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Abstract

Cancer cells must overcome telomere erosion to achieve unlimited replicative potential, thus requiring the acquisition of a telomere maintenance mechanism. The majority of tu- mors exploits telomerase reverse-transcriptase, the telomerase enzyme, while between 5% and 10% of cancers are telomerase-negative, ensuring the maintenance of telomeric ho- meostasis through alternative means. Critically short telomeres have been shown to induce chromosomal fusions, structures that persist throughout the cell cycle and that can lead to the formation of anaphase chromatin bridges; when broken with cytokinesis, these atypical mitotic structures often result in the formation of chromosomal abnormalities. Neverthe- less, the broader impact of telomere maintenance and defects on the generation of struc- tural variation remains to be elucidated. Alternative lengthening of telomeres (ALT) employs an error-prone homology-directed DNA repair mechanism, namely break-induced replica- tion (BIR); the elongation of short telomeres occurs via recombination and synthesis on telomere repeat-containing sites, both at telomeres and interspersed on chromosomes, in the absence of telomerase. Interestingly, BIR appears to also take place on chromosomes that experienced bridge breakage, leading to the segregation of the affected genetic materi- al into micronuclei, sites where complex genomic rearrangements (CGRs) have been proven to accumulate, in the subsequent cell cycle. Additionally, ALT tumors often present mutually exclusive, truncating mutations of ATRX or DAXX, epigenetic modifiers responsible for al- lowing the compaction in heterochromatin of repetitive sequences of the genome, including telomeres. This project investigated the effect of telomere maintenance and associated factors on the formation and repertoire of structural variants. ALT, intrinsically elevating recombination rates, could promote chromosomal instability and the emergence of specific CGR patterns. Preliminary analyses on the PCAWG dataset highlighted significant correlations between ALT(-related) features and CGRs in cancer samples. To mimic different subsets of the PCAWG dataset, knock-out models of ALT-characteristic mutations and a gene silencing model of ALT induction were generated. Nuclear atypia is a well established marker of ongo- ing genetic instability, hence type and frequency of atypia across models were scored, iden- tifying micronucleated cells as the population to enrich for through the aid of the MAGIC (Machine learning-Assisted Genomics and Imaging Convergence) platform, to maximise the capturing of chromosomal alterations. Strand-seq allowed the assessment of the contribu- tion of each element in the formation of the spectrum of rearrangements. While the ALT- characteristic truncating mutations of ATRX increased the number of de novo SVs found in the cells, the simultaneous absence of p53 was fundamental to boost the complexity of the observed rearrangements. By itself, ALT induction also resulted in the accumulation of dis- tinct classes of complex rearrangements, the breakpoints of which were particularly en- riched in susceptible sequences found in the genome, as G-quadruplex forming regions and both late- and early-replicating regions. By characterising CGRs in cells with diverse genetic backgrounds and telomere maintenance mechanisms, this study confirmed the role of ALT as a strong mutagenic force, proving its ability to induce the de novo formation of distinct patterns of structural variants. Taken together, these findings will aid the identification of novel potential vulnerabilities of ALT cancers.