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The advent of CRISPR-Cas9 genetic engineering technologies has vastly improved our ability to interrogate genetic interactions in high-throughput studies. CRISPR-Cas9 screens have rapidly evolved to become the main approach for unbiased, genome-wide, forward genetic studies. This work seeks to harness the innate potential of CRISPR-Cas9 screens to identify functional genetic interactions. First, we clarified the role of the master tumour suppressor, p53, in the feasibility of CRISPR-Cas9 screening by performing parallel focused screens in wild-type and TP53KO human RPE-1 cells and conducting downstream analysis of screen performance. Our results demonstrated that functional p53 negatively impacts the sensitivity of CRISPR-Cas9 screens. Moreover, we established other important factors that affect screen sensitivity, including appropriate guide-RNA representation, sufficient sequencing depth, and the use of multiple high-editing clones. Careful consideration of these factors in screen design and execution allows successful CRISPR-Cas9 screens to be carried out in both p53-proficient and p53-deficient cells, thereby fostering new biological insights. Second, using focused and whole-genome CRISPR-Cas9 screens we identified RAD54L2 as a novel factor involved in cellular responses to the topoisomerase II (TOP2) poison etoposide. Through downstream biochemical studies, we demonstrated that RAD54L2 can counter TOP2-DNA genotoxic adducts, ultimately preventing DNA double-strand break formation and adding a layer of intricacy to the cellular mechanisms that safeguard the genome from TOP2-mediated DNA damage. Since etoposide is widely used for cancer chemotherapy, these findings suggest RAD54L2-mediated resolution of TOP2-DNA adducts could represent a mechanism for tumour adaptation and, therefore, raise RAD54L2 as a potential biomarker for chemotherapy and an attractive candidate for drug discovery. Third, we performed whole-genome CRISPR-Cas9 screens in cancer models of microsatellite instability (MSI) and made headway into identifying novel genetic interactions that can drive resistance or hypersensitivity to WRN-depletion. This work amplifies the evidence for WRN-depletion as a promising new avenue for chemotherapy in cancer models with MSI. Altogether, this work demonstrates the potential of CRISPR-Cas9 screening technologies in identifying functional genetic interactions in a variety of cellular contexts. Additionally, this work contributes to the growing fields of p53 and TOP2 biology and highlights the potential of WRN inhibition as a promising chemotherapeutic agent.

Background: Lynch syndrome (LS) is the most common inherited cause of Endometrial Cancer (EC), however testing for LS in EC is not routine. Identifying LS determines eligibility for immunotherapy trials, enables surveillance to reduce colorectal cancer deaths, and promotes cascade testing of relatives. Methods: I performed a systematic review with meta-analysis of the prevalence of LS associated EC. Next, LS testing was offered to all women presenting with EC to our institution between 2015-2017. Tumours were tested for microsatellite instability (MSI) and mismatch repair (MMR) deficiency immunohistochemistry (IHC), with MLH1 hypermethylation testing and/or germline next generation sequencing (NGS) as indicated. I assessed the acceptability of LS testing at various stages of the cancer pathway. Costs were identified using micro-costing, whereby clinical/laboratory staff time and consumables/capital equipment costs were calculated. The immunological and molecular landscape of proven LS-EC was compared to sporadic EC by IHC and NGS. Results: The systematic review included 12,633 EC cases and found 3% were LS-associated. This was supported by our prospective cohort study of 500 patients, of whom only less than 1% declined testing, when offered on the day of surgery. Consenting women for LS testing in follow-up was associated with less cancer worry. Sensitivity and specificity for microsatellite instability testing with targeted MLH1 promotor hypermethylation analysis was 62.5% and 97.3% respectively. For immunohistochemistry with targeted MLH1 promotor hypermethylation analysis the sensitivity and specificity were 100% and 98.6% respectively. Sixteen women had LS-EC, of whom only three were known to have LS. Eleven variants of unknown significance were found. Of those tumours with unexplained microsatellite instability or immunohistochemistry mismatch protein loss, 16% and 92% were found to have a somatic path_MMR mutation. The cheapest testing strategy cost just £42.01/EC. LS15 EC (n=63) had significantly higher densities of infiltrating immune cells than sporadic MMR-deficient (n=95)/proficient EC (n=402), and its distinct molecular profile reflected its unique heritage. Conclusion: Unselected testing of EC for LS is cheap, acceptable to women and by directing tailored treatment and risk-reducing interventions, will save lives. The immunological and somatic mutational landscape of LS associated EC is distinct from other etiologies of EC MMR deficiency; this should inform clinical treatment trials exploring the efficacy of checkpoint inhibition in MMR deficient cancers. These results pioneered international LS-EC guidance and opened discussion with UK policy makers.

A cancer genome carries the historic mutagenic activity that has occurred throughout the development of a tumour. While driver mutations were the main focus of cancer research for a long time, passenger mutational signatures – the imprints of DNA damage and DNA repair processes that have been operative during tumourigenesis – are also biologically informative. Since the inception of the field, many mutational signatures have been uncovered from the analysis of primary tumours. However, the causes and the mechanisms underlying many of these signatures are not fully understood nor have they been established with experimental evidence. Several pioneering experimental studies have shown that mutational signatures are not just mathematical abstraction; they can be recreated in vitro using isogenic experimental model systems. In this dissertation, I build on these pioneering efforts to demonstrate the concept of mutational signatures by engineering gene knockouts in various isogenic human cell line models (i.e. HAP-1 cells and human-induced pluripotent stem cells) and using whole-genome sequencing as readouts to study their associated mutational patterns. In doing so, I have successfully validated some of the most widely-applied signatures, including those that are associated with deficiencies in homologous recombination-based repair and mismatch repair. During this process, I have also identified key issues in designing and performing such mutational signature studies, and hence devised a set of guidelines for future experiments and analyses. The work described in this dissertation paves the way for future investigations aiming to understand the relationships between DNA repair and the causes of somatic mutations in human cancers. The experimental evidence established for some of the mutational signatures here may hopefully support their clinical applications henceforth.

Low-grade and early-stage endometrioid endometrial carcinomas (EECs) have an overall good prognosis but biomarkers identifying patients at risk of relapse are still lacking. Recently, CTNNB1 exon 3 mutation has been identified as a potential risk factor of recurrence in these patients. We evaluate the prognostic value of CTNNB1 mutation in a single-centre cohort of 218 low-grade, early-stage EECs, and the correlation with beta-catenin and LEF1 immunohistochemistry as candidate surrogate markers. CTNNB1 exon 3 hotspot mutations were evaluated by Sanger sequencing. Immunohistochemical staining of mismatch repair proteins (MLH1, PMS2, MSH2, and MSH6), p53, beta-catenin, and LEF1 was performed in representative tissue microarrays. Tumours were also reviewed for mucinous and squamous differentiation, and MELF pattern. Nineteen (8.7%) tumours harboured a mutation in CTNNB1 exon 3. Nuclear beta-catenin and LEF1 were significantly associated with CTNNB1 mutation, showing nuclear beta-catenin a better specificity and positive predictive value for CTNNB1 mutation. Tumours with CTNNB1 exon 3 mutation were associated with reduced disease-free survival (p = 0.010), but no impact on overall survival was found (p = 0.807). The risk of relapse in tumours with CTNNB1 exon 3 mutation was independent of FIGO stage, tumour grade, mismatch repair protein expression, or the presence of lymphovascular space invasion. CTNNB1 exon 3 mutation has a negative impact on disease-free survival in low-grade, early-stage EECs. Nuclear beta-catenin shows a higher positive predictive value than LEF1 for CTNNB1 exon 3 mutation in these tumours.

Cancers begin through acquisition of genomic alterations in normal cells, leading to uncontrolled cell division. However, cancer cells continue to accumulate alterations throughout the disease course. Therefore cancer in a patient is a continually changing entity subject to microevolution. This leads to tumor heterogeneity, or genetic diversity within a cancer case. Heterogeneity significantly complicates cancer diagnosis and treatment, as different cancer cells in the same patient may behave and respond differently. Current research and clinical paradigms of cancer inspect the disease at one or a few fixed time points, for instance at a patient's oncologist visits. Furthermore, most clinical and research assays of cancer in patients analyze only the small subsets of cancer cells obtained through singular biopsies. Through studying mutation and heterogeneity, we can formulate models of cancer as the dynamic process that it is, and explore the temporal and spatial gaps in between the currently studied snapshots of cancer.In this work, we first investigate microsatellite instability (MSI), a prolific mutational pattern present in subsets of human cancers. We introduce a new software algorithm, MANTIS, which uses next-generation sequencing data to quantify the accumulation of MSI rather than its mere presence or absence. Applying this to a large cohort of 11,139 cancers from 39 types, we identify MSI in 3.8% of cases, including adrenocortical carcinoma where MSI had not previously been characterized. These findings have direct clinical applicability, as tumors with MSI are frequently sensitive to checkpoint inhibitor immunotherapy. Our analysis of such a diverse cohort highlights the potential of large-scale sequencing to identify and characterize rare phenotypes which may benefit subsets of patients.Another means to assess changes in tumor heterogeneity over time is through subclonal modeling. Subclones provide a theoretical framework to approximate tumor heterogeneity and cancer microevolution through gradual accumulation of mutations. Here we infer tumor subclones and estimate their phylogeny in patients with cholangiocarcinoma, small-cell lung cancer, interdigitating dendritic cell sarcoma, and cancers with MSI. We leverage rapid research autopsy to obtain multiple high-quality tumor samples, and formulate subclonal models which identify shifts in cancer cell populations in response to treatment and at various metastatic sites. Furthermore, we provide extensions of the subclonal inference tool Canopy which provide ordering of mutations within phylogenetic branches, as well as quantification of MSI accumulation and microsatellite length distributions within tumor subclones. These findings demonstrate the power of subclonal modeling to provide insights into the timing and distribution of mutational events leading to tumor heterogeneity, and their contributions to treatment resistance and metastasis.Taken together, these studies suggest large-cohort next-generation sequencing and refining subclonal analysis algorithms as future pathways for expanding the "predictability horizon" of cancer. Further understanding of the dynamics of cancer mutation, especially intratumor and interpatient heterogeneity, may permit for instance elucidation of the effects of treatments on tumor genetic composition, and prediction of genetic distinctions between primary and metastatic sites of disease. Such a view of cancer as a process hence has the potential to improve precision medicine care of this disease.

Accumulation of genomic mutations in hematopoietic and mesenchymal progenitor cells can lead to such diseases as stem cell derived leukemias and lymphomas and sporadic tumors of the gastrointestinal organs. One source of mutation accretion is the dysfunction of DNA Mismatch Repair (MMR), whereby failure to correct specific replication errors may cause the fixation of inappropriate genomic sequences. Microsatellites are repetitive regions of DNA that are highly vulnerable to mutation, so microsatellite instability (MSI) can serve as a marker for MMR dysfunction. We hypothesize that the frequency of MSI in both human mesenchymal stem cells (hMSCs) and human hematopoietic stem cells (hHSCs) increases with age. We have isolated DNA from 12 hMSC colony forming units (CFU) and 6 hHSC CFU of the same patient (ages 30-92). Utilizing PCR with fluorescent primer pairs, five microsatellite loci were amplified for 24 samples from each CFU. The microsatellites were then visualized using polyacrylamide gel electrophoresis and a Typhoon 9200 phosphoimager. A sample was scored positive for MSI if the banding appeared deviant from normal. Each CFU sample was then characterized as MSI-Stable, MSI-Low, or MSI-High. Both hMSCs and hHSCs demonstrated an increase in MSI frequency with age, with a significant increase among the hHSCs that I studied when combined with a larger pool of data of other research (p=0.0034). This pattern suggests an increasing level of MMR dysfunction. The comparison between MSI frequency in hMSCs and hHSCs suggests that the level of accumulation of genomic instability is similar between cell types. This assessment identifies aging hMSCs and hHSCs as a risk factor for downstream tumorigenesis and bone degradation. These cells should be further analyzed as a target for the understanding and treatment of bone and muscle related disorders, but the field of stem cell regenerative medicine should be cautious of the risks of MMR failure.

Multicellular organisms are mosaic in nature because of genetic alterations that occur in somatic cells. There are many factors that can contribute to the formation of such alterations including aberrant DNA repair, environmental insults, epigenetic modification, errors in DNA replication and errors in chromosome duplication/segregation. To further the study of the distributions, frequencies and rates at which some alterations can occur, mouse reporter models were implemented. The Tg(âA-G11PLAP) transgenic mutation reporter mouse harbors an allele (G11 PLAP ) that is rendered incapable of producing its functional enzyme because of a reading frame shift caused by an insertion of 11 G:C basepairs. Spontaneous deletion of one G:C basepair from this mononucleotide repeat restores gene function, and cells with PLAP activity can be detected histochemically. G11 PLAP mice enable mutant cells to be visualized in situ and were used to study variation during early development, in the germline, under oxidative stress and in solid tumors. To study LOH in diverse cell types in the body another reporter model was implemented. Mice that carry two different fluorescent protein genes as alleles of a locus were generated to address this issue because LOH would change a cell’s phenotype from bichrome to monochrome. As a step in assessing the utility of this approach, we derived MEF and ES cell lines from mice that carried two different fluorescent protein genes as alleles at the chromosome 6 locus, ROSA26. FACS showed that the vast majority of cells in each line expressed the two marker proteins at similar levels, but populations exhibited extrinsic and intrinsic noise with respect to expression. In addition, cells with a monochrome phenotype were frequent (10-4). In ES cells, all monochrome events were accompanied by allele loss. Mitotic recombination appeared to be the major cause, although UPD also appeared to have contributed to LOH. These cells provided a novel assay for studying genetic/karyotypic stability of cultured ES cells. Results obtained from studies with these cells support the need for caution regarding the use of cultured stem cells in therapy.

Variation in melanoma susceptibility varies widely among Caucasian populations. This dissertation evaluated germline and somatic variation in melanoma and geographic heterogeneity in nevi density in populations of European ancestry. We investigated a single nucleotide polymorphism in the epidermal growth factor (EGF) gene that had been previously described as a risk factor for melanoma. We conducted case-control analyses using participants collected as part of the Genes, Environment and Melanoma Study, a large, international case-control study of melanoma using two different study designs including a classic comparison of incident cases to unaffected controls and a novel design comparing multiple primary melanoma cases to those with single primary melanoma. The two analytic approaches used in this study provide evidence against a strong association between EGF 61*G and susceptibility to incident primary melanoma. In a second study, we combined molecular tools and epidemiologic techniques to evaluate independent histologic and phenotypic predictors of microsatellite instability in primary melanoma. We found that the MSI-High phenotype is rare in melanoma, and that the MSI-Low phenotype is observed in a subset (36%) of primary melanoma. Furthermore, histologic characteristics and phenotypic factors did not differ with respect to the presence of either low or high levels of microsatellite instability in primary melanoma. Although, high level instability was significantly associated with tumor location. Because there are geographic gradients in the density of benign nevi that serve as markers of melanoma risk, this dissertation also investigated geographic and population variation in nevi density among European populations. In multivariable analysis, study center, ancestral origin and degree of freckling were independent predictors of nevi density. Genetic cluster analysis was unable to differentiate European subpopulations in simulations representative of the underlying allele frequencies in the study sample. Thus, self-defined ancestral labels may provide more useful information regarding environmental and phenotypic risk factors for benign nevi and melanoma than genetic cluster analysis, suggesting that strategies to account for population stratification may not require extensive genotyping. Further work is required to characterize germline and somatic variation in melanoma, and the relevance of heterogeneity observed in nevi density among European populations.