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1.
Virulence ; 13(1): 890-902, 2022 12.
Article in English | MEDLINE | ID: mdl-35587156

ABSTRACT

Antibodies to SARS-CoV-2 are central to recovery and immunity from COVID-19. However, the relationship between disease severity and the repertoire of antibodies against specific SARS-CoV-2 epitopes an individual develops following exposure remains incompletely understood. Here, we studied seroprevalence of antibodies to specific SARS-CoV-2 and other betacoronavirus antigens in a well-annotated, community sample of convalescent and never-infected individuals obtained in August 2020. One hundred and twenty-four participants were classified into five groups: previously exposed but without evidence of infection, having no known exposure or evidence of infection, seroconverted without symptoms, previously diagnosed with symptomatic COVID-19, and recovered after hospitalization with COVID-19. Prevalence of IgGs specific to the following antigens was compared between the five groups: recombinant SARS-CoV-2 and betacoronavirus spike and nucleocapsid protein domains, peptides from a tiled array of 22-mers corresponding to the entire spike and nucleocapsid proteins, and peptides corresponding to predicted immunogenic regions from other proteins of SARS-CoV-2. Antibody abundance generally correlated positively with severity of prior illness. A number of specific immunogenic peptides and some that may be associated with milder illness or protection from symptomatic infection were identified. No convincing association was observed between antibodies to Receptor Binding Domain(s) (RBDs) of less pathogenic betacoronaviruses HKU1 or OC43 and COVID-19 severity. However, apparent cross-reaction with SARS-CoV RBD was evident and some predominantly asymptomatic individuals had antibodies to both MERS-CoV and SARS-CoV RBDs. Findings from this pilot study may inform development of diagnostics, vaccines, and therapeutic antibodies, and provide insight into viral pathogenic mechanisms.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , Epitopes , Humans , Pilot Projects , Seroepidemiologic Studies , Spike Glycoprotein, Coronavirus
2.
Cell ; 170(3): 577-592.e10, 2017 Jul 27.
Article in English | MEDLINE | ID: mdl-28753431

ABSTRACT

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.


Subject(s)
Neoplasms/genetics , Neoplasms/pathology , RNA Interference , Cell Line, Tumor , Gene Library , Gene Regulatory Networks , Humans , Multiprotein Complexes/metabolism , Neoplasms/metabolism , Oncogenes , RNA, Small Interfering , Signal Transduction , Transcription Factors/metabolism
3.
Science ; 351(6278): 1208-13, 2016 Mar 11.
Article in English | MEDLINE | ID: mdl-26912361

ABSTRACT

5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA-mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in an MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP-deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p16/metabolism , Methionine/metabolism , Neoplasms/metabolism , Protein-Arginine N-Methyltransferases/metabolism , Purine-Nucleoside Phosphorylase/metabolism , Cell Line, Tumor , Cell Survival , Cyclin-Dependent Kinase Inhibitor p16/genetics , Deoxyadenosines/metabolism , Gene Deletion , Humans , Neoplasms/drug therapy , Neoplasms/genetics , Protein-Arginine N-Methyltransferases/genetics , Purine-Nucleoside Phosphorylase/genetics , RNA, Small Interfering/genetics , Thionucleosides/metabolism
5.
Cancer Res ; 76(2): 390-402, 2016 Jan 15.
Article in English | MEDLINE | ID: mdl-26577700

ABSTRACT

The introduction of MAPK pathway inhibitors paved the road for significant advancements in the treatment of BRAF-mutant (BRAF(MUT)) melanoma. However, even BRAF/MEK inhibitor combination therapy has failed to offer a curative treatment option, most likely because these pathways constitute a codependent signaling network. Concomitant PTEN loss of function (PTEN(LOF)) occurs in approximately 40% of BRAF(MUT) melanomas. In this study, we sought to identify the nodes of the PTEN/PI3K pathway that would be amenable to combined therapy with MAPK pathway inhibitors for the treatment of PTEN(LOF)/BRAF(MUT) melanoma. Large-scale compound sensitivity profiling revealed that PTEN(LOF) melanoma cell lines were sensitive to PI3Kß inhibitors, albeit only partially. An unbiased shRNA screen (7,500 genes and 20 shRNAs/genes) across 11 cell lines in the presence of a PI3Kß inhibitor identified an adaptive response involving the IGF1R-PI3Kα axis. Combined inhibition of the MAPK pathway, PI3Kß, and PI3Kα or insulin-like growth factor receptor 1 (IGF1R) synergistically sustained pathway blockade, induced apoptosis, and inhibited tumor growth in PTEN(LOF)/BRAF(MUT) melanoma models. Notably, combined treatment with the IGF1R inhibitor, but not the PI3Kα inhibitor, failed to elevate glucose or insulin signaling. Taken together, our findings provide a strong rationale for testing combinations of panPI3K, PI3Kß + IGF1R, and MAPK pathway inhibitors in PTEN(LOF)/BRAF(MUT) melanoma patients to achieve maximal response.


Subject(s)
MAP Kinase Signaling System/genetics , Melanoma/genetics , PTEN Phosphohydrolase/metabolism , Proto-Oncogene Proteins B-raf/genetics , Receptor, IGF Type 1/metabolism , Apoptosis , Cell Death , Cell Proliferation , Humans , Melanoma/pathology , Proteomics
6.
PLoS One ; 10(9): e0138486, 2015.
Article in English | MEDLINE | ID: mdl-26378449

ABSTRACT

Death Receptor 5 (DR5) agonists demonstrate anti-tumor activity in preclinical models but have yet to demonstrate robust clinical responses. A key limitation may be the lack of patient selection strategies to identify those most likely to respond to treatment. To overcome this limitation, we screened a DR5 agonist Nanobody across >600 cell lines representing 21 tumor lineages and assessed molecular features associated with response. High expression of DR5 and Casp8 were significantly associated with sensitivity, but their expression thresholds were difficult to translate due to low dynamic ranges. To address the translational challenge of establishing thresholds of gene expression, we developed a classifier based on ratios of genes that predicted response across lineages. The ratio classifier outperformed the DR5+Casp8 classifier, as well as standard approaches for feature selection and classification using genes, instead of ratios. This classifier was independently validated using 11 primary patient-derived pancreatic xenograft models showing perfect predictions as well as a striking linearity between prediction probability and anti-tumor response. A network analysis of the genes in the ratio classifier captured important biological relationships mediating drug response, specifically identifying key positive and negative regulators of DR5 mediated apoptosis, including DR5, CASP8, BID, cFLIP, XIAP and PEA15. Importantly, the ratio classifier shows translatability across gene expression platforms (from Affymetrix microarrays to RNA-seq) and across model systems (in vitro to in vivo). Our approach of using gene expression ratios presents a robust and novel method for constructing translatable biomarkers of compound response, which can also probe the underlying biology of treatment response.


Subject(s)
Cell Lineage/genetics , Gene Expression Regulation, Neoplastic/genetics , Gene Expression/genetics , Pancreatic Neoplasms/genetics , Protein Biosynthesis/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Animals , Apoptosis/genetics , Caspase 8/genetics , Cell Line, Tumor , Humans , Mice , Xenograft Model Antitumor Assays/methods
7.
Cancer Res ; 74(12): 3294-305, 2014 Jun 15.
Article in English | MEDLINE | ID: mdl-24747911

ABSTRACT

Tankyrases (TNKS) play roles in Wnt signaling, telomere homeostasis, and mitosis, offering attractive targets for anticancer treatment. Using unbiased combination screening in a large panel of cancer cell lines, we have identified a strong synergy between TNKS and MEK inhibitors (MEKi) in KRAS-mutant cancer cells. Our study uncovers a novel function of TNKS in the relief of a feedback loop induced by MEK inhibition on FGFR2 signaling pathway. Moreover, dual inhibition of TNKS and MEK leads to more robust apoptosis and antitumor activity both in vitro and in vivo than effects observed by previously reported MEKi combinations. Altogether, our results show how a novel combination of TNKS and MEK inhibitors can be highly effective in targeting KRAS-mutant cancers by suppressing a newly discovered resistance mechanism.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Proto-Oncogene Proteins/genetics , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Tankyrases/metabolism , ras Proteins/genetics , Acetamides/administration & dosage , Aminopyridines/administration & dosage , Aniline Compounds/administration & dosage , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Cell Line, Tumor , Drug Synergism , Erlotinib Hydrochloride , Feedback, Physiological , Female , Humans , MAP Kinase Kinase Kinases/antagonists & inhibitors , MAP Kinase Kinase Kinases/metabolism , Mice , Mice, Nude , Morpholines/administration & dosage , Mutation , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins p21(ras) , Pyrimidinones/administration & dosage , Quinazolines/administration & dosage , Receptor, Fibroblast Growth Factor, Type 2/antagonists & inhibitors , Signal Transduction , Sulfonamides/administration & dosage , Tankyrases/antagonists & inhibitors , Thiazoles/administration & dosage , Xenograft Model Antitumor Assays
8.
Int J Cancer ; 107(2): 229-37, 2003 Nov 01.
Article in English | MEDLINE | ID: mdl-12949799

ABSTRACT

The beta-catenin pathway is involved in growth, differentiation and tumor formation. Suppression of pathway activity by expressed inhibitors can cause growth arrest or apoptosis in certain colon carcinoma lines. We compare the effects of 2 pathway inhibitors, a VE-cadherin cytoplasmic domain fragment (Cad5CD) and a truncated, dominant-negative Tcf4 (TcfDN), using a microplate assay for cell death and microarray gene expression analysis. The cell-lethal assay shows that Cad5CD, when expressed in HT29 human colon tumor cells and 3 non-colon lines, selectively kills the HT29 cells. Cad5CD overexpression inhibits beta-catenin/Tcf4 transcriptional activity, as determined by results from microarray experiments. Our results support the view that beta-catenin is an attractive anti-cancer target, especially if the Cad5CD binding site or Cad5CD itself can be exploited for drug development. In addition, therapeutically relevant phenotypes such as drug selectivity may be difficult to predict from gene expression analysis alone. Other more specialized phenotypic tests such as cell-lethal assays may be required.


Subject(s)
Apoptosis , Cadherins/genetics , Colorectal Neoplasms/pathology , Cytoskeletal Proteins/metabolism , Gene Expression Regulation, Neoplastic/physiology , Trans-Activators/metabolism , Transcription Factors/genetics , Antigens, CD , Binding Sites , Cadherins/metabolism , Cell Adhesion , Cell Division , Cell Movement , Colorectal Neoplasms/metabolism , Gene Expression Profiling , Humans , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Protein Structure, Tertiary , Signal Transduction , TCF Transcription Factors , Transcription Factor 7-Like 2 Protein , Transcription Factors/metabolism , Transcription, Genetic , Transfection , Tumor Cells, Cultured , beta Catenin
9.
J Biomol Screen ; 8(3): 283-91, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12857382

ABSTRACT

Screens for cytostasis/cytoxicity have considerable value for the discovery of therapeutic agents and the investigation of the biology of apoptosis. For instance, genetic screens for proteins, protein fragments, peptides, RNAs, or chemicals that kill tissue culture cells may aid in identifying new cancer therapeutic targets. A microplate assay for cell death is needed to achieve throughputs sufficient to sift through thousands of agents from expression or chemical libraries. The authors describe a homogeneous assay for cell death in tissue culture cells compatible with 96- or 384-well plates. In combination with a previously described system for retroviral packaging and transduction, nearly 6000 expression library clones could be screened per week in a 96-well plate format. The screening system may also prove useful for chemical screens.


Subject(s)
Apoptosis , Cell Death , Cell Line , Cells, Cultured , Culture Techniques , DNA, Complementary/metabolism , Drug Screening Assays, Antitumor , Gene Library , Genes, Reporter , Humans , Luminescent Proteins , Microscopy, Video , Peptide Library , Plasmids/metabolism , Retroviridae/genetics , Time Factors , Transfection
10.
Biochem Genet ; 40(11-12): 359-78, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12463345

ABSTRACT

Transdominant genetic selections can yield protein fragment and peptide modulators of specific biochemical pathways. In yeast, such screens have been highly successful in targeting the MAP (mitogen-activated protein) kinase growth-control pathway. We performed a similar type of selection aimed at recovery of modulators of the mammalian MAP kinase cascade. Two pathway activators were identified, fragments of the TrkB and Raf-1 kinases. In a second selection directed at the beta-catenin growth-control pathway, three different clones encoding cadherin fragments were recovered. In neither selection were peptide inhibitors observed. We conclude that some transdominant selections in mammalian cells can readily yield high-penetrance protein fragments, but may be less amenable to isolation of peptide inhibitors.


Subject(s)
Cytoskeletal Proteins/genetics , Mitogen-Activated Protein Kinases/genetics , Selection, Genetic , Signal Transduction/genetics , Trans-Activators/genetics , 3T3 Cells , Animals , Biological Assay , Cytoskeletal Proteins/physiology , In Vitro Techniques , Mice , Mitogen-Activated Protein Kinases/physiology , Signal Transduction/physiology , Trans-Activators/physiology , beta Catenin
11.
J Biomol Screen ; 7(3): 275-80, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12097190

ABSTRACT

Retroviruses are useful for genetics studies to deliver genes that express proteins, peptides, and RNAs. Several steps, including DNA preparation, transfection, packaging, transduction, and assay, are required to execute screens using retroviral constructs. Unlike screens with purified components, whole-cell assays using retroviral constructs need a large number of steps with microplate manipulations. The nature of these steps, especially the involvement of cultured mammalian cells, limits the throughput of such screens. To improve the efficiency of genetic experiments with retroviral expression vectors, an automated system for retroviral screening in microplates was devised and tested. The system, called Somata, provides high throughputs and robust, reproducible performance.


Subject(s)
Genetic Vectors/analysis , Retroviridae/metabolism , Flow Cytometry , Humans , Retroviridae/genetics
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