Precision oncology : oncogenic stat3 signalling in brain tumours

1 1.0 INTRODUCTION 2 1.1. Classification of Glioblastomas 2 1.1.1. Histological Classification of Glioblastomas 3 1.1.2. Molecular Classification of Glioblastomas 5 1.2. GBM Cell Biology 7 1.3. Glioblastoma-Propagating Cells (GPCs) 8 1.3.1. Functional Validation of GPCs 10 1.4. Mouse Models of Gliomas 11 1.4.1. Genetically Engineered Mouse Models (GEMMs) 12 1.4.2. Xenograft Mouse Models 12 1.5. Signalling Pathways Regulating GBM Biology 13 1.5. 1. STAT3 Signalling Pathway 14 1.5. 1.1. STAT3 Signalling in Glioblastoma 16 1.5. 1.2. STAT3 in Tumourigenesis 17 1.5. 1.3. STAT3 Inhibitors 18 1.5. 2. IGF-1R Signalling Pathway 20 1.5. 2.1. IGF-1R Inhibitors 21 1.6. Mechanisms of Chemoresistance in Glioblastoma 22 1.6. 1. STAT3 in Treatment Resistance 23 1.7. Gap in Knowledge and Objectives of Study 24 2.0 MATERIALS AND METHODS 26 2.1. Tissue Collection and Primary Gliomasphere Culture 26 2.2. Small Molecule Inhibitors and Reagents 26 2.3. Cell Viability Assays 27 2.3.1. Cell Viability Assessment Post-treatment with Small Molecule Inhibitors 27 Oncogenic STAT3 Signalling in Brain Tumours Melanie Tan (G1303305G) iii 2.3.2. Dose-response Curves and IC50 Calculations 27 2.3.3. Gliomasphere Formation Assay 27 2.3.4. Invasion Assay 28 2.4. Combination Index (CI Values) 28 2.5. Protein Analysis 28 2.5. 1. Immunoblot 28 2.5. 2. Co-immunoprecipitation 29 2.5. 3. Enzyme-Linked Immunosorbent Assay (ELISA) 29 2.5. 4. Immunohistochemistry 30 2.6. Quantitative Real Time RT-PCR 30 2.7. Lentiviral-mediated Knockdown and Over-expression 31 2.8. Stereotaxic Intracranial Implantations 31 2.9. Statistical Analysis 32 2.9. 1. Kaplan-Meier Analysis 32 2.10. Bioinformatics Analyses 33 (With help from Mr Edwin Sandanaraj) 2.10. 1. Microarray Data Processing and Analysis 33 2.10. 2. Predictive Database Analysis 33 2.10. 3. Bayesian Information Criterion Analysis 34 2.10. 4. Relative Odds Estimation 34 2.10. 5. Functional Gene Module Analysis 35 2.10. 6. PamChip Kinome Analysis 35 2.10. 7. SynergySeq 36 3.0 RESULTS 38 3.1. STAT3 Functionally-tuned Gene Signature 38 3.2. Pharmacological Inhibition of STAT3 Mitigates Glioma Cell Growth 44 3.3. Mechanism Contributing to STAT3-resistant Profile 49 3.4. IGF-1R Signalling Pathway Contributes to Chemoresistance Mechanism in STAT3-low GPCs 51 3.5. Depletion of IGFBP2 and C-terminal IGF-1R Restores Sensitivity 55 3.6. Utility of STAT3 Functionally-tuned Gene Signature 62 3.7. Chemosensitisation of STAT3-stratified Cells Synergises Oncogenic STAT3 Signalling in Brain Tumours Melanie Tan (G1303305G) iv with Standard of Care Temozolomide 63 4.0 DISCUSSION 68 4.1. STAT3 Functionally-tuned Gene Signature as a Prognostic Indicator 68 4.2. Understanding Mechanisms underlying STAT3-stratified Patients 69 4.3. Tumour Recurrence 70 4.4. Clinical Significance 72 5.0 FUTURE DIRECTIONS 74 6.0 CONCLUSION 76 7.0 REFERENCES 77 8.0 APPENDICES 98 A. List of Genes Comprising the STAT3 Functionally-tuned Gene Signature 98 B. Contingency Table, Univariate and Multivariate Analyses 103 I. Contingency analysis 103 II. Univariate and multivariate analyses 104 C. IC50 Curves of STAT3-stratified GPCs Treated with Various STAT3 Inhibitors 105 D. Winnowed List of Genes Contributing to Resistance in STAT3-low Cohorts 106 E. List of Protein Tyrosine Kinase 108 F. H-score and IC50 Values of Patient Tumours 110 I. H-score 110 II. IC50 curves of STAT3-stratified GPCs treated with AZD1480 111 9.0 PUBLICATIONS 112 10.0 POSTERS 113 Oncogenic STAT3 Signalling in Brain Tumours Melanie Tan (G1303305G)