RESUMO
Selectively ablating damaged cells is an evolving therapeutic approach for age-related disease. Current methods for genome-wide screens to identify genes whose deletion might promote the death of damaged or senescent cells are generally underpowered because of the short timescales of cell death as well as the difficulty of scaling non-dividing cells. Here, we establish "Death-seq," a positive-selection CRISPR screen optimized to identify enhancers and mechanisms of cell death. Our screens identified synergistic enhancers of cell death induced by the known senolytic ABT-263. The screen also identified inducers of cell death and senescent cell clearance in models of age-related diseases by a related compound, ABT-199, which alone is not senolytic but exhibits less toxicity than ABT-263. Death-seq enables the systematic screening of cell death pathways to uncover molecular mechanisms of regulated cell death subroutines and identifies drug targets for the treatment of diverse pathological states such as senescence, cancer, and fibrosis.
Assuntos
Senescência Celular , Senoterapia , Senescência Celular/genética , Morte Celular , Compostos de AnilinaRESUMO
Ammonia is a ubiquitous, toxic by-product of cell metabolism. Its high membrane permeability and proton affinity causes ammonia to accumulate inside acidic lysosomes in its poorly membrane-permeant form: ammonium (NH 4 + ). Ammonium buildup compromises lysosomal function, suggesting the existence of mechanisms that protect cells from ammonium toxicity. Here, we identified SLC12A9 as a lysosomal ammonium exporter that preserves lysosomal homeostasis. SLC12A9 knockout cells showed grossly enlarged lysosomes and elevated ammonium content. These phenotypes were reversed upon removal of the metabolic source of ammonium or dissipation of the lysosomal pH gradient. Lysosomal chloride increased in SLC12A9 knockout cells and chloride binding by SLC12A9 was required for ammonium transport. Our data indicate that SLC12A9 is a chloride-driven ammonium co-transporter that is central in an unappreciated, fundamental mechanism of lysosomal physiology that may have special relevance in tissues with elevated ammonia, such as tumors.
RESUMO
Lysosomes degrade macromolecules and recycle their nutrient content to support cell function and survival. However, the machineries involved in lysosomal recycling of many nutrients remain to be discovered, with a notable example being choline, an essential metabolite liberated via lipid degradation. Here, we engineered metabolic dependency on lysosome-derived choline in pancreatic cancer cells to perform an endolysosome-focused CRISPR-Cas9 screen for genes mediating lysosomal choline recycling. We identified the orphan lysosomal transmembrane protein SPNS1 as critical for cell survival under choline limitation. SPNS1 loss leads to intralysosomal accumulation of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Mechanistically, we reveal that SPNS1 is a proton gradient-dependent transporter of LPC species from the lysosome for their re-esterification into phosphatidylcholine in the cytosol. Last, we establish that LPC efflux by SPNS1 is required for cell survival under choline limitation. Collectively, our work defines a lysosomal phospholipid salvage pathway that is essential under nutrient limitation and, more broadly, provides a robust platform to deorphan lysosomal gene function.
Assuntos
Colina , Fosfolipídeos , Colina/metabolismo , Sobrevivência Celular , Fosfolipídeos/metabolismo , Fosfatidilcolinas/metabolismo , Lisossomos/metabolismoRESUMO
Monoclonal antibody therapies targeting tumour antigens drive cancer cell elimination in large part by triggering macrophage phagocytosis of cancer cells1-7. However, cancer cells evade phagocytosis using mechanisms that are incompletely understood. Here we develop a platform for unbiased identification of factors that impede antibody-dependent cellular phagocytosis (ADCP) using complementary genome-wide CRISPR knockout and overexpression screens in both cancer cells and macrophages. In cancer cells, beyond known factors such as CD47, we identify many regulators of susceptibility to ADCP, including the poorly characterized enzyme adipocyte plasma membrane-associated protein (APMAP). We find that loss of APMAP synergizes with tumour antigen-targeting monoclonal antibodies and/or CD47-blocking monoclonal antibodies to drive markedly increased phagocytosis across a wide range of cancer cell types, including those that are otherwise resistant to ADCP. Additionally, we show that APMAP loss synergizes with several different tumour-targeting monoclonal antibodies to inhibit tumour growth in mice. Using genome-wide counterscreens in macrophages, we find that the G-protein-coupled receptor GPR84 mediates enhanced phagocytosis of APMAP-deficient cancer cells. This work reveals a cancer-intrinsic regulator of susceptibility to antibody-driven phagocytosis and, more broadly, expands our knowledge of the mechanisms governing cancer resistance to macrophage phagocytosis.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/genética , Sistemas CRISPR-Cas , Citofagocitose/genética , Macrófagos/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Animais , Anticorpos Monoclonais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos de Neoplasias/imunologia , Antígeno CD47/antagonistas & inibidores , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Edição de Genes , Técnicas de Inativação de Genes , Humanos , Linfoma de Células T/imunologia , Linfoma de Células T/patologia , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Receptores Acoplados a Proteínas G/metabolismoRESUMO
A central question in the post-genomic era is how genes interact to form biological pathways. Measurements of gene dependency across hundreds of cell lines have been used to cluster genes into 'co-essential' pathways, but this approach has been limited by ubiquitous false positives. In the present study, we develop a statistical method that enables robust identification of gene co-essentiality and yields a genome-wide set of functional modules. This atlas recapitulates diverse pathways and protein complexes, and predicts the functions of 108 uncharacterized genes. Validating top predictions, we show that TMEM189 encodes plasmanylethanolamine desaturase, a key enzyme for plasmalogen synthesis. We also show that C15orf57 encodes a protein that binds the AP2 complex, localizes to clathrin-coated pits and enables efficient transferrin uptake. Finally, we provide an interactive webtool for the community to explore our results, which establish co-essentiality profiling as a powerful resource for biological pathway identification and discovery of new gene functions.
Assuntos
Redes Reguladoras de Genes , Genes , Genoma , Clatrina/metabolismo , Endocitose , Epigênese Genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Anotação de Sequência Molecular , Neoplasias/genética , Plasmalogênios/biossíntese , Transdução de Sinais/genéticaRESUMO
Activating mutations in RAS GTPases drive many cancers, but limited understanding of less-studied RAS interactors, and of the specific roles of different RAS interactor paralogs, continues to limit target discovery. We developed a multistage discovery and screening process to systematically identify genes conferring RAS-related susceptibilities in lung adenocarcinoma. Using affinity purification mass spectrometry, we generated a protein-protein interaction map of RAS interactors and pathway components containing hundreds of interactions. From this network, we constructed a CRISPR dual knockout library targeting 119 RAS-related genes that we screened for KRAS-dependent genetic interactions (GI). This approach identified new RAS effectors, including the adhesion controller RADIL and the endocytosis regulator RIN1, and >250 synthetic lethal GIs, including a potent KRAS-dependent interaction between RAP1GDS1 and RHOA. Many GIs link specific paralogs within and between gene families. These findings illustrate the power of multiomic approaches to uncover synthetic lethal combinations specific for hitherto untreatable cancer genotypes. SIGNIFICANCE: We establish a deep network of protein-protein and genetic interactions in the RAS pathway. Many interactions validated here demonstrate important specificities and redundancies among paralogous RAS regulators and effectors. By comparing synthetic lethal interactions across KRAS-dependent and KRAS-independent cell lines, we identify several new combination therapy targets for RAS-driven cancers.This article is highlighted in the In This Issue feature, p. 1775.
Assuntos
Neoplasias Pulmonares/genética , Proteômica/métodos , Proteínas ras/genética , HumanosRESUMO
Cancer genomics studies have identified thousands of putative cancer driver genes1. Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif2 from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities.
Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Cultura de Células/métodos , Proliferação de Células/genética , Genoma Humano/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Esferoides Celulares/patologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Motivos de Aminoácidos , Animais , Carboxipeptidases/antagonistas & inibidores , Carboxipeptidases/deficiência , Carboxipeptidases/genética , Carboxipeptidases/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Terapia de Alvo Molecular , Mutação , Fenótipo , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Esferoides Celulares/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pooled CRISPR-Cas9 screens are a powerful method for functionally characterizing regulatory elements in the non-coding genome, but off-target effects in these experiments have not been systematically evaluated. Here, we investigate Cas9, dCas9, and CRISPRi/a off-target activity in screens for essential regulatory elements. The sgRNAs with the largest effects in genome-scale screens for essential CTCF loop anchors in K562 cells were not single guide RNAs (sgRNAs) that disrupted gene expression near the on-target CTCF anchor. Rather, these sgRNAs had high off-target activity that, while only weakly correlated with absolute off-target site number, could be predicted by the recently developed GuideScan specificity score. Screens conducted in parallel with CRISPRi/a, which do not induce double-stranded DNA breaks, revealed that a distinct set of off-targets also cause strong confounding fitness effects with these epigenome-editing tools. Promisingly, filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded sgRNAs and enabled identification of essential regulatory elements.