Chromoanagenesis and cancer: mechanisms and consequences of localized, complex chromosomal rearrangements

Abstract

Next-generation sequencing of DNA from human tumors or individuals with developmental abnormalities has led to the discovery of a process we term chromoanagenesis, in which large numbers of complex rearrangements occur at one or a few chromosomal loci in a single catastrophic event. Two mechanisms underlie these rearrangements, both of which can be facilitated by a mitotic chromosome segregation error to produce a micronucleus containing the chromosome to undergo rearrangement. In the first, chromosome shattering (chromothripsis) is produced by mitotic entry before completion of DNA replication within the micronucleus, with a failure to disassemble the micronuclear envelope encapsulating the chromosomal fragments for random reassembly in the subsequent interphase. Alternatively, locally defective DNA replication initiates serial, microhomology-mediated template switching (chromoanasynthesis) that produces local rearrangements with altered gene copy numbers. Complex rearrangements are present in a broad spectrum of tumors and in individuals with congenital or developmental defects, highlighting the impact of chromoanagenesis on human disease.

Figure 1. Mechanism for the creation of complex chromosomal rearrangements by non-homologous end joining after chromosome shattering

Chromothripsis results in the shattering of one or a few chromosomes (or a chromosome arm) leading to the simultaneous creation of many double strand breaks. Most of the shattered fragments are stitched back together though Non-Homologous End Joining (NHEJ) leading to chromoanagenesis: the creation of a chromosome with complex, high-localized chromosomal rearrangements. The rearranged chromosome contains two copy number states: a high copy number state for each religated fragment and a low copy number state for fragments not-reincorporated and therefore lost. Broken DNA fragments may also be joined together to form circular, extrachromosomal double minute chromosomes that often harbor oncogenes and are frequently amplified, resulting in a dramatically increased copy number of DNA fragments on these chromosomes.

Figure 2. Mitotic errors produce micronuclei and subsequent chromoanagenesis

(A) Cells with extra centrosomes form multi-polar mitotic spindles. In many instances centrosomes coalesce into two groups prior to anaphase. (B) Centrosome clustering increases the frequency of merotelic attachments, the situation in which a duplicated chromosome attaches through its kinetochore to microtubules arising from both mitotic spindle poles^,. (C) If not corrected before anaphase merotelically attached chromosomes may lag in the middle of the mitotic spindle. (D) Lagging chromosomes are sometimes excluded from both daughter nuclei and instead form a micronucleus in one of the daughter cells in the subsequent interphase. (E) Micronuclei often contain fewer nuclear pore complexes, impairing nuclear import and (F) delaying DNA replication of chromosome(s) in the micronucleus. Chromoanagensis (the creation of complex, localized chromosomal rearrangements) can arise in micronuclei through two distinct mechanisms. (G) The predominant pathway known as chromothripsis, involves chromosome shattering following mitotic entry while the micronucleus is still replicating its DNA. The incompletely replicated micronuclear DNA undergoes premature chromosome condensation that results in pulverization of the trapped chromosome(s). Often the nuclear envelope of the micronucleus fails to disassemble during the next mitosis and the intact micronucleus randomly segregates at mitotic exit into one of the daughter cells. (H) During the subsequent interphase, shattered chromosome pieces within the micronucleus are repaired by NHEJ. (I) A second pathway known as chromoanasynthesis, leads to the creation of complex rearrangements in micronuclei through a replication-based mechanism, such as Microhomology Mediated Break-Induced Replication (MMBIR). In this phenomenon, defective DNA replication in the micronucleus leads to a collapsed replication fork that initiates microhomology-dependent priming of DNA replication and serial template switching. MMBIR can result in chromoanagenesis, with the creation of complex chromosomal rearrangements at genomic regions surrounding the collapsed replication fork. (J) The micronucleus nuclear envelope eventually disassembles during a subsequent mitosis, releasing the rearranged chromosome. (K) The rearranged chromosome is segregated on the mitotic spindle and reincorporated into the major nucleus of the cell.

Figure 3. Mechanism for complex chromosomal rearrangements as a result of Fork-Stalling and Template Switching (FoSTes) and Microhomology Mediated Break-Induced Replication (MMBIR)

Fork-Stalling and Template Switching (FoSTes) occurs when a replication fork stalls at a DNA lesion while MMBIR is initiated following replication fork collapse. FoSTes and MMBIR lead to microhomology-dependent priming of DNA replication and serial template switching. This leads to chromoanagenesis: the creation of complex chromosomal rearrangements in a genomic region surrounding the collapsed replication fork. In addition to the deletion and retention of DNA fragments, FoSTes and MMBIR can also lead to duplication and triplication of DNA sequences. Therefore, FoSTes and MMBIR can result in more than two copy number states on the rearranged chromosome. Modified from reference.

Figure 4. Chromoanagenesis may create oncogenic lesions

The complex chromosomal rearrangements created by chromoanagenesis can be oncogenic. (A) Initial chromosomal shattering followed by rejoining by NHEJ may create circular fragments of DNA harboring oncogenes, such as MYC. Amplification of these extrachromosomal “double minute” chromosomes can provide a growth advantage. Other pieces of a shattered chromosome may be joined together to create a highly rearranged chromosome. (B) Chromoanagenesis can lead to the loss or disruption of regions containing tumor suppressor genes, such as FBXW7. (C) Rearrangements may also create oncogenic fusion genes by joining the coding sequence two normal genes together, for example, the fusion of the MLL and the FOXR1 genes.