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1.
J Mol Diagn ; 22(1): 72-80, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31733350

RESUMO

Chromosomal rearrangements resulting in fusion transcripts have been reported in precursor B-cell acute lymphoblastic leukemia (B-ALL). The identification of fusion events is crucial in the diagnosis of B-ALL. In this study, we used NanoString nCounter technology to design, validate, and evaluate a multiplex panel for the detection of B-ALL fusion transcripts. Fifty-one B-ALL fusion transcripts reported in children in the literature were included in the design of the NanoString panel. Twenty-six fusion transcripts were validated using 64 positive-control samples and 74 negative-control samples with 100% sensitivity and 99% specificity in comparison to RT-PCR. Our results support a potential role of NanoString's technology as a robust and cost-effective technique that could be used in the detection of fusion transcripts and implemented into the diagnostic algorithm of B-ALL.

2.
Nat Commun ; 10(1): 4343, 2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31554817

RESUMO

Infant gliomas have paradoxical clinical behavior compared to those in children and adults: low-grade tumors have a higher mortality rate, while high-grade tumors have a better outcome. However, we have little understanding of their biology and therefore cannot explain this behavior nor what constitutes optimal clinical management. Here we report a comprehensive genetic analysis of an international cohort of clinically annotated infant gliomas, revealing 3 clinical subgroups. Group 1 tumors arise in the cerebral hemispheres and harbor alterations in the receptor tyrosine kinases ALK, ROS1, NTRK and MET. These are typically single-events and confer an intermediate outcome. Groups 2 and 3 gliomas harbor RAS/MAPK pathway mutations and arise in the hemispheres and midline, respectively. Group 2 tumors have excellent long-term survival, while group 3 tumors progress rapidly and do not respond well to chemoradiation. We conclude that infant gliomas comprise 3 subgroups, justifying the need for specialized therapeutic strategies.


Assuntos
Neoplasias Encefálicas/genética , Metilação de DNA , Epigenômica/métodos , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Receptores Proteína Tirosina Quinases/genética , Quinase do Linfoma Anaplásico/genética , Quinase do Linfoma Anaplásico/metabolismo , Neoplasias Encefálicas/classificação , Neoplasias Encefálicas/metabolismo , Feminino , Glioma/classificação , Glioma/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptor trkA/genética , Receptor trkA/metabolismo , Análise de Sobrevida , Sequenciamento Completo do Exoma/métodos
3.
Am J Surg Pathol ; 43(9): 1203-1211, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31290759

RESUMO

Neuroblastoma is the most common extracranial solid tumor of childhood with a median age of presentation of 17 months. A common theme in high-risk neuroblastoma is maintenance of telomeres, one mechanism for which involves alternate lengthening of telomeres (ALT) associated with ATRX gene mutations. Mutations are believed to result in loss of ATRX protein, and therefore immunohistochemistry is used to detect mutations. We screened 133 cases of neuroblastoma by ATRX immunohistochemistry, and found 9 cases with partial to total absence of ATRX. Sequencing for ATRX mutations detected a mutation in 1 of 9 cases, suggesting immunostaining was not reliable for detecting mutations. To correlate immunostaining with ALT, fluorescence in situ hybridization (FISH) for ALT was performed in 6 of these cases and 5 (from 4 patients) showed ALT, implying impaired ATRX protein function, despite the failure to identify a mutation. Two other cases with large deletions in the ATRX gene showed diffusely positive staining for ATRX protein but showed ALT by FISH. Four of the 6 patients with ALT-positive tumors were over 5 years old. Therefore, 29 additional patients 5 years old and above with ATRX-positive tumors were screened for ALT by FISH and 6 additional cases with ALT were detected, bringing the total to 29% (10/34) of children 5 years old and above, 70% of which showed positive ATRX immunohistochemistry. Patients with ATRX mutations in neuroblastoma tend to have a more chronic and progressive course of disease. Screening neuroblastoma tumors at diagnosis for ATRX mutations may help identify patients who might benefit from personalized therapy directed against ALT. However, relaying on negative immunohistochemistry for ATRX protein to identify ALT in neuroblastoma may miss a significant proportion of patients. The addition of FISH for ALT as part of the diagnostic workup, especially for older children (5 y old and above), would help ensure that patients are correctly identified for anti-ALT therapy.

5.
Methods Mol Biol ; 1869: 57-67, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30324513

RESUMO

The use of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) has become commonplace in the study of neuronal development, physiology, disease modelling, and therapy development. Due to the transient nature of working with these cells, it is important to regularly confirm the cell status as a naive stem cell versus a more defined neural progenitor cell (NPC). Classically, this has been done using a panel of specific antibodies to test for the expression of transcription factors known to be observed in ESCs, but not NPCs. However, this method is both time consuming and expensive. Here, we describe the use of the NanoString nCounter system for determining the levels of expression of key transcription factors that will effectively aid in determining the state of your stem cell cultures.


Assuntos
Nanotecnologia/métodos , Células-Tronco/metabolismo , Animais , Biomarcadores/metabolismo , Sondas de DNA/metabolismo , Análise de Dados , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo
6.
Pediatr Dev Pathol ; 22(3): 205-213, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30089422

RESUMO

BACKGROUND: NanoString technology is an innovative barcode-based system that requires less tissue than traditional techniques and can test for multiple fusion transcripts in a single reaction. The objective of this study was to determine the utility of NanoString technology in the detection of sarcoma-specific fusion transcripts in pediatric sarcomas. DESIGN: Probe pairs for the most common pediatric sarcoma fusion transcripts were designed for the assay. The NanoString assay was used to test 22 specific fusion transcripts in 45 sarcoma samples that had exhibited one of these fusion genes previously by reverse transcription polymerase chain reaction (RT-PCR). A mixture of frozen (n = 18), formalin-fixed, paraffin-embedded (FFPE) tissue (n = 23), and rapid extract template (n = 4) were used for testing. RESULTS: Each of the 22 transcripts tested was detected in at least one of the 45 tumor samples. The results of the NanoString assay were 100% concordant with the previous RT-PCR results for the tumor samples, and the technique was successful using both FFPE and rapid extract method. CONCLUSION: Multiplexed interrogation for sarcoma-specific fusion transcripts using NanoString technology is a reliable approach for molecular diagnosis of pediatric sarcomas and works well with FFPE tissues. Future work will involve validating additional sarcoma fusion transcripts as well as determining the optimal workflow for diagnostic purposes.


Assuntos
Código de Barras de DNA Taxonômico , Fusão Gênica/genética , Nanotecnologia , Sarcoma/diagnóstico , Sondas de DNA/genética , Formaldeído , Humanos , Inclusão em Parafina , Pediatria , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma/classificação , Sarcoma/genética
7.
J Neurosurg Pediatr ; 21(2): 145-152, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29219788

RESUMO

OBJECTIVE Metastatic dissemination is a major treatment challenge and cause of death in patients with medulloblastoma. However, the influence of molecular biology on the pattern of metastatic dissemination at diagnosis is not known. In this study, the authors sought to define the location, pattern, and imaging characteristics of medulloblastoma metastases across subgroups at diagnosis. METHODS A consecutive cohort of patients with metastatic medulloblastoma at The Hospital for Sick Children and the University Hospital Motol, who underwent up-front MRI of the craniospinal axis, was assembled and allocated to subgroups using NanoString limited gene-expression profiling. Radiological characteristics (including location, morphology, size, diffusion restriction, and contrast enhancement) were discerned through a retrospective review. RESULTS Forty metastatic medulloblastomas were identified with up-front neuroimaging of the craniospinal axis: 5 sonic hedgehog (SHH), 16 Group 3, and 19 Group 4 metastases. Significant subgroup-specific differences were observed, particularly with respect to tumor location, size, and morphology. Group 3 metastases were most frequently laminar compared with a more nodular pattern in Group 4 (14 of 16 in Group 3 vs 8 of 19 in Group 4; p = 0.0004). Laminar metastases were not observed in patients with SHH medulloblastoma. Suprasellar metastases are highly specific to Group 4 (p = 0.016). Two of the 5 SHH cases had multifocal lesions in the cerebellum, raising the possibility that these were in fact synchronous primary tumors and not true metastases. A minority of patients with Group 4 metastases harbored metastatic deposits that did not enhance on MRI after contrast administration, often in patients whose primary tumor did not enhance. CONCLUSIONS The location, morphology, and imaging characteristics of metastatic medulloblastoma differ across molecular subgroups, with implications for diagnosis and management. This suggests that the biology of leptomeningeal dissemination differs among medulloblastoma subgroups.


Assuntos
Neoplasias Cerebelares/patologia , Meduloblastoma/patologia , Criança , Diagnóstico Diferencial , Humanos , Imagem por Ressonância Magnética/métodos , Metástase Neoplásica , Neoplasias Primárias Múltiplas/patologia , Neuroimagem/métodos , Estudos Retrospectivos
8.
J Neuropathol Exp Neurol ; 76(7): 562-570, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28863456

RESUMO

Previous studies identified recurrent fusion and duplication events in pediatric low-grade glioma (pLGG). In addition to their role in diagnosis, the presence of these events aid in dictating therapy and predicting patient survival. Clinically, BRAF alterations are most commonly identified using fluorescent in situ hybridization (FISH). However, this method is costly, labor-intensive and does not identify nonBRAF events. Here, we evaluated the NanoString nCounter gene expression system for detecting 32 of the most commonly reported fusion/duplication events in pLGG. The assay was validated on 90 pLGG samples using FISH as the gold standard and showed sensitivity and specificity of 97% and 98%, respectively. We next profiled formalin-fixed paraffin-embedded preserved biopsy specimens from 429 pLGG cases. 171 (40%) of the cases within our cohort tested positive for a fusion or duplication event contained within our panel. These events, in order of prevalence, were KIAA1549-BRAF 16;9 (89/171, 52.0%), KIAA1549-BRAF 15;9 (42/171, 24.6%), KIAA1549-BRAF 16;11 (14/171, 8.2%), FGFR1-TACC1 17;7 (13/171, 7.6%), MYBL1 duplication (5/171, 2.9%), KIAA1549-BRAF 18;10 (4/171, 2.3%), KIAA1549-BRAF 15;11 (2/171, 1.2%), FAM131B-BRAF 2;9 (1/171, 0.6%), and RNF130-BRAF 3;9 (1/171, 0.6%). This work introduces NanoString as a viable clinical replacement for the detection of fusion and duplication events in pLGG.


Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas B-raf/genética , Biomarcadores Tumorais/genética , Biópsia , Neoplasias Encefálicas/diagnóstico , Estudos de Coortes , Hibridização Genômica Comparativa , Feminino , Glioma/diagnóstico , Humanos , Hibridização in Situ Fluorescente , Masculino , Mutação/genética , Pediatria , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética
9.
J Clin Oncol ; 35(25): 2934-2941, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28727518

RESUMO

Purpose BRAF V600E is a potentially highly targetable mutation detected in a subset of pediatric low-grade gliomas (PLGGs). Its biologic and clinical effect within this diverse group of tumors remains unknown. Patients and Methods A combined clinical and genetic institutional study of patients with PLGGs with long-term follow-up was performed (N = 510). Clinical and treatment data of patients with BRAF V600E mutated PLGG (n = 99) were compared with a large international independent cohort of patients with BRAF V600E mutated-PLGG (n = 180). Results BRAF V600E mutation was detected in 69 of 405 patients (17%) with PLGG across a broad spectrum of histologies and sites, including midline locations, which are not often routinely biopsied in clinical practice. Patients with BRAF V600E PLGG exhibited poor outcomes after chemotherapy and radiation therapies that resulted in a 10-year progression-free survival of 27% (95% CI, 12.1% to 41.9%) and 60.2% (95% CI, 53.3% to 67.1%) for BRAF V600E and wild-type PLGG, respectively ( P < .001). Additional multivariable clinical and molecular stratification revealed that the extent of resection and CDKN2A deletion contributed independently to poor outcome in BRAF V600E PLGG. A similar independent role for CDKN2A and resection on outcome were observed in the independent cohort. Quantitative imaging analysis revealed progressive disease and a lack of response to conventional chemotherapy in most patients with BRAF V600E PLGG. Conclusion BRAF V600E PLGG constitutes a distinct entity with poor prognosis when treated with current adjuvant therapy.


Assuntos
Neoplasias Encefálicas/enzimologia , Glioma/enzimologia , Proteínas Proto-Oncogênicas B-raf/genética , Adolescente , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Neoplasias do Tronco Encefálico/enzimologia , Neoplasias do Tronco Encefálico/genética , Neoplasias do Tronco Encefálico/patologia , Neoplasias do Tronco Encefálico/terapia , Criança , Pré-Escolar , Estudos de Coortes , Diencéfalo/enzimologia , Diencéfalo/patologia , Feminino , Glioma/genética , Glioma/patologia , Glioma/terapia , Humanos , Lactente , Masculino , Mutação , Gradação de Tumores , Prognóstico
10.
Acta Neuropathol Commun ; 4(1): 93, 2016 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-27577993

RESUMO

Paediatric brain tumours arising in the thalamus present significant diagnostic and therapeutic challenges to physicians due to their sensitive midline location. As such, genetic analysis for biomarkers to aid in the diagnosis, prognosis and treatment of these tumours is needed. Here, we identified 64 thalamic gliomas with clinical follow-up and characterized targeted genomic alterations using newly optimized droplet digital and NanoString-based assays. The median age at diagnosis was 9.25 years (range, 0.63-17.55) and median survival was 6.43 (range, 0.01-27.63) years. Our cohort contained 42 and 22 tumours reviewed as low and high grade gliomas, respectively. Five (12 %) low grade and 11 (50 %) high grade gliomas were positive for the H3F3A/HIST1H3B K27M (H3K27M) mutation. Kaplan-Meier survival analysis revealed significantly worse overall survival for patients harbouring the H3K27M mutation versus H3F3A/HIST1H3B wild type (H3WT) samples (log-rank p < 0.0001) with a median survival of 1.02 vs. 9.12 years. Mitogen-activated protein kinase (MAPK) pathway activation via BRAF or FGFR1 hotspot mutations or fusion events were detected in 44 % of patients, and was associated with long-term survival in the absence of H3K27M (log-rank p < 0.0001). Multivariate analysis demonstrated H3K27M status and high grade histology to be the most significant independent predictors of poor overall survival with hazard ratios of 6.945 and 7.721 (p < 0.0001), respectively. In contrast, MAPK pathway activation is a predictor of favourable patient outcome, although not independent of other clinical factors. Importantly, we show that low grade malignancies may harbour H3K27M mutations and that these tumours show a dismal survival compared to low grade H3WT cases. Our data strongly supports the inclusion of targeted genetic testing in childhood thalamic tumours to most accurately stratify patients into appropriate risk groups.


Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Sistema de Sinalização das MAP Quinases/genética , Tálamo , Adolescente , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/cirurgia , Criança , Pré-Escolar , Feminino , Seguimentos , Glioma/patologia , Glioma/cirurgia , Humanos , Lactente , Estimativa de Kaplan-Meier , Masculino , Análise Multivariada , Mutação , Gradação de Tumores , Prognóstico , Modelos de Riscos Proporcionais , Tálamo/patologia , Tálamo/cirurgia
11.
J Clin Oncol ; 34(29): 3537-3543, 2016 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-27573663

RESUMO

Purpose Vinblastine monotherapy has shown promising activity and a low-toxicity profile in patients with pediatric low-grade glioma (PLGG) who experienced treatment failure after initial treatment with chemotherapy and/or radiation. The aim of this study was to assess the activity of vinblastine in therapy-naïve children. Patients and Methods Patients < 18 years old with unresectable and/or progressive therapy-naïve PLGG were eligible. Vinblastine was administered once per week at a dose of 6 mg/m2 intravenously over a period of 70 weeks. Vision, quality of life, neurofibromatosis type 1 (NF1) status, and BRAF mutation/fusion status were also determined and correlated with outcome. Results Fifty-four patients were enrolled onto the study, with a median age of 8 years (range, 0.7 to 17.2 years). Most patients had chiasmatic/hypothalamic tumors (55.5%), and 13 patients (24.1%) had NF1. The most common histology was pilocytic astrocytoma (46.3%). Seventeen patients were diagnosed using radiologic criteria alone. Best response to chemotherapy was centrally reviewed with a response rate (complete, partial, or minor response) of 25.9%. Disease stabilization (complete, partial, or minor response or stable disease) was achieved in 47 patients (87.0%). Visual improvement was observed in 20% of patients with optic pathway glioma. Five-year overall survival and progression-free survival (PFS) rates were 94.4% (95% CI, 88.5% to 100%) and 53.2% (95% CI, 41.3% to 68.5%), respectively, for the entire cohort. Patients with NF1 had a significantly better PFS (85.1%; 95% CI, 68.0% to 100%) when compared with patients without NF1 (42.0%; 95% CI, 29.1% to 60.7%; P = .012). Age< 3 years or > 10 years was not associated with poor outcome. Treatment was well tolerated, and quality of life was not affected during treatment. In this trial, there was no correlation between BRAF alterations and outcome. Conclusion Vinblastine administered once per week is well tolerated in children with treatment naïve PLGG. Overall survival and PFS are comparable to current therapies, with a favorable toxicity profile and a maintained quality of life.


Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Astrocitoma/tratamento farmacológico , Neoplasias Encefálicas/tratamento farmacológico , Neurofibromatose 1/tratamento farmacológico , Vimblastina/administração & dosagem , Adolescente , Antineoplásicos Fitogênicos/efeitos adversos , Astrocitoma/complicações , Astrocitoma/genética , Astrocitoma/patologia , Neoplasias Encefálicas/complicações , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Canadá , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Humanos , Lactente , Masculino , Mutação , Gradação de Tumores , Neurofibromatose 1/patologia , Fusão Oncogênica , Proteínas Proto-Oncogênicas B-raf/genética , Qualidade de Vida , Taxa de Sobrevida , Vimblastina/efeitos adversos , Transtornos da Visão/etiologia
12.
Genome Med ; 6(4): 32, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24944581

RESUMO

Target identification is a critical step in the lengthy and expensive process of drug development. Here, we describe a genome-wide screening platform that uses systematic overexpression of pooled human ORFs to understand drug mode-of-action and resistance mechanisms. We first calibrated our screen with the well-characterized drug methotrexate. We then identified new genes involved in the bioactivity of diverse drugs including antineoplastic agents and biologically active molecules. Finally, we focused on the transcription factor RHOXF2 whose overexpression conferred resistance to DNA damaging agents. This approach represents an orthogonal method for functional screening and, to our knowledge, has never been reported before.

13.
Biochem Biophys Res Commun ; 445(4): 746-56, 2014 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-24561123

RESUMO

G-protein coupled receptors (GPCRs) are involved in a variety of disease processes and comprise major drug targets. However, the complexity of integral membrane proteins such as GPCRs makes the identification of their interacting partners and subsequent drug development challenging. A comprehensive understanding of GPCR protein interaction networks is needed to design effective therapeutic strategies to inhibit these drug targets. Here, we developed a novel split-ubiquitin membrane yeast two-hybrid (MYTH) technology called CHIP-MYTH, which allows the unbiased characterization of interaction partners of full-length GPCRs in a drug-dependent manner. This was achieved by coupling DNA microarray technology to the MYTH approach, which allows a quantitative evaluation of interacting partners of a given integral membrane protein in the presence or absence of drug. As a proof of principle, we applied the CHIP-MYTH approach to the human ß2-adrenergic receptor (ß2AR), a target of interest in the treatment of asthma, chronic obstructive pulmonary disease (COPD), neurological disease, cardiovascular disease, and obesity. A CHIP-MYTH screen was performed in the presence or absence of salmeterol, a long-acting ß2AR-agonist. Our results suggest that ß2AR activation with salmeterol can induce the dissociation of heterotrimeric G-proteins, Gαßγ, into Gα and Gßγ subunits, which in turn activates downstream signaling cascades. Using CHIP-MYTH, we confirmed previously known and identified novel ß2AR interactors involved in GPCR-mediated signaling cascades. Several of these interactions were confirmed in mammalian cells using LUminescence-based Mammalian IntERactome (LUMIER) and co-immunoprecipitation assays. In summary, the CHIP-MYTH approach is ideal for conducting comprehensive protein-protein interactions (PPI) screenings of full-length GPCRs in the presence or absence of drugs, thus providing a valuable tool to further our understanding of GPCR-mediated signaling.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Albuterol/análogos & derivados , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteômica/métodos , Receptores Adrenérgicos beta 2/metabolismo , Albuterol/farmacologia , Animais , Células HEK293 , Humanos , Modelos Moleculares , Receptores Acoplados a Proteínas-G/metabolismo , Xinafoato de Salmeterol , Transdução de Sinais/efeitos dos fármacos , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo
14.
G3 (Bethesda) ; 3(8): 1375-87, 2013 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-23797109

RESUMO

The application of new proteomics and genomics technologies support a view in which few drugs act solely by inhibiting a single cellular target. Indeed, drug activity is modulated by complex, often incompletely understood cellular mechanisms. Therefore, efforts to decipher mode of action through genetic perturbation such as RNAi typically yields "hits" that fall into several categories. Of particular interest to the present study, we aimed to characterize secondary activities of drugs on cells. Inhibiting a known target can result in clinically relevant synthetic phenotypes. In one scenario, drug perturbation could, for example, improperly activate a protein that normally inhibits a particular kinase. In other cases, additional, lower affinity targets can be inhibited as in the example of inhibition of c-Kit observed in Bcr-Abl-positive cells treated with Gleevec. Drug transport and metabolism also play an important role in the way any chemicals act within the cells. Finally, RNAi per se can also affect cell fitness by more general off-target effects, e.g., via the modulation of apoptosis or DNA damage repair. Regardless of the root cause of these unwanted effects, understanding the scope of a drug's activity and polypharmacology is essential for better understanding its mechanism(s) of action, and such information can guide development of improved therapies. We describe a rapid, cost-effective approach to characterize primary and secondary effects of small-molecules by using small-scale libraries of virally integrated short hairpin RNAs. We demonstrate this principle using a "minipool" composed of shRNAs that target the genes encoding the reported protein targets of approved drugs. Among the 28 known reported drug-target pairs, we successfully identify 40% of the targets described in the literature and uncover several unanticipated drug-target interactions based on drug-induced synthetic lethality. We provide a detailed protocol for performing such screens and for analyzing the data. This cost-effective approach to mammalian knockdown screens, combined with the increasing maturation of RNAi technology will expand the accessibility of similar approaches in academic settings.


Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Piperazinas/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , Linhagem Celular Tumoral , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mesilato de Imatinib , Lentivirus/genética , Miniaturização , Proteínas/antagonistas & inibidores , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo
15.
BMC Genomics ; 12: 213, 2011 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-21548937

RESUMO

BACKGROUND: Genome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP) is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens. RESULTS: Experiments with specially constructed lentiviral-based plasmid pools containing ~78,000 shRNAs demonstrated that the GMAP is capable of deconvolving genome-wide shRNA "dropout" screens. Further experiments with a larger, ~90,000 shRNA pool demonstrate that equivalent results are obtained from plasmid pools and from genomic DNA derived from lentivirus infected cells. Parallel testing of large shRNA pools using GMAP and next-generation sequencing methods revealed that the two methods provide valid and complementary approaches to deconvolution of genome-wide shRNA screens. Additional experiments demonstrated that GMAP is equivalent to similar microarray-based products when used for deconvolution of open reading frame over-expression screens. CONCLUSION: Herein, we demonstrate four major applications for the GMAP resource, including deconvolution of pooled RNAi screens in cells with at least 90,000 distinct shRNAs. We also provide detailed methodologies for pooled shRNA screen readout using GMAP and compare next-generation sequencing to GMAP (i.e. microarray) based deconvolution methods.


Assuntos
Testes Genéticos/métodos , Genômica/métodos , Animais , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta/genética , Controle de Qualidade , Interferência de RNA , Saccharomyces cerevisiae/genética , Software
16.
PLoS Genet ; 4(2): e1000005, 2008 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-18454192

RESUMO

Pseudomonas aeruginosa is an opportunistic human pathogen that is a key factor in the mortality of cystic fibrosis patients, and infection represents an increased threat for human health worldwide. Because resistance of Pseudomonas aeruginosa to antibiotics is increasing, new inhibitors of pharmacologically validated targets of this bacterium are needed. Here we demonstrate that a cell-based yeast phenotypic assay, combined with a large-scale inhibitor screen, identified small molecule inhibitors that can suppress the toxicity caused by heterologous expression of selected Pseudomonas aeruginosa ORFs. We identified the first small molecule inhibitor of Exoenzyme S (ExoS), a toxin involved in Type III secretion. We show that this inhibitor, exosin, modulates ExoS ADP-ribosyltransferase activity in vitro, suggesting the inhibition is direct. Moreover, exosin and two of its analogues display a significant protective effect against Pseudomonas infection in vivo. Furthermore, because the assay was performed in yeast, we were able to demonstrate that several yeast homologues of the known human ExoS targets are likely ADP-ribosylated by the toxin. For example, using an in vitro enzymatic assay, we demonstrate that yeast Ras2p is directly modified by ExoS. Lastly, by surveying a collection of yeast deletion mutants, we identified Bmh1p, a yeast homologue of the human FAS, as an ExoS cofactor, revealing that portions of the bacterial toxin mode of action are conserved from yeast to human. Taken together, our integrated cell-based, chemical-genetic approach demonstrates that such screens can augment traditional drug screening approaches and facilitate the discovery of new compounds against a broad range of human pathogens.


Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Pseudomonas aeruginosa/enzimologia , ADP Ribose Transferases/genética , Inibidores de Adenilil Ciclases , Adenilil Ciclases/genética , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Toxinas Bacterianas/genética , Células CHO , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Expressão Gênica , Genes Bacterianos , Humanos , Testes de Sensibilidade Microbiana , Mutação , Fases de Leitura Aberta , Fenótipo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Virulência/genética
17.
Mol Cell ; 26(1): 15-25, 2007 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-17434123

RESUMO

The ATP binding cassette (ABC) transporters are important in human health and disease and represent the largest family of transmembrane proteins; however, their highly hydrophobic nature complicates the use of standard biochemical approaches to identify interacting proteins. Here, we report the development of a modified version of the split-ubiquitin membrane yeast two-hybrid (MYTH) technology using genomically integrated "bait" constructs, hence the designation iMYTH. We used iMYTH in a library-screening format and identified six potential interacting partners of the yeast ABC transporter Ycf1p. Strains deleted for several of these genes result in arsenite sensitivity similar to a Deltaycf1 strain. Transport assays show that one of these, Tus1p, a guanine nucleotide exchange factor (GEF) for the small GTPase Rho1p, is a Rho1p-dependent-positive regulator of Ycf1p. Our study provides proof of principle that iMYTH is an ideal methodology to identify physiological interactors and regulators of ABC transporters and other yeast transmembrane proteins.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Membrana Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transporte Biológico , Citosol/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Metais Pesados/farmacologia , Modelos Biológicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Deleção de Sequência , Proteínas rho de Ligação ao GTP/metabolismo
18.
Genome Res ; 13(7): 1744-53, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12840049

RESUMO

Analysis of membrane protein interactions is difficult because of the hydrophobic nature of these proteins, which often renders conventional biochemical and genetic assays fruitless. This is a substantial problem because proteins that are integral or associated with membranes represent approximately one-third of all proteins in a typical eukaryotic cell. We have shown previously that the modified split-ubiquitin system can be used as a genetic assay for the in vivo detection of interactions between the two characterized yeast transmembrane proteins, Ost1p and Wbp1p. This so-called split-ubiquitin membrane yeast two-hybrid (YTH) system uses the split-ubiquitin approach in which reconstitution of two ubiquitin halves is mediated by a protein-protein interaction. Here we converted the split-ubiquitin membrane YTH system into a generally applicable in vivo screening approach to identify interacting partners of a particular mammalian transmembrane protein. We have demonstrated the effectiveness of this approach by using the mammalian ErbB3 receptor as bait and have identified three previously unknown ErbB3-interacting proteins. In addition, we have confirmed one of the newly found interactions between ErbB3 and the membrane-associated RGS4 protein by coimmunoprecipitating the two proteins from human cells. We expect the split-ubiquitin membrane YTH technology to be valuable for the identification of potential interacting partners of integral membrane proteins from many model organisms.


Assuntos
Proteínas RGS/metabolismo , Receptor ErbB-3/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Biblioteca Gênica , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Humanos , Rim/química , Rim/embriologia , Rim/metabolismo , Substâncias Macromoleculares , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mapeamento de Peptídeos , Mapeamento de Interação de Proteínas , Proteínas RGS/química , Proteínas RGS/genética , Ratos , Receptor ErbB-3/biossíntese , Receptor ErbB-3/química , Receptor ErbB-3/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Ubiquitina/biossíntese , Ubiquitina/genética
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