Loss of FAM111B protease mutated in hereditary fibrosing poikiloderma negatively regulates telomere length.

Hereditary fibrosing poikiloderma (HFP) is a rare human dominant negative disorder caused by mutations in the FAM111B gene that encodes a nuclear trypsin-like serine protease. HFP patients present with symptoms including skin abnormalities, tendon contractures, myopathy and lung fibrosis. We characterized the cellular roles of human FAM111B using U2OS and MCF7 cell lines and report here that the protease interacts with components of the nuclear pore complex. Loss of FAM111B expression resulted in abnormal nuclear shape and reduced telomeric DNA content suggesting that FAM111B protease is required for normal telomere length; we show that this function is independent of telomerase or recombination driven telomere extension. Even though FAM111B-deficient cells were proficient in DNA repair, they showed hallmarks of genomic instability such as increased levels of micronuclei and ultra-fine DNA bridges. When mutated as in HFP, FAM111B was more frequently localized to the nuclear envelope, suggesting that accumulation of the mutated protease at the nuclear periphery may drive the disease pathology.

RNA-seq analysis of the human surfactant air-liquid interface culture reveals alveolar type II cell-like transcriptome.

Understanding pulmonary diseases requires robust culture models that are reproducible, sustainable in long-term culture, physiologically relevant, and suitable for assessment of therapeutic interventions. Primary human lung cells are physiologically relevant but cannot be cultured in vitro long term and, although engineered organoids are an attractive choice, they do not phenotypically recapitulate the lung parenchyma; overall, these models do not allow for the generation of reliable disease models. Recently, we described a new cell culture platform based on H441 cells that are grown at the air-liquid interface to produce the SALI culture model, for studying and correcting the rare interstitial lung disease surfactant protein B (SPB) deficiency. Here, we report the characterization of the effects of SALI culture conditions on the transcriptional profile of the constituent H441 cells. We further analyze the transcriptomics of the model in the context of surfactant metabolism and the disease phenotype through SFTPB knockout SALI cultures. By comparing the gene expression profile of SALI cultures with that of human lung parenchyma obtained via single-cell RNA sequencing, we found that SALI cultures are remarkably similar to human alveolar type II cells, implying clinical relevance of the SALI culture platform as a non-diseased human lung alveolar cell model.

Manufacturing a chimpanzee adenovirus-vectored SARS-CoV-2 vaccine to meet global needs.

Manufacturing has been the key factor limiting rollout of vaccination during the COVID-19 pandemic, requiring rapid development and large-scale implementation of novel manufacturing technologies. ChAdOx1 nCoV-19 (AZD1222, Vaxzevria) is an efficacious vaccine against SARS-CoV-2, based upon an adenovirus vector. We describe the development of a process for the production of this vaccine and others based upon the same platform, including novel features to facilitate very large-scale production. We discuss the process economics and the "distributed manufacturing" approach we have taken to provide the vaccine at globally-relevant scale and with international security of supply. Together, these approaches have enabled the largest viral vector manufacturing campaign to date, providing a substantial proportion of global COVID-19 vaccine supply at low cost.

Aluminum Enters Mammalian Cells and Destabilizes Chromosome Structure and Number.

Chromosome instability (CIN) consists of high rates of structural and numerical chromosome abnormalities and is a well-known hallmark of cancer. Aluminum is added to many industrial products of frequent use. Yet, it has no known physiological role and is a suspected human carcinogen. Here, we show that V79 cells, a well-established model for the evaluation of candidate chemical carcinogens in regulatory toxicology, when cultured in presence of aluminum-in the form of aluminum chloride (AlCl3) and at concentrations in the range of those measured in human tissues-incorporate the metal in a dose-dependent manner, predominantly accumulating it in the perinuclear region. Intracellular aluminum accumulation rapidly leads to a dose-dependent increase in DNA double strand breaks (DSB), in chromosome numerical abnormalities (aneuploidy) and to proliferation arrest in the G2/M phase of the cell cycle. During mitosis, V79 cells exposed to aluminum assemble abnormal multipolar mitotic spindles and appear to cluster supernumerary centrosomes, possibly explaining why they accumulate chromosome segregation errors and damage. We postulate that chronic aluminum absorption favors CIN in mammalian cells, thus promoting carcinogenesis.

Native-like SARS-CoV-2 Spike Glycoprotein Expressed by ChAdOx1 nCoV-19/AZD1222 Vaccine.

Vaccine development against the SARS-CoV-2 virus focuses on the principal target of the neutralizing immune response, the spike (S) glycoprotein. Adenovirus-vectored vaccines offer an effective platform for the delivery of viral antigen, but it is important for the generation of neutralizing antibodies that they produce appropriately processed and assembled viral antigen that mimics that observed on the SARS-CoV-2 virus. Here, we describe the structure, conformation, and glycosylation of the S protein derived from the adenovirus-vectored ChAdOx1 nCoV-19/AZD1222 vaccine. We demonstrate native-like post-translational processing and assembly, and reveal the expression of S proteins on the surface of cells adopting the trimeric prefusion conformation. The data presented here confirm the use of ChAdOx1 adenovirus vectors as a leading platform technology for SARS-CoV-2 vaccines.

Efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine against SARS-CoV-2 variant of concern 202012/01 (B.1.1.7): an exploratory analysis of a randomised controlled trial.

BACKGROUND: A new variant of SARS-CoV-2, B.1.1.7, emerged as the dominant cause of COVID-19 disease in the UK from November, 2020. We report a post-hoc analysis of the efficacy of the adenoviral vector vaccine, ChAdOx1 nCoV-19 (AZD1222), against this variant. METHODS: Volunteers (aged ≥18 years) who were enrolled in phase 2/3 vaccine efficacy studies in the UK, and who were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 or a meningococcal conjugate control (MenACWY) vaccine, provided upper airway swabs on a weekly basis and also if they developed symptoms of COVID-19 disease (a cough, a fever of 37·8°C or higher, shortness of breath, anosmia, or ageusia). Swabs were tested by nucleic acid amplification test (NAAT) for SARS-CoV-2 and positive samples were sequenced through the COVID-19 Genomics UK consortium. Neutralising antibody responses were measured using a live-virus microneutralisation assay against the B.1.1.7 lineage and a canonical non-B.1.1.7 lineage (Victoria). The efficacy analysis included symptomatic COVID-19 in seronegative participants with a NAAT positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to vaccine received. Vaccine efficacy was calculated as 1 - relative risk (ChAdOx1 nCoV-19 vs MenACWY groups) derived from a robust Poisson regression model. This study is continuing and is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137. FINDINGS: Participants in efficacy cohorts were recruited between May 31 and Nov 13, 2020, and received booster doses between Aug 3 and Dec 30, 2020. Of 8534 participants in the primary efficacy cohort, 6636 (78%) were aged 18-55 years and 5065 (59%) were female. Between Oct 1, 2020, and Jan 14, 2021, 520 participants developed SARS-CoV-2 infection. 1466 NAAT positive nose and throat swabs were collected from these participants during the trial. Of these, 401 swabs from 311 participants were successfully sequenced. Laboratory virus neutralisation activity by vaccine-induced antibodies was lower against the B.1.1.7 variant than against the Victoria lineage (geometric mean ratio 8·9, 95% CI 7·2-11·0). Clinical vaccine efficacy against symptomatic NAAT positive infection was 70·4% (95% CI 43·6-84·5) for B.1.1.7 and 81·5% (67·9-89·4) for non-B.1.1.7 lineages. INTERPRETATION: ChAdOx1 nCoV-19 showed reduced neutralisation activity against the B.1.1.7 variant compared with a non-B.1.1.7 variant in vitro, but the vaccine showed efficacy against the B.1.1.7 variant of SARS-CoV-2. FUNDING: UK Research and Innovation, National Institute for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midlands NIHR Clinical Research Network, and AstraZeneca.

Endogenous aldehyde accumulation generates genotoxicity and exhaled biomarkers in esophageal adenocarcinoma.

Volatile aldehydes are enriched in esophageal adenocarcinoma (EAC) patients' breath and could improve early diagnosis, however the mechanisms of their production are unknown. Here, we show that weak aldehyde detoxification characterizes EAC, which is sufficient to cause endogenous aldehyde accumulation in vitro. Two aldehyde groups are significantly enriched in EAC biopsies and adjacent tissue: (i) short-chain alkanals, and (ii) medium-chain alkanals, including decanal. The short-chain alkanals form DNA-adducts, which demonstrates genotoxicity and confirms inadequate detoxification. Metformin, a putative aldehyde scavenger, reduces this toxicity. Tissue and breath concentrations of the medium-chain alkanal decanal are correlated, and increased decanal is linked to reduced ALDH3A2 expression, TP53 deletion, and adverse clinical features. Thus, we present a model for increased exhaled aldehydes based on endogenous accumulation from reduced detoxification, which also causes therapeutically actionable genotoxicity. These results support EAC early diagnosis trials using exhaled aldehyde analysis.

When the genome bluffs: a tandem duplication event during generation of a novel Agmo knockout mouse model fools routine genotyping.

BACKGROUND: Genome editing in mice using either classical approaches like homologous recombination or CRISPR/Cas9 has been reported to harbor off target effects (insertion/deletion, frame shifts or gene segment duplications) that lead to mutations not only in close proximity to the target site but also outside. Only the genomes of few engineered mouse strains have been sequenced. Since the role of the ether-lipid cleaving enzyme alkylglycerol monooxygenase (AGMO) in physiology and pathophysiology remains enigmatic, we created a knockout mouse model for AGMO using EUCOMM stem cells but unforeseen genotyping issues that did not agree with Mendelian distribution and enzyme activity data prompted an in-depth genomic validation of the mouse model. RESULTS: We report a gene segment tandem duplication event that occurred during the generation of an Agmo knockout-first allele by homologous recombination. Only low homology was seen between the breakpoints. While a single copy of the recombinant 18 kb cassette was integrated correctly around exon 2 of the Agmo gene, whole genome nanopore sequencing revealed a 94 kb duplication in the Agmo locus that contains Agmo wild-type exons 1-3. The duplication fooled genotyping by routine PCR, but could be resolved using qPCR-based genotyping, targeted locus amplification sequencing and nanopore sequencing. Despite this event, this Agmo knockout mouse model lacks AGMO enzyme activity and can therefore be used to study its physiological role. CONCLUSIONS: A duplication event occurred at the exact locus of the homologous recombination and was not detected by conventional quality control filters such as FISH or long-range PCR over the recombination sites. Nanopore sequencing provides a cost convenient method to detect such underrated off-target effects, suggesting its use for additional quality assessment of gene editing in mice and also other model organisms.

Single-dose administration and the influence of the timing of the booster dose on immunogenicity and efficacy of ChAdOx1 nCoV-19 (AZD1222) vaccine: a pooled analysis of four randomised trials.

BACKGROUND: The ChAdOx1 nCoV-19 (AZD1222) vaccine has been approved for emergency use by the UK regulatory authority, Medicines and Healthcare products Regulatory Agency, with a regimen of two standard doses given with an interval of 4-12 weeks. The planned roll-out in the UK will involve vaccinating people in high-risk categories with their first dose immediately, and delivering the second dose 12 weeks later. Here, we provide both a further prespecified pooled analysis of trials of ChAdOx1 nCoV-19 and exploratory analyses of the impact on immunogenicity and efficacy of extending the interval between priming and booster doses. In addition, we show the immunogenicity and protection afforded by the first dose, before a booster dose has been offered. METHODS: We present data from three single-blind randomised controlled trials-one phase 1/2 study in the UK (COV001), one phase 2/3 study in the UK (COV002), and a phase 3 study in Brazil (COV003)-and one double-blind phase 1/2 study in South Africa (COV005). As previously described, individuals 18 years and older were randomly assigned 1:1 to receive two standard doses of ChAdOx1 nCoV-19 (5 × 1010 viral particles) or a control vaccine or saline placebo. In the UK trial, a subset of participants received a lower dose (2·2 × 1010 viral particles) of the ChAdOx1 nCoV-19 for the first dose. The primary outcome was virologically confirmed symptomatic COVID-19 disease, defined as a nucleic acid amplification test (NAAT)-positive swab combined with at least one qualifying symptom (fever ≥37·8°C, cough, shortness of breath, or anosmia or ageusia) more than 14 days after the second dose. Secondary efficacy analyses included cases occuring at least 22 days after the first dose. Antibody responses measured by immunoassay and by pseudovirus neutralisation were exploratory outcomes. All cases of COVID-19 with a NAAT-positive swab were adjudicated for inclusion in the analysis by a masked independent endpoint review committee. The primary analysis included all participants who were SARS-CoV-2 N protein seronegative at baseline, had had at least 14 days of follow-up after the second dose, and had no evidence of previous SARS-CoV-2 infection from NAAT swabs. Safety was assessed in all participants who received at least one dose. The four trials are registered at ISRCTN89951424 (COV003) and ClinicalTrials.gov, NCT04324606 (COV001), NCT04400838 (COV002), and NCT04444674 (COV005). FINDINGS: Between April 23 and Dec 6, 2020, 24 422 participants were recruited and vaccinated across the four studies, of whom 17 178 were included in the primary analysis (8597 receiving ChAdOx1 nCoV-19 and 8581 receiving control vaccine). The data cutoff for these analyses was Dec 7, 2020. 332 NAAT-positive infections met the primary endpoint of symptomatic infection more than 14 days after the second dose. Overall vaccine efficacy more than 14 days after the second dose was 66·7% (95% CI 57·4-74·0), with 84 (1·0%) cases in the 8597 participants in the ChAdOx1 nCoV-19 group and 248 (2·9%) in the 8581 participants in the control group. There were no hospital admissions for COVID-19 in the ChAdOx1 nCoV-19 group after the initial 21-day exclusion period, and 15 in the control group. 108 (0·9%) of 12 282 participants in the ChAdOx1 nCoV-19 group and 127 (1·1%) of 11 962 participants in the control group had serious adverse events. There were seven deaths considered unrelated to vaccination (two in the ChAdOx1 nCov-19 group and five in the control group), including one COVID-19-related death in one participant in the control group. Exploratory analyses showed that vaccine efficacy after a single standard dose of vaccine from day 22 to day 90 after vaccination was 76·0% (59·3-85·9). Our modelling analysis indicated that protection did not wane during this initial 3-month period. Similarly, antibody levels were maintained during this period with minimal waning by day 90 (geometric mean ratio [GMR] 0·66 [95% CI 0·59-0·74]). In the participants who received two standard doses, after the second dose, efficacy was higher in those with a longer prime-boost interval (vaccine efficacy 81·3% [95% CI 60·3-91·2] at ≥12 weeks) than in those with a short interval (vaccine efficacy 55·1% [33·0-69·9] at <6 weeks). These observations are supported by immunogenicity data that showed binding antibody responses more than two-fold higher after an interval of 12 or more weeks compared with an interval of less than 6 weeks in those who were aged 18-55 years (GMR 2·32 [2·01-2·68]). INTERPRETATION: The results of this primary analysis of two doses of ChAdOx1 nCoV-19 were consistent with those seen in the interim analysis of the trials and confirm that the vaccine is efficacious, with results varying by dose interval in exploratory analyses. A 3-month dose interval might have advantages over a programme with a short dose interval for roll-out of a pandemic vaccine to protect the largest number of individuals in the population as early as possible when supplies are scarce, while also improving protection after receiving a second dose. FUNDING: UK Research and Innovation, National Institutes of Health Research (NIHR), The Coalition for Epidemic Preparedness Innovations, the Bill & Melinda Gates Foundation, the Lemann Foundation, Rede D'Or, the Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca.

Native-like SARS-CoV-2 spike glycoprotein expressed by ChAdOx1 nCoV-19/AZD1222 vaccine.

Vaccine development against the SARS-CoV-2 virus focuses on the principal target of the neutralizing immune response, the spike (S) glycoprotein. Adenovirus-vectored vaccines offer an effective platform for the delivery of viral antigen, but it is important for the generation of neutralizing antibodies that they produce appropriately processed and assembled viral antigen that mimics that observed on the SARS-CoV-2 virus. Here, we describe the structure, conformation and glycosylation of the S protein derived from the adenovirus-vectored ChAdOx1 nCoV-19/AZD1222 vaccine. We demonstrate native-like post-translational processing and assembly, and reveal the expression of S proteins on the surface of cells adopting the trimeric prefusion conformation. The data presented here confirms the use of ChAdOx1 adenovirus vectors as a leading platform technology for SARS-CoV-2 vaccines.

Phase 1/2 trial of SARS-CoV-2 vaccine ChAdOx1 nCoV-19 with a booster dose induces multifunctional antibody responses.

More than 190 vaccines are currently in development to prevent infection by the novel severe acute respiratory syndrome coronavirus 2. Animal studies suggest that while neutralizing antibodies against the viral spike protein may correlate with protection, additional antibody functions may also be important in preventing infection. Previously, we reported early immunogenicity and safety outcomes of a viral vector coronavirus vaccine, ChAdOx1 nCoV-19 (AZD1222), in a single-blinded phase 1/2 randomized controlled trial of healthy adults aged 18-55 years ( NCT04324606 ). Now we describe safety and exploratory humoral and cellular immunogenicity of the vaccine, from subgroups of volunteers in that trial, who were subsequently allocated to receive a homologous full-dose (SD/SD D56; n = 20) or half-dose (SD/LD D56; n = 32) ChAdOx1 booster vaccine 56 d following prime vaccination. Previously reported immunogenicity data from the open-label 28-d interval prime-boost group (SD/SD D28; n = 10) are also presented to facilitate comparison. Additionally, we describe volunteers boosted with the comparator vaccine (MenACWY; n = 10). In this interim report, we demonstrate that a booster dose of ChAdOx1 nCoV-19 is safe and better tolerated than priming doses. Using a systems serology approach we also demonstrate that anti-spike neutralizing antibody titers, as well as Fc-mediated functional antibody responses, including antibody-dependent neutrophil/monocyte phagocytosis, complement activation and natural killer cell activation, are substantially enhanced by a booster dose of vaccine. A booster dose of vaccine induced stronger antibody responses than a dose-sparing half-dose boost, although the magnitude of T cell responses did not increase with either boost dose. These data support the two-dose vaccine regime that is now being evaluated in phase 3 clinical trials.

T cell and antibody responses induced by a single dose of ChAdOx1 nCoV-19 (AZD1222) vaccine in a phase 1/2 clinical trial.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), has caused a global pandemic, and safe, effective vaccines are urgently needed1. Strong, Th1-skewed T cell responses can drive protective humoral and cell-mediated immune responses2 and might reduce the potential for disease enhancement3. Cytotoxic T cells clear virus-infected host cells and contribute to control of infection4. Studies of patients infected with SARS-CoV-2 have suggested a protective role for both humoral and cell-mediated immune responses in recovery from COVID-19 (refs. 5,6). ChAdOx1 nCoV-19 (AZD1222) is a candidate SARS-CoV-2 vaccine comprising a replication-deficient simian adenovirus expressing full-length SARS-CoV-2 spike protein. We recently reported preliminary safety and immunogenicity data from a phase 1/2 trial of the ChAdOx1 nCoV-19 vaccine (NCT04400838)7 given as either a one- or two-dose regimen. The vaccine was tolerated, with induction of neutralizing antibodies and antigen-specific T cells against the SARS-CoV-2 spike protein. Here we describe, in detail, exploratory analyses of the immune responses in adults, aged 18-55 years, up to 8 weeks after vaccination with a single dose of ChAdOx1 nCoV-19 in this trial, demonstrating an induction of a Th1-biased response characterized by interferon-γ and tumor necrosis factor-α cytokine secretion by CD4+ T cells and antibody production predominantly of IgG1 and IgG3 subclasses. CD8+ T cells, of monofunctional, polyfunctional and cytotoxic phenotypes, were also induced. Taken together, these results suggest a favorable immune profile induced by ChAdOx1 nCoV-19 vaccine, supporting the progression of this vaccine candidate to ongoing phase 2/3 trials to assess vaccine efficacy.

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK.

BACKGROUND: A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. METHODS: This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. FINDINGS: Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0-75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4-97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8-80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3-4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. INTERPRETATION: ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, Bill & Melinda Gates Foundation, Lemann Foundation, Rede D'Or, Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca.

Genomic Instability Is an Early Event in Aluminium-Induced Tumorigenesis.

Genomic instability is generally considered as a hallmark of tumorigenesis and a prerequisite condition for malignant transformation. Aluminium salts are suspected environmental carcinogens that transform mammary epithelial cells in vitro through unknown mechanisms. We report here that long-term culture in the presence of aluminium chloride (AlCl3) enables HC11 normal mouse mammary epithelial cells to form tumours and metastases when injected into the syngeneic and immunocompetent BALB/cByJ strain. We demonstrate that AlCl3 rapidly increases chromosomal structural abnormalities in mammary epithelial cells, while we failed to detect direct modulation of specific mRNA pathways. Our observations provide evidence that clastogenic activity-a well-recognized inducer of genomic instability-might account in part for the transforming abilities of aluminium in mammary epithelial cells.

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.

Targeting TRIM37-driven centrosome dysfunction in 17q23-amplified breast cancer.

Genomic instability is a hallmark of cancer, and has a central role in the initiation and development of breast cancer1,2. The success of poly-ADP ribose polymerase inhibitors in the treatment of breast cancers that are deficient in homologous recombination exemplifies the utility of synthetically lethal genetic interactions in the treatment of breast cancers that are driven by genomic instability3. Given that defects in homologous recombination are present in only a subset of breast cancers, there is a need to identify additional driver mechanisms for genomic instability and targeted strategies to exploit these defects in the treatment of cancer. Here we show that centrosome depletion induces synthetic lethality in cancer cells that contain the 17q23 amplicon, a recurrent copy number aberration that defines about 9% of all primary breast cancer tumours and is associated with high levels of genomic instability4-6. Specifically, inhibition of polo-like kinase 4 (PLK4) using small molecules leads to centrosome depletion, which triggers mitotic catastrophe in cells that exhibit amplicon-directed overexpression of TRIM37. To explain this effect, we identify TRIM37 as a negative regulator of centrosomal pericentriolar material. In 17q23-amplified cells that lack centrosomes, increased levels of TRIM37 block the formation of foci that comprise pericentriolar material-these foci are structures with a microtubule-nucleating capacity that are required for successful cell division in the absence of centrosomes. Finally, we find that the overexpression of TRIM37 causes genomic instability by delaying centrosome maturation and separation at mitotic entry, and thereby increases the frequency of mitotic errors. Collectively, these findings highlight TRIM37-dependent genomic instability as a putative driver event in 17q23-amplified breast cancer and provide a rationale for the use of centrosome-targeting therapeutic agents in treating these cancers.

A living biobank of ovarian cancer ex vivo models reveals profound mitotic heterogeneity.

High-grade serous ovarian carcinoma is characterised by TP53 mutation and extensive chromosome instability (CIN). Because our understanding of CIN mechanisms is based largely on analysing established cell lines, we developed a workflow for generating ex vivo cultures from patient biopsies to provide models that support interrogation of CIN mechanisms in cells not extensively cultured in vitro. Here, we describe a "living biobank" of ovarian cancer models with extensive replicative capacity, derived from both ascites and solid biopsies. Fifteen models are characterised by p53 profiling, exome sequencing and transcriptomics, and karyotyped using single-cell whole-genome sequencing. Time-lapse microscopy reveals catastrophic and highly heterogeneous mitoses, suggesting that analysis of established cell lines probably underestimates mitotic dysfunction in advanced human cancers. Drug profiling reveals cisplatin sensitivities consistent with patient responses, demonstrating that this workflow has potential to generate personalized avatars with advantages over current pre-clinical models and the potential to guide clinical decision making.

CRISPR-Cas9 Causes Chromosomal Instability and Rearrangements in Cancer Cell Lines, Detectable by Cytogenetic Methods.

CRISPR-Cas9 has quickly become the method of choice for genome editing, with multiple publications describing technical advances and novel applications. It has been widely adopted as a tool for basic research and has significant translational and clinical potential. However, its usage has outpaced the establishment of essential and rigorous controls for unwanted off-target effects, manifested as small mutations, large deletions of target loci, or large-scale chromosomal rearrangements. A common application of CRISPR-Cas9 is as a tool for creating isogenic cell-line models to study the effects of precise mutations, or variants, on disease traits. Here, we describe the effect of standard CRISPR-Cas9 mutagenesis protocols on well characterized cancer cell lines. We demonstrate that commonly used methods for detecting correctly mutated clones fail to uncover large-scale rearrangements. We show that simple cytogenetic methods can be used to identify clones carrying chromosomal abnormalities and large mutations at target loci. These methods are quick and cost-efficient, and we suggest that such controls should be performed prior to publication of studies based on novel CRISPR-Cas9 mutated cancer cell lines.