RESUMO
Tunicamycin (TM) inhibits eukaryotic asparagine-linked glycosylation, protein palmitoylation, ganglioside production, proteoglycan synthesis, 3-hydroxy-3-methylglutaryl coenzyme-A reductase activity, and cell wall biosynthesis in bacteria. Treatment of cells with TM elicits endoplasmic reticulum stress and activates the unfolded protein response. Although widely used in laboratory settings for many years, it is unknown how TM enters cells. Here, we identify in an unbiased genetic screen a transporter of the major facilitator superfamily, major facilitator domain containing 2A (MFSD2A), as a critical mediator of TM toxicity. Cells without MFSD2A are TM-resistant, whereas MFSD2A-overexpressing cells are hypersensitive. Hypersensitivity is associated with increased cellular TM uptake concomitant with an enhanced endoplasmic reticulum stress response. Furthermore, MFSD2A mutant analysis reveals an important function of the C terminus for correct intracellular localization and protein stability, and it identifies transmembrane helical amino acid residues essential for mediating TM sensitivity. Overall, our data uncover a critical role for MFSD2A by acting as a putative TM transporter at the plasma membrane.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Tunicamicina/farmacologia , Western Blotting , Linhagem Celular , Sobrevivência Celular , Glicosilação/efeitos dos fármacos , Humanos , Imunoprecipitação , Microscopia Confocal , Simportadores , Proteínas Supressoras de Tumor/genéticaRESUMO
Induced pluripotent stem cells (iPSCs) can be generated from various differentiated cell types by the expression of a set of defined transcription factors. So far, iPSCs have been generated from primary cells, but it is unclear whether human cancer cell lines can be reprogrammed. Here we describe the generation and characterization of iPSCs derived from human chronic myeloid leukemia cells. We show that, despite the presence of oncogenic mutations, these cells acquired pluripotency by the expression of 4 transcription factors and underwent differentiation into cell types derived of all 3 germ layers during teratoma formation. Interestingly, although the parental cell line was strictly dependent on continuous signaling of the BCR-ABL oncogene, also termed oncogene addiction, reprogrammed cells lost this dependency and became resistant to the BCR-ABL inhibitor imatinib. This finding indicates that the therapeutic agent imatinib targets cells in a specific epigenetic differentiated cell state, and this may contribute to its inability to fully eradicate disease in chronic myeloid leukemia patients.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Benzamidas , Células Cultivadas , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/genética , Humanos , Mesilato de Imatinib , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Pirimidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de SinaisRESUMO
CD3-engaging bispecific antibodies (BsAbs) enable the formation of an immune synapse between T cells and tumor cells, resulting in robust target cell killing not dependent on a preexisting tumor specific T cell receptor. While recent studies have shed light on tumor cell-specific factors that modulate BsAb sensitivity, the T cell-intrinsic determinants of BsAb efficacy and response durability are poorly understood. To better clarify the genes that shape BsAb-induced T cell responses, we conducted targeted analyses and a large-scale unbiased in vitro CRISPR/Cas9-based screen to identify negative regulators of BsAb-induced T cell proliferation. These analyses revealed that CD8+ T cells are dependent on CD4+ T cell-derived signaling factors in order to achieve sustained killing in vitro. Moreover, the mammalian target of rapamycin (mTOR) pathway and several other candidate genes were identified as intrinsic regulators of BsAb-induced T cell proliferation and/or activation, highlighting promising approaches to enhancing the utility of these potent therapeutics.
Assuntos
Anticorpos Biespecíficos , Neoplasias , Anticorpos Biespecíficos/farmacologia , Formação de Anticorpos , Humanos , Ativação Linfocitária/genética , Receptores de Antígenos de Linfócitos TRESUMO
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/metabolismoRESUMO
SHP2 is a ubiquitous tyrosine phosphatase involved in regulating both tumor and immune cell signaling. In this study, we discovered a novel immune modulatory function of SHP2. Targeting this protein with allosteric SHP2 inhibitors promoted anti-tumor immunity, including enhancing T cell cytotoxic function and immune-mediated tumor regression. Knockout of SHP2 using CRISPR/Cas9 gene editing showed that targeting SHP2 in cancer cells contributes to this immune response. Inhibition of SHP2 activity augmented tumor intrinsic IFNγ signaling resulting in enhanced chemoattractant cytokine release and cytotoxic T cell recruitment, as well as increased expression of MHC Class I and PD-L1 on the cancer cell surface. Furthermore, SHP2 inhibition diminished the differentiation and inhibitory function of immune suppressive myeloid cells in the tumor microenvironment. SHP2 inhibition enhanced responses to anti-PD-1 blockade in syngeneic mouse models. Overall, our study reveals novel functions of SHP2 in tumor immunity and proposes that targeting SHP2 is a promising strategy for cancer immunotherapy.
Assuntos
Imunidade Celular , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Transdução de Sinais/genéticaRESUMO
Nitrogen-containing bisphosphonates (N-BPs), such as alendronate, are the most widely prescribed medications for diseases involving bone, with nearly 200 million prescriptions written annually. Recently, widespread use of N-BPs has been challenged due to the risk of rare but traumatic side effects such as atypical femoral fracture (AFF) and osteonecrosis of the jaw (ONJ). N-BPs bind to and inhibit farnesyl diphosphate synthase, resulting in defects in protein prenylation. Yet, it remains poorly understood what other cellular factors might allow N-BPs to exert their pharmacological effects. Here, we performed genome-wide studies in cells and patients to identify the poorly characterized gene, ATRAID Loss of ATRAID function results in selective resistance to N-BP-mediated loss of cell viability and the prevention of alendronate-mediated inhibition of prenylation. ATRAID is required for alendronate inhibition of osteoclast function, and ATRAID-deficient mice have impaired therapeutic responses to alendronate in both postmenopausal and senile (old age) osteoporosis models. Last, we performed exome sequencing on patients taking N-BPs that suffered ONJ or an AFF. ATRAID is one of three genes that contain rare nonsynonymous coding variants in patients with ONJ or an AFF that is also differentially expressed in poor outcome groups of patients treated with N-BPs. We functionally validated this patient variation in ATRAID as conferring cellular hypersensitivity to N-BPs. Our work adds key insight into the mechanistic action of N-BPs and the processes that might underlie differential responsiveness to N-BPs in people.
Assuntos
Difosfonatos , Nitrogênio , Alendronato/farmacologia , Animais , Osso e Ossos , Difosfonatos/farmacologia , Difosfonatos/uso terapêutico , Humanos , Camundongos , OsteoclastosRESUMO
Two major mRNA isoforms arise via alternative splicing in the 5'-UTR of Drosophila splicing assembly factor rnp-4f pre-mRNA, designated "long" (unspliced) and "short" (alternatively spliced). The coding potential for the two isoforms is identical, raising interesting questions as to the control mechanism and functional significance of this 5'-UTR intronic splicing decision. Developmental Northerns show that two temporally distinct rnp-4f mRNA degradation episodes occur during embryogenesis. The first occurs at the midblastula transition (MBT) stage and involves degradation of both maternally-derived transcripts, while the second involves only the long mRNA isoform and occurs during late embryo stages. Immunostaining of ovaries and staged embryos combined with results from developmental Westerns shows that maternal RNP-4F protein persists into fertilized eggs at high levels, associated with a burst of long isoform-specific transcription which begins just after the MBT and peaks in mid-embryo stages. These observations are discussed in support of a putative negative feedback control model for modulation of RNP-4F translation. In situ hybridization shows that the long isoform is relatively abundant throughout the developing embryonic germ band and central nervous system (CNS), especially along the dorsal roof of the ventral nerve cord. Long rnp-4f mRNA knockdown via RNAi reveals a variety of CNS abnormalities, which leads us to postulate that this isoform acts upstream of other genes which have been shown to be important for normal CNS development.
Assuntos
Regiões 5' não Traduzidas , Processamento Alternativo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Sistema Nervoso Central/embriologia , Sistema Nervoso Central/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Íntrons , Interferência de RNA , RNA Mensageiro/genética , Ribonucleoproteínas/genéticaRESUMO
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/métodosRESUMO
Proteasomes are central regulators of protein homeostasis in eukaryotes. Proteasome function is vulnerable to environmental insults, cellular protein imbalance and targeted pharmaceuticals. Yet, mechanisms that cells deploy to counteract inhibition of this central regulator are little understood. To find such mechanisms, we reduced flux through the proteasome to the point of toxicity with specific inhibitors and performed genome-wide screens for mutations that allowed cells to survive. Counter to expectation, reducing expression of individual subunits of the proteasome's 19S regulatory complex increased survival. Strong 19S reduction was cytotoxic but modest reduction protected cells from inhibitors. Protection was accompanied by an increased ratio of 20S to 26S proteasomes, preservation of protein degradation capacity and reduced proteotoxic stress. While compromise of 19S function can have a fitness cost under basal conditions, it provided a powerful survival advantage when proteasome function was impaired. This means of rebalancing proteostasis is conserved from yeast to humans.
Assuntos
Células Epiteliais/fisiologia , Hepatócitos/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Linhagem Celular , Sobrevivência Celular , HumanosRESUMO
We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using a biotinylated N-linked glycosylation reporter peptide attached to CTA1, we provide direct evidence that ~12% of the internalized CTA1 pool reaches the ER. We also explored the sortase labeling method to attach the catalytic subunit of diphtheria toxin as a toxic warhead to CTA1, thus converting CTx into a cytolethal toxin. This new toxin conjugate enabled us to conduct a genetic screen in human cells, which identified ST3GAL5, SLC35A2, B3GALT4, UGCG, and ELF4 as genes essential for CTx intoxication. The first four encode proteins involved in the synthesis of gangliosides, which are known receptors for CTx. Identification and isolation of the ST3GAL5 and SLC35A2 mutant clonal cells uncover a previously unappreciated differential contribution of gangliosides to intoxication by CTx.
Assuntos
Toxina da Cólera/toxicidade , Toxina Diftérica/química , Transporte Proteico/genética , Aminoaciltransferases/química , Aminoaciltransferases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Células Cultivadas , Cólera/fisiopatologia , Toxina da Cólera/química , Toxina da Cólera/genética , Clonagem Molecular , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Gangliosídeos/metabolismo , Gangliosídeos/fisiologia , Engenharia Genética , Haploidia , Humanos , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas de Transporte de Monossacarídeos/fisiologia , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/toxicidade , Análise de Sequência de Proteína , Sialiltransferases/química , Sialiltransferases/metabolismo , Sialiltransferases/fisiologiaRESUMO
Insertional mutagenesis in a haploid background can disrupt gene function. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT.
Assuntos
Estudos de Associação Genética/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Fenótipo , Alelos , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Linhagem Celular Tumoral , Cromossomos Humanos , Clonagem Molecular , Genoma Humano , Bactérias Gram-Negativas/patogenicidade , Haploidia , Humanos , Mutagênese Insercional , Mutação , Interferência de RNA , Sitios de Sequências Rotuladas , Fatores de Virulência/análise , Fatores de Virulência/metabolismoRESUMO
Loss-of-function genetic screens in model organisms have elucidated numerous biological processes, but the diploid genome of mammalian cells has precluded large-scale gene disruption. We used insertional mutagenesis to develop a screening method to generate null alleles in a human cell line haploid for all chromosomes except chromosome 8. Using this approach, we identified host factors essential for infection with influenza and genes encoding important elements of the biosynthetic pathway of diphthamide, which are required for the cytotoxic effects of diphtheria toxin and exotoxin A. We also identified genes needed for the action of cytolethal distending toxin, including a cell-surface protein that interacts with the toxin. This approach has both conceptual and practical parallels with genetic approaches in haploid yeast.