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.

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.

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.

Emphysematous Pyelonephritis: A single center review.

BACKGROUND AND OBJECTIVE: Emphysematous pyelonephritis (EPN) is a rare, life-threatening necrotizing renal parenchymal infection. Traditional management of EPN with nephrectomy had a mortality of 40-50%. The purpose of this case series was to assess the management, biochemical factors, and outcome of EPN patients. METHODS: In this retrospective study, patients admitted to The Indus Hospital, Karachi with a diagnosis of EPN from January 2010 to February 2019, were grouped according to the Huang Tseng Classification (HTC). Their biochemical parameters, sensorium states and outcomes were recorded and analysed. RESULTS: Twenty patients were reviewed (9 males). No mortality was recorded. 11 patients (55%) were treated conservatively with only intravenous antibiotics and eight patients underwent an intervention: minimally invasive with drain placement in six patients, and invasive in four patients (two underwent subsequent nephrectomy, and two patients had nephrectomy only). One patient left against medical advice. All patients had decreased serum creatinine levels and total leucocyte counts on discharge. CONCLUSION: EPN can be successfully managed conservatively for HTC Grade-1 and 2. Conservative treatment may be tried in higher grades, but poor response should lead to prompt escalation of treatment.

Non-random Mis-segregation of Human Chromosomes.

A common assumption is that human chromosomes carry equal chances of mis-segregation during compromised cell division. Human chromosomes vary in multiple parameters that might generate bias, but technological limitations have precluded a comprehensive analysis of chromosome-specific aneuploidy. Here, by imaging specific centromeres coupled with high-throughput single-cell analysis as well as single-cell sequencing, we show that aneuploidy occurs non-randomly following common treatments to elevate chromosome mis-segregation. Temporary spindle disruption leads to elevated mis-segregation and aneuploidy of a subset of chromosomes, particularly affecting chromosomes 1 and 2. Unexpectedly, we find that a period of mitotic delay weakens centromeric cohesion and promotes chromosome mis-segregation and that chromosomes 1 and 2 are particularly prone to suffer cohesion fatigue. Our findings demonstrate that inherent properties of individual chromosomes can bias chromosome mis-segregation and aneuploidy rates, with implications for studies on aneuploidy in human disease.

Rif1 Regulates the Fate of DNA Entanglements during Mitosis.

Clearance of entangled DNA from the anaphase mid-region must accurately proceed in order for chromosomes to segregate with high fidelity. Loss of Taz1 (fission yeast ortholog of human TRF1/TRF2) leads to stalled telomeric replication forks that trigger telomeric entanglements; the resolution of these entanglements fails at ≤20°C. Here, we investigate these entanglements and their promotion by the conserved replication/repair protein Rif1. Rif1 plays no role in taz1Δ fork stalling. Rather, Rif1 localizes to the anaphase mid-region and regulates the resolution of persisting DNA structures. This anaphase role for Rif1 is genetically separate from the role of Rif1 in S/G2, though both roles require binding to PP1 phosphatase, implying spatially and temporally distinct Rif1-regulated phosphatase substrates. Rif1 thus acts as a double-edged sword. Although it inhibits the resolution of taz1Δ telomere entanglements, it promotes the resolution of non-telomeric ultrafine anaphase bridges at ≤20°C. We suggest a unifying model for Rif1's seemingly diverse roles in chromosome segregation in eukaryotes.

DNA barcode based wildlife forensics for resolving the origin of claw samples using a novel primer cocktail.

Excessive wildlife hunting for commercial purposes can have negative impacts on biodiversity and may result in species extinction. To ensure compliance with legal statutes, forensic identification approaches relying on molecular markers may be used to identify the species of origin of animal material from hairs, claw, blood, bone, or meat. Using this approach, DNA sequences from the COI "barcoding" gene have been used to identify material from a number of domesticated animal species. However, many wild species of carnivores still present great challenges in generating COI barcodes using standard "universal" primer pairs. In the work presented here, the mitochondrial COI gene was successfully amplified using a novel primer cocktail, and the products were sequenced to determine the species of twenty one unknown samples of claw material collected as part of forensic wildlife case investigations. Sixteen of the unknown samples were recognized to have originated from either Panthera leo or P. pardus individuals. The remaining five samples could be identified only to the family level due to the absence of reference animal sequences. This is the first report on the use of COI sequences for the identification of P. pardus and P. leo from claw samples as part of forensic investigations in India. The study also highlights the need for adequate reference material to aid in the resolution of suspected cases of illegal wildlife harvesting.

Replication stress links structural and numerical cancer chromosomal instability.

Cancer chromosomal instability (CIN) results in an increased rate of change of chromosome number and structure and generates intratumour heterogeneity. CIN is observed in most solid tumours and is associated with both poor prognosis and drug resistance. Understanding a mechanistic basis for CIN is therefore paramount. Here we find evidence for impaired replication fork progression and increased DNA replication stress in CIN(+) colorectal cancer (CRC) cells relative to CIN(-) CRC cells, with structural chromosome abnormalities precipitating chromosome missegregation in mitosis. We identify three new CIN-suppressor genes (PIGN (also known as MCD4), MEX3C (RKHD2) and ZNF516 (KIAA0222)) encoded on chromosome 18q that are subject to frequent copy number loss in CIN(+) CRC. Chromosome 18q loss was temporally associated with aneuploidy onset at the adenoma-carcinoma transition. CIN-suppressor gene silencing leads to DNA replication stress, structural chromosome abnormalities and chromosome missegregation. Supplementing cells with nucleosides, to alleviate replication-associated damage, reduces the frequency of chromosome segregation errors after CIN-suppressor gene silencing, and attenuates segregation errors and DNA damage in CIN(+) cells. These data implicate a central role for replication stress in the generation of structural and numerical CIN, which may inform new therapeutic approaches to limit intratumour heterogeneity.