Publications by authors named "John G Doench"

87 Publications

Author Correction: Cytochrome P450 oxidoreductase contributes to phospholipid peroxidation in ferroptosis.

Nat Chem Biol 2021 Mar 2. Epub 2021 Mar 2.

Chemical Biology and Therapeutics Science Program, Broad Institute, Cambridge, MA, USA.

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http://dx.doi.org/10.1038/s41589-021-00767-wDOI Listing
March 2021

Massively parallel assessment of human variants with base editor screens.

Cell 2021 Feb;184(4):1064-1080.e20

Genetic Perturbation Platform, Broad Institute, Cambridge, MA 02142, USA. Electronic address:

Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors, identifying point mutations that confer drug sensitivity or resistance. We also create a library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.
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http://dx.doi.org/10.1016/j.cell.2021.01.012DOI Listing
February 2021

Targeting oncoproteins with a positive selection assay for protein degraders.

Sci Adv 2021 Feb 5;7(6). Epub 2021 Feb 5.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.

Most intracellular proteins lack hydrophobic pockets suitable for altering their function with drug-like small molecules. Recent studies indicate that some undruggable proteins can be targeted by compounds that can degrade them. For example, thalidomide-like drugs (IMiDs) degrade the critical multiple myeloma transcription factors IKZF1 and IKZF3 by recruiting them to the cereblon E3 ubiquitin ligase. Current loss of signal ("down") assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. Here, we describe a gain of signal ("up") assay for degraders. In arrayed chemical screens, we identified novel IMiD-like IKZF1 degraders and Spautin-1, which, unlike the IMiDs, degrades IKZF1 in a cereblon-independent manner. In a pooled CRISPR-Cas9-based screen, we found that CDK2 regulates the abundance of the ASCL1 oncogenic transcription factor. This methodology should facilitate the identification of drugs that directly or indirectly degrade undruggable proteins.
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http://dx.doi.org/10.1126/sciadv.abd6263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864573PMC
February 2021

In vivo screens using a selective CRISPR antigen removal lentiviral vector system reveal immune dependencies in renal cell carcinoma.

Immunity 2021 Jan 20. Epub 2021 Jan 20.

Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, MA, USA; Center for Cancer Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA. Electronic address:

CRISPR-Cas9 genome engineering has increased the pace of discovery for immunology and cancer biology, revealing potential therapeutic targets and providing insight into mechanisms underlying resistance to immunotherapy. However, endogenous immune recognition of Cas9 has limited the applicability of CRISPR technologies in vivo. Here, we characterized immune responses against Cas9 and other expressed CRISPR vector components that cause antigen-specific tumor rejection in several mouse cancer models. To avoid unwanted immune recognition, we designed a lentiviral vector system that allowed selective CRISPR antigen removal (SCAR) from tumor cells. The SCAR system reversed immune-mediated rejection of CRISPR-modified tumor cells in vivo and enabled high-throughput genetic screens in previously intractable models. A pooled in vivo screen using SCAR in a CRISPR-antigen-sensitive renal cell carcinoma revealed resistance pathways associated with autophagy and major histocompatibility complex class I (MHC class I) expression. Thus, SCAR presents a resource that enables CRISPR-based studies of tumor-immune interactions and prevents unwanted immune recognition of genetically engineered cells, with implications for clinical applications.
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http://dx.doi.org/10.1016/j.immuni.2021.01.001DOI Listing
January 2021

Tumor-suppressor function of Beclin 1 in breast cancer cells requires E-cadherin.

Proc Natl Acad Sci U S A 2021 Feb;118(5)

Center for Autophagy Research, University of Texas Southwestern Medical Center, Dallas, TX 75390.

Beclin 1, an autophagy and haploinsufficient tumor-suppressor protein, is frequently monoallelically deleted in breast and ovarian cancers. However, the precise mechanisms by which Beclin 1 inhibits tumor growth remain largely unknown. To address this question, we performed a genome-wide CRISPR/Cas9 screen in MCF7 breast cancer cells to identify genes whose loss of function reverse Beclin 1-dependent inhibition of cellular proliferation. Small guide RNAs targeting and , tumor-suppressor genes that encode cadherin/catenin complex members E-cadherin and alpha-catenin, respectively, were highly enriched in the screen. CRISPR/Cas9-mediated knockout of or reversed Beclin 1-dependent suppression of breast cancer cell proliferation and anchorage-independent growth. Moreover, deletion of or inhibited the tumor-suppressor effects of Beclin 1 in breast cancer xenografts. Enforced Beclin 1 expression in MCF7 cells and tumor xenografts increased cell surface localization of E-cadherin and decreased expression of mesenchymal markers and beta-catenin/Wnt target genes. Furthermore, CRISPR/Cas9-mediated knockout of and the autophagy class III phosphatidylinositol kinase complex 2 (PI3KC3-C2) gene, , but not PI3KC3-C1-specific or other autophagy genes , , or , resulted in decreased E-cadherin plasma membrane and increased cytoplasmic E-cadherin localization. Taken together, these data reveal previously unrecognized cooperation between Beclin 1 and E-cadherin-mediated tumor suppression in breast cancer cells.
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http://dx.doi.org/10.1073/pnas.2020478118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865132PMC
February 2021

UFMylation inhibits the proinflammatory capacity of interferon-γ-activated macrophages.

Proc Natl Acad Sci U S A 2021 Jan;118(1)

Department of Pathology and Immunology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110;

Macrophages activated with interferon-γ (IFN-γ) in combination with other proinflammatory stimuli, such as lipopolysaccharide or tumor necrosis factor-α (TNF-α), respond with transcriptional and cellular changes that enhance clearance of intracellular pathogens at the risk of damaging tissues. IFN-γ effects must therefore be carefully balanced with inhibitory mechanisms to prevent immunopathology. We performed a genome-wide CRISPR knockout screen in a macrophage cell line to identify negative regulators of IFN-γ responses. We discovered an unexpected role of the ubiquitin-fold modifier (Ufm1) conjugation system (herein UFMylation) in inhibiting responses to IFN-γ and lipopolysaccharide. Enhanced IFN-γ activation in UFMylation-deficient cells resulted in increased transcriptional responses to IFN-γ in a manner dependent on endoplasmic reticulum stress responses involving Ern1 and Xbp1. Furthermore, UFMylation in myeloid cells is required for resistance to influenza infection in mice, indicating that this pathway modulates in vivo responses to infection. These findings provide a genetic roadmap for the regulation of responses to a key mediator of cellular immunity and identify a molecular link between the UFMylation pathway and immune responses.
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http://dx.doi.org/10.1073/pnas.2011763118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817147PMC
January 2021

Intercellular Mitochondria Transfer to Macrophages Regulates White Adipose Tissue Homeostasis and Is Impaired in Obesity.

Cell Metab 2021 Feb 4;33(2):270-282.e8. Epub 2020 Dec 4.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA; Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address:

Recent studies suggest that mitochondria can be transferred between cells to support the survival of metabolically compromised cells. However, whether intercellular mitochondria transfer occurs in white adipose tissue (WAT) or regulates metabolic homeostasis in vivo remains unknown. We found that macrophages acquire mitochondria from neighboring adipocytes in vivo and that this process defines a transcriptionally distinct macrophage subpopulation. A genome-wide CRISPR-Cas9 knockout screen revealed that mitochondria uptake depends on heparan sulfates (HS). High-fat diet (HFD)-induced obese mice exhibit lower HS levels on WAT macrophages and decreased intercellular mitochondria transfer from adipocytes to macrophages. Deletion of the HS biosynthetic gene Ext1 in myeloid cells decreases mitochondria uptake by WAT macrophages, increases WAT mass, lowers energy expenditure, and exacerbates HFD-induced obesity in vivo. Collectively, this study suggests that adipocytes and macrophages employ intercellular mitochondria transfer as a mechanism of immunometabolic crosstalk that regulates metabolic homeostasis and is impaired in obesity.
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http://dx.doi.org/10.1016/j.cmet.2020.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7858234PMC
February 2021

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

Cell 2021 01 20;184(1):76-91.e13. Epub 2020 Oct 20.

Department of Laboratory Medicine, Yale School of Medicine, New Haven, CT 06520, USA; Department of Immunobiology, Yale School of Medicine, New Haven, CT 06520, USA; Yale Cancer Center, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address:

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.
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http://dx.doi.org/10.1016/j.cell.2020.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574718PMC
January 2021

CRISPR-Cas9 Genome-Wide Knockout Screen Identifies Mechanism of Selective Activity of Dehydrofalcarinol in Mesenchymal Stem-like Triple-Negative Breast Cancer Cells.

J Nat Prod 2020 10 6;83(10):3080-3092. Epub 2020 Oct 6.

Natural Products Discovery Group, Institute for Natural Products Applications and Research Technologies, Department of Chemistry & Biochemistry, Stephenson Life Science Research Center, University of Oklahoma, Norman, Oklahoma 73019, United States.

There are no targeted therapies available for triple-negative breast cancers (TNBCs) in part because they represent a heterogeneous group of tumors with diverse oncogenic drivers. Our goal is to identify targeted therapies for subtypes of these cancers using a mechanism-blind screen of natural product extract libraries. An extract from was 4-fold more potent for cytotoxicity against MDA-MB-231 cells, which represent the mesenchymal stem-like (MSL) subtype, as compared to cells of other TNBC subtypes. Bioassay-guided fractionation led to the isolation of six polyacetylenes, and subsequent investigations of plant sources known to produce polyacetylenes yielded six additional structurally related compounds. A subset of these compounds retained selective cytotoxic effects in MSL subtype cells. Studies suggest that these selective effects do not appear to be due to PPARγ agonist activities that have previously been reported for polyacetylenes. A CRISPR-Cas9-mediated gene knockout screen was employed to identify the mechanism of selective cytotoxic activity of the most potent and selective compound, dehydrofalcarinol (). This genomic screen identified , the gene encoding the enzyme 17β-hydroxysteroid dehydrogenase type 11, as a mediator of the selective cytotoxic effects of in MDA-MB-231 cells that express high levels of this protein. The Project Achilles cancer dependency database further identified a subset of Ewing sarcoma cell lines as highly dependent on expression, and it was found these were also highly sensitive to . This report demonstrates the value of CRISPR-Cas9 genome-wide screens to identify the mechanisms underlying the selective activities of natural products.
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http://dx.doi.org/10.1021/acs.jnatprod.0c00642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722265PMC
October 2020

Illuminating Host-Mycobacterial Interactions with Genome-wide CRISPR Knockout and CRISPRi Screens.

Cell Syst 2020 Sep;11(3):239-251.e7

Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore 138602, Singapore; Synthetic Biology Group, MIT Synthetic Biology Center, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA; Research Laboratory of Electronics, MIT, Cambridge, MA 02139, USA; Broad Institute, Cambridge, MA 02139, USA; Department of Electrical Engineering and Computer Science, MIT, Cambridge, MA 02139, USA; Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, MIT, Cambridge, MA 02139, USA. Electronic address:

Existing antibiotics are inadequate to defeat tuberculosis (TB), a leading cause of death worldwide. We sought potential targets for host-directed therapies (HDTs) by investigating the host immune response to mycobacterial infection. We used high-throughput CRISPR knockout and CRISPR interference (CRISPRi) screens to identify perturbations that improve the survival of human phagocytic cells infected with Mycobacterium bovis BCG (Bacillus Calmette-Guérin), as a proxy for Mycobacterium tuberculosis (Mtb). Many of these perturbations constrained the growth of intracellular mycobacteria. We identified over 100 genes associated with diverse biological pathways as potential HDT targets. We validated key components of the type I interferon and aryl hydrocarbon receptor signaling pathways that respond to the small-molecule inhibitors cerdulatinib and CH223191, respectively; these inhibitors enhanced human macrophage survival and limited the intracellular growth of Mtb. Thus, high-throughput functional genomic screens, by elucidating highly complex host-pathogen interactions, can serve to identify HDTs to potentially improve TB treatment.
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http://dx.doi.org/10.1016/j.cels.2020.08.010DOI Listing
September 2020

Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection.

bioRxiv 2020 Jun 17. Epub 2020 Jun 17.

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-β signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.
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http://dx.doi.org/10.1101/2020.06.16.155101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457610PMC
June 2020

Optimization of AsCas12a for combinatorial genetic screens in human cells.

Nat Biotechnol 2021 01 13;39(1):94-104. Epub 2020 Jul 13.

Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Cas12a RNA-guided endonucleases are promising tools for multiplexed genetic perturbations because they can process multiple guide RNAs expressed as a single transcript, and subsequently cleave target DNA. However, their widespread adoption has lagged behind Cas9-based strategies due to low activity and the lack of a well-validated pooled screening toolkit. In the present study, we describe the optimization of enhanced Cas12a from Acidaminococcus (enAsCas12a) for pooled, combinatorial genetic screens in human cells. By assaying the activity of thousands of guides, we refine on-target design rules and develop a comprehensive set of off-target rules to predict and exclude promiscuous guides. We also identify 38 direct repeat variants that can substitute for the wild-type sequence. We validate our optimized AsCas12a toolkit by screening for synthetic lethalities in OVCAR8 and A375 cancer cells, discovering an interaction between MARCH5 and WSB2. Finally, we show that enAsCas12a delivers similar performance to Cas9 in genome-wide dropout screens but at greatly reduced library size, which will facilitate screens in challenging models.
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http://dx.doi.org/10.1038/s41587-020-0600-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854777PMC
January 2021

Discovering the anti-cancer potential of non-oncology drugs by systematic viability profiling.

Nat Cancer 2020 Feb 20;1(2):235-248. Epub 2020 Jan 20.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Anti-cancer uses of non-oncology drugs have occasionally been found, but such discoveries have been serendipitous. We sought to create a public resource containing the growth inhibitory activity of 4,518 drugs tested across 578 human cancer cell lines. We used PRISM, a molecular barcoding method, to screen drugs against cell lines in pools. An unexpectedly large number of non-oncology drugs selectively inhibited subsets of cancer cell lines in a manner predictable from the cell lines' molecular features. Our findings include compounds that killed by inducing PDE3A-SLFN12 complex formation; vanadium-containing compounds whose killing depended on the sulfate transporter SLC26A2; the alcohol dependence drug disulfiram, which killed cells with low expression of metallothioneins; and the anti-inflammatory drug tepoxalin, which killed via the multi-drug resistance protein ABCB1. The PRISM drug repurposing resource (https://depmap.org/repurposing) is a starting point to develop new oncology therapeutics, and more rarely, for potential direct clinical translation.
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http://dx.doi.org/10.1038/s43018-019-0018-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7328899PMC
February 2020

Author Correction: Cas9 activates the p53 pathway and selects for p53-inactivating mutations.

Nat Genet 2020 Jul;52(7):748-749

Cancer Program, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41588-020-0663-9DOI Listing
July 2020

Defective NADPH production in mitochondrial disease complex I causes inflammation and cell death.

Nat Commun 2020 06 1;11(1):2714. Epub 2020 Jun 1.

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.

Electron transport chain (ETC) defects occurring from mitochondrial disease mutations compromise ATP synthesis and render cells vulnerable to nutrient and oxidative stress conditions. This bioenergetic failure is thought to underlie pathologies associated with mitochondrial diseases. However, the precise metabolic processes resulting from a defective mitochondrial ETC that compromise cell viability under stress conditions are not entirely understood. We design a whole genome gain-of-function CRISPR activation screen using human mitochondrial disease complex I (CI) mutant cells to identify genes whose increased function rescue glucose restriction-induced cell death. The top hit of the screen is the cytosolic Malic Enzyme (ME1), that is sufficient to enable survival and proliferation of CI mutant cells under nutrient stress conditions. Unexpectedly, this metabolic rescue is independent of increased ATP synthesis through glycolysis or oxidative phosphorylation, but dependent on ME1-produced NADPH and glutathione (GSH). Survival upon nutrient stress or pentose phosphate pathway (PPP) inhibition depends on compensatory NADPH production through the mitochondrial one-carbon metabolism that is severely compromised in CI mutant cells. Importantly, this defective CI-dependent decrease in mitochondrial NADPH production pathway or genetic ablation of SHMT2 causes strong increases in inflammatory cytokine signatures associated with redox dependent induction of ASK1 and activation of stress kinases p38 and JNK. These studies find that a major defect of CI deficiencies is decreased mitochondrial one-carbon NADPH production that is associated with increased inflammation and cell death.
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http://dx.doi.org/10.1038/s41467-020-16423-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264245PMC
June 2020

Cas9 activates the p53 pathway and selects for p53-inactivating mutations.

Nat Genet 2020 07 18;52(7):662-668. Epub 2020 May 18.

Cancer Program, Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Cas9 is commonly introduced into cell lines to enable CRISPR-Cas9-mediated genome editing. Here, we studied the genetic and transcriptional consequences of Cas9 expression itself. Gene expression profiling of 165 pairs of human cancer cell lines and their Cas9-expressing derivatives revealed upregulation of the p53 pathway upon introduction of Cas9, specifically in wild-type TP53 (TP53-WT) cell lines. This was confirmed at the messenger RNA and protein levels. Moreover, elevated levels of DNA repair were observed in Cas9-expressing cell lines. Genetic characterization of 42 cell line pairs showed that introduction of Cas9 can lead to the emergence and expansion of p53-inactivating mutations. This was confirmed by competition experiments in isogenic TP53-WT and TP53-null (TP53) cell lines. Lastly, Cas9 was less active in TP53-WT than in TP53-mutant cell lines, and Cas9-induced p53 pathway activation affected cellular sensitivity to both genetic and chemical perturbations. These findings may have broad implications for the proper use of CRISPR-Cas9-mediated genome editing.
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http://dx.doi.org/10.1038/s41588-020-0623-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343612PMC
July 2020

DNA methylation enzymes and PRC1 restrict B-cell Epstein-Barr virus oncoprotein expression.

Nat Microbiol 2020 08 18;5(8):1051-1063. Epub 2020 May 18.

Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA.

To accomplish the remarkable task of lifelong infection, the Epstein-Barr virus (EBV) switches between four viral genome latency and lytic programmes to navigate the B-cell compartment and evade immune responses. The transforming programme, consisting of highly immunogenic EBV nuclear antigen (EBNA) and latent membrane proteins (LMPs), is expressed in newly infected B lymphocytes and in post-transplant lymphomas. On memory cell differentiation and in most EBV-associated Burkitt's lymphomas, all but one viral antigen are repressed for immunoevasion. To gain insights into the epigenetic mechanisms that restrict immunogenic oncoprotein expression, a genome-scale CRISPR-Cas9 screen was performed in EBV and Burkitt's lymphoma cells. Here, we show that the ubiquitin ligase ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) and its DNA methyltransferase partner DNA methyltransferase I (DNMT1) are critical for the restriction of EBNA and LMP expression. All UHRF1 reader and writer domains were necessary for silencing and DNMT3B was identified as an upstream viral genome CpG methylation initiator. Polycomb repressive complex I exerted a further layer of control over LMP expression, suggesting a second mechanism for latency programme switching. UHRF1, DNMT1 and DNMT3B are upregulated in germinal centre B cells, the Burkitt's lymphoma cell of origin, providing a molecular link between B-cell state and the EBV latency programme. These results suggest rational therapeutic targets to manipulate EBV oncoprotein expression.
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http://dx.doi.org/10.1038/s41564-020-0724-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7462085PMC
August 2020

MYC Controls the Epstein-Barr Virus Lytic Switch.

Mol Cell 2020 05 20;78(4):653-669.e8. Epub 2020 Apr 20.

Division of Infectious Disease, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA; Department of Microbiology, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Harvard Graduate Program in Virology, Boston, MA 02115, USA. Electronic address:

Epstein-Barr virus (EBV) is associated with multiple human malignancies. To evade immune detection, EBV switches between latent and lytic programs. How viral latency is maintained in tumors or in memory B cells, the reservoir for lifelong EBV infection, remains incompletely understood. To gain insights, we performed a human genome-wide CRISPR/Cas9 screen in Burkitt lymphoma B cells. Our analyses identified a network of host factors that repress lytic reactivation, centered on the transcription factor MYC, including cohesins, FACT, STAGA, and Mediator. Depletion of MYC or factors important for MYC expression reactivated the lytic cycle, including in Burkitt xenografts. MYC bound the EBV genome origin of lytic replication and suppressed its looping to the lytic cycle initiator BZLF1 promoter. Notably, MYC abundance decreases with plasma cell differentiation, a key lytic reactivation trigger. Our results suggest that EBV senses MYC abundance as a readout of B cell state and highlights Burkitt latency reversal therapeutic targets.
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http://dx.doi.org/10.1016/j.molcel.2020.03.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7245572PMC
May 2020

Design and analysis of CRISPR-Cas experiments.

Nat Biotechnol 2020 07 13;38(7):813-823. Epub 2020 Apr 13.

Broad Institute of Harvard and MIT, Cambridge, MA, USA.

A large and ever-expanding set of CRISPR-Cas systems now enables the rapid and flexible manipulation of genomes in both targeted and large-scale experiments. Numerous software tools and analytical methods have been developed for the design and analysis of CRISPR-Cas experiments, including resources to design optimal guide RNAs for various modes of manipulation and to analyze the results of such experiments. A major recent focus has been the development of comprehensive tools for use on data from large-scale CRISPR-based genetic screens. As this field continues to progress, a clear ongoing challenge is not only to innovate, but to actively maintain and improve existing tools so that researchers across disciplines can rely on a stable set of excellent computational resources for CRISPR-Cas experiments.
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http://dx.doi.org/10.1038/s41587-020-0490-7DOI Listing
July 2020

Cytochrome P450 oxidoreductase contributes to phospholipid peroxidation in ferroptosis.

Nat Chem Biol 2020 03 17;16(3):302-309. Epub 2020 Feb 17.

Chemical Biology and Therapeutics Science Program, Broad Institute, Cambridge, MA, USA.

Ferroptosis is widely involved in degenerative diseases in various tissues including kidney, liver and brain, and is a targetable vulnerability in multiple primary and therapy-resistant cancers. Accumulation of phospholipid hydroperoxides in cellular membranes is the hallmark and rate-limiting step of ferroptosis; however, the enzymes contributing to lipid peroxidation remain poorly characterized. Using genome-wide, CRISPR-Cas9-mediated suppressor screens, we identify cytochrome P450 oxidoreductase (POR) as necessary for ferroptotic cell death in cancer cells exhibiting inherent and induced susceptibility to ferroptosis. By genetic depletion of POR in cancer cells, we reveal that POR contributes to ferroptosis across a wide range of lineages and cell states, and in response to distinct mechanisms of ferroptosis induction. Using systematic lipidomic profiling, we further map POR's activity to the lipid peroxidation step in ferroptosis. Hence, our work suggests that POR is a key mediator of ferroptosis and potential druggable target for developing antiferroptosis therapeutics.
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http://dx.doi.org/10.1038/s41589-020-0472-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7353921PMC
March 2020

Genetic screens in isogenic mammalian cell lines without single cell cloning.

Nat Commun 2020 02 6;11(1):752. Epub 2020 Feb 6.

Genetic Perturbation Platform, Broad Institute of MIT and Harvard, 75 Ames Street, Cambridge, MA, 02142, USA.

Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs of mammalian cells, however, is labor-intensive, time-consuming, and, in some cell types, essentially impossible. Here, we present an approach to create isogenic pairs of cells that avoids single cell cloning, and screen these pairs with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We query the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, identifying both expected and uncharacterized buffering and synthetic lethal interactions. Additionally, we compare acute CRISPR-based knockout, single cell clones, and small-molecule inhibition. We observe that, while the approaches provide largely overlapping information, differences emerge, highlighting an important consideration when employing genetic screens to identify and characterize potential drug targets. We anticipate that this methodology will be broadly useful to comprehensively study gene function across many contexts.
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http://dx.doi.org/10.1038/s41467-020-14620-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005275PMC
February 2020

Genome-wide In Vivo CNS Screening Identifies Genes that Modify CNS Neuronal Survival and mHTT Toxicity.

Neuron 2020 04 30;106(1):76-89.e8. Epub 2020 Jan 30.

Department of Brain and Cognitive Sciences, MIT, Cambridge, MA 02139, USA; Picower Institute for Learning and Memory, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

Unbiased in vivo genome-wide genetic screening is a powerful approach to elucidate new molecular mechanisms, but such screening has not been possible to perform in the mammalian central nervous system (CNS). Here, we report the results of the first genome-wide genetic screens in the CNS using both short hairpin RNA (shRNA) and CRISPR libraries. Our screens identify many classes of CNS neuronal essential genes and demonstrate that CNS neurons are particularly sensitive not only to perturbations to synaptic processes but also autophagy, proteostasis, mRNA processing, and mitochondrial function. These results reveal a molecular logic for the common implication of these pathways across multiple neurodegenerative diseases. To further identify disease-relevant genetic modifiers, we applied our screening approach to two mouse models of Huntington's disease (HD). Top mutant huntingtin toxicity modifier genes included several Nme genes and several genes involved in methylation-dependent chromatin silencing and dopamine signaling, results that reveal new HD therapeutic target pathways.
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http://dx.doi.org/10.1016/j.neuron.2020.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181458PMC
April 2020

Genome-wide CRISPR screen identifies host dependency factors for influenza A virus infection.

Nat Commun 2020 01 9;11(1):164. Epub 2020 Jan 9.

Broad Institute of MIT and Harvard, 415 Main Street, Cambridge, MA, 02142, USA.

Host dependency factors that are required for influenza A virus infection may serve as therapeutic targets as the virus is less likely to bypass them under drug-mediated selection pressure. Previous attempts to identify host factors have produced largely divergent results, with few overlapping hits across different studies. Here, we perform a genome-wide CRISPR/Cas9 screen and devise a new approach, meta-analysis by information content (MAIC) to systematically combine our results with prior evidence for influenza host factors. MAIC out-performs other meta-analysis methods when using our CRISPR screen as validation data. We validate the host factors, WDR7, CCDC115 and TMEM199, demonstrating that these genes are essential for viral entry and regulation of V-type ATPase assembly. We also find that CMTR1, a human mRNA cap methyltransferase, is required for efficient viral cap snatching and regulation of a cell autonomous immune response, and provides synergistic protection with the influenza endonuclease inhibitor Xofluza.
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http://dx.doi.org/10.1038/s41467-019-13965-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952391PMC
January 2020

Agreement between two large pan-cancer CRISPR-Cas9 gene dependency data sets.

Nat Commun 2019 12 20;10(1):5817. Epub 2019 Dec 20.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

Genome-scale CRISPR-Cas9 viability screens performed in cancer cell lines provide a systematic approach to identify cancer dependencies and new therapeutic targets. As multiple large-scale screens become available, a formal assessment of the reproducibility of these experiments becomes necessary. We analyze data from recently published pan-cancer CRISPR-Cas9 screens performed at the Broad and Sanger Institutes. Despite significant differences in experimental protocols and reagents, we find that the screen results are highly concordant across multiple metrics with both common and specific dependencies jointly identified across the two studies. Furthermore, robust biomarkers of gene dependency found in one data set are recovered in the other. Through further analysis and replication experiments at each institute, we show that batch effects are driven principally by two key experimental parameters: the reagent library and the assay length. These results indicate that the Broad and Sanger CRISPR-Cas9 viability screens yield robust and reproducible findings.
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http://dx.doi.org/10.1038/s41467-019-13805-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925302PMC
December 2019

Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy.

Nature 2019 12 11;576(7787):471-476. Epub 2019 Dec 11.

Department of Immunology, St Jude Children's Research Hospital, Memphis, TN, USA.

Adoptive cell therapy represents a new paradigm in cancer immunotherapy, but it can be limited by the poor persistence and function of transferred T cells. Here we use an in vivo pooled CRISPR-Cas9 mutagenesis screening approach to demonstrate that, by targeting REGNASE-1, CD8 T cells are reprogrammed to long-lived effector cells with extensive accumulation, better persistence and robust effector function in tumours. REGNASE-1-deficient CD8 T cells show markedly improved therapeutic efficacy against mouse models of melanoma and leukaemia. By using a secondary genome-scale CRISPR-Cas9 screening, we identify BATF as the key target of REGNASE-1 and as a rheostat that shapes antitumour responses. Loss of BATF suppresses the increased accumulation and mitochondrial fitness of REGNASE-1-deficient CD8 T cells. By contrast, the targeting of additional signalling factors-including PTPN2 and SOCS1-improves the therapeutic efficacy of REGNASE-1-deficient CD8 T cells. Our findings suggest that T cell persistence and effector function can be coordinated in tumour immunity and point to avenues for improving the efficacy of adoptive cell therapy for cancer.
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http://dx.doi.org/10.1038/s41586-019-1821-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6937596PMC
December 2019

A Compendium of Genetic Modifiers of Mitochondrial Dysfunction Reveals Intra-organelle Buffering.

Cell 2019 11;179(5):1222-1238.e17

Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114, USA, Massachusetts General Hospital, Boston, MA 02114, USA; Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA. Electronic address:

Mitochondrial dysfunction is associated with a spectrum of human conditions, ranging from rare, inborn errors of metabolism to the aging process. To identify pathways that modify mitochondrial dysfunction, we performed genome-wide CRISPR screens in the presence of small-molecule mitochondrial inhibitors. We report a compendium of chemical-genetic interactions involving 191 distinct genetic modifiers, including 38 that are synthetic sick/lethal and 63 that are suppressors. Genes involved in glycolysis (PFKP), pentose phosphate pathway (G6PD), and defense against lipid peroxidation (GPX4) scored high as synthetic sick/lethal. A surprisingly large fraction of suppressors are pathway intrinsic and encode mitochondrial proteins. A striking example of such "intra-organelle" buffering is the alleviation of a chemical defect in complex V by simultaneous inhibition of complex I, which benefits cells by rebalancing redox cofactors, increasing reductive carboxylation, and promoting glycolysis. Perhaps paradoxically, certain forms of mitochondrial dysfunction may best be buffered with "second site" inhibitors to the organelle.
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http://dx.doi.org/10.1016/j.cell.2019.10.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053407PMC
November 2019

CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation.

Cell Rep 2019 07;28(5):1307-1322.e8

Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, 181 Longwood Avenue, Boston, MA 02115, USA; Department of Microbiology, Harvard Medical School, Boston, MA 02115, USA; Department of Biostatistics and Bioinformatics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA; Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. Electronic address:

CD40 has major roles in B cell development, activation, and germinal center responses. CD40 hypoactivity causes immunodeficiency whereas its overexpression causes autoimmunity and lymphomagenesis. To systematically identify B cell autonomous CD40 regulators, we use CRISPR/Cas9 genome-scale screens in Daudi B cells stimulated by multimeric CD40 ligand. These highlight known CD40 pathway components and reveal multiple additional mechanisms regulating CD40. The nuclear ubiquitin ligase FBXO11 supports CD40 expression by targeting repressors CTBP1 and BCL6. FBXO11 knockout decreases primary B cell CD40 abundance and impairs class-switch recombination, suggesting that frequent lymphoma monoallelic FBXO11 mutations may balance BCL6 increase with CD40 loss. At the mRNA level, CELF1 controls exon splicing critical for CD40 activity, while the N6-adenosine methyltransferase WTAP negatively regulates CD40 mRNA abundance. At the protein level, ESCRT negatively regulates activated CD40 levels while the negative feedback phosphatase DUSP10 limits downstream MAPK responses. These results serve as a resource for future studies and highlight potential therapeutic targets.
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http://dx.doi.org/10.1016/j.celrep.2019.06.079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684324PMC
July 2019

Autophagy genes in myeloid cells counteract IFNγ-induced TNF-mediated cell death and fatal TNF-induced shock.

Proc Natl Acad Sci U S A 2019 08 25;116(33):16497-16506. Epub 2019 Jul 25.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110;

Host inflammatory responses must be tightly regulated to ensure effective immunity while limiting tissue injury. IFN gamma (IFNγ) primes macrophages to mount robust inflammatory responses. However, IFNγ also induces cell death, and the pathways that regulate IFNγ-induced cell death are incompletely understood. Using genome-wide CRISPR/Cas9 screening, we identified autophagy genes as central mediators of myeloid cell survival during the IFNγ response. Hypersensitivity of autophagy gene-deficient cells to IFNγ was mediated by tumor necrosis factor (TNF) signaling via receptor interacting protein kinase 1 (RIPK1)- and caspase 8-mediated cell death. Mice with myeloid cell-specific autophagy gene deficiency exhibited marked hypersensitivity to fatal systemic TNF administration. This increased mortality in myeloid autophagy gene-deficient mice required the IFNγ receptor, and mortality was completely reversed by pharmacologic inhibition of RIPK1 kinase activity. These findings provide insight into the mechanism of IFNγ-induced cell death via TNF, demonstrate a critical function of autophagy genes in promoting cell viability in the presence of inflammatory cytokines, and implicate this cell survival function in protection against mortality during the systemic inflammatory response.
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http://dx.doi.org/10.1073/pnas.1822157116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697821PMC
August 2019

6-Phosphogluconate Dehydrogenase Links Cytosolic Carbohydrate Metabolism to Protein Secretion via Modulation of Glutathione Levels.

Cell Chem Biol 2019 09 13;26(9):1306-1314.e5. Epub 2019 Jun 13.

Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA. Electronic address:

The proteinaceous extracellular matrix (ECM) is vital for the survival, proliferation, migration, and differentiation of many types of cancer. However, little is known regarding metabolic pathways required for ECM secretion. By using an unbiased computational approach, we searched for enzymes whose suppression may lead to disruptions in protein secretion. Here, we show that 6-phosphogluconate dehydrogenase (PGD), a cytosolic enzyme involved in carbohydrate metabolism, is required for ER structural integrity and protein secretion. Chemical inhibition or genetic suppression of PGD activity led to cell stress accompanied by significantly expanded ER volume and was rescued by compensating endogenous glutathione supplies. Our results also suggest that this characteristic ER-dilation phenotype may be a general marker indicating increased ECM protein congestion inside cells and decreased secretion. Thus, PGD serves as a link between cytosolic carbohydrate metabolism and protein secretion.
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http://dx.doi.org/10.1016/j.chembiol.2019.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754299PMC
September 2019

Gene-centric functional dissection of human genetic variation uncovers regulators of hematopoiesis.

Elife 2019 05 9;8. Epub 2019 May 9.

Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, United States.

Genome-wide association studies (GWAS) have identified thousands of variants associated with human diseases and traits. However, the majority of GWAS-implicated variants are in non-coding regions of the genome and require in depth follow-up to identify target genes and decipher biological mechanisms. Here, rather than focusing on causal variants, we have undertaken a pooled loss-of-function screen in primary hematopoietic cells to interrogate 389 candidate genes contained in 75 loci associated with red blood cell traits. Using this approach, we identify 77 genes at 38 GWAS loci, with most loci harboring 1-2 candidate genes. Importantly, the hit set was strongly enriched for genes validated through orthogonal genetic approaches. Genes identified by this approach are enriched in specific and relevant biological pathways, allowing regulators of human erythropoiesis and modifiers of blood diseases to be defined. More generally, this functional screen provides a paradigm for gene-centric follow up of GWAS for a variety of human diseases and traits.
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http://dx.doi.org/10.7554/eLife.44080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534380PMC
May 2019