ABSTRACT
Immune health requires innate and adaptive immune cells to engage precisely balanced pro- and anti-inflammatory forces. We employ the concept of chemical immunophenotypes to classify small molecules functionally or mechanistically according to their patterns of effects on primary innate and adaptive immune cells. The high-specificity, low-toxicity cyclin-dependent kinase 8 (CDK8) inhibitor 16-didehydro-cortistatin A (DCA) exerts a distinct tolerogenic profile in both innate and adaptive immune cells. DCA promotes regulatory T cells (Treg) and Th2 differentiation while inhibiting Th1 and Th17 differentiation in both murine and human cells. This unique chemical immunophenotype led to mechanistic studies showing that DCA promotes Treg differentiation in part by regulating a previously undescribed CDK8-GATA3-FOXP3 pathway that regulates early pathways of Foxp3 expression. These results highlight previously unappreciated links between Treg and Th2 differentiation and extend our understanding of the transcription factors that regulate Treg differentiation and their temporal sequencing. These findings have significant implications for future mechanistic and translational studies of CDK8 and CDK8 inhibitors.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cyclin-Dependent Kinase 8/antagonists & inhibitors , Forkhead Transcription Factors/metabolism , GATA3 Transcription Factor/metabolism , Heterocyclic Compounds, 4 or More Rings/pharmacology , Immune Tolerance/drug effects , Immunophenotyping , Isoquinolines/pharmacology , Adolescent , Adult , Animals , CD4-Positive T-Lymphocytes/drug effects , Cell Differentiation/drug effects , Cyclin-Dependent Kinase 8/metabolism , Humans , Immunity, Innate/drug effects , Mice, Inbred BALB C , Middle Aged , Phosphorylation/drug effects , Proto-Oncogene Proteins c-jun/metabolism , STAT Transcription Factors/metabolism , Signal Transduction/drug effects , Young AdultABSTRACT
Cancer is often seen as a disease of mutations and chromosomal abnormalities. However, some cancers, including pediatric rhabdoid tumors (RTs), lack recurrent alterations targetable by current drugs and need alternative, informed therapeutic options. To nominate potential targets, we performed a high-throughput small-molecule screen complemented by a genome-scale CRISPR-Cas9 gene-knockout screen in a large number of RT and control cell lines. These approaches converged to reveal several receptor tyrosine kinases (RTKs) as therapeutic targets, with RTK inhibition effective in suppressing RT cell growth in vitro and against a xenograft model in vivo. RT cell lines highly express and activate (phosphorylate) different RTKs, creating dependency without mutation or amplification. Downstream of RTK signaling, we identified PTPN11, encoding the pro-growth signaling protein SHP2, as a shared dependency across all RT cell lines. This study demonstrates that large-scale perturbational screening can uncover vulnerabilities in cancers with "quiet" genomes.
Subject(s)
Antineoplastic Agents/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Rhabdoid Tumor/genetics , Animals , Antineoplastic Agents/therapeutic use , CRISPR-Cas Systems , Cell Line, Tumor , Female , HEK293 Cells , Humans , Mice , Mice, Nude , Mutation , Protein Kinase Inhibitors/therapeutic use , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Rhabdoid Tumor/drug therapy , Small Molecule Libraries/pharmacologyABSTRACT
This Letter is being retracted owing to issues with Fig. 1d and Supplementary Fig. 31b, and the unavailability of original data for these figures that raise concerns regarding the integrity of the figures. Nature published two previous corrections related to this Letter1,2. These issues in aggregate undermine the confidence in the integrity of this study. Authors Michael Foley, Monica Schenone, Nicola J. Tolliday, Todd R. Golub, Steven A. Carr, Alykhan F. Shamji, Andrew M. Stern and Stuart L. Schreiber agree with the Retraction. Authors Lakshmi Raj, Takao Ide, Aditi U. Gurkar, Anna Mandinova and Sam W. Lee disagree with the Retraction. Author Xiaoyu Li did not respond.
ABSTRACT
We previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs, and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high-throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.
Subject(s)
Gene Expression Profiling/methods , Cell Line, Tumor , Drug Resistance, Neoplasm , Gene Expression Profiling/economics , Humans , Neoplasms/drug therapy , Organ Specificity , Pharmaceutical Preparations/metabolism , Sequence Analysis, RNA/economics , Sequence Analysis, RNA/methods , Small Molecule LibrariesABSTRACT
Advances in human genetics have dramatically expanded our understanding of complex heritable diseases. Genome-wide association studies have identified an allelic series of CARD9 variants associated with increased risk of or protection from inflammatory bowel disease (IBD). The predisposing variant of CARD9 is associated with increased NF-κB-mediated cytokine production. Conversely, the protective variant lacks a functional C-terminal domain and is unable to recruit the E3 ubiquitin ligase TRIM62. Here, we used biochemical insights into CARD9 variant proteins to create a blueprint for IBD therapeutics and recapitulated the mechanism of the CARD9 protective variant using small molecules. We developed a multiplexed bead-based technology to screen compounds for disruption of the CARD9-TRIM62 interaction. We identified compounds that directly and selectively bind CARD9, disrupt TRIM62 recruitment, inhibit TRIM62-mediated ubiquitinylation of CARD9, and demonstrate cellular activity and selectivity in CARD9-dependent pathways. Taken together, small molecules targeting CARD9 illustrate a path toward improved IBD therapeutics.
Subject(s)
CARD Signaling Adaptor Proteins/antagonists & inhibitors , CARD Signaling Adaptor Proteins/genetics , Enzyme-Linked Immunosorbent Assay/methods , Genetic Variation , Inflammatory Bowel Diseases/genetics , Drug Evaluation, Preclinical , Genetic Markers , High-Throughput Screening Assays , Humans , Inflammatory Bowel Diseases/drug therapy , Protein Binding , Sensitivity and Specificity , Tripartite Motif Proteins/antagonists & inhibitors , Tripartite Motif Proteins/genetics , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/geneticsABSTRACT
Enhancing production of the anti-inflammatory cytokine interleukin-10 (IL-10) is a promising strategy to suppress pathogenic inflammation. To identify new mechanisms regulating IL-10 production, we conducted a phenotypic screen for small molecules that enhance IL-10 secretion from activated dendritic cells. Mechanism-of-action studies using a prioritized hit from the screen, BRD6989, identified the Mediator-associated kinase CDK8, and its paralog CDK19, as negative regulators of IL-10 production during innate immune activation. The ability of BRD6989 to upregulate IL-10 is recapitulated by multiple, structurally differentiated CDK8 and CDK19 inhibitors and requires an intact cyclin C-CDK8 complex. Using a highly parallel pathway reporter assay, we identified a role for enhanced AP-1 activity in IL-10 potentiation following CDK8 and CDK19 inhibition, an effect associated with reduced phosphorylation of a negative regulatory site on c-Jun. These findings identify a function for CDK8 and CDK19 in regulating innate immune activation and suggest that these kinases may warrant consideration as therapeutic targets for inflammatory disorders.
Subject(s)
Cyclin-Dependent Kinase 8/metabolism , Interleukin-10/biosynthesis , Myeloid Cells/drug effects , Small Molecule Libraries/pharmacology , Animals , Cells, Cultured , Cyclin-Dependent Kinase 8/immunology , Dose-Response Relationship, Drug , Humans , Interleukin-10/immunology , Mice , Mice, Inbred C57BL , Molecular Structure , Myeloid Cells/immunology , Myeloid Cells/metabolism , Small Molecule Libraries/chemistry , Structure-Activity RelationshipABSTRACT
Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFß-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.
Subject(s)
Glutathione Peroxidase/metabolism , Lipid Peroxidation/drug effects , Neoplasms/drug therapy , Neoplasms/enzymology , Cadherins/metabolism , Cell Death , Cell Line, Tumor , Cell Lineage , Cell Transdifferentiation , Drug Resistance, Neoplasm/genetics , Epithelial-Mesenchymal Transition , Humans , Iron/metabolism , Lipid Peroxides/metabolism , Male , Melanoma/drug therapy , Melanoma/enzymology , Melanoma/metabolism , Melanoma/pathology , Mesoderm/drug effects , Mesoderm/enzymology , Mesoderm/metabolism , Mesoderm/pathology , Neoplasms/genetics , Neoplasms/pathology , Phospholipid Hydroperoxide Glutathione Peroxidase , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Proteomics , Proto-Oncogene Proteins B-raf/genetics , Reproducibility of Results , Zinc Finger E-box-Binding Homeobox 1/geneticsABSTRACT
Background: Large-scale image sets acquired by automated microscopy of perturbed samples enable a detailed comparison of cell states induced by each perturbation, such as a small molecule from a diverse library. Highly multiplexed measurements of cellular morphology can be extracted from each image and subsequently mined for a number of applications. Findings: This microscopy dataset includes 919 265 five-channel fields of view, representing 30 616 tested compounds, available at "The Cell Image Library" (CIL) repository. It also includes data files containing morphological features derived from each cell in each image, both at the single-cell level and population-averaged (i.e., per-well) level; the image analysis workflows that generated the morphological features are also provided. Quality-control metrics are provided as metadata, indicating fields of view that are out-of-focus or containing highly fluorescent material or debris. Lastly, chemical annotations are supplied for the compound treatments applied. Conclusions: Because computational algorithms and methods for handling single-cell morphological measurements are not yet routine, the dataset serves as a useful resource for the wider scientific community applying morphological (image-based) profiling. The dataset can be mined for many purposes, including small-molecule library enrichment and chemical mechanism-of-action studies, such as target identification. Integration with genetically perturbed datasets could enable identification of small-molecule mimetics of particular disease- or gene-related phenotypes that could be useful as probes or potential starting points for development of future therapeutics.
Subject(s)
Image Processing, Computer-Assisted , Small Molecule Libraries , Cell Line , Cells/drug effects , Cells/ultrastructure , HumansABSTRACT
Evidence suggests that specific mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) are critical for the initiation and maintenance of certain tumor types and that inhibiting these mutant enzymes with small molecules may be therapeutically beneficial. In order to discover mutant allele-selective IDH1 inhibitors with chemical features distinct from existing probes, we screened a collection of small molecules derived from diversity-oriented synthesis. The assay identified compounds that inhibit the IDH1-R132H mutant allele commonly found in glioma. Here, we report the discovery of a potent (IC50 = 50 nM) series of IDH1-R132H inhibitors having 8-membered ring sulfonamides as exemplified by the compound BRD2879. The inhibitors suppress (R)-2-hydroxyglutarate production in cells without apparent toxicity. Although the solubility and pharmacokinetic properties of the specific inhibitor BRD2879 prevent its use in vivo, the scaffold presents a validated starting point for the synthesis of future IDH1-R132H inhibitors having improved pharmacological properties.
ABSTRACT
Identifying therapeutic targets in rare cancers remains challenging due to the paucity of established models to perform preclinical studies. As a proof-of-concept, we developed a patient-derived cancer cell line, CLF-PED-015-T, from a paediatric patient with a rare undifferentiated sarcoma. Here, we confirm that this cell line recapitulates the histology and harbours the majority of the somatic genetic alterations found in a metastatic lesion isolated at first relapse. We then perform pooled CRISPR-Cas9 and RNAi loss-of-function screens and a small-molecule screen focused on druggable cancer targets. Integrating these three complementary and orthogonal methods, we identify CDK4 and XPO1 as potential therapeutic targets in this cancer, which has no known alterations in these genes. These observations establish an approach that integrates new patient-derived models, functional genomics and chemical screens to facilitate the discovery of targets in rare cancers.
Subject(s)
Cyclin-Dependent Kinase 4/genetics , Karyopherins/genetics , Rare Diseases/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Sarcoma/genetics , A549 Cells , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , CRISPR-Cas Systems , Cell Cycle , Cell Line, Tumor , Doxorubicin/administration & dosage , Drug Screening Assays, Antitumor , Exome , Female , Genomics , Humans , Hydrazines/administration & dosage , Mice , Mice, Nude , Neoplasm Metastasis , Neoplasm Recurrence, Local , Neoplasm Transplantation , Piperazines/administration & dosage , Pyridines/administration & dosage , RNA Interference , Rare Diseases/drug therapy , Sarcoma/drug therapy , Sequence Analysis, RNA , Triazoles/administration & dosage , Exportin 1 ProteinABSTRACT
Salt-inducible kinases (SIKs) are promising therapeutic targets for modulating cytokine responses during innate immune activation. The study of SIK inhibition in animal models of disease has been limited by the lack of selective small-molecule probes suitable for modulating SIK function in vivo. We used the pan-SIK inhibitor HG-9-91-01 as a starting point to develop improved analogs, yielding a novel probe 5 (YKL-05-099) that displays increased selectivity for SIKs versus other kinases and enhanced pharmacokinetic properties. Well-tolerated doses of YKL-05-099 achieve free serum concentrations above its IC50 for SIK2 inhibition for >16 h and reduce phosphorylation of a known SIK substrate in vivo. While in vivo active doses of YKL-05-099 recapitulate the effects of SIK inhibition on inflammatory cytokine responses, they did not induce metabolic abnormalities observed in Sik2 knockout mice. These results identify YKL-05-099 as a useful probe to investigate SIK function in vivo and further support the development of SIK inhibitors for treatment of inflammatory disorders.
Subject(s)
Molecular Probes/chemistry , Protein Serine-Threonine Kinases/metabolism , Animals , Cells, Cultured , Inhibitory Concentration 50 , Mice , Mice, Knockout , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/chemistryABSTRACT
Radiotherapy is not currently informed by the genetic composition of an individual patient's tumour. To identify genetic features regulating survival after DNA damage, here we conduct large-scale profiling of cellular survival after exposure to radiation in a diverse collection of 533 genetically annotated human tumour cell lines. We show that sensitivity to radiation is characterized by significant variation across and within lineages. We combine results from our platform with genomic features to identify parameters that predict radiation sensitivity. We identify somatic copy number alterations, gene mutations and the basal expression of individual genes and gene sets that correlate with the radiation survival, revealing new insights into the genetic basis of tumour cellular response to DNA damage. These results demonstrate the diversity of tumour cellular response to ionizing radiation and establish multiple lines of evidence that new genetic features regulating cellular response after DNA damage can be identified.
Subject(s)
DNA Damage , Neoplasms/genetics , Cell Line, Tumor , Cell Survival/radiation effects , DNA Copy Number Variations/radiation effects , DNA Damage/radiation effects , Gene Expression Regulation, Neoplastic/radiation effects , Humans , Neoplasms/metabolism , Neoplasms/physiopathology , Neoplasms/radiotherapy , Radiation Tolerance , Radiation, IonizingABSTRACT
Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large numbers of compounds against large numbers of cell lines is currently impractical, and such experiments are often difficult to control. Here we report a method called PRISM that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM revealed the expected patterns of cell killing seen in conventional (unpooled) assays. In a screen of 102 cell lines across 8,400 compounds, PRISM led to the identification of BRD-7880 as a potent and highly specific inhibitor of aurora kinases B and C. Cell line pools also efficiently formed tumors as xenografts, and PRISM recapitulated the expected pattern of erlotinib sensitivity in vivo.
Subject(s)
DNA Barcoding, Taxonomic/methods , Drug Resistance, Neoplasm/genetics , Genotyping Techniques/methods , High-Throughput Nucleotide Sequencing/methods , Neoplasms/classification , Neoplasms/genetics , Animals , Cell Line, Tumor , Humans , MiceABSTRACT
PURPOSE: We used human stem and progenitor cells to develop a genetically accurate novel model of MYC-driven Group 3 medulloblastoma. We also developed a new informatics method, Disease-model Signature versus Compound-Variety Enriched Response ("DiSCoVER"), to identify novel therapeutics that target this specific disease subtype. EXPERIMENTAL DESIGN: Human neural stem and progenitor cells derived from the cerebellar anlage were transduced with oncogenic elements associated with aggressive medulloblastoma. An in silico analysis method for screening drug sensitivity databases (DiSCoVER) was used in multiple drug sensitivity datasets. We validated the top hits from this analysis in vitro and in vivo RESULTS: Human neural stem and progenitor cells transformed with c-MYC, dominant-negative p53, constitutively active AKT and hTERT formed tumors in mice that recapitulated Group 3 medulloblastoma in terms of pathology and expression profile. DiSCoVER analysis predicted that aggressive MYC-driven Group 3 medulloblastoma would be sensitive to cyclin-dependent kinase (CDK) inhibitors. The CDK 4/6 inhibitor palbociclib decreased proliferation, increased apoptosis, and significantly extended the survival of mice with orthotopic medulloblastoma xenografts. CONCLUSIONS: We present a new method to generate genetically accurate models of rare tumors, and a companion computational methodology to find therapeutic interventions that target them. We validated our human neural stem cell model of MYC-driven Group 3 medulloblastoma and showed that CDK 4/6 inhibitors are active against this subgroup. Our results suggest that palbociclib is a potential effective treatment for poor prognosis MYC-driven Group 3 medulloblastoma tumors in carefully selected patients. Clin Cancer Res; 22(15); 3903-14. ©2016 AACR.
Subject(s)
Cerebellar Neoplasms/genetics , Computational Biology/methods , Genetic Predisposition to Disease , Medulloblastoma/genetics , Models, Biological , Animals , Apoptosis/drug effects , Biomarkers , Cell Line, Tumor , Computer Simulation , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/metabolism , Disease Models, Animal , Drug Discovery , Gene Expression Profiling , Humans , Medulloblastoma/drug therapy , Medulloblastoma/metabolism , Medulloblastoma/pathology , Mice , Neural Stem Cells/metabolism , Phosphorylation , Piperazines/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-myc/genetics , Pyridines/pharmacology , Transcriptome , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor AssaysABSTRACT
High cancer death rates indicate the need for new anticancer therapeutic agents. Approaches to discovering new cancer drugs include target-based drug discovery and phenotypic screening. Here, we identified phosphodiesterase 3A modulators as cell-selective cancer cytotoxic compounds through phenotypic compound library screening and target deconvolution by predictive chemogenomics. We found that sensitivity to 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, across 766 cancer cell lines correlates with expression of the gene PDE3A, encoding phosphodiesterase 3A. Like DNMDP, a subset of known PDE3A inhibitors kill selected cancer cells, whereas others do not. Furthermore, PDE3A depletion leads to DNMDP resistance. We demonstrated that DNMDP binding to PDE3A promotes an interaction between PDE3A and Schlafen 12 (SLFN12), suggestive of a neomorphic activity. Coexpression of SLFN12 with PDE3A correlates with DNMDP sensitivity, whereas depletion of SLFN12 results in decreased DNMDP sensitivity. Our results implicate PDE3A modulators as candidate cancer therapeutic agents and demonstrate the power of predictive chemogenomics in small-molecule discovery.
Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 3/metabolism , Cytotoxins/pharmacology , Neoplasms/therapy , Pyridazines/chemistry , Pyridazines/pharmacology , Cell Line , Cell Line, Tumor , Cell Survival/drug effects , Cytotoxins/chemistry , Cytotoxins/isolation & purification , Drug Delivery Systems , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Genomics , Humans , ImmunoblottingABSTRACT
Changes in cellular gene expression in response to small-molecule or genetic perturbations have yielded signatures that can connect unknown mechanisms of action (MoA) to ones previously established. We hypothesized that differential basal gene expression could be correlated with patterns of small-molecule sensitivity across many cell lines to illuminate the actions of compounds whose MoA are unknown. To test this idea, we correlated the sensitivity patterns of 481 compounds with â¼19,000 basal transcript levels across 823 different human cancer cell lines and identified selective outlier transcripts. This process yielded many novel mechanistic insights, including the identification of activation mechanisms, cellular transporters and direct protein targets. We found that ML239, originally identified in a phenotypic screen for selective cytotoxicity in breast cancer stem-like cells, most likely acts through activation of fatty acid desaturase 2 (FADS2). These data and analytical tools are available to the research community through the Cancer Therapeutics Response Portal.
Subject(s)
Gene Expression Regulation, Neoplastic/drug effects , Small Molecule Libraries/pharmacology , Aflatoxins/chemistry , Aflatoxins/pharmacology , Blotting, Western , Breast Neoplasms/drug therapy , Cell Line, Tumor , Computer Simulation , Drug Delivery Systems , Female , Humans , Molecular Structure , Principal Component Analysis , Real-Time Polymerase Chain ReactionABSTRACT
UNLABELLED: Identifying genetic alterations that prime a cancer cell to respond to a particular therapeutic agent can facilitate the development of precision cancer medicines. Cancer cell-line (CCL) profiling of small-molecule sensitivity has emerged as an unbiased method to assess the relationships between genetic or cellular features of CCLs and small-molecule response. Here, we developed annotated cluster multidimensional enrichment analysis to explore the associations between groups of small molecules and groups of CCLs in a new, quantitative sensitivity dataset. This analysis reveals insights into small-molecule mechanisms of action, and genomic features that associate with CCL response to small-molecule treatment. We are able to recapitulate known relationships between FDA-approved therapies and cancer dependencies and to uncover new relationships, including for KRAS-mutant cancers and neuroblastoma. To enable the cancer community to explore these data, and to generate novel hypotheses, we created an updated version of the Cancer Therapeutic Response Portal (CTRP v2). SIGNIFICANCE: We present the largest CCL sensitivity dataset yet available, and an analysis method integrating information from multiple CCLs and multiple small molecules to identify CCL response predictors robustly. We updated the CTRP to enable the cancer research community to leverage these data and analyses.
Subject(s)
Computational Biology/methods , Drug Resistance, Neoplasm/genetics , Drug Screening Assays, Antitumor , Gene Expression Regulation, Neoplastic/drug effects , Neoplasms/genetics , Small Molecule Libraries , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cluster Analysis , Datasets as Topic , Dose-Response Relationship, Drug , Drug Synergism , Humans , Mutation , Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacologyABSTRACT
CARD9 is a central component of anti-fungal innate immune signaling via C-type lectin receptors, and several immune-related disorders are associated with CARD9 alterations. Here, we used a rare CARD9 variant that confers protection against inflammatory bowel disease as an entry point to investigating CARD9 regulation. We showed that the protective variant of CARD9, which is C-terminally truncated, acted in a dominant-negative manner for CARD9-mediated cytokine production, indicating an important role for the C terminus in CARD9 signaling. We identified TRIM62 as a CARD9 binding partner and showed that TRIM62 facilitated K27-linked poly-ubiquitination of CARD9. We identified K125 as the ubiquitinated residue on CARD9 and demonstrated that this ubiquitination was essential for CARD9 activity. Furthermore, we showed that similar to Card9-deficient mice, Trim62-deficient mice had increased susceptibility to fungal infection. In this study, we utilized a rare protective allele to uncover a TRIM62-mediated mechanism for regulation of CARD9 activation.
Subject(s)
CARD Signaling Adaptor Proteins/physiology , Candidiasis, Invasive/immunology , Receptors, Angiotensin/physiology , Receptors, Endothelin/physiology , Ubiquitin-Protein Ligases/physiology , Adjuvants, Immunologic/pharmacology , Animals , CARD Signaling Adaptor Proteins/chemistry , CARD Signaling Adaptor Proteins/deficiency , CARD Signaling Adaptor Proteins/genetics , Candidiasis, Invasive/genetics , Colitis/chemically induced , Colitis/genetics , Colitis/prevention & control , Cytokines/biosynthesis , Dendritic Cells/immunology , Dendritic Cells/metabolism , Genes, Dominant , Genetic Predisposition to Disease , HEK293 Cells , HeLa Cells , Humans , Inflammatory Bowel Diseases/genetics , Mice , Mice, 129 Strain , Mice, Knockout , Protein Interaction Mapping , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/physiology , Protein Processing, Post-Translational , Protein Structure, Tertiary , Receptors, Angiotensin/chemistry , Receptors, Angiotensin/deficiency , Receptors, Endothelin/chemistry , Receptors, Endothelin/deficiency , Recombinant Fusion Proteins/metabolism , Signal Transduction , Specific Pathogen-Free Organisms , Tripartite Motif Proteins , Ubiquitin-Protein Ligases/chemistry , UbiquitinationABSTRACT
Studies of human genetics and pathophysiology have implicated the regulation of autophagy in inflammation, neurodegeneration, infection, and autoimmunity. These findings have motivated the use of small-molecule probes to study how modulation of autophagy affects disease-associated phenotypes. Here, we describe the discovery of the small-molecule probe BRD5631 that is derived from diversity-oriented synthesis and enhances autophagy through an mTOR-independent pathway. We demonstrate that BRD5631 affects several cellular disease phenotypes previously linked to autophagy, including protein aggregation, cell survival, bacterial replication, and inflammatory cytokine production. BRD5631 can serve as a valuable tool for studying the role of autophagy in the context of cellular homeostasis and disease.