Journal articles on the topic 'Genomic screens'
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Gaillochet, Christophe, Ward Develtere, and Thomas B. Jacobs. "CRISPR screens in plants: approaches, guidelines, and future prospects." Plant Cell 33, no. 4 (April 1, 2021): 794–813. http://dx.doi.org/10.1093/plcell/koab099.
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Abstract Clustered regularly interspaced short palindromic repeat (CRISPR)-associated systems have revolutionized genome engineering by facilitating a wide range of targeted DNA perturbations. These systems have resulted in the development of powerful new screens to test gene functions at the genomic scale. While there is tremendous potential to map and interrogate gene regulatory networks at unprecedented speed and scale using CRISPR screens, their implementation in plants remains in its infancy. Here we discuss the general concepts, tools, and workflows for establishing CRISPR screens in plants and analyze the handful of recent reports describing the use of this strategy to generate mutant knockout collections or to diversify DNA sequences. In addition, we provide insight into how to design CRISPR knockout screens in plants given the current challenges and limitations and examine multiple design options. Finally, we discuss the unique multiplexing capabilities of CRISPR screens to investigate redundant gene functions in highly duplicated plant genomes. Combinatorial mutant screens have the potential to routinely generate higher-order mutant collections and facilitate the characterization of gene networks. By integrating this approach with the numerous genomic profiles that have been generated over the past two decades, the implementation of CRISPR screens offers new opportunities to analyze plant genomes at deeper resolution and will lead to great advances in functional and synthetic biology.
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Rorth, P., K. Szabo, A. Bailey, T. Laverty, J. Rehm, G. M. Rubin, K. Weigmann, et al. "Systematic gain-of-function genetics in Drosophila." Development 125, no. 6 (March 15, 1998): 1049–57. http://dx.doi.org/10.1242/dev.125.6.1049.
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A modular misexpression system was used to carry out systematic gain-of-function genetic screens in Drosophila. The system is based on inducible expression of genes tagged by insertion of a P-element vector carrying a GAL4-regulated promoter oriented to transcribe flanking genomic sequences. To identify genes involved in eye and wing development, the 2300 independent lines were screened for dominant phenotypes. Among many novel genes, the screen identified known genes, including hedgehog and decapentaplegic, implicated in these processes. A genetic interaction screen for suppressors of a cell migration defect in a hypomorphic slow border cells mutant identified known genes with likely roles in tyrosine kinase signaling and control of actin cytoskeleton, among many novel genes. These studies demonstrate the ability of the modular misexpression system to identify developmentally important genes and suggest that it will be generally useful for genetic interaction screens.
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Sheel, Ankur, and Wen Xue. "Genomic Amplifications Cause False Positives in CRISPR Screens." Cancer Discovery 6, no. 8 (August 2016): 824–26. http://dx.doi.org/10.1158/2159-8290.cd-16-0665.
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Hart, Traver, Kevin R. Brown, Fabrice Sircoulomb, Robert Rottapel, and Jason Moffat. "Measuring error rates in genomic perturbation screens: gold standards for human functional genomics." Molecular Systems Biology 10, no. 7 (July 2014): 733. http://dx.doi.org/10.15252/msb.20145216.
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Salamon, Hugh, Midori Kato-Maeda, Peter M. Small, Jorg Drenkow, and Thomas R. Gingeras. "Detection of Deleted Genomic DNA Using a Semiautomated Computational Analysis of GeneChip Data." Genome Research 10, no. 12 (November 21, 2000): 2044–54. http://dx.doi.org/10.1101/gr.152900.
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Genomic diversity within and between populations is caused by single nucleotide mutations, changes in repetitive DNA systems, recombination mechanisms, and insertion and deletion events. The contribution of these sources to diversity, whether purely genetic or of phenotypic consequence, can only be investigated if we have the means to quantitate and characterize diversity in many samples. With the advent of complete sequence characterization of representative genomes of different species, the possibility of developing protocols to screen for genetic polymorphism across entire genomes is actively being pursued. The large numbers of measurements such approaches yield demand that we pay careful attention to the numerical analysis of data. In this paper we present a novel application of an Affymetrix GeneChip to perform genome-wide screens for deletion polymorphism. A high-density oligonucleotide array formatted for mRNA expression and targeted at a fully sequenced 4.4-million–base pair Mycobacterium tuberculosis standard strain genome was adapted to compare genomic DNA. Hybridization intensities to 111,000 probe pairs (perfect complement and mismatch complement) were measured for genomic DNA from a clinical strain and from a vaccine organism. Because individual probe-pair hybridization intensities exhibit limited sensitivity/specificity characteristics to detect deletions, data-analytical methodology to exploit measurements from multiple probes in tandem locations across the genome was developed. The TSTEP (Tandem Set Terminal Extreme Probability) algorithm designed specifically to analyze the tandem hybridization measurements data was applied and shown to discover genomic deletions with high sensitivity. The TSTEP algorithm provides a foundation for similar efforts to characterize deletions in many hybridization measures in similar-sized and larger genomes. Issues relating to the design of genome content screening experiments and the implications of these methods for studying population genomics and the evolution of genomes are discussed.
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Jilderda, Laura J., Lin Zhou, and Floris Foijer. "Understanding How Genetic Mutations Collaborate with Genomic Instability in Cancer." Cells 10, no. 2 (February 6, 2021): 342. http://dx.doi.org/10.3390/cells10020342.
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Chromosomal instability is the process of mis-segregation for ongoing chromosomes, which leads to cells with an abnormal number of chromosomes, also known as an aneuploid state. Induced aneuploidy is detrimental during development and in primary cells but aneuploidy is also a hallmark of cancer cells. It is therefore believed that premalignant cells need to overcome aneuploidy-imposed stresses to become tumorigenic. Over the past decade, some aneuploidy-tolerating pathways have been identified through small-scale screens, which suggest that aneuploidy tolerance pathways can potentially be therapeutically exploited. However, to better understand the processes that lead to aneuploidy tolerance in cancer cells, large-scale and unbiased genetic screens are needed, both in euploid and aneuploid cancer models. In this review, we describe some of the currently known aneuploidy-tolerating hits, how large-scale genome-wide screens can broaden our knowledge on aneuploidy specific cancer driver genes, and how we can exploit the outcomes of these screens to improve future cancer therapy.
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The Transatlantic Multiple Sclerosis Genetics Cooperative. "A meta-analysis of genomic screens in multiple sclerosis." Multiple Sclerosis 7, no. 1 (February 1, 2001): 3–11. http://dx.doi.org/10.1191/135245801669625359.
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XU, AXIANG, and SHENGKUN SUN. "Genomic profiling screens small molecules of metastatic prostate carcinoma." Oncology Letters 10, no. 3 (July 8, 2015): 1402–8. http://dx.doi.org/10.3892/ol.2015.3472.
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Ashoti, Ator, Francesco Limone, Melissa van Kranenburg, Anna Alemany, Mirna Baak, Judith Vivié, Frederica Piccioni, et al. "Considerations and practical implications of performing a phenotypic CRISPR/Cas survival screen." PLOS ONE 17, no. 2 (February 17, 2022): e0263262. http://dx.doi.org/10.1371/journal.pone.0263262.
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Genome-wide screens that have viability as a readout have been instrumental to identify essential genes. The development of gene knockout screens with the use of CRISPR-Cas has provided a more sensitive method to identify these genes. Here, we performed an exhaustive genome-wide CRISPR/Cas9 phenotypic rescue screen to identify modulators of cytotoxicity induced by the pioneer transcription factor, DUX4. Misexpression of DUX4 due to a failure in epigenetic repressive mechanisms underlies facioscapulohumeral muscular dystrophy (FHSD), a complex muscle disorder that thus far remains untreatable. As the name implies, FSHD generally starts in the muscles of the face and shoulder girdle. Our CRISPR/Cas9 screen revealed no key effectors other than DUX4 itself that could modulate DUX4 cytotoxicity, suggesting that treatment efforts in FSHD should be directed towards direct modulation of DUX4 itself. Our screen did however reveal some rare and unexpected genomic events, that had an important impact on the interpretation of our data. Our findings may provide important considerations for planning future CRISPR/Cas9 phenotypic survival screens.
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Kaplow, Irene M., Rohit Singh, Adam Friedman, Chris Bakal, Norbert Perrimon, and Bonnie Berger. "RNAiCut: automated detection of significant genes from functional genomic screens." Nature Methods 6, no. 7 (July 2009): 476–77. http://dx.doi.org/10.1038/nmeth0709-476.
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Lovejoy, C. A., X. Xu, C. E. Bansbach, G. G. Glick, R. Zhao, F. Ye, B. M. Sirbu, L. C. Titus, Y. Shyr, and D. Cortez. "Functional genomic screens identify CINP as a genome maintenance protein." Proceedings of the National Academy of Sciences 106, no. 46 (November 4, 2009): 19304–9. http://dx.doi.org/10.1073/pnas.0909345106.
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Borcherding, Dana C., Kevin He, Neha V. Amin, and Angela C. Hirbe. "TYK2 in Cancer Metastases: Genomic and Proteomic Discovery." Cancers 13, no. 16 (August 19, 2021): 4171. http://dx.doi.org/10.3390/cancers13164171.
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Advances in genomic analysis and proteomic tools have rapidly expanded identification of biomarkers and molecular targets important to cancer development and metastasis. On an individual basis, personalized medicine approaches allow better characterization of tumors and patient prognosis, leading to more targeted treatments by detection of specific gene mutations, overexpression, or activity. Genomic and proteomic screens by our lab and others have revealed tyrosine kinase 2 (TYK2) as an oncogene promoting progression and metastases of many types of carcinomas, sarcomas, and hematologic cancers. TYK2 is a Janus kinase (JAK) that acts as an intermediary between cytokine receptors and STAT transcription factors. TYK2 signals to stimulate proliferation and metastasis while inhibiting apoptosis of cancer cells. This review focuses on the growing evidence from genomic and proteomic screens, as well as molecular studies that link TYK2 to cancer prevalence, prognosis, and metastasis. In addition, pharmacological inhibition of TYK2 is currently used clinically for autoimmune diseases, and now provides promising treatment modalities as effective therapeutic agents against multiple types of cancer.
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Condurat, Alexandra-Larisa, Jill Jones, Elizabeth Gonzalez, Jeyna Doshi, Kevin Zhou, Jessica W. Tsai, Prasidda Khadka, et al. "LGG-45. Genetic dependencies inMYB/MYBL1-driven pediatric low-grade glioma models." Neuro-Oncology 24, Supplement_1 (June 1, 2022): i98. http://dx.doi.org/10.1093/neuonc/noac079.357.
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Abstract AIM: Pediatric low-grade gliomas (pLGGs) are a heterogenous group of tumors, diverse in their localization, histology, mutational landscape, clinical behavior, and treatment response. Genomic alterations impacting the MYB family of transcription factors were identified in two distinct pLGG subtypes: Angiocentric Gliomas (AG) and Diffuse Astrocytomas (DA). The molecular profiles and therapeutic vulnerabilities associated with these genomic alterations remain unexplored. In this study we highlight the use of genome-wide CRISPR/Cas9 knock-out screens for an unbiased identification of translatable therapeutic targets. METHODOLOGY: Given the lack of patient-derived pLGG cell lines, we engineered in vitro pLGG mouse and human neural stem cell (NSC) models to harbor pLGG-relevant genomic alterations. We performed single cell RNA sequencing to investigate the transcriptional profiles driven by these mutations and to dissect the central regulatory networks enabling tumorigenesis. Specific genetic dependencies associated with MYB/MYBL1 mutations were screened using the Brie genome-wide mouse CRISPR lentiviral knock-out pooled library, consisting of 78,637 single guide RNAs (sgRNAs) targeting 19,674 mouse genes. RESULTS: We have successfully generated in vitro NSC-based pLGG models crucial to deepening our knowledge on pLGG biology and the identification of translatable therapeutic targets. Genome-scale CRISPR/Cas9 knock-out screens in isogenic NSCs models, expressing distinct MYB/MYBL1 alterations or a control transgene, revealed several differential genetic dependencies. Among the top identified dependencies are regulators of cell-stress response, cell-cycle progression, and modulators of the ubiquitin-proteasome degradation pathway. CONCLUSION: Genome-wide CRISPR knock-out screens are a powerful tool for the unbiased identification of mutation-specific genetic dependencies that can be explored as candidates for precision medicine approaches.
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Carvajal-Gonzalez, Jose Maria, Sonia Mulero-Navarro, Michael Smith, and Marek Mlodzik. "A Novel Frizzled-Based Screening Tool Identifies Genetic Modifiers of Planar Cell Polarity in Drosophila Wings." G3 Genes|Genomes|Genetics 6, no. 12 (December 1, 2016): 3963–73. http://dx.doi.org/10.1534/g3.116.035535.
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Abstract Most mutant alleles in the Fz-PCP pathway genes were discovered in classic Drosophila screens looking for recessive loss-of-function (LOF) mutations. Nonetheless, although Fz-PCP signaling is sensitive to increased doses of PCP gene products, not many screens have been performed in the wing under genetically engineered Fz overexpression conditions, mostly because the Fz phenotypes were strong and/or not easy to score and quantify. Here, we present a screen based on an unexpected mild Frizzled gain-of-function (GOF) phenotype. The leakiness of a chimeric Frizzled protein designed to be accumulated in the endoplasmic reticulum (ER) generated a reproducible Frizzled GOF phenotype in Drosophila wings. Using this genotype, we first screened a genome-wide collection of large deficiencies and found 16 strongly interacting genomic regions. Next, we narrowed down seven of those regions to finally test 116 candidate genes. We were, thus, able to identify eight new loci with a potential function in the PCP context. We further analyzed and confirmed krasavietz and its interactor short-stop as new genes acting during planar cell polarity establishment with a function related to actin and microtubule dynamics.
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Gorodkin, Jan, and Ivo L. Hofacker. "From Structure Prediction to Genomic Screens for Novel Non-Coding RNAs." PLoS Computational Biology 7, no. 8 (August 4, 2011): e1002100. http://dx.doi.org/10.1371/journal.pcbi.1002100.
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Hu, G., and J. Luo. "A primer on using pooled shRNA libraries for functional genomic screens." Acta Biochimica et Biophysica Sinica 44, no. 2 (January 23, 2012): 103–12. http://dx.doi.org/10.1093/abbs/gmr116.
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Chiu, Yu-Chiao, Siyuan Zheng, Li-Ju Wang, Brian S. Iskra, Manjeet K. Rao, Peter J. Houghton, Yufei Huang, and Yidong Chen. "Predicting and characterizing a cancer dependency map of tumors with deep learning." Science Advances 7, no. 34 (August 2021): eabh1275. http://dx.doi.org/10.1126/sciadv.abh1275.
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Genome-wide loss-of-function screens have revealed genes essential for cancer cell proliferation, called cancer dependencies. It remains challenging to link cancer dependencies to the molecular compositions of cancer cells or to unscreened cell lines and further to tumors. Here, we present DeepDEP, a deep learning model that predicts cancer dependencies using integrative genomic profiles. It uses a unique unsupervised pretraining that captures unlabeled tumor genomic representations to improve the learning of cancer dependencies. We demonstrated DeepDEP’s improvement over conventional machine learning methods and validated the performance with three independent datasets. By systematic model interpretations, we extended the current dependency maps with functional characterizations of dependencies and a proof-of-concept in silico assay of synthetic essentiality. We applied DeepDEP to pan-cancer tumor genomics and built the first pan-cancer synthetic dependency map of 8000 tumors with clinical relevance. In summary, DeepDEP is a novel tool for investigating cancer dependency with rapidly growing genomic resources.
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Trent, Jeffrey, John Carpten, Michael Reich, Ted Liefeld, Jonathan Keats, Spyro Mousses, William Hahn, et al. "The Multiple Myeloma Research Consortium Genomics Initiative." Blood 110, no. 11 (November 16, 2007): 2498. http://dx.doi.org/10.1182/blood.v110.11.2498.2498.
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Abstract The Multiple Myeloma Research Consortium (MMRC) Genomics Initiative is a three-year program to analyze tumor tissue from hundreds of multiple myeloma (MM) patients via gene expression profiling (GEP), comparative genomic hybridization (aCGH), and exon re-sequencing. In addition, RNAi knockdown of selected genes in MM tumor cell lines is being evaluated to identify potential new targets. All genomic data generated is scheduled for placement in an open-access Multiple Myeloma Genomics Portal pre-publication and in near real-time (www.broad.mit.edu/mmgp). Additionally, samples are also destined for drug validation and correlative science on clinical protocols as this study moves forward. This comprehensive project is spearheaded by the MMRC and conducted via collaboration with the Eli and Edythe L. Broad Institute of MIT and Harvard, the Translational Genomics Research Institute (TGen), Mayo Clinic Arizona, and The Dana-Farber Cancer Center. The study is supported by the collection from member institutions of the MMRC of bone marrow aspirates and matched peripheral blood samples from over 1000 patients. Specific genomic technologies that are currently being employed across this sample set include GEP using Affymetrix Human Genome U133A 2.0 Plus Arrays, and, in parallel, efforts to identify regions of genomic gain and loss are using Agilent Human Genome CGH arrays. In contrast to other large-scale genomic projects based on exon-sequencing of targeted gene sets, this project will be the first to perform genome-scale single molecule sequencing (SMS) of DNA from patient specimens. Results will be targeted against candidate classes of genes (e.g. kinases, phosphatases, known oncogenes and tumor suppressors), and genes from GEP or within candidate regions of copy gain or loss identified by the aCGH experiments. Mutations will be further validated in an independent set of patient specimens. Finally we will attempt to identify points of vulnerability of MM through systematic loss-of-function screens in myeloma cell lines using high-throughput RNA interference (using both shRNA and siRNA platforms). Importantly, data generated from this genomics initiative will ultimately be made public pre-publication through the established MMRC Multiple Myeloma Genomics Portal. Data from all aspects of this project (sample collection and analyte isolation, GEP, aCGH, SMS, RNAi and bioinformatics) will be described in this presentation. The power of this study is the comprehensive collection of gene expression, CGH, and genome sequencing on a single reference set of clinically annotated samples. The addition of RNAi screens makes this a very important and unique data resource, which we hope will help expedite the discovery of novel targeted agents for MM scientific community.
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Baumgarten, Nina, Florian Schmidt, Martin Wegner, Marie Hebel, Manuel Kaulich, and Marcel H. Schulz. "Computational prediction of CRISPR-impaired non-coding regulatory regions." Biological Chemistry 402, no. 8 (March 2, 2021): 973–82. http://dx.doi.org/10.1515/hsz-2020-0392.
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Abstract Genome-wide CRISPR screens are becoming more widespread and allow the simultaneous interrogation of thousands of genomic regions. Although recent progress has been made in the analysis of CRISPR screens, it is still an open problem how to interpret CRISPR mutations in non-coding regions of the genome. Most of the tools concentrate on the interpretation of mutations introduced in gene coding regions. We introduce a computational pipeline that uses epigenomic information about regulatory elements for the interpretation of CRISPR mutations in non-coding regions. We illustrate our analysis protocol on the analysis of a genome-wide CRISPR screen in hTERT-RPE1 cells and reveal novel regulatory elements that mediate chemoresistance against doxorubicin in these cells. We infer links to established and to novel chemoresistance genes. Our analysis protocol is general and can be applied on any cell type and with different CRISPR enzymes.
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Deschênes, Astrid, Pascal Belleau, Dennis Plenker, Amber Habowski, Hardik Patel, Youngkyu Park, Hervé Tiriac, Lindsey A. Baker, Alexander Krasnitz, and David A. Tuveson. "Abstract 4042: Genomic and pharmaco-genomic profiling of pancreatic cancer using patient-derived organoids." Cancer Research 82, no. 12_Supplement (June 15, 2022): 4042. http://dx.doi.org/10.1158/1538-7445.am2022-4042.
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Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal common malignancies, with little improvement in patient outcomes over the past decades. We have developed a novel methodology to culture organoids from both human healthy pancreatic ductal epithelial tissues and PDAC. A collection of patient-derived organoids (PDO), grown using this protocol, numbers over 100 models to date. The 100% neoplastic purity of the organoid cultures facilitates molecular characterization that has been traditionally challenging in the pauci-cellular state of primary pancreatic tumors. These PDO open new opportunities for deep genomic and transcriptomic studies of the disease, and for individualized drug screens. Here we demonstrate that accurate predictive models of response to pharmacological treatments of PDAC can be developed using data from such screens alongside molecular profiles of the PDO. Methods: Molecular analysis of the PDO library yielded genomic and transcriptional profiles of the cultures, including those of copy number variation (CNV), mutations in the exome and mRNA expression. From these, we drew features for prediction of drug responses. Using molecular features drawn from these profiles, we developed a panel of predictive models for response to standard-of-care cytotoxic agents and a number of targeted treatments. We employed Random Forest (RF) regression as a machine-learning tool for this purpose. Results: PDO are faithful models of PDAC, whose molecular features closely resemble those of PDAC tumor specimens. Using a subset of these features, we were able to accurately learn PDO responses to cytotoxic agents: for each of the five agents considered, the predicted drug response correlated strongly (p < 10-7) with the observed value. A similar accuracy of prediction was achieved for a number of targeted agents. Conclusion: PDOs are a valuable resource for molecular and pharmacological characterization of PDAC, with a potential to guide clinical decisions with regard to treatment. Citation Format: Astrid Deschênes, Pascal Belleau, Dennis Plenker, Amber Habowski, Hardik Patel, Youngkyu Park, Hervé Tiriac, Lindsey A. Baker, Alexander Krasnitz, David A. Tuveson. Genomic and pharmaco-genomic profiling of pancreatic cancer using patient-derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4042.
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Jain, Shushant, David Sondervan, Patrizia Rizzu, Zoltan Bochdanovits, Daniel Caminada, and Peter Heutink. "The Complete Automation of Cell Culture." Journal of Biomolecular Screening 16, no. 8 (July 20, 2011): 932–39. http://dx.doi.org/10.1177/1087057111413920.
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Genomic approaches provide enormous amounts of raw data with regard to genetic variation, the diversity of RNA species, and protein complement. High-throughput (HT) and high-content (HC) cellular screens are ideally suited to contextualize the information gathered from other “omic” approaches into networks and can be used for the identification of therapeutic targets. Current methods used for HT–HC screens are laborious, time-consuming, and prone to human error. The authors thus developed an automated high-throughput system with an integrated fluorescent imager for HC screens called the AI.CELLHOST. The implementation of user-defined culturing and assay plate setup parameters allows parallel operation of multiple screens in diverse mammalian cell types. The authors demonstrate that such a system is able to successfully maintain different cell lines in culture for extended periods of time as well as significantly increasing throughput, accuracy, and reproducibility of HT and HC screens.
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Hagen, F. K. "Genomic and RNA interference screens targeted at O-glycosylation in C. elegans." Biochemical Society Transactions 30, no. 1 (February 1, 2002): A1. http://dx.doi.org/10.1042/bst030a001c.
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Zimmermann, Michal, Olga Murina, Martin A. M. Reijns, Angelo Agathanggelou, Rachel Challis, Žygimantė Tarnauskaitė, Morwenna Muir, et al. "CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions." Nature 559, no. 7713 (July 2018): 285–89. http://dx.doi.org/10.1038/s41586-018-0291-z.
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Daouti, S., B. Latario, S. Nagulapalli, F. Buxton, S. Uziel-Fusi, G. W. Chirn, D. Bodian, et al. "Development of comprehensive functional genomic screens to identify novel mediators of osteoarthritis." Osteoarthritis and Cartilage 13, no. 6 (June 2005): 508–18. http://dx.doi.org/10.1016/j.joca.2005.02.003.
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Thu, K. L., J. Silvester, M. J. Elliott, W. Ba-alawi, M. H. Duncan, A. C. Elia, A. S. Mer, et al. "Disruption of the anaphase-promoting complex confers resistance to TTK inhibitors in triple-negative breast cancer." Proceedings of the National Academy of Sciences 115, no. 7 (January 29, 2018): E1570—E1577. http://dx.doi.org/10.1073/pnas.1719577115.
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TTK protein kinase (TTK), also known as Monopolar spindle 1 (MPS1), is a key regulator of the spindle assembly checkpoint (SAC), which functions to maintain genomic integrity. TTK has emerged as a promising therapeutic target in human cancers, including triple-negative breast cancer (TNBC). Several TTK inhibitors (TTKis) are being evaluated in clinical trials, and an understanding of the mechanisms mediating TTKi sensitivity and resistance could inform the successful development of this class of agents. We evaluated the cellular effects of the potent clinical TTKi CFI-402257 in TNBC models. CFI-402257 induced apoptosis and potentiated aneuploidy in TNBC lines by accelerating progression through mitosis and inducing mitotic segregation errors. We used genome-wide CRISPR/Cas9 screens in multiple TNBC cell lines to identify mechanisms of resistance to CFI-402257. Our functional genomic screens identified members of the anaphase-promoting complex/cyclosome (APC/C) complex, which promotes mitotic progression following inactivation of the SAC. Several screen candidates were validated to confer resistance to CFI-402257 and other TTKis using CRISPR/Cas9 and siRNA methods. These findings extend the observation that impairment of the APC/C enables cells to tolerate genomic instability caused by SAC inactivation, and support the notion that a measure of APC/C function could predict the response to TTK inhibition. Indeed, an APC/C gene expression signature is significantly associated with CFI-402257 response in breast and lung adenocarcinoma cell line panels. This expression signature, along with somatic alterations in genes involved in mitotic progression, represent potential biomarkers that could be evaluated in ongoing clinical trials of CFI-402257 or other TTKis.
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Bahrami, Ehsan, Martin Becker, Anna-Katharina Wirth, Jan Phillip Schmid, Tobias Herold, Rupert Öllinger, Roland Rad, and Irmela Jeremias. "A CRISPR/Cas9 Library Screen in Patients' Leukemia Cells In Vivo." Blood 134, Supplement_1 (November 13, 2019): 3945. http://dx.doi.org/10.1182/blood-2019-124288.
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Background: Functional genomic screens elegantly increase our understanding of biology of leukemias. So far, CRISPR/Cas9 screens are widely performed in cell lines and in genetically engineered mouse models, in vitro and in vivo; here, we extended their use to patient-derived leukemia cells in vivo. Methods Serially transplantable patient-derived xenograft (PDX) models were generated from children and adults with acute lymphoblastic leukemia (ALL). Cas9 was stably expressed in PDX ALL cells using a split form of Cas9 assembled by inteins, facilitating lentiviral-mediated gene delivery. Customized sgRNA library was generated using golden gate cloning, at 5 sgRNAs per target gene. The sgRNA vector additionally expressed a fluorochrome marker and a tag, for sequential magnetic-activated cell sorting (MACS) and flow cytometry (FACS) enrichment of sgRNA transduced PDX cells. Highly enriched Cas9/sgRNA double transgenic cells were transplanted into NSG mice and animals sacrificed after different periods of time. Cells were re-isolated from bone marrow, purified and subjected to PCR-based amplification of sgRNA library followed by next generation sequencing. Differential sgRNA distributions were analysed using a MAGeCK pipeline. Results We aimed to establish a comprehensive CRISPR screen pipeline allowing functional genomic screens in patients' acute leukemia cells. We investigated surface molecules required for cell homing and growth in mice, using a distinct customized sgRNA library. Quality controls of the sgRNA plasmid pool as well as transgenic PDX input samples verified standard distribution of all sgRNAs. As knockout was required at the time point of transplantation, conditions for prolonged culture of PDX ALL cells in vitro were optimized. Before injection into NSG mice, transduced PDX ALL cells were successfully enriched to above 95% using MACS and FACS. Over time in vivo, deep sequencing of re-isolated PDX cells revealed unchanged distribution of control sgRNAs, but strong loss of sgRNAs targeting CXCR4 and ITGB1, suggesting that CXCR4 and ITGB1 might be required for PDX ALL cell homing and engraftment. To validate the findings of drop-out CRISPR screen, we analyzed single sgRNAs targeting CXCR4 and ITGB1 in PDX cells. Competitive in vivo assays monitored by recombinant fluorochrome markers showed that the cells with CXCR4 or ITGB1 knockout had a significant disadvantage in vivo with respect to homing and growth in mice, compared to the control population. Taken together, we established a comprehensive workflow for CRISPR screen in PDX model of ALL in vivo. Our data identify and validate that CXCR4 and ITGB1 are required for homing and growth of PDX ALL cells in mice. Conclusion We show that CRISPR/Cas9 functional genetic screens are feasible in PDX acute leukemia models in vivo and report the first such screen, as far as to our knowledge. Extending CRISPR/Cas9 screens to patients' cells will greatly facilitate our understanding of individual leukemia biology and therapeutic targets in the future. Disclosures Becker: AVA Lifescience GmbH: Consultancy.
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Lew, A. E., L. A. Jackson, and M. I. Bellgard. "Comparative genomic analysis of non-coding sequences and the application of RNA interference tools for bovine functional genomics." Australian Journal of Experimental Agriculture 45, no. 8 (2005): 995. http://dx.doi.org/10.1071/ea05057.
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Non-coding (nc) RNAs are important regulators of developmental genes, and essential for the modification of cellular DNA and chromatin through a process known as RNA interference (RNAi). The mediators of RNAi can be in the form of short double stranded (ds) RNAs, micro (mi) RNAs or small interfering (si) RNAs. miRNAs are involved in a translation repression pathway that inhibits protein translation in mRNA targets. Comparative genomic screens have revealed conserved regulatory non-coding sequences, which assist to predict the function of endogenous miRNAs. Only a few comparative studies include bovine genomic sequence, and RNAi has yet to be applied in bovine genome functional screens. siRNAs target homologous mRNAs for degradation, and thereby, silence specific genes. The use of synthetic siRNAs facilitates the elucidation of gene pathways by specific gene knockdown. A survey of the literature identifies a small number of reports using RNAi to examine immune pathways in bovine cell lines; however, they do not target genes involved in specific production traits. Applications of RNAi to elucidate bovine immune pathways for relevant bacterial and parasite diseases are yet to be reported. The inhibition of viral replication using RNAi has been demonstrated with bovine RNA viruses such as pestivirus and foot and mouth disease virus signifying the potential of RNAi as an antiviral therapeutic. RNAi approaches combined with genome data for protozoan parasites, insects and nematodes, will expedite the identification of novel targets for the treatment and prevention of economically important parasitic infections. This review will examine the approaches used in mammalian RNAi research, the current status of its applications to livestock systems and will discuss potential applications in beef cattle programs.
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Baetz, Kristin K., Nevan J. Krogan, Andrew Emili, Jack Greenblatt, and Philip Hieter. "The ctf13-30/CTF13 Genomic Haploinsufficiency Modifier Screen Identifies the Yeast Chromatin Remodeling Complex RSC, Which Is Required for the Establishment of Sister Chromatid Cohesion." Molecular and Cellular Biology 24, no. 3 (February 1, 2004): 1232–44. http://dx.doi.org/10.1128/mcb.24.3.1232-1244.2003.
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ABSTRACT The budding yeast centromere-kinetochore complex ensures high-fidelity chromosome segregation in mitosis and meiosis by mediating the attachment and movement of chromosomes along spindle microtubules. To identify new genes and pathways whose function impinges on chromosome transmission, we developed a genomic haploinsufficiency modifier screen and used ctf13-30, encoding a mutant core kinetochore protein, as the reference point. We demonstrate through a series of secondary screens that the genomic modifier screen is a successful method for identifying genes that encode nonessential proteins required for the fidelity of chromosome segregation. One gene isolated in our screen was RSC2, a nonessential subunit of the RSC chromatin remodeling complex. rsc2 mutants have defects in both chromosome segregation and cohesion, but the localization of kinetochore proteins to centromeres is not affected. We determined that, in the absence of RSC2, cohesin could still associate with chromosomes but fails to achieve proper cohesion between sister chromatids, indicating that RSC has a role in the establishment of cohesion. In addition, numerous subunits of RSC were affinity purified and a new component of RSC, Rtt102, was identified. Our work indicates that only a subset of the nonessential RSC subunits function in maintaining chromosome transmission fidelity.
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Hutz, Janna E., Thomas Nelson, Hua Wu, Gregory McAllister, Ioannis Moutsatsos, Savina A. Jaeger, Somnath Bandyopadhyay, et al. "The Multidimensional Perturbation Value." Journal of Biomolecular Screening 18, no. 4 (November 29, 2012): 367–77. http://dx.doi.org/10.1177/1087057112469257.
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Screens using high-throughput, information-rich technologies such as microarrays, high-content screening (HCS), and next-generation sequencing (NGS) have become increasingly widespread. Compared with single-readout assays, these methods produce a more comprehensive picture of the effects of screened treatments. However, interpreting such multidimensional readouts is challenging. Univariate statistics such as t-tests and Z-factors cannot easily be applied to multidimensional profiles, leaving no obvious way to answer common screening questions such as “Is treatment X active in this assay?” and “Is treatment X different from (or equivalent to) treatment Y?” We have developed a simple, straightforward metric, the multidimensional perturbation value (mp-value), which can be used to answer these questions. Here, we demonstrate application of the mp-value to three data sets: a multiplexed gene expression screen of compounds and genomic reagents, a microarray-based gene expression screen of compounds, and an HCS compound screen. In all data sets, active treatments were successfully identified using the mp-value, and simulations and follow-up analyses supported the mp-value’s statistical and biological validity. We believe the mp-value represents a promising way to simplify the analysis of multidimensional data while taking full advantage of its richness.
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Spreafico, Roberto, Leah B. Soriaga, Johannes Grosse, Herbert W. Virgin, and Amalio Telenti. "Advances in Genomics for Drug Development." Genes 11, no. 8 (August 15, 2020): 942. http://dx.doi.org/10.3390/genes11080942.
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Drug development (target identification, advancing drug leads to candidates for preclinical and clinical studies) can be facilitated by genetic and genomic knowledge. Here, we review the contribution of population genomics to target identification, the value of bulk and single cell gene expression analysis for understanding the biological relevance of a drug target, and genome-wide CRISPR editing for the prioritization of drug targets. In genomics, we discuss the different scope of genome-wide association studies using genotyping arrays, versus exome and whole genome sequencing. In transcriptomics, we discuss the information from drug perturbation and the selection of biomarkers. For CRISPR screens, we discuss target discovery, mechanism of action and the concept of gene to drug mapping. Harnessing genetic support increases the probability of drug developability and approval.
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Lin, Shan, Clement Larrue, Nastassja K. Scheidegger, Bo Kyung A. Seong, Neekesh V. Dharia, Caroline Wechsler, Guillaume Kugener, et al. "An In Vivo CRISPR Screening Platform to Identify New Therapeutic Targets in AML." Blood 138, Supplement 1 (November 5, 2021): 266. http://dx.doi.org/10.1182/blood-2021-153039.
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Abstract First-generation, large-scale functional genomic screens have revealed hundreds of potential genetic vulnerabilities in acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because these large-scale genetic screens were primarily performed in vitro in established AML cell lines, their translational relevance has been debated. Therefore, we established a protocol for CRISPR screening in orthotopic xenograft models of human AML, including patient-derived-xenograft (PDX) models that are tractable for CRISPR-editing. We first defined experimental conditions necessary for an optimal in vivo screen via barcoding experiments. We determined that sub-lethal irradiation was necessary for improved barcode representation in bone marrow and to reduce mouse-to-mouse variation. Moreover, it was critical to combine samples from multiple mice to achieve complete in vivo library representation. Next, using the Broad DepMap and other publicly available functional genomic screen data, we identified 200 genes that were stronger dependencies in AML cell lines compared to all other cancer types screened. Using this list, we created a secondary library and performed parallel in vivo and in vitro screens using the MV4-11 and U937 cell lines and a PDX model. In vitro and in vivo hits were surprisingly well correlated, although a modest number of targets did not score well in vivo. Notably, dependencies identified across AML cell line models were strongly recapitulated in the PDX model, validating the application of AML cell lines for dependency discovery. Our in vivo screens nominated the mitochondria-localized RING-type ubiquitin E3 ligase MARCH5 as a potential therapeutic target in AML. Using CRISPR, we first validated this in vitro dependency on MARCH5 and determined that MARCH5 is a critical guardian to prevent apoptosis in AML. MARCH5 depletion activates the canonical mitochondrial apoptosis pathway in a BAX/BAK1-dependent manner. Multiple genome-wide screens revealed that a dependency on MARCH5 is strongly correlated with a dependency on MCL1, but not other anti-apoptotic BCL2-family members, across the AML cell lines in the screen. As observed with MCL1 inhibition, MARCH5 depletion sensitized AML cells to venetoclax, a BCL2-specific inhibitor FDA-approved in combination with a hypomethylating agent for the treatment of older adults with AML. Importantly, MARCH5 depletion diminished the venetoclax resistance induced by MCL1 overexpression but not that caused by BCLXL overexpression. Altogether, these results suggest that MARCH5 is required for maintaining MCL1 activity specifically. Since there are no small molecule inhibitors directed against MARCH5, we deployed a dTAG system as an approximation of pharmacological inhibition. This approach uses a hetero-bifunctional small molecule that binds the FKBP12 F36V-fused MARCH5 and the E3 ligase VHL, leading to the ubiquitination and proteasome-mediated degradation of the fusion protein. dTAG-MARCH5 cells were established via deleting endogenous MARCH5 by CRISPR and expressing exogenous FKBP-tagged MARCH5 protein. MARCH5 degradation with the dTAG molecule dTAG V-1 markedly impaired cell growth in vitro. Additionally, we demonstrated the utility of dTAG system in vivo using a PDX model. The combination treatment of dTAG V-1 and venetoclax elicited a much stronger anti-leukemic effect compared to the treatment with only venetoclax or dTAG V-1, further highlighting MARCH5 as a promising synergistic target for enhancing the efficacy of venetoclax in AML. Taken together, our in vivo screening approach, coupled with CRISPR-competent PDX models and dTAG-directed protein degradation, constitute a useful platform for prioritizing AML targets emerging from in vitro screens to serve as the starting point for therapy development. Disclosures Dharia: Genentech: Current Employment. Piccioni: Merck Research Laboratories: Current Employment. Stegmaier: Bristol Myers Squibb: Consultancy; KronosBio: Consultancy; AstraZeneca: Consultancy; Auron Therapeutics: Consultancy, Current equity holder in publicly-traded company; Novartis: Research Funding.
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Safikhani, Zhaleh, Nehme El-Hachem, Rene Quevedo, Petr Smirnov, Anna Goldenberg, Nicolai Juul Birkbak, Christopher Mason, et al. "Assessment of pharmacogenomic agreement." F1000Research 5 (May 9, 2016): 825. http://dx.doi.org/10.12688/f1000research.8705.1.
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In 2013 we published an analysis demonstrating that drug response data and gene-drug associations reported in two independent large-scale pharmacogenomic screens, Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Cell Line Encyclopedia (CCLE), were inconsistent. The GDSC and CCLE investigators recently reported that their respective studies exhibit reasonable agreement and yield similar molecular predictors of drug response, seemingly contradicting our previous findings. Reanalyzing the authors’ published methods and results, we found that their analysis failed to account for variability in the genomic data and more importantly compared different drug sensitivity measures from each study, which substantially deviate from our more stringent consistency assessment. Our comparison of the most updated genomic and pharmacological data from the GDSC and CCLE confirms our published findings that the measures of drug response reported by these two groups are not consistent. We believe that a principled approach to assess the reproducibility of drug sensitivity predictors is necessary before envisioning their translation into clinical settings.
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Banerji, Versha, Kenneth N. Ross, Loretta S. Li, Stacey M. Frumm, Anna C. Schinzel, Cynthia K. Hahn, Rose M. Kakoza, et al. "Intersecting Chemical Genomic and Genetic Screens Identifies Glycogen Synthase Kinase-3α (GSK-3α) as a Modulator of Differentiation In Acute Myeloid Leukemia." Blood 116, no. 21 (November 19, 2010): 1000. http://dx.doi.org/10.1182/blood.v116.21.1000.1000.
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Abstract Abstract 1000 The treatment of acute myeloid leukemia (AML) poses a vexing challenge despite an improved understanding of its molecular pathogenesis. A two-hit theory has been proposed for the pathogenesis of AML where the first hit imparts a proliferation defect and the second a block in differentiation. Drug discovery efforts for AML, however, have largely focused on the proliferation defect. Using the intersection of chemical biology and high-throughput genetic screening we sought to identify new AML differentiation targets by measuring the induction of a complex gene expression signature of myeloid maturation. We performed two independent small molecule library screens and a high-throughput shRNA screen for perturbations that induce differentiation in AML cells. We measured this differentiation signature using the previously described gene expression-based high-throughput screening (GE-HTS) approach in which gene expression signatures serve as surrogates for different biological states. Glycogen Synthase Kinase-3 (GSK-3) emerged as a target at the intersection of these three screens. GSK-3 is a multifunctional serine threonine kinase involved in diverse cellular processes including differentiation, signal transduction, cell cycle regulation and proliferation, with an emerging role in human leukemia. We demonstrate that the GSK-3 inhibitors scoring in the primary screens indeed induce the differentiation signature with a dose-response in AML cell lines. In order to further validate GSK-3 as a target, we extended testing to lithium chloride and SB216763, two commonly used GSK-3 inhibitors not in the original screens. Both of these molecules induced AML differentiation as measured by gene expression and morphological changes in multiple AML cell lines and in primary patient blasts in vitro. GSK-3 is expressed as two highly homologous but non-redundant isoforms, GSK-3α and GSK-3β, with small molecule inhibitors non-selectively reported to target both. In order to further validate the findings of the small molecule library screens, we performed a high-throughput shRNA screen targeting the human kinome for shRNAs that induce differentiation. Multiple hairpins against GSK-3α scored. In secondary testing of four AML cell lines, we found that genetic loss of GSK-3α induced differentiation as measured by induction of the complex gene expression signature, alterations in genome-wide expression, and morphological changes associated with maturation. Moreover, colony formation in methylcellulose was impeded. These effects could be rescued with a GSK-3α cDNA immune to the effects of the shRNA, further supporting the on-target activity of the hairpin. In contrast, GSK-3β-directed hairpins induced minimal differentiation. We next extended testing to an in vivo U937 orthotopic model of AML. While pan-GSK-3 inhibition with lithium chloride treatment did not demonstrate efficacy in this model, inhibition of GSK-3α with shRNA attenuated development of disease compared to an shRNA control. We hypothesize that the rise in β-catenin with pan-inhibition of GSK-3 may attenuate the response to lithium in vivo. In contrast, isolated knockdown of GSK-3α does not induce β-catenin. While much of the prior cancer literature has focused on the role of GSK-3β in human malignancy, these studies suggest a role for GSK-3α in AML differentiation and support a role for GSK-3α-directed targeted therapy. Disclosures: No relevant conflicts of interest to declare.
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Brown, Jennifer R., Ross Levine, Elke Raderschall, Christina Thompson, D. Gary Gilliland, and Arnold S. Freedman. "Systematic Genomic Screen for Tyrosine Kinase Mutations in CLL." Blood 110, no. 11 (November 16, 2007): 2069. http://dx.doi.org/10.1182/blood.v110.11.2069.2069.
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Abstract Genomic screens have identified activating somatic mutations in tyrosine kinases in a spectrum of acute and chronic hematologic malignancies. These mutations lead to increased cellular proliferation and/or impairment of apoptosis. Chronic lymphocytic leukemia is a common chronic leukemia of adults that is thought to result at least in part from failure of apoptosis. Recent data suggest that CLL cell turnover is higher than previously thought and may correlate with disease progression, suggesting that somatic mutations that activate signal transduction may contribute to CLL pathogenesis and/or progression. The purpose of this study was to look systematically for activating mutations in protein tyrosine kinases in CLL using high-throughput DNA resequencing. 95 CLL patients were selected to encompass the range of prognostic groups defined by somatic hypermutation of the immunoglobulin heavy chain gene, ZAP-70 status and FISH cytogenetics. DNA was isolated from Ficoll separated peripheral blood mononuclear cells, and germline DNA was obtained from buccal swabs and/or saliva samples. 70 tyrosine kinases were selected for resequencing based on their involvement in lymphocyte biology, known altered gene expression in CLL, or location at a site of chromosomal gain, loss or mutation in CLL. All coding exons of ZAP-70 were included. Nested M13 tailed primers were designed to amplify and sequence the exons encoding the activation loops and JM domains. Bidirectional sequence analysis was performed using Mutation Surveyor version 2.28 (SoftGenetics). Candidate mutations were reamplified and sequenced from the original CLL DNA sample for verification; in parallel, the same exon was amplified and resequenced from the paired germline sample to assess whether non-synonymous mutations not known to be SNPs were present in the germline. A total of 178 exons were sequenced per sample. Analysis of 6.21 megabases of sequence data identified nine novel non-synonymous alterations in seven different genes; at least seven of these nine alterations are present in the germline, with the other two presently undergoing analysis. Although these non-synonymous alleles may have functional relevance to the pathogenesis of CLL, these data indicate that a significant proportion of non-synonymous alleles identified in high-throughput genomic screens are non-synonymous polymorphisms. These findings further suggest that the rate of acquired somatic mutation in the tyrosine kinome in CLL is low, and demonstrate the importance of matched germline DNA to allow assessment of candidate non-synonymous mutations in human cancer samples.
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Hong-Geller, Elizabeth, and Sofiya N. Micheva-Viteva. "Functional Gene Discovery Using RNA Interference-Based Genomic Screens to Combat Pathogen Infection." Current Drug Discovery Technologies 7, no. 2 (June 1, 2010): 86–94. http://dx.doi.org/10.2174/157016310793180657.
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Reyes, Luis H., Maria P. Almario, and Katy C. Kao. "Genomic Library Screens for Genes Involved in n-Butanol Tolerance in Escherichia coli." PLoS ONE 6, no. 3 (March 8, 2011): e17678. http://dx.doi.org/10.1371/journal.pone.0017678.
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Roti, Giovanni, Anne Carlton, Kenneth N. Ross, Michele Markstein, Kostandin Pajcini, Angela H. Su, Norbert Perrimon, et al. "Complementary Genomic Screens Identify SERCA as a Therapeutic Target in NOTCH1 Mutated Cancer." Cancer Cell 23, no. 3 (March 2013): 390–405. http://dx.doi.org/10.1016/j.ccr.2013.01.015.
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Fromont-Racine, Micheline, Andrew E. Mayes, Adeline Brunet-Simon, Jean-Christophe Rain, Alan Colley, Ian Dix, Laurence Decourty, et al. "Genome-Wide Protein Interaction Screens Reveal Functional Networks Involving Sm-Like Proteins." Yeast 1, no. 2 (January 1, 2000): 95–110. http://dx.doi.org/10.1155/2000/919260.
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A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (L¯ike Sm¯) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein–protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale.
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Fromont-Racine, Micheline, Andrew E. Mayes, Adeline Brunet-Simon, Jean-Christophe Rain, Alan Colley, Ian Dix, Laurence Decourty, et al. "Genome-Wide Protein Interaction Screens Reveal Functional Networks Involving Sm-Like Proteins." Yeast 1, no. 2 (2000): 95–110. http://dx.doi.org/10.1002/1097-0061(20000630)17:2<95::aid-yea16>3.0.co;2-h.
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A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence ofSaccharomyces cerevisiaehas identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (L¯ike Sm¯) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein–protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale.
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Shen, Yaoqing, Cameron J. Grisdale, Sumaiya A. Islam, Pinaki Bose, Jake Lever, Eric Y. Zhao, Natalie Grinshtein, et al. "Comprehensive genomic profiling of glioblastoma tumors, BTICs, and xenografts reveals stability and adaptation to growth environments." Proceedings of the National Academy of Sciences 116, no. 38 (August 30, 2019): 19098–108. http://dx.doi.org/10.1073/pnas.1813495116.
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Glioblastoma multiforme (GBM) is the most deadly brain tumor, and currently lacks effective treatment options. Brain tumor-initiating cells (BTICs) and orthotopic xenografts are widely used in investigating GBM biology and new therapies for this aggressive disease. However, the genomic characteristics and molecular resemblance of these models to GBM tumors remain undetermined. We used massively parallel sequencing technology to decode the genomes and transcriptomes of BTICs and xenografts and their matched tumors in order to delineate the potential impacts of the distinct growth environments. Using data generated from whole-genome sequencing of 201 samples and RNA sequencing of 118 samples, we show that BTICs and xenografts resemble their parental tumor at the genomic level but differ at the mRNA expression and epigenomic levels, likely due to the different growth environment for each sample type. These findings suggest that a comprehensive genomic understanding of in vitro and in vivo GBM model systems is crucial for interpreting data from drug screens, and can help control for biases introduced by cell-culture conditions and the microenvironment in mouse models. We also found that lack of MGMT expression in pretreated GBM is linked to hypermutation, which in turn contributes to increased genomic heterogeneity and requires new strategies for GBM treatment.
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Wiles, Amy M., Dashnamoorthy Ravi, Selvaraj Bhavani, and Alexander J. R. Bishop. "An Analysis of Normalization Methods for Drosophila RNAi Genomic Screens and Development of a Robust Validation Scheme." Journal of Biomolecular Screening 13, no. 8 (July 25, 2008): 777–84. http://dx.doi.org/10.1177/1087057108323125.
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Genome-wide RNA interference (RNAi) screening allows investigation of the role of individual genes in a process of choice. Most RNAi screens identify a large number of genes with a continuous gradient in the assessed phenotype. Screeners must decide whether to examine genes with the most robust phenotype or the full gradient of genes that cause an effect and how to identify candidate genes. The authors have used RNAi in Drosophila cells to examine viability in a 384-well plate format and compare 2 screens, untreated control and treatment. They compare multiple normalization methods, which take advantage of different features within the data, including quantile normalization, background subtraction, scaling, cellHTS2 (Boutros et al. 2006), and interquartile range measurement. Considering the false-positive potential that arises from RNAi technology, a robust validation method was designed for the purpose of gene selection for future investigations. In a retrospective analysis, the authors describe the use of validation data to evaluate each normalization method. Although no method worked ideally, a combination of 2 methods, background subtraction followed by quantile normalization and cellHTS2, at different thresholds, captures the most dependable and diverse candidate genes. Thresholds are suggested depending on whether a few candidate genes are desired or a more extensive systems-level analysis is sought. The normalization approaches and experimental design to perform validation experiments are likely to apply to those high-throughput screening systems attempting to identify genes for systems-level analysis. ( Journal of Biomolecular Screening 2008:777-784)
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Mühleisen, Thomas W., Andreas J. Forstner, Per Hoffmann, and Sven Cichon. "Brain imaging genomics: influences of genomic variability on the structure and function of the human brain." Medizinische Genetik 32, no. 1 (May 1, 2020): 47–56. http://dx.doi.org/10.1515/medgen-2020-2007.
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Abstract Brain imaging genomics is an emerging discipline in which genomic and brain imaging data are integrated in order to elucidate the molecular mechanisms that underly brain phenotypes and diseases, including neuropsychiatric disorders. As with all genetic analyses of complex traits and diseases, brain imaging genomics has evolved from small, individual candidate gene investigations towards large, collaborative genome-wide association studies. Recent investigations, mostly population-based, have studied well-powered cohorts comprising tens of thousands of individuals and identified multiple robust associations of single-nucleotide polymorphisms and copy number variants with structural and functional brain phenotypes. Such systematic genomic screens of millions of genetic variants have generated initial insights into the genetic architecture of brain phenotypes and demonstrated that their etiology is polygenic in nature, involving multiple common variants with small effect sizes and rare variants with larger effect sizes. Ongoing international collaborative initiatives are now working to obtain a more complete picture of the underlying biology. As in other complex phenotypes, novel approaches – such as gene–gene interaction, gene–environment interaction, and epigenetic analyses – are being implemented in order to investigate their contribution to the observed phenotypic variability. An important consideration for future research will be the translation of brain imaging genomics findings into clinical practice.
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Schubert, Max G., Daniel B. Goodman, Timothy M. Wannier, Divjot Kaur, Fahim Farzadfard, Timothy K. Lu, Seth L. Shipman, and George M. Church. "High-throughput functional variant screens via in vivo production of single-stranded DNA." Proceedings of the National Academy of Sciences 118, no. 18 (April 27, 2021): e2018181118. http://dx.doi.org/10.1073/pnas.2018181118.
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Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.
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Sun, Claire, Caroline Drinkwater, Dhanya Sooraj, Gabrielle Bradshaw, Claire Shi, Dasun Fernando, Sarah Parackal, Daniel Gough, Jason Cain, and Ron Firestein. "MODL-21. INTEGRATIVE APPROACHES IN FUNCTIONAL GENOMICS TO IDENTIFY GENETIC DEPENDENCIES IN PEDIATRIC BRAIN CANCER." Neuro-Oncology 22, Supplement_3 (December 1, 2020): iii415. http://dx.doi.org/10.1093/neuonc/noaa222.594.
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Abstract The precise decoding of human genomes facilitated by the advancements in next-generation sequencing has led to a better understanding of genetic underpinnings of pediatric brain cancers. Indeed, it is now evident that tumours of the same type harbour distinct driving mutations and molecular aberrations that can result in different prognosis and treatment outcomes. The profounder insight into the the identity, amount and types of molecular aberrations has paved the way for the advent of targeted therapies in precision medicine. Nevertheless, less than 10% of pediatric cancer patients harbour actionable mutations. Strictly limited therapeutic options that are firstly available for brain cancers and secondly acceptable for children’s development further impede the breakthrough in the survival rate in pediatric brain cancers. This underscores a desperate need to delve beyond genomic sequencing to identify biomarker coupled therapies that not only featured with treatment efficacy in the central nervous system but also acceptable side effects for children. The Hudson-Monash Paediatric Precision Medicine (HMPPM) Program focuses on utilising genetic profiles of patients’ tumour models to identify new therapeutic targets and repurpose existing ones using high-throughput functional genomics screens (2220 drugs and CRISPR screen of 300 oncogenic genes). Using a large compendium of over sixty patient derived paediatric brain cancer models, we provide proof-of-concept data that shows an integrative pipeline for functional genomics with multi-omics datasets to perform genotype-phenotype correlations and, therefore, identify genetic dependencies. Herein, using several examples in ATRT, DIPG and HGG, we show how functional interrogations can better define molecular subclassification of tumours and identify unique vulnerabilities.
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Young, Ryan M., James D. Phelan, Arthur L. Shaffer, George W. Wright, Da Wei Huang, Roland Schmitz, Calvin Johnson, Thomas Oellerich, Wyndham Wilson, and Louis M. Staudt. "Taming the Heterogeneity of Aggressive Lymphomas for Precision Therapy." Annual Review of Cancer Biology 3, no. 1 (March 4, 2019): 429–55. http://dx.doi.org/10.1146/annurev-cancerbio-030518-055734.
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Genomic analyses of diffuse large B cell lymphoma (DLBCL) are revealing the genetic and phenotypic heterogeneity of these aggressive lymphomas. In part, this heterogeneity reflects the existence of distinct genetic subtypes that acquire characteristic constellations of somatic genetic alterations to converge on the DLBCL phenotype. In parallel, functional genomic screens and proteomic analyses have identified multiprotein assemblies that coordinate oncogenic survival signaling in DLBCL. In this review, we merge these recent insights into a unified conceptual framework with implications for the design of precision medicine trials in DLBCL.
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Mark-Danieli, Michal, Nihay Laham, Michal Kenan-Eichler, Asher Castiel, Daniel Melamed, Meytal Landau, Nicole M. Bouvier, Matthew J. Evans, and Eran Bacharach. "Single Point Mutations in the Zinc Finger Motifs of the Human Immunodeficiency Virus Type 1 Nucleocapsid Alter RNA Binding Specificities of the Gag Protein and Enhance Packaging and Infectivity." Journal of Virology 79, no. 12 (June 15, 2005): 7756–67. http://dx.doi.org/10.1128/jvi.79.12.7756-7767.2005.
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ABSTRACT A specific interaction between the nucleocapsid (NC) domain of the Gag polyprotein and the RNA encapsidation signal (Ψ) is required for preferential incorporation of the retroviral genomic RNA into the assembled virion. Using the yeast three-hybrid system, we developed a genetic screen to detect human immunodeficiency virus type 1 (HIV-1) Gag mutants with altered RNA binding specificities. Specifically, we randomly mutated full-length HIV-1 Gag or its NC portion and screened the mutants for an increase in affinity for the Harvey murine sarcoma virus encapsidation signal. These screens identified several NC zinc finger mutants with altered RNA binding specificities. Furthermore, additional zinc finger mutants that also demonstrated this phenotype were made by site-directed mutagenesis. The majority of these mutants were able to produce normal virion-like particles; however, when tested in a single-cycle infection assay, some of the mutants demonstrated higher transduction efficiencies than that of wild-type Gag. In particular, the N17K mutant showed a seven- to ninefold increase in transduction, which correlated with enhanced vector RNA packaging. This mutant also packaged larger amounts of foreign RNA. Our results emphasize the importance of the NC zinc fingers, and not other Gag sequences, in achieving specificity in the genome encapsidation process. In addition, the described mutations may contribute to our understanding of HIV diversity resulting from recombination events between copackaged viral genomes and foreign RNA.
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Bonangelino, Cecilia J., Edna M. Chavez, and Juan S. Bonifacino. "Genomic Screen for Vacuolar Protein Sorting Genes inSaccharomyces cerevisiae." Molecular Biology of the Cell 13, no. 7 (July 2002): 2486–501. http://dx.doi.org/10.1091/mbc.02-01-0005.
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The biosynthetic sorting of hydrolases to the yeast vacuole involves transport along two distinct routes referred to as the carboxypeptidase Y and alkaline phosphatase pathways. To identify genes involved in sorting to the vacuole, we conducted a genome-wide screen of 4653 homozygous diploid gene deletion strains ofSaccharomyces cerevisiae for missorting of carboxypeptidase Y. We identified 146 mutant strains that secreted strong-to-moderate levels of carboxypeptidase Y. Of these, only 53 of the corresponding genes had been previously implicated in vacuolar protein sorting, whereas the remaining 93 had either been identified in screens for other cellular processes or were only known as hypothetical open reading frames. Among these 93 were genes encoding: 1) the Ras-like GTP-binding proteins Arl1p and Arl3p, 2) actin-related proteins such as Arp5p and Arp6p, 3) the monensin and brefeldin A hypersensitivity proteins Mon1p and Mon2p, and 4) 15 novel proteins designated Vps61p-Vps75p. Most of the novel gene products were involved only in the carboxypeptidase Y pathway, whereas a few, including Mon1p, Mon2p, Vps61p, and Vps67p, appeared to be involved in both the carboxypeptidase Y and alkaline phosphatase pathways. Mutants lacking some of the novel gene products, including Arp5p, Arp6p, Vps64p, and Vps67p, were severely defective in secretion of mature α-factor. Others, such as Vps61p, Vps64p, and Vps67p, displayed defects in the actin cytoskeleton at 30°C. The identification and phenotypic characterization of these novel mutants provide new insights into the mechanisms of vacuolar protein sorting, most notably the probable involvement of the actin cytoskeleton in this process.
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Hadjidekova, S., and D. Toncheva. "Array-Based Comparative Genomic Hybridization Application for Revealing Genomic Micro Imbalances in Congenital Malformations." Balkan Journal of Medical Genetics 12, no. 1 (January 1, 2009): 3–8. http://dx.doi.org/10.2478/v10034-009-0001-0.
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Array-Based Comparative Genomic Hybridization Application for Revealing Genomic Micro Imbalances in Congenital MalformationsBirth defects affect 3-5% of live births and are a major cause of fetal, neonatal and infant morbidity and mortality in all industrialized countries. Some 40-60% of congenital physical anomalies in humans have no cause, 20% that seem to be multifactorial, 10-13% environmental and 12-25% genetic.Classical cytogenetic or common comparative genomic hybridization (CGH) methods have limited use in investigation of the whole genome because of their low resolution (5-10 Mb). Fluorescence in situ hybridization (FISH) and quantitative fluorescence polymerase chain reaction (QF-PCR) have higher resolution but do not allow genome-wide screening and require some prior knowledge regarding the suspected chromosomal abnormality and its genomic location.Because of these limitations, the impact of genetic micro imbalances as etiological factors for the development of congenital malformations (CM) is underestimated. Array-based techniques have enabled higher resolution screens for genomic imbalances in CM as they permit identification of micro aberrations with a size between 60 bp and several hundred kilobases. They make possible screening of the whole genome and detection of novel unbalanced micro structural rearrangements in a single reaction and also effective screening of new dose-dependent genes. In addition, the application of the aCGH technology has the potential to improve our understanding of the normal quantitative variants of the human genome.
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Laffitte, Marie-Claude N., Philippe Leprohon, Barbara Papadopoulou, and Marc Ouellette. "Plasticity of the Leishmania genome leading to gene copy number variations and drug resistance." F1000Research 5 (September 20, 2016): 2350. http://dx.doi.org/10.12688/f1000research.9218.1.
Full textAbstract:
Leishmania has a plastic genome, and drug pressure can select for gene copy number variation (CNV). CNVs can apply either to whole chromosomes, leading to aneuploidy, or to specific genomic regions. For the latter, the amplification of chromosomal regions occurs at the level of homologous direct or inverted repeated sequences leading to extrachromosomal circular or linear amplified DNAs. This ability of Leishmania to respond to drug pressure by CNVs has led to the development of genomic screens such as Cos-Seq, which has the potential of expediting the discovery of drug targets for novel promising drug candidates.
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Gimeno, Marian, Edurne San José-Enériz, Angel Rubio, Leire Garate, Estíbaliz Miranda, Carlos Castilla, Xabier Agirre, Felipe Prosper, and Fernando Carazo. "Identifying Lethal Dependencies with HUGE Predictive Power." Cancers 14, no. 13 (July 1, 2022): 3251. http://dx.doi.org/10.3390/cancers14133251.
Full textAbstract:
Recent functional genomic screens—such as CRISPR-Cas9 or RNAi screening—have fostered a new wave of targeted treatments based on the concept of synthetic lethality. These approaches identified LEthal Dependencies (LEDs) by estimating the effect of genetic events on cell viability. The multiple-hypothesis problem is related to a large number of gene knockouts limiting the statistical power of these studies. Here, we show that predictions of LEDs from functional screens can be dramatically improved by incorporating the “HUb effect in Genetic Essentiality” (HUGE) of gene alterations. We analyze three recent genome-wide loss-of-function screens—Project Score, CERES score and DEMETER score—identifying LEDs with 75 times larger statistical power than using state-of-the-art methods. Using acute myeloid leukemia, breast cancer, lung adenocarcinoma and colon adenocarcinoma as disease models, we validate that our predictions are enriched in a recent harmonized knowledge base of clinical interpretations of somatic genomic variants in cancer (AUROC > 0.87). Our approach is effective even in tumors with large genetic heterogeneity such as acute myeloid leukemia, where we identified LEDs not recalled by previous pipelines, including FLT3-mutant genotypes sensitive to FLT3 inhibitors. Interestingly, in-vitro validations confirm lethal dependencies of either NRAS or PTPN11 depending on the NRAS mutational status. HUGE will hopefully help discover novel genetic dependencies amenable for precision-targeted therapies in cancer. All the graphs showing lethal dependencies for the 19 tumor types analyzed can be visualized in an interactive tool.