Abstract PR12: Genome-wide in-vivo tumor xenograft CRISPR knockout screening for identifying KRAS mutant synthetic lethal interactions

2017 ◽  
Author(s):  
Edwin H. Yau ◽  
Tariq Rana
Keyword(s):  
Tumor Xenograft ◽  
Synthetic Lethal ◽  
Genome Wide ◽  
Synthetic Lethal Interactions

scholarly journals Multiplex enCas12a screens show functional buffering by paralogs is systematically absent from genome-wide CRISPR/Cas9 knockout screens

2020 ◽  
Author(s):  
Merve Dede ◽  
Megan McLaughlin ◽  
Eiru Kim ◽  
Traver Hart
Keyword(s):  
Cell Lines ◽  
Protein Complexes ◽  
Essential Genes ◽  
Systematic Analysis ◽  
Synthetic Lethal ◽  
Genome Wide ◽  
Synthetic Lethal Interactions ◽  
Crispr Screen ◽  
A Cell ◽  
Genetic Buffering

AbstractMajor efforts on pooled library CRISPR knockout screening across hundreds of cell lines have identified genes whose disruption leads to fitness defects, a critical step in identifying candidate cancer targets. However, the number of essential genes detected from these monogenic knockout screens are very low compared to the number of constitutively expressed genes in a cell, raising the question of why there are so few essential genes. Through a systematic analysis of screen data in cancer cell lines generated by the Cancer Dependency Map, we observed that half of all constitutively-expressed genes are never hits in any CRISPR screen, and that these never-essentials are highly enriched for paralogs. We investigated paralog buffering through systematic dual-gene CRISPR knockout screening by testing algorithmically defined ~400 candidate paralog pairs with the enCas12a multiplex knockout system in three cell lines. We observed 24 synthetic lethal paralog pairs which have escaped detection by monogenic knockout screens at stringent thresholds. Nineteen of 24 (79%) synthetic lethal interactions were present in at least two out of three cell lines and 14 of 24 (58%) were present in all three cell lines tested, including alternate subunits of stable protein complexes as well as functionally redundant enzymes. Together these observations strongly suggest that paralogs represent a targetable set of genetic dependencies that are systematically under-represented among cell-essential genes due to genetic buffering in monogenic CRISPR-based mammalian functional genomics approaches.

Synthetic-lethal interactions between ATR inhibition and functional genetic and proteomic biomarkers to predict responses in endometriosis-associated cancers.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e18000-e18000
Author(s):  
Oren Gilad ◽  
Dansu Li ◽  
Erin George ◽  
Rakesh Chettier ◽  
Fiona Simpkins ◽  
...  
Keyword(s):  
Synthetic Lethality ◽  
In Vivo Studies ◽  
Multiple Cancer ◽  
Driver Genes ◽  
Synthetic Lethal ◽  
Cancer Driver ◽  
Synthetic Lethal Interactions ◽  
Synthetically Lethal

e18000 Background: Endometriosis is a common gynecologic disorder proven to be a precursor to several cancer types. We developed a potent and selective inhibitor (ATRN-119) of a critical DNA damage response (DDR) protein kinase: the ataxia telangiectasia and Rad3-related protein (ATR). Treatment with ATRN-119 is synthetically lethal with multiple cancer-associated changes in DDR pathways, representing a new and effective strategy to treat cancer. The objective of this study is to evaluate the overlap of DDR genes that respond to ATRN-119 and those mutated in endometriosis. Methods: We sequenced the exomes of 2,932 unrelated women with surgically-confirmed endometriosis (GERMLINE) and 274 tissue blocks containing endometriosis lesions (LESION). DNA was extracted using standard methods. Missense and truncation variants were analyzed. These data were compared to analysis of a whole proteome screen for factors that respond to exposure to ATRN-119 and may influence responsiveness to treatment. Factors observed in both methods were considered high-priority biomarker candidates and were experimentally tested for synthetic lethality with ATRN-119 treatment. Results: Analysis of endometriosis patients found 89% of the LESION samples had 2 or more DDR mutations vs 83% of the GERMLINE samples. There is an excess of DDR mutations per sample in LESION (5.5 mutations) vs GERMLINE (3.89 mutations) [p = 4.66x10-6, Mann Whitney test]. In parallel, we identified 92 genes as protein responders to ATRN-119 treatment. Mutations in 21 of these 92 genes show nominal association with surgical endometriosis (p < 0.05). However, of these responsive genes, 18 are known TIER 1 cancer-driver genes and well-characterized mutations were found in three dominant genes in the LESION tissue (ATM, DDB1, and ARID1A). Overall 20% of the patients who’s LESION we examined subsequently developed an endometriosis-associated cancer. Both in vitro and in vivo studies confirmed synthetic-lethal interactions between ATRN-119 treatment and alteration of these genes. Conclusions: The overlap between DDR genes responding to ATRN-119 and those mutated in endometriosis-associated cancer suggest that genetic markers underlying response and resistance will be critical to extend the use of these drugs while increasing efficacy and minimizing toxicities. Furthermore, our data support the inclusion of endometriosis-associated cancer patients in planned ATRN-119 clinical trials.

scholarly journals A Genome-wide RNAi Screen Identifies Multiple Synthetic Lethal Interactions with the Ras Oncogene

Cell ◽  
2009 ◽  
Vol 137 (5) ◽  
pp. 835-848 ◽  
Author(s):  
Ji Luo ◽  
Michael J. Emanuele ◽  
Danan Li ◽  
Chad J. Creighton ◽  
Michael R. Schlabach ◽  
...  
Keyword(s):  
Rnai Screen ◽  
Ras Oncogene ◽  
Synthetic Lethal ◽  
Genome Wide ◽  
A Genome ◽  
Synthetic Lethal Interactions

scholarly journals Genome-wide RNAi screen for synthetic lethal interactions with the C. elegans kinesin-5 homolog BMK-1

2015 ◽  
Vol 2 (1) ◽  
Author(s):  
André F. Maia ◽  
Marvin E. Tanenbaum ◽  
Matilde Galli ◽  
Daphne Lelieveld ◽  
David A. Egan ◽  
...  
Keyword(s):  
Rnai Screen ◽  
Synthetic Lethal ◽  
C Elegans ◽  
Genome Wide ◽  
Synthetic Lethal Interactions

scholarly journals Characterization of the Yeast Amphiphysins Rvs161p and Rvs167p Reveals Roles for the Rvs Heterodimer In Vivo

2006 ◽  
Vol 17 (3) ◽  
pp. 1306-1321 ◽  
Author(s):  
Helena Friesen ◽  
Christine Humphries ◽  
Yuen Ho ◽  
Oliver Schub ◽  
Karen Colwill ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Vegetative Growth ◽  
Vesicle Trafficking ◽  
Sh3 Domain ◽  
Cell Integrity ◽  
Synthetic Lethal ◽  
Synthetic Lethal Interactions ◽  
Src Homology

We have used comprehensive synthetic lethal screens and biochemical assays to examine the biological role of the yeast amphiphysin homologues Rvs161p and Rvs167p, two proteins that play a role in regulation of the actin cytoskeleton, endocytosis, and sporulation. We found that unlike some forms of amphiphysin, Rvs161p-Rvs167p acts as an obligate heterodimer during vegetative growth and neither Rvs161p nor Rvs167p forms a homodimer in vivo. RVS161 and RVS167 have an identical set of 49 synthetic lethal interactions, revealing functions for the Rvs proteins in cell polarity, cell wall synthesis, and vesicle trafficking as well as a shared role in mating. Consistent with these roles, we show that the Rvs167p-Rvs161p heterodimer, like its amphiphysin homologues, can bind to phospholipid membranes in vitro, suggesting a role in vesicle formation and/or fusion. Our genetic screens also reveal that the interaction between Abp1p and the Rvs167p Src homology 3 (SH3) domain may be important under certain conditions, providing the first genetic evidence for a role for the SH3 domain of Rvs167p. Our studies implicate heterodimerization of amphiphysin family proteins in various functions related to cell polarity, cell integrity, and vesicle trafficking during vegetative growth and the mating response.

Abstract C221: A genome-wide RNA interference screen identifies synthetic lethal interactions with theBRAFoncogene.

2013 ◽  
Author(s):  
Chengyin Min ◽  
David J. Konieczkowski ◽  
Christine Kwon ◽  
Marika Linja ◽  
Krishna Vasudevan ◽  
...  
Keyword(s):  
Rna Interference ◽  
Synthetic Lethal ◽  
Genome Wide ◽  
A Genome ◽  
Synthetic Lethal Interactions

scholarly journals Genetic screens in isogenic mammalian cell lines without single cell cloning

2019 ◽  
Author(s):  
Peter C DeWeirdt ◽  
Kendall R Sanson ◽  
Ruth E Hanna ◽  
Mudra Hegde ◽  
Annabel K Sangree ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Genetic Interaction ◽  
Cell Types ◽  
Repair Gene ◽  
Synthetic Lethal ◽  
Mammalian Cell Lines ◽  
Genome Wide ◽  
Single Cell Cloning ◽  
Synthetic Lethal Interactions

Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs for mammalian cells, however, is labor-intensive, time-consuming, and impossible in some cell types. Here we present an approach to create isogenic pairs of cells and screen them with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We queried the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, via 25 genome-wide screens across 4 cell lines. For all three genes, we identify a rich set of both expected and novel buffering and synthetic lethal interactions. Further, we compare the interactions observed in genetic space to those found when targeting these genes with small molecules and identify hits that may inform the clinical uses for these inhibitors. We anticipate that this methodology will be broadly useful to comprehensively study genes of interest across many cell types.

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