Disentangling the roles of aneuploidy, chromosomal instability and tumour heterogeneity in developing resistance to cancer therapies.

Aneuploidy is defined as the cellular state of having a number of chromosomes that deviates from a multiple of the normal haploid chromosome number of a given organism. Aneuploidy can be present in a static state: Down syndrome individuals stably maintain an extra copy of chromosome 21 in their cells. In cancer cells, however, aneuploidy is usually present in combination with chromosomal instability (CIN) which leads to a continual generation of new chromosomal alterations and the development of intratumour heterogeneity (ITH). The prevalence of cells with specific chromosomal alterations is further shaped by evolutionary selection, for example, during the administration of cancer therapies. Aneuploidy, CIN and ITH have each been individually associated with poor prognosis in cancer, and a wealth of evidence suggests they contribute, either alone or in combination, to cancer therapy resistance by providing a reservoir of potential resistant states, or the ability to rapidly evolve resistance. A full understanding of the contribution and interplay between aneuploidy, CIN and ITH is required to tackle therapy resistance in cancer patients. However, these characteristics often co-occur and are intrinsically linked, presenting a major challenge to defining their individual contributions. Moreover, their accurate measurement in both experimental and clinical settings is a technical hurdle. Here, we attempt to deconstruct the contribution of the individual and combined roles of aneuploidy, CIN and ITH to therapy resistance in cancer, and outline emerging approaches to measure and disentangle their roles as a step towards integrating these principles into cancer therapeutic strategy.

Targeted assembly of ectopic kinetochores to induce chromosome-specific segmental aneuploidies.

Cancer cells display persistent underlying chromosomal instability, with individual tumour types intriguingly exhibiting characteristic subsets of whole, and subchromosomal aneuploidies. Few methods to induce specific aneuploidies will exist, hampering investigation of functional consequences of recurrent aneuploidies, as well as the acute consequences of specific chromosome mis-segregation. We therefore investigated the possibility of sabotaging the mitotic segregation of specific chromosomes using nuclease-dead CRISPR-Cas9 (dCas9) as a cargo carrier to specific genomic loci. We recruited the kinetochore-nucleating domain of centromere protein CENP-T to assemble ectopic kinetochores either near the centromere of chromosome 9, or the telomere of chromosome 1. Ectopic kinetochore assembly led to increased chromosome instability and partial aneuploidy of the target chromosomes, providing the potential to induce specific chromosome mis-segregation events in a range of cell types. We also provide an analysis of putative endogenous repeats that could support ectopic kinetochore formation. Overall, our findings provide new insights into ectopic kinetochore biology and represent an important step towards investigating the role of specific aneuploidy and chromosome mis-segregation events in diseases associated with aneuploidy.

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

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.

The multifaceted role of micronuclei in tumour progression: A whole organism perspective.

Within most tumour types, cancerous cells exist in a state of aneuploidy, an incorrect chromosome number or structure. Additionally, tumour cells frequently exhibit chromosomal instability; the ongoing loss or gain of whole or parts of chromosomes during cell division. Chromosomal instability results in a high rate of chromosome segregation defects, and a constantly changing genomic landscape. A second consequence of recurrent chromosome segregation defects is the exclusion of mis-segregated chromatin from the newly reforming nucleus. Chromosomes, or chromosome fragments that are not incorporated into the main nucleus are often packaged into extranuclear structures called micronuclei. While the initial impact of micronucleus formation is an imbalance or loss of genetic material in the resulting daughter cells, several other downstream consequences are now known to result from this process. In this review, we discuss the further consequences of micronucleus formation, including how structural changes to the micronuclear envelope, and the rupturing of micronuclear membranes can contribute to metastasis, immune cell activation and overall, tumour progression.

Cells on lockdown: long-term consequences of CDK4/6 inhibition.

Inhibition of cyclin-dependent kinases Cdk4/6 is emerging as a useful anti-proliferative chemotherapy, but it remains unclear how durable inhibition of cancer cell proliferation is achieved to promote a long-lasting response in patients, or how toxicity is limited to cancer cells with minimal side effects. Two recent papers in The EMBO Journal investigating senescence induction following prolonged Cdk4/6 inhibitor treatment now reveal important insights into ways to increase anti-tumour effects of Cdk4/6 inhibition and to reduce therapy-induced side effects of senescence induction.

Diversity in chromosome numbers promotes resistance to chemotherapeutics.

In this issue of Developmental Cell, papers from Ippolito et al. and from Lukow et al. show that increasing the range of aneuploidy states in cells increases their chance of developing resistance when they are subjected to chemotherapy.

Chromosome Instability through the Ages: Parallels between Speciation and Somatic (Cancer) Evolution.

Cancer research has recently benefitted from the integration of evolutionary theory to study somatic genome evolution during tumor development. Here, we explore how investigating mechanistic principles of chromosomal instability during both species and somatic evolution can reciprocally inform each field.

Trends in antimicrobial resistance legislation 2011-2019: A review of the US policy response to the antimicrobial resistance threat and its public health Impact.

BACKGROUND: Antimicrobial resistance is a continued global threat to public health. In recognition of this threat, the executive branch of the United States government consolidated guidelines through the President's Council of Advisors on Science and Technology and Executive Order 13676. METHODS: An analysis was conducted to assess the US government's response to this growing threat. Human and animal health legislation from 2011 to 2019 was reviewed using the congressional legislative database where 28 pieces of antimicrobial distinct legislation was identified. RESULTS: The majority of the bills identified were human health related and common themes found were incentivizing the development of new products, biodefense, expanded scope of use, and stewardship. Much fewer were identified pertaining to animal health; of those identified, common themes included stricter veterinary oversight, barriers to approval pathways for new products, and stewardship. CONCLUSIONS: The public health impact of these legislative efforts is in part related to a broader political tension of the role of government vs local authorities in managing regulatory enforcement and guidelines.

Specific Mechanisms of Chromosomal Instability Indicate Therapeutic Sensitivities in High-Grade Serous Ovarian Carcinoma.

Chromosomal instability (CIN) comprises continual gain and loss of chromosomes or parts of chromosomes and occurs in the majority of cancers, often conferring poor prognosis. Because of a scarcity of functional studies and poor understanding of how genetic or gene expression landscapes connect to specific CIN mechanisms, causes of CIN in most cancer types remain unknown. High-grade serous ovarian carcinoma (HGSC), the most common subtype of ovarian cancer, is the major cause of death due to gynecologic malignancy in the Western world, with chemotherapy resistance developing in almost all patients. HGSC exhibits high rates of chromosomal aberrations and knowledge of causative mechanisms would represent an important step toward combating this disease. Here we perform the first in-depth functional characterization of mechanisms driving CIN in HGSC in seven cell lines that accurately recapitulate HGSC genetics. Multiple mechanisms coexisted to drive CIN in HGSC, including elevated microtubule dynamics and DNA replication stress that can be partially rescued to reduce CIN by low doses of paclitaxel and nucleoside supplementation, respectively. Distinct CIN mechanisms indicated relationships with HGSC-relevant therapy including PARP inhibition and microtubule-targeting agents. Comprehensive genomic and transcriptomic profiling revealed deregulation of various genes involved in genome stability but were not directly predictive of specific CIN mechanisms, underscoring the importance of functional characterization to identify causes of CIN. Overall, we show that HGSC CIN is complex and suggest that specific CIN mechanisms could be used as functional biomarkers to indicate appropriate therapy. SIGNIFICANCE: These findings characterize multiple deregulated mechanisms of genome stability that lead to CIN in ovarian cancer and demonstrate the benefit of integrating analysis of said mechanisms into predictions of therapy response.

Towards restoring proper chromosome segregation and preventing ageing.

The natural progressive dysfunction of most living organisms-ageing-has captured the attention of several studies, with the intention to develop rejuvenation strategies. Evidence is emerging of a positive correlation between natural ageing and chromosomal instability (CIN). In this issue of EMBO Reports, Barroso-Vilares et al [1] now show a link between ageing and the erroneous assembly of the apparatus required for a proper cellular division. They compare this mechanism in young and naturally aged human cells and describe a strategy to delay age-related CIN.

Virtual reconstruction of unilateral pelvic fractures by using pelvic symmetry.

PURPOSE: Pelvic fractures are known to be one of the most difficult injuries to treat. The objective of this study is to introduce a novel technique for virtual unilateral pelvic fracture reconstruction. Since the pelvis exhibits remarkable left-right symmetry, the contralateral hemipelvis can be used as a template for rebuilding the fractured hemipelvis. METHODS: CT scan data of the pelvic region of eight subjects with acute unilateral pelvic fractures were involved in this study. Computer-aided design software was used to create 3D models of these pelvises. The contralateral hemipelvis of each subject was then reflected across the sagittal plane, and the fractured hemipelvis was rebuilt by aligning the bone fragments with their equivalent location on the reflected side. To evaluate the quality of this reduction process, a 3D deviation analysis was conducted to calculate the differences between the reflected intact hemipelvis and the reconstructed hemipelvis. RESULTS: Results showed that the average root mean square (RMS) of deviations and average percentage of points within a ± 2 mm predefined threshold was 1.32 ± 0.22 mm and 88.4 ± 3.78%, respectively. The deviation color maps obtained indicated that the largest differences were along the fracture lines and on the non-articular surfaces of the pelvises. CONCLUSION: These results allowed us to conclude the validity of this procedure, since the average RMS difference was below 2 mm and the average percentage of points within ± 2 mm was high. The proposed technique will allow surgeons to provide their patients with more accurate reconstruction procedures which can potentially improve surgical outcomes.

Left Atrial Intramural Hematoma Post-Ablation of Supraventricular Tachycardia: A Rare Complication Treated Successfully.

We report successful management of left atrial hematoma after ablation of supraventricular tachycardia. A 43-year-old female patient experienced chest pain immediately after radiofrequency ablation of a symptomatic left posterolateral accessory pathway. Transthoracic echocardiography demonstrated a large mass occupying the left atrium. Computed tomography and transesophageal echocardiography results were consistent with posterolateral intramural hematoma. She became hemodynamically unstable, requiring emergent surgery. The mass resolved completely by 6 weeks. (Level of Difficulty: Beginner.).

Human chromosome-specific aneuploidy is influenced by DNA-dependent centromeric features.

Intrinsic genomic features of individual chromosomes can contribute to chromosome-specific aneuploidy. Centromeres are key elements for the maintenance of chromosome segregation fidelity via a specialized chromatin marked by CENP-A wrapped by repetitive DNA. These long stretches of repetitive DNA vary in length among human chromosomes. Using CENP-A genetic inactivation in human cells, we directly interrogate if differences in the centromere length reflect the heterogeneity of centromeric DNA-dependent features and whether this, in turn, affects the genesis of chromosome-specific aneuploidy. Using three distinct approaches, we show that mis-segregation rates vary among different chromosomes under conditions that compromise centromere function. Whole-genome sequencing and centromere mapping combined with cytogenetic analysis, small molecule inhibitors, and genetic manipulation revealed that inter-chromosomal heterogeneity of centromeric features, but not centromere length, influences chromosome segregation fidelity. We conclude that faithful chromosome segregation for most of human chromosomes is biased in favor of centromeres with high abundance of DNA-dependent centromeric components. These inter-chromosomal differences in centromere features can translate into non-random aneuploidy, a hallmark of cancer and genetic diseases.

Single-cell approaches to understand genome organisation throughout the cell cycle.

Mammalian genomes are ordered at several scales, ranging from nucleosomes (beads on a string), to topologically associated domains (TADs), laminar associated domains (LADs), and chromosome territories. These are described briefly below and we refer the reader to some recent comprehensive reviews on genome architecture summarising the current state of knowledge of the organisational principles of the nucleus [1,2]. Biological observations from populations of millions of individual cells can reveal consensus behaviour. New methods to study and interpret biological data at the single-cell level have recently been instrumental in revealing new understanding of cell-to-cell variation and novel biology. Here we will summarise the recent advances in single-cell technology that have provided insights into the behaviour of the mammalian genome during a cell cycle. We will focus on the interphase domain structure of chromosomes, including TADs and LADs, and how chromosome architecture changes during the cell cycle. The role of genome architecture relating to gene expression has been reviewed elsewhere [3].

The emerging links between chromosomal instability (CIN), metastasis, inflammation and tumour immunity.

Many cancers possess an incorrect number of chromosomes, a state described as aneuploidy. Aneuploidy is often caused by Chromosomal Instability (CIN), a process of continuous chromosome mis-segregation. CIN is believed to endow tumours with enhanced evolutionary capabilities due to increased intratumour heterogeneity, and facilitating adaptive resistance to therapies. Recently, however, additional consequences and associations with CIN have been revealed, prompting the need to understand this universal hallmark of cancer in a multifaceted context. This review is focused on the investigation of possible links between CIN, metastasis and the host immune system in cancer development and treatment. We specifically focus on these links since most cancer deaths are due to the consequences of metastasis, and immunotherapy is a rapidly expanding novel avenue of cancer therapy.