Pervasive lesion segregation shapes cancer genome evolution

Cancers arise through the acquisition of oncogenic mutations and grow through clonal expansion. Here we reveal that most mutagenic DNA lesions are not resolved as mutations within a single cell-cycle. Instead, DNA lesions segregate unrepaired into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterise this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multi-allelic and combinatorial genetic diversity. The phasing of lesions enables the accurate measurement of strand biased repair processes, quantification of oncogenic selection, and fine mapping of sister chromatid exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.This work was supported by: Cancer Research UK (20412, 22398), the European Research Council (615584, 682398), the Wellcome Trust (WT108749/Z/15/Z, WT106563/Z/14/A, WT202878/B/16/Z), the European Molecular Biology Laboratory, the MRC Human Genetics Unit core funding programme grants (MC_UU_00007/11, MC_UU_00007/16), and the ERDF/Spanish Ministry of Science, Innovation and Universities-Spanish State Research Agency/DamReMap Project (RTI2018-094095-B-I00)

Oncogenic selection of germline and somatic variants

Most cancers are caused by a combination of inherited germline variants and somatically acquired mutations that drive cellular dysregulation. Cancer genomics aims to distinguish functional cancer-causing genetic variants from the millions of benign passenger mutations. This thesis incorporates measures of evolutionary selection into genetic analysis to identify germline and somatic variants that are preferentially retained during cancer development. Loss-of-heterozygosity (LOH) describes when one copy of a locus is either deleted or replaced by the other copy. By studying patterns of LOH across almost 10,000 human cancers from The Cancer Genome Atlas (TCGA), this thesis quantifies preferential allelic imbalance, and in doing so identifies germline variants that are important to the developing tumour. Initial analysis discovered widespread sequencing biases and cross-sample contamination in the TCGA dataset. Consequently, a rigorous filtering pipeline was developed to mitigate their influence. In total, 1,678 pairs of tumour-normal samples (14.7%) were found to be contaminated or affected by experimental error. Subsequent analysis identified damaging variants in DNA double strand break repair genes as the most common targets of biased LOH. This result suggests a ratchet-like process where heterozygous germline mutations in these genes reduce the efficacy of DNA double-strand break repair, increasing the likelihood of a second hit. Recent work revealed that strand-specific DNA damage segregates independently into daughter cells resulting in the chromosome-scale phasing of mutations. By analysing strand inheritance in genomic segments with and without a potential driver mutation, this thesis developed and applied a statistical approach to quantify oncogenic selection of somatic mutations in a manner agnostic to functional annotation and without the requirement of LOH. Together this work provides insight into the interplay of mutation and selection in cancer evolution, revealing common routes to oncogenesis, and delineating the early stages of cancer development

GenomeArtiFinder variant include list

Background: The loss of genetic diversity in segments over a genome (loss-of-heterozygosity, LOH) is a common occurrence in many types of cancer. By analysing patterns of preferential allelic retention during LOH in approximately 10,000 cancer samples from The Cancer Genome Atlas (TCGA), we sought to systematically identify genetic polymorphisms currently segregating in the human population that are preferentially selected for, or against during cancer development. Results: Experimental batch effects and cross-sample contamination were found to be substantial confounders in this widely used and well studied dataset. To mitigate these we developed a generally applicable classifier to quantify contamination and other abnormalities. We provide these results as a resource to aid further analysis of TCGA whole exome sequencing data. In total, 1,678 pairs of samples (14.7%) were found to be contaminated or affected by systematic experimental error. After filtering, our analysis of LOH revealed an overall trend for biased retention of cancer-associated risk alleles previously identified by genome wide association studies. Analysis of predicted damaging germline variants identified highly significant oncogenic selection for recessive tumour suppressor alleles. These are enriched for biological pathways involved in genome maintenance and stability. Conclusions: Our results identified predicted damaging germline variants in genes responsible for the repair of DNA strand breaks and homologous repair as the most common targets of allele biased LOH. This suggests a ratchet-like process where heterozygous germline mutations in these genes reduce the efficacy of DNA double-strand break repair, increasing the likelihood of a second hit at the locus removing the wild-type allele and triggering an oncogenic mutator phenotype.Luft, Juliet; Taylor, Martin. (2020). GenomeArtiFinder variant include list, [dataset]. University of Edinburgh, MRC IGMM, MRC Human Genetics Unit. https://doi.org/10.7488/ds/2860

Strand-resolved mutagenicity of DNA damage and repair.

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution

Strand-resolved mutagenicity of DNA damage and repair.

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution