Publications

Nature Communications | 2020.08.27

Multiplexed single-cell transcriptional response profiling to define cancer vulnerabilities and therapeutic mechanism of action

James M. McFarland, Brenton R. Paolella, Allison Warren, Kathryn Geiger-Schuller,... Aviv Regev, Andrew J. Aguirre, Francisca Vazquez, & Aviad Tsherniak

Assays to study cancer cell responses to pharmacologic or genetic perturbations are typically restricted to using simple phenotypic readouts such as proliferation rate. Information-rich assays, such as gene-expression profiling, have generally not permitted efficient profiling of a given perturbation across multiple cellular contexts. Here, we develop MIX-Seq, a method for multiplexed transcriptional profiling of post-perturbation responses across a mixture of samples with single-cell resolution, using SNP-based computational demultiplexing of single-cell RNA-sequencing data. We show that MIX-Seq can be used to profile responses to chemical or genetic perturbations across pools of 100 or more cancer cell lines. We combine it with Cell Hashing to further multiplex additional experimental conditions, such as post-treatment time points or drug doses. Analyzing the high-content readout of scRNA-seq reveals both shared and context-specific transcriptional response components that can identify drug mechanism of action and enable prediction of long-term cell viability from short-term transcriptional responses to treatment.

doi.org/10.1038/s41467-020-17440-w
Nature Cancer | 2020.01.20

Discovering the anticancer potential of non-oncology drugs by systematic viability profiling

Steven M. Corsello, Rohith T. Nagari, Ryan D. Spangler, Jordan Rossen, Mustafa Kocak,... Jesse S. Boehm, Christopher C. Mader, Aviad Tsherniak & Todd R. Golub

Anticancer 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 (profiling relative inhibition simultaneously in mixtures), 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 molecular features of the cell lines. Our findings include compounds that killed by inducing phosphodiesterase 3A-Schlafen 12 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 multidrug resistance protein ATP-binding cassette subfamily B member 1 (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.

doi.org/10.1038/s43018-019-0018-6
Cell | 2019.10.17

Optical Pooled Screens in Human Cells

David Feldman, Avtar Singh, Jonathan L. Schmid-Burgk, Rebecca J Carlson,... Anja Mezger, Anthony J. Garrity, Feng Zhang, & Paul C. Blainey

Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.

doi.org/10.1016/j.cell.2019.09.016
Cell Reports | 2019.08.27

Small-Molecule and CRISPR Screening Converge to Reveal Receptor Tyrosine Kinase Dependencies in Pediatric Rhabdoid Tumors

Elaine M. Oberlick, Matthew G. Rees, Brinton Seashore-Ludlow, Francisca Vazquez,... William C. Hahn, Elizabeth A. Stewart, Stuart L. Schreiber, & Charles W.M. Roberts

Cancer is often seen as a disease of mutations and chromosomal abnormalities. However, some cancers, including pediatric rhabdoid tumors (RTs), lack recurrent alterations targetable by current drugs and need alternative, informed therapeutic options. To nominate potential targets, we performed a high-throughput small-molecule screen complemented by a genome-scale CRISPR-Cas9 gene-knockout screen in a large number of RT and control cell lines. These approaches converged to reveal several receptor tyrosine kinases (RTKs) as therapeutic targets, with RTK inhibition effective in suppressing RT cell growth in vitro and against a xenograft model in vivo. RT cell lines highly express and activate (phosphorylate) different RTKs, creating dependency without mutation or amplification. Downstream of RTK signaling, we identified PTPN11, encoding the pro-growth signaling protein SHP2, as a shared dependency across all RT cell lines. This study demonstrates that large-scale perturbational screening can uncover vulnerabilities in cancers with “quiet” genomes.

doi.org/10.1016/j.celrep.2019.07.021
Nature | 2019.05.08

Next-generation characterization of the Cancer Cell Line Encyclopedia

Mahmoud Ghandi, Franklin W. Huang, Judit Jané-Valbuena, Gregory V. Kryukov,... Todd R. Golub, Levi A. Garraway & William R. Sellers

Large panels of comprehensively characterized human cancer models, including the Cancer Cell Line Encyclopedia (CCLE), have provided a rigorous framework with which to study genetic variants, candidate targets, and small-molecule and biological therapeutics and to identify new marker-driven cancer dependencies. To improve our understanding of the molecular features that contribute to cancer phenotypes, including drug responses, here we have expanded the characterizations of cancer cell lines to include genetic,...

doi.org/10.1038/s41586-019-1186-3
Nature Medicine | 2019.05.08

The landscape of cancer cell line metabolism

Haoxin Li, Shaoyang Ning, Mahmoud Ghandi, Gregory V. Kryukov,... Stuart L. Schreiber, Clary B. Clish, Levi A. Garraway & William R. Sellers

Despite considerable efforts to identify cancer metabolic alterations that might unveil druggable vulnerabilities, systematic characterizations of metabolism as it relates to functional genomic features and associated dependencies remain uncommon. To further understand the metabolic diversity of cancer, we profiled 225 metabolites in 928 cell lines from more than 20 cancer types in the Cancer Cell Line Encyclopedia (CCLE) using liquid chromatography–mass spectrometry (LC-MS). This resource enables unbiased association analysis...

doi.org/10.1038/s41591-019-0404-8
Nature | 2019.04.10

WRN helicase is a synthetic lethal target in microsatellite unstable cancers

Edmond M. Chan, Tsukasa Shibue, James M. McFarland, Benjamin Gaeta,... Todd R. Golub, Aviad Tsherniak, Francisca Vazquez & Adam J. Bass

Synthetic lethality—an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not—can be exploited for cancer therapeutics. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach. Hypothesizing that other DNA repair defects,...

doi.org/10.1038/s41586-019-1102-x
Nature | 2018.12.17

Loss of ADAR1 in tumours overcomes resistance to immune checkpoint blockade

Jeffrey J. Ishizuka, Robert T. Manguso, Collins K. Cheruiyot, Kevin Bi,... John G. Doench, David Kozono, Erez Y. Levanon & W. Nicholas Haining

Most patients with cancer either do not respond to immune checkpoint blockade or develop resistance to it, often because of acquired mutations that impair antigen presentation. Here we show that loss of function of the RNA-editing enzyme ADAR1 in tumour cells profoundly sensitizes tumours to immunotherapy and overcomes resistance to checkpoint blockade. In the absence of ADAR1, A-to-I editing of interferon-inducible RNA species is reduced, leading to double-stranded RNA ligand sensing by PKR and MDA5; this results in growth inhibition...

doi.org/10.1038/s41586-018-0768-9
Nature Communications | 2018.11.02

Improved estimation of cancer dependencies from large-scale RNAi screens using model-based normalization and data integration

James M. McFarland, Zandra V. Ho, Guillaume Kugener, Joshua M. Dempster,... Todd R. Golub, William C. Hahn, David E. Root & Aviad Tsherniak

The availability of multiple datasets comprising genome-scale RNAi viability screens in hundreds of diverse cancer cell lines presents new opportunities for understanding cancer vulnerabilities. Integrated analyses of these data to assess differential dependency across genes and cell lines are challenging due to confounding factors such as batch effects and variable screen quality, as well as difficulty assessing gene dependency on an absolute scale. To address these issues, we incorporated cell line screen-quality parameters and hierarchical Bayesian inference into DEMETER2, an analytical framework...

doi.org/10.1038/s41467-018-06916-5
Nature Genetics | 2017.10.30

Computational correction of copy number effect improves specificity of CRISPR-Cas9 essentiality screens in cancer cells

Robin M. Meyers, Jordan G. Bryan, James M. McFarland, Barbara A. Weir,... David E. Root, William C. Hahn, & Aviad Tsherniak

The CRISPR–Cas9 system has revolutionized gene editing both at single genes and in multiplexed loss-of-function screens, thus enabling precise genome-scale identification of genes essential for proliferation and survival of cancer cells1,2. However, previous studies have reported that a gene-independent antiproliferative effect of Cas9-mediated DNA cleavage confounds such measurement of genetic dependency, thereby leading to false-positive results in copy number–amplified regions3,4. We developed CERES, a computational method...

doi.org/10.1038/ng.3984
Cell | 2017.07.27

Defining a Cancer Dependency Map

Aviad Tsherniak, Francisca Vazquez, Phillip G. Montgomery, Barbara A. Weir,... Todd R. Golub, Jesse S. Boehm, & William C. Hahn

Most human epithelial tumors harbor numerous alterations, making it difficult to predict which genes are required for tumor survival. To systematically identify cancer dependencies, we analyzed 501 genome-scale loss-of-function screens performed in diverse human cancer cell lines. We developed DEMETER, an analytical framework that segregates on- from off-target effects of RNAi. 769 genes were differentially required in subsets of these cell lines at a threshold of six SDs from the mean. We found predictive models for 426 dependencies....

doi.org/10.1016/j.cell.2017.06.010
Nature | 2017.07.19

In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target

Robert T. Manguso, Hans W. Pope, Margaret D. Zimmer, Flavian D. Brown,... David E. Root, Arlene H. Sharpe, John G. Doench & W. Nicholas Haining

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR–Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune...

doi.org/10.1038/nature23270
Nature Medicine | 2017.04.07

The Drug Repurposing Hub: a next-generation drug library and information resource

Steven M. Corsello, Joshua A. Bittker, Zihan Liu, Joshua Gould,... Christopher C. Mader, Aravind Subramanian, & Todd R. Golub

Drug repurposing, the application of an existing therapeutic to a new disease indication, holds the promise of rapid clinical impact at a lower cost than de novo drug development. To date there has not been a systematic effort to identify such opportunities, limited in part by the lack of a comprehensive library of clinical compounds suitable for testing. To address this challenge, we hand-curated a collection of 4,707 compounds, experimentally confirmed their identity, and annotated them with literature-reported targets...

doi.org/10.1038/nm.4306
Cell | 2016.12.15

Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens

Atray Dixit, Oren Parnas, Biyu Li, Jenny Chen,... Eric S. Lander, Jonathan S. Weissman, Nir Friedman, & Aviv Regev

Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes-such as transcriptional profiles-at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells...

10.1016/j.cell.2016.11.038
Science | 2016.03.11

MTAP deletion confers enhanced dependency on the PRMT5 arginine methyltransferase in cancer cells

Gregory V. Kryukov, Frederick H. Wilson, Jason R. Ruth, Joshiawa Paulk,... Clary B. Clish, James E. Bradner, William C. Hahn, Levi A. Garraway

The discovery of cancer dependencies has the potential to inform therapeutic strategies and to identify putative drug targets. Integrating data from comprehensive genomic profiling of cancer cell lines and from functional characterization of cancer cell dependencies, we discovered that loss of the enzyme methylthioadenosine phosphorylase (MTAP) confers a selective dependence on protein arginine methyltransferase 5 (PRMT5) and its binding partner WDR77. MTAP is frequently lost due to its proximity to the commonly deleted tumor...

doi.org/10.1126/science.aad5214
Nature Biotechnology | 2016.02.29

High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines

Channing Yu, Aristotle M. Mannan, Griselda Metta Yvone, Kenneth N. Ross,... Stuart L. Schreiber, Andrew L. Kung, & Todd R. Golub

Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large numbers of compounds against large numbers of cell lines is currently impractical, and such experiments are often difficult to control1,2,3,4. Here we report a method called PRISM that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM revealed the expected patterns of cell killing seen in conventional (unpooled) assays...

doi.org/10.1038/nbt.3460
Nature Chemical Biology | 2015.12.14

Correlating chemical sensitivity and basal gene expression reveals mechanism of action

Matthew G. Rees, Brinton Seashore-Ludlow, Jaime H. Cheah, Drew J. Adams,... Paul A. Clemons, Alykhan F. Shamji, & Stuart L. Schreiber

Changes in cellular gene expression in response to small-molecule or genetic perturbations have yielded signatures that can connect unknown mechanisms of action (MoA) to ones previously established. We hypothesized that differential basal gene expression could be correlated with patterns of small-molecule sensitivity across many cell lines to illuminate the actions of compounds whose MoA are unknown. To test this idea, we correlated the sensitivity patterns of 481 compounds with ∼19,000 basal transcript levels across 823 different...

doi.org/10.1038/nchembio.1986
Nature | 2015.11.16

Pharmacogenomic agreement between two cancer cell line data sets

The Cancer Cell Line Encyclopedia Consortium & The Genomics of Drug Sensitivity in Cancer Consortium

Large cancer cell line collections broadly capture the genomic diversity of human cancers and provide valuable insight into anti-cancer drug response. Here we show substantial agreement and biological consilience between drug sensitivity measurements and their associated genomic predictors from two publicly available large-scale pharmacogenomics resources: The Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer databases....

doi.org/10.1038/nature15736
Nature | 2012.03.28

The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity

TJordi Barretina, Giordano Caponigro, Nicolas Stransky, Kavitha Venkatesan,... William R. Sellers, Robert Schlegel, & Levi A. Garraway

The systematic translation of cancer genomic data into knowledge of tumour biology and therapeutic possibilities remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacological annotation is available1. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacological profiles for 24 anticancer drugs across 479 of the cell lines....

doi.org/10.1038/nature11003