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1.
Commun Biol ; 4(1): 736, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34127790

RESUMO

Aggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future.


Assuntos
Redes e Vias Metabólicas/genética , Neurônios/metabolismo , Proteínas tau/metabolismo , Animais , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Edição de Genes , Genes/genética , Genes/fisiologia , Testes Genéticos/métodos , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Neuroblastoma/metabolismo , Ratos , Serina-Treonina Quinases TOR/metabolismo
2.
Cell Stem Cell ; 25(1): 39-53.e10, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31080135

RESUMO

Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEC-like organoids, suggesting that RSPO-LGR4/5-mediated WNT/ß-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPR-based loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/ß-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ductos Biliares/patologia , Proteínas de Ciclo Celular/metabolismo , Células Epiteliais/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína Axina/genética , Proteína Axina/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Modelos Animais de Doenças , Humanos , Regeneração Hepática , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Piridinas/toxicidade , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Trombospondinas/genética , Trombospondinas/metabolismo , Via de Sinalização Wnt , Proteínas de Sinalização YAP
3.
Cell Rep ; 27(2): 616-630.e6, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970262

RESUMO

Human pluripotent stem cells (hPSCs) generate a variety of disease-relevant cells that can be used to improve the translation of preclinical research. Despite the potential of hPSCs, their use for genetic screening has been limited by technical challenges. We developed a scalable and renewable Cas9 and sgRNA-hPSC library in which loss-of-function mutations can be induced at will. Our inducible mutant hPSC library can be used for multiple genome-wide CRISPR screens in a variety of hPSC-induced cell types. As proof of concept, we performed three screens for regulators of properties fundamental to hPSCs: their ability to self-renew and/or survive (fitness), their inability to survive as single-cell clones, and their capacity to differentiate. We identified the majority of known genes and pathways involved in these processes, as well as a plethora of genes with unidentified roles. This resource will increase the understanding of human development and genetics. This approach will be a powerful tool to identify disease-modifying genes and pathways.


Assuntos
Sistemas CRISPR-Cas/genética , Testes Genéticos/métodos , Genoma/genética , Células-Tronco Pluripotentes/metabolismo , Humanos
4.
Methods Mol Biol ; 1888: 153-174, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30519946

RESUMO

Chemogenomic profiling is a powerful and unbiased approach to elucidate pharmacological targets and the mechanism of bioactive compounds. It is based on identifying cellular hypersensitivity and resistance caused by individual gene modulations with genome-wide coverage. Due to the requirement of bar-coded, genome-wide deletion collections, high-resolution experiments of this nature have historically been limited to fungal systems. Pooled RNAi reagents have enabled similar attempts in mammalian cells but efforts have been hampered by significant off-target effects and experimental noise. The CRISPR/Cas9 system for the first time enables precise DNA editing at defined loci in a genome-wide fashion. Here we present the detailed protocol that leverages the CRISPR/Cas9 system for chemogenomic profiling and target identification of diverse chemical probes.


Assuntos
Sistemas CRISPR-Cas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Genômica , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Relação Dose-Resposta a Droga , Edição de Genes , Perfilação da Expressão Gênica/métodos , Vetores Genéticos/genética , Genômica/métodos , Haploinsuficiência , Humanos , Lentivirus/genética , RNA Guia de Cinetoplastídeos
5.
EMBO Rep ; 19(9)2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30126924

RESUMO

Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens, or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well-characterized autophagy-related proteins orchestrates the biogenesis of autophagosomes; however, the origin of the required membranes is incompletely understood. Here, we have applied sensitized pooled CRISPR screens and identify the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2- and DFCP1-positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and ß-oxidation of fatty acids. Immunostaining and interaction proteomics data suggest that TMEM41B localizes to the endoplasmic reticulum (ER). Taken together, we propose that TMEM41B is a novel ER-localized regulator of autophagosome biogenesis and lipid mobilization.


Assuntos
Autofagia/fisiologia , Mobilização Lipídica/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Autofagossomos/metabolismo , Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Retículo Endoplasmático/metabolismo , Ácidos Graxos/metabolismo , Técnicas de Inativação de Genes , Células HeLa , Homeostase , Humanos , Lentivirus , Gotículas Lipídicas/metabolismo , Mobilização Lipídica/genética , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo
6.
Nat Med ; 24(7): 939-946, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29892062

RESUMO

CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells1-3. Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells3-13. Here, using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), we achieved an average insertion or deletion (indel) efficiency greater than 80%. This high efficiency of indel generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs. In previous studies, the toxicity of Cas9 in hPSCs was less apparent because of low transfection efficiency and subsequently low DSB induction3. The toxic response to DSBs was P53/TP53-dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. Moreover, as hPSCs can acquire P53 mutations14, cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Engenharia Genética , Células-Tronco Pluripotentes/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Quebras de DNA de Cadeia Dupla , Deleção de Genes , Humanos , RNA Guia de Cinetoplastídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Receptor fas/genética , Receptor fas/metabolismo
7.
J Cell Biol ; 217(6): 1941-1955, 2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29588376

RESUMO

The multiciliated cell (MCC) is an evolutionarily conserved cell type, which in vertebrates functions to promote directional fluid flow across epithelial tissues. In the conducting airway, MCCs are generated by basal stem/progenitor cells and act in concert with secretory cells to perform mucociliary clearance to expel pathogens from the lung. Studies in multiple systems, including Xenopus laevis epidermis, murine trachea, and zebrafish kidney, have uncovered a transcriptional network that regulates multiple steps of multiciliogenesis, ultimately leading to an MCC with hundreds of motile cilia extended from their apical surface, which beat in a coordinated fashion. Here, we used a pool-based short hairpin RNA screening approach and identified TRRAP, an essential component of multiple histone acetyltransferase complexes, as a central regulator of MCC formation. Using a combination of immunofluorescence, signaling pathway modulation, and genomic approaches, we show that (a) TRRAP acts downstream of the Notch2-mediated basal progenitor cell fate decision and upstream of Multicilin to control MCC differentiation; and (b) TRRAP binds to the promoters and regulates the expression of a network of genes involved in MCC differentiation and function, including several genes associated with human ciliopathies.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cílios/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem da Célula , Epigênese Genética , Células Epiteliais/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Humanos , Pulmão/citologia , RNA Interferente Pequeno/metabolismo , Receptor Notch2 , Transdução de Sinais , Fatores de Transcrição
8.
Proc Natl Acad Sci U S A ; 115(2): E180-E189, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29269392

RESUMO

PARKIN, an E3 ligase mutated in familial Parkinson's disease, promotes mitophagy by ubiquitinating mitochondrial proteins for efficient engagement of the autophagy machinery. Specifically, PARKIN-synthesized ubiquitin chains represent targets for the PINK1 kinase generating phosphoS65-ubiquitin (pUb), which constitutes the mitophagy signal. Physiological regulation of PARKIN abundance, however, and the impact on pUb accumulation are poorly understood. Using cells designed to discover physiological regulators of PARKIN abundance, we performed a pooled genome-wide CRISPR/Cas9 knockout screen. Testing identified genes individually resulted in a list of 53 positive and negative regulators. A transcriptional repressor network including THAP11 was identified and negatively regulates endogenous PARKIN abundance. RNAseq analysis revealed the PARKIN-encoding locus as a prime THAP11 target, and THAP11 CRISPR knockout in multiple cell types enhanced pUb accumulation. Thus, our work demonstrates the critical role of PARKIN abundance, identifies regulating genes, and reveals a link between transcriptional repression and mitophagy, which is also apparent in human induced pluripotent stem cell-derived neurons, a disease-relevant cell type.


Assuntos
Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Genoma Humano/genética , Mitofagia/genética , Proteínas Repressoras/genética , Ubiquitina-Proteína Ligases/genética , Linhagem Celular Tumoral , Células Cultivadas , Células HCT116 , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Recém-Nascido , Neurônios/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
9.
Cell ; 170(3): 577-592.e10, 2017 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-28753431

RESUMO

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.


Assuntos
Neoplasias/genética , Neoplasias/patologia , Interferência de RNA , Linhagem Celular Tumoral , Biblioteca Gênica , Redes Reguladoras de Genes , Humanos , Complexos Multiproteicos/metabolismo , Neoplasias/metabolismo , Oncogenes , RNA Interferente Pequeno , Transdução de Sinais , Fatores de Transcrição/metabolismo
10.
Sci Rep ; 7: 42728, 2017 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-28205648

RESUMO

Chemogenomic profiling is a powerful and unbiased approach to elucidate pharmacological targets and the mechanism of bioactive compounds. Until recently, genome-wide, high-resolution experiments of this nature have been limited to fungal systems due to lack of mammalian genome-wide deletion collections. With the example of a novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, we demonstrate that the CRISPR/Cas9 system enables the generation of transient homo- and heterozygous deletion libraries and allows for the identification of efficacy targets and pathways mediating hypersensitivity and resistance relevant to the compound mechanism of action.


Assuntos
Sistemas CRISPR-Cas , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Células Cultivadas , Inibidores Enzimáticos/química , Deleção de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Testes Farmacogenômicos/métodos
11.
Mol Cell ; 63(4): 633-646, 2016 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-27499295

RESUMO

The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods. Furthermore, the repair outcomes are determined by the protospacer sequence rather than genomic context, indicating that DNA repair profiling in cell lines can be used to anticipate repair outcomes in primary cells. Chemical inhibition of DNA-PK enabled dissection of the DNA repair profiles into contributions from c-NHEJ and MMEJ. Finally, this work elucidates a strategy for using "error-prone" DNA-repair machinery to generate precise edits.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Endonucleases/metabolismo , Edição de Genes , Perfilação da Expressão Gênica/métodos , Proteínas de Bactérias/genética , Proteína 9 Associada à CRISPR , Endonucleases/genética , Células HCT116 , Células HEK293 , Humanos , Células K562 , Interferência de RNA , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Fatores de Tempo , Transfecção
12.
Elife ; 52016 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-27351204

RESUMO

SQSTM1 is an adaptor protein that integrates multiple cellular signaling pathways and whose expression is tightly regulated at the transcriptional and post-translational level. Here, we describe a forward genetic screening paradigm exploiting CRISPR-mediated genome editing coupled to a cell selection step by FACS to identify regulators of SQSTM1. Through systematic comparison of pooled libraries, we show that CRISPR is superior to RNAi in identifying known SQSTM1 modulators. A genome-wide CRISPR screen exposed MTOR signalling and the entire macroautophagy machinery as key regulators of SQSTM1 and identified several novel modulators including HNRNPM, SLC39A14, SRRD, PGK1 and the ufmylation cascade. We show that ufmylation regulates SQSTM1 by eliciting a cell type-specific ER stress response which induces SQSTM1 expression and results in its accumulation in the cytosol. This study validates pooled CRISPR screening as a powerful method to map the repertoire of cellular pathways that regulate the fate of an individual target protein.


Assuntos
Regulação da Expressão Gênica , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Proteína Sequestossoma-1/metabolismo , Autofagia , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Citometria de Fluxo , Marcação de Genes , Testes Genéticos , Humanos , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
13.
Science ; 351(6278): 1208-13, 2016 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-26912361

RESUMO

5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA-mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in an MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP-deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Metionina/metabolismo , Neoplasias/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Purina-Núcleosídeo Fosforilase/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Inibidor p16 de Quinase Dependente de Ciclina/genética , Desoxiadenosinas/metabolismo , Deleção de Genes , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína-Arginina N-Metiltransferases/genética , Purina-Núcleosídeo Fosforilase/genética , RNA Interferente Pequeno/genética , Tionucleosídeos/metabolismo
14.
Proc Natl Acad Sci U S A ; 113(1): 182-7, 2016 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-26677873

RESUMO

Macroautophagy is a key stress-response pathway that can suppress or promote tumorigenesis depending on the cellular context. Notably, Kirsten rat sarcoma (KRAS)-driven tumors have been reported to rely on macroautophagy for growth and survival, suggesting a potential therapeutic approach of using autophagy inhibitors based on genetic stratification. In this study, we evaluated whether KRAS mutation status can predict the efficacy to macroautophagy inhibition. By profiling 47 cell lines with pharmacological and genetic loss-of-function tools, we were unable to confirm that KRAS-driven tumor lines require macroautophagy for growth. Deletion of autophagy-related 7 (ATG7) by genome editing completely blocked macroautophagy in several tumor lines with oncogenic mutations in KRAS but did not inhibit cell proliferation in vitro or tumorigenesis in vivo. Furthermore, ATG7 knockout did not sensitize cells to irradiation or to several anticancer agents tested. Interestingly, ATG7-deficient and -proficient cells were equally sensitive to the antiproliferative effect of chloroquine, a lysosomotropic agent often used as a pharmacological tool to evaluate the response to macroautophagy inhibition. Moreover, both cell types manifested synergistic growth inhibition when treated with chloroquine plus the tyrosine kinase inhibitors erlotinib or sunitinib, suggesting that the antiproliferative effects of chloroquine are independent of its suppressive actions on autophagy.


Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Cloroquina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Autofagia/genética , Proteína 7 Relacionada à Autofagia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Cloridrato de Erlotinib/farmacologia , Técnicas de Inativação de Genes , Humanos , Indóis/farmacologia , Mutação , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Tolerância a Radiação/genética , Sunitinibe , Enzimas Ativadoras de Ubiquitina/genética
15.
Proc Natl Acad Sci U S A ; 112(1): E21-9, 2015 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-25524627

RESUMO

The mammalian target of rapamycin complex 1 (mTORC1) integrates multiple signals from growth factors, nutrients, and cellular energy status to control a wide range of metabolic processes, including mRNA biogenesis; protein, nucleotide, and lipid synthesis; and autophagy. Deregulation of the mTORC1 pathway is found in cancer as well as genetic disorders such as tuberous sclerosis complex (TSC) and sporadic lymphangioleiomyomatosis. Recent studies have shown that the mTORC1 inhibitor rapamycin and its analogs generally suppress proliferation rather than induce apoptosis. Therefore, it is critical to use alternative strategies to induce death of cells with activated mTORC1. In this study, a small-molecule screen has revealed that the combination of glutaminase (GLS) and heat shock protein 90 (Hsp90) inhibitors selectively triggers death of TSC2-deficient cells. At a mechanistic level, high mTORC1-driven translation rates in TSC1/2-deficient cells, unlike wild-type cells, sensitizes these cells to endoplasmic reticulum (ER) stress. Thus, Hsp90 inhibition drives accumulation of unfolded protein and ER stress. When combining proteotoxic stress with oxidative stress by depletion of the intracellular antioxidant glutathione by GLS inhibition, acute cell death is observed in cells with activated mTORC1 signaling. This study suggests that this combination strategy may have the potential to be developed into a therapeutic use for the treatment of mTORC1-driven tumors.


Assuntos
Glutaminase/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Esclerose Tuberosa/metabolismo , Esclerose Tuberosa/patologia , Animais , Apoptose/efeitos dos fármacos , Benzoquinonas/farmacologia , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glutamato Desidrogenase/antagonistas & inibidores , Glutamato Desidrogenase/metabolismo , Glutaminase/metabolismo , Glutamina/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Modelos Biológicos , Oxirredução/efeitos dos fármacos , Fenótipo , Sirolimo/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfetos/farmacologia , Tiadiazóis/farmacologia , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Proc Natl Acad Sci U S A ; 111(8): 3128-33, 2014 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-24520176

RESUMO

Defects in epigenetic regulation play a fundamental role in the development of cancer, and epigenetic regulators have recently emerged as promising therapeutic candidates. We therefore set out to systematically interrogate epigenetic cancer dependencies by screening an epigenome-focused deep-coverage design shRNA (DECODER) library across 58 cancer cell lines. This screen identified BRM/SMARCA2, a DNA-dependent ATPase of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex, as being essential for the growth of tumor cells that harbor loss of function mutations in BRG1/SMARCA4. Depletion of BRM in BRG1-deficient cancer cells leads to a cell cycle arrest, induction of senescence, and increased levels of global H3K9me3. We further demonstrate the selective dependency of BRG1-mutant tumors on BRM in vivo. Genetic alterations of the mSWI/SNF chromatin remodeling complexes are the most frequent among chromatin regulators in cancers, with BRG1/SMARCA4 mutations occurring in ∼10-15% of lung adenocarcinomas. Our findings position BRM as an attractive therapeutic target for BRG1 mutated cancers. Because BRG1 and BRM function as mutually exclusive catalytic subunits of the mSWI/SNF complex, we propose that such synthetic lethality may be explained by paralog insufficiency, in which loss of one family member unveils critical dependence on paralogous subunits. This concept of "cancer-selective paralog dependency" may provide a more general strategy for targeting other tumor suppressor lesions/complexes with paralogous subunits.


Assuntos
DNA Helicases/deficiência , Epigênese Genética/fisiologia , Complexos Multiproteicos/genética , Neoplasias/genética , Proteínas Nucleares/deficiência , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Western Blotting , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Senescência Celular/genética , Técnicas de Silenciamento de Genes , Biblioteca Gênica , Histonas/metabolismo , Humanos , Imunoprecipitação , Complexos Multiproteicos/metabolismo , RNA Interferente Pequeno/genética , Fatores de Transcrição/metabolismo
17.
Mol Cell ; 49(1): 172-85, 2013 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-23142078

RESUMO

The metabolism of glucose and glutamine, primary carbon sources utilized by mitochondria to generate energy and macromolecules for cell growth, is directly regulated by mTORC1. We show that glucose and glutamine, by supplying carbons to the TCA cycle to produce ATP, positively feed back to mTORC1 through an AMPK-, TSC1/2-, and Rag-independent mechanism by regulating mTORC1 assembly and its lysosomal localization. We discovered that the ATP-dependent TTT-RUVBL1/2 complex was disassembled and repressed by energy depletion, resulting in its decreased interaction with mTOR. The TTT-RUVBL complex was necessary for the interaction between mTORC1 and Rag and formation of mTORC1 obligate dimers. In cancer tissues, TTT-RUVBL complex mRNAs were elevated and positively correlated with transcripts encoding proteins of anabolic metabolism and mitochondrial function-all mTORC1-regulated processes. Thus, the TTT-RUVBL1/2 complex responds to the cell's metabolic state, directly regulating the functional assembly of mTORC1 and indirectly controlling the nutrient signal from Rags to mTORC1.


Assuntos
Metabolismo Energético , Lisossomos/metabolismo , Proteínas/metabolismo , Estresse Fisiológico , ATPases Associadas a Diversas Atividades Celulares , Trifosfato de Adenosina/metabolismo , Adenilato Quinase/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Ciclo do Ácido Cítrico , DNA Helicases/genética , DNA Helicases/metabolismo , Feminino , Glucose/deficiência , Glutamina/deficiência , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transdução de Sinais , Estatísticas não Paramétricas , Serina-Treonina Quinases TOR , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
18.
Science ; 332(6035): 1322-6, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21659605

RESUMO

The evolutionarily conserved serine-threonine kinase mammalian target of rapamycin (mTOR) plays a critical role in regulating many pathophysiological processes. Functional characterization of the mTOR signaling pathways, however, has been hampered by the paucity of known substrates. We used large-scale quantitative phosphoproteomics experiments to define the signaling networks downstream of mTORC1 and mTORC2. Characterization of one mTORC1 substrate, the growth factor receptor-bound protein 10 (Grb10), showed that mTORC1-mediated phosphorylation stabilized Grb10, leading to feedback inhibition of the phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated, mitogen-activated protein kinase (ERK-MAPK) pathways. Grb10 expression is frequently down-regulated in various cancers, and loss of Grb10 and loss of the well-established tumor suppressor phosphatase PTEN appear to be mutually exclusive events, suggesting that Grb10 might be a tumor suppressor regulated by mTORC1.


Assuntos
Proteína Adaptadora GRB10/metabolismo , Insulina/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Dados de Sequência Molecular , Complexos Multiproteicos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR
19.
Curr Protoc Chem Biol ; 3(1): 39-52, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23836588

RESUMO

The in-cell western (ICW) technique is a cell-based immunoassay method for quantitative measurement of protein expression or phosphorylation levels that can be used for both small molecule and siRNA screening. The method involves growth of cells in microplates, fixation, permeabilization, and staining with specific antibodies and/or cell labeling dyes. ICW assays take advantage of the properties of near-infrared dyes to achieve higher signal-to-noise ratios than are possible for methods utilizing fluorophores in the visible range of the spectrum, and typically involve measurements using two fluorescent channels: one to measure levels of the target of interest, and one to measure total cell number for normalization. The ICW method is readily adaptable to high-throughput format and has been successfully used with a variety of targets and cell lines. The protocols in this unit describe an ICW procedure for quantitative measurement of rpS6-phosphorylation as an endpoint for monitoring mTORC1 signaling in HeLa cells. This assay can be used for small molecule or siRNA screening, and with modification is adaptable to other cell lines and targets. Curr. Protoc. Chem. Biol. 3:39-52 © 2011 by John Wiley & Sons, Inc.

20.
Assay Drug Dev Technol ; 8(2): 186-99, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20085456

RESUMO

The mTORC1 pathway is a central regulator of cell growth, and defective mTORC1 regulation plays a causative role in a variety of human diseases, including cancer, tumor syndromes such as the tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), and metabolic diseases such as diabetes and obesity. Given the importance of mTORC1 signaling in these diseases, there has been significant interest in developing screening methods suitable for identifying inhibitors of mTORC1 activation. To this end, we have developed a high-throughput, cell-based assay for the detection of rpS6-phosphorylation as a measure of mTORC1 signaling. This assay takes advantage of the "In Cell Western" (ICW) technique using the Aerius infrared imaging system (LI-COR Biosciences). The ICW procedure involves fixation and immunostaining of cells in a manner similar to standard immunofluorescence methods but takes advantage of secondary antibodies conjugated to infrared-excitable fluorophores for quantitative detection by the Aerius scanner. In addition, the cells are stained with an infrared-excitable succinimidyl ester dye, which covalently modifies free amine groups in fixed cells and provides a quantitative measure of cell number. We present validation data and pilot screens in a 384-well format demonstrating that this assay provides a statistically robust method for both small molecule and siRNA screening approaches designed to identify inhibitors of mTORC1 signaling.


Assuntos
RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Aminas/química , Aminas/efeitos da radiação , Especificidade de Anticorpos , Western Blotting , Contagem de Células , Sobrevivência Celular , Avaliação Pré-Clínica de Medicamentos , Determinação de Ponto Final , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa , Humanos , Raios Infravermelhos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , Proteínas , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas , Serina-Treonina Quinases TOR , Transfecção
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