CMD1LL, Transcriptional regulator LVNC8

233

Histone methyltransferase that PRDM2 7799 specifically methylates Lys-9 of histone H3

Polymerase that synthesizes small RNA primers for the made during discontinuous DNA P49, DNA PRIM1 5557 replication. Subunit 48

Polymerase that synthesizes small RNA primers for the Okazaki fragments made during discontinuous DNA PRIM2 5558 replication. PRIM2A, P58

Involved in and PRKCA 5578 transcription regulation PKCA, AAG6

Involved in cell cycle checkpoint and PRKCB2, PKCB, PRKCB 5579 transcription regulation PRKCB1

DNA-dependent protein kinase involved HYRC, HYRC1, XRCC7, PRKDC 5591 in DNA repair DNAPK

HRMT1L2, IR1B4, PRMT1 3276 Arginine methyltransferase ANM1, HCP1

PRMT2 3275 Methylates H4 HRMT1L1

Methylates (mono and asymmetric dimethylation) the guanidino nitrogens PRMT3 10196 of arginyl residues in some proteins. HRMT1L3

PRMT5 10419 Chromatin regulator SKB1, HRMT1L5

PRMT6 55170 Methyltransferase activity HRMT1L6

Involved in pre-mRNA splicing and DNA PSO4, PRP19, PRPF19 27339 repair. NMP200, UBOX4

PSIP2, DFS70, LEDGF, PSIP1 11168 Transcriptional coactivator P75, P52

234

PTGES3 10728 Involved in transcriptional regulation CPGES, TEBP, P23

MRD31, PUR1, PUR- PURA 5813 Transcriptional activator ALPHA

Involved in signal transduction through PYGO1 26108 the Wnt pathway.

RAD1 5810 Involved in DNA damage response REC1, HRAD1

Involved in cell growth, maintenance of chromosomal stability, and ATR- dependent checkpoint activation upon RAD17 5884 DNA damage. RAD24, CCYC, R24L

Involved in ubiquitination and DNA RAD18 56852 damage repair RNF73

Involved in cell cycle, DNA repair and CDLS4, SCC1, MCD, RAD21 5885 apoptosis. NXP1 1

RAD23B 5887 DNA repair gene P58

Component of the MRN complex, which plays a central role in double-strand break (DSB) repair, DNA recombination, maintenance of telomere integrity and RAD50 10111 meiosis. NBSLD

RAD51A, RECA, BRCC5, Homologous recombination DNA FANCR, HsT16930, RAD51 5888 damage response MRMV2

RAD51AP1 10635 Involved in DNA damage response PIR51

Involved in DNA damage repair via RAD51B 5890 homologous recombination RAD51L1, REC2

Involved in DNA damage repair via RAD51C 5889 homologous recombination FANCO, BROVCA3

235

Involved in DNA damage repair via RAD51L3, BROVCA4, RAD51D 5892 homologous recombination TRAD, R51H3

Involved in double-stranded break RAD52 5893 repair.

Involved in DNA repair and mitotic RDH54 RAD54B 25788 recombination.

Involved in DNA repair and mitotic RAD54A, HR54 RAD54L 8438 recombination.

RAD9A 5883 Involved in DNA damage response RAD9

Regulates signalling pathways that RAP1A 5906 affect cell proliferation and adhesion RAP1

Involved in signalling cascade that RAPGEF1 2889 induces apoptosis GRF2, C3G

Involved in cell cycle and cell death RDA32,123F2, RASSF1 11186 pathways NORE2A, REH3P21

Involved in chromatin regulation, cell RB1 5925 cycle and is a tumour suppressor gene OSRC

Involved in histone acetylation and RBBP4 5928 chromatin assembly. Component NuRD RbAp48

Involved in regulating gene induction RBBP5 5929 and H3 Lys-4 methylation RBQ3, SWD

Core histone-binding subunit, NuRD RBBP7 5931 complex RbAp46

RBBP8 5932 DNA damage repair CtIP, SCKL2

Involved in cell division and RBL1 5933 heterochromatin formation

Involved in cell division and RBL2 5934 heterochromatin formation

236

May confer resistance to the antitumor RDM1 201299 agent cisplatin. Binds to DNA and RNA. RAD52B

RECQL 5965 DNA helicase RECQ1

RECQL4 9401 DNA-dependent ATPase

DNA helicase, role in DNA replication, RECQL5 9400 transcription and repair.

RELA 5970 Chromatin remodeller NFKB3

Deoxycytidyl transferase involved in REV1 51455 DNA repair. REV1L

Interacts with MAD2L2 to form the error prone DNA polymerase zeta REV3L 5980 involved in translesion DNA synthesis. POLZ

Component of a complex that possesses RFC1 5981 DNA-dependent ATPase activity

Component of a complex that possesses RFC2 5982 DNA-dependent ATPase activity

Component of a complex that possesses RFC3 5983 DNA-dependent ATPase activity

Component of a complex that possesses RFC4 5984 DNA-dependent ATPase activity

Component of a complex that possesses RFC5 5985 DNA-dependent ATPase activity

RIF1 55183 Conserved DNA damage response gene

RARRES3 5920 Tumour suppressor or growth regulator. RIG1, TIG3

RING1 6015 Involved in chromatin regulation

Involved in the dissolution of double RMI1 80010 Holliday junctions

237

RMI2 116028 Involved in homologous recombination C16orf75

Involved in ubiquitination and DNA RNF168 165918 damage repair

RNF2 6045 Chromatin modifier

RNF20 56254 Chromatin modifier

Chromatin modifier, regulates RNF4 6047 degradation of PML

RNF40 9810 Chromatin modifier

RNF8 9025 Involved in DNA damage response

Involved in DNA replication and damage RPA1 6117 response

Involved in DNA replication and damage RPA2 6118 response

Involved in DNA replication and damage RPA3 6119 response

Binds ssDNA and probably plays a role RPA4 29935 in DNA repair.

RPAIN 84268 Participates in DNA metabolism RIP, HRIP

RRP8 23378 Chromatin regulator KIAA0409, NML

TP-dependent DNA helicase implicated in telomere-length regulation, DNA C20orf41, NHL, repair and the maintenance of genomic DKCA4, DKCB5, RTEL1 51750 stability. PFBMFT3

DNA-dependent ATPase and DNA helicase activities, involved in chromatin TIP49, PONTIN, ECP54, RUVBL1 8607 modification TIH1

238

DNA-dependent ATPase and DNA helicase activities, involved in chromatin TIP48, REPTIN, ECP51, RUVBL2 10856 modification TIH2

HOMS1, Snu66, SART1 9092 Role in mRNA splicing SNRNP110, Ara1

SATB1 6304 Chromatin remodeller

Histone methyltransferase that specifically monomethylates Lys-4 of SETD7 80854 histone H3 SET9, SET7, KMT7

Regulates histone methylation, gene silencing, and transcriptional repression-trimethylates Lys-9 of SETDB1 9869 histone H3

SETMAR 6419 DNA repair METNASE, Mar1

Required for double-strand break repair SFR1 119392 via homologous recombination C10orf78, MEIR5

Role in chromosome cohesion during SGOL1, CAID, NY-BR- SGO1 151648 mitosis 85, SGO

SHFD1, DSS1, SHSF1, SHFM1 7979 Involved in DNA damage response SEM1, ECD

E3 ubiquitin-protein ligase involved in SHPRH 257218 DNA repair.

SIN3A 25942 Acts as a transcriptional repressor.

Acts as a transcriptional repressor, SIN3B 23309 NuRD associated

Transcriptional regulator involved in cell cycle, response to DNA damage, SIRT1 23411 metabolism, apoptosis and autophagy. Sirtuin 1

239

SIRT2 22933 NAD-dependent protein deacetylase SIR2L

SIRT3 23410 NAD-dependent protein deacetylase

NAD-dependent lysine demalonylase, SIRT5 23408 desuccinylase and deglutarylase

SIRT6 51548 NAD-dependent protein deacetylase.

AD-dependent protein deacetylase that specifically mediates deacetylation of SIRT7 51547 histone H3 at Lys-18

SLX1A 548593 Involved in DNA damage repair GIYD1

SLX1B 79008 Involved in DNA damage repair GIYD2

Structure-specific endonuclease MUS312, FANCP, SLX4 84464 subunit. BTBD12

SMAD3 4088 Transcription factor

SNF2L1, SNF2L, SNF2- SMARCA1 6594 Involved in chromatin remodelling. Like 1

SWI/SNF complex, transcription SNF2L2, SNF2-Alpha, SMARCA2 6595 activator BAF190B

SWI/SNF complex, transcription SMARCA4 6597 activator

HEL1, ETL1, ADERM, SMARCAD1 56916 SNF protein, required for DNA repair ATP-Dependent and heterochromatin organization. Helicase 1

SWI/SNF complex, chromatin SNF5L1, INI1, SNF5, SMARCB1 6598 remodeller HSNF5, BAF47

CRACC1, BAF155, SMARCC1 6599 Involved in chromatin remodelling. SWI3

SMARCC2 6601 Involved in chromatin remodelling. CRACC2, BAF170

240

SMARCD1 6602 Involved in chromatin remodelling. CRACD1, BAF60A

SMARCD2 6603 Involved in chromatin remodelling. CRACD2, BAF60B

SMARCD3 6604 Involved in chromatin remodelling. CRACD3, BAF60C

Involved in chromosome cohesion SMC1L1, CDLS2, SMC1A 8243 during cell cycle and in DNA repair. DXS423E

Cell cycle, DNA replication and DNA SMC2 10592 repair SMC2L1, CAP-E

Cell cycle, DNA replication and DNA SMC3 9126 repair CSPG6

Cell cycle, DNA replication and DNA SMC4 10051 repair SMC4L1, CAP-C

Involved in DNA double-strand breaks SMC5 23137 by homologous recombination. SMC5L1

Involved in DNA double-strand breaks SMC6 79677 by homologous recombination. SMC6L1

RNA degradation and DNA damage SMG1 23049 response

Involved in nonsense-mediated mRNA SMG5 23381 decay, telomerase associated protein Est1p-Like Protein B

Involved in nonsense-mediated mRNA C17orf31, EST1-Like SMG6 23293 decay Protein A

Plays a role in nonsense-mediated C1orf16, EST1-Like SMG7 9887 mRNA decay. Protein C

SMUG1 23583 DNA repair gene FDG

Methylates both histones and non- histone proteins, including p53/TP53 KMT3C, ZMYND14, SMYD2 56950 and RB1. HSKM-B

241

Chromatin regulator, DNA damage SP1 6667 response

SP100 6672 Component of PML body

SP110 3431 Transcription factor IFI41, IFI75, VODI

SPATA43, TOPVIA, Required for meiotic recombination SPO11 23626 CT35

c1orf124, PRO4323, DNA repair SPRTN 83932 DVC1

SRCAP 10847 Chromatin regulator

SSB 6741 Involved in RNA metabolism LARP3

involved in mitochondrial DNA SSBP1 6742 replication, binds ssDNA SSBP, SOSS-B1, MtSSB

FACT, T160, SSRP1, SSRP1 6749 Chromatin remodeller T160

SSU72 29101 Role in RNA processing and termination. HSPC182, PNAS-120

STAG1 10274 DNA replication SCC3A, SA1

Chromatin Modifier/epigenetic STAG2 10735 remodeller SCC3B, SA2

Involved in DNA damage repair and STRA13 201254 genome maintenance. CENPX, MHF2, MHF2

Involved in telomere attachment to SUN1 23353 and gametogenesis. UNC84A, KIAA0810

CDC68, SPT16, SPT16, SUPT16H 11198 Chromatin remodeller FACTP140

Involved in mRNA processing and SUPT5H 6829 transcription elongation SPT5H

242

SUV39H, HTDG, KMT1A, MG44, Histone H3-K9 SUV39H1 6839 Chromatin remodeller Methyltransferase 1

KMT1B, Histone H3-K9 SUV39H2 79723 Chromatin remodeller Methyltransferase 2

SUZ12 23512 Chromatin remodeller CHET9, JJAZ1

SWI5 375757 Involved in homologous recombination C9orf119, SAE3

Involved in histone mRNA 3-end SYMPK 8189 processing. SYM, SPK

DYT3, CCGS, CCG1, TAF1 6872 Transcription and phosphorylation BA2R, TAF2A

TBK1 29110 Ubiquitination NAK, T2K

TBP 6908 Transcription TF2D, GTF2D, SCA17

Assembly of RNA Polymerase-II TCEA1 6917 Initiation Complex TFIIS, GTF2S

TCEB3 6924 Transcription TCEB3A

TDG 6996 Chromatin Structure and Modification

TDP1 55775 DNA repair gene

TDRD1 56165 piRNA metabolic process

Nucleic acid binding and nucleotide TDRD10 126668 binding.

TDRD12 91646 ATP-dependent RNA helicase activity. ECAT8

poly(A) RNA binding and chromatin TDRD3 81550 binding.

TDRD5 163589 Involved in DNA integrity TUDOR3

243

Involved in proper precursor and SPATA36, TDR2, TDRD6 221400 mature miRNA expression BA446F17.4

TDRD7 23424 Post-transcriptional regulation CATC4, TRAP

Nucleic acid binding and helicase TDRD9 122402 activity. C14orf75, NET54

TDRKH 11022 Nucleic acid binding and RNA binding. TDRD2

TELO2 9894 Regulator of the DNA damage response TEL2, CLK2, HCLK2

TEP1 7011 Telomerase TP1, TLP1, P240

TERC 7012 Telomerase TRC3, DKCA1, TR

TERF1 7013 Shelterin complex TRF1, TRBF1, PIN2

TERF2 7014 Shelterin complex TRF2, TRBF2

Shelterin complex and as a transcription RAP1, TERF2 TERF2IP 54386 regulator. Interacting Protein

TCS1, EST2, TP2, TERT 7015 Telomerase DKCA2, TRT

Chromatin Modifier/epigenetic TET1 80312 remodeller, DNA demethylation CXXC6

Chromatin Modifier/epigenetic TET2 54790 remodeller, DNA demethylation KIAA1546

Mitochondrial transcription regulation, 7019 TFAM replication and repair. TCF6, TCF6L2

Controls proliferation, differentiation, TGFB1 7040 adhesion, migration and other functions TGFB, DPD1

RNA binding protein involved in translational control, splicing and TIAL1 7073 apoptosis. TIAR, TCBP

244

Protein kinase binding and signal TICAM1 148022 transducer activity. TRIF

TIN2, DKCA3, TERF1 (TRF1)-Interacting TINF2 26277 Shelterin complex Nuclear Factor 2

Sensor of cytosolic DNA from bacteria STING, ERIS, HMITA, TMEM173 340061 and viruses MITA, SAVI, NET23

TMEM201 199953 Mitosis SAMP1, NET5

Nuclear organisation and DNA TMPO 7112 replication LAP2

Wnt signalling pathway, telomerase and TNKS 8658 vesicle trafficking. TIN1, TANK1, PARP5A

Wnt signalling pathway, telomerase and TNKS-2, TANK2, TNKS2 80351 vesicle trafficking. PARP5B

TOP1 7150 Involved in DNA transcription TOPI

Involved in DNA replication and TOP2A 7153 transcription TOP2

Involved in DNA replication and TOP2B 7155 transcription Topo II Beta

TOP3A 7156 Involved in DNA replication Topo III-Alpha

Involved in DNA replication and TOP3B 8940 transcription TOP3B1

TOPBP1 11073 Conserved DNA damage response gene TOP2BP1

Conserved DNA damage response gene, TP53 7157 induces growth arrest or apoptosis

TP53BP1 7158 Conserved DNA damage response gene 53BP1

245

TP53 INDUCED TP53I13 90313 Tumour suppressor PROTEIN 13

TP53 INDUCED TP53I3 9540 Member of TP53 signalling pathway PROTEIN

TREX1 11277 Editing and processing nucleases DNase III

TREX2 11219 Editing and processing nucleases

Chromatin Modifier/epigenetic TRIM28 10155 remodeller RNF96, KAP1, TIF1B

TRIM32 22954 E3 activity. HT2A, LGMD2H

TRIM33 51592 E3 ubiquitin-protein ligase. TIF1G, RFG7

poly(A) RNA binding and ubiquitin- TRIM56 81844 protein transferase activity. RNF109

Chromatin Modifier/epigenetic TRIP13 9319 remodeller

Chromatin Modifier/epigenetic TRRAP 8295 remodeller TRA1, STAF40, PAF400

SMG10, KIAA0406, TELO2 Interacting TTI1 9675 Regulator of the DNA damage response. Protein 1

C8orf41, MRT39, TELO2 Interacting TTI2 80185 Regulator of the DNA damage response. Protein 2

TUBA1B 10376 Chromatin, cell cycle, apoptosis Tubulin

Chromatin Modifier/epigenetic TYMP 1890 remodeller

Ubiquitin gene, Ubiquitin A-52 Residue UBA52 7311 Ribosomal Protein Fusion Product 1 RPL40

246

UBB 7314 Ubiquitin gene, ubiquitin B HEL-S-50

UBC 7316 Ubiquitin gene, ubiquitin C HMG20

UBE2A 7319 Ubiquitination and modification RAD6A

UBE2B 7320 Ubiquitination and modification RAD6B

UBE2N 7334 Ubiquitination and modification UBC13

DNA damage repair and Fanconi UBE2T 29089 anaemia FANCT

UBE2V2 7336 Ubiquitination and modification MMS2

UBR5 51366 Transcriptional regulation

Chromatin Modifier/epigenetic UHRF1 29128 remodeller

RAP80, X2HRIP110, UIMC1 51720 DNA repair RXRIP110

RNA-dependent helicase, involved in decay of mRNA containing premature UPF1 5976 stop codons RENT1, NORF1

Negative regulator of DNA damage USP1 7398 repair HUBP

Related to cell cycle and DNA damage VCP 7415 repair

Regulator of deubiquitinating WDR48 57599 complexes. UAF1, P80

Chromatin Modifier/epigenetic WDR77 79084 remodeller MEP50, P44

WEE1 7465 Cell cycle

WRAP53 55135 Telomerase and Cajal bodies TCAB1, DKCB3

WRN 7486 DNA helicase RECQL2, RECQ3

247

WRNIP1 56897 DNA synthesis

XAB2 56949 DNA repair gene HCNP, SYF1

XPA 7507 DNA repair gene XP1, XPAC

XPCC, P125, RAD4, XPC 7508 DNA repair gene XP3

XRCC1 7515 DNA repair gene RCC

XRCC2 7516 Homologous recombination RAD51-Like

XRCC3 7517 Homologous recombination RAD51-Like, CMM6

XRCC4 7518 DNA repair gene

KU80, KUB2, Ku86, XRCC5 7520 DNA repair gene NFIV, KARP1

Single-stranded DNA-dependent ATP- dependent helicase, involved in DNA XRCC6 2547 non-homologous end joining. G22P1, KU70

XRCC6P2 389901 DNA repair gene

Transcription regulation and DNA ZBTB32 27033 binding (involved in Fanconi anaemia) ZNF538

ZBTB44 29068 Transcription regulation ZNF851

Nucleic acid binding and transcription ZBTB48 3104 factor activity HKR3, ZNF855, TZAP

ZCCHC7 84186 RNA degradation

ZMYM2 7750 Transcription factor ZNF198

Involved in transcriptional regulation, DNA replication and probably DNA INO80B ZNHIT4 83444 repair.

ZSWIM7 125150 Involved in homologous recombination SWS1

248

Table A2.6. Genetic events identified by whole genome sequencing.

Refer- Alter- Allelic Sample Gene Variation Gene ence native Fracti- Protein ID Symbol Chromosome Type Region Start site End site Allele Allele on Variant chr1: chr1: LA-N-6 AIM2 1 SNV:DEL Exon 159032486 159032486 65.38 chrx: chrx: CHLA-90 ATRX X SV:DEL 76935119 76959029 chrx: chrx: PARKNP ATRX X SV:DEL 76924445 77028694 chrx: chrx: PAISZV ATRX X SV:DEL 76937155 76983145 chr2: chr2: PAPTAN BARD1 2 SNV:DEL Exon 215645502 215645522 42.86 chr2: chr2: PARXFT BARD1 2 SNV:DEL Exon 215645502 215645522 45.45 chr17: chr2: PARGIW BRIP1 17 SV:TRA Intron 59796638 14401098 chr19: chr19: SK-N-BE2c CARM1 19 SNV Exon 11031414 11031414 C A 46.67 P472T chr2: chr12: PAIFXV CCNT1 12 SV:DEL 25957192 104379378 chr22: chr22: SH-SY5Y CHEK2 22 SNV:DEL Exon 29091856 29091856 75 chr16: chr16: CHLA-90 CREBBP 16 SV:DUP Intron 3885945 3919341 chr7: COG-N-291 DDX60 7 SV:TRA Intron 116809191 chr1: chr1: COG-N-291 1 SV:DEL 3801505 30723742 chr16: chr16: LA-N-6 EME2 16 SNV:DEL Exon 1823389 1823389 43.48 Intron/bas e before chr3: chr3: PAPTAN FOXP1 3 SNV exon 71408396 71408396 C T 46.67 chr2: chr2: COG-N-291 GEN1 2 SV:DEL 1485200 27254414 chr6: chr6: PARGIW HIST1H1B 6 SNV:DEL Exon 27834678 27834692 38.46 chr1: chr1: PAISZV HIST2H2BC 1 SV:DEL 110246334 181216287 chr3: chr3: COG-N-291 HLTF 3 SNV Exon 148789089 148789089 C A 61.54 D282Y chr2: chr2: PARXFT IDH1 2 SNV:DEL Promoter 209120302 209120302 35 Promoter; chr18: chr18: PARGIW INO80C 18 SNV:DEL 5'UTR 33077852 33077859 55.56 chr17: chr17: PARXFT KDM6B 17 SNV:DEL Exon 7751858 7751863 54.55 chr12: chr12: SH-SY5Y KRAS 12 SNV Exon 25398284 25398284 C A 45.45 G12V chr8: chr8: COG-N-291 MCM4 8 SNV Exon 48875559 48875559 T G 45.83 F218V

249

chr2: chr2: CHW_11 MCM6 2 SNV Exon 136624255 136624255 A T 52 I220N chr11: chr11: SH-SY5Y MRE11A 11 SNV Exon 94226952 94226952 C G 56.25 chr2: chr2: PARKNP MSH2 2 SNV:DEL Exon 47693814 47693815 69.23 chr2: chr2: SK-N-FI NCL 2 SNV:DEL Exon 232325414 232325416 57.89 chr2: chr2: COG-N-291 NCL 2 SNV:DEL Exon 232325414 232325414 48.15 chr2: chr2: PAIFXV NCL 2 SNV:DEL Exon 232325414 232325414 100 chr2: chr2: PAIFXV NCL 2 SNV:DEL Exon 232326655 232326655 39.47 chr20: chr20: PAIFXV NCOA3 20 SNV:DEL Exon 46279814 46279822 44.44 chr20: chr20: PARGIW NCOA3 20 SNV:DEL Exon 46279863 46279865 100 chr17: chr17: PAISZV NCOR1 17 SV:DEL 16095144 16095626 chr12: chr12: PAPTAN NCOR2 12 SNV:DEL Exon 124887058 124887069 63.64 chr8: chr8: PAPFSL NSMCE2 8 SNV:DEL Exon 126114595 126114595 66.67 chr7: chr7: LA-N-6 POLM 7 SV:DEL 31199193 64005420 chr3: chr3: PAIFXV POLQ 3 SNV:DEL Exon 121208703 121208705 50 chr17: chr17: PAIFXV POLR2A 17 SNV Exon 7405325 7405325 G A 22.22 S819N chr8: chr8: SK-N-FI PPP2R2A 8 SNV:DEL Intron 26150637 26150637 60 chr1: chr1: SK-N-FI PRDM2 1 SNV:DEL Intron 14074949 14074950 57.14 chr21: chr21: SK-N-BE2c PRMT2 21 SNV Intron 48056789 48056789 C A 40 chr14: chr14: SK-N-BE2c PRMT5 14 SNV Intron 23393931 23393931 G T 14.81 chr5: chr5: PARKNP RAD17 5 SNV:DEL Intron 68667048 68667050 46.67 chr9: chr9: PAPFSL RAD23B 9 SNV:DEL Intron 110046745 110046747 60 chr9: chr9: SH-SY5Y RAD23B 9 SNV:DEL Intron 110046745 110046747 100 chr14: chr14: SH-SY5Y RAD51B 14 SV:INV Intron 68907987 69297996 chr13: chr13: PARGIW RB1 13 SNV:DEL Exon 48878084 48878092 40 chr18: chr18: PARGIW RBBP8 18 SNV:DEL Intron 20513542 20513549 64.71 chr20: PARGIW RBL1 20 SV:TRA Intron 35627929 chr8: chr8: PAPFSL RECQL4 8 SNV Exon 145738660 145738660 C T 30.77 V802M SH-SY5Y RECQL5 17 SNV Exon chr17: chr17: G A 80 R175C

250

73658807 73658807 chr2: chr8: PARGIW RIF1 2 SNV:DEL Exon 152320880 152320882 23.08 chr1: chr1: PAISZV SETDB1 1 SNV Exon 150934631 150934631 C A 29.03 S1052* chr15: chr15: PAPTAN SMAD3 15 SV:DEL Intron 67404674 67405604 chr9: chr9: PARKNP SMARCA2 9 SV:DEL Intron 2147643 2151529 chr19: chr19: SH-SY5Y SMARCA4 19 SNV Exon 11134251 11134251 C T 44.44 R973W R1587*; R1590*; R1653*; R1621*; chr19: chr19: R1588*; SK-N-FI SMARCA4 19 SNV Exon 11170813 11170813 C T 100 R1591* Splice Site; chr10: chr10: SH-SY5Y SMC3 10 SNV:DEL Intron 112360304 112360305 60 chr16: chr16: SK-N-BE2c SMG1 16 SNV Exon 18827823 18827823 C A 36.36 S3368I L431P; chr12: chr12: L438P; LA-N-6 SP1 12 SNV Exon 53777044 53777044 T C 46.15 L390P chr19: SK-N-BE2c SRCAP 19 SV:TRA Intron 2599286 chr3: chr3: CHLA-90 STAG1 3 SV:DEL 135906165 136313178 chr1: COG-N-291 TCEB3 1 SV:DEL 30723742 chr19: chr19: PARKNP TDRD12 19 SNV Exon 33222697 33222697 G A 45.83 D31N chr5: chr5: SH-SY5Y TERT 5 SNV Promoter 1295228 1295228 G A 60 chr1: chr1: PAPTAN TMEM201 1 SNV Exon 9671877 9671877 C G 80 S611C E127K; E247K; chr17: chr17: E154K; CHLA-90 TP53 17 SNV Exon 7577082 7577082 C T 100 E286K M246R; M114R; chr17: chr17: M207R; SK-N-FI TP53 17 SNV Exon 7577544 7577544 A C 100 M87R C96F; chr17: chr17: C3F; SK-N-BE2c TP53 17 SNV Exon 7578526 7578526 C A 100 C135F chr17: chr17: CHLA-90 TP53 17 SNV:DEL Intron 7588679 7588679 100 chr7: chr7: G3004W; SH-SY5Y TRRAP 7 SNV Exon 98581778 98581778 G T 28.57 G3033W chr8: chr8: SH-SY5Y TTI2 8 SNV Exon 33364825 33364825 C A 52.63 L283F chr5: PAPTAN UIMC1 5 SV:TRA Intron 176387600

251

chr9: chr9: SK-N-BE2c VCP 9 SNV Exon 35064279 35064279 C A 37.5 E194* chr6: chr6: PAPFSL WRNIP1 6 SNV:DEL Exon 2766363 2766374 37.5 chr13: chr13: CHW_11 ZMYM2 13 SNV Exon 20580560 20580560 G T 32 S449I SNV, single nucleotide variation; SNV:DEL, deletion of 1-20 bp; SV:DEL, structural variation: deletion; SV:TRA, structural variation: translocation; SV:INV, structural variation: inversion.

252