We propose to definitively characterise the somatic genetics of matched pair breast cancer cell lines through generation of comprehensive... Show More
We propose to definitively characterise the somatic genetics of matched pair breast cancer cell lines through generation of comprehensive catalogues of somatic mutations by high coverage genome sequencing coupled with integrated transcriptomic and methylation analyses.
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This study includes 3 datasets:
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The disordered transcriptomes of cancerÂ encompass direct effects of somatic mutation on transcription; co-ordinatedÂ secondary alterations in transcriptional pathways; and increasedÂ transcriptional noise. To catalogue the rules governing how somatic mutationÂ Overall, 59% of 6980 exonicÂ substitutions wereÂ expressed. Compared to other classes, nonsense mutations showed lowerÂ expression levels than expected withÂ patterns characteristic ofÂ nonsense-mediated decay. 14% of 4234 genomic rearrangements causedÂ transcriptional abnormalities,Â including exon skips, exon reusage, fusionÂ transcripts and premature poly-adenylation. We found productive, stableÂ transcriptionÂ from sense-to-antisense gene fusions and gene-to-intergenicÂ rearrangements, suggesting that these mutation classes may drive moreÂ transcriptional disruption than previously suspected. Systematic integration ofÂ transcriptome with genome data therefore reveals theÂ rules by whichÂ transcriptional machinery interprets somatic mutation.
CRISPR-Cas9 loss-of-function screens are instrumental to systematically identify genes important for cellular fitness in cancer cells. While structural rearrangements are a ubiquitous feature in cancer, their impact on CRISPR-Cas9 response has not yet been systematically assessed. Utilising data for 163 CRISPR-Cas9 screened cancer cell lines, we demonstrate that targeting tandem amplified regions is highly detrimental to cellular fitness, in stark contrast to amplifications arising from chromosomal duplications, which have little to no effect. In addition, high ploidy leads to decreased CRISPR-Cas9 loss of fitness effects in a gene-independent way. Using whole-genome sequencing and fluorescent in situ hybridisation we confirm that clustered Cas9 double-strand DNA cuts in a single chromosome, contrary to multiple chromosomes, are associated with a strong decrease in cell fitness. We propose this as a novel way to exploit collateral vulnerabilities introduced by structural rearrangements in cancer cells, by systematically identifying tissue non-expressed genes that are tandem amplified. 25% of the screened cell lines have at least one putative collateral essentiality, showing that this is a generalizable way to selectively kill tumour cells. Lastly, we present a flexible computational tool, Crispy, to perform association analysis of different types of genomic alterations in CRISPR-Cas9 screens. Our results demonstrate the importance of structural rearrangements in mediating the effect of CRISPR-Cas9-induced DNA damage on cell fitness, and how this could be harnessed to create selective cancer therapies, especially in tumours enriched for tandem duplications.