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2. Post-translational modification-centric base editor screens to assess phosphorylation site functionality in high throughput

Author

Kennedy, Patrick H., Alborzian Deh Sheikh, Amin, Balakar, Matthew, Jones, Alexander C., Olive, Meagan E., Hegde, Mudra, Matias, Maria I., Pirete, Natan, Burt, Rajan, Levy, Jonathan, Little, Tamia, Hogan, Patrick G., Liu, David R., Doench, John G., Newton, Alexandra C., Gottschalk, Rachel A., de Boer, Carl G., Alarcón, Suzie, Newby, Gregory A., and Myers, Samuel A.

Published
2024
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3. Three million images and morphological profiles of cells treated with matched chemical and genetic perturbations

Author

Chandrasekaran, Srinivas Niranj, Cimini, Beth A., Goodale, Amy, Miller, Lisa, Kost-Alimova, Maria, Jamali, Nasim, Doench, John G., Fritchman, Briana, Skepner, Adam, Melanson, Michelle, Kalinin, Alexandr A., Arevalo, John, Haghighi, Marzieh, Caicedo, Juan C., Kuhn, Daniel, Hernandez, Desiree, Berstler, James, Shafqat-Abbasi, Hamdah, Root, David E., Swalley, Susanne E., Garg, Sakshi, Singh, Shantanu, and Carpenter, Anne E.

Published
2024
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8. DYRK1A promotes viral entry of highly pathogenic human coronaviruses in a kinase-independent manner

Author

Strine, Madison S, Cai, Wesley L, Wei, Jin, Alfajaro, Mia Madel, Filler, Renata B, Biering, Scott B, Sarnik, Sylvia, Chow, Ryan D, Patil, Ajinkya, Cervantes, Kasey S, Collings, Clayton K, DeWeirdt, Peter C, Hanna, Ruth E, Schofield, Kevin, Hulme, Christopher, Konermann, Silvana, Doench, John G, Hsu, Patrick D, Kadoch, Cigall, Yan, Qin, and Wilen, Craig B

Subjects
Biodefense, Emerging Infectious Diseases, Pneumonia & Influenza, Pneumonia, Infectious Diseases, Prevention, Vaccine Related, Genetics, Lung, Aetiology, 2.1 Biological and endogenous factors, Underpinning research, 1.1 Normal biological development and functioning, Infection, Good Health and Well Being, Animals, Humans, Angiotensin-Converting Enzyme 2, COVID-19, Dipeptidyl Peptidase 4, Middle East Respiratory Syndrome Coronavirus, SARS-CoV-2, Severe acute respiratory syndrome-related coronavirus, Virus Internalization, Biological Sciences, Agricultural and Veterinary Sciences, Medical and Health Sciences, Developmental Biology
Abstract

Identifying host genes essential for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has the potential to reveal novel drug targets and further our understanding of Coronavirus Disease 2019 (COVID-19). We previously performed a genome-wide CRISPR/Cas9 screen to identify proviral host factors for highly pathogenic human coronaviruses. Few host factors were required by diverse coronaviruses across multiple cell types, but DYRK1A was one such exception. Although its role in coronavirus infection was previously undescribed, DYRK1A encodes Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A and is known to regulate cell proliferation and neuronal development. Here, we demonstrate that DYRK1A regulates ACE2 and DPP4 transcription independent of its catalytic kinase function to support SARS-CoV, SARS-CoV-2, and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) entry. We show that DYRK1A promotes DNA accessibility at the ACE2 promoter and a putative distal enhancer, facilitating transcription and gene expression. Finally, we validate that the proviral activity of DYRK1A is conserved across species using cells of nonhuman primate and human origin. In summary, we report that DYRK1A is a novel regulator of ACE2 and DPP4 expression that may dictate susceptibility to multiple highly pathogenic human coronaviruses.

Published
2023

9. Mammalian SWI/SNF complex activity regulates POU2F3 and constitutes a targetable dependency in small cell lung cancer

Author

Duplaquet, Leslie, So, Kevin, Ying, Alexander W., Pal Choudhuri, Shreoshi, Li, Xinyue, Xu, Grace D., Li, Yixiang, Qiu, Xintao, Li, Rong, Singh, Shilpa, Wu, Xiaoli S., Hamilton, Seth, Chien, Victor D., Liu, Qi, Qi, Jun, Somerville, Tim D.D., Heiling, Hillary M., Mazzola, Emanuele, Lee, Yenarae, Zoller, Thomas, Vakoc, Christopher R., Doench, John G., Forrester, William C., Abrams, Tinya, Long, Henry W., Niederst, Matthew J., Drapkin, Benjamin J., Kadoch, Cigall, and Oser, Matthew G.

Published
2024
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12. Leukemia-intrinsic determinants of CAR-T response revealed by iterative in vivo genome-wide CRISPR screening

Author

Ramos, Azucena, Koch, Catherine E., Liu-Lupo, Yunpeng, Hellinger, Riley D., Kyung, Taeyoon, Abbott, Keene L., Fröse, Julia, Goulet, Daniel, Gordon, Khloe S., Eidell, Keith P., Leclerc, Paul, Whittaker, Charles A., Larson, Rebecca C., Muscato, Audrey J., Yates, Kathleen B., Dubrot, Juan, Doench, John G., Regev, Aviv, Vander Heiden, Matthew G., Maus, Marcela V., Manguso, Robert T., Birnbaum, Michael E., and Hemann, Michael T.

Published
2023
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15. A genome-wide CRISPR screen identifies WDFY3 as a regulator of macrophage efferocytosis

Author

Shi, Jianting, Wu, Xun, Wang, Ziyi, Li, Fang, Meng, Yujiao, Moore, Rebecca M, Cui, Jian, Xue, Chenyi, Croce, Katherine R, Yurdagul, Arif, Doench, John G, Li, Wei, Zarbalis, Konstantinos S, Tabas, Ira, Yamamoto, Ai, and Zhang, Hanrui

Subjects
Biochemistry and Cell Biology, Biological Sciences, Underpinning research, 1.1 Normal biological development and functioning, 2.1 Biological and endogenous factors, Aetiology, Generic health relevance, Inflammatory and immune system, Animals, Humans, Mice, Adaptor Proteins, Signal Transducing, Apoptosis, Autophagy-Related Proteins, Cells, Cultured, Clustered Regularly Interspaced Short Palindromic Repeats, Macrophages, Mice, Inbred C57BL, Phagocytosis
Abstract

Phagocytic clearance of dying cells, termed efferocytosis, is essential for maintaining tissue homeostasis, yet our understanding of efferocytosis regulation remains incomplete. Here we perform a FACS-based, genome-wide CRISPR knockout screen in primary mouse macrophages to search for novel regulators of efferocytosis. The results show that Wdfy3 knockout in macrophages specifically impairs uptake, but not binding, of apoptotic cells due to defective actin disassembly. Additionally, WDFY3 interacts with GABARAP, thus facilitating LC3 lipidation and subsequent lysosomal acidification to permit the degradation of apoptotic cell components. Mechanistically, while the C-terminus of WDFY3 is sufficient to rescue the impaired degradation induced by Wdfy3 knockout, full-length WDFY3 is required to reconstitute the uptake of apoptotic cells. Finally, WDFY3 is also required for efficient efferocytosis in vivo in mice and in vitro in primary human macrophages. This work thus expands our knowledge of the mechanisms of macrophage efferocytosis, as well as supports genome-wide CRISPR screen as a platform for interrogating complex functional phenotypes in primary macrophages.

Published
2022

17. Transcriptional control of leukemogenesis by the chromatin reader SGF29

Author

Barbosa, Karina, Deshpande, Anagha, Perales, Marlenne, Xiang, Ping, Murad, Rabi, Pramod, Akula Bala, Minkina, Anna, Robertson, Neil, Schischlik, Fiorella, Lei, Xue, Sun, Younguk, Brown, Adam, Amend, Diana, Jeremias, Irmela, Doench, John G., Humphries, R. Keith, Ruppin, Eytan, Shendure, Jay, Mali, Prashant, Adams, Peter D., and Deshpande, Aniruddha J.

Published
2024
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18. MEN1 mutations mediate clinical resistance to menin inhibition

Author

Perner, Florian, Stein, Eytan M., Wenge, Daniela V., Singh, Sukrit, Kim, Jeonghyeon, Apazidis, Athina, Rahnamoun, Homa, Anand, Disha, Marinaccio, Christian, Hatton, Charlie, Wen, Yanhe, Stone, Richard M., Schaller, David, Mowla, Shoron, Xiao, Wenbin, Gamlen, Holly A., Stonestrom, Aaron J., Persaud, Sonali, Ener, Elizabeth, Cutler, Jevon A., Doench, John G., McGeehan, Gerard M., Volkamer, Andrea, Chodera, John D., Nowak, Radosław P., Fischer, Eric S., Levine, Ross L., Armstrong, Scott A., and Cai, Sheng F.

Published
2023
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19. Pharmacological disruption of mSWI/SNF complex activity restricts SARS-CoV-2 infection

Author

Wei, Jin, Patil, Ajinkya, Collings, Clayton K., Alfajaro, Mia Madel, Liang, Yu, Cai, Wesley L., Strine, Madison S., Filler, Renata B., DeWeirdt, Peter C., Hanna, Ruth E., Menasche, Bridget L., Ökten, Arya, Peña-Hernández, Mario A., Klein, Jon, McNamara, Andrew, Rosales, Romel, McGovern, Briana L., Luis Rodriguez, M., García-Sastre, Adolfo, White, Kris M., Qin, Yiren, Doench, John G., Yan, Qin, Iwasaki, Akiko, Zwaka, Thomas P., Qi, Jun, Kadoch, Cigall, and Wilen, Craig B.

Published
2023
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22. In vivo CRISPR screens reveal the landscape of immune evasion pathways across cancer

Author

Dubrot, Juan, Du, Peter P., Lane-Reticker, Sarah Kate, Kessler, Emily A., Muscato, Audrey J., Mehta, Arnav, Freeman, Samuel S., Allen, Peter M., Olander, Kira E., Ockerman, Kyle M., Wolfe, Clara H., Wiesmann, Fabius, Knudsen, Nelson H., Tsao, Hsiao-Wei, Iracheta-Vellve, Arvin, Schneider, Emily M., Rivera-Rosario, Andrea N., Kohnle, Ian C., Pope, Hans W., Ayer, Austin, Mishra, Gargi, Zimmer, Margaret D., Kim, Sarah Y., Mahapatra, Animesh, Ebrahimi-Nik, Hakimeh, Frederick, Dennie T., Boland, Genevieve M., Haining, W. Nicholas, Root, David E., Doench, John G., Hacohen, Nir, Yates, Kathleen B., and Manguso, Robert T.

Published
2022
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25. Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal HCoVs

Author

Rebendenne, Antoine, Roy, Priyanka, Bonaventure, Boris, Chaves Valadão, Ana Luiza, Desmarets, Lowiese, Arnaud-Arnould, Mary, Rouillé, Yves, Tauziet, Marine, Giovannini, Donatella, Touhami, Jawida, Lee, Yenarae, DeWeirdt, Peter, Hegde, Mudra, Urbach, Serge, Koulali, Khadija El, de Gracia, Francisco Garcia, McKellar, Joe, Dubuisson, Jean, Wencker, Mélanie, Belouzard, Sandrine, Moncorgé, Olivier, Doench, John G., and Goujon, Caroline

Published
2022
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26. Phosphate dysregulation via the XPR1–KIDINS220 protein complex is a therapeutic vulnerability in ovarian cancer

Author

Bondeson, Daniel P., Paolella, Brenton R., Asfaw, Adhana, Rothberg, Michael V., Skipper, Thomas A., Langan, Carly, Mesa, Gabriel, Gonzalez, Alfredo, Surface, Lauren E., Ito, Kentaro, Kazachkova, Mariya, Colgan, William N., Warren, Allison, Dempster, Joshua M., Krill-Burger, John M., Ericsson, Maria, Tang, Andrew A., Fung, Iris, Chambers, Emily S., Abdusamad, Mai, Dumont, Nancy, Doench, John G., Piccioni, Federica, Root, David E., Boehm, Jesse, Hahn, William C., Mannstadt, Michael, McFarland, James M., Vazquez, Francisca, and Golub, Todd R.

Published
2022
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28. Discovering metabolic disease gene interactions by correlated effects on cellular morphology.

Author

Jiao, Yang, Ahmed, Umer, Sim, MF Michelle, Bejar, Andrea, Zhang, Xiaolan, Talukder, M Mesbah Uddin, Rice, Robert, Flannick, Jason, Podgornaia, Anna I, Reilly, Dermot F, Engreitz, Jesse M, Kost-Alimova, Maria, Hartland, Kate, Mercader, Josep-Maria, Georges, Sara, Wagh, Vilas, Tadin-Strapps, Marija, Doench, John G, Edwardson, J Michael, Rochford, Justin J, Rosen, Evan D, and Majithia, Amit R

Subjects
Cells, Cultured, Adipocytes, Humans, Diabetes Mellitus, Insulin Resistance, Acyltransferases, GTP-Binding Protein gamma Subunits, CCAAT-Enhancer-Binding Proteins, Phenotype, Adipogenesis, Gene Regulatory Networks, HEK293 Cells, Transcriptome, Protein Interaction Maps, Perilipin-1, Functional genomics, Gene discovery, Genetic screen, High content imaging, Insulin resistance, Lipodystrophy, Metabolic syndrome, Type 2 diabetes, Cells, Cultured, Biochemistry and Cell Biology, Physiology
Abstract

OBJECTIVE:Impaired expansion of peripheral fat contributes to the pathogenesis of insulin resistance and Type 2 Diabetes (T2D). We aimed to identify novel disease-gene interactions during adipocyte differentiation. METHODS:Genes in disease-associated loci for T2D, adiposity and insulin resistance were ranked according to expression in human adipocytes. The top 125 genes were ablated in human pre-adipocytes via CRISPR/CAS9 and the resulting cellular phenotypes quantified during adipocyte differentiation with high-content microscopy and automated image analysis. Morphometric measurements were extracted from all images and used to construct morphologic profiles for each gene. RESULTS:Over 107 morphometric measurements were obtained. Clustering of the morphologic profiles accross all genes revealed a group of 14 genes characterized by decreased lipid accumulation, and enriched for known lipodystrophy genes. For two lipodystrophy genes, BSCL2 and AGPAT2, sub-clusters with PLIN1 and CEBPA identifed by morphological similarity were validated by independent experiments as novel protein-protein and gene regulatory interactions. CONCLUSIONS:A morphometric approach in adipocytes can resolve multiple cellular mechanisms for metabolic disease loci; this approach enables mechanistic interrogation of the hundreds of metabolic disease loci whose function still remains unknown.

Published
2019

36. Paralog knockout profiling identifies DUSP4 and DUSP6 as a digenic dependence in MAPK pathway-driven cancers

Author

Ito, Takahiro, Young, Michael J., Li, Ruitong, Jain, Sidharth, Wernitznig, Andreas, Krill-Burger, John M., Lemke, Christopher T., Monducci, Davide, Rodriguez, Diego J., Chang, Liang, Dutta, Sanjukta, Pal, Debjani, Paolella, Brenton R., Rothberg, Michael V., Root, David E., Johannessen, Cory M., Parida, Laxmi, Getz, Gad, Vazquez, Francisca, Doench, John G., Zamanighomi, Mahdi, and Sellers, William R.

Published
2021
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37. An alternative splicing switch in FLNB promotes the mesenchymal cell state in human breast cancer.

Author

Li, Ji, Choi, Peter S, Chaffer, Christine L, Labella, Katherine, Hwang, Justin H, Giacomelli, Andrew O, Kim, Jong Wook, Ilic, Nina, Doench, John G, Ly, Seav Huong, Dai, Chao, Hagel, Kimberly, Hong, Andrew L, Gjoerup, Ole, Goel, Shom, Ge, Jennifer Y, Root, David E, Zhao, Jean J, Brooks, Angela N, Weinberg, Robert A, and Hahn, William C

Subjects
Cell Line, Tumor, Mesenchymal Stem Cells, Animals, Humans, Mice, Nude, Breast Neoplasms, RNA-Binding Proteins, Neoplasm Proteins, Protein Isoforms, RNA, Messenger, Reproducibility of Results, Gene Expression Regulation, Neoplastic, Alternative Splicing, Base Sequence, Open Reading Frames, Genome, Human, Exons, Female, Epithelial-Mesenchymal Transition, Filamins, Hyaluronan Receptors, EMT, FLNB, QKI, RBFOX1, alternative splicing, basal-like breast cancer, cancer biology, cell biology, human, mouse, Cell Line, Tumor, Mice, Nude, RNA, Messenger, Gene Expression Regulation, Neoplastic, Genome, Human, Cancer, Breast Cancer, Genetics, Human Genome, 1.1 Normal biological development and functioning, 2.1 Biological and endogenous factors, Generic Health Relevance, Biochemistry and Cell Biology
Abstract

Alternative splicing of mRNA precursors represents a key gene expression regulatory step and permits the generation of distinct protein products with diverse functions. In a genome-scale expression screen for inducers of the epithelial-to-mesenchymal transition (EMT), we found a striking enrichment of RNA-binding proteins. We validated that QKI and RBFOX1 were necessary and sufficient to induce an intermediate mesenchymal cell state and increased tumorigenicity. Using RNA-seq and eCLIP analysis, we found that QKI and RBFOX1 coordinately regulated the splicing and function of the actin-binding protein FLNB, which plays a causal role in the regulation of EMT. Specifically, the skipping of FLNB exon 30 induced EMT by releasing the FOXC1 transcription factor. Moreover, skipping of FLNB exon 30 is strongly associated with EMT gene signatures in basal-like breast cancer patient samples. These observations identify a specific dysregulation of splicing, which regulates tumor cell plasticity and is frequently observed in human cancer.

Published
2018

38. A Next Generation Connectivity Map: L1000 Platform and the First 1,000,000 Profiles

Author

Subramanian, Aravind, Narayan, Rajiv, Corsello, Steven M, Peck, David D, Natoli, Ted E, Lu, Xiaodong, Gould, Joshua, Davis, John F, Tubelli, Andrew A, Asiedu, Jacob K, Lahr, David L, Hirschman, Jodi E, Liu, Zihan, Donahue, Melanie, Julian, Bina, Khan, Mariya, Wadden, David, Smith, Ian C, Lam, Daniel, Liberzon, Arthur, Toder, Courtney, Bagul, Mukta, Orzechowski, Marek, Enache, Oana M, Piccioni, Federica, Johnson, Sarah A, Lyons, Nicholas J, Berger, Alice H, Shamji, Alykhan F, Brooks, Angela N, Vrcic, Anita, Flynn, Corey, Rosains, Jacqueline, Takeda, David Y, Hu, Roger, Davison, Desiree, Lamb, Justin, Ardlie, Kristin, Hogstrom, Larson, Greenside, Peyton, Gray, Nathanael S, Clemons, Paul A, Silver, Serena, Wu, Xiaoyun, Zhao, Wen-Ning, Read-Button, Willis, Wu, Xiaohua, Haggarty, Stephen J, Ronco, Lucienne V, Boehm, Jesse S, Schreiber, Stuart L, Doench, John G, Bittker, Joshua A, Root, David E, Wong, Bang, and Golub, Todd R

Subjects
Biological Sciences, Bioinformatics and Computational Biology, Genetics, Aetiology, 2.1 Biological and endogenous factors, Cell Line, Tumor, Drug Resistance, Neoplasm, Gene Expression Profiling, Humans, Neoplasms, Organ Specificity, Pharmaceutical Preparations, Sequence Analysis, RNA, Small Molecule Libraries, Functional genomics, chemical biology, gene expression profiling, Medical and Health Sciences, Developmental Biology, Biological sciences, Biomedical and clinical sciences
Abstract

We previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs, and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high-throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.

Published
2017

39. Modular vector assembly enables rapid assessment of emerging CRISPR technologies

Author

McGee, Abby V., primary, Liu, Yanjing V., additional, Griffith, Audrey L., additional, Szegletes, Zsofia M., additional, Wen, Bronte, additional, Kraus, Carolyn, additional, Miller, Nathan W., additional, Steger, Ryan J., additional, Escude Velasco, Berta, additional, Bosch, Justin A., additional, Zirin, Jonathan D., additional, Viswanatha, Raghuvir, additional, Sontheimer, Erik J., additional, Goodale, Amy, additional, Greene, Matthew A., additional, Green, Thomas M., additional, and Doench, John G., additional

Published
2024
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40. Framework for in vivo T cell screens

Author

Milling, Lauren E., primary, Markson, Samuel C., additional, Tjokrosurjo, Qin, additional, Derosia, Nicole M., additional, Streeter, Ivy S.L., additional, Hickok, Grant H., additional, Lemmen, Ashlyn M., additional, Nguyen, Thao H., additional, Prathima, Priyamvada, additional, Fithian, William, additional, Schwartz, Marc A., additional, Hacohen, Nir, additional, Doench, John G., additional, LaFleur, Martin W., additional, and Sharpe, Arlene H., additional

Published
2024
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41. Mammalian SWI/SNF complex activity regulates POU2F3 and constitutes a targetable dependency in small cell lung cancer

Author

Duplaquet, Leslie, primary, So, Kevin, additional, Ying, Alexander W., additional, Li, Xinyue, additional, Li, Yixiang, additional, Qiu, Xintao, additional, Li, Rong, additional, Singh, Shilpa, additional, Wu, Xiaoli S., additional, Liu, Qi, additional, Qi, Jun, additional, Somerville, Tim D.D., additional, Heiling, Hillary, additional, Mazzola, Emanuele, additional, Lee, Yenarae, additional, Zoller, Thomas, additional, Vakoc, Christopher R., additional, Doench, John G, additional, Forrester, William C., additional, Abrams, Tinya, additional, Long, Henry W, additional, Niederst, Matthew J., additional, Kadoch, Cigall, additional, and Oser, Matthew G., additional

Published
2024
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42. Epigenetic silencing by SETDB1 suppresses tumour intrinsic immunogenicity

Author

Griffin, Gabriel K., Wu, Jingyi, Iracheta-Vellve, Arvin, Patti, James C., Hsu, Jeffrey, Davis, Thomas, Dele-Oni, Deborah, Du, Peter P., Halawi, Aya G., Ishizuka, Jeffrey J., Kim, Sarah Y., Klaeger, Susan, Knudsen, Nelson H., Miller, Brian C., Nguyen, Tung H., Olander, Kira E., Papanastasiou, Malvina, Rachimi, Suzanna, Robitschek, Emily J., Schneider, Emily M., Yeary, Mitchell D., Zimmer, Margaret D., Jaffe, Jacob D., Carr, Steven A., Doench, John G., Haining, W. Nicholas, Yates, Kathleen B., Manguso, Robert T., and Bernstein, Bradley E.

Published
2021
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43. Intercellular Mitochondria Transfer to Macrophages Regulates White Adipose Tissue Homeostasis and Is Impaired in Obesity

Author

Brestoff, Jonathan R., Wilen, Craig B., Moley, John R., Li, Yongjia, Zou, Wei, Malvin, Nicole P., Rowen, Marina N., Saunders, Brian T., Ma, Hongming, Mack, Madison R., Hykes, Barry L., Jr., Balce, Dale R., Orvedahl, Anthony, Williams, Jesse W., Rohatgi, Nidhi, Wang, Xiaoyan, McAllaster, Michael R., Handley, Scott A., Kim, Brian S., Doench, John G., Zinselmeyer, Bernd H., Diamond, Michael S., Virgin, Herbert W., Gelman, Andrew E., and Teitelbaum, Steven L.

Published
2021
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44. Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway

Author

Viswanathan, Vasanthi S, Ryan, Matthew J, Dhruv, Harshil D, Gill, Shubhroz, Eichhoff, Ossia M, Seashore-Ludlow, Brinton, Kaffenberger, Samuel D, Eaton, John K, Shimada, Kenichi, Aguirre, Andrew J, Viswanathan, Srinivas R, Chattopadhyay, Shrikanta, Tamayo, Pablo, Yang, Wan Seok, Rees, Matthew G, Chen, Sixun, Boskovic, Zarko V, Javaid, Sarah, Huang, Cherrie, Wu, Xiaoyun, Tseng, Yuen-Yi, Roider, Elisabeth M, Gao, Dong, Cleary, James M, Wolpin, Brian M, Mesirov, Jill P, Haber, Daniel A, Engelman, Jeffrey A, Boehm, Jesse S, Kotz, Joanne D, Hon, Cindy S, Chen, Yu, Hahn, William C, Levesque, Mitchell P, Doench, John G, Berens, Michael E, Shamji, Alykhan F, Clemons, Paul A, Stockwell, Brent R, and Schreiber, Stuart L

Subjects
Cancer, Cadherins, Cell Death, Cell Line, Tumor, Cell Lineage, Cell Transdifferentiation, Drug Resistance, Neoplasm, Epithelial-Mesenchymal Transition, Glutathione Peroxidase, Humans, Iron, Lipid Peroxidation, Lipid Peroxides, Male, Melanoma, Mesoderm, Neoplasms, Phospholipid Hydroperoxide Glutathione Peroxidase, Prostatic Neoplasms, Proteomics, Proto-Oncogene Proteins B-raf, Reproducibility of Results, Zinc Finger E-box-Binding Homeobox 1, General Science & Technology
Abstract

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFβ-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.

Published
2017

46. Genetic and Proteomic Interrogation of Lower Confidence Candidate Genes Reveals Signaling Networks in β-Catenin-Active Cancers

Author

Rosenbluh, Joseph, Mercer, Johnathan, Shrestha, Yashaswi, Oliver, Rachel, Tamayo, Pablo, Doench, John G, Tirosh, Itay, Piccioni, Federica, Hartenian, Ella, Horn, Heiko, Fagbami, Lola, Root, David E, Jaffe, Jacob, Lage, Kasper, Boehm, Jesse S, and Hahn, William C

Subjects
Biochemistry and Cell Biology, Biological Sciences, Cancer, Rare Diseases, Biotechnology, Genetics, 2.1 Biological and endogenous factors, Aetiology, CRISPR-Cas Systems, Clustered Regularly Interspaced Short Palindromic Repeats, Gene Editing, Genetic Therapy, Humans, Neoplasms, Proteomics, RNA, Guide, Kinetoplastida, RNA, Small Interfering, beta Catenin, CRISPR-Cas9, RNAi, genetic interactions, protein-protein interactions, β-catenin, Biochemistry and cell biology
Abstract

Genome-scale expression studies and comprehensive loss-of-function genetic screens have focused almost exclusively on the highest confidence candidate genes. Here, we describe a strategy for characterizing the lower confidence candidates identified by such approaches. We interrogated 177 genes that we classified as essential for the proliferation of cancer cells exhibiting constitutive β-catenin activity and integrated data for each of the candidates, derived from orthogonal short hairpin RNA (shRNA) knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated gene editing knockout screens, to yield 69 validated genes. We then characterized the relationships between sets of these genes using complementary assays: medium-throughput stable isotope labeling by amino acids in cell culture (SILAC)-based mass spectrometry, yielding 3,639 protein-protein interactions, and a CRISPR-mediated pairwise double knockout screen, yielding 375 combinations exhibiting greater- or lesser-than-additive phenotypic effects indicating genetic interactions. These studies identify previously unreported regulators of β-catenin, define functional networks required for the survival of β-catenin-active cancers, and provide an experimental strategy that may be applied to define other signaling networks.

Published
2016

47. Systematic Functional Interrogation of Rare Cancer Variants Identifies Oncogenic Alleles

Author

Kim, Eejung, Ilic, Nina, Shrestha, Yashaswi, Zou, Lihua, Kamburov, Atanas, Zhu, Cong, Yang, Xiaoping, Lubonja, Rakela, Tran, Nancy, Nguyen, Cindy, Lawrence, Michael S, Piccioni, Federica, Bagul, Mukta, Doench, John G, Chouinard, Candace R, Wu, Xiaoyun, Hogstrom, Larson, Natoli, Ted, Tamayo, Pablo, Horn, Heiko, Corsello, Steven M, Lage, Kasper, Root, David E, Subramanian, Aravind, Golub, Todd R, Getz, Gad, Boehm, Jesse S, and Hahn, William C

Subjects
Biological Sciences, Biomedical and Clinical Sciences, Bioinformatics and Computational Biology, Genetics, Oncology and Carcinogenesis, Cancer, Human Genome, Biotechnology, 2.1 Biological and endogenous factors, Aetiology, Alleles, Animals, Cell Line, Tumor, Cell Transformation, Neoplastic, Disease Models, Animal, Gene Expression Profiling, Genetic Association Studies, Genetic Predisposition to Disease, Genetic Variation, Heterografts, High-Throughput Screening Assays, Humans, Male, Mice, Neoplasms, Oncogenes, Reproducibility of Results, Biochemistry and cell biology, Oncology and carcinogenesis
Abstract

UnlabelledCancer genome characterization efforts now provide an initial view of the somatic alterations in primary tumors. However, most point mutations occur at low frequency, and the function of these alleles remains undefined. We have developed a scalable systematic approach to interrogate the function of cancer-associated gene variants. We subjected 474 mutant alleles curated from 5,338 tumors to pooled in vivo tumor formation assays and gene expression profiling. We identified 12 transforming alleles, including two in genes (PIK3CB, POT1) that have not been shown to be tumorigenic. One rare KRAS allele, D33E, displayed tumorigenicity and constitutive activation of known RAS effector pathways. By comparing gene expression changes induced upon expression of wild-type and mutant alleles, we inferred the activity of specific alleles. Because alleles found to be mutated only once in 5,338 tumors rendered cells tumorigenic, these observations underscore the value of integrating genomic information with functional studies.SignificanceExperimentally inferring the functional status of cancer-associated mutations facilitates the interpretation of genomic information in cancer. Pooled in vivo screen and gene expression profiling identified functional variants and demonstrated that expression of rare variants induced tumorigenesis. Variant phenotyping through functional studies will facilitate defining key somatic events in cancer. Cancer Discov; 6(7); 714-26. ©2016 AACR.See related commentary by Cho and Collisson, p. 694This article is highlighted in the In This Issue feature, p. 681.

Published
2016

49. Targetable leukemia dependency on noncanonical PI3Kγ signaling

Author

Luo, Qingyu, primary, Raulston, Evangeline G., additional, Prado, Miguel A., additional, Wu, Xiaowei, additional, Gritsman, Kira, additional, Yan, Kezhi, additional, Booth, Christopher A. G., additional, Xu, Ran, additional, van Galen, Peter, additional, Doench, John G., additional, Shimony, Shai, additional, Long, Henry W., additional, Neuberg, Donna S., additional, Paulo, Joao A., additional, and Lane, Andrew A., additional

Published
2023
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50. X-CHIME enables combinatorial, inducible, lineage-specific and sequential knockout of genes in the immune system

Author

LaFleur, Martin W., primary, Lemmen, Ashlyn M., additional, Streeter, Ivy S. L., additional, Nguyen, Thao H., additional, Milling, Lauren E., additional, Derosia, Nicole M., additional, Hoffman, Zachary M., additional, Gillis, Jacob E., additional, Tjokrosurjo, Qin, additional, Markson, Samuel C., additional, Huang, Amy Y., additional, Anekal, Praju V., additional, Montero Llopis, Paula, additional, Haining, W. Nicholas, additional, Doench, John G., additional, and Sharpe, Arlene H., additional

Published
2023
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