Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
DSpace
UBCV
Wong, Derek
Doctor of Philosophy - PhD
University of British Columbia
Mutations in the Capicua (CIC) gene, located on chromosome 19q, were first identified in up to 70% of 1p19q-codeleted, IDH-mutated Oligodendrogliomas (ODGs), a subtype of diffuse low grade glioma (DLGG). Since then, several studies have highlighted that loss or dysregulation of CIC is associated with tumour progression, worse prognosis, and treatment resistance in multiple cancer types. One CIC binding partner, Ataxin-1-Like (ATXN1L), has been implicated as an important regulator of CIC function in murine development, but little is known in the context of cancer. In this thesis, we characterize the functional interaction between CIC and ATXN1L and their relationship in regulating pathways involved in tumourigenesis. The function of ATXN1L in relation to CIC was investigated by assessing and comparing the transcriptomic consequences of ATXN1L knockout (KO) and CIC KO using isogenic cell lines. Loss of either CIC or ATXN1L led to concordant dysregulation of gene sets in each context which converged upon several pathways involved in differentiation and activation of the mitogen activated protein kinase (MAPK) pathway as a result of decreased CIC-DNA binding. Further analysis of cancers harboring deletions in CIC and ATXN1L, namely, stomach adenocarcinoma, prostate adenocarcinoma, and astrocytoma, resulted in convergent dysregulation of several pathways involved in cell proliferation and growth. In addition to decreased CIC-DNA binding, loss of ATXN1L resulted in increased CIC protein instability as a result of proteasomal degradation which was found to be independent of ERK activity, the canonical CIC degradation pathway. Instead, loss of ATXN1L was found to promote CIC instability through ubiquitination by the E3-ligase TRIM25, a novel CIC interactor. Transcriptomic analyses of breast carcinomas with TRIM25 amplification and liver hepatocellular carcinomas with high TRIM25 expression revealed dysregulation of genes and pathways reminiscent of CIC loss, supporting its role in the post-translational regulation of CIC. The results of these studies illuminate our understanding and the intricacy of mechanisms which regulate normal CIC function in cancer; and further supports the role of CIC as a potent tumour suppressor in a multitude of cancer types.
Functional Investigations of Capicua (CIC) and ATAXIN-1-Like (ATXN1L) in cancer. by Derek Wong A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2020 ©Derek Wong, 2020ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Functional Investigations of Capicua (CIC) and ATAXIN-1-Like (ATXN1L) in cancer. submitted by Derek Wong in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pathology and Laboratory Medicine Examining Committee: Dr. Stephen Yip MD, PhD, FRCPC, Pathology and Laboratory Medicine Supervisor Dr. David Huntsman, MD, FRCPC, FCCMG, Pathology and Laboratory Medicine Co-supervisor Dr. Poul Sorensen, MD, PhD,FRCPC, Pathology and Laboratory Medicine University Examiner Dr. Amina Zoubeidi, PhD, Urologic Sciences University Examiner Additional Supervisory Committee Members: Dr. Marcel Bally, PhD, Experimental Therapeutics Supervisory Committee Member Dr. Marco Marra, PhD, FRS(C), FCAHS, OBC, Medical Genetics Supervisory Committee Member iii Abstract Mutations in the Capicua (CIC) gene, located on chromosome 19q, were first identified in up to 70% of 1p19q-codeleted, IDH-mutated Oligodendrogliomas (ODGs), a subtype of diffuse low grade glioma (DLGG). Since then, several studies have highlighted that loss or dysregulation of CIC is associated with tumour progression, worse prognosis, and treatment resistance in multiple cancer types. One CIC binding partner, Ataxin-1-Like (ATXN1L), has been implicated as an important regulator of CIC function in murine development, but little is known in the context of cancer. In this thesis, we characterize the functional interaction between CIC and ATXN1L and their relationship in regulating pathways involved in tumourigenesis. The function of ATXN1L in relation to CIC was investigated by assessing and comparing the transcriptomic consequences of ATXN1L knockout (KO) and CIC KO using isogenic cell lines. Loss of either CIC or ATXN1L led to concordant dysregulation of gene sets in each context which converged upon several pathways involved in differentiation and activation of the mitogen activated protein kinase (MAPK) pathway as a result of decreased CIC-DNA binding. Further analysis of cancers harboring deletions in CIC and ATXN1L, namely, stomach adenocarcinoma, prostate adenocarcinoma, and astrocytoma, resulted in convergent dysregulation of several pathways involved in cell proliferation and growth. In addition to decreased CIC-DNA binding, loss of ATXN1L resulted in increased CIC protein instability as a result of proteasomal degradation which was found to be independent of ERK activity, the canonical CIC degradation pathway. Instead, loss of ATXN1L was found to promote CIC instability through ubiquitination by the E3-ligase TRIM25, a novel CIC interactor. Transcriptomic analyses of breast carcinomas with TRIM25 amplification and liver hepatocellular carcinomas with high TRIM25 expression revealed dysregulation of genes and pathways reminiscent of CIC loss, supporting its role in the post-translational regulation of CIC. The results of these studies illuminate our understanding and the intricacy of mechanisms which regulate normal CIC function in cancer; and further supports the role of CIC as a potent tumour suppressor in a multitude of cancer types. iv Lay Summary Brain tumours are generally associated with poor outcomes due to the difficulty in surgical resection and their close proximity to important cognitive structures. Research in the past decade has uncovered several mutations associated with specific subtypes of brain tumours that can be used to predict their clinical outcomes. However, much research is still needed to understand the biology of each mutation. Oligodendrogliomas are a type of brain tumour associated with mutations in Capicua (CIC), a gene involved in development. In this thesis, we explore how normal CIC function is regulated by ATXN1L and ways in which cancers can modulate their function in order to promote aggressive behaviors. By understanding the regulators and function of CIC, future research can investigate methods to modulate CIC function to better treat a plethora of tumours which rely on diminished CIC function for metastasis, progression, and treatment resistance. v Preface This dissertation is original and independent work by the author Derek Wong. The research in this thesis was conducted with ethics approval from the UBC – BC Cancer Research Ethics Board, Certificate Number: H08-02838. Portions of Chapter 1 have been published as: • Derek Wong, Stephen Yip (2019) Chapter 6 - Pathology of Primary Brain Tumors – Gliomas. In Kaisorn Chaichana and Alfredo Quiñones-Hinojosa, Comprehensive Overview of Modern Surgical Approaches to Intrinsic Brain Tumors. Pages 121-137 • Derek Wong, Stephen Yip (2020) Making heads or tails – The emergence of Capicua in cancer. Journal of Pathology. 250(5):532-540. I am the first author of these two manuscripts. I wrote and performed the majority of literature research for these manuscripts. Portions of Chapter 3 and 4 have been published as: • Derek Wong, Kohl Lounsbury, Amy Lum, Jungeun Song, Susanna Chan, Veronique LeBlanc, Suganthi Chittaranjan, Marco Marra, Stephen Yip (2018) Transcriptomic analysis of CIC and ATXN1L reveal a functional relationship exploited by cancer. Oncogene. 38(2):273-290. I am the first author of this manuscript. I wrote, designed and performed the majority of experiments and analyses for this manuscript. Portions of Chapter 4 have been submitted as: • Derek Wong, Samantha S Lee, Lisa Sogerer, Victor Wong, Amy Lum, Adrian B. Levine, Marco A Marra, Stephen Yip (2020) Loss of ATXN1L promotes ERK independent proteasomal degradation of Capicua through TRIM25 in cancer. BMC Biology. 18(154). I am the first author of this manuscript. I wrote, designed and performed the majority of experiments and analyses for this manuscript. vi Table of Contents Abstract .......................................................................................................................................... iii Lay Summary ................................................................................................................................. iv Preface ............................................................................................................................................. v Table of Contents ........................................................................................................................... vi List of Tables................................................................................................................................. xii List of Figures .............................................................................................................................. xiii List of Abbreviations.................................................................................................................... xiv Acknowledgements ...................................................................................................................... xvi Chapter 1: Introductions .................................................................................................................. 1 1.1 Low Grade Gliomas ......................................................................................................... 1 1.1.1 Etiology and Epidemiology....................................................................................... 1 1.1.2 Molecular Alterations and Histopathology ............................................................... 1 1.2 IDH Mutations .................................................................................................................. 4 1.3 Capicua (CIC) ................................................................................................................... 4 1.3.1 CIC Aberrations in Cancer ........................................................................................... 4 1.3.2 CIC in Mammalian Development and Oncogenesis..................................................... 6 1.4 ATXN1L in Development ................................................................................................ 8 1.5 Thesis ................................................................................................................................ 9 1.6 Objectives and Hypothesis ............................................................................................... 9 1.7 Specific Aims and Thesis Outline .................................................................................... 9 Chapter 2: Materials and Methods ................................................................................................ 11 2.1 Immunoprecipitation ...................................................................................................... 11 2.2 Cell Lysate Preparation .................................................................................................. 11 vii 2.3 Western Blot ................................................................................................................... 11 2.4 Real-Time quantitative PCR (RT-qPCR) ....................................................................... 12 2.5 Cell Culture Conditions .................................................................................................. 12 2.6 ATXN1L CRISPR Knockout ......................................................................................... 13 2.7 siRNA Transfection ........................................................................................................ 14 2.8 Microarray Expression Profiling .................................................................................... 14 2.9 TCGA Expression Analyses ........................................................................................... 15 2.10 Gene Set Enrichment Analysis ....................................................................................... 16 2.11 Targeted Chromatin Immunoprecipitation-PCR ............................................................ 16 2.12 Proximity Ligation Assay ............................................................................................... 17 2.13 Site Directed Mutagenesis .............................................................................................. 17 2.14 Antibody Generation ...................................................................................................... 18 2.15 Immunofluorescence ...................................................................................................... 18 2.16 Cellular Fractionation ..................................................................................................... 18 2.17 Fractionation Immunoprecipitation ................................................................................ 18 2.18 Phosphatase Treatment ................................................................................................... 19 2.19 Drug treatments .............................................................................................................. 19 2.20 In Silico Protein Analysis ............................................................................................... 19 2.22 mRNA sequencing ......................................................................................................... 20 2.23 Immunoprecipitation Mass Spectrometry ...................................................................... 20 2.24 TRIM25 gene expression analyses ................................................................................. 20 2.25 Immunohistochemistry ................................................................................................... 21 Chapter 3: CIC and ATXN1L Regulate the Cell Cycle ................................................................ 22 3.1 Introduction .................................................................................................................... 22 3.2 Results ............................................................................................................................ 23 3.2.1 ATXN1L is a functional interacting partner of CIC ............................................... 23 viii 3.2.3 Generation of ATXN1L CRISPR Knockout Cell Lines ......................................... 24 3.2.3 Transcriptomic analysis of ATXN1LKO and CICKO isogenic cell lines result in convergence on activation of the MAPK pathway ................................................................ 25 3.2.4 Deletions in ATXN1/ATXN1L or CIC lead to convergent transcriptomic changes. 27 3.2.5 Deletions in ATXN1/ATXN1L or CIC lead to convergent dysregulation of cellular processes and gene set ........................................................................................................... 30 3.2.6 CIC-ATXN1L interaction facilitates CIC-DNA binding ........................................ 34 3.3 Discussion ...................................................................................................................... 35 Chapter 4: CIC and ATXN1L Interaction Forms a Post-Translational Functional and Reciprocal Relationship .................................................................................................................................. 37 4.1 Introduction .................................................................................................................... 37 4.2 Results ............................................................................................................................ 38 4.2.1 Loss of ATXN1L promotes the proteasomal degradation of CIC .......................... 38 4.2.3 Loss of CIC leads to dysregulation of ATXN1L .................................................... 40 4.2.3 ATXN1L mediated CIC instability is independent of ERK activity ...................... 42 4.2.4 Inactivation of CIC by ERK remains intact and independent of ATXN1L ............ 44 4.2.5 CIC degradation is mediated by the E3-ligase TRIM25 ......................................... 46 4.2.6 Glioblastoma cell lines exhibit ATXN1L mediated CIC instability and TRIM25 mediated degradation ............................................................................................................. 48 4.2.7 TRIM25 amplification and CIC deletion dysregulate similar gene sets .................. 50 4.3 Discussion ...................................................................................................................... 53 Chapter 5: The Effects of IDH mutation on CIC and ATXN1L loss ............................................. 56 5.1 Introduction .................................................................................................................... 56 5.2 Results ............................................................................................................................ 57 5.2.1 ATXN1L loss in IDH1-R132H mutant cells leads to derepression of CIC target genes 57 5.2.2 IDH mutation dampens the transcriptomic effects of ATXN1L and CIC loss ........ 58 ix 5.3 Discussion ...................................................................................................................... 60 Chapter 6: Conclusions ................................................................................................................. 61 6.1 Summary of study and findings ...................................................................................... 61 6.2 Study strengths and limitations ...................................................................................... 63 6.3 Future directions ............................................................................................................. 63 6.3.1 Investigate the role of ATXN1/ATXN1L in relation to CIC isoforms ................... 64 6.3.2 Investigate the role of ATXN1/ATXN1L in different CIC complexes .................. 64 6.3.3 Determine the amino acid residues of CIC required for degradation ..................... 64 6.3.4 Explore the use of small molecule inhibitors to re-establish CIC function ............ 65 References ..................................................................................................................................... 66 Appendices .................................................................................................................................... 73 Appendix A ............................................................................................................................... 73 Appendix B ................................................................................................................................ 75 Supplemental Figure 1 – CIC-ATXN1L interaction ............................................................. 75 Supplemental Figure 2 – ATXN1 function only partially compensates for ATXN1L loss. . 76 Supplemental Figure 3 – ATXN1L loss leads to decreased CIC protein expression ............ 77 Supplemental Figure 4 – Reintroduction of ATXN1L .......................................................... 77 Supplemental Figure 5 - ATXN1LKO cell lines show derepression of the MAPK pathway. . 78 Supplemental Figure 6 – TCGA Pathway Analyses .............................................................. 79 Supplemental Figure 7 – ATXN1LKO cells exhibit decreased CIC-DNA binding ................. 80 Supplemental Figure 8 – Loss of ATXN1L leads to CIC instability ...................................... 80 Supplemental Figure 9 – Loss of ATXN1L leads to CIC ubiquitination and degradation ... 81 Supplemental Figure 10 – in Silico protein analysis of CIC and ATXN1L .......................... 82 Supplemental Figure 11 – Loss of CIC leads to ATXN1L dysregulation ............................. 83 Supplemental Figure 12 – ERK mediates CIC function ........................................................ 84 Supplemental Figure 13 – EGF/FGF treatment results in increased CIC expression ........... 85 x Supplemental Figure 14 – CIC interactors in the absence of ATXN1L ................................ 86 Supplemental Figure 15 – Characterization of glioma cell lines ........................................... 87 Supplemental Figure 16 – Validation of CIC-ATXN1L-TRIM25 in glioma cells ............... 88 Supplemental Figure 17 – TRIM25 pathways in cell lines ................................................... 89 Appendix C (Tables are hyperlinked) ....................................................................................... 90 Supplemental Table 1 – TCGA samples................................................................................ 90 Supplemental Table 2 – HEK Differential Expression Analysis ........................................... 90 Supplemental Table 3 – NHA Differential Expression Analysis .......................................... 90 Supplemental Table 4 – Type I LGG Differential Expression Analysis ............................... 90 Supplemental Table 5 – HEK and NHA vs Type I LGG Functional Enrichment................. 90 Supplemental Table 6 – Prostate Adenocarcinoma Differential Expression Analysis .......... 90 Supplemental Table 7 – Type II LGG Differential Expression Analysis .............................. 90 Supplemental Table 8 – Stomach Adenocarcinoma Differential Expression Analysis......... 90 Supplemental Table 9 – TCGA Functional Enrichment ........................................................ 90 Supplemental Table 10 – TCGA Shared Genes and Functional Enrichment ........................ 90 Supplemental Table 11 – CIC IP-MS results in ATXN1LKO NHA ........................................ 90 Supplemental Table 12 – TRIM25 knockdown differentially expressed genes .................... 91 Supplemental Table 13 – Differentially expressed ATXN1LKO NHA genes ......................... 91 Supplemental Table 14 – TRIM25 knockdown vs CIC-ATXN1L knockout DE genes ......... 91 Supplemental Table 15 – In vitro TRIM25-CIC-ATXN1L Functional Enrichment ............... 91 Supplemental Table 16 – TCGA BRCA TRIM25 Differential Expression Analysis ............ 91 Supplemental Table 17 – TCGA BRCA vs LGG/PRAD/STAD Functional Enrichment ..... 91 Supplemental Table 18 – IDH-WT and IDH-R132H Mutant CICKO Differential Expression Analysis ................................................................................................................................. 91 Supplemental Table 19 - IDH-WT and IDH-R132H Mutant ATXN1LKO Differential Expression Analysis ............................................................................................................... 91 xi Supplemental Table 20 – Shared Differentially Expressed Genes in IDH-WT and IDH-R132H Mutant CICKO and ATXN1LKO cell lines ................................................................... 91 Supplemental Table 21 –IDH-R132H mutant CICKO and ATXN1LKO Functional Enrichment ............................................................................................................................................... 91 Appendix D ............................................................................................................................... 92 Supplemental Materials 1 – Antibodies ................................................................................. 92 Supplemental Materials 2 – RT-qPCR Primers ..................................................................... 93 Supplemental Materials 3 – siRNA sequences ...................................................................... 93 Supplemental Materials 4 – CRISPR derived Cell Lines and Genetic Backgrounds ............ 93 Supplemental Materials 5 – Other Cell Lines ........................................................................ 94 Supplemental Materials 6 – ChIP-PCR Primers .................................................................... 94 xii List of Tables Table 1.1 – Molecular Subtypes of LGG ........................................................................................ 3 Table 3.1 – TCGA analysis cohorts .............................................................................................. 27 Table 3.2 – Shared DE Genes across TCGA cohorts analysed .................................................... 32 xiii List of Figures Figure 1.1 – Clinical Presentation of Oligodendroglioma .............................................................. 2 Figure 1.2 – Frequency of CIC mutations and copy number loss in cancer ................................... 6 Figure 1.3 – The role of CIC in murine development ..................................................................... 7 Figure 3.1 – CIC and ATXN1L physically and functionally interact. .......................................... 23 Figure 3.2 – ATXN1LKO cell lines show derepression of CIC target genes. ................................. 24 Figure 3.3 – Loss of CIC or ATXN1L dysregulate similar genes and gene sets in vitro. ............ 26 Figure 3.4 – Differentially expressed genes identified in tumours with ATXN1 and/or ATXN1L aberrations and CIC aberrations show overlap and directional concordance. .............................. 29 Figure 3.5 –Tumours with ATXN1 and/or ATXN1L aberrations and CIC aberrations display overlap in gene enrichment signatures. ......................................................................................... 31 Figure 3.6 – ATXN1L interaction with CIC facilitates CIC binding to promoter regions of CIC target genes.................................................................................................................................... 34 Figure 4.1 – Loss of ATXN1L results in CIC instability .............................................................. 39 Figure 4.2 – Loss of CIC is associated with ATXN1L dysregulation .......................................... 41 Figure 4.3 – ATXN1L mediated CIC instability is independent of ERK activity ........................ 43 Figure 4.4 – ERK activity relieves CIC repressive function ......................................................... 45 Figure 4.5 – CIC Interacts with the E3-Ligase TRIM25 .............................................................. 47 Figure 4.6 – TRIM25 and ATXN1L mediated CIC stability in GBM cell lines .......................... 49 Figure 4.7 – CIC, ATXN1L, and TRIM25 regulate cell cycle in vitro and in TCGA patient data ....................................................................................................................................................... 52 Figure 4.8 – Proposed mechanistic model of CIC-ATXN1L-TRIM25 interaction ...................... 54 Figure 5.1 – Loss of ATXN1L in IDH mutant cells dysregulate CIC function ............................. 58 Figure 5.2 – Transcriptomic effects of IDH mutation on CIC and ATXN1L loss ......................... 59 xiv List of Abbreviations5mC αKG 2HG ABD ATXN1L ATXN1 AXH BCCA BCCRC BCGSC BRCA ChIP CIC CNV CRISPR DE DEA DNA E2F EBD ETS FFPE GBM GEO GO GSEA HEK HMG HOG IDH 5-methylcytosine α-ketoglutarate 2-Hydroxyglutarate Ataxin Binding Domain Ataxin-1-Like Ataxin1 Ataxin‐1 and HMG‐box Protein 1 British Columbia Cancer Agency British Columbia Cancer Research Center British Columbia Genome Sciences Center Breast Carcinoma Chromatin Immunoprecipitation Capicua Copy Number Variation Clustered Regularly Interspaced Short Palindromic Repeats Differentially Expressed Differential Expression Analysis Deoxyribonucleic Acid E2 Transcription Factor ERK Binding Domain E-Twenty-Six Transcription Factor Formalin Fixed Paraffin Embedded Glioblastoma Gene Expression Omnibus Gene Ontology Gene Set Enrichment Analysis Human Embryonic Kidney High Mobility Group Human Oligodendroglial Isocitrate Dehydrogenase xv IHC IP LGG LIHC MAPK mTOR NHA ODG PBS PRAD PTM Rb RNA RTK RT-qPCR SCA1 SD STAD TBS TCGA WHO Immunohistochemistry Immunoprecipitation Low Grade Glioma Liver Hepatocellular Carcinoma Mitogen Activated Protein Kinase Mammalian Target of Rapamycin Normal Human Astrocyte Oligodendroglioma Phosphate Buffered Saline Prostate Adenocarcinoma Post Translational Modification Retinoblastoma Transcriptional Corepressor Ribonucleic Acid Receptor Tyrosine Kinase Real-Time Quantitative Polymerase Chain Reaction Spinocerebellar Ataxia 1 Standard Deviation Stomach Adenocarcinoma Tris Buffered Saline The Cancer Genome Atlas World Health Organization xvi Acknowledgements The completion of this dissertation was achieved through the support and help of a number of people. I would like to thank my supervisor Dr. Stephen Yip who gave me guidance but also the freedom to explore scientifically, my doctoral supervisory committee (Dr. Marco Marra, Dr. David Huntsman, Dr. Marcel Bally, Dr. William Lockwood) for providing their valuable time and advice, and past and present members of the Stephen Yip lab (Amy Lum, Adrian Levine, Tae Hoon Lee, Kohl Lounsbury, Samantha Lee, Lisa Sogerer, and Victor Wong) and Marco Marra lab (Veronique LeBlanc, and Suganthi Chittaranjan) who contributed technically and scientifically to varying degrees. I would also like to thank members of Dr Gregory Cairncross, Dr Jennifer Chan, Dr Kenneth Aldape, and Dr. David Huntsman laboratories for helpful discussions and advice. I am thankful to my friends, family, and my partner Philip for their support throughout my studies. Finally, I would like to acknowledge all the patients who donated samples and that make this research possible. The research presented in this thesis was funded by the Canadian Cancer Society and the BC Cancer Foundation through BrainCare BC. 1 Chapter 1: Introductions 1.1 Low Grade Gliomas 1.1.1 Etiology and Epidemiology Primary brain tumors, by strict definition, arise from the intrinsic non-neoplastic cellular elements of the brain. As postulated initially by Harvey Cushing and Percival Bailey in the 1920s - the normal endogenous cellular constituents of the brain have neoplastic counterparts [1]. This formed the intellectual construct for brain tumor ontogeny which has also influenced the nomenclature of the tumors today. Diffuse and infiltrative growth is a cardinal feature of the vast majority of glioma, especially those with World Health Organization (WHO) grades of II to IV classification. Infiltrating glioma (WHO II-IV), as the name suggests, displays distant spread along vasculature and white matter tracts which contribute to recurrence locally and in distal sites. Scherer first described these histological features in the 1930s, now known as Scherer’s secondary structures [2], which are some of the defining histological features of infiltrating glioma. Low grade gliomas (LGGs) encompass grade II and III tumours with an incidence rate of roughly 1.43/100,000 people [3], are less common than glioblastoma (GBM), the grade IV counterpart. Compared to GBM, LGGs grow slowly and are generally associated with a favorable outcome. However, many LGGs inevitably progress through malignant transformation towards GBM. Although many advances in understanding the molecular landscape of gliomas, in particular LGGs, have been made in the past three decades, the treatment options and survival for these tumours have stagnated. Despite having a generally high 5 year survival rate of between 32-81% (depending on subtype), compared to GBM (8%), LGG patients are often diagnosed at a much younger age (mean = 41 years), compared to GBM patients (mean = 66 years) [3] resulting in a greater loss of productive life in these individuals. 1.1.2 Molecular Alterations and Histopathology The diagnostic algorithm for LGG in the post WHO 2016 Classification era is much more reliant on the molecular information of IDH1/2, ATRX, and copy number status of chromosomes 1p/19q and represents a marked departure from the 2007 histology based classification. LGGs 2 can be separated into three major molecular subtypes which provide superior prognostic information compared to traditional histological classification (Table 1.1) [4-6]. Figure 1.1 – Clinical Presentation of Oligodendroglioma Anaplastic oligodendroglioma with foci of microvascular proliferation is characterized by tumor cells with round to ovoid nuclei associated with nil to small amount of cytoplasm and perinuclear clearing (A). The tumor cells strongly express the IDH1 R132H mutant protein (B) but retain nuclear expression of ATRX(C). This is confirmed by 1p19q FISH showing co-deletion (D, only 1p FISH shown). (E) MRI imaging of anaplastic oligodendroglioma, IDH mutant, 1p19q co-deleted (WHO III). (Left) Circumscribed T2 FLAIR signal abnormality within the left frontal lobe on the axial plane is indicative of a solid/cystic intra-axial tumor. (Middle) The tumor shows scant intra-lesional contrast enhancement on T1, especially in the anterior aspect on axial plane. (Right) However, the vast majority of the tumor is non-enhancing on coronal plane. 3 Table 1.1 – Molecular Subtypes of LGG Type Histology IDH Status 1p19q Status Type I Oligodendroglioma Mutated Co-deleted Type II Astrocytoma Mutated Retained Type III IDH Wildtype Astrocytoma Wildtype Retained In addition to providing superior prognostic data, each molecular subtype can also be generally associated with a histologically defined entity. Type I LGG which harbour concurrent losses of the entire short arm of chromosome 1 (1p) and long arm of chromosome 19 (19q) [1p19q co-deletion], is most often associated with oligodendroglial histology [7, 8]. Virtually all 1p19q co-deleted tumours harbor IDH mutations. The arm-wise losses are the result of unbalanced translocation events and are generally accompanied by a relatively quiet genome [9, 10]. The histological features frequently described in ODG include perinuclear clearing in FFPE tissues and are known to have a “fried-egg” appearance. It is likely the result of “washing out” of cytoplasmic glycogen by organic solvent during tissue processing and is not evident in tissue frozen sections (Figure 1.1). Moreover, tumor cells from ODG tend to display round, regular nuclear features with very little morphological pleomorphism and no associated cytoplasmic content. This is reminiscent of non-neoplastic oligodendrocytes. The presence of delicate branching blood vessels, known colloquially as “chicken-wire” appearance, is also associated with low-grade ODG. These vessels can undergo proliferation in anaplastic ODG, and predominance of this histological finding is one of the diagnostic criteria for WHO III tumor. Conversely, Type II LGGs are most often associated with Astrocytomas and have a high frequency of ATRX immunonegativity by immunohistochemical staining (IHC) as a result of ATRX loss of function genomic aberrations. Astrocytoma cells frequently display elongated, hyperchromatic nuclei in association with variable amount of eosinophilic cytoplasm reminiscent of non-neoplastic astrocytes. Astrocytic tumors can display cytological features of astrocytoma, oligodendroglioma, or a combination of the two. However, absence of 1p19q co-deletion itself remains the most important molecular identifier of astrocytomas. Lastly, Type III tumours are a small percentage of LGGs which do not exhibit IDH mutations or 1p19q co-deletion. These tumours, referred to as “triple negative tumours”, carry the worst prognosis and are associated with genomic aberrations reminiscent of GBM such as homozygous loss of CDKN2A/B and PTEN as well as EGFR amplification [11]. 4 1.2 IDH Mutations There have been many important discoveries on the molecular pathogenesis of infiltrating glioma in the past 10 years. However, the identification and characterization of recurrent mutations in the genes IDH1/2 remain the most significant with impact on all spheres in the clinical management of patients with infiltrating gliomas. Initially identified in young GBM patients, it was later verified to be arising from preceding LGGs [12]. Follow-up studies confirmed that recurrent heterozygous IDH mutations occur early during gliomagenesis and are found in a large percentage of LGG [11]. A vast majority of IDH mutations occur in codon 132 of IDH1, which leads to arginine to histidine amino acid change (IDH1 p.R132H) that accounts for >83% of all mutations in LGG. A minority of IDH1 mutations in codon 132 lead to other amino acid changes. Mutations affecting codon 172 of IDH2 are only found in approximately 5% of gliomas with IDH mutations. All IDH mutations result in the aberrant overproduction of the oncometabolite 2-hydroxyglutarate (2HG), which is normally produced in very small amounts in extreme physiological conditions and has profound effects on the epigenome of the cancer cell [13]. Specifically, 2-HG inhibits α-ketoglutarate (αKG)-dependent dioxygenases including TET2 and also lowers the levels of 5-hydroxymethylcytosine (5mC), which lead to alterations of genome-wide histone and DNA methylation [14-16]. Together, these changes have significant impact on the cancer epigenome [17, 18]. 2HG also affects other cellular pathways, including those that regulate the normal physiological degradation of hypoxia inducible factor-1 (HIF1a) [19]. Taken together, overexpression of 2HG as a result of IDH mutations is essential for gliomagenesis. 1.3 Capicua (CIC) 1.3.1 CIC Aberrations in Cancer Capicua (CIC), a gene located on human chromosome 19q13.3, encodes for an evolutionarily conserved member of the High Mobility Group (HMG) box protein superfamily involved in DNA binding. CIC was first identified as a transcriptional repressor, with two isoforms (CIC-S and CIC-L), responsible for regulating growth and proliferation of specific tissues during the development of Drosophila melanogaster [20-25]. In general, CIC acts as a repressor of receptor tyrosine kinase (RTK) responsive genes and is inactivated through phosphorylation by ERK, a member of the MAPK cascade [21, 24-27]. The most well characterized mammalian CIC target genes are the ETS transcription factor family genes 5 ETV1/4/5 which have been found to be upregulated in multiple cancers with CIC aberrations and to drive many oncogenic phenotypes [28-33]. The status of CIC has recently become a topic of interest in cancer because CIC aberrations have been associated with various cellular processes involved in worse overall patient outcome [29, 31, 34, 35]. Aberrations in CIC were first identified in undifferentiated small round cell sarcoma, also referred to as Ewing-like sarcoma (ELS). A subset of these tumours were found to harbor a CIC-DUX4 translocation event resulting in the fusion of the C-terminal of CIC (exon 20) to the trans-activating domain of DUX4 (exon 1), transforming CIC from a transcriptional repressor to an activator [32, 36]. CIC fusion events involving other partners have also been identified in ELS (CIC-FOXO4) [37, 38], peripheral neuroectodermal tumours (CIC-NUTM1) [39, 40], and angiosarcoma (CIC-LEUTX) [41]. One fusion event involving the CIC interactor ATXN1 (ATXN1-NUTM1) has also been identified in a primitive brain tumour [42]. Although CIC-DUX4 fusions are the most prevalent, CIC rearrangements have been found to behave promiscuously as many tumours with CIC rearrangements do not have known partners [43]. Recurrent mutations in CIC were later identified in ODG, or Type I LGG (Figure 1.2) [5, 7, 8]. Subsequent studies have identified CIC mutations in stomach adenocarcinoma [28], lung adenocarcinoma [31], breast carcinoma [44], colorectal carcinoma [45], melanoma [46], and multiple myeloma [47]. Interestingly, recurrent missense mutations in the HMG-box DNA binding domain and C1 motif have only been observed in ODG, suggesting that the biology of CIC mutation in ODG may be unique. Additionally, clonally distinct CIC mutations, in an otherwise low mutation burden tumour, have also been identified in ODG suggesting that there is a high selective pressure towards clones which escape CIC regulatory function [4, 48]. Interestingly, CIC mutations have not been identified in other glioma subtypes [5, 7, 8, 49]. In ODG, CIC mutations have been associated with a more aggressive phenotype and poorer prognosis [34, 50, 51]; similar outcomes have also been observed in other cancer types harbouring CIC aberrations which may be due to upregulation of the MAPK signaling pathway and oncogenic ETS transcription factors ETV1/4/5 [28-34, 36, 52]. CIC mutations have also been found to cooperate with IDH mutation to regulate 2HG levels [53], an oncometabolite produced by mutant IDH that is central to initiation and maintenance of the glioma hypermethylator phenotype [17]. 6 Homozygous and heterozygous deletions of CIC have been observed in prostate adenocarcinoma [54], stomach adenocarcinoma [28], neuroblastoma [55], and astrocytoma [54]. Lastly, reduced CIC protein expression has been observed in prostate adenocarcinoma [56], hepatocellular carcinoma [33] and glioblastoma [57]. Altogether, the high prevalence of CIC aberrations and CIC dysregulation in a plethora of different cancer types supports CIC’s role as a transcription factor with potent pan-cancer tumour suppressor functions [58]. Figure 1.2 – Frequency of CIC mutations and copy number loss in cancer 1.3.2 CIC in Mammalian Development and Oncogenesis Following the discovery of recurrent CIC mutations in ODG, several mouse models have been used to investigate the role of CIC mutation in gliomagenesis. The mouse (Cic) and human (CIC) orthologs were first identified in developing granule neurons as a transiently expressed factor [20]. Patients which harbor germline heterozygous loss of function CIC mutations have presented with several neurodevelopmental disorders [59]. Similarly, homozygous deletion of Cic in the developing mouse forebrain (EMX1-Cre) or hypothalamus/amygdala (Otp-Cre) result in a spectrum of neurodevelopmental disorders [59]. Lineage specific knockout of Cic in embryonic neural precursor cells compromised the differentiation of neuroblasts to neurons (Nes-Cre) and promoted arrest as OLIG2 positive oligodendroglial progenitor cell-like state 7 (FOXG1-Cre) [60]. In both studies, loss of Cic resulted in reduced cortical thickness, decreased brain size, hyperproliferation of the sub-ventricular zone, and partial post-natal lethality [59-62]. While Cic loss was found to result in the onset of cellular behaviors characteristic of gliomas, the spontaneous formation of glioma was not observed; supporting the notion that preceding events such as IDH mutation are required for transformation [4, 17, 63], and that loss of CIC in ODG occurs later [64]. Figure 1.3 – The role of CIC in murine development In addition to neurogenesis, CIC has also been identified as an important developmental regulator of other organ systems such as lung, liver, and immune. Homozygous embryonic deletion of Cic or destabilization of CIC through homozygous deletion of ATXN1 and/or ATXN1L resulted in defective lung alveolarization, [65] impaired alveolar maturation, and insufficient surfactant production [52]. Lung alveolarization defects were due to the derepression of ETV4 and MMP9, a matrix metallopeptidase responsible for breakdown of extracellular matrix [65]. Homozygous Cic knockout mice also displayed impaired bile acid homeostasis and metabolism, which was attributed to the downregulation of several hepatic transcriptional 8 regulators, upregulation of proinflammatory cytokines [66], and overexpansion of liver-resident memory T-lymphocytes (T-cells) [67]. Spontaneous T-cell activation and enhanced follicular helper T-cell differentiation, mediated by ETV5 overexpression, was also observed in hematopoietic lineage specific (Vav1-Cre) and T-cell specific (CD4-Cre) Cic knockout mice which establishes CIC as an important factor in lineage specification and maturation of T-cells. Lastly, the only malignant tumour found to be initiated by Cic loss in murine models is the induction of T-cell acute lymphoblastic leukemia (T-ALL) (Tek-Cre) which was mediated by ETV4 derepression and activation of an oncogenic RAS transcriptomic signature [52, 68]. Together, these studies provide strong evidence of the potent tumour suppressor function of CIC in regulating proliferation and differentiation in various stem and progenitor lineages across all three primary germ layers (Figure 1.3). 1.4 ATXN1L in Development Dysregulation of CIC target genes has also been implicated in the neuropathology of spinocerebellar ataxia 1 (SCA1) through aberrant binding of CIC to mutant Ataxin1 (ATXN1) [59, 69-71]. The neurotoxicity caused by mutant ATXN1-CIC complexes was later shown to be relieved through competitive binding of CIC to the ATXN1 homologue, Ataxin-1-Like (ATXN1L) [72, 73]. ATXN1 and ATXN1L are members of the Ataxin protein family and share an ataxin‐1 and HMG‐box protein 1 (AXH) domain required for interaction with CIC [73, 74] and have been reported to interact with CIC in multiple contexts including development and disease state [59, 65, 66, 72]. Previous studies utilizing mouse models to study development and SCA1 pathology have indicated some level of functional redundancy between ATXN1 and ATXN1L, which is also supported by the high degree of protein homology [73]. These studies also highlighted that loss of ATXN1L affected CIC function much more profoundly and robustly compared to loss of ATXN1 in murine models [65, 66]. ATXN1 and ATXN1L have also been reported as regulators of Notch signalling through repression of HEY1, a gene downstream of Notch activation [75]. One study has also suggested that ATXN1L may be involved in maintaining hematopoietic stem cell quiescence and renewal through the regulation of HOXA7 and HOXA9 [76]. However, additional characterized targets of ATXN1L remain elusive and its role in regulating CIC function in cancer remains unclear. 9 1.5 Thesis Although significant progress has been made in the past two decades in understanding the molecular alterations involved in gliomagenesis, treatment options remain limited and rarely involve targeted therapy. In order to develop targeted therapies, a deeper understanding towards the molecular mechanisms and downstream consequences of these alterations is required. Alterations in CIC were first discovered in ODGs, however, it is clear now that loss or altered function of this tumour suppressor is a potent driver of pathways associated with poor prognosis such as metastasis, invasion, treatment resistance, and progression in several cancers. One aspect of CIC function is the molecular relationships CIC forms between its interacting partners and how they may modulate CIC function. By studying the regulation of CIC at a post-translational level, we may find targets to potentially re-establish normal CIC function as a therapeutic target. 1.6 Objectives and Hypothesis The primary objective of this thesis is to investigate the functional relationship between CIC and ATXN1L with the goal of further understanding the role of ATXN1L as a CIC interacting partner and their relationship in cancer. I hypothesize that the interaction between CIC and ATXN1L is vital to the normal function and stability of CIC and that loss of either proteins can be leveraged by cancers to drive cancer progression. 1.7 Specific Aims and Thesis Outline To address the objective and hypothesis, the following specific aims were devised: Aim 1: Comprehensively characterize and compare the transcriptomic effects of ATXN1L loss and CIC loss in in vitro cell lines and patient tumours. Chapter 3 describes the in vitro establishment of the interaction between CIC and ATXN1L, generation of ATXN1LKO cell lines, downstream transcriptomic investigations, validation using TCGA patient cohorts, and in vitro assessment of the mechanism. Comprehensive and comparative transcriptomic analysis of CICKO and ATXN1LKO cell lines revealed for the first time, the extent that CIC loss and ATXN1L loss converge transcriptomically both at the gene and pathway level which were also validated in three TCGA cohorts. This data 10 suggests that the loss of ATXN1L may be a mechanism of modulating CIC function in several cancer types in addition to the CIC aberrations already reported in the literature. Aim 2: Investigate the post-translational regulation of CIC by ATXN1L and vice versa. Chapter 4 describes in vitro experiments that establish a reciprocal functional relationship between CIC and ATXN1L whereby loss of one interacting partner results in the post-translational dysregulation of the other partner. CIC and ATXN1L were found to promote each other’s protein stability and cellular localization. Loss of ATXN1L promoted ERK independent proteasomal degradation of CIC through ubiquitination by the E3-ligase TRIM25. These observations were further validated using transcriptomic analyses of the TCGA breast carcinoma and liver hepatocellular carcinoma cohorts. This study establishes the mechanism of CIC dysregulation through ATXN1L loss as well as reveal a novel CIC degradation pathway independent of canonical ERK mediated CIC degradation. Aim 3: Investigate the role and effects of IDH mutation on the functional relationship between CIC and ATXN1L. Chapter 5 describes and explores the effects of IDH1-R132H mutation on the transcriptomic relationship between CIC and ATXN1L. Comparative transcriptomic analysis of CICKO and ATXN1LKO cell lines in IDH1 wildtype and IDH1-R132H mutant backgrounds was performed. IDH was found to “dampen” the effects of CIC or ATXN1L loss on the global transcriptome, though did not affect the dysregulation of pathways related to MAPK and cellular growth. 11 Chapter 2: Materials and Methods 2.1 Immunoprecipitation For each cell line and replicate, one to two 15cm plate at roughly 70-80% confluency were harvested and lysed using Pierce™ IP Lysis Buffer (Thermofisher Scientific, Waltham, MA, USA [ThermoSci]) supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail (ThermoSci). Lysates were incubated on ice for 30 minutes and homogenized by passing through a 25 gauge needle 15-20 times followed by incubation on ice for 30 minutes. Insoluble cell debris was pelleted down by centrifugation at 13000 rpm for 15 minutes at 4⁰C. 1mg of protein for each replicate was blocked using 40µL of Sepharose® 4B beads (Sigma Aldrich, St. Louis, MO, USA [Sigma]). Pierce™ Protein A/G Plus Agarose Beads or Dynabeads™ (ThermoSci) were preblocked using 1% BSA, rotating for 1 hour at 4⁰C. Conjugation of anti-CIC, anti-ATXN1L, anti-FLAG or control IgG antibody to agarose/Dynabeads (ThermoSci) beads was performed by adding 1-7.5 µg of antibody to the preblocked beads in 1mL of 1% BSA. Conjugated beads were pelleted down. Blocked lysate was added to conjugated beads and incubated, rotating for 3 hours at 4⁰C. Beads were pelleted down and washed 3x in IP lysis buffer followed by suspension in 4x loading buffer and 10x reducing agent. Beads were boiled at 95⁰C for 8 minutes to release captured protein. 2.2 Cell Lysate Preparation Whole cell lysate was prepared by resuspending cell pellets with 3-5x ice cold RIPA buffer (Santa Cruz Biotechnology, Dallas, TX, USA [SantaCruz]) according to manufacturer’s protocol with fresh supplemented Halt™ Protease and Phosphatase Inhibitor Cocktail (ThermoSci). Lysates were incubated on ice for 1 hour and insoluble cell debris was pelleted at 13000rpm for 15 minutes at 4⁰C. Protein was quantified using BCA assay (ThermoSci) and normalized samples were prepared with 4x loading buffer (ThermoSci) and 10x reducing agent (ThermoSci). 2.3 Western Blot Samples were separated using gel electrophoresis on NuPage 3-8% Tris-Acetate gels run (Life Technologies, Carlsbad, CA, USA [Life Tech]) in 1x Tris-Acetate running buffer (Life Tech) or NuPage 10% Bis-Tris gels (Life Tech) run in 1x MOPS running buffer (Life Tech). 12 Running buffers were supplemented with oxidizing agent (Life Tech). Electrophoresis was run at 100V for 25minutes followed by 150V for 1-2.5 hours. Separated proteins were transferred onto methanol activated PVDF membrane (Millipore, Burlington, MA, USA) for 70-120 minutes at 100V in 1x transfer buffer (Life Tech) with 20% methanol. Membranes were blocked with 5% skim milk in TBS with 0.01% Tween (TBS-T) for one hour at room temperature followed by overnight incubation in primary antibody. Membranes were incubated with HRP conjugated secondary antibody (SantaCruz) for 1 hour at room temperature and washed 3x 30 minutes in TBS-T before and after secondary antibody incubation. Protein signal detection was performed using either ECL substrate (Bio-Rad, Hercules, CA, USA [BioRad]) or SuperSignal West Femto substrate (ThermoSci). Antibody information can be found in Supplemental Materials 1. Images were captured using a ChemiDoc MP Imager (BioRad) or ImageQuant LAS4000 (GE Healthcare, Chicago, IL, USA) and protein quantifications were performed using ImageJ software. 2.4 Real-Time quantitative PCR (RT-qPCR) RNA extraction was performed using the RNeasy Mini Kit (74104, Qiagen, Hilden, Germany) according to manufacturer’s protocol. 1µg of template RNA was used for cDNA conversion using SuperScript™ III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s protocol. RT-qPCR was performed using 4-12.5ng of template cDNA with PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Beverly Hills, CA, USA) according to manufacturer’s recommended reaction component amounts and cycling conditions. RT-qPCR reactions were run on a QuantStudio 6 Flex Real-Time PCR System using QuantStudio™ Real-Time PCR Software to generate CT values. Analysis of relative mRNA expression was performed using the 2-ΔΔCT method with TATA-box binding protein (TBP) expression as an endogenous control. Sequences of the RT-qPCR primers used are provided in Supplemental Materials 2. 2.5 Cell Culture Conditions The HEK293a cell line was obtained from Dr Gregg Morin (Canada’s Michael Smith Genome Sciences Centre, Vancouver, BC, Canada) and authenticated by Genetica DNA Laboratories (Cincinnati, Ohio, USA). Wildtype IDH1-stable NHA cell line [77] was purchased from Applied Biological Materials (ABM) Inc. (T3022, Richmond, BC, Canada). U251 and U343 glioblastoma cells were obtained from Dr Shoukat Dedhar (BC Cancer Research Center, 13 Vancouver, BC, Canada) and LN18, LN229, and U87-MG glioblastoma cells were obtained from Dr Kevin Bennewith (BC Cancer Research Center, Vancouver, BC, Canada). The CIC-DUX4 sarcoma line (NCC-CDS-X1-C1) [78] was obtained from Dr Tadashi Kondo (National Cancer Center, Tokyo, Japan). HEK293a, NHA, HOG, U87-MG, U251, and U343 cell lines were cultured in Dulbeccos modified Eagle’s medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and 5% FBS for LN18 and LN229 cell lines. NCC-CDS-X1-C1 cell line was cultured in RPMI (Life Technologies) supplemented with 10% FBS. BTIC cell lines MGG119 [79], BT54, and BT88 [80] were cultured in serum free Neural Basal Media (Life Technologies) supplemented with N2/B27 (Gibco) and EGF/FGF (20ng/mL; Peprotech). All cultures were maintained in a 37⁰C, 5% CO2 incubator. Cells were tested and verified to be mycoplasma free using MycoFluor™ Mycoplasma Detection Kit (Invitrogen). Unless otherwise stated, cells were washed once with PBS and harvested via scraping or centrifugation at 70-90% confluency. ATXN1LKO cell lines were generated using the CRISPR/Cas9 genome editing technology as previously described [54]. A table of cell lines used and their genetic background can be found in Supplemental Materials 4 and 5. 2.6 ATXN1L CRISPR Knockout HEK derived ATXN1LWT (N4 and N8), ATXN1LKO (A10, A30 and B21) and NHA derived ATXN1LKO (B82, B16, and B21) cell lines were generated using CRISPR/Cas9 genome editing technology. CRISPR guide sequences against exon 1 of ATXN1L were designed using the online CRISPR Design tool (http://crispr.mit.edu). CRISPR/Cas9 guide sequences were cloned into the construct pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene, Watertown, MA, USA) following the Zhang protocol (http://www.genome-engineering.org/crispr/wpcontent/uploads/2014/05/ CRISPR-Reagent-Description-Rev20140509.pdf). ATXN1L target sequences are as follows: ATXN1L_Exon1_A (5’-CACCGACCTGTTCATGAAAGGAGTC-3’) and ATXN1L_Exon1_B (5’- CACCGCAGCCGCTGGACCTTGCTCC-3’). HEK cells were transfected with the CRISPR/Cas9 construct targeted against ATXN1L using TurboFect™ Transfection Reagent (ThermoSci) and selected in media containing Puromycin. NHA cells were transfected with the CRISPR/Cas9 construct targeted against ATXN1L using TransIT-X2® Dynamic Delivery System (Mirus Bio LLC, Madison, WI, USA) and selected in media containing puromycin. Cells 14 were selected for 72 hours and then seeded at 200 cells/15cm dish. Single colonies were selected using cloning cylinders 7-14 days post seeding and transferred into 96 well plates. Each clone was screened for ATXN1L expression using Western Blot. Clones without detectable ATXN1L protein expression were further Sanger sequenced to confirm genome editing at the targeted site. 2.7 siRNA Transfection Cells were transfected at 70% confluency with Silencer Select (Life Tech) and Stealth RNAi (Invitrogen) targeted at ATXN1L, ATXN1, or TRIM25. Targeted siRNA information can be found in Supplemental Materials 3. Each siRNA was tested independently prior to pooled siRNA experiments (data not shown). Control transfections were performed with Stealth RNAi™ siRNA Negative Control, Med GC (12935300, ThermoSci) or BLOCK-iT Fluorescent Oligo (13750062, Invitrogen). Transfections were performed using Lipofectamine® RNAiMAX Transfection Reagent (Life Tech) for HEK cells or TransIT-X2® Dynamic Delivery System (Mirus Bio LLC, Madison, WI, USA) for all other adherent cell lines. Neurosphere cell lines (MGG119, BT054, BT088) were transfected using nucleofection (Mouse Neural Stem Cell Nucleofector Kit: VPG-1004, Lonza, Basel, Switzerland) according to manufacturer’s protocol. FLAG-tagged ATXN1L construct was purchased from Addgene (#33242) [72]. Cells were harvested 24-72 hours post transfection for experiments. 2.8 Microarray Expression Profiling The following cell lines were subjected to microarray expression profiling using GeneChip Human Gene 2.0 ST array (Affymetrix, Santa Clara, CA, USA) at the Centre for Applied Genomics (The Hospital for Sick Children, Toronto, Canada) in triplicate (three separate passages): three HEK derived ATXN1LWT cell lines (parental HEK, and two scramble treated CRISPR controls N4 and N8), three HEK derived ATXN1LKO cell lines (A10, A30 and B21), the parental CICWT/ATXN1LWT NHA cell line, two NHA derived CICKO cell lines (A2 and H9), and one NHA derived ATXN1LKO cell line (B82). Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Halden, Germany) according to manufacturer’s protocol. Gene annotation was performed with the R/Bioconductor package hugene20sttranscriptcluster.db (version 8.5.0), and only transcript clusters that mapped to single genes were retained for further analyses. Multiple transcript clusters that mapped to identical genes were aggregated using mean expression values. Expression values for each gene were averaged across all HEK or NHA ATXN1LWT/CICWT cell lines and replicates, respective to the analysis. Differentially expressed genes were identified 15 using fold-change (FC) differences in gene expression between each individual ATXN1LKO/CICKO replicate and the average ATXN1LWT/CICWT expression. Genes with an FC value of >1.5 in at least 4/9 (HEK ATXN1LKO), 3/6 (NHA CICKO), and 2/3 (NHA ATXN1LKO) comparisons were considered to be differentially expressed [28, 81]. The data are accessible through the Gene Expression Omnibus (dataset GSE103515). 2.9 TCGA Expression Analyses RNA-sequencing results were obtained from The Cancer Genome Atlas (TCGA) using http://firebrowse.org/ (doi:10.7908/C11G0KM9) and mutation and copy number data were obtained from TCGA using http://www.cbioportal.org/ [82, 83]. Differential expression analysis of Lower Grade Gliomas (LGG), Prostate Adenocarcinoma (PRAD), Stomach Adenocarcinoma (STAD), and Breast Carcinoma (BRCA) TCGA cohorts was performed using the R package DEseq2 [84]. The LGG cohort was stratified into three subgroups based on IDH mutation and 1p19q-codeletion status [4]. Tumours harbouring IDH mutation and 1p19q-codeletion were grouped as Type I tumours and differential expression analysis was performed between CIC mutated (Type I LGGCIC-Mut) (Truncating and Nonsense mutations, n=39) and Type I LGGCIC-WT (n=68) samples. IDH mutated, 1p19q retained tumours were grouped as Type II tumours and differential expression analysis was performed comparing samples with heterozygous/homozygous deletions in ATXN1 or ATXN1L without CIC copy number variations (deletions and amplifications) (CNVs) (Type II-LGGATXN1/ATXN1L-Del, n=9), or samples with heterozygous/homozygous deletions in CIC without ATXN1 or ATXN1L CNVs (Type II-LGGCIC-Del) (n=30), with samples harbouring no CNVs in ATXN1L, ATXN1 or CIC (Type II-LGGWT) (n=66). Type III tumours, defined as IDH wildtype, were not analyzed in this study. In the PRAD cohort, Differential expression analysis was performed comparing samples with homozygous/heterozygous deletions in CIC without ATXN1L CNVs (PRADCIC-Del) (n=18), or samples with homozygous/heterozygous deletions in ATXN1L without CIC CNVs (PRADATXN1L-Del) (n=80), with samples harbouring no CIC, ATXN1 or ATXN1L CNVs (PRADWT) (n=143). In the STAD cohort, differential expression analysis was performed using samples with homozygous/heterozygous deletions in CIC (STADCIC-Del) (n=10), ATXN1L (STADATXN1L-Del) (n=23), ATXN1 (STADATXN1-Del) (n=20), or deletions in at least 2/4 alleles of ATXN1L/ATXN1 (PRADATXN1/ATXN1L-Del) (n=14), with samples harbouring no CNVs in CIC, ATXN1L or ATXN1 (STADWT) (n=95). In the LGG, PRAD, and STAD cohorts, only wildtype samples with ATXN1, 16 ATXN1L or CIC expression, respective to the analysis, above the first quartile were used to reduce the possibility of secondary mechanisms other than CNVs affecting transcriptional regulation of ATXN1, ATXN1L and CIC [85]. Lastly, for the BRCA cohort, differential expression analysis was performed comparing samples with TRIM25 amplification (n=73) with samples with copy neutral TRIM25 (n=449). A table of all TCGA Samples used for analyses can be found in Supplemental Table 1. 2.10 Gene Set Enrichment Analysis The Metascape software (http:/metascape.org) was used to perform functional enrichments using the multiple gene lists mode [86]. Gene ontology (GO) Biological Processes, Hallmark Gene Sets and Oncogenic Signatures were used for enrichment analyses of all DE genes, with a p-value cut-off of 0.05, and a minimum enrichment of 1.5. Only terms with a BH-adjusted p-value < 0.05 were retained. 2.11 Targeted Chromatin Immunoprecipitation-PCR For each cell line (HEK, D10, A30) and replicate, two ~70-80% confluent 15cm plates were treated with 1% formaldehyde (Sigma Aldrich, St. Louis, MO, USA) in PBS for 12 minutes with gentle rocking, followed by treatment with 0.125M glycine (Sigma) for five minutes. Crosslinked cells were combined and pelleted using centrifugation at 1200rpm for 5 minutes at room temperature, resuspended in 450μL ChIP lysis buffer (50mM Tris-HCl pH 8.0, 1% SDS, 10mM EDTA, 1X Phosphatase and Protease Inhibitor cocktail (PIC) [ThermoSci]), and lysed on ice for 30 minutes. Cells were homogenized by 6 passages through a 20-gauge needle, and nuclei were pelleted by centrifugation at 5000rpm for 10 minutes at 4⁰C. The pellet was resuspended in 900μL shearing buffer (10mM Tris-HCl pH 8.0, 0.1% SDS, 1mM EDTA, 1X PIC) and transferred to a 1mL milliTUBE with AFA fiber (D-Mark Biosciences, Toronto, ON, Canada) using a 30-gauge needle. Chromatin was sonicated using a Covaris S2 sonicator using the following settings: 10% duty cycle, 6 intensity, 500 burst, 16 cycles (20s on, 40s off) at 4-6⁰C. Insoluble debris and unfragmented chromatin were removed by centrifugation at 14000rpm for 12 minutes at 4⁰C. An aliquot of chromatin was de-crosslinked overnight at 68⁰C (0.2M NaCl, 0.05mg/mL RNase), treated with proteinase K for 30 min at 42⁰C, and purified using the MinElute PCR Purification Kit (28006, Qiagen, Halden, Germany) to allow determination of chromatin concentration using the Qubit dsDNA high sensitivity assay (Life Tech) and confirmation of sufficient DNA fragmentation to a 150-300 bp size range on a bioanalyzer using 17 High Sensitivity DNA Chips (Agilent Technologies, Santa Clara, CA, USA). Protein A Dynabeads (Life Tech) blocked with bovine serum albumin and salmon sperm DNA for 3h were used to pre-clear chromatin for 2h. For the immunoprecipitation, three volumes of IP buffer (10mM Tris-HCl pH 8.0, 1% Triton X-100, 0.1% deoxycholate, 0.1% SDS, 90mM NaCl, 2mM EDTA, 1x PIC) were added to 22.5μg of chromatin, which was then incubated with 2.5μg of anti-CIC antibody for 1h at 4⁰C. Blocked Protein A Dynabeads (25μL) were then added to the chromatin and antibody and incubated overnight at 4⁰C. The samples were then washed twice in low salt buffer (20mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2mM EDTA, 150mM NaCl) and twice in high salt buffer (same as low salt buffer except 300mM NaCl). DNA was eluted in 100mM sodium bicarbonate with 1% SDS and 0.05mg/mL RNase at 68⁰C for 6 hours, followed by treatment with proteinase K for 30 min at 42⁰C. For each IP, 100ng of chromatin was subject to the same treatment to serve as input controls. Eluted DNA was purified using the MinElute PCR. Purification Kit, and qPCR analysis was performed as described above using the PowerUp SYBR® Green PCR Master Mix (Life Tech) according to the manufacturer’s recommended reaction component amounts and cycling conditions. Percent input values were calculated for each site, and reported as fold-change differences compared to NCR1. Two-sided Student’s t-tests were used to assess differential expression. Sequences of the qPCR primers used are provided in Supplemental Materials 6. 2.12 Proximity Ligation Assay Cells were seeded and cultured on glass coverslips for 24-72 hours and fixed directly to the coverslip by adding equal volume of 4% PFA to media and incubating for 20 minutes at room temperature followed by fresh 4% PFA and incubated at room temperature for a further 20 minutes. Fixed cells were washed in PBS and permeabilized using 0.2% Triton X-100 at room temperature for 20 minutes. Proximity ligation assay was performed according to protocol using Duolink® PLA Technology (Sigma Aldrich, St. Louis, MO, USA). 2.13 Site Directed Mutagenesis FLAG-tagged CIC-S (V41G) and ATXN1L (V485A) construct was generated using the QuikChange Lightning Site-Directed Mutagenesis Kit (210518, Agilent Technologies, Santa Clara, California, USA) according to manufacturer’s protocol. The primer sequence was targeted at residue 41 of CIC-S (5’- CCTTGGCACTCCTTAGGCCCCTTCCTGGCACCC - 3’). The 18 wildtype FLAG-tagged CIC-S used was previously described [53]. The primer sequence was targeted at residue 485 of ATXN1L (5’ – GAGCTGAAGCGGGCGGAGGACCTCCAG – 3’). 2.14 Antibody Generation ATNX1L custom antibody was generated in Rabbit against residues 275-295 and 619-636 of ATXN1L by ProSci – Custom Antibodies (Poway, CA, USA). Antibodies were affinity purified from the rabbit serum and validated using siRNA knockdown controls. 2.15 Immunofluorescence Cells were seeded and cultured on glass coverslips for 24-72 hours and fixed directly to the coverslip by adding equal volume of 4% PFA to media and incubating for 20 minutes at room temperature followed by fresh 4% PFA and incubated at room temperature for a further 20 minutes. Fixed cells were washed in PBS and permeabilized using 0.2% Triton X-100 at room temperature for 20 minutes. Fixed cells were blocked in 2.5% BSA for one hour followed by overnight incubation at 4⁰C in primary antibody diluted in blocking solution. Fixed cells were washed 3x with PBS and incubated at room temperature with Alexa Fluor 488 or Alexa Fluor 546 conjugated secondary antibody (Invitrogen), diluted in blocking solution for one hour. Stained cells were washed in PBS and incubated in DAPI (Invitrogen), diluted in PBS for 5 minutes. Stained cells were mounted onto a slide using ProLong™ Gold Antifade Mountant (Invitrogen). Images were captured on Zeiss LSM800 confocal microscope using Airyscan or Colibri Upright LED based microscope using ZEN microscope software. Antibody information can be found in Supplemental Materials 1. 2.16 Cellular Fractionation Nuclear and cytoplasmic fractionations were performed using the NE-PER™ Nuclear and Cytoplasmic Extraction kit (ThermoSci) according to manufacturer’s protocol. 20 µg of cytoplasmic protein and 5µg of nuclear protein were prepared for western blot. 2.17 Fractionation Immunoprecipitation For each cell line, two 15cm plates at roughly 70-80% confluency were harvested and lysed using Cytoplasmic Lysis Buffer (10mM Tris-HCl, 10mM NaCl, 2mM MgCl2, 1mM EDTA, 0.05% NP-40, and 1X Phosphatase and Protease Inhibitor cocktail (PIC) [ThermoSci]) 19 on ice for 10 minutes with agitation every 2 minutes. Nuclei were pelleted at 500rpm for 5 minutes and washed 3x with Wash Buffer (10mM Tris-HCl, 10mM NaCl, 2mM MgCl2, 1mM EDTA, and 1X Phosphatase and Protease Inhibitor cocktail (PIC) [ThermoSci]). Nuclei were lysed using Nuclear Lysis Buffer (250mM NaCl, 20mM Na3PO4, 30mM Na4P2O7, 5mM EDTA, 10mM NaF, 10% Glycerol, 1% NP-40, 1mM DTT, and 1X Phosphatase and Protease Inhibitor cocktail (PIC) [ThermoSci]) on ice. Nuclei were passed through a 25 gage needle 20 times and incubated on ice for 30 minutes with agitation every 5 minutes. Lysates were cleared by centrifugation at 13000rpm for 15 minutes and immunoprecipitation was performed as described above. 2.18 Phosphatase Treatment 40µg of protein lysate prepared using NP40 lysis buffer (150mM NaCl, 1.0% NP-40, 50mM Tris-HCl, pH 8.0) was treated with 1µL (400U) of Lambda Protein Phosphatase (New England BioLabs, Ipswich, MA, USA) at 30⁰C for 2 hours using manufacturer’s recommended reagent quantities. Treated lysates were suspended in loading buffer and reducing agent and denatured at 95⁰C for 8 minutes. 2.19 Drug treatments The HEK and NHA cell lines were treated with 25µg/mL and 100µg/mL of Cycloheximide, respectively. Cells were harvested at the specified time points. Both HEK and NHA cell lines were treated overnight for 16 hours with 10µM of MG132 prior to harvesting. Cell lines were treated with 20ng/mL of recombinant EGF/FGF (Peprotech, Rocky Hill, New Jersey, United States) following >16 hours of serum starvation. Samples were harvested at specified times. LY3214996 was used for ERK inhibition at a concentration of 10nM. Trametinib was used for MEK inhibition at a concentration of 5nM. Both drugs were dissolved in DMSO. Cell lines were treated for the specified amount of time, or 16 hours. 2.20 In Silico Protein Analysis ATXN1L nuclear localization signal prediction was performed using http://nls-mapper.iab.keio.ac.jp with a cut-off score of 3.0 across the whole amino acid sequence [87]. ATXN1L Phosphorylation site prediction was performed using http://scansite3.mit.edu/. CIC-S ubiquitination site prediction was performed using HCKSAAP-UbSite [88]. 20 2.22 mRNA sequencing Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Halden, Germany) according to manufacturer’s protocol. Sample quality control was performed using the Agilent 2100 Bioanalyzer. Samples were then prepped following the standard protocol for the NEBnext Ultra ii Stranded mRNA (New England Biolabs). Sequencing was performed on the Illumina NextSeq 500 with Paired End 42bp × 42bp reads. De-multiplexed read sequences were then aligned to the reference sequence using STAR aligners [89]. Assembly was estimated using Cufflinks (http://cole-trapnelllab.github.io/cufflinks/) [90] through bioinformatics apps available on Illumina Sequence Hub. Two independent monoclonal knockouts with 3 biological replicates from different passages were sequenced for each knockout condition. NHA CICKO cell lines were generated as per LeBlanc et al. [28], and NHA ATXN1LKO cell lines were generated per above from Wong et al. Raw mRNA sequencing files are also available in the Gene Expression Omnibus (GEO) repository (accession number: GSE140471). 2.23 Immunoprecipitation Mass Spectrometry IP-MS of ATXN1LKO NHA cell line B82 was performed using an endogenous CIC antibody. 3 replicates were performed and run alongside IgG negative control IPs. Following IP, proteins were purified and digested using the SP3 method [91]. Replicates and controls were tandem mass tag labeled using a TMT 11-plex reagent set and run through a liquid chromatography/mass spectrometry protocol on an Orbitrap Fusion Tribrid MS platform (Thermo Scientific). Data from the Orbitrap Fusion were processed using the Proteome Discoverer Software. MS2 spectra were searched using Sequest HT [92] against a combined UniProt human proteome database appended to a list of common contaminants. Data were filtered at the peptide spectral match-level to control for false discoveries using a q-value cut off of 0.01 as determined by Percolator [93]. Contaminants were removed prior to downstream analysis. Perseus [94] was used to perform two-sided t-tests to identify proteins that were significantly enriched in each IP experiment compared to their respective IgG controls (FDR < 0.05). 2.24 TRIM25 gene expression analyses siTRIM25 RNA Sequencing data was obtained from Walsh et al. (https://doi.org/10.1016/j.celrep.2017.07.052) [95] and only genes with FDR < 0.05 and 21 directional concordance in both cell lines (BT549 and MDA-MB-231) were considered differentially expressed and used for downstream analyses. 2.25 Immunohistochemistry Slides were deparaffinised using xylene and a decreasing ethanol gradient. Slides were stained using the Ventana BenchMark automated stainer (Roche, Basel, Switzerland). Normal brain and a normal tissue TMA were used as controls. Stained slides were blind scored by two independent neuropathologists. 22 Chapter 3: CIC and ATXN1L Regulate the Cell Cycle 3.1 Introduction CIC has recently been shown have potent tumour suppressor function through the inhibition of several target genes, mainly ETV1/4/5 which are heavily involved in oncogenic and tumour progression processes. Deficiency or dysregulation of CIC has been shown to promote the development of T-cell acute lymphoblastic leukemia [68], Ewing sarcoma [36] and primitive neuroectodermal tumours [39]; lead to increased metastatic potential or progression in hepatocellular carcinoma [33], lung cancer [31], glioblastoma (GBM) [57], prostate cancer [96], neuroblastoma [55]; and treatment resistance in pancreatic adenocarcinoma [29], multiple myeloma [47], and melanoma [46]. The interaction between CIC and ATXN1L has previously been reported in the context of murine lung development which suggested that loss of ATXN1L result in defective lung development through dysregulation of CIC and its downstream target gene ETV4 [65]. However, the interaction and role of ATXN1L in regard to CIC function has not been well characterized nor investigated at a global transcriptomic level. Furthermore, genomic aberrations in ATXN1L have not been previously reported nor investigated in any cancer contexts. In this chapter, we explore the physical and functional association between CIC and ATXN1L using novel isogenic cell line models and bioinformatic analyses of patient data from The Cancer Genome Atlas (TCGA) consortium. ATXN1L loss was found to negatively affect the transcriptional repression of known CIC target genes through dysfunctional CIC promoter binding in vitro. Transcriptome analyses of ATXN1LKO and CICKO cell lines demonstrated upregulation of the MAPK signalling cascade. Transcriptome analyses of TCGA low grade glioma, prostate adenocarcinoma, and stomach adenocarcinoma cohorts with loss of CIC or ATXN1/ATXN1L converged towards upregulation of the mitotic cell cycle through the myc and E2F pathways. This study establishes the CIC-ATXN1-ATXN1L axis as an important regulator of a broad array of pathways that converge upon regulation of the cell cycle. 23 3.2 Results 3.2.1 ATXN1L is a functional interacting partner of CIC To validate the CIC-ATXN1L interaction, we performed immunoprecipitation (IP) of endogenous CIC and ATXN1L human embryonic kidney (HEK), human oligodendroglial (HOG) [97], and normal human astrocytes (NHAs) stably expressing IDH1WT [17] (Figure 3.1A-B and Supplemental Figure 1A-D). Proximity ligation assay was also performed using CICKO NHA cells stably expressing FLAG tagged CIC-S. Interactions between FLAG tagged CIC-S and ATXN1L were found to be present in both the cytoplasm and the nucleus (Figure 3.1C). To investigate the functional redundancy of ATXN1 and ATXN1L in relation to their interactions with CIC, ATXN1 and ATXN1L were independently knocked down in HEK and NHA cell lines using pooled targeted siRNAs (Supplemental Materials 3). Following knockdown of ATXN1L, the relative expression of CIC target genes (ETV1/4/5, DUSP6, and SPRY4) [28] was found to be affected at a greater frequency and to a greater degree compared to ATXN1 knockdown, suggesting that the interaction of CIC and ATXN1L may be more relevant for the repression of CIC target genes (Figure 3.1D-E and Supplemental Figure 1E-F). Figure 3.1 – CIC and ATXN1L physically and functionally interact. (A) Representative western blot of endogenous CIC IP showing co-IP of ATXN1L in HEK cells. A CIC IP in a CICKO cell line (D10) and an IgG IP in the parental HEK were performed as negative controls. (B) Representative western blot of endogenous ATXN1L IP showing co-IP of CIC in HEK cells. An ATXN1L IP in our ATXN1LKO cell line (A30) and IgG IP in the parental HEK were performed as negative controls. (C) Immunofluorescence images of FLAG-ATXN1L PLA in CICKO cell lines stably expressing FLAG or FLAG-tagged CIC-S. White bars denote 10µm. (D) Representative western blot of ATXN1L and ATXN1 knockdown using targeted siRNA in HEK cells. A 24 scrambled siRNA (siCtrl) was used as a negative control and vinculin was used as a loading control. (E) Tukey boxplots showing relative mRNA transcript levels of ATXN1L, ATXN1, CIC, and CIC target genes ETV1/4/5, DUSP6 and SPRY4 measured by RT-qPCR following independent siRNA knockdown of ATXN1L or ATXN1 for 48 hours in HEK cells. Data were collected from 3 independent experiments and p-values were calculated using the two-tailed independent Student’s t-test comparing siATXN1L and siATXN1 to siCtrl. 3.2.3 Generation of ATXN1L CRISPR Knockout Cell Lines Based on our above observations, we sought to further characterize the transcriptional network of ATXN1L. Using CRISPR/Cas9 genome editing technology [98, 99] targeted at exon 1 of ATXN1L, three ATXN1LKO HEK-derived (A10, A30, B21) and one ATXN1LKO NHA-derived (B82) cell lines were generated that showed no detectable ATXN1L protein expression (Figure 3.2A-B). Genome editing at the CRISPR target site were confirmed by Sanger sequencing (data not shown). CIC target genes ETV1/4/5 in HEK ATXN1LKO (Figure 3C) and ETV1 in NHA ATXN1LKO cells (Figure 3.2D) were found to be derepressed, consistent with our ATXN1L siRNA observations. Further, siRNA knockdown of ATXN1 in ATXN1LKO NHA cells did not result in derepression of ETV4/5 (Supplemental Figure 2) which suggests that regulation of ETV4/5 in NHA cells by CIC may be independent of ATXN1/ATXN1L or under additional compensatory regulatory networks. CIC-S protein expression was also found to be reduced in ATXN1LKO lines (Supplemental Figure 3) despite observing no significant decrease in CIC mRNA expression (Figure 3.2C-D). These findings were consistent with previously published data [29, 59, 65]. Reintroduction of ATXN1L-FLAG resulted in slight repression of ETV4 and DUSP6 in HEK cells (Supplemental Figure 4). Figure 3.2 – ATXN1LKO cell lines show derepression of CIC target genes. 25 (A) Representative western blots of CIC and ATXN1L expression in three ATXN1LWT (HEK, N4, N8) and three ATXN1LKO (A10, A30, B21) HEK-derived cell lines generated using CRISPR/Cas9. HEK cells were also treated with a “scrambled” non-targeting siRNA (siCtrl) and an ATXN1L siRNA (siATXN1L) to confirm antibody specificity. Vinculin was used as a loading control. (B) Representative western blots showing CIC and ATXN1L expression in the parental NHA and one ATXN1LKO (B82) NHA-derived cell line using CRISPR/Cas9. Vinculin was used as a loading control. (C) Tukey boxplots showing relative mRNA expression levels of ATXN1L, CIC, ATXN1 and the known CIC target genes ETV1/4/5 in HEK ATXN1LWT (HEK, N4, N8) and ATXN1LKO (A10, A30, B21) cell lines. Data were collected from 3 independent experiments and p-values were calculated using the two-tailed independent Student’s t-test. (D) Tukey boxplots showing relative mRNA expression levels of ATXN1L, CIC, ATXN1 and the known CIC target genes ETV1/4/5 in parental NHA and ATXN1LKO (B82) NHA cell lines. Data were collected from 3 independent experiments and p-values were calculated using the two-tailed independent Student’s t-test. 3.2.3 Transcriptomic analysis of ATXN1LKO and CICKO isogenic cell lines result in convergence on activation of the MAPK pathway To further investigate the reciprocal relationship between CIC and ATXN1L on a transcriptomic level, differential expression analysis (DEA) comparing CICKO or ATXN1LKO cell lines with CICWT/ATXN1LWT cell lines was performed using Affymetrix gene expression microarray data. Genes that were identified to be differentially expressed (DE) in ATXN1LKO HEK (n=619) (Supplemental Table 2A) were compared to DE genes previously identified in CICKO HEK cell lines (n=583) (Supplemental Table 2B) [28]. A total of 110 genes were found to be DE in both contexts (Figure 3.3A and Supplemental Table 2C) including confirmed (ETV1/4/5, DUSP6, SPRY4) and putative (SCN9A, LPAR6, ENPP2, EDIL3, COL2A1) targets of CIC transcriptional regulation [28]. Similarly, comparison of DE genes identified in ATXN1LKO NHA cells (n=914) (Supplemental Table 3A) and CICKO NHA cell lines (n=1163) (Supplemental Table 3B) yielded 299 shared DE genes (Figure 3.3B and Supplemental Table 3C). SHC3 was the only confirmed CIC target gene found to be DE in both NHA CICKO and ATXN1LKO cell lines[28]. Surprisingly, several previously described CIC target genes that were found to be DE in CICKO NHA cells (ETV1/4/5, DUSP6, SPRY4, GPR3) were not found to be DE in NHA ATXN1LKO cells which may be due to compensation by ATXN1 [28, 30, 100], or to regulation by additional regulatory networks independent of ATXN1/ATXN1L. Regardless, the large number of shared DE genes identified in our ATXN1LKO and CICKO NHA cell lines suggests that the relationship between CIC and ATXN1L, in this cellular context, may still be important on a global transcriptomic level. 26 Lastly, DE genes identified in our HEK and NHA ATXN1LKO cell lines were compared to DE genes identified in Type I LGGs (IDH mutated, 1p19q-codeleted) [28] with truncating or nonsense CIC mutations (Type I LGGCIC-Mut) (n=703, Supplemental Table 4A, FDR < 5%), a relevant tumour context. 27 DE genes (p = 0.32) were shared with the HEK dataset, including multiple confirmed and putative CIC target genes (ETV1/4/5, DUSP6, SPRY4, EDIL3, ENPP2, SCN9A) [28] (Supplemental Table 4B), and 32 DE genes (p = 0.79) were shared with the NHA dataset, of which, SHC3 was the only confirmed CIC target gene[28] (Supplemental Table 4C). Gene set enrichment analysis (GSEA) of genes identified as upregulated upon loss of CIC or ATXN1L in all three contexts (HEK, NHA, LGG) converged upon gene signatures indicative of MAPK pathway activation (Figure 3.3C-D, and Supplemental Table 5C). Relative mRNA transcript levels of CIC target genes downstream of MAPK activation showed significant upregulation in our HEK (DUSP6, SPRY4, SPRED2, PTPN9, SHC3) and NHA (SPRY4, SHC3, SPRED1) ATXN1LKO cell lines, while protein expression (ETV4, DUSP6, SPRY4, PTPN9, pMEK, pERK) was variable (Supplemental Figure 5). Figure 3.3 – Loss of CIC or ATXN1L dysregulate similar genes and gene sets in vitro. (A) Scatter plot of shared DE genes identified in HEK ATXN1LKO (x-axis) and HEK CICKO (y-axis) (Supplemental Table 2C). Labeled genes are genes also found to be DE in TCGA Type I LGGs harbouring CIC truncating mutations. (B) Scatter plot of shared DE genes identified in NHA ATXN1LKO (x-axis) and NHA CICKO (y-axis) (Supplemental Table 3C). Labeled genes are genes also found to be DE in TCGA Type I LGGs harbouring CIC truncating mutations. p-values (A/B) were calculated using Fisher’s exact test. (C) UpSet plot showing intersections of terms enriched for in upregulated DE genes identified in NHA CICKO, NHA ATXN1LKO, HEK CICKO, HEK ATXN1LKO and TCGA Type I LGGs with CIC truncating mutations (Supplemental Table 5C). Analysis was performed using GO biological 27 processes terms, Hallmark Gene Sets, and Oncogenic signatures. (D) Heat map showing the top 20 enriched terms for upregulated DE genes identified in NHA CICKO, NHA ATXN1LKO, HEK CICKO, HEK ATXN1LKO and TCGA LGG Type I with CIC truncating mutations using Hallmark gene sets and Oncogenic signatures. Term IDs from MSigDB are shown. Terms related to a MAPK activation signature are denoted with a red arrow. 3.2.4 Deletions in ATXN1/ATXN1L or CIC lead to convergent transcriptomic changes. CIC aberrations have recently been associated with poor prognosis in multiple cancer types such as Type I LGG [28, 30], pancreatic [29], lung [31], sarcoma [32, 36], hepatocellular carcinoma [33], prostate adenocarcinoma (PRAD) [96, 100], and stomach adenocarcinoma (STAD) [101]. CIC alterations in these cancers have been correlated with increased metastatic potential, treatment resistance, and invasiveness. Based on our in vitro CIC-ATXN1L observations, we hypothesized that aberrations in ATXN1L may be exploited by multiple cancer types resulting in biological consequences similar to aberrations in CIC. Analysis of TCGA identified a high frequency of ATXN1L copy number variations (CNVs) including heterozygous and homozygous deletions observed in the PRAD (173/492), LGG (12/283), and STAD (69/287) cohorts. To explore the relationship between CIC and ATXN1L alterations in primary cancer settings, copy number and gene expression data were obtained from TCGA and differential expression analyses between samples with copy number loss of CIC or ATXN1/ATXN1L and samples with intact CIC and ATXN1L were performed. Table 3.1 describes the CIC, ATXN1, and ATXN1L status of each cohort used for DEA. Table 3.1 – TCGA analysis cohorts Label Cohort ATXN1 Status ATXN1L Status CIC Status Type I LGGCIC-Mut Low Grade Glioma Wildtype Wildtype Truncating and Nonsense Mutations * Type I LGGCIC-WT Low Grade Glioma Wildtype Wildtype Wildtype * Type II LGGCIC-Del Low Grade Glioma Wildtype Wildtype Homozygous and Heterozygous Deletions Type II LGGATXN1/ATXN1L-Del Low Grade Glioma Homozygous and Heterozygous Deletions Homozygous and Heterozygous Deletions Wildtype ** Type II LGGWT Low Grade Glioma Wildtype Wildtype Wildtype PRADCIC-Del Prostate Adenocarcinoma Wildtype Wildtype Homozygous and Heterozygous Deletions PRADATXN1L-Del Prostate Adenocarcinoma Wildtype Homozygous and Heterozygous Deletions Wildtype 28 PRADWT Prostate Adenocarcinoma Wildtype Wildtype Wildtype STADCIC-Del Stomach Adenocarcinoma Wildtype Wildtype Homozygous and Heterozygous Deletions STADATXN1-Del Stomach Adenocarcinoma Homozygous and Heterozygous Deletions Wildtype Wildtype STADATXN1L-Del Stomach Adenocarcinoma Wildtype Homozygous and Heterozygous Deletions Wildtype STADATXN1/ATXN1L-Del Stomach Adenocarcinoma Homozygous and Heterozygous Deletions Homozygous and Heterozygous Deletions Wildtype *** STADWT Stomach Adenocarcinoma Wildtype Wildtype Wildtype *CIC Status is for the remaining allele and exists in a hemizygous state due to the 1p19q co-deletion **Homozygous/Heterozygous deletions in ATXN1 or ATXN1L ***Homozygous deletions in ATXN1 or ATXN1L,or Heterozygous deletions in ATXN1 and ATXN1L DEA comparing PRADCIC-Del (n=34) and PRADWT (n=190) identified 842 DE genes (Supplemental Table 6A, FDR < 5%, fold change > 1.5). Samples with homozygous deletions of CIC were included in this analysis regardless of ATXN1L status due to the complete loss of CIC. Similarly, DEA comparing PRADATXN1L-Del (n=90) and PRADWT samples identified 811 DE genes (Supplemental Table 6B, FDR < 5%, fold change > 1.5). A total of 232 genes were shared between the two analyses (Figure 3.4A/Supplemental Table 6C). Prior to DEA of the LGG cohort, tumours were stratified into Type I (IDH mutated, 1p19q-codeleted), Type II (IDH mutated, 1p19q-retained), and Type III (IDH wildtype) tumours based on canonical genomic signatures [4, 11]. DEA investigating samples with ATXN1L deletions was not performed in Type I LGGs due to the lack of a normal comparator caused by 1p19q co-deletion (loss or aberration in at least one allele of CIC in every sample). However, ATXN1 (n=7) and ATXN1L (n=4) deletions were identified at similar rates and found to be mutually exclusive in Type II LGG, suggesting that, in this context, loss of either gene may result in CIC instability. Therefore, DEA was performed comparing Type II-LGGATXN1/ATXN1L-Del (n=11) and Type II LGGWT (n=88) samples which identified 611 DE genes (Supplemental Table 7A, FDR < 5%). Similarly, DEA was performed comparing Type II-LGGCIC-Del (n=30) and Type II LGGWT samples which identified 1538 DE genes (Supplemental Table 7B, FDR < 5%). A total of 132 genes were shared between the two analyses (Figure 3.4B/Supplemental Table 7C). 29 Figure 3.4 – Differentially expressed genes identified in tumours with ATXN1 and/or ATXN1L aberrations and CIC aberrations show overlap and directional concordance. (A) Scatter plot of shared DE genes identified in PRADATXN1L-Del (x-axis) and PRADCIC-Del samples (y-axis) (Supplemental Table 6C). (B) Scatter plot of shared DE genes identified in Type II-LGGATXN1/ATXN1L-Del (x-axis) and Type II-LGGCIC-Del (y-axis) (Supplemental Table 7C). (C) Scatter plot of shared DE genes identified in STADATXN1L-Del (x-axis) and STADCIC-Del (y-axis) (Supplemental Table 8E). (D) Scatter plot of shared DE genes identified in STADATXN1-Del (x-axis) and STADCIC-Del (y-axis) (Supplemental Table 8F). (E) Scatter plot of shared DE genes identified in STADATXN1/ATXN1L-Del (x-axis) and STADCIC-Del (y-axis) (Supplemental Table 8G). *Labeled genes in each scatter plot (A-E), were found to be DE and shared in at least 2/3 cancer contexts. (Table 3.2). p-values were calculated using Fisher’s exact test. Lastly, deletions in ATXN1 and ATXN1L were identified in 118/287 samples in the TCGA STAD cohort but were not found to be mutually exclusive with CIC deletions (49/287). DEA comparing STADCIC-Del (n=10) and STADWT (n=126) identified 3223 DE genes, including the canonical CIC target genes ETV4/5 (Supplemental Table 8A, FDR < 5% and fold change > 1.5). Due to the comparable frequency and large number of ATXN1 (n=49/287) and ATXN1L (n=69/287) deletions in the STAD cohort, three independent DEAs were performed comparing STADATXN1-Del (n=20), STADATXN1L-Del (n=22), or STADATXN1/ATXN1L-Del (n=15) and STADWT to investigate the differences and similarities between ATXN1 and ATXN1L function within the context of STADs. DEA of STADATXN1-Del, STADATXN1L-Del, and STADATXN1/ATXN1L-Del identified 2072, 410, and 1557 DE genes, respectively (Supplemental Tables 8B, 8C, 8D, FDR < 5%, fold change > 1.5). Surprisingly, differential expression of CIC target genes (ETV1/4/5) was not 30 identified in these analyses. Comparisons between STADCIC-Del versus STADATXN1-Del, STADATXN1L-Del, and STADATXN1/ATXN1L-Del yielded 1153, 148, and 537 shared DE genes, respectively (Figure 3.4C-E/Supplemental Tables 8E, 8F, 8G). Shared DE genes identified in all three cohorts (PRAD, LGG, STAD) displayed a high degree of directional concordance. 3.2.5 Deletions in ATXN1/ATXN1L or CIC lead to convergent dysregulation of cellular processes and gene set Using multi-list GSEAs, we observed a high degree of intersection for terms enriched in genes found to be upregulated in tumours with CIC loss and tumours with ATXN1/ATXN1L loss (Figure 3.5A-C). In the PRAD and Type II LGG cohorts, these terms converged upon processes associated with nuclear division, DNA replication, and progression through the mitotic cycle (Supplemental Figure 6A, 6B/Supplemental Table 9A, 9B). Similarly, terms enriched for genes identified as upregulated in the STAD cohort related to upregulation of myc and mTOR (Supplemental Figure 6C/Supplemental Table 9C). GSEA of upregulated DE genes identified in all three cohorts (in conjunction), yielded terms related to cellular mitosis, DNA replication, and activation of E2F, myc and mTOR (Figure 3.5D-E/Supplemental Table 9D). Upregulation of polycomb repressive complex (PRC2) targets and terms related to DNA conformation were also enriched. Across the three TCGA cohorts investigated, 49 shared DE genes were seen in at least two of the three cohorts analysed (Table 3.2). Amongst these 49 genes, we observed upregulation in several genes related to nuclear division and cytokinesis including AURKA, CDK4, NUSAP1, BUB1, TPX2, NUF2 and CDCA3. Of these 49 shared genes, the majority (45, 92%) also showed directional concordance between cohorts. Gene enrichment analysis of upregulated shared DE genes once again revealed terms related to mitotic cell cycle progression/nuclear division, hallmark E2F targets and regulation of chromatin conformation (Figure 3.5F/Supplemental Table 10). 31 Figure 3.5 –Tumours with ATXN1 and/or ATXN1L aberrations and CIC aberrations display overlap in gene enrichment signatures. (A) UpSet plot showing intersections of terms enriched in upregulated DE genes identified in Type II-LGGATXN1/ATXN1L-Del and Type II-LGGCIC-Del (Supplemental Table 9A). (B) UpSet plot showing intersections of terms enriched in upregulated genes identified in PRADATXN1L-Del and PRADCIC-Del (Supplemental Table 9B). (C) UpSet plot showing intersections of terms enriched in upregulated genes identified in STADATXN1L-Del, STADATXN1-Del, STADATXN1/ATXN1L-Del, and STADCIC-Del (Supplemental Table 9C). *Gene set enrichment analyses for (A), (B), and (C) were performed using GO biological processes terms, hallmark gene sets, and oncogenic signatures. (D) UpSet plot showing intersections of GO biological processes terms enriched in upregulated genes identified in all three TCGA cohorts analyzed with ATXN1 and/or ATXN1L, or CIC loss (Supplemental Table 9D). (E) Heat map showing the top 20 enriched terms for upregulated DE genes identified in all three TCGA cohorts analyzed with ATXN1 and/or ATXN1L, or CIC loss using only hallmark gene sets and oncogenic signatures. Term IDs from MSigDB are shown. (F) Terms enriched for upregulated shared genes that were identified in at least 2/3 cohorts analyzed (Supplemental Table 10). Terms from GO Biological Process terms, Hallmark gene sets and Oncogenic signatures were used. Table 3.2 – Shared DE Genes across TCGA cohorts analysed Log2Fold Change Type II LGG PRAD STAD Cohort Frequency Gene Entrez.ID CIC-Del ATXN1/ATXN1L-Del CIC-Del ATXN1L-Del CIC-Del ATXN1-Del ATXN1L-Del ATXN1/ATXN1L-Del Up Down ACTN2 88 - - -0.91 -1.18 -2.95 -1.38 -1.09 - 0 2 ADAMTSL3 57188 -0.78 -0.85 - - -2.13 -1 - - 0 2 ATOH7 220202 - - 0.7 0.91 1.72 - - 1.09 2 0 ATP1A2 477 - - -0.83 -0.64 -3.8 -2.81 - - 0 2 AURKA 6790 1.04 0.65 - - 0.87 0.75 0.67 1.07 2 0 BUB1 699 1.17 0.83 0.71 0.66 - - - - 2 0 CAMK2A 815 - - -0.8 -0.86 -2.05 -1.33 - - 0 2 CASQ1 844 - - -1.31 -0.9 -2.76 -2.3 - - 0 2 CDCA3 83461 0.59 0.82 0.63 0.6 - - - - 2 0 CDK4 1019 0.61 0.82 - - 0.73 0.64 - 0.82 2 0 CDON 50937 - - 0.65 0.6 -1.42 -1.55 - -1.39 1 1 CHI3L2 1117 - - -0.99 -0.76 -2.56 -1.47 -0.93 -1.39 0 2 COL4A6 1288 -0.65 -0.89 -0.68 -0.76 -2.85 -2.11 -0.96 - 0 3 DNMT3B 1789 - - 1.14 0.68 2.11 0.8 - - 2 0 EME1 146956 - - 0.58 0.65 1.14 0.69 - - 2 0 FAM107A 11170 - - -0.58 -0.67 -2.41 -1.4 -1.16 0 2 FAM124A 220108 - - -0.71 -0.66 -1.92 -1.36 - - 0 2 FLNC 2318 - - -0.66 -0.66 -3.28 -2.77 - - 0 2 HES6 55502 - - 0.71 1.34 1.36 1.24 2 0 LDB3 11155 - - -0.73 -0.64 -2.91 -2.88 - - 0 2 LINGO3 645191 - - 0.75 0.59 -1.94 -1.24 -0.98 - 1 1 LIX1 167410 - - -1.33 -0.84 -1.99 -1.5 - - 0 2 MTBP 27085 0.63 0.72 0.74 0.59 - - - - 2 0 MYBPC2 4606 - - -0.77 -0.97 -2.51 -1.47 - - 0 2 MYOCD 93649 - - -0.7 -0.61 -3.21 -2.43 - - 0 2 MYOT 9499 - - -1.18 -1.12 -1.59 -2.02 - - 0 2 MYOZ2 51778 - - -0.85 -1.29 -2.33 -1.5 - - 0 2 NHLH1 4807 - - 0.58 0.58 1.59 0.68 0.88 0.94 2 0 NKAPL 222698 - - -0.77 -0.62 -1.97 -1.02 - - 0 2 NTN1 9423 - - -0.65 -0.58 -1.51 -1.51 - -1.41 0 2 NUF2 83540 1.17 0.77 0.89 1.02 - - - - 2 0 NUSAP1 51203 1.17 0.9 0.85 0.63 - - - - 2 0 PCP4L1 654790 - - -0.86 -0.7 -3.2 -2.74 - - 0 2 PGM5 5239 - - -0.74 -0.65 -3.19 -2.54 - - 0 2 PPARGC1A 10891 - - -0.64 -0.7 2.28 1.32 - - 1 1 33 PRIMA1 145270 - - -0.84 -0.59 -2.71 -2.29 - - 0 2 RAE1 8480 0.14 0.27 - - 1.00 0.72 0.34 0.86 2 0 RNFT2 84900 - - 1.13 0.6 1.37 0.85 - - 2 0 SLC2A5 6518 - - -0.78 -1.14 -2.1 -0.77 - - 0 2 SNRPB 6628 0.32 0.21 - - 0.82 0.56 - 0.88 2 0 SSPN 8082 -0.45 -0.65 - - -1.47 -0.92 - - 0 2 SYNPO2 171024 -0.56 -0.71 -0.60 -0.54 -3.90 -2.92 - - 1 2 SYNPO2L 79933 0.57 1.08 -0.72 -0.87 - - - - 1 1 TGFBR3 7049 -0.44 -0.71 - - -1.14 -1.13 - - 0 2 TMEM47 83604 -0.42 -0.61 - - -1.37 -1.09 - - 0 2 TPX2 22974 1.29 0.81 0.87 0.62 - - - - 2 0 UBE2C 11065 1.23 0.82 - - 1.5 0.82 - 1.32 2 0 VGLL3 389136 - - -1.03 -1.03 -1.34 - - -1.05 0 2 YJEFN3 374887 - - 0.87 0.6 0.81 0.88 0.74 1.23 2 0 3.2.6 CIC-ATXN1L interaction facilitates CIC-DNA binding In addition to promoting CIC protein stability, ATXN1L may also facilitate the promoter binding efficiency of CIC. To assess this, targeted chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP-qPCR) was performed in parental HEK, CICKO (D10) and ATXN1LKO (A30) cells. Enrichment of CIC binding to several sites within the promoter regions of ETV4, DUSP6 and SPRY4 were found to be significantly reduced in ATXN1LKO (HEK-A30) cells compared to the parental HEK cell line (Supplemental Figure 7). ChIP-PCR was also performed using a FLAG-tagged, V41G CIC-S mutant (CIC-SV41G) which lacks the ability to interact with ATXN1L (Figure 3.6A) [102]. Enrichment of CIC-SV41G binding to the promoter regions of CIC target genes (ETV4, DUSP6, SPRY4) was comparable to the enrichment observed in the ATXN1LKO cell line A30 (Figure 3.6B), further indicating that interaction with ATXN1L contributes to CIC’s binding to the promoter regions of target genes. Therefore, de-repression of CIC target genes observed in ATXN1LKO cells, described above, may be attributed to a decrease in CIC binding to the promoter regions of these genes. Figure 3.6 – ATXN1L interaction with CIC facilitates CIC binding to promoter regions of CIC target genes. (A) FLAG-tagged Immunoprecipitation of wildtype CIC-S (WT) and V41G mutant CIC-S (V41G) showing lack of both ATXN1L and ATXN1 co-IP with the mutant form of CIC. An IgG IP was used as a control. (B) Tukey boxplots showing enrichment of FLAG-tagged wildtype CIC (WT) and V41G mutant CIC (V41G) binding to promoter regions of CIC target genes (ETV4, DUSP6, SPRY4) relative to a negative control region (NCR1) 35 following FLAG-tagged CIC ChIP-qPCR in CICKO HEK cells stably expressing wildtype CIC-S (WT), FLAG (FLAG), and V41G mutant CIC-S (V41G). Data were collected over 3 independent experiments and p-values were calculated using the two-tailed independent Student’s t-test. p-values are displayed in the table to the right. 3.3 Discussion This study is the first to have explored, in depth, the functional relationship between ATXN1L and CIC at the proteomic and transcriptomic level in isogenic cell lines and several distinct primary cancer settings. Functional in vitro studies and transcriptomic profiling of CICKO and ATXN1LKO isogenic cell lines show that the post-translational stability and transcriptionally repressive activity of CIC relies heavily on its physical interaction with ATXN1L. Upregulation of multiple CIC target genes downstream of the MAPK signaling cascade were observed in our ATXN1LKO transcriptomic analyses, indicating that ATXN1L may be an important mediator of CIC in feedback loops responsible for regulating MAPK signalling [28, 29]. The importance of the CIC-ATXN1-ATXN1L axis in cancer is becoming increasingly evident as one that can be exploited by RTK-driven cancers to subvert inhibition of upstream druggable targets such as EGFR and MEK, as already seen in pancreatic cancer [29, 35]. Establishing the status of this axis may become a powerful prognostic indicator in a multitude of RTK driven cancers. Although the exact mechanism through which ATXN1L stabilizes CIC is unknown, our results indicate that proteasomal degradation of CIC, in the absence of ATXN1L, may be a potential candidate of targeted therapies aimed at increasing CIC stability and repressive function. Further highlighting the context-dependent relationship and interplay between CIC, ATXN1, and ATXN1L, DEA and GSEA of three TCGA cohorts (PRAD, Type II LGG, and STAD) also showed that tumours with loss of ATXN1 and/or ATXN1L demonstrated transcriptomic changes similar to those found in tumours with CIC loss. In the PRAD cohort, ATXN1L deletions were observed at a much higher frequency compared to ATXN1 deletions, indicating the possibility that, in this context, ATXN1L may be the primary interactor of CIC. Conversely, ATXN1 and ATXN1L deletions were seen at similar frequencies in both Type II LGG and STAD suggesting that, in these contexts, both proteins may play equally important roles in regulating CIC function. Notably, hemizygous loss of ATXN1 or ATXN1L in STAD did not appear sufficient to dysregulate CIC function, while homozygous loss of ATXN1 or ATXN1L, or hemizygous loss of both ATXN1 and ATXN1L did have more important effects, which may indicate that in the context of STAD, a higher threshold of ATXN1/ATXN1L loss is required for CIC dysfunction. The distinct differences in the relationship between CIC and ATXN1/ATXN1L 36 between these three cohorts may indicate that the CIC-ATXN1-ATXN1L axis may be more complex than previously appreciated and greatly dependent on cellular context. Although our TCGA analyses of Type II LGGs, PRAD and STAD samples did not yield gene signatures indicative of MAPK activation as expected [28, 29, 35, 52], our data instead suggests that aberrations in the CIC-ATXN1-ATXN1L axis may contribute to cellular growth and proliferation through the upregulation of myc and E2F targets. The E2F transcription factor family has been implicated in promoting cell cycle transitions and driving aberrant cellular proliferation in multiple contexts including prostate cancer and gliomas [103-107]. Aberrant activation of myc has also been described in up to 40% of gastric cancers and is associated with poor prognosis due to promotion of a cancer stem like state [101, 108, 109]. GSEA also identified many terms related to cellular division; including chromosome condensation and segregation, DNA replication, and G2/S phase transitions. Together, these gene signatures suggest that aberrations in the CIC-ATXN1-ATXN1L axis may highlight a subpopulation of E2F-dependent tumours that may be candidates for targeted treatment through the cyclin/Rb/E2F pathway [110, 111]. Terms related to upregulation of polycomb group (PRC1/PRC2) targets were also found to be enriched in all three cohorts. The polycomb group complexes are chromatin modifying complexes essential for embryonic development and stem cell self-renewal and thus are frequently dysregulated in multiple cancers [112, 113]. Several polycomb group target genes were found to be dysregulated in our differential expression analyses including several members of the HOX family (HOXA3, HOXA4, HOXB3, HOXB9, HOXD10) and the MT1 family (MT1A, MT1E, MT1G, MT1H) and members of the polycomb group complexes (EZH2, CBX8) [114]. Here, we have shown aberrations in ATXN1/ATXN1L or CIC in three distinct contexts are associated with gene signatures indicative of cellular growth and proliferation independent of the MAPK activation previously identified in many CIC inactivated contexts [28, 29, 31, 35, 52], which may indicate that CIC and ATXN1L act as regulators of cell cycle in a much broader context than previously appreciated. This study has expanded on the current knowledge of CIC and ATXN1/ATXN1L in cancer biology and highlighted the unique, context-specific, reciprocal relationship between CIC and ATXN1/ATXN1L. Further study into this interaction may provide insight into the mechanisms responsible for mediating the functional relationship between CIC and ATXN1/ATXN1L and further our understanding of the prognostic and therapeutic value of the CIC-ATXN1/ATXN1L axis. 37 Chapter 4: CIC and ATXN1L Interaction Forms a Post-Translational Functional and Reciprocal Relationship 4.1 Introduction In the previous chapter, chapter 3, we revealed a reciprocal functional transcriptomic relationship between CIC and ATXN1L frequently exploited by cancer to dysregulate the cell cycle. In addition to these transcriptomic changes, we also observed a decrease in CIC protein which was not reflective of CIC mRNA expression in our ATXN1LKO cell lines. While many studies have investigated the role of CIC loss of function mutations in cancer, far fewer studies have investigated the role that post-translational CIC dysregulation plays in promoting oncogenesis or cancer progression. In glioblastoma, post-translational CIC inactivation has been observed as a result of ERK phosphorylation and subsequent degradation through interaction with the E3-ligase PJA1 [57]. Conversely, post-translational dysregulation of CIC through loss of ATXN1L has been observed in pancreatic cancer and melanoma resulting in a treatment resistant phenotype [29]. ATXN1L is a member of the Ataxin protein family which has been shown to be a potent regulator of CIC function in both murine development and cancer [29, 54, 65]. Previous studies investigating murine development have indicated some level of functional redundancy between ATXN1L and its homologue ATXN1. However, these studies have also highlighted that loss of ATXN1L affected CIC function much more profoundly and robustly compared to loss of ATXN1, which is also consistent in cancer [65, 66]. Although several studies have consistently reported that the loss of ATXN1L results in the post-translational dysregulation of CIC stability and function, the exact mechanism responsible for this phenomenon remains unknown. In this chapter, we explore the physical relationship between CIC and ATXN1L, and interrogate the mechanism responsible for CIC instability following the loss of ATXN1L. We uncover a reciprocal physical relationship between CIC and ATXN1L; and an ERK independent mechanism whereby the interaction between CIC and ATXN1L protects CIC from degradation by the E3-ligase TRIM25. Utilising both in vitro gene expression data from genetically modified cell lines and patient derived gene expression data from The Cancer Genome Atlas (TCGA), TRIM25 and CIC-ATXN1L were found to antagonistically regulate similar gene sets related to the cell cycle. 38 4.2 Results 4.2.1 Loss of ATXN1L promotes the proteasomal degradation of CIC Several studies in human cell lines and mouse studies have reported decreased CIC protein expression following loss of ATXN1L [29, 54, 65]. In concordance with these studies, decreased CIC protein expression was observed in our ATXN1LKO cell lines [54] which could be partially rescued to levels similar to the untreated parental cell line following treatment with MG132, an inhibitor of the 20S subunit of the proteasome (Figure 4.1A), or ectopic expression of a FLAG-tagged ATXN1L construct (Figure 4.1B). Inhibition of translation using cycloheximide also revealed a decrease in CIC protein half-life in ATXN1LKO cells compared to ATXN1LWT cells (Supplemental Figure 8). This decrease in CIC protein expression was found to be exclusive to ATXN1L loss and was not observed following siRNA knockdown of ATXN1, a homologue of ATXN1L (Figure 4.1C). Additionally, introduction of mutant FLAG-tagged ATXN1L-V485A, which is unable to interact with CIC [102] (Supplemental Figure 9A), also did not rescue CIC protein expression (Figure 4.1D). Density gradient fractionation of ATXN1LWT and ATXN1LKO cell lysates revealed an increase of CIC in higher molecular weight fractions that also contain SUG1, a proteasome subunit (Supplemental Figure 9B). Using immunoprecipitation, an increased accumulation of ubiquitin associated with CIC was also observed in ATXN1LKO cells which have decreased CIC expression levels (Figure 4.1E, F). Similarly, using proximity ligation assay (PLA), interaction between CIC and ubiquitin was observed to increase following treatment with MG132 in ATXN1LKO (Supplemental Figure 9C, D) or siRNA knockdown of ATXN1L in ATXN1LWT cells (Figure 4.1G, H). 39 Figure 4.1 – Loss of ATXN1L results in CIC instability (A) Representative Western blot of ATXN1LWT (HEK) and ATXN1LKO cell lines (A10, A30, B21) treated with MG132. DMSO used as negative control. Below: barplots showing quantification of CIC expression. (B) Representative Western blot of ATXN1LWT (HEK) and ATXN1LKO cell lines (A10, A30, B21) cell lines ectopically over-expressing FLAG-tagged ATXN1L. Below: barplots showing quantification of CIC expression. (C) Representative Western blot of siRNA knockdown of ATXN1 and ATXN1L in NHA. Scrambled siRNA used as negative control. Below: barplots showing quantification of CIC expression. (D) Representative Western blot of ATXN1LWT (HEK) and ATXN1LKO cell lines (A30) ectopically over-expressing FLAG-tagged WT ATXN1L or mutant ATXN1L-V485A. (E) Barplots showing quantification of Ubiquitin Western blots normalized to CIC Western blots. (F) Representative Western blot of CIC immunoprecipitation in ATXN1LWT (NHA) and ATXN1LKO (B82) cell lines treated with MG132. DMSO used as negative control. (G) Immunofluorescence images of proximity ligation assay showing CIC-Ubiquitin interaction in ATXN1LWT (NHA) and ATXN1LKO (B82) cell lines treated with MG132. DMSO used as negative control. White bars denote 10µm. (H) Tukey boxplots showing quantification of number of foci/cell. (*) Western blot quantifications were collected from three independent experiments and were normalized to vinculin unless specified otherwise. Error bars represent one standard deviation. PLA quantifications were collected from 65 individual cells. p-values were calculated using the two-tailed independent Student’s t-test. Statistically significant values are denoted (* = p < 0.05, ** = p < 0.01, *** = p < 0.001). 40 4.2.3 Loss of CIC leads to dysregulation of ATXN1L Previous studies have investigated the effects of ATXN1/ATXN1L loss on CIC function without further probing the potential reciprocal effects of CIC loss on ATXN1/ATXN1L. Western blots of previously generated CICKO cell lines (HEK-D10, HOG-F11, NHA-A2) [28], revealed a decrease in the apparent molecular weight of ATXN1L compared to their respective CICWT parental cell lines (Figure 4.2A). In silico analysis of ATXN1L peptide sequence (Supplemental Figure 10) suggested that this decrease may be attributed to the loss of post-translational modification(s) (PTMs) such as phosphorylation [115]. Treatment of CICWT (HEK, NHA) cell lysates with lambda protein phosphatase (LPP), an indiscriminate phosphatase, resulted in a reduction of ATXN1L apparent molecular weight consistent with that observed in CICKO cell lines, suggesting that the absence of CIC results in loss or lack of ATXN1L phosphorylation (Figure 4.2B). Cellular fractionation also revealed that ATXN1L exhibited cytoplasmic localization in CICKO cells (HEK-D10, NHA-A2, HOG-F11) compared to their respective parental cell lines where ATXN1L was mostly localized to the nucleus (Figure 4.2C). This was also confirmed through immunofluorescence of endogenous ATXN1L (Figure 4.2D and Supplemental Figure 11A-B). To further investigate the relationship between CIC loss and ATXN1L phosphorylation/localization, endogenous ATXN1L IP was performed following cellular fractionation of CICWT (HEK and NHA) and CICKO (HEK-D10 and NHA-A2) cells. Surprisingly, phosphorylated (Serine/Threonine) ATXN1L appeared to localize primarily to the cytoplasm (Figure 4.2E and Supplemental Figure 11C). Higher molecular weight ATXN1L was still detected in the nuclear fraction of CICWT cells which may indicate that the nuclear localization of ATXN1L is regulated by an alternative, unknown PTM. In addition to decreased molecular weight, CIC loss also appeared to affect the protein stability of ATXN1L. CICKO cells (HEK-D10 and NHA-A2) treated with the protein synthesis inhibitor cycloheximide (CHX) resulted in decreased ATXN1L protein expression at 24 hours post treatment, compared to the treated CICWT parental cell lines (Figure 4.2F and Supplemental Figure 11D). Lastly, to investigate the potential downstream consequences of ATXN1L dysregulation resulting from CIC loss, the relative expression of the known ATXN1L target gene, HEY1 [75], was assessed and found to be derepressed in the NHA CICKO cell line A2, but significantly repressed in the HEK CICKO cell line D10 (Figure 4.2G). In contrast, HEY1 was derepressed in the ATXN1LKO cell lines HEK-A30 and NHA-B82 (Supplemental Figure 11E). Reintroduction of 41 a FLAG-tagged short isoform of CIC (CIC-S) into CICKO cells (HEK-D10) rescued the decrease in ATXN1L molecular weight, restored some nuclear ATXN1L localization (Figure 4.2H), and returned HEY1 expression to levels comparable to that observed in the parental HEK cell line (Figure 4.2I). Although the role of ATXN1L phosphorylation remains enigmatic, these results indicate that loss of CIC leads to the dysregulation of ATXN1L localization, phosphorylation/PTM status, and protein stability. Figure 4.2 – Loss of CIC is associated with ATXN1L dysregulation (A) Representative western blots of parental (HEK, NHA, HOG) and CICKO (HEK-D10, NHA-A2, HOG-F11) cell lines showing a reduction in apparent molecular weight of ATXN1L in CICKO cell lines. Vinculin was used as a loading control. (B) Representative western blot of parental (HEK, NHA) and CICKO (HEK-D10, NHA-A2) cell lines showing the apparent molecular weight of ATXN1L following control or phosphatase treatment (+LPP). Vinculin was used as a loading control. (C) Representative western blots of parental (HEK, NHA, HOG) and CICKO (HEK-D10, NHA-A2, HOG-F11) cell lines following fractionation showing differences in the apparent molecular weight and localization of ATXN1L in the cytoplasmic (Cyto) and nuclear (Nuc) fractions. Vinculin was used as a cytoplasmic control and Lamin-B1 was used as a nuclear control. (D) Immunofluorescent imaging of endogenous ATXN1L (orange) in CICWT (NHA) and CICKO (NHA-A2) cell lines showing ATXN1L localization. Tubulin (green) was stained to show cell morphology and nuclei were stained with DAPI (blue). White bars seen on the merged image represent 10μm. (E) Western blot of endogenous ATXN1L IP following cellular fractionation (Cytoplasm and Nuclear) of CICWT (HEK) and CICKO (D10) cells. Phosphorylated Serine/Threonine (pSer/Thr) was blotted to show the localization of phosphorylated ATXN1L in the presence or absence of CIC. An IgG IP was 42 performed as a negative control. Vinculin was blotted as a cytoplasmic control and Lamin-B1 was blotted as a nuclear control. (F) Representative Western blot of CICWT (NHA) and CICKO (A2) cells treated with 100µM of cycloheximide over a 24 hour time course showing ATXN1L stability in the presence and absence of CIC. Vinculin was blotted as a loading control and P53 and CHK1 were blotted as protein synthesis inhibition controls. (G) Tukey boxplots showing relative mRNA transcript levels of HEY1 in parental (HEK, NHA, HOG) and CICKO (HEK-D10, NHA-A2, HOG-F11) cell lines. Data were collected from 3 independent experiments (passages) and p-values were calculated using the two tailed independent Student’s t-test comparing CICKO cell lines to their respective parental cell line. (H) Representative western blot of ATXN1L showing apparent molecular weight and cellular localization following reintroduction of CIC-S-FLAG into HEK-D10 (CICKO) cells. Vinculin was used as a cytoplasmic control and Lamin-B1 was used as a nuclear control. (I) Tukey boxplots showing relative mRNA transcript levels of HEY1 following reintroduction of CIC-S-FLAG into HEK-D10 (CICKO) cells. Data were collected from 3 independent experiments and p-values were calculated using the two tailed independent Student’s t-test comparing FLAG and CIC-S-FLAG treated cells to the parental HEK cells. 4.2.3 ATXN1L mediated CIC instability is independent of ERK activity Previous studies have established ERK as a downstream component of the mitogen activated protein kinase (MAPK) pathway important for regulating the protein expression, localization, and function of CIC through phosphorylation [21, 25-27, 57, 116]. Additionally, transcriptomic analyses of ATXN1LKO cell lines and The Cancer Genome Atlas (TCGA) patient samples have shown upregulation of gene signatures related to MAPK signaling (Figure 4.3A) [54]. To further interrogate the mechanism of CIC degradation following ATXN1L loss, we investigated whether ERK activity contributes to the regulation of CIC function in relation to ATXN1L status. Phosphorylated ERK (pThr202/Tyr204) was found to be decreased in our ATXN1LKO cell lines compared to the ATXN1LWT cell lines, detected using ELISA (Figure 4.3B) and Western Blot (Figure 4.3C). Despite the lack of increased ERK activity, ATXN1LKO cell lines displayed mRNA upregulation of the many CIC target genes (ETV1/4/5, DUSP6, SPRY4) which are downstream of ERK signaling (Figure 4.3D), consistent with previously published data [54]. Further, dual inhibition of MEK and ERK using small the molecule inhibitors trametinib and LY3214996, respectively; did not rescue CIC protein expression in ATXN1LKO cell lines (Figure 4.3E) or following siRNA knockdown of ATXN1L in ATXN1LWT cell lines (Figure 4.3F). 43 Figure 4.3 – ATXN1L mediated CIC instability is independent of ERK activity (A) Heatmap showing the top 20 upregulated gene sets in ATXN1LKO NHA and HEK cell lines. Terms related to the MAPK pathway are bolded. (B) ELISA quantification of phosphorylated ERK (pThr202/Tyr204) in ATXN1LWT and ATXN1LKO NHA and HEK cell lines. Quantifications were normalized to total ERK. (C) Representative Western blot of phosphorylated ERK (pThr202/Tyr204) in ATXN1LWT (NHA) and ATXN1LKO (B82, B16, B21) cell lines. (D) Relative mRNA expression of CIC target genes ETV1/4/5, DUSP6, SPRY4 in ATXN1LWT (NHA) and ATXN1LKO (B82, B16, B21) cell lines. Gene expression was normalized to TBP and the parental ATXN1LWT (NHA) cell line was used as a relative control. (E) Representative Western blot of ATXN1LWT (HEK) and ATXN1LKO (A10, A30, B21) cell lines treated with MEK/ERK inhibitors trametinib/LY3214996. DMSO was used as a negative control. Below: barplot quantifications of CIC expression. Quantifications were normalized to Vinculin. (F) Representative Western blot of ATXN1LWT (HEK) cells treated with MEK/ERK inhibitor and ATXN1L siRNA. DMSO and scrambled siRNA used as negative controls. Right: barplot quantification of CIC expression. Quantifications were normalized to Vinculin. *Western blot, ELISA, and RT-qPCR quantifications were collected from three independent experiments. Error bars represent one standard deviation. p-values were calculated using the two-tailed independent Student’s t-test. Statistically significant values are denoted (* = p < 0.05, ** = p < 0.01). 44 4.2.4 Inactivation of CIC by ERK remains intact and independent of ATXN1L Despite observing no involvement of ERK in the reduction of CIC protein expression in ATXN1LKO cells, we were interested to determine if ERK mediated CIC dysregulation remained intact in our cell system. Treatment of ATXN1LWT and ATXN1LKO cells with epidermal growth factor (EGF)/fibroblast growth factor (FGF) following serum starvation resulted in an increase of CIC protein expression in both ATXN1LWT and ATXN1LKO cells (Figure 4.4A, Supplemental Figure 12A, B), which could be rescued with dual MEK/ERK inhibition (Figure 4.4B, Supplemental Figure 12C). CIC localization was also not affected by EGF/FGF treatment as observed using immunofluorescence (Figure 4.4C). Although CIC protein expression was observed to increase in our cell systems, derepression of several CIC target genes (ETV1/4/5, DUSP6, SPRY4; Figure 4.4D and Supplemental Figure 12D, E) and decreased CIC binding to the promoter region of target genes (ETV4, DUSP6, SPRY4; Figure 3E) was observed following EGF/FGF treatment. The increase in CIC protein expression in our cell systems following EGF/FGF treatment was unexpected and thus were further validated in glioblastoma (GBM) cell lines (Supplemental Figure 13A, B) and primary brain tumour initiating cells (BTIC; Supplemental Figure 13C). All cell systems exhibited behavior consistent with our previous observations. 45 Figure 4.4 – ERK activity relieves CIC repressive function (A) Representative Western blot of ATXN1LWT (HEK) and ATXN1LKO (A30) cell lines treated with FGF/EGF over 0-24 hours following serum starvation. FBS control was cultured in FBS for the duration of the timecourse. Below: barplot quantifications of CIC expression. Quantifications were normalized to Vinculin. (B) Representative Western blot of ATXN1LWT (HEK) and ATXN1LKO (A30) cell lines treated with FGF/EGF and MEK/ERK inhibitors trametinib/LY3214996. (C) Immunofluorescence images of NHA cells cultured in FBS, serum starvation, and EGF/FGF. White bars denote 10µm. (D) Relative mRNA expression of ATXN1L, CIC, and CIC target genes DUSP6, SPRY4, and ETV1/4/5 in ATXN1LWT (NHA) and ATXN1LKO (B82) cell lines treated with FGF/EGF for 8 46 hours following serum starvation. Gene expression was normalized to TBP and serum starved parental ATXN1LWT (NHA) cell line was used as a relative control. (E) ChIP-PCR showing relative enrichment of CIC-DNA interaction following serum starvation or treatment with EGF/FGF (1 hour) in ATXN1LWT (HEK) and ATXN1LKO (A30) cell lines. Relative enrichment was normalized to a control region. (*) Western blot, RT-qPCR, and ChIP-PCR quantifications were collected from three independent experiments. Error bars represent one standard deviation. p-values were calculated using the two-tailed independent Student’s t-test. Statistically significant values are denoted (* = p < 0.05, ** = p < 0.01). 4.2.5 CIC degradation is mediated by the E3-ligase TRIM25 To further investigate CIC interactors which may be responsible for mediating the degradation of CIC protein following ATXN1L loss, CIC immunoprecipitation was performed in ATXN1LKO cell lines treated with MG132 and interacting proteins were identified using mass spectrometry. Previously identified CIC interactors such as 14-3-3 regulatory proteins and the ATXN1L homologue ATXN1 were highly enriched (Figure 4.5A and Supplemental Table S11). Using CICKO cell lines with stably expressing FLAG-tagged CIC-S [117], CIC interaction with 14-3-3 regulatory proteins and the nuclear pore protein TPR were observed to increase following siRNA knockdown of ATXN1L using PLA (Supplemental Figure 14A, B, C). CIC was not found to interact with many previously identified proteins involved in transcriptional regulation such as SIN3A, HDAC1/2, and members of the SWI/SNF complex in our ATXN1LKO background [117-119]. Amongst the most highly enriched CIC interactors was the RING-finger E3-ligase TRIM25. CIC interaction with TRIM25 was validated using immunoprecipitation (Figure 4.5B and Supplemental Figure 14D) and PLA (Figure 4.5C) and was found to be elevated in both ATXN1LKO and ATXN1L knockdown cell systems. Knockdown of TRIM25 using siRNA was able to induce increased protein expression of both CIC isoforms in ATXN1LWT and ATXN1LKO cells (Figure 4.5D); and decrease mRNA expression of CIC target genes ETV1/4/5 (Figure 4.5E). Ectopic overexpression of FLAG-tagged TRIM25 resulted in a further decrease in CIC protein expression in ATXN1LKO cells (Figure 4.5F). TRIM25 was observed to localize to both the cytoplasm and nucleus (Supplemental Figure 14F). 47 Figure 4.5 – CIC Interacts with the E3-Ligase TRIM25 (A) Volcano plot showing CIC interacting proteins identified using CIC immunoprecipitation followed by mass spectrometry in ATXN1LKO NHA cells. Red data points are high confidence interactors. (B) Representative Western blot of CIC immunoprecipitation showing interaction with TRIM25 in ATXN1LWT (NHA) and ATXN1LKO (B82) cell lines. Right: barplot showing quantifications of TRIM25 Western blots. Quantifications were normalized to CIC Western blots. (C) Immunofluorescence images of proximity ligation assay showing CIC-S-FLAG-TRIM25 interaction in ATXN1LWT cells treated with ATXN1L siRNA. Scrambled siRNA was used as a negative control. White bars denote 10µm. Right: Tukey boxplots showing quantification of number of foci/cell. (D) Representative Western blot of ATXN1LWT (HEK) and ATXN1LKO (A10, A30, B21) cell lines treated with TRIM25 siRNA. Scrambled siRNA was used as a negative control. Below: barplot quantifications of CIC expression. Quantifications were normalized to Vinculin. (E) Relative mRNA expression of CIC and CIC target genes ETV1/4/5 following treatment with TRIM25 siRNA for 48 hours. Scrambled siRNA was used as a negative control. Gene expression was normalized to TBP. (F) Representative Western blot of ATXN1LWT (NHA) and ATXN1LKO (B82) cell lines ectopically overexpressing FLAG-tagged TRIM25. Empty FLAG vector was used as a negative control. *Western blot and RT-qPCR quantifications were collected from three independent experiments. PLA quantifications were collected from 65 individual cells. Error bars represent one standard deviation. p-values were calculated using the 48 two-tailed independent Student’s t-test. Statistically significant values are denoted (* = p < 0.05, ** = p < 0.01, *** = p < 0.001). 4.2.6 Glioblastoma cell lines exhibit ATXN1L mediated CIC instability and TRIM25 mediated degradation To further validate our observations, we investigated the relationship between ERK, CIC, ATXN1L, and TRIM25 in additional cellular and biological systems. CIC was found to be expressed in several GBM and primary brain tumour initiating cell (BTIC) lines (Figure 4.6A, B) despite exhibiting increased expression of CIC target genes downstream of MAPK (ETV1/4/5, DUSP6, SPRY4: Supplemental Figure 15A, B), compared to NHA cells. Strong expression of CIC was not expected in GBM cell lines [57]. Therefore, CIC expression was verified in both low and high- grade patient glioma samples using IHC (n=17) and found to be expressed across all subtypes, with lower expression in oligodendroglioma (Figure 4.6C, D). To determine the role of ERK and ATXN1L in regulating CIC protein expression, GBM and BTIC cell lines were treated with dual MEK and ERK inhibition which did not increase CIC expression, consistent with our previous observations (Figure 4.6E, Supplemental Figure 16A). Similarly, knockdown of ATXN1L did result in decreased CIC protein expression in both GBM (Supplemental Figure 16B) and BTIC (Figure 4.6F) cell lines. Derepression of CIC target genes (ETV1/4/5, DUSP6, SPRY4) was also observed following siRNA knockdown of ATXN1L in GBM (Figure 4.6G, Supplemental Figure 16C) and BTIC (Supplemental Figure 16D) cell lines; while siRNA knockdown of TRIM25 resulted in further repression. Dual inhibition of MEK and ERK were not sufficient to rescue decreases in CIC protein expression following siRNA knockdown of ATXN1L (Figure 4.6H, Supplemental Figure 16E), whereas, concurrent knockdown of ATXN1L and TRIM25 was able to rescue decreased CIC protein (Figure 4.6I, Supplemental Figure 16F). 49 Figure 4.6 – TRIM25 and ATXN1L mediated CIC stability in GBM cell lines (A) Representative Western blot of CIC, ATXN1L, and phosphorylated ERK (pThr202/Tyr204) expression in GBM cell lines. (B) Representative Western blot of CIC, ATXN1L, and phosphorylated ERK (pThr202/Tyr204) expression in BTIC cell lines. (C) Tukey boxplots showing H-scores of CIC immunohistochemistry staining on 50 glioma samples. (D) Immunohistochemistry images of CIC staining on glioma samples. Black bars denote 200µm. (E) Representative Western blot of BTIC cell lines in standard EGF/FGF culture conditions and following 16 hours of MEK/ERK inhibition. (F) Representative Western blot of BTIC cell lines following siRNA knockdown of ATXN1L or TRIM25. Fluorescent RNA was used as a negative control. (G) Relative mRNA expression of CIC and CIC target genes (DUSP6, SPRY4, ETV1/4/5) following treatment with ATXN1L or TRIM25 siRNA for 48 hours in LN18 and U251 cell lines. Scrambled siRNA was used as a negative control. Gene expression was normalized to TBP. (H) Representative Western blot of LN229 and U343 cell lines treated with MEK/ERK inhibitors trametinib/LY3214996 and/or ATXN1L siRNA. DMSO and scrambled siRNA were used as negative control. Below: barplot quantifications of CIC protein expression. (I) Representative Western blot of U251 and U343 cell lines treated with ATXN1L and/or TRIM25 siRNA. Scrambled siRNA were used as negative control. Below: barplot quantifications of CIC protein expression. *Western blot and RT-qPCR quantifications were collected from three independent experiments. Error bars represent one standard deviation. p-values were calculated using the two-tailed independent Student’s t-test. Statistically significant values are denoted (* = p < 0.05, ** = p < 0.01). 4.2.7 TRIM25 amplification and CIC deletion dysregulate similar gene sets Probing the TCGA database, alterations in TRIM25 were observed in several cancer types, most notably, amplifications in the invasive breast carcinoma (BRCA) and mesothelioma data sets (Figure 4.7A). To determine if TRIM25 may regulate the transcription factor functions of CIC, differentially expressed (DE) genes using published gene expression data of TRIM25 knockdown in breast cancer cell lines (BT549 and MDA-MB-231, Supplemental Table 12) from Walsh et al. (https://doi.org/10.1016/j.celrep.2017.07.052) [95] were compared to DE genes identified in CICKO and ATXN1LKO NHA cell lines (Supplemental Table 13A, B). As TRIM25 was knocked down, we expected directional discordance between DE genes identified in TRIM25 knockdown versus CICKO/ATXN1LKO. Of the 1531 DE genes identified in the TRIM25 knockdown cell lines (FDR < 0.05, concordant direction), 161 (131 discordant) were shared with CICKO and 122 (104 discordant) were shared with ATXN1LKO DE genes (Figure 4.7B and Supplemental Tables 14A, B). Gene set enrichment analysis of discordant genes identified several terms related to cell growth/proliferation, and cell attachment/organization which is consistent with CIC/ATXN1L’s function as regulators of the cell cycle and metastasis (Supplemental Figure 17 and Supplemental Table 15A, B). To further validate the relationship between TRIM25 and CIC, differential expression analysis was performed using the TCGA BRCA cohort comparing samples with TRIM25 amplification versus samples with copy number neutral TRIM25. A total of 2789 DE genes were identified (FDR < 0.01, Fold change > 1.5; Supplemental Table 16A) which were then compared to previously identified DE genes in TCGA type II low grade glioma (Type II LGG, n=1538), prostate adenocarcinoma (PRAD, n=842), and stomach adenocarcinoma (STAD, n=3223) with CIC copy number loss (Supplemental Tables 6, 7, 8), expecting directional concordance. 347 genes (321 concordant), 290 (221 concordant), and 682 (553 concordant) DE genes were shared 51 between BRCA and Type II LGG, PRAD, and STAD, respectively (Figure 4.7C, Supplemental Table 16B, C, D). Gene set enrichment analysis of upregulated and directionally concordant DE genes shared between BRCA and Type II LGG/PRAD/STAD identified several terms related to cell cycle, consistent with previously published work and our cell line analyses (Figure 4.7D, Supplemental Table 17). BRCA patients which expressed high TRIM25 expression (top 25%) also showed decreased overall survival (Figure 4.7E). In addition to the TCGA BRCA cohort, TRIM25 was further investigated in the TCGA liver hepatocellular carcinoma (LIHC) cohort in which CIC dysregulation, at the proteomic level, has been described as a negative prognostic factor [33, 120]. Patients with high TRIM25 expression (top 25%) were found to have higher expression of the CIC target genes ETV1/4/5 (Figure 4.7F) as well as decreased overall survival (Figure 4.7G). 52 Figure 4.7 – CIC, ATXN1L, and TRIM25 regulate cell cycle in vitro and in TCGA patient data (A) Barplot displaying frequency of TRIM25 alterations in the TCGA pan-cancer study. Bars are based upon cancer subtype and split based on alteration type. (B) Scatter plot showing Log2Fold change of differentially expressed genes shared between TRIM25 siRNA in BT549 and MDA-MB-231 breast cancer cell lines and CIC/ATXN1L knockout in NHA cell lines. Differentially expressed genes with directionally discordant change are colored (red/blue). (C) Scatter plot showing Log2Fold change of differentially expressed genes shared between TCGA BRCA samples with TRIM25 amplification and TCGA Type II LGG, PRAD, and STAD samples with CIC deletions. Differentially expressed genes with directionally concordant change are colored. Red terms relate to cell cycle, growth, and proliferation. Blue terms relate to cell structure, organization, and adhesion. (D) Heatmap 53 showing the top 20 enriched gene sets for directionally concordant upregulated differentially expressed genes shared between TCGA BRCA samples with TRIM25 amplification and TCGA Type II LGG, PRAD, and STAD samples with CIC deletions. Highlighted terms are color coded respective to the cluster used for gene set enrichment analysis in panel D. Red terms are related to cell cycle, growth, and proliferation. (E) Kaplan-Meier curve showing overall survival of TCGA BRCA patients with high (top 25%) and low (bottom 75%) TRIM25 expression. (F) Tukey barplots showing expression of TRIM25, ETV1, ETV4, and ETV5 in TCGA LIHC samples with high (top 25%) and low (bottom 75%) TRIM25 expression. (G) Kaplan-Meier curve showing overall survival of TCGA LIHC patients with high (top 25%) and low (bottom 75%) TRIM25 expression. (*) Statistically significant values are denoted (* = p < 0.05, ** = p < 0.01, *** = p < 0.001). 4.3 Discussion In this study we explore the post-translational regulation of CIC in the context of ATXN1L loss and uncover a novel mechanism of CIC protein regulation independent of ERK activity in several cellular systems and contexts (Figure 4.8). The role of ATXN1L as a mediator of CIC repressor function has been well established through several functional studies, both in vitro and in vivo [29, 54, 65]; though little has been explored in regards to the mechanisms of ATXN1L mediated post-translational stability of CIC. Using isogenic ATXN1LKO cell lines, CIC instability was observed to be a downstream consequence of increased ubiquitin accumulation and that inhibition of ERK did not affect this CIC instability. Studies investigating the regulation of CIC protein expression in Drosophila melanogaster have established ERK as an important mediator of CIC stability through phosphorylation [21, 24-27]. However, unlike mammalian cells, Drosophila melanogaster lack an orthologue of ATXN1L suggesting that mammalian cells may have evolved several independent pathways to intricately regulate the post-translational function and stability of CIC. This can be evidenced by the temporal differences in CIC inactivation following loss of ATXN1L (hours) [54] versus phosphorylation by ERK (minutes) [31], which may suggest that CIC instability resulting from loss of ATXN1L may be due to an increased rate of normal CIC turnover. In this study, ERK activity was observed to alleviate CIC repressive function; but, contrary to several studies, CIC protein expression increased or remained constant. This discrepancy may be due to context specific and complex regulatory networks involving CIC in mammalian cells that have not been fully explored. As most mammalian studies investigating the role of ERK in regulating CIC stability have been performed in the context of cancers driven heavily by MAPK activation (melanoma, lung adenocarcinoma, glioblastoma), our observations may be due to post-translational feedback loops that are protective against cytotoxic ERK hyperactivation [121] in cell systems that are not heavily driven by or adapted to high levels of 54 MAPK activation. This study also establishes that the regulation of CIC function and protein stability is regulated by independent pathways, dependent on the cellular context. Figure 4.8 – Proposed mechanistic model of CIC-ATXN1L-TRIM25 interaction (A) CIC-ATXN1L form a repressive complex which stabilizes CIC from degradation by TRIM25 and protects ATXN1L from loss of an unknown post-translational modification. (B) In the absence of ATXN1L, CIC is targeted by TRIM25 for degradation by ubiquitination and transported for degradation through the nuclear pore by 14-3-3 chaperones. (C) In the absence of CIC, ATXN1L undergoes loss of an unknown post-translational modification and exclusion from the nucleus. In the cytoplasm, phosphorylated ATXN1L is targeted by an unknown phosphatase resulting in degradation and instability. In addition, this study identified the RING-finger E3-ligase TRIM25, an important mediator of anti-viral activity [122, 123], as a mediator of CIC post-translational stability. Interestingly, PJA1, another E3-ligase involved in the post-translational regulation of CIC also contains a RING-finger domain and has been implicated in the innate immune system. Overexpression of TRIM25 was able to reduce CIC protein expression while siRNA knockdown of TRIM25 was able to stabilize CIC and promote its tumour suppressor function. Utilising previously published gene expression data and TCGA cohorts, TRIM25 amplification in breast carcinoma was found to dysregulate several shared gene sets, related to cell growth and proliferation, with CIC loss. Interestingly, CIC loss has been linked to increased cancer stem cell-like properties in breast cancer cells [124]. Decreased overall survival was also observed in patients with higher TRIM25 expression in both the BRCA and LIHC TCGA cohorts further supporting the status of CIC function as a prognostic indicator. Although the majority of TRIM25 studies have been focussed on its role as a negative regulator of viral replication, several studies have also suggested that overexpression of TRIM25 may promote tumour 55 progression in hepatocellular carcinoma [125] and lung cancer [126], cell growth and proliferation in prostate adenocarcinoma [127] and breast cancer [128], and metastasis in breast cancer [95], supporting the role of TRIM25 as an oncogene. CIC, which has now been established as a potent tumour suppressor gene, has also been identified as a positive regulator of viral replication [129] which suggests that the CIC-ATXN1L-TRIM25 axis may be relevant in several different cellular and pathological contexts. 56 Chapter 5: The Effects of IDH mutation on CIC and ATXN1L loss 5.1 Introduction One of the defining molecular features of LGG are the recurrent R132 and R172 missense mutations of IDH1 and IDH2, respectively [130]. The most common mutation being the IDH1 R132H mutation. Under normal physiological conditions, IDH enzymes, which are involved in the citric acid cycle, are responsible for the enzymatic conversion of isocitrate to α-ketoglutarate, a co-factor important for the normal function of dioxygenase dependent enzymes such as the DNA demethylases TET1/2 and several histone demethylases. The recurrent R132 and R172 missense mutations produce a neomorphic IDH enzyme which can further reduce α-ketoglutarate into 2-hydroxyglutarate (2HG) [131], an oncometabolite which is a competitive inhibitor of dioxygenase dependent demethylases [15, 16] resulting in DNA and histone hypermethylation termed the “hypermethylator phenotype” [17, 18]. In LGG, CIC mutations occur exclusively in IDH mutated, 1p19q co-deleted tumours suggesting that there may be a functional relationship between CIC and IDH mutations [11]. One study found that the recurrent missense mutations in the HMG box and C1 motifs co-operatively regulate 2HG levels in cell lines [53]. Interestingly, multiple distinct CIC mutations have been found in recurrent IDH mutated LGG samples suggesting there is a strong selective pressure for clones to abrogate CIC function [132]. Contrastingly, loss of the mutant IDH allele has also been observed in recurrent LGG samples which may suggest that IDH mutations are dispensable post initiation and co-operation with CIC mutations is non-essential [133]. In this chapter, we explore the effects of IDH mutation on the transcriptional network of CIC and ATXN1L. 57 5.2 Results 5.2.1 ATXN1L loss in IDH1-R132H mutant cells leads to derepression of CIC target genes To establish the functional interaction between CIC and ATXN1L in IDH mutant cell lines, NHA cells overexpressing mutant IDH1-R132H were treated with siRNA targeted at ATXN1 or ATXN1L. Similar to in our IDH wildtype cell lines, knockdown of ATXN1L led to a greater and more consistent reduction in CIC protein and derepression of CIC target genes (Figure 5.1A/B). Of note, knockdown of ATXN1L in this cell system also resulted in CIC downregulation which was not seen in our IDH wildtype cells. ATXN1 knockdown did not affect CIC expression. 58 Figure 5.1 – Loss of ATXN1L in IDH mutant cells dysregulate CIC function (A) Representative Western blot IDH-R132H mutant cell line (F8) treated with ATXN1 or ATXN1L siRNA. Scrambled siRNA was used as a negative control. Vinculin was used as a loading control (B) Relative mRNA expression of CIC, ATXN1, ATXN1L, and CIC target genes (ETV1/4/5, DUSP6, SPRY4) following siRNA knockdown of ATXN1 or ATXN1L in IDH-R132H mutant cells (F8). Expression was normalized to TBP and Scrambled siRNA control was used as a relative control. (C) Representative Western blot of IDH1-R132H mutant ATXN1LKO cell lines (B18, C22). Vinculin used as a loading control. (D) Relative mRNA expression of ATXN1L, CIC, and CIC target genes (DUSP6, SPRY4, ETV1/4/5) in IDH1-R132H mutant ATXN1LKO cell lines. Expression was normalized to TBP and the parental cell line (F8) was used as a relative control. 5.2.2 IDH mutation dampens the transcriptomic effects of ATXN1L and CIC loss To investigate the effects of IDH mutation on the functional transcriptomic relationship between CIC and ATXN1L, differential expression analysis (DEA) was performed comparing IDH mutant CICKO or ATXN1LKO cell lines with IDH wildtype cell lines using RNA sequencing data. In the CICKO cell lines, 1258 (724 up, 534 down) DE genes were identified in IDH wildtype cell lines, while 265 (170 up, 94 down) were identified in IDH-R132H mutant cell lines (Figure 5.2A, Supplemental Table 18). Similarly, in the ATXN1LKO cell lines, 957 (648 up, 309 down) DE genes were identified in IDH wildtype cell lines, while 251 (106 up, 145 down) were identified in IDH-R132H mutant cell lines (Figure 5.2B, Supplemental Table 19). In both knockout systems, the presence of IDH mutation reduced the number of DE genes, however, the difference in magnitude of differential expression was found to be variable. Comparison of the DE genes identified in CICKO and ATXN1LKO cell lines, in both IDH contexts, identified 223 shared DE genes in IDH wildtype (Figure 5.2C and Supplemental Table 20A) and 46 shared DE genes in IDH-R132H mutant cell lines (Figure 5.2C, Supplemental Table 20B). Directional changes in expression in both IDH wildtype and IDH mutant cell lines were highly concordant between CICKO and ATXN1LKO cell lines. Interestingly, in the IDH mutant contexts, the canonical CIC target genes ETV1, ETV4, and CCND1 were DE in both CICKO and ATXN1LKO cell lines, but in IDH wildtype contexts, these genes were only identified in CICKO cell lines. Gene set enrichment analysis of shared upregulated DE genes between IDH-R132H mutant CICKO and ATXN1LKO cell lines converged upon activation of the MAPK pathway which was also identified in the IDH wildtype cell lines (Figure 5.2D, Supplemental Table 21). Other gene sets that were also found to be convergently upregulated in both IDH contexts include activation of the TNFα, NF-κB, IL2/STAT5, Hypoxia, and epithelial to mesenchymal transition. The convergence of gene sets in both IDH contexts highly suggests that the presence of IDH mutation does not greatly affect the pathways dysregulated by loss of CIC or ATXN1L. 59 Figure 5.2 – Transcriptomic effects of IDH mutation on CIC and ATXN1L loss (A) Volcano plot of differentially expressed genes identified in IDH wildtype and IDH-R132H mutant CICKO cell lines. (B) Volcano plot of differentially expressed genes identified in IDH wildtype and IDH-R132H mutant ATXN1LKO cell lines. (C) Scatter plot of genes which were found to be differentially expressed and shared between CICKO and ATXN1LKO cell lines in IDH wildtype cells (top) and IDH-R132H mutant cells (bottom). (D) Heatmap showing the top 20 enriched terms identified in IDH-R132H mutant CICKO and ATXN1LKO cell lines. Terms in bold italics were terms also identified in IDH wildtype CICKO and ATXN1LKO cell lines. 60 5.3 Discussion In this chapter, we explore the effects of the neomorphic IDH mutations found in low grade gliomas on the transcriptomic changes we previously observed in CICKO and ATXN1LKO NHA cell lines. Similar to in IDH wildtype NHA cells, siRNA knockdown of ATXN1L resulted in a more robust dysregulation of CIC target genes than ATXN1 knockdown. These observations were consistent with IDH wildtype and R132H mutant ATXN1LKO CRISPR cell lines suggesting that the presence of IDH mutation does not affect the transcriptomic relationship between CIC and ATXN1L. Upon further transcriptomic interrogation, a significant reduction in the number of DE genes in the IDH-R132H mutant backgrounds was observed, which supports the notion that the CIC-ATXN1L complex may partially regulate context dependent targets through interaction with the epigenome as the production of 2HG is known to greatly alter the epigenomic landscape in LGG and acute myeloid leukemia [18, 134]. Thus, the presence of IDH mutation may create a “dampening” effect of CIC-ATXN1L in relation to the epigenome and global transcriptome. One interesting thing to note is that while IDH mutation reduced the total number of DE genes, many of the canonical CIC target genes were still observed to be DE in IDH-R132H mutant cell lines suggesting that IDH mutation does not affect the binding of the CIC-ATXN1L to target genes that interact directly with the complex. Additionally, the pathways dysregulated in both IDH contexts were found to converge upon activation of oncogenic pathways such as MAPK, hypoxia, and epithelial to mesenchymal transition. Pathways that were highly enriched only in the IDH mutant contexts such as vasculature development, and downregulation of TP53/DNA damage response are also heavily implicated in glioma biology. In contrast, terms related to the epigenome identified in IDH wildtype cell lines, such as activation of EZH2 targets, were not observed to be highly enriched in IDH-R132H cell lines which may be due to the effects of 2HG on influencing the epigenomic landscape. In conclusion, IDH mutation “dampens” the breadth of CIC-ATXN1L’s transcriptomic network but does not appear to greatly affect the functional relationship between CIC and ATXN1L nor the pathways it regulates. 61 Chapter 6: Conclusions 6.1 Summary of study and findings The role of CIC function as a prognostic indicator has been highlighted by several biological and clinical studies in a multitude of cancer types through the dysregulation of several oncogenic pathways related to the cell cycle, metastasis, and treatment resistance. Most studies have investigated these phenotypes in relation to ETV1/4/5, which are the most widely studied and canonical CIC target genes, however, understanding of the mechanisms and interacting partners which govern CIC function are lacking. Additionally, as ectopic restoration of CIC function has been shown to be a powerful tumour suppressor in vitro, comprehension of the mechanisms pertinent to CIC activity and regulation may also reveal druggable targets in an effort to restore CIC function in patient tumours. Dysregulation of CIC function has not only been found to lead to aberrant expression of oncogenic pathways in cancer, it has also been found to promote the pathology of SCA1, a neurodegenerative disease. The importance of normal CIC function in unrelated pathologies suggest that intact CIC is important for not only supressing oncogenic pathways, but also in maintaining normal cellular function. In SCA1, the dysregulation of CIC is strongly linked to a functional interaction with ATXN1 and ATXN1L, though this relationship had previously not been thoroughly explored in cancer. In this thesis, we explored the relationship between CIC and ATXN1L on both the transcriptomic and proteomic level in several cancer types and delve into the mechanisms of CIC regulation in relation to ATXN1L. Utilising CRISPR/Cas9 generated knockout cell systems and TCGA patient cohorts; in Chapter 3, we reveal a reciprocal transcriptomic relationship between CIC and ATXN1/ATXN1L whereby loss or dysregulation of CIC or ATXN1/ATXN1L resulted in the dysregulation of highly overlapping gene sets and pathways related to the cell cycle. These pathways included activation of the MAPK, E2F, mTOR, and myc pathways and downregulation of Rb. In addition to cell cycle, dysregulation of the epigenome, particularly, gene sets related to the polycomb repressive complex were highly enriched in TCGA patient cohorts suggesting that the CIC-ATXN1/ATXN1L complex may also play a role in regulating the epigenome. In vitro, the dysregulation of CIC target genes, upon loss of ATXN1L, was found to be a result of both decreased CIC protein expression as well as decreased CIC binding to the DNA. While our in 62 vitro cell lines displayed a reliance of CIC on ATXN1L for expression and function, the reliance of CIC on ATXN1 and/or ATXN1L was found to be driven by the cellular context in the patient cohorts analysed (low grade glioma, prostate adenocarcinoma, and stomach adenocarcinoma). As a downstream effector of the MAPK pathway, previous studies had established the role of ERK phosphorylation in regulating both CIC function and protein stability. However, much of these studies were performed in Drosophila melanogaster which lack an ATXN1L ortholog. In Chapter 4, we further delve into the mechanisms involved in the post-translational regulation of mammalian CIC following loss of ATXN1L in vitro. While the loss of ATXN1L was found to activate gene sets indicative of MAPK activation, the role of ERK in regulating CIC protein expression and function was found to be independent from ATXN1L. Compared to ERK signaling, which occurs on the scale of minutes, loss of ATXN1L was found to promote CIC ubiquitination and proteasomal degradation through an increased rate of passive degradation on the order of hours. This passive degradation of CIC was found to be mediated by TRIM25, a RING-finger E3-ligase previously implicated in anti-viral replication. Transcriptomic analyses of TCGA patient cohorts also showed that patients with high expression of TRIM25 had decreased overall survival in breast carcinoma, and increased expression of ETV1/4/5 in liver hepatocellular carcinoma. Lastly, recurrent CIC mutations were first identified in oligodendroglioma which are molecularly characterized by IDH mutation and 1p19q co-deletion. In Chapter 5, we utilised isogenic IDH1-R132H mutant, CRISPR-Cas9 generated cell lines to investigate the role of IDH mutation on the transcriptomic relationship between CIC and ATXN1/ATXN1L. Similar to IDH wildtype cell lines, loss of ATXN1L in IDH1-R132H cell lines resulted in the dysregulation of several CIC target genes. However, on a global transcriptomic scale, IDH1-R132H mutation was found to dramatically reduce the total number of dysregulated genes in both CIC knockout and ATXN1L knockout cell lines suggesting that IDH1-R132H mutation may “dampen” the effects of CIC/ATXN1L loss through interaction with the epigenome. In summary, the main contributions of this thesis for the field of oncology research are: 1. It provides further support of the tumour suppressor potential of CIC in several cancer types. 63 2. It establishes a reciprocal relationship between CIC and ATXN1/ATXN1L on a global transcriptomic level in mammalian cells and the role of the CIC-ATXN1/ATXN1L complex in regulating cellular pathways involved in the cell cycle. 3. It led to the discovery of TRIM25 as a novel, ERK independent, post-translational regulator of CIC protein expression and function. 4. It demonstrates that the regulation of CIC function and post-translational stability in mammalian cells is more intricate and complex than previously appreciated. 6.2 Study strengths and limitations Many of the experiments in this thesis utilised immortalized normal cell lines, such as HEK and NHA, to investigate the normal function of CIC as a regulator of the cell cycle; and while immortalized cell lines may not be fully transformed, many of the genomic alterations and adaptations required for the establishment of these cell lines are known to affect pathways related to senescence and cell cycle. Cell lines are also not able to recapitulate the inter and intra tumoural heterogeneity of patient tumours. Therefore, the use of TCGA patient cohorts was an important validation of our in vitro observations. In the cell lines used for this study, the majority displayed a sole reliance on ATXN1L for CIC protein expression, while in patient cohorts, transcriptomic analyses revealed a differential reliance on ATXN1 and/or ATXN1L on CIC function. Further experiments using cell lines from different cellular contexts such as prostate adenocarcinoma and stomach adenocarcinoma as well as patient derived primary cell lines would have strengthened our in vitro and in silico observations had they been available. Unfortunately, the only primary cell lines available for these studies were glioma derived neurosphere cell lines which are non-adherent and slow growing, making them experimentally challenging. Had patient FFPE with matched genomic and transcriptomic profiles been available, further validation of the reliance of CIC expression on ATXN1/ATXN1L could have been performed using CIC IHC. Given these limitations, this study is strengthened by the consistency of our observations in several different cancer types suggesting that the mechanisms uncovered apply generally to CIC biology. 6.3 Future directions The discoveries made in this study have generated several additional research avenues which could be pursued in the future to further our understanding of CIC biology and the 64 intricacy of CIC regulation both in normal cells and in cancer. Below are some future directions, in relation to this thesis, which may further our understanding of CIC biology and regulation: 6.3.1 Investigate the role of ATXN1/ATXN1L in relation to CIC isoforms In this study and others, ATXN1 and ATXN1L were found to interact with both the short (CIC-S) and long (CIC-L) isoforms of CIC. However, it is unclear to what extent the functions and molecular mechanisms of CIC-S-ATXN1/ATXN1L and CIC-L-ATXN1/ATXN1L complexes differ. Studies thus far have been unable to differentiate the two isoforms or focus solely on the short isoform due to a lack of reagents which uniquely target only one isoform. CIC-L and CIC-S isoforms only differ at the first exon of each isoform which compounds this issue. Loss of ATXN1L in our in vitro experiments was found to affect the stability of CIC-S much more profoundly than CIC-L suggesting that there is differential regulation of each isoform. The two CIC isoforms have also found to localize differentially with CIC-S localizing to the both the nucleus and cytoplasm and CIC-L primarily localizing to the nucleus. 6.3.2 Investigate the role of ATXN1/ATXN1L in different CIC complexes It is unclear whether all CIC proteins in the cell exist in a complex with ATXN1/ATXN1L or if there are ATXN1/ATXN1L independent mechanisms for CIC stability. CIC has also been shown to interact with several different protein complexes with various cellular functions such as the SIN3-HDAC repressive complex, the SWI/SNF chromatin remodeling complex, and mitochondrial ACLY involved in metabolism. In our study, CIC was not found to interact with these complexes in ATXN1L knockout cell lines. However, it is unknown whether ATXN1L is an important mediator which facilitates the assembly of these CIC complexes. The role or presence of ATXN1/ATXN1L in these CIC complexes is unknown and may provide further insight into the mechanisms of CIC activity. 6.3.3 Determine the amino acid residues of CIC required for degradation While our study was able to identify a novel E3-ligase involved in CIC regulation, it was not able to identify the exact residues which are necessary for ubiquitination. Several other studies have also not been able to identify ubiquitination residues on CIC. So far, the only domain that has been identified to be important for CIC protein regulation is the ERK binding domain. However, it is unclear whether this domain is also required for ERK independent regulation of CIC. It will be important for future studies to identify additional domains and 65 residues required for CIC degradation which will provide further insight into our understanding of CIC protein regulation. 6.3.4 Explore the use of small molecule inhibitors to re-establish CIC function Loss of function CIC mutations have now been identified in several different cancer types as a means of promoting more aggressive behaviors. Interestingly, in other cancer subtypes CIC dysregulation, through several mechanisms involving ERK and ATXN1L, rather than loss of function mutations, have been identified as a means to circumvent CIC activity. In these tumours, in theory, it may be possible to restore CIC function through the use of small molecular inhibitors. ERK inhibitors have been shown to be effective in reducing CIC phosphorylation and increasing the repression of CIC target genes. However, this mechanism requires the presence of ATXN1L which protects CIC from TRIM25 degradation. While TRIM25 small molecular inhibitors are currently not available, inhibition of the TRIM25 degradation pathway in conjunction with ERK may be a means to restore CIC activity and stability. Future research should identify the binding pockets required for CIC-TRIM25 interaction in order to design competitive small molecule inhibitors. Conversely, protein-protein interactor stabilizers could also be employed to strengthen the CIC-ATXN1L interaction as the interfaces that facilitate this interaction have already been mapped. 66 References 1. Bailey, P. and H. Cushing, A classification of the tumours of the glioma group on a histogenetic basis, with a correlated study of prognosis. By Percival Bailey and Harvey Cushing. Medium 8vo. 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Mazor, T., et al., Clonal expansion and epigenetic reprogramming following deletion or amplification of mutant IDH1. Proc Natl Acad Sci U S A, 2017. 114(40): p. 10743-10748. 134. Sasaki, M., et al., IDH1(R132H) mutation increases murine haematopoietic progenitors and alters epigenetics. Nature, 2012. 488(7413): p. 656-9. 73 Appendices Appendix A This appendix lists all of the publications I contributed to during my PhD studies. First or co-first authorships are underlined 1. Derek Wong, Samantha S Lee, Lisa Sogerer, Victor Wong, Amy Lum, Marco A Marra, Stephen Yip. (2020) Loss of ATXN1L promotes ERK independent proteasomal degradation of Capicua through TRIM25 in cancer. BMC Biology. 18 (154) 2. Adrian B. Levine, Derek Wong, Mostafa Fatehi, Stephen Yip. (2020) Ependymoma and Chordoma. Neurosurgery. doi:10.1093/neuros/nyaa329 3. Derek Wong, Stephen Yip (2020) Making heads or tails – The emergence of Capicua in cancer. The Journal of Pathology. 250 (5), 532-540 4. Derek Wong, Stephen Yip (2019) Finding a four-leaf clover—identifying long-term survivors in IDH-wildtype glioblastoma. Neuro-oncology. 21 (11), 1352-1353 5. Nisreen Mohammad, Derek Wong, Amy Lum, Jonah Lin, Julie Ho, Cheng-han Lee, Tony Ng, Stephen Yip. (2019) Characterization of IDH1/IDH2 Mutations and D-2-Hydroxyglutarate Oncometabolite Level in Dedifferentiated Chondrosarcoma. American Journal of Surgical Pathology. doi:10.1111/his.14018 6. Derek Wong, Stephen Yip, and Poul Sorensen. (2019) Methods for identifying patients with tropomyosin receptor kinase (TRK) fusion cancer. Pathology and Oncology Research. doi:10.1007/s12253-019-00685-2 7. Derek Wong, Yaoqing Shen, Erin Pleasance, Adrian Levine, Martin Jones, Brian Thiessen, Brian Toyota, Janessa Laskins, Steve Jones, Marco Marra, Stephen Yip. The Pivotal Role of Sampling Recurrent Tumors in the Precision Care of Patients with Tumors of the Central Nervous System. (2019) Molecular Case Studies. 5: a004143 8. Derek Wong, Stephen Yip (2019) Chapter 6 - Pathology of Primary Brain Tumors – Gliomas. In Kaisorn Chaichana and Alfredo Quiñones-Hinojosa, Comprehensive Overview of Modern Surgical Approaches to Intrinsic Brain Tumors. Pages 121-137 9. Patel N, Wang J, Shiozawa K, Jones KB, Zhang Y, Prokop JW, Davenport GG, Nihira NT, Hao Z, Wong D, Brandsmeier L, Meadows SK, Sampaio AV, Werff RV, Endo M, Capecchi MR, McNagny KM, Mak TW, Nielsen TO, Underhill TM, Myers RM, Kondo T, Su L 74 (2019) HDAC2 regulates site-specific acetylation of MDM2 and its ubiquitination signaling in tumor suppression. iScience. 13, 43-54. 10. Derek Wong, Kohl Lounsbury, Amy Lum, Jungeun Song, Susanna Chan, Veronique LeBlanc, Suganthi Chittaranjan, Marco Marra, Stephen Yip (2018) Transcriptomic analysis of CIC and ATXN1L reveal a functional relationship exploited by cancer. Oncogene. 38(2):273-290. 11. Derek Wong and Stephen Yip (2018) Machine learning classifies cancer. Nature. 555, 446-447. 75 Appendix B Supplemental Figure 1 – CIC-ATXN1L interaction (A) Representative western blot of endogenous CIC IP showing co-IP of ATXN1L in NHA cells. A CIC IP in a CICKO cell line (A2) and an IgG IP in the parental NHA were performed as negative controls. (B) Representative western blot of endogenous ATXN1L IP showing co-IP of CIC in NHA cells. An ATXN1L IP in our ATXN1LKO cell line (B82) and IgG IP in the parental NHA were performed as negative controls. (C) Representative western blot of endogenous CIC IP showing co-IP of ATXN1L in HOG cells. A CIC IP in a CICKO cell line (F11) and an IgG IP in the parental HOG were performed as negative controls. (D) Representative western blot of endogenous ATXN1L IP showing co-IP of CIC in HOG cells. An ATXN1L IP in our CICKO cell line (F11) and IgG IP in the parental HOG were performed as negative controls. (E) Representative western blot of ATXN1L and ATXN1 knockdown using targeted siRNA in NHA cells. A scrambled siRNA (siCtrl) was used as a negative control and vinculin was used as a loading control. (F) Tukey boxplots showing relative mRNA transcript levels of ATXN1L, ATXN1, CIC, and CIC target genes ETV1/4/5, DUSP6 and SPRY4 measured by RT-qPCR following independent siRNA knockdown of ATXN1L or ATXN1 for 48 hours. Data were collected from 3 independent experiments and p-values were calculated using the two-tailed independent Student’s t-test comparing siATXN1L and siATXN1 to siCtrl. 76 Supplemental Figure 2 – ATXN1 function only partially compensates for ATXN1L loss. (A) Representative western blot of ATXN1 knockdown using targeted siRNA in ATXN1LKO HEK-A30, A10, and B21 cells. A scrambled siRNA (siCtrl) was used as a negative control and vinculin was used as a loading control. (B) Tukey boxplots showing relative mRNA transcript levels of ATXN1L, ATXN1, CIC, and CIC target genes ETV1/4/5, DUSP6 and SPRY4 measured by RT-qPCR following siRNA knockdown of ATXN1 for 48 hours in ATXN1LKO HEK-A10, A30, and B21 cells. Data were collected from 3 independent experiments and p-values were calculated using the two-tailed independent Student’s t-test. p-values directly above each boxplot are referenced to the no treatment parental cell line. All other p-values represent t-tests compared to siCtrl. (C) Representative western blot of ATXN1 knockdown using targeted siRNA in ATXN1LKO NHA-B82 cells. A scrambled siRNA (siCtrl) was used as a negative control and vinculin was used as a loading control. (D) Tukey boxplots showing relative mRNA transcript levels of ATXN1L, ATXN1, CIC, and CIC target genes ETV1/4/5, DUSP6 and SPRY4 measured by RT-77 qPCR following siRNA knockdown of ATXN1 for 48 hours in ATXN1LKO NHA-B82 cells. Data were collected from 3 independent experiments and p-values were calculated using the two-tailed independent Student’s t-test. Supplemental Figure 3 – ATXN1L loss leads to decreased CIC protein expression (A) Representative western blot showing decreased CIC protein expression in ATXN1LKO cell line HEK-A30. Vinculin was blotted as a loading control. (B) Protein quantification of CIC-S and CIC-L protein expression in the parental HEK and NHA, and ATXN1LKO cell lines HEK-A30 and NHA-B82. Values were normalized to the loading control Vinculin and their respective parental ATXN1LWT cell lines. Data were collected from 3 independent experiments (passages) and p-values were calculated using the two tailed independent Student’s t-test comparing ATXN1LKO cell lines to their respective parental cell line. Supplemental Figure 4 – Reintroduction of ATXN1L (A) Representative western blots showing introduction of ATXN1L-FLAG into the parental HEK and ATXN1LKO HEK-A30 cell line. Vinculin was used as a loading control. (B) Tukey boxplots showing relative mRNA transcript levels of ATXN1L, CIC, and CIC target genes ETV4 and DUSP6 following introduction of FLAG or ATXN1L-FLAG constructs into the parental HEK and ATXN1LKO HEK-A30 cell lines for 48 hours. Data were collected from 3 independent experiments and p-values were calculated using the two-tailed independent Student’s t-test. Note: the y-axis on the ATXN1L plot is shown as log-scale. 78 Supplemental Figure 5 - ATXN1LKO cell lines show derepression of the MAPK pathway. (A) Tukey boxplots showing relative mRNA transcript levels of genes downstream of MAPK activation (DUSP6, SPRY4, SHC3, SPRED1, SPRED2, PTPN9) measured by RT-qPCR in ATXN1LWT (HEK, N8) and ATXN1LKO (A30, B21) cell lines. Data were collected from 3 independent experiments and p-values were calculated using the two-tailed independent Student’s t-test comparing isogenic cell lines (N8, A30, B21) to the parental HEK cell line. (B) Tukey boxplots showing relative mRNA transcript levels of genes downstream of MAPK activation (DUSP6, SPRY4, SHC3, SPRED1, SPRED2, PTPN9) measured by RT-qPCR in ATXN1LWT (NHA) and ATXN1LKO (B82) cell lines. Data were collected from 3 independent experiments and p-values were calculated using the two-tailed independent Student’s t-test comparing isogenic cell lines (N8, A30, B21) to the parental HEK cell line. (C) Representative western blots showing protein expression of ATXN1, ETV4, and genes downstream of MAPK activation (PTPN9, DUSP6, SPRY4, pMEK, MEK, pERK, ERK) in ATXN1LWT (HEK, N4, N8) and ATXN1LKO (A10, A30, B21) cell lines. Tubulin and Vinculin were used as loading controls. (D) Representative western blots showing protein expression of ATXN1, ETV4, and genes downstream of MAPK activation (PTPN9, DUSP6, SPRY4, pMEK, MEK, pERK, ERK) in ATXN1LWT (NHA) and ATXN1LKO (B82) cell lines. Tubulin and Vinculin were used as loading controls. 79 Supplemental Figure 6 – TCGA Pathway Analyses (A) Heat map showing the top 20 enriched terms for upregulated DE genes identified in Type II-LGGATXN1/ATXN1L-Del and Type II-LGGCIC-Del. (B) Heat map showing the top 20 enriched terms for upregulated DE genes identified in PRADATXN1L-Del and PRADCIC-Del. (C) Heat map showing the top 20 enriched terms for upregulated DE genes identified in STADATXN1L-Del, STADATXN1-Del, STADATXN1/ATXN1L-Del and STADCIC-Del. *Gene set enrichment analyses were performed using gene ontology biological processes, hallmark gene sets, and oncogenic signatures. 80 Supplemental Figure 7 – ATXN1LKO cells exhibit decreased CIC-DNA binding Tukey boxplots showing enrichment of CIC binding to promoter regions of CIC target genes (ETV4, DUSP6, SPRY4) relative to a negative control region (NCR1) following CIC ChIP-qPCR in CICWT (HEK), CICKO (D10) and ATXN1LKO (A30) cell lines. Data were collected over 3 independent experiments and p-values were calculated using the two-tailed independent Student’s t-test. Supplemental Figure 8 – Loss of ATXN1L leads to CIC instability (A) Immunofluorescent visualization of endogenous CIC (orange) and the proteasomal subunit PSMC5 (green) localization following siRNA knockdown of ATXN1L in NHA cells. Scrambled siRNA was used as a control and DAPI (blue) was used to stain cell nuclei. White bars seen on the merged image denote 10μm. (B) Representative western blots of ATXN1LWT (NHA) and ATXN1LKO (B82) cells treated with 100µM of cycloheximide over a 24 hour timecourse showing CIC stability in the presence and absence of ATXN1L. Vinculin was blotted as a loading control and P53 and CHK1 were blotted as protein synthesis inhibition controls. 81 Supplemental Figure 9 – Loss of ATXN1L leads to CIC ubiquitination and degradation (A) Representative Western blot of FLAG immunoprecipitation of wildtype ATXN1L-FLAG and mutant ATXN1L-V485A-FLAG in HEK cells. (B) Representative Western blot of cellular fractionation across a sucrose gradient of 20-40% showing localization of CIC. SUG1 was used as a proteasome marker. Quantification of CIC protein/fraction are displayed below. Values were normalized to the cumulative total of CIC in all fractions. (C) Immunoflourescence images of proximity ligation assay showing CIC-Ubiquitin interaction in ATXN1LWT (NHA) cell lines treated with ATXN1L siRNA. Scrambled siRNA used as negative control. White bars denote 10µm. (D) Tukey boxplots showing quantification of number of foci/cell. 82 Supplemental Figure 10 – in Silico protein analysis of CIC and ATXN1L (A) Protein analysis of ATXN1L showing high confidence putative nuclear localization motifs (green) and high confidence putative phosphorylation residues (red). The AXH domain required for CIC interaction is highlighted in yellow. (B) Protein analysis of CIC-S showing high confidence putative ubiquitination residues (red). The ATXN binding domain required for ATXN1L interaction is highlighted in yellow, the HMG box DNA binding motif is highlighted in green, and the C1 protein interacting motif is highlighted in grey. 83 Supplemental Figure 11 – Loss of CIC leads to ATXN1L dysregulation (A) Immunofluorescent imaging of endogenous ATXN1L (orange) in CICWT (HEK) and CICKO (HEK-D10) cell lines showing ATXN1L localization. Tubulin (green) was stained to show cell morphology and nuclei were stained with DAPI (blue). White bars seen on the merged image represent 10μm. (B) Immunofluorescent imaging of endogenous ATXN1L (orange) in CICWT (HOG) and CICKO (HOG-F11) cell lines showing ATXN1L localization. Tubulin (green) was stained to show cell morphology and nuclei were stained with DAPI (blue). White bars seen on the merged image represent 10μm. (C) Western blot of endogenous ATXN1L IP following cellular fractionation (Cytoplasm and Nuclear) of CICWT (NHA) and CICKO (A2) cells. Phosphorylated Serine/Threonine (pSer/Thr) was blotted to show the localization of phosphorylated ATXN1L in the presence or absence of CIC. An IgG IP was performed as a negative control. Vinculin was blotted as a cytoplasmic control and Lamin-B1 was blotted as a nuclear control. (D) Representative Western blot of CICWT (HEK) and CICKO (D10) cells treated with 25µM of cycloheximide over a 24 hour timecourse showing ATXN1L stability in the presence and absence of CIC. Vinculin was blotted as a loading control and P53 and CHK1 were blotted as protein synthesis inhibition controls. (E) Tukey 84 boxplots showing relative mRNA transcript levels of HEY1 and HEY2 in parental HEK and NHA, and ATXN1LKO HEK (A10, A30, B21) and NHA (B82) cell lines. Data were collected from 3 independent experiments (passages) and p-values were calculated using the two tailed independent Student’s t-test comparing ATXN1LKO cell lines to their respective parental cell line. (F) Tukey boxplots showing relative mRNA transcript levels of HEY1 and HEY2 in no treatment parental HEK and NHA, and ATXN1LKO HEK (A10, A30, B21) and NHA (B82) cell lines treated with either siCtrl (Blue) or siATXN1 (Red). Data were collected from 3 independent experiments and p-values were calculated using the two tailed independent Student’s t-test. p-values directly above each boxplot are referenced to the no treatment parental cell line. All other p-values represent t-tests compared to siCtrl. Supplemental Figure 12 – ERK mediates CIC function (A) Representative Western blot of ATXN1LWT (NHA) and ATXN1LKO (B82) cell lines treated with FGF/EGF over 0-24 hours following serum starvation. FBS control was cultured in FBS for the duration of the timecourse. (B) Relative mRNA expression of ATXN1L, CIC, and CIC target genes DUSP6, SPRY4, and ETV1/4/5 in ATXN1LWT (HEK) and ATXN1LKO (A30) cell lines treated with FGF/EGF for 8 hours following serum starvation. Gene expression was normalized to TBP and serum starved parental ATXN1LWT (HEK) cell line was used as a relative 85 control. (C) Representative Western blot of ATXN1LWT (NHA) and ATXN1LKO (B82) cell lines treated with FGF/EGF and MEK/ERK inhibitors trametinib/LY3214996. *RT-qPCR quantifications were collected from three independent experiments. Error bars represent one standard deviation. p-values were calculated using the two-tailed independent Student’s t-test. Statistically significant values are denoted (* = p < 0.05, ** = p < 0.01). Supplemental Figure 13 – EGF/FGF treatment results in increased CIC expression (A) Representative Western blot of GBM cells following serum starvation and EGF/FGF treatment (1 hour). (B) Representative Western blot of GBM cells following serum starvation and EGF/FGF treatment (16 hours). (C) Representative Western blot of BTIC MGG119 following EGF/FGF starvation (16 hours) and EGF/FGF treatment over 120 minutes. 86 Supplemental Figure 14 – CIC interactors in the absence of ATXN1L (A) Representative Western blot of CICKO cells with FLAG tagged CIC-S (NHA-S) reintroduced treated with ATXN1L siRNA over 72 hours. Scrambled siRNA was used as a negative control. (B) Immunofluorescence images of proximity ligation assay showing CIC-S-FLAG-14-3-3 interaction in NHA-S cells treated with ATXN1L siRNA. Scrambled siRNA was used as a negative control. White bars denote 10µm. Right: Tukey boxplots showing quantification of number of foci/cell. (C) Immunofluorescence images of proximity ligation assay showing CIC-S-FLAG-TPR interaction in NHA-S cells treated with ATXN1L siRNA. Scrambled siRNA was used as a negative control. White bars denote 10µm. Right: Tukey boxplots showing quantification of number of foci/cell. (D) Representative Western blot of CIC immunoprecipitation showing interaction with TRIM25 in ATXN1LWT (HEK) and ATXN1LKO (A30) cell lines. (E) Immunofluorescence images showing localization of TRIM25 in NHA cells. White bars denote 10µm. (*) PLA quantifications were collected from 65 individual cells. Error bars represent one standard deviation. p-values were calculated using the two-tailed independent Student’s t-test. Statistically significant values are denoted (* = p < 0.05, ** = p < 0.01, *** = p < 0.001). 87 Supplemental Figure 15 – Characterization of glioma cell lines (A) Relative mRNA expression of CIC target genes ETV1/4/5, DUSP6, and SPRY4 in GBM cell lines. Expression was normalized to TBP and NHA was used as a relative control. (B) Relative mRNA expression of CIC target genes ETV1/4/5, DUSP6, and SPRY4 in BTIC lines. Expression was normalized to TBP and NHA was used as a relative control. 88 Supplemental Figure 16 – Validation of CIC-ATXN1L-TRIM25 in glioma cells (A) Representative Western blot of GBM cell lines treated with MEK/ERK inhibitors trametinib/LY3214996. DMSO was used as a negative control. (B) Representative Western blot of GBM cell lines treated with ATXN1L siRNA for 48 hours. Scrambled siRNA was used as negative control. (C) Relative mRNA expression of CIC and CIC target genes ETV1/4/5, DUSP6, and SPRY4 in GBM cell lines following siRNA knockdown of ATXN1L or 89 TRIM25 for 48 hours. Expression was normalized to TBP and scrambled siRNA was used as a negative control. (D) Relative mRNA expression of CIC and CIC target genes ETV1/4/5, DUSP6, and SPRY4 in BTIC cell lines following siRNA knockdown of ATXN1L or TRIM25 for 48 hours. Expression was normalized to TBP and fluorescent RNA was used as a negative control. (E) Representative Western blot of GBM cell lines treated with MEK/ERK inhibitors trametinib/LY3214996 and ATXN1L siRNA. DMSO and scrambled siRNA were used as negative control. Below: barplot quantifications of CIC expression. (F) Representative Western blot of GBM cell lines treated with ATXN1L and TRIM25 siRNA. Scrambled siRNA were used as negative control. Below: barplot quantifications of CIC expression. (*) RT-qPCR quantifications were collected from three independent experiments. Error bars represent one standard deviation. p-values were calculated using the two-tailed independent Student’s t-test. Statistically significant values are denoted (* = p < 0.05, ** = p < 0.01). Supplemental Figure 17 – TRIM25 pathways in cell lines (A) Heatmap showing the top 20 enriched gene sets for directionally discordant differentially expressed genes shared between TRIM25 siRNA in BT549 and MDA-MB-231 breast cancer cell lines and CIC/ATXN1L knockout in NHA cell lines. 90 Appendix C (Tables are hyperlinked) Supplemental Table 1 – TCGA samples TCGA Patient Samples Supplemental Table 2 – HEK Differential Expression Analysis HEK CICKO and ATXN1LKO DEA Supplemental Table 3 – NHA Differential Expression Analysis NHA CICKO and ATXN1LKO DEA Supplemental Table 4 – Type I LGG Differential Expression Analysis Type I LGG DEA Supplemental Table 5 – HEK and NHA vs Type I LGG Functional Enrichment In vitro GSEA Supplemental Table 6 – Prostate Adenocarcinoma Differential Expression Analysis PRAD DE Genes Supplemental Table 7 – Type II LGG Differential Expression Analysis Type II LGG DE Genes Supplemental Table 8 – Stomach Adenocarcinoma Differential Expression Analysis STAD DE Genes Supplemental Table 9 – TCGA Functional Enrichment TCGA GSEA Supplemental Table 10 – TCGA Shared Genes and Functional Enrichment TCGA Shared GSEA Supplemental Table 11 – CIC IP-MS results in ATXN1LKO NHA CIC IP MS results 91 Supplemental Table 12 – TRIM25 knockdown differentially expressed genes TRIM25 DE Genes Supplemental Table 13 – Differentially expressed ATXN1LKO NHA genes NHA ATXN1LKO DE Genes (RNA-seq) Supplemental Table 14 – TRIM25 knockdown vs CIC-ATXN1L knockout DE genes TRIM25 knockdown vs CIC-ATXN1L knockout Supplemental Table 15 – In vitro TRIM25-CIC-ATXN1L Functional Enrichment CIC-ATXN1L-TRIM25 in vitro GSEA Supplemental Table 16 – TCGA BRCA TRIM25 Differential Expression Analysis BRCA DE Genes Supplemental Table 17 – TCGA BRCA vs LGG/PRAD/STAD Functional Enrichment TCGA Shared GSEA Supplemental Table 18 – IDH-WT and IDH-R132H Mutant CICKO Differential Expression Analysis IDH-WT/Mut CIC DE Supplemental Table 19 - IDH-WT and IDH-R132H Mutant ATXN1LKO Differential Expression Analysis IDH-WT/Mut ATXN1L DE Supplemental Table 20 – Shared Differentially Expressed Genes in IDH-WT and IDH-R132H Mutant CICKO and ATXN1LKO cell lines CIC vs ATXN1L shared Supplemental Table 21 –IDH-R132H mutant CICKO and ATXN1LKO Functional Enrichment CIC vs ATXN1L GSEA 92 Appendix D Supplemental Materials 1 – Antibodies Target Company Catalogue # Western IF IP IHC Alpha Tubulin Abcam ab7291 1:5000 1:500 ATXN1 NeuroMab 75-122 1:1000 ATXN1L ProSci Custom 1:500 1:100 Yes CHK1 Santa Cruz sc-8408 1:1000 CIC Sigma HPA044341 1:500 1:200 Yes 1:200 CIC Bethyl Labs A301-203A Yes DUSP6 Novus NB100-79998 1:1000 ETV1 Sigma SAB5300473-100UL 1:1000 ETV4 Novus NBP1-28856 1:1000 ETV5 Sigma WH0002119M2 1:1000 FLAG Santa Cruz F3165 1:5000 Yes Goat anti-mouse IgG-488 ThermoFisher A-11029 1:500 Goat anti-mouse IgG-HRP Santa Cruz SC-2005 1:5000 Goat anti-rabbit IgG-546 ThermoFisher A-11035 1:500 Goat anti-rabbit IgG-HRP Santa Cruz SC-2004 1:5000 HDAC1 Abcam ab7028 1:1000 IDH1-R132H Dianova DIA-H09 1:1000 Lamin B1 Abcam ab133741 1:1000 Mouse IgG Santa Cruz sc-2025 Yes P53 Santa Cruz sc-126 1:1000 Pan 14-3-3 Millipore AB9748-I 1:100 Phospho – (Ser/Thr) Abcam ab17464 1:1000 Phospho ERK Cell Signaling 9101 1:1000 Phospho MEK Cell Signaling 9121 1:1000 PJA1 SantaCruz Sc-517068 1:1000 PSMC5 (SUG1) Invitrogen MA3-087 1:500 Rabbit IgG Cell Signaling 2729 Yes SPRY4 Pierce PA5-31401 1:1000 Total ERK Cell Signaling 4695 1:1000 Total MEK Cell Signaling 9122 1:1000 TPR Abcam ab84516 1:100 TRIM25 Abcam ab167154 1:2000 1:100 Ubiquitin Abcam ab7254 1:1000 Vinculin Abcam ab129002 1:5000 93 Supplemental Materials 2 – RT-qPCR Primers Gene Forward primer sequence (5’ to 3’) Reverse primer sequence (5’ to 3’) CIC GCGTCTCCAAATGACACA CCGACTTCTTGTCCGAATAC ATXN1L TGACCAGCGAGGATATGG GGACCATTCAGAAGCAC ATXN1 CGTGGCCGTGATACAGTTCG GGAACACGGCAAATCAAAGAGC DUSP6 CCAGTGGAGATCTTGCCCTTC TGTACTTGATGCCGAATTCCTCC ETV1 ACTGCTGAAGACAGACATGGAAC CATGTAGGCCATGCTCTCATCAA ETV4 AGGCGGAGGTTGAAGAAAGG GGGCAGAAGAAAGGCAAAGG ETV5 AGGGAAATCTCGATCTGAGGAATG GCTAACCAAGCCTCTTGAAGTTGA SPRY4 CAACGGCTCTTAGACCACATGG GATGCACACTCCTTGCATTTACAC HEY1 CCTCCGCCTCCAAACTGT TATAGGGCTTGCCAAGGTTTGC HEY2 GCGTCGGGATCGGATAAATAAC CTGTTAGGCACTCTCGGAATCC SPRED1 GTCAGGATGCTCCAGACCCT CTCCCTCTGAGTCTGACATACAATGA SPRED2 CTCATCCATGGTGAACGACAGAA GTCAAAGGCTCGGGCATC PTPN9 TGGTACTTCAGGCTCTGTTTTACTTG CCCTTCAGCAGGTTTAGGACTTTC Supplemental Materials 3 – siRNA sequences Target Company siRNA ID#/Catalogue# siRNA Sequence ATXN1L Life Technologies s230554 CGAGAGCGAAAUUUAGUAAtt ATXN1L Life Technologies s230553 CCAUCUGUGGUGAAUAUGAtt ATXN1L Life Technologies ATXN1LHSS155872 GCCUUCCCUAUGCUGUGCCACCUAA ATXN1 Life Technologies n294053 AUAAGCAGAUACAAAAAAtt ATXN1 Life Technologies ATXN1HSS109491 UUUGGAACACGGCAAAUCAAAGAGC TRIM25 Life Technologies 118751 CCUCGACAAGGAAGAUAAAtt TRIM25 Life Technologies s15204 CCAUAGACCUCAAAACGAtt TRIM25 Life Technologies s15205 CAACACCGCCCACAACAAAtt Supplemental Materials 4 – CRISPR derived Cell Lines and Genetic Backgrounds Name Parental CIC ATXN1L IDH1 HEK - - - - D10 HEK KO - - A10 HEK - KO - A30 HEK - KO - B21 HEK - KO - HOG - - - - F11 HOG KO - - NHA - - - WT OE A2 NHA KO - WT OE H9 NHA KO - WT OE B82 NHA - KO WT OE B16 NHA - KO WT OE B21 NHA - KO WT OE 94 F8 NHA - - R132H C5 F8 KO - R132H E10 F8 KO - R132H B18 F8 - KO R132H C22 F8 - KO R132H A2-WT A2 CIC-S OE - - Supplemental Materials 5 – Other Cell Lines Name Tissue Derived U87-MG Glioblastoma U251 Glioblastoma U343 Glioblastoma LN18 Glioblastoma LN229 Glioblastoma MGG119 Glioblastoma BT054 Oligodendroglioma BT088 Oligodendroglioma Supplemental Materials 6 – ChIP-PCR Primers Gene Forward primer sequence (5' to 3') Reverse primer sequence (5' to 3') NCR1 TGGTCTCCATGACAACGAAGAAGAG GTCCCATCCTACTGAAGTGCCC NCR2 TGGGAATGGTCGCAGAGGTT GGTCTCATCGGTGAACCCCC ETV4 Site A GCGGGGGCCAATCAGAATGTA AACTCCAGCCGCGAGTTTTA ETV4 Site B GCTTCATTCAGTGCTCCGGCT CGTGGCGAGACGGTGAAGA ETV4 Site C CGGGAGGGTGGGCTCATTCA ATGCCCTTGAACTCCTGCCC ETV4 Site D AGAACTAGCAAGAACCATGAGCCT TGTGTCTGGTCCCTATCCCCAC DUSP6 Site A TCTTGGACTCAGCCTCGCAC TTCCGCAGCACGCTCATTGG DUSP6 Site B CGAGGCAGCTCCTCAATGGAT CTCATTGGCTGGCCCGGAGA DUSP6 Site C CCGGCTGGAACAGGTTGTGTT GGGCTTATCCGGAGCGGAAAT SPRY4 Site A GCTGTCAGCTCAGCTCGCTA CGCGCTCAGGGTCTGTTGT SPRY4 Site B CGTGCCGTTCGCGATTCTAT GCCAGTCCCAGCAATCACG SPRY4 Site C GGCACAACAGACCCTGAGCG AGCCAGTCCCAGCAATCACG
Thesis/Dissertation
eng
Pathology and Laboratory Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Functional Investigations of Capicua (CIC) and ATAXIN-1-Like (ATXN1L) in cancer.
Text