RESUMO
Interrogation of cellular metabolism with high-throughput screening approaches can unravel contextual biology and identify cancer-specific metabolic vulnerabilities. To systematically study the consequences of distinct metabolic perturbations, we assemble a comprehensive metabolic drug library (CeMM Library of Metabolic Drugs; CLIMET) covering 243 compounds. We, next, characterize it phenotypically in a diverse panel of myeloid leukemia cell lines and primary patient cells. Analysis of the drug response profiles reveals that 77 drugs affect cell viability, with the top effective compounds targeting nucleic acid synthesis, oxidative stress, and the PI3K/mTOR pathway. Clustering of individual drug response profiles stratifies the cell lines into five functional groups, which link to specific molecular and metabolic features. Mechanistic characterization of selective responses to the PI3K inhibitor pictilisib, the fatty acid synthase inhibitor GSK2194069, and the SLC16A1 inhibitor AZD3965, bring forth biomarkers of drug response. Phenotypic screening using CLIMET represents a valuable tool to probe cellular metabolism and identify metabolic dependencies at large.
Assuntos
Leucemia Mieloide/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Análise por Conglomerados , Ácidos Graxos/biossíntese , Genótipo , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Transportadores de Ácidos Monocarboxílicos/genética , Fenótipo , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Pirimidinonas/metabolismo , Pirimidinonas/farmacologia , Pirrolidinas/metabolismo , Pirrolidinas/farmacologia , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/classificação , Simportadores/genética , Análise de Sistemas , Tiofenos/metabolismo , Tiofenos/farmacologia , Triazóis/metabolismo , Triazóis/farmacologia , Células Tumorais CultivadasRESUMO
About a thousand genes in the human genome encode for membrane transporters. Among these, several solute carrier proteins (SLCs), representing the largest group of transporters, are still orphan and lack functional characterization. We reasoned that assessing genetic interactions among SLCs may be an efficient way to obtain functional information allowing their deorphanization. Here we describe a network of strong genetic interactions indicating a contribution to mitochondrial respiration and redox metabolism for SLC25A51/MCART1, an uncharacterized member of the SLC25 family of transporters. Through a combination of metabolomics, genomics and genetics approaches, we demonstrate a role for SLC25A51 as enabler of mitochondrial import of NAD, showcasing the potential of genetic interaction-driven functional gene deorphanization.
Assuntos
Epistasia Genética , Mitocôndrias/metabolismo , NAD/metabolismo , Proteína Desacopladora 1/metabolismo , Transporte Biológico , Humanos , Mitocôndrias/genética , Oxirredução , Proteína Desacopladora 1/genéticaRESUMO
Macrophages represent the first line of immune defense against pathogens, and phagosome acidification is a necessary step in pathogen clearance. Here, we identified the bicarbonate transporter SLC4A7, which is strongly induced upon macrophage differentiation, as critical for phagosome acidification. Loss of SLC4A7 reduced acidification of phagocytosed beads or bacteria and impaired the intracellular microbicidal capacity in human macrophage cell lines. The phenotype was rescued by wild-type SLC4A7, but not by SLC4A7 mutants, affecting transport capacity or cell surface localization. Loss of SLC4A7 resulted in increased cytoplasmic acidification during phagocytosis, suggesting that SLC4A7-mediated, bicarbonate-driven maintenance of cytoplasmic pH is necessary for phagosome acidification. Altogether, we identify SLC4A7 and bicarbonate-driven cytoplasmic pH homeostasis as an important element of phagocytosis and the associated microbicidal functions in macrophages.