Replication stress generates distinctive landscapes of DNA copy number alterations and chromosome scale losses

Abstract

Background A major driver of cancer chromosomal instability is replication stress, the slowing or stalling of DNA replication. How replication stress and genomic instability are connected is not known. Aphidicolin-induced replication stress induces breakages at common fragile sites, but the exact causes of fragility are debated, and acute genomic consequences of replication stress are not fully explored. Results We characterize DNA copy number alterations (CNAs) in single, diploid non-transformed cells, caused by one cell cycle in the presence of either aphidicolin or hydroxyurea. Multiple types of CNAs are generated, associated with different genomic regions and features, and observed copy number landscapes are distinct between aphidicolin and hydroxyurea-induced replication stress. Coupling cell type-specific analysis of CNAs to gene expression and single-cell replication timing analyses pinpointed the causative large genes of the most recurrent chromosome-scale CNAs in aphidicolin. These are clustered on chromosome 7 in RPE1 epithelial cells but chromosome 1 in BJ fibroblasts. Chromosome arm level CNAs also generate acentric lagging chromatin and micronuclei containing these chromosomes. Conclusions Chromosomal instability driven by replication stress occurs via focal CNAs and chromosome arm scale changes, with the latter confined to a very small subset of chromosome regions, potentially heavily skewing cancer genome evolution. Different inducers of replication stress lead to distinctive CNA landscapes providing the opportunity to derive copy number signatures of specific replication stress mechanisms. Single-cell CNA analysis thus reveals the impact of replication stress on the genome, providing insights into the molecular mechanisms which fuel chromosomal instability in cancer.

Chromosome Inequality: Causes and Consequences of Non-Random Segregation Errors in Mitosis and Meiosis

“…Larger chromosomes were also found to missegregate more frequently in human cells when attachment error correction was compromised by inhibiting Aurora B kinase, when microtubules were stabilized by low concentrations of taxol, or when chromosome congression was compromised by inhibition of the mitotic kinesin CENP-E [ 49 , 50 ]. Interestingly, generating DNA damage in human cells during the interphase, using ionizing radiation or by causing replication stress, specifically elevated structural rearrangements and micronuclear incorporation of larger chromosomes as well [ 33 , 51 , 52 ]. Lastly, loss of endogenous CENP-A in human cells increased the segregation error frequency of chromosomes 6, X, and Y [ 53 , 54 ].…”

“…For example, random DNA damage, such as ionizing radiation, induces structural rearrangements and the formation of micronuclei of larger chromosomes more often [ 33 , 52 ]. Furthermore, replication stress relatively frequently induces the formation of micronuclei containing larger chromosomes, because these are more likely to contain sequences sensitive to replication stress [ 6 , 51 , 99 ]. Replication stress is a common phenotype in cancer as it is induced by overexpression of various oncogenes [ 100 ].…”

Single-cell DNA replication dynamics in genomically unstable cancers

“…The copyright holder for this preprint (which this version posted April 10, 2023. ; https://doi.org/10.1101/2023.04.10.536250 doi: bioRxiv preprint PERT's methodological improvements allowed for quantification of clonal and subclonal RT shifts and estimation of the relative proliferation rate of tumor subclones. Our finding that losses preferentially emerged from late RT loci and gains from early RT loci suggests that certain gains and losses might result from mechanisms of over-and under-replication or reflect differential fitness of gains vs losses in gene-rich early vs gene-poor late RT loci [37,38,40]. Similarly, our finding that bins with subclonal losses shift to earlier RT and bins with gains shift to later RT could be explained by transcriptional buffering of large CNAs as early RT is linked with high transcription rate [12,43].…”

“…S11h). These findings suggest that the RT of a genomic locus influences its gain and loss rates [37,40].…”

Tracking genome evolution in single cell clones reveals the rates and features of copy number alterations generated by ongoing chromosomal instability in cancer

“…For example, ATR inhibitor AZD6738 is effective against high replication stress cancer cells, while PARP inhibitors act upon tumours with homologous recombination deficiency (HRD) (Forment and O’Connor, 2018; Bryant et al ., 2005; Farmer et al ., 2005; Ubhi and Brown, 2019). Recent studies have unveiled characteristic genomic alteration patterns resulting from chromosomal instability processes (Shaikh et al ., 2022b), offering an opportunity for tailored cancer treatments based on individual tumour genomic alterations. HRD genomic “scars” in BRCA-deficient tumours serve as a prime example, guiding the use of PARP inhibitors in clinical decisions.…”