Chromosomal instability as a source of genomic plasticity

Abstract

Chromosomal instability (CIN) is a hallmark of the most aggressive malignancies. Features of these tumors include complex genomic rearrangements, the presence of mis-segregated chromosomes in micronuclei, and extrachromosomal DNA (ecDNA) formation. Here, we review the development of CIN, and examine CIN in the context of cancer evolution, tumor genomic evolution, and therapeutic resistance. We also discuss the role of whole-genome duplications, breakage-fusion-bridge cycles, ecDNA or double minutes in gene amplification promoting tumor evolution.

Keywords: Cancer Evolution; Chromosomal Instability; Micronuclei; ecDNA.

Figure 1:. Evolution of Tumor Cells with Copy Number Alterations

The specific genomic alterations that are characteristic of a subset of tumor cells with copy number alterations. Diploid cells first undergo loss of heterozygosity (LOH) in tumor suppressor genes such as p53 loss and mutations in the DNA damage response (DDR). Cells may undergo different trajectories: 1. (red dotted line) stochastic chromosomal gains and losses at high fitness cost due to the risk of nullisomy 2. (green dotted line) Whole genome duplication results in reduced fitness cost. 3.Stochastic chromosomal gains and losses results in selection of chromosomes with higher expression of oncogenes and lower expression of tumor suppressor genes. 4. This generates a near triploid karyotype, a state better poised for further evolution. 5. Later events in tumor evolution typically, but not exclusively, result in gene amplification events such as extrachromosomal (ecDNA) formation, bridge fusion break cycles (BFB) to form fold back inversions and new, post-whole genome duplication, LOH events to promote immune evasion, treatment resistance and metastasis (some icons made in Biorender).

Figure 2:. Chromosome Bridges Facilitate Gene Amplification

A. Dicentric Chromosomes form after fusion events such as after (1) critical telomere shortening, exposing chromosome ends and resulting in fusion of chromosomes with two centromeres (2). Upon cell division, dicentric chromosomes are attached to both centromeres (gold) impairing completion of cytokinesis resulting in an elongated connection between the two daughter cells called a chromosome bridge (3), resulting in breakage of the dicentric chromosome. Repair of the broken chromosome may result in (4) fusion of the broken chromosomes creating another dicentric chromosome, containing a duplication. A subsequent cycle of chromosome bridge formation, chromosome breakage (5) and fusion (6) results in fold back inversions and further gene amplification. Break-Fusion Bridge Cycles (7) can then generate large amplifications such as homogenously staining regions (HSR). B. An alternative model of the gene amplification through chromosome bridge formation. An actomyosin force results in fracture and separation of the dicentric chromosomes into the daughter cells. Broken ends undergo aberrant replication. At the subsequent mitosis, the chromosome that underwent a fusion-break cycle is at risk for further missegregation due to impaired replication. Localization into the micronucleus results in an increased risk of chromothripsis [26], facilitating gene amplification through HSR or ecDNA formation.

Figure 3:. Micronuclear Membrane Rupture Results in Chromothripsis Promoting Gene Amplification Through Formation of Homogeneously Staining Region and ecDNA

A. In a normal mitosis, chromosomes will align at the metaphase plate and progress to anaphase. A merotelic attachment results in a chromosome (light blue) that becomes localized into a spontaneously forming micronucleus (MN)[31]. B. Chromosomes can be localized to the Micronucleus (MN) due to improper mitotic segregation or due to dicentric bridge breakage events. Upon spontaneous MN membrane rupture, a chromosome fracturing event called chromothripsis can occur. Upon Non-homologous end joining (NHEJ) dependent religation, circular fragments can be created of fragments of the oncogene or drug resistance gene [30,31] C. Based on the model presented in Dunphy et al, circular fragments generated after chromothripsis can undergo circular recombination resulting in gene amplification forming an Homogeneously Staining Region (HSR) or ecDNA [43]

Figure 4:. Chromosomally Unstable Cells Exhibit Karyotypic Diversity and Evolve Genomic structures such as ecDNA

A. Chromosomal instability facilitates resistance to oncogene addiction and chemotherapeutic agents likely through initial genomic heterogeneity followed by iterations of genomic instability facilitating treatment resistance through gene amplification of drug-specific resistance genes [40,41]. Upon selective pressure, gene amplification can result in HSR formation, further selective pressure is associated with localization of an HSR into a MN and transition of an HSR to ecDNA [11]. B. ecDNA increase gene expression through increased copy number and through genomic interactions promoting increased gene expression of genes located on the ecDNA through interaction with enhancers localized to chromosomes. Conversely, ecDNAs can function as super enhancers (blue circle) and promote expression of chromosomally localized genes (grey promotor) [35,36]