Publications by authors named "Jason Moffat"

134 Publications

Analysis of combinatorial CRISPR screens with the Orthrus scoring pipeline.

Nat Protoc 2021 Sep 10. Epub 2021 Sep 10.

Bioinformatics and Computational Biology Graduate Program, University of Minnesota-Twin Cities, Minneapolis, MN, USA.

The continued improvement of combinatorial CRISPR screening platforms necessitates the development of new computational pipelines for scoring combinatorial screening data. Unlike for single-guide RNA (sgRNA) pooled screening platforms, combinatorial scoring for multiplexed systems is confounded by guide design parameters such as the number of gRNAs per construct, the position of gRNAs along constructs, and additional features that may impact gRNA expression, processing or capture. In this protocol we describe Orthrus, an R package for processing, scoring and analyzing combinatorial CRISPR screening data that addresses these challenges. This protocol walks through the application of Orthrus to previously published combinatorial screening data from the CHyMErA experimental system, a platform we recently developed that pairs Cas9 with Cas12a gRNAs and enables programmed targeting of multiple genomic sites. We demonstrate Orthrus' features for screen quality assessment and two distinct scoring modes for dual guide RNAs (dgRNAs) that target the same gene twice or dgRNAs that target two different genes. Running Orthrus requires basic R programming experience, ~5-10 min of computational time and 15-60 min total.
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http://dx.doi.org/10.1038/s41596-021-00596-0DOI Listing
September 2021

Paralogous synthetic lethality underlies genetic dependencies of the cancer-mutated gene .

Life Sci Alliance 2021 11 30;4(11). Epub 2021 Aug 30.

Michael Smith Laboratories, University of British Columbia, Vancouver, Canada

, a component of the mitotically essential cohesin complex, is highly mutated in several different tumour types, including glioblastoma and bladder cancer. Whereas cohesin has roles in many cancer-related pathways, such as chromosome instability, DNA repair and gene expression, the complex nature of cohesin function has made it difficult to determine how loss might either promote tumorigenesis or be leveraged therapeutically across divergent cancer types. Here, we have performed whole-genome CRISPR-Cas9 screens for -dependent genetic interactions in three distinct cellular backgrounds. Surprisingly, , the paralog of , was the only negative genetic interaction that was shared across all three backgrounds. We also uncovered a paralogous synthetic lethal mechanism behind a genetic interaction between and the iron regulatory gene Finally, investigation of an unusually strong context-dependent genetic interaction in HAP1 cells revealed factors that could be important for alleviating cohesin loading stress. Together, our results reveal new facets of STAG2 and cohesin function across a variety of genetic contexts.
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http://dx.doi.org/10.26508/lsa.202101083DOI Listing
November 2021

LRRC8A-containing chloride channel is crucial for cell volume recovery and survival under hypertonic conditions.

Proc Natl Acad Sci U S A 2021 06;118(23)

Department of Experimental and Health Sciences, Universitat Pompeu Fabra, 08003 Barcelona, Spain.

Regulation of cell volume is essential for tissue homeostasis and cell viability. In response to hypertonic stress, cells need rapid electrolyte influx to compensate water loss and to prevent cell death in a process known as regulatory volume increase (RVI). However, the molecular component able to trigger such a process was unknown to date. Using a genome-wide CRISPR/Cas9 screen, we identified , which encodes a chloride channel subunit, as the gene most associated with cell survival under hypertonic conditions. Hypertonicity activates the p38 stress-activated protein kinase pathway and its downstream MSK1 kinase, which phosphorylates and activates LRRC8A. LRRC8A-mediated Cl efflux facilitates activation of the with-no-lysine (WNK) kinase pathway, which in turn, promotes electrolyte influx via Na/K/2Cl cotransporter (NKCC) and RVI under hypertonic stress. LRRC8A-S217A mutation impairs channel activation by MSK1, resulting in reduced RVI and cell survival. In summary, LRRC8A is key to bidirectional osmotic stress responses and cell survival under hypertonic conditions.
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http://dx.doi.org/10.1073/pnas.2025013118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8201826PMC
June 2021

A method for benchmarking genetic screens reveals a predominant mitochondrial bias.

Mol Syst Biol 2021 05;17(5):e10013

Department of Computer Science and Engineering, University of Minnesota - Twin Cities, Minneapolis, MN, USA.

We present FLEX (Functional evaluation of experimental perturbations), a pipeline that leverages several functional annotation resources to establish reference standards for benchmarking human genome-wide CRISPR screen data and methods for analyzing them. FLEX provides a quantitative measurement of the functional information captured by a given gene-pair dataset and a means to explore the diversity of functions captured by the input dataset. We apply FLEX to analyze data from the diverse cell line screens generated by the DepMap project. We identify a predominant mitochondria-associated signal within co-essentiality networks derived from these data and explore the basis of this signal. Our analysis and time-resolved CRISPR screens in a single cell line suggest that the variable phenotypes associated with mitochondria genes across cells may reflect screen dynamics and protein stability effects rather than genetic dependencies. We characterize this functional bias and demonstrate its relevance for interpreting differential hits in any CRISPR screening context. More generally, we demonstrate the utility of the FLEX pipeline for performing robust comparative evaluations of CRISPR screens or methods for processing them.
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http://dx.doi.org/10.15252/msb.202010013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138267PMC
May 2021

NOX4 links metabolic regulation in pancreatic cancer to endoplasmic reticulum redox vulnerability and dependence on PRDX4.

Sci Adv 2021 May 7;7(19). Epub 2021 May 7.

Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

There is an urgent need to identify vulnerabilities in pancreatic ductal adenocarcinoma (PDAC). PDAC cells acquire metabolic changes that augment NADPH production and cytosolic redox homeostasis. Here, we show that high NADPH levels drive activity of NADPH oxidase 4 (NOX4) expressed in the endoplasmic reticulum (ER) membrane. NOX4 produces HO metabolized by peroxiredoxin 4 (PRDX4) in the ER lumen. Using functional genomics and subsequent in vitro and in vivo validations, we find that PDAC cell lines with high NADPH levels are dependent on PRDX4 for their growth and survival. PRDX4 addiction is associated with increased reactive oxygen species, a DNA-PKcs-governed DNA damage response and radiosensitivity, which can be rescued by depletion of NOX4 or NADPH. Hence, this study has identified NOX4 as a protein that paradoxically converts the reducing power of the cytosol to an ER-specific oxidative stress vulnerability in PDAC that may be therapeutically exploited by targeting PRDX4.
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http://dx.doi.org/10.1126/sciadv.abf7114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104867PMC
May 2021

Colorectal Cancer Cells Enter a Diapause-like DTP State to Survive Chemotherapy.

Cell 2021 01;184(1):226-242.e21

Princess Margaret Cancer Center, University Health Network, Toronto, ON M5G 1L7, Canada; Ontario Institute for Cancer Research, Toronto, ON M5G 0A3, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S 1A8, Canada; Department of Surgery, University Health Network, Toronto, ON M5G 1L7, Canada. Electronic address:

Cancer cells enter a reversible drug-tolerant persister (DTP) state to evade death from chemotherapy and targeted agents. It is increasingly appreciated that DTPs are important drivers of therapy failure and tumor relapse. We combined cellular barcoding and mathematical modeling in patient-derived colorectal cancer models to identify and characterize DTPs in response to chemotherapy. Barcode analysis revealed no loss of clonal complexity of tumors that entered the DTP state and recurred following treatment cessation. Our data fit a mathematical model where all cancer cells, and not a small subpopulation, possess an equipotent capacity to become DTPs. Mechanistically, we determined that DTPs display remarkable transcriptional and functional similarities to diapause, a reversible state of suspended embryonic development triggered by unfavorable environmental conditions. Our study provides insight into how cancer cells use a developmentally conserved mechanism to drive the DTP state, pointing to novel therapeutic opportunities to target DTPs.
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http://dx.doi.org/10.1016/j.cell.2020.11.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8437243PMC
January 2021

Digital microfluidic isolation of single cells for -Omics.

Nat Commun 2020 11 11;11(1):5632. Epub 2020 Nov 11.

Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON, M5S 3H6, Canada.

We introduce Digital microfluidic Isolation of Single Cells for -Omics (DISCO), a platform that allows users to select particular cells of interest from a limited initial sample size and connects single-cell sequencing data to their immunofluorescence-based phenotypes. Specifically, DISCO combines digital microfluidics, laser cell lysis, and artificial intelligence-driven image processing to collect the contents of single cells from heterogeneous populations, followed by analysis of single-cell genomes and transcriptomes by next-generation sequencing, and proteomes by nanoflow liquid chromatography and tandem mass spectrometry. The results described herein confirm the utility of DISCO for sequencing at levels that are equivalent to or enhanced relative to the state of the art, capable of identifying features at the level of single nucleotide variations. The unique levels of selectivity, context, and accountability of DISCO suggest potential utility for deep analysis of any rare cell population with contextual dependencies.
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http://dx.doi.org/10.1038/s41467-020-19394-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7658233PMC
November 2020

Functional genomic landscape of cancer-intrinsic evasion of killing by T cells.

Nature 2020 10 23;586(7827):120-126. Epub 2020 Sep 23.

Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.

The genetic circuits that allow cancer cells to evade destruction by the host immune system remain poorly understood. Here, to identify a phenotypically robust core set of genes and pathways that enable cancer cells to evade killing mediated by cytotoxic T lymphocytes (CTLs), we performed genome-wide CRISPR screens across a panel of genetically diverse mouse cancer cell lines that were cultured in the presence of CTLs. We identify a core set of 182 genes across these mouse cancer models, the individual perturbation of which increases either the sensitivity or the resistance of cancer cells to CTL-mediated toxicity. Systematic exploration of our dataset using genetic co-similarity reveals the hierarchical and coordinated manner in which genes and pathways act in cancer cells to orchestrate their evasion of CTLs, and shows that discrete functional modules that control the interferon response and tumour necrosis factor (TNF)-induced cytotoxicity are dominant sub-phenotypes. Our data establish a central role for genes that were previously identified as negative regulators of the type-II interferon response (for example, Ptpn2, Socs1 and Adar1) in mediating CTL evasion, and show that the lipid-droplet-related gene Fitm2 is required for maintaining cell fitness after exposure to interferon-γ (IFNγ). In addition, we identify the autophagy pathway as a conserved mediator of the evasion of CTLs by cancer cells, and show that this pathway is required to resist cytotoxicity induced by the cytokines IFNγ and TNF. Through the mapping of cytokine- and CTL-based genetic interactions, together with in vivo CRISPR screens, we show how the pleiotropic effects of autophagy control cancer-cell-intrinsic evasion of killing by CTLs and we highlight the importance of these effects within the tumour microenvironment. Collectively, these data expand our knowledge of the genetic circuits that are involved in the evasion of the immune system by cancer cells, and highlight genetic interactions that contribute to phenotypes associated with escape from killing by CTLs.
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http://dx.doi.org/10.1038/s41586-020-2746-2DOI Listing
October 2020

Systematic mapping of genetic interactions for de novo fatty acid synthesis identifies C12orf49 as a regulator of lipid metabolism.

Nat Metab 2020 06 1;2(6):499-513. Epub 2020 Jun 1.

Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.

The de novo synthesis of fatty acids has emerged as a therapeutic target for various diseases, including cancer. Because cancer cells are intrinsically buffered to combat metabolic stress, it is important to understand how cells may adapt to the loss of de novo fatty acid biosynthesis. Here, we use pooled genome-wide CRISPR screens to systematically map genetic interactions (GIs) in human HAP1 cells carrying a loss-of-function mutation in fatty acid synthase (FASN), whose product catalyses the formation of long-chain fatty acids. FASN-mutant cells show a strong dependence on lipid uptake that is reflected in negative GIs with genes involved in the LDL receptor pathway, vesicle trafficking and protein glycosylation. Further support for these functional relationships is derived from additional GI screens in query cell lines deficient in other genes involved in lipid metabolism, including LDLR, SREBF1, SREBF2 and ACACA. Our GI profiles also identify a potential role for the previously uncharacterized gene C12orf49 (which we call LUR1) in regulation of exogenous lipid uptake through modulation of SREBF2 signalling in response to lipid starvation. Overall, our data highlight the genetic determinants underlying the cellular adaptation associated with loss of de novo fatty acid synthesis and demonstrate the power of systematic GI mapping for uncovering metabolic buffering mechanisms in human cells.
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http://dx.doi.org/10.1038/s42255-020-0211-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566881PMC
June 2020

Functional genomics identifies new synergistic therapies for retinoblastoma.

Oncogene 2020 07 22;39(31):5338-5357. Epub 2020 Jun 22.

Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Sinai Health, Toronto, ON, Canada.

Local intravitreal or intra-arterial chemotherapy has improved therapeutic success for the pediatric cancer retinoblastoma (RB), but toxicity remains a major caveat. RB initiates primarily with RB1 loss or, rarely, MYCN amplification, but the critical downstream networks are incompletely understood. We set out to uncover perturbed molecular hubs, identify synergistic drug combinations to target these vulnerabilities, and expose and overcome drug resistance. We applied dynamic transcriptomic analysis to identify network hubs perturbed in RB versus normal fetal retina, and performed in vivo RNAi screens in RB1 and RB1;MYCN orthotopic xenografts to pinpoint essential hubs. We employed in vitro and in vivo studies to validate hits, define mechanism, develop new therapeutic modalities, and understand drug resistance. We identified BRCA1 and RAD51 as essential for RB cell survival. Their oncogenic activity was independent of BRCA1 functions in centrosome, heterochromatin, or ROS regulation, and instead linked to DNA repair. RAD51 depletion or inhibition with the small molecule inhibitor, B02, killed RB cells in a Chk1/Chk2/p53-dependent manner. B02 further synergized with clinically relevant topotecan (TPT) to engage this pathway, activating p53-BAX mediated killing of RB but not human retinal progenitor cells. Paradoxically, a B02/TPT-resistant tumor exhibited more DNA damage than sensitive RB cells. Resistance reflected dominance of the p53-p21 axis, which mediated cell cycle arrest instead of death. Deleting p21 or applying the BCL2/BCL2L1 inhibitor Navitoclax re-engaged the p53-BAX axis, and synergized with B02, TPT or both to override resistance. These data expose new synergistic therapies to trigger p53-induced killing in diverse RB subtypes.
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http://dx.doi.org/10.1038/s41388-020-1372-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7391301PMC
July 2020

The Rational Development of CD133-Targeting Immunotherapies for Glioblastoma.

Cell Stem Cell 2020 06 27;26(6):832-844.e6. Epub 2020 May 27.

McMaster Stem Cell and Cancer Research Institute, McMaster University, Hamilton, ON, L8S 4K1, Canada; Departments of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada; Surgery, Faculty of Health Sciences, McMaster University, 1200 Main Street West, Hamilton, ON L8N 3Z5, Canada. Electronic address:

CD133 marks self-renewing cancer stem cells (CSCs) in a variety of solid tumors, and CD133+ tumor-initiating cells are known markers of chemo- and radio-resistance in multiple aggressive cancers, including glioblastoma (GBM), that may drive intra-tumoral heterogeneity. Here, we report three immunotherapeutic modalities based on a human anti-CD133 antibody fragment that targets a unique epitope present in glycosylated and non-glycosylated CD133 and studied their effects on targeting CD133+ cells in patient-derived models of GBM. We generated an immunoglobulin G (IgG) (RW03-IgG), a dual-antigen T cell engager (DATE), and a CD133-specific chimeric antigen receptor T cell (CAR-T): CART133. All three showed activity against patient-derived CD133+ GBM cells, and CART133 cells demonstrated superior efficacy in patient-derived GBM xenograft models without causing adverse effects on normal CD133+ hematopoietic stem cells in humanized CD34+ mice. Thus, CART133 cells may be a therapeutically tractable strategy to target CD133+ CSCs in human GBM or other treatment-resistant primary cancers.
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http://dx.doi.org/10.1016/j.stem.2020.04.008DOI Listing
June 2020

Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform.

Nat Biotechnol 2020 05 16;38(5):638-648. Epub 2020 Mar 16.

Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.

Systematic mapping of genetic interactions (GIs) and interrogation of the functions of sizable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR (clustered regularly interspaced short palindromic repeats)-based screening system for combinatorial genetic manipulation that employs coexpression of CRISPR-associated nucleases 9 and 12a (Cas9 and Cas12a) and machine-learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive GIs and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemogenetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for GI mapping and the functional analysis of sizable genomic regions, such as alternative exons.
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http://dx.doi.org/10.1038/s41587-020-0437-zDOI Listing
May 2020

Direct interaction between CEP85 and STIL mediates PLK4-driven directed cell migration.

J Cell Sci 2020 04 23;133(8). Epub 2020 Apr 23.

Lunenfeld-Tanenbaum Research Institute, University of Toronto, 600 University Avenue, Toronto M5G 1X5, Canada

PLK4 has emerged as a prime target for cancer therapeutics, and its overexpression is frequently observed in various types of human cancer. Recent studies have further revealed an unexpected oncogenic activity of PLK4 in regulating cancer cell migration and invasion. However, the molecular basis behind the role of PLK4 in these processes still remains only partly understood. Our previous work has demonstrated that an intact CEP85-STIL binding interface is necessary for robust PLK4 activation and centriole duplication. Here, we show that CEP85 and STIL are also required for directional cancer cell migration. Mutational and functional analyses reveal that the interactions between CEP85, STIL and PLK4 are essential for effective directional cell motility. Mechanistically, we show that PLK4 can drive the recruitment of CEP85 and STIL to the leading edge of cells to promote protrusive activity, and that downregulation of CEP85 and STIL leads to a reduction in ARP2 (also known as ACTR2) phosphorylation and reorganization of the actin cytoskeleton, which in turn impairs cell migration. Collectively, our studies provide molecular insight into the important role of the CEP85-STIL complex in modulating PLK4-driven cancer cell migration.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.238352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7183410PMC
April 2020

IPO11 mediates βcatenin nuclear import in a subset of colorectal cancers.

J Cell Biol 2020 02;219(2)

Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.

Activation of Wnt signaling entails βcatenin protein stabilization and translocation to the nucleus to regulate context-specific transcriptional programs. The majority of colorectal cancers (CRCs) initiate following APC mutations, resulting in Wnt ligand-independent stabilization and nuclear accumulation of βcatenin. The mechanisms underlying βcatenin nucleocytoplasmic shuttling remain incompletely defined. Using a novel, positive selection, functional genomic strategy, DEADPOOL, we performed a genome-wide CRISPR screen and identified IPO11 as a required factor for βcatenin-mediated transcription in APC mutant CRC cells. IPO11 (Importin-11) is a nuclear import protein that shuttles cargo from the cytoplasm to the nucleus. IPO11-/- cells exhibit reduced nuclear βcatenin protein levels and decreased βcatenin target gene activation, suggesting IPO11 facilitates βcatenin nuclear import. IPO11 knockout decreased colony formation of CRC cell lines and decreased proliferation of patient-derived CRC organoids. Our findings uncover a novel nuclear import mechanism for βcatenin in cells with high Wnt activity.
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http://dx.doi.org/10.1083/jcb.201903017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7041691PMC
February 2020

A CD133-AKT-Wnt signaling axis drives glioblastoma brain tumor-initiating cells.

Oncogene 2020 02 6;39(7):1590-1599. Epub 2019 Nov 6.

McMaster Stem Cell and Cancer Research Institute, McMaster University, Hamilton, ON, L8S 4K1, Canada.

Mechanistic insight into signaling pathways downstream of surface receptors has been revolutionized with integrated cancer genomics. This has fostered current treatment modalities, namely immunotherapy, to capitalize on targeting key oncogenic signaling nodes downstream of a limited number of surface markers. Unfortunately, rudimentary mechanistic understanding of most other cell surface proteins has reduced the clinical utility of these markers. CD133 has reproducibly been shown to correlate with disease progression, recurrence, and poor overall survivorship in the malignant adult brain tumor, glioblastoma (GBM). Using several patient-derived CD133 and CD133 GBMs we describe intrinsic differences in determinants of stemness, which we owe to a CD133-AKT-Wnt signaling axis in which CD133 functions as a putative cell surface receptor for AKT-dependent Wnt activation. These findings may have implications for personalized oncology trials targeting PI3K/AKT or Wnt as both pathways may be activated independent of their canonical drivers, leading to treatment resistance and disease relapse.
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http://dx.doi.org/10.1038/s41388-019-1086-xDOI Listing
February 2020

Forward genetic screen in human podocytes identifies diphthamide biosynthesis genes as regulators of adhesion.

Am J Physiol Renal Physiol 2019 12 30;317(6):F1593-F1604. Epub 2019 Sep 30.

Feinberg Cardiovascular and Renal Research Institute, Northwestern University Feinberg School of Medicine, Chicago, Illinois.

Podocyte function is tightly linked to the complex organization of its cytoskeleton and adhesion to the underlying glomerular basement membrane. Adhesion of cultured podocytes to a variety of substrates is reported to correlate with podocyte health. To identify novel genes that are important for podocyte function, we designed an in vitro genetic screen based on podocyte adhesion to plates coated with either fibronectin or soluble Fms-like tyrosine kinase-1 (sFLT1)/Fc. A genome-scale pooled RNA interference screen on immortalized human podocytes identified 77 genes that increased adhesion to fibronectin, 101 genes that increased adhesion to sFLT1/Fc, and 44 genes that increased adhesion to both substrates when knocked down. Multiple shRNAs against diphthamide biosynthesis protein 1-4 (DPH1-DPH4) were top hits for increased adhesion. Immortalized human podocyte cells stably expressing these hairpins displayed increased adhesion to both substrates. We then used CRISPR-Cas9 to generate podocyte knockout cells for , , or , which also displayed increased adhesion to both fibronectin and sFLT1/Fc, as well as a spreading defect. Finally, we showed that nephrocyte-specific knockdown of Dph1, Dph2, and Dph4 resulted in altered nephrocyte function. In summary, we report here a novel high-throughput method to identify genes important for podocyte function. Given the central role of podocyte adhesion as a marker of podocyte health, these data are a rich source of candidate regulators of glomerular disease.
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http://dx.doi.org/10.1152/ajprenal.00195.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962514PMC
December 2019

High-throughput genome-wide phenotypic screening via immunomagnetic cell sorting.

Nat Biomed Eng 2019 10 23;3(10):796-805. Epub 2019 Sep 23.

Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada.

Genome-scale functional genetic screens are used to identify key genetic regulators of a phenotype of interest. However, the identification of genetic modifications that lead to a phenotypic change requires sorting large numbers of cells, which increases operational times and costs and limits cell viability. Here, we introduce immunomagnetic cell sorting facilitated by a microfluidic chip as a rapid and scalable high-throughput method for loss-of-function phenotypic screening using CRISPR-Cas9. We used the method to process an entire genome-wide screen containing more than 10 cells in less than 1 h-considerably surpassing the throughput achieved by fluorescence-activated cell sorting, the gold-standard technique for phenotypic cell sorting-while maintaining high levels of cell viability. We identified modulators of the display of CD47, which is a negative regulator of phagocytosis and an important cell-surface target for immuno-oncology drugs. The top hit of the screen, the glutaminyl cyclase QPCTL, was validated and shown to modify the N-terminal glutamine of CD47. The method presented could bridge the gap between fluorescence-activated cell sorting and less flexible yet higher-throughput systems such as magnetic-activated cell sorting.
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http://dx.doi.org/10.1038/s41551-019-0454-8DOI Listing
October 2019

Pooled CRISPR-Based Genetic Screens in Mammalian Cells.

J Vis Exp 2019 09 4(151). Epub 2019 Sep 4.

Donnelly Centre, University of Toronto; Department of Molecular Genetics, University of Toronto; Institute for Biomaterials and Biomedical Engineering, University of Toronto;

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.
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http://dx.doi.org/10.3791/59780DOI Listing
September 2019

CRISPR screens are feasible in TP53 wild-type cells.

Mol Syst Biol 2019 08;15(8):e8679

Donnelly Centre, University of Toronto, Toronto, ON, Canada.

A recent study by Haapaniemi et al (2018) reported that intact p53 signaling hampers CRISPR-based functional genomic screens. Brown et al report good performance of genome-scale screens in TP53 wild-type cells and reiterate best practices for CRISPR screening.
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http://dx.doi.org/10.15252/msb.20188679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6686785PMC
August 2019

Tailored tetravalent antibodies potently and specifically activate Wnt/Frizzled pathways in cells, organoids and mice.

Elife 2019 08 27;8. Epub 2019 Aug 27.

Donnelly Centre, University of Toronto, Toronto, Canada.

Secreted Wnt proteins regulate development and adult tissue homeostasis by binding and activating cell-surface Frizzled receptors and co-receptors including LRP5/6. The hydrophobicity of Wnt proteins has complicated their purification and limited their use in basic research and as therapeutics. We describe modular tetravalent antibodies that can recruit Frizzled and LRP5/6 in a manner that phenocopies the activities of Wnts both in vitro and in vivo. The modular nature of these synthetic Frizzled and LRP5/6 Agonists, called FLAgs, enables tailored engineering of specificity for one, two or multiple members of the Frizzled family. We show that FLAgs underlie differentiation of pluripotent stem cells, sustain organoid growth, and activate stem cells in vivo. Activation of Wnt signaling circuits with tailored FLAgs will enable precise delineation of functional outcomes directed by distinct receptor combinations and could provide a new class of therapeutics to unlock the promise of regenerative medicine.
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http://dx.doi.org/10.7554/eLife.46134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6711705PMC
August 2019

Identifying chemogenetic interactions from CRISPR screens with drugZ.

Genome Med 2019 08 22;11(1):52. Epub 2019 Aug 22.

Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Background: Chemogenetic profiling enables the identification of gene mutations that enhance or suppress the activity of chemical compounds. This knowledge provides insights into drug mechanism of action, genetic vulnerabilities, and resistance mechanisms, all of which may help stratify patient populations and improve drug efficacy. CRISPR-based screening enables sensitive detection of drug-gene interactions directly in human cells, but until recently has primarily been used to screen only for resistance mechanisms.

Results: We present drugZ, an algorithm for identifying both synergistic and suppressor chemogenetic interactions from CRISPR screens. DrugZ identifies synthetic lethal interactions between PARP inhibitors and both known and novel members of the DNA damage repair pathway, confirms KEAP1 loss as a resistance factor for ERK inhibitors in oncogenic KRAS backgrounds, and defines the genetic context for temozolomide activity.

Conclusions: DrugZ is an open-source Python software for the analysis of genome-scale drug modifier screens. The software accurately identifies genetic perturbations that enhance or suppress drug activity. Interestingly, analysis of new and previously published data reveals tumor suppressor genes are drug-agnostic resistance genes in drug modifier screens. The software is available at github.com/hart-lab/drugz .
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http://dx.doi.org/10.1186/s13073-019-0665-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706933PMC
August 2019

Bmi1 regulates human glioblastoma stem cells through activation of differential gene networks in CD133+ brain tumor initiating cells.

J Neurooncol 2019 Jul 21;143(3):417-428. Epub 2019 May 21.

Stem Cell and Cancer Research Institute, McMaster University, 1280 Main Street West, Hamilton, ON, L8S 4K1, Canada.

Purpose: Glioblastoma (GBM) is the most aggressive adult brain cancer, with a 15 month median survivorship attributed to the existence of treatment-refractory brain tumor initiating cells (BTICs). In order to better understand the mechanisms regulating the tumorigenic properties of this population, we studied the role of the polycomb group member BMI1 in our patient-derived GBM BTICs and its relationship with CD133, a well-established marker of BTICs.

Methods: Using gain and loss-of-function studies for Bmi1 in neural stem cells (NSCs) and patient-derived GBM BTICs respectively, we assessed in vitro self-renewal and in vivo tumor formation in these two cell populations. We further explored the BMI1 transcriptional regulatory network through RNA sequencing of different GBM BTIC populations that were knocked down for Bmi1.

Results: There is a differential role of BMI1 in CD133-positive cells, notably involving cell metabolism. In addition, we identified pivotal targets downstream of BMI1 in CD133+ cells such as integrin alpha 2 (ITGA2), that may contribute to regulating GBM stem cell properties.

Conclusions: Our work sheds light on the association of three genes with CD133-BMI1 circuitry, their importance as downstream effectors of the BMI1 signalling pathway, and their potential as future targets for tackling GBM treatment-resistant cell populations.
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http://dx.doi.org/10.1007/s11060-019-03192-1DOI Listing
July 2019

Genetic interaction networks in cancer cells.

Curr Opin Genet Dev 2019 02 8;54:64-72. Epub 2019 Apr 8.

Donnelly Centre, University of Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, ON, Canada. Electronic address:

The genotype-to-phenotype relationship in health and disease is complex and influenced by both an individual's environment and their unique genome. Personal genetic variants can modulate gene function to generate a phenotype either through a single gene effect or through genetic interactions involving two or more genes. The relevance of genetic interactions to disease phenotypes has been particularly clear in cancer research, where an extreme genetic interaction, synthetic lethality, has been exploited as a therapeutic strategy. The obvious benefits of unmasking genetic background-specific vulnerabilities, coupled with the power of systematic genome editing, have fueled efforts to translate genetic interaction mapping from model organisms to human cells. Here, we review recent developments in genetic interaction mapping, with a focus on CRISPR-based genome editing technologies and cancer.
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http://dx.doi.org/10.1016/j.gde.2019.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6820710PMC
February 2019

Essential Gene Profiles for Human Pluripotent Stem Cells Identify Uncharacterized Genes and Substrate Dependencies.

Cell Rep 2019 04;27(2):599-615.e12

Donnelly Centre, University of Toronto, Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada; Canadian Institute for Advanced Research, Toronto, ON, Canada; Institute for Biomaterials and BioMedical Engineering, University of Toronto, ON, Canada. Electronic address:

Human pluripotent stem cells (hPSCs) provide an invaluable tool for modeling diseases and hold promise for regenerative medicine. For understanding pluripotency and lineage differentiation mechanisms, a critical first step involves systematically cataloging essential genes (EGs) that are indispensable for hPSC fitness, defined as cell reproduction in this study. To map essential genetic determinants of hPSC fitness, we performed genome-scale loss-of-function screens in an inducible Cas9 H1 hPSC line cultured on feeder cells and laminin to identify EGs. Among these, we found FOXH1 and VENTX, genes that encode transcription factors previously implicated in stem cell biology, as well as an uncharacterized gene, C22orf43/DRICH1. hPSC EGs are substantially different from other human model cell lines, and EGs in hPSCs are highly context dependent with respect to different growth substrates. Our CRISPR screens establish parameters for genome-wide screens in hPSCs, which will facilitate the characterization of unappreciated genetic regulators of hPSC biology.
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http://dx.doi.org/10.1016/j.celrep.2019.02.041DOI Listing
April 2019

Global Genetic Networks and the Genotype-to-Phenotype Relationship.

Cell 2019 03;177(1):85-100

The Donnelly Centre, University of Toronto, 160 College Street, Toronto ON, Canada; Department of Molecular Genetics, University of Toronto, 1 Kings College Circle, Toronto ON, Canada. Electronic address:

Genetic interactions identify combinations of genetic variants that impinge on phenotype. With whole-genome sequence information available for thousands of individuals within a species, a major outstanding issue concerns the interpretation of allelic combinations of genes underlying inherited traits. In this Review, we discuss how large-scale analyses in model systems have illuminated the general principles and phenotypic impact of genetic interactions. We focus on studies in budding yeast, including the mapping of a global genetic network. We emphasize how information gained from work in yeast translates to other systems, and how a global genetic network not only annotates gene function but also provides new insights into the genotype-to-phenotype relationship.
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http://dx.doi.org/10.1016/j.cell.2019.01.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817365PMC
March 2019

Structure-guided design fine-tunes pharmacokinetics, tolerability, and antitumor profile of multispecific frizzled antibodies.

Proc Natl Acad Sci U S A 2019 04 20;116(14):6812-6817. Epub 2019 Mar 20.

Program in Molecular Medicine, Hospital for Sick Children Research Institute, Toronto, ON M5G 0A4, Canada;

Aberrant activation of Wnt/β-catenin signaling occurs frequently in cancer. However, therapeutic targeting of this pathway is complicated by the role of Wnt in stem cell maintenance and tissue homeostasis. Here, we evaluated antibodies blocking 6 of the 10 human Wnt/Frizzled (FZD) receptors as potential therapeutics. Crystal structures revealed a common binding site for these monoclonal antibodies (mAbs) on FZD, blocking the interaction with the Wnt palmitoleic acid moiety. However, these mAbs displayed gastrointestinal toxicity or poor plasma exposure in vivo. Structure-guided engineering was used to refine the binding of each mAb for FZD receptors, resulting in antibody variants with improved in vivo tolerability and developability. Importantly, the lead variant mAb significantly inhibited tumor growth in the HPAF-II pancreatic tumor xenograft model. Taken together, our data demonstrate that anti-FZD cancer therapeutic antibodies with broad specificity can be fine-tuned to navigate in vivo exposure and tolerability while driving therapeutic efficacy.
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http://dx.doi.org/10.1073/pnas.1817246116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452705PMC
April 2019

Yeast Two-Hybrid Analysis for Ubiquitin Variant Inhibitors of Human Deubiquitinases.

J Mol Biol 2019 03 11;431(6):1160-1171. Epub 2019 Feb 11.

Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 3E1, Canada; Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, Toronto, ON, M5S3E1, Canada; Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

We applied a yeast-two-hybrid (Y2H) analysis to screen for ubiquitin variant (UbV) inhibitors of a human deubiquitinase (DUB), ubiquitin-specific protease 2 (USP2). The Y2H screen used USP2 as the bait and a prey library consisting of UbVs randomized at four specific positions, which were known to interact with USP2 from phage display analysis. The screen yielded numerous UbVs that bound to USP2 both as a Y2H interaction in vivo and as purified proteins in vitro. The Y2H-derived UbVs inhibited the catalytic activity of USP2 in vitro with nanomolar-range potencies, and they bound and inhibited USP2 in human cells. Mutational and structural analysis showed that potent and selective inhibition could be achieved by just two substitutions in a UbV, which exhibited improved hydrophobic and hydrophilic contacts compared to the wild-type ubiquitin interaction with USP2. Our results establish Y2H as an effective platform for the development of UbV inhibitors of DUBs in vivo, providing an alternative strategy for the analysis of DUBs that are recalcitrant to phage display and other in vitro methods.
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http://dx.doi.org/10.1016/j.jmb.2019.02.007DOI Listing
March 2019

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15.

Structure 2019 04 31;27(4):590-605.e5. Epub 2019 Jan 31.

The Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor β pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation.
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http://dx.doi.org/10.1016/j.str.2019.01.002DOI Listing
April 2019

A rapid in vitro methodology for simultaneous target discovery and antibody generation against functional cell subpopulations.

Sci Rep 2019 01 29;9(1):842. Epub 2019 Jan 29.

Donnelly Centre, University of Toronto, Toronto, M5S 3E1, Canada.

Cell surface antigen discovery is of great interest for biomedical research both for isolation of rare cell populations and therapeutic targeting. We developed a rapid, cost-effective, fully in vitro technology which facilities the simultaneous target discovery and human antibody generation on the surface of virtually any cell population of interest. We apply our technique to human colorectal cancer-initiating cells (CICs) and identify hundreds of unique human antibodies. We characterized the top three antibody candidates targeting these CICs and identify their protein targets as integrin α7 (ITGA7), HLA-A1 and integrin β6 (ITGB6). We demonstrate that these antibodies can be used to isolate self-renewing colorectal CICs, and that the integrin α7 antibody can prospectively identify glioblastoma brain tumor initiating cells as well as human muscle stem cells. We also demonstrate that genetic ablation of integrin β6 impedes colorectal CIC function. The methodology can be readily applied to other cell populations including stem cells, cancer, or immune cells to facilitate the rapid identification of novel targets and simultaneous generation of potent and specific antibodies with therapeutic potential.
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http://dx.doi.org/10.1038/s41598-018-37462-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6351593PMC
January 2019

Functional genomic characterization of a synthetic anti-HER3 antibody reveals a role for ubiquitination by RNF41 in the anti-proliferative response.

J Biol Chem 2019 01 6;294(4):1396-1409. Epub 2018 Dec 6.

Banting and Best Department of Medical Research and Department of Medical Genetics, The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada. Electronic address:

Dysregulation of the ErbB family of receptor tyrosine kinases is involved in the progression of many cancers. Antibodies targeting the dimerization domains of family members EGFR and HER2 are approved cancer therapeutics, but efficacy is restricted to a subset of tumors and resistance often develops in response to treatment. A third family member, HER3, heterodimerizes with both EGFR and HER2 and has also been implicated in cancer. Consequently, there is strong interest in developing antibodies that target HER3, but to date, no therapeutics have been approved. To aid the development of anti-HER3 antibodies as cancer therapeutics, we combined antibody engineering and functional genomics screens to identify putative mechanisms of resistance or synthetic lethality with antibody-mediated anti-proliferative effects. We developed a synthetic antibody called IgG 95, which binds to HER3 and promotes ubiquitination, internalization, and receptor down-regulation. Using an shRNA library targeting enzymes in the ubiquitin proteasome system, we screened for genes that effect response to IgG 95 and uncovered the E3 ubiquitin ligase RNF41 as a driver of IgG 95 anti-proliferative activity. RNF41 has been shown previously to regulate HER3 levels under normal conditions and we now show that it is also responsible for down-regulation of HER3 upon treatment with IgG 95. Moreover, our findings suggest that down-regulation of RNF41 itself may be a mechanism for acquired resistance to treatment with IgG 95 and perhaps other anti-HER3 antibodies. Our work deepens our understanding of HER3 signaling by uncovering the mechanistic basis for the anti-proliferative effects of potential anti-HER3 antibody therapeutics.
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http://dx.doi.org/10.1074/jbc.RA118.004420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349115PMC
January 2019
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