Although the genes essential for life have been identified in less complex model organisms, their elucidation in human cells has been hindered by technical barriers. We used extensive mutagenesis in ...haploid human cells to identify approximately 2000 genes required for optimal fitness under culture conditions. To study the principles of genetic interactions in human cells, we created a synthetic lethality network focused on the secretory pathway based exclusively on mutations. This revealed a genetic cross-talk governing Golgi homeostasis, an additional subunit of the human oligosaccharyltransferase complex, and a phosphatidylinositol 4-kinase β adaptor hijacked by viruses. The synthetic lethality map parallels observations made in yeast and projects a route forward to reveal genetic networks in diverse aspects of human cell biology.
•Currently a vast heterogeneity in synergistic drug action scoring and analysis exists.•Novel cellular models offer robust ways to identify relevant combination treatments.•New pharmacological and ...genetic tools improve odds for effective combination therapies.
Treatment of complex diseases such as cancer, cardiovascular disease, diabetes or neurological disorders frequently warrants the utilization of drug combinations for therapeutic intervention. In fact, the most successful example is the current standard of care for HIV patients. However, identification of successful drug cocktails is not a simple task and is hampered by lack of standardization in terminology, experimental protocols and models as well as data analysis. Here we discuss the most recent developments in combinatorial drug screening by covering technological advancements in screening strategies, cellular model systems as well as novel drug classes. We believe the research progress being made provides promising basis to build on and identify, develop and optimize efficacious clinically relevant combinatorial drug treatments.
Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and ...amino acids to modulate metabolic status and cellular responses. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H(+)-ATPase. Here we describe the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator-RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR.
Toll-like receptors (TLRs) have a crucial role in the recognition of pathogens and initiation of immune responses
. Here we show that a previously uncharacterized protein encoded by CXorf21-a gene ...that is associated with systemic lupus erythematosus
-interacts with the endolysosomal transporter SLC15A4, an essential but poorly understood component of the endolysosomal TLR machinery also linked to autoimmune disease
. Loss of this type-I-interferon-inducible protein, which we refer to as 'TLR adaptor interacting with SLC15A4 on the lysosome' (TASL), abrogated responses to endolysosomal TLR agonists in both primary and transformed human immune cells. Deletion of SLC15A4 or TASL specifically impaired the activation of the IRF pathway without affecting NF-κB and MAPK signalling, which indicates that ligand recognition and TLR engagement in the endolysosome occurred normally. Extensive mutagenesis of TASL demonstrated that its localization and function relies on the interaction with SLC15A4. TASL contains a conserved pLxIS motif (in which p denotes a hydrophilic residue and x denotes any residue) that mediates the recruitment and activation of IRF5. This finding shows that TASL is an innate immune adaptor for TLR7, TLR8 and TLR9 signalling, revealing a clear mechanistic analogy with the IRF3 adaptors STING, MAVS and TRIF
. The identification of TASL as the component that links endolysosomal TLRs to the IRF5 transcription factor via SLC15A4 provides a mechanistic explanation for the involvement of these proteins in systemic lupus erythematosus
.
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.
In genetic screens aimed at understanding drug resistance mechanisms in chronic myeloid leukemia cells, inactivation of the cullin 3 adapter protein-encoding leucine zipper-like transcription ...regulator 1 (
) gene led to enhanced mitogen-activated protein kinase (MAPK) pathway activity and reduced sensitivity to tyrosine kinase inhibitors. Knockdown of the
ortholog
resulted in a Ras-dependent gain-of-function phenotype. Endogenous human LZTR1 associates with the main RAS isoforms. Inactivation of
led to decreased ubiquitination and enhanced plasma membrane localization of endogenous KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog). We propose that LZTR1 acts as a conserved regulator of RAS ubiquitination and MAPK pathway activation. Because
disease mutations failed to revert loss-of-function phenotypes, our findings provide a molecular rationale for
involvement in a variety of inherited and acquired human disorders.
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.
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•CRISPR screen identifies SLC4A7 to be required for phagosome acidification•Intracellular bacterial killing by macrophages is impaired in absence of SLC4A7•Bicarbonate transport activity of SLC4A7 is required for phagosome acidification•SLC4A7 regulates phagosome acidification via homeostatic control of cytoplasmic pH
Phagosome acidification is a necessary step in pathogen clearance by macrophages. Sedlyarov et al. identify the bicarbonate transporter SLC4A7 as essential for phagosome acidification. SLC4A7-mediated bicarbonate import ensures homeostatic regulation of cytoplasmic pH. In the absence of SLC4A7, the macrophage cytoplasm acidifies, which perturbs phagosome maturation and impairs bacterial killing by macrophages.
Regulation of cell and tissue homeostasis by programmed cell death is a fundamental process with wide physiological and pathological implications. The advent of scalable somatic cell genetic ...technologies creates the opportunity to functionally map such essential pathways, thereby identifying potential disease-relevant components. We investigated the genetic basis underlying necroptotic cell death by performing a complementary set of loss-of-function and gain-of-function genetic screens. To this end, we established FADD-deficient haploid human KBM7 cells, which specifically and efficiently undergo necroptosis after a single treatment with either TNFα or the SMAC mimetic compound birinapant. A series of unbiased gene-trap screens identified key signaling mediators, such as TNFR1, RIPK1, RIPK3, and MLKL. Among the novel components, we focused on the zinc transporter SLC39A7, whose knock-out led to necroptosis resistance by affecting TNF receptor surface levels. Orthogonal, solute carrier (SLC)-focused CRISPR/Cas9-based genetic screens revealed the exquisite specificity of SLC39A7, among ~400 SLC genes, for TNFR1-mediated and FAS-mediated but not TRAIL-R1-mediated responses. Mechanistically, we demonstrate that loss of SLC39A7 resulted in augmented ER stress and impaired receptor trafficking, thereby globally affecting downstream signaling. The newly established cellular model also allowed genome-wide gain-of-function screening for genes conferring resistance to necroptosis via the CRISPR/Cas9-based synergistic activation mediator approach. Among these, we found cIAP1 and cIAP2, and characterized the role of TNIP1, which prevented pathway activation in a ubiquitin-binding dependent manner. Altogether, the gain-of-function and loss-of-function screens described here provide a global genetic chart of the molecular factors involved in necroptosis and death receptor signaling, prompting further investigation of their individual contribution and potential role in pathological conditions.
Small-molecule drugs may complement antibody-based therapies in an immune-oncology setting, yet systematic methods for the identification and characterization of the immunomodulatory properties of ...these entities are lacking. We surveyed the immumomodulatory potential of 1,402 small chemical molecules, as defined by their ability to alter the cell-cell interactions among peripheral mononuclear leukocytes ex vivo, using automated microscopy and population-wide single-cell image analysis. Unexpectedly, ∼10% of the agents tested affected these cell-cell interactions differentially. The results accurately recapitulated known immunomodulatory drug classes and revealed several clinically approved drugs that unexpectedly harbor the ability to modulate the immune system, which could potentially contribute to their physiological mechanism of action. For instance, the kinase inhibitor crizotinib promoted T cell interactions with monocytes, as well as with cancer cells, through inhibition of the receptor tyrosine kinase MSTR1 and subsequent upregulation of the expression of major histocompatibility complex molecules. The approach offers an attractive platform for the personalized identification and characterization of immunomodulatory therapeutics.
Tandem affinity purification–mass spectrometry (TAP-MS) is a popular strategy for the identification of protein–protein interactions, characterization of protein complexes, and entire networks. Its ...employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression.
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