Understanding How Single Cell Errors Generate Cancer Genome Complexity

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Summary

The BFB (breakage-fusion-bridge) cycle, also known as the chromosomal breakage-fusion-bridge cycle, is a mutational process that results in the amplification of genes and instability of the genome. In cancer genomes, signatures of BFB cycles can be found with chromothripsis, which is another catastrophic mutational event. They explain this correlation by explaining a mutational cascade that is launched by a single cell division mistake known as chromosomal bridge creation, which quickly increases genomic complexity. This discovery allows them to explain the relationship between the two variables. They demonstrate that actomyosin forces are necessary for the early breakdown of the bridge. Chromothripsis begins with an abnormality in the interphase replication of bridge DNA and continues until it causes an accumulation. Extensive DNA damage is produced as a result of a subsequent burst of DNA replication that occurs during the succeeding mitosis. In the course of the second cell division, damaged bridge chromosomes commonly fail to segregate properly, resulting in the formation of micronuclei and an increase in the rate of chromothripsis. They hypothesize that repeated instances of this mutational cascade are responsible for the ongoing evolution and subclonal heterogeneity that are hallmarks of many human malignancies.

Fig.1 Graphical abstract. (Umbreit, 2020)

Research Criteria

They created essential steps of the BFB cycle in a defined system, enabling mechanistic studies and determination of the immediate and long-term genomic consequences of bridge formation. To identify the immediate outcomes of bridge breakage, they used live-cell imaging coupled with single-cell whole-genome sequencing (Look-Seq).

Sample Type

Human cell lines.

Result—The Extension of Chromosome Bridges Is Required for Their Breakage

To determine whether bridge extension is required for breaking, they constrained cell migration and bridge extension using rectangular fibronectin "micropatterns". When plated on lengthy patterns, newly created chromosomal bridges in RPE-1 (retinal pigmented epithelial) cells extended to an average of 160 m, and they ruptured during interphase with a kinetic identical to an unconstrained cell. On the other hand, limiting bridge extension using brief micropatterns limited bridge extension to less than 50 m and essentially avoided bridge breaking (only 10% bridge cleavage prior to entry into the next mitosis). Additionally, the Lamin B1 knockdown failed to hasten bridge breakage, which resulted in a significant decrease in spontaneous nuclear envelope ruptures (NE) of over eightfold. In order to shatter them, chromosomal bridge extension rather than NE rupture is required.

Fig.2 Extension of chromosome bridges is required for their breakage. (Umbreit, 2020)

Result—Single-Cell Sequencing to Determine the Immediate Effect of Chromosome Bridge Breakage

They use live-cell imaging and single-cell whole-genome sequencing (Look-Seq) to identify the immediate effects of bridge breakage without confounding genomic alterations during cell divisions. Induced chromosome bridges were monitored, and the two daughter cells were isolated ~8 hours after bridge breakage for sequencing. Sequencing covered ~90% of the specific sequence of each homologous chromosome with one or more reads at 25x genome coverage.

Because dicentric fusions of sister chromatids or single chromatids from different chromosomes break, the BFB model predicts reciprocal terminal chromosome segment gain and loss patterns in daughter cells. After bridge breakage, reciprocal exchange affected one or more chromosome arms (>2.5Mb) in all 20 daughter cell pairs. Haplotype copy number analysis revealed the broken homologous chromosomes. They found that four daughter cell pairs exchanged internal chromosome segments. Bridge breakage when dicentric fusions of replicated chromatids from two chromosomes form explains this pattern.

Fig.3 Immediate effect of chromosome bridge breakage on DNA copy number. (Umbreit, 2020)

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Reference

1. Umbreit, N.T.; et al. Mechanisms generating cancer genome complexity from a single cell division error. Science. 2020, 368(6488): eaba0712.