Showing papers by "Allan Bradley published in 2019"
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TL;DR: It is established that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions.
Abstract: Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation.
118 citations
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TL;DR: It is demonstrated that Sall1 augments reprogramming of mouse EpiSCs and embryonic fibroblasts and that these induced pluripotent stem cells are indeed fully pluripotency including formation of chimeric mice.
Abstract: Primed epiblast stem cells (EpiSCs) can be reverted to a pluripotent embryonic stem cell (ESC)-like state by expression of single reprogramming factor. We used CRISPR activation to perform a genome-scale, reprogramming screen in EpiSCs and identified 142 candidate genes. Our screen validated a total of 50 genes, previously not known to contribute to reprogramming, of which we chose Sall1 for further investigation. We show that Sall1 augments reprogramming of mouse EpiSCs and embryonic fibroblasts and that these induced pluripotent stem cells are indeed fully pluripotent including formation of chimeric mice. We also demonstrate that Sall1 synergizes with Nanog in reprogramming and that overexpression in ESCs delays their conversion back to EpiSCs. Lastly, using RNA sequencing, we identify and validate Klf5 and Fam189a2 as new downstream targets of Sall1 and Nanog. In summary, our work demonstrates the power of using CRISPR technology in understanding molecular mechanisms that mediate complex cellular processes such as reprogramming.
42 citations
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TL;DR: A genome-scale CRISPR knockout library screen of THP-1 human macrophages was performed to identify loss-of-function mutations conferring resistance to Salmonella uptake, indicating a role for the relatively uncharacterized gene NHLRC2 in bothSalmonella invasion and macrophage differentiation.
Abstract: A genome-scale CRISPR knockout library screen of THP-1 human macrophages was performed to identify loss-of-function mutations conferring resistance to Salmonella uptake. The screen identified 183 candidate genes, from which 14 representative genes involved in actin dynamics (ACTR3, ARPC4, CAPZB, TOR3A, CYFIP2, CTTN, and NHLRC2), glycosaminoglycan metabolism (B3GNT1), receptor signaling (PDGFB and CD27), lipid raft formation (CLTCL1), calcium transport (ATP2A2 and ITPR3), and cholesterol metabolism (HMGCR) were analyzed further. For some of these pathways, known chemical inhibitors could replicate the Salmonella resistance phenotype, indicating their potential as targets for host-directed therapy. The screen indicated a role for the relatively uncharacterized gene NHLRC2 in both Salmonella invasion and macrophage differentiation. Upon differentiation, NHLRC2 mutant macrophages were hyperinflammatory and did not exhibit characteristics typical of macrophages, including atypical morphology and inability to interact and phagocytose bacteria/particles. Immunoprecipitation confirmed an interaction of NHLRC2 with FRYL, EIF2AK2, and KLHL13. IMPORTANCESalmonella exploits macrophages to gain access to the lymphatic system and bloodstream to lead to local and potentially systemic infections. With an increasing number of antibiotic-resistant isolates identified in humans, Salmonella infections have become major threats to public health. Therefore, there is an urgent need to identify alternative approaches to anti-infective therapy, including host-directed therapies. In this study, we used a simple genome-wide screen to identify 183 candidate host factors in macrophages that can confer resistance to Salmonella infection. These factors may be potential therapeutic targets against Salmonella infections.
40 citations
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Technische Universität München1, Wellcome Trust Sanger Institute2, University of Western Australia3, Sir Charles Gairdner Hospital4, University of Tübingen5, Russian National Research Medical University6, Russian Academy of Sciences7, Max Planck Society8, German Cancer Research Center9, Li Ka Shing Faculty of Medicine, University of Hong Kong10, Charité11, University of Cambridge12, University of Oviedo13
TL;DR: The development of transposon tools to perform genome-wide recessive screens in vivo and validate identified putative tumor suppressor genes using a CRISPR/Cas9 validation platform are reported.
Abstract: B-cell lymphoma (BCL) is the most common hematologic malignancy. While sequencing studies gave insights into BCL genetics, identification of non-mutated cancer genes remains challenging. Here, we describe PiggyBac transposon tools and mouse models for recessive screening and show their application to study clonal B-cell lymphomagenesis. In a genome-wide screen, we discover BCL genes related to diverse molecular processes, including signaling, transcriptional regulation, chromatin regulation, or RNA metabolism. Cross-species analyses show the efficiency of the screen to pinpoint human cancer drivers altered by non-genetic mechanisms, including clinically relevant genes dysregulated epigenetically, transcriptionally, or post-transcriptionally in human BCL. We also describe a CRISPR/Cas9-based in vivo platform for BCL functional genomics, and validate discovered genes, such as Rfx7, a transcription factor, and Phip, a chromatin regulator, which suppress lymphomagenesis in mice. Our study gives comprehensive insights into the molecular landscapes of BCL and underlines the power of genome-scale screening to inform biology.
34 citations
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University of Strasbourg1, Wellcome Trust Sanger Institute2, Baylor College of Medicine3, University of Cambridge4, National Research Council5, Research Triangle Park6, Nanjing University7, European Bioinformatics Institute8, Technische Universität München9, Academy of Sciences of the Czech Republic10, Soochow University (Suzhou)11, University of California, Berkeley12, Ewha Womans University13, AstraZeneca14, Fordham University15, Seoul National University16, Wolfson Centre for Age-Related Diseases17
TL;DR: The International Mouse Phenotyping Consortium reports the generation of new mouse mutant strains for over 5,000 genes from targeted embryonic stem cells on the C57BL/6N genetic background, which nearly triples the number of genes with reporter alleles and almost doubles theNumber of conditional alleles available to the scientific community.
Abstract: The International Mouse Phenotyping Consortium reports the generation of new mouse mutant strains for over 5,000 genes from targeted embryonic stem cells on the C57BL/6N genetic background. This includes 2,850 null alleles for which no equivalent mutant mouse line exists, 2,987 novel conditional-ready alleles, and 4,433 novel reporter alleles. This nearly triples the number of genes with reporter alleles and almost doubles the number of conditional alleles available to the scientific community. When combined with more than 30 years of community effort, the total mutant allele mouse resource covers more than half of the genome. The extensively validated collection is archived and distributed through public repositories, facilitating availability to the worldwide biomedical research community, and expanding our understanding of gene function and human disease.
30 citations
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TL;DR: Mechanistically, data indicate a new mechanism, how NPM1c mislocalizes FLT3-TKD and changes its signal transduction ability, and aberrant STAT5 activation not only occurs in primary murine cells but also in AML patients with combined FLT 3-TkD and N PM1c mutations.
12 citations
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TL;DR: QiSeq is developed, a method for (semi-)quantitative transposon insertion site sequencing, which overcomes biasing limitations of previous library preparation methods for candidate cancer gene discovery and gives insights into the clonal distribution of insertions for the study of genetic tumor evolution.
Abstract: While sequencing and array-based studies are creating catalogues of genetic alterations in cancer, discriminating cancer drivers among the large sets of epigenetically, transcriptionally or posttranslationally dysregulated genes remains a challenge. Transposon-based genetic screening in mice has proven to be a powerful approach to address this challenge. Insertional mutagenesis directly flags biologically relevant genes and, combined with the transposon's unique molecular fingerprint, facilitates the recovery of insertion sites. We have generated transgenic mouse lines harboring different versions of PiggyBac-based oncogenic transposons, which in conjunction with PiggyBac transposase mice can be used for whole-body or tissue-specific insertional mutagenesis screens. We have also developed QiSeq, a method for (semi-)quantitative transposon insertion site sequencing, which overcomes biasing limitations of previous library preparation methods. QiSeq can be used in multiplexed high-throughput formats for candidate cancer gene discovery and gives insights into the clonal distribution of insertions for the study of genetic tumor evolution.
6 citations
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TL;DR: KY1049 demonstrates a dose-dependent reduction in clotting time and a doses-dependent increase in thrombin burst, thereby functionally restoring the hemostatic activity of F.VIII mimetic bispecific antibody with activities comparable to a SIA of Hemlibra.
1 citations