Age, DNA Methylation and the Malignant Potential of the Serrated Neoplasia Pathway Lochlan John Fennell B. Biomed Sci A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2020 Faculty of Medicine ORC ID: 0000-0003-3214-3527 1 Abstract Colorectal cancer is the third most common cancer in Australia and is responsible for the death of over four thousand Australians each year. There are two overarching molecular pathways leading to colorectal cancer. The conventional pathway, which is responsible for ~75% of colorectal cancer diagnoses, occurs in a step-wise manner and is the consequence of a series of genetic alterations including mutations of tumour suppressor genes and gross chromosomal abnormalities. This pathway has been extensively studied over the past three decades. The serrated neoplasia pathway is responsible for the remaining colorectal cancers. This pathway is triggered by oncogenic BRAF mutation and these cancers accumulate epigenetic alterations while progressing to invasive cancer. DNA methylation is important in serrated neoplasia, however the extent and role of DNA methylation on the initiation and progression of serrated lesions is not clear. DNA methylation accumulates in tissues with age, and advanced serrated lesions and cancers occur almost exclusively in elderly patients. How this methylation affects serrated lesions is unknown. In this thesis I set out to address three key research questions related to DNA methylation, age and serrated colorectal neoplasia. First, what is the extent of DNA methylation in colorectal cancers?; Second, Does age-related hypermethylation, and namely that occurring at the loci encoding tumour suppressor genes, increase the risk of serrated colorectal neoplasia?; and if true, how can we reconcile this with the existence of early onset serrated colorectal cancer? In the first chapter of this thesis, I examine the DNA methylation and transcriptional architecture of 216 colorectal cancer samples collected consecutively at the Royal Brisbane and Women’s hospital. Clustering analysis of DNA methylation data revealed five distinct subtypes of colorectal cancers, including two clusters with high levels of DNA methylation, two with intermediate levels of DNA methylation, and one devoid of DNA methylation alterations. This study highlighted striking associations between DNA methylation and age, gender and tumour location. Oncogenic BRAF mutations were common in CIMP-H1, but rare in CIMP-H2. The inverse was true for CIMP-H2. These analyses were robustly verified using data from TCGA. Using exome capture sequencing data from TCGA, we assessed the frequency of mutations in genes encoding epigenetic regulators in cancers by methylation subtype. The frequency of 2 mutation in epigenetic regulators increased concomitant with increasing genomic hypermethylation, and the spectrum of mutations differed by methylation subtype. Mutations in epigenetic regulators may provide insight into the epigenetic aberrancies observed in these highly methylated cancers. In the second chapter of this thesis, I investigate the role of age and age-associated DNA methylation and the associated transcriptional changes, in the risk of serrated neoplastic transformation using a murine model of serrated neoplasia driven by oncogenic Braf. First, the DNA methylation landscape of wild type animals was assessed over the lifespan of the animals via reduced representation bisulphite sequencing. This revealed pervasive age-associated DNA methylation, with a tendency for methylation to accumulate at the promoters of genes encoding WNT signalling regulators. Activating the Braf oncogene at wean induces a remarkable acceleration of DNA methylation accumulation, with a stronger still enrichment at WNT signalling loci. Lastly, we activated the Braf mutation in mice at wean for five months, and at nine months of age for the same period. We observe a 10.5 fold relative risk of advanced serrated lesions in animals induced at nine months compared with the younger animals, despite the same period of oncogenic exposure. These data directly implicate the aging process in determining the malignant threat of Braf induced neoplasia. Methylation analysis of these animals revealed differences in loci identified as age-associated, and those accelerated by oncogenic Braf. This study strongly implicates age and DNA methylation as a risk factors for serrated neoplastic progression and may have implications for patient surveillance. In the third chapter of this thesis, I attempt to elucidate the role of mutations in WNT signalling genes in serrated colorectal cancers, hypothesising that genetic alterations of WNT regulators will underpin early onset BRAF mutant cancers. Large exome sequencing data sets from earlier studies were collated to establish a cohort of 199 BRAF mutant colorectal cancers with somatic mutation data. This study reveals a mosaic of WNT pathway mutations, including several potential novel driver genes. APC mutations in the setting of BRAF mutant cancers were associated with poor patient outcome, and importantly, younger age at diagnosis. These data indicates that mutation of APC could provide an alternate avenue for progressing to invasive cancer, abrogating the need for excessive DNA methylation alterations. 3 To conclude, this thesis has comprehensively evaluated the role of DNA methylation and age in the risk of serrated colorectal neoplasia. These data improve our understanding of the role of DNA methylation in colorectal carcinogenesis and may provide an impetus for investigating patient-centric surveillance regimens according to the age of patients at index colonoscopy. 4 Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. 5 Publications included in this thesis 1) Fennell, L., Dumenil, T., Wockner, L., Hartel, G., Nones, K., Bond, C., Borowsky, J., Liu, C., McKeone, D., Bowdler, L., Montgomery, G., Klein, K., Hoffmann, I., Patch, A.- M., Kazakoff, S., Pearson, J., Waddell, N., Wirapati, P., Lochhead, P., Imamura, Y., Ogino, S., Shao, R., Tejpar, S., Leggett, B., Whitehall, V., 2019. Integrative Genome- Scale DNA Methylation Analysis of a Large and Unselected Cohort Reveals 5 Distinct Subtypes of Colorectal Adenocarcinomas. Cellular and Molecular Gastroenterology and Hepatology 8, 269–290. https://doi.org/10.1016/j.jcmgh.2019.04.002 2) Fennell, L.J., Kane, A., Liu, C., McKeone, D., Fernando, W., Su, C., Bond, C., Jamieson, S., Dumenil, T., Patch, A.-M., Kazakoff, S.H., Pearson, J.V., Waddell, N., Leggett, B., Whitehall, V.L.J., 2020. APC Mutation Marks an Aggressive Subtype of BRAF Mutant Colorectal Cancers. Cancers 12, 1171. 6 Submitted manuscripts included in this thesis 1) Fennell, L.J, Kane, A., Liu, C., McKeone D., Hartel, G., Bond, C.E., Bettington, M., Leggett, B.A. and Whitehall, V.L.J., 2020, Braf mutation induces rapid neoplastic transformation in the aged and extensively hypermethylated intestinal epithelium Submitted Other publications during candidature 1) Kane A., Liu, C., Fennell, L.J., McKeone, D., Bond, C., Pollock, P., Young, G., Leggett, B., and Whitehall, V. 2021 Aspirin reduces the incidence of metastasis in a preclinical study of Braf mutant serrated colorectal neoplasia. In Press, British J Cancer. Contribution: Bioinformatic analysis of RNA-Seq data 2) Kane, A., Fennell, L. J., Liu, C., Borowsky, J., Mckeone, D., Bond, C., Kazakoff, S., Patch, A., Koufariotis, L., Pearson, J., Waddell, N., Leggett, B., and Whitehall, V. 2019. Alterations in signaling pathways that accompany spontaneous transition to malignancy in a mouse model of BRAF mutant microsatellite stable colorectal cancer. In Press Neoplasia. Contribution: Bioinformatic analysis of RNA-Seq data 3) Liu, C., Fennell, L.J., Bettington, M.L., Walker, N.I., Dwine, J., Leggett, B.A. & Whitehall, V.L.J 2019 DNA Methylation changes that precede onset of dysplasia in advanced sessile serrated