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1.
Nat Commun ; 12(1): 1302, 2021 02 26.
Article in English | MEDLINE | ID: mdl-33637726

ABSTRACT

Genetic redundancy has evolved as a way for human cells to survive the loss of genes that are single copy and essential in other organisms, but also allows tumours to survive despite having highly rearranged genomes. In this study we CRISPR screen 1191 gene pairs, including paralogues and known and predicted synthetic lethal interactions to identify 105 gene combinations whose co-disruption results in a loss of cellular fitness. 27 pairs influence fitness across multiple cell lines including the paralogues FAM50A/FAM50B, two genes of unknown function. Silencing of FAM50B occurs across a range of tumour types and in this context disruption of FAM50A reduces cellular fitness whilst promoting micronucleus formation and extensive perturbation of transcriptional programmes. Our studies reveal the fitness effects of FAM50A/FAM50B in cancer cells.


Subject(s)
CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Genome , Proteins/genetics , Animals , Apoptosis , Cell Line, Tumor , DNA-Binding Proteins/genetics , Gene Knockout Techniques , Heterografts , Humans , Mice , Mice, Inbred NOD , Mice, SCID , RNA-Binding Proteins/genetics , Transcriptome
2.
Cancer Res ; 77(21): e39-e42, 2017 11 01.
Article in English | MEDLINE | ID: mdl-29092936

ABSTRACT

Multiomics experiments are increasingly commonplace in biomedical research and add layers of complexity to experimental design, data integration, and analysis. R and Bioconductor provide a generic framework for statistical analysis and visualization, as well as specialized data classes for a variety of high-throughput data types, but methods are lacking for integrative analysis of multiomics experiments. The MultiAssayExperiment software package, implemented in R and leveraging Bioconductor software and design principles, provides for the coordinated representation of, storage of, and operation on multiple diverse genomics data. We provide the unrestricted multiple 'omics data for each cancer tissue in The Cancer Genome Atlas as ready-to-analyze MultiAssayExperiment objects and demonstrate in these and other datasets how the software simplifies data representation, statistical analysis, and visualization. The MultiAssayExperiment Bioconductor package reduces major obstacles to efficient, scalable, and reproducible statistical analysis of multiomics data and enhances data science applications of multiple omics datasets. Cancer Res; 77(21); e39-42. ©2017 AACR.


Subject(s)
Genomics , Neoplasms/genetics , Software , Computational Biology , Datasets as Topic , Genome, Human , Humans
3.
Cancer Res ; 77(18): 4961-4972, 2017 09 15.
Article in English | MEDLINE | ID: mdl-28760853

ABSTRACT

Head and neck squamous cell carcinoma (HNSCC) includes epithelial cancers of the oral and nasal cavity, larynx, and pharynx and accounts for ∼350,000 deaths per year worldwide. Smoking-related HNSCC is associated with few targetable mutations but is defined by frequent copy-number alteration, the most common of which is gain at 3q. Critical 3q target genes have not been conclusively determined for HNSCC. Here, we present data indicating that MAP3K13 (encoding LZK) is an amplified driver gene in HNSCC. Copy-number gain at 3q resulted in increased MAP3K13 mRNA in HNSCC tumor samples and cell lines. Silencing LZK reduced cell viability and proliferation of HNSCC cells with 3q gain but not control cell lines. Inducible silencing of LZK caused near-complete loss of colony-forming ability in cells harboring 3q gain. These results were validated in vivo by evidence that LZK silencing was sufficient to reduce tumor growth in a xenograft model of HNSCC. Our results establish LZK as critical for maintaining expression of mutant stabilized p53. Cancer Res; 77(18); 4961-72. ©2017 AACR.


Subject(s)
Carcinoma, Squamous Cell/pathology , Cell Proliferation , Head and Neck Neoplasms/pathology , MAP Kinase Kinase Kinases/metabolism , Mutant Proteins/metabolism , Mutation/genetics , Tumor Suppressor Protein p53/metabolism , Animals , Apoptosis , Biomarkers, Tumor , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Female , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/metabolism , Humans , MAP Kinase Kinase Kinases/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Mutant Proteins/chemistry , Mutant Proteins/genetics , Protein Stability , Tumor Cells, Cultured , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/genetics , Xenograft Model Antitumor Assays
4.
Cancer Res ; 76(3): 724-35, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-26637668

ABSTRACT

MLK4 is a member of the mixed-lineage family of kinases that regulate the JNK, p38, and ERK kinase signaling pathways. MLK4 mutations have been identified in various human cancers, including frequently in colorectal cancer, where their function and pathobiological importance have been uncertain. In this study, we assessed the functional consequences of MLK4 mutations in colon tumorigenesis. Biochemical data indicated that a majority of MLK4 mutations are loss-of-function (LOF) mutations that can exert dominant-negative effects. In seeking to understand the abrogated activity of these mutants, we elucidated a new MLK4 catalytic domain structure. To determine whether MLK4 is required to maintain tumorigenic phenotypes, we reconstituted its signaling axis in colon cancer cells harboring MLK4-inactivating mutations. We found that restoring MLK4 activity reduced cell viability, proliferation, and colony formation in vitro and delayed tumor growth in vivo. Mechanistic investigations established that restoring the function of MLK4 selectively induced the JNK pathway and its downstream targets, cJUN, ATF3, and the cyclin-dependent kinase inhibitors CDKN1A and CDKN2B. Our work indicates that MLK4 is a novel tumor-suppressing kinase harboring frequent LOF mutations that lead to diminished signaling in the JNK pathway and enhanced proliferation in colon cancer.


Subject(s)
Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , MAP Kinase Kinase Kinases/genetics , MAP Kinase Kinase Kinases/metabolism , MAP Kinase Signaling System/genetics , Animals , Carcinogenesis , Colonic Neoplasms/pathology , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Mutation , Signal Transduction , Xenograft Model Antitumor Assays
5.
Cancer Res ; 74(22): 6390-6396, 2014 Nov 15.
Article in English | MEDLINE | ID: mdl-25256751

ABSTRACT

Cancer genome sequencing is being used at an increasing rate to identify actionable driver mutations that can inform therapeutic intervention strategies. A comparison of two of the most prominent cancer genome sequencing databases from different institutes (Cancer Cell Line Encyclopedia and Catalogue of Somatic Mutations in Cancer) revealed marked discrepancies in the detection of missense mutations in identical cell lines (57.38% conformity). The main reason for this discrepancy is inadequate sequencing of GC-rich areas of the exome. We have therefore mapped over 400 regions of consistent inadequate sequencing (cold-spots) in known cancer-causing genes and kinases, in 368 of which neither institute finds mutations. We demonstrate, using a newly identified PAK4 mutation as proof of principle, that specific targeting and sequencing of these GC-rich cold-spot regions can lead to the identification of novel driver mutations in known tumor suppressors and oncogenes. We highlight that cross-referencing between genomic databases is required to comprehensively assess genomic alterations in commonly used cell lines and that there are still significant opportunities to identify novel drivers of tumorigenesis in poorly sequenced areas of the exome. Finally, we assess other reasons for the observed discrepancy, such as variations in dbSNP filtering and the acquisition/loss of mutations, to give explanations as to why there is a discrepancy in pharmacogenomic studies, given recent concerns with poor reproducibility of data.


Subject(s)
Mutation , Neoplasms/genetics , Sequence Analysis, DNA , Cell Line, Tumor , Exome , Genes, Tumor Suppressor , Humans , Oncogenes , Pharmacogenetics , Polymorphism, Single Nucleotide , p21-Activated Kinases/genetics
6.
Metabolism ; 58(8): 1161-9, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19428033

ABSTRACT

Lean healthy individuals are characterized by the ability to rapidly adapt metabolism to acute changes in substrate availability and metabolic rate. However, in glucose-intolerance/insulin-resistant conditions, such as that induced by starvation, the flexibility of tissues to rapidly respond to change in substrate availability is diminished. We asked whether the conundrum of increased glucose demand by the contracting skeletal muscle during prolonged exercise and the glucose intolerance of starvation would result in the obstruction of oxidative disposal of ingested (13)C-labeled glucose during exercise. Seven lean, healthy, physically active individuals (2 women, 5 men) completed a randomized crossover study comparing the effects of the normal-fed condition vs a 67-hour water-only fast on the metabolic response to carbohydrate ingestion during 80 minutes of exercise at 56% of maximum oxygen uptake. Compared with the normal condition, fasting resulted in a large overall increase in the rate of fat oxidation (mean effect, 71%; 95% confidence limit, +/-22%) and moderate reductions in both exogenous (-54%, +/-10%) and endogenous (-40%, +/-19%) glucose oxidation rates during exercise. Over the course of exercise, fat oxidation was impermeable to change in the fasting condition, but increased moderately (33%, +/-19%) in the normal condition. These changes were associated with a large increase in plasma free fatty-acid concentration (120%, +/-64%) and a moderate increase in blood lactate concentration (58%, +/-50%). In contrast, large reductions in resting blood glucose (-21%, +/-14%) and moderate reductions in plasma insulin concentrations (-47%, +/-26%) were observed in the fast condition; but this effect was reversed for glucose (30%, +/- 24%) and negated for insulin by the end of exercise. To conclude, a 67-hour fast leads to an impermeable increase in fat oxidation, suppression of both exogenous and endogenous carbohydrate oxidation, and a metabolic response consistent with resistance to contraction-induced exogenous glucose uptake and oxidation.


Subject(s)
Dietary Carbohydrates/administration & dosage , Dietary Carbohydrates/metabolism , Exercise , Glucose/administration & dosage , Glucose/metabolism , Starvation , Adult , Blood Glucose/metabolism , Carbon Isotopes , Cross-Over Studies , Ergometry , Female , Glucose Intolerance/metabolism , Humans , Lipid Peroxidation , Male , Oxidation-Reduction , Oxygen Consumption , Time Factors
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