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1.
Cell ; 173(4): 864-878.e29, 2018 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-29681454

RESUMEN

Diversity in the genetic lesions that cause cancer is extreme. In consequence, a pressing challenge is the development of drugs that target patient-specific disease mechanisms. To address this challenge, we employed a chemistry-first discovery paradigm for de novo identification of druggable targets linked to robust patient selection hypotheses. In particular, a 200,000 compound diversity-oriented chemical library was profiled across a heavily annotated test-bed of >100 cellular models representative of the diverse and characteristic somatic lesions for lung cancer. This approach led to the delineation of 171 chemical-genetic associations, shedding light on the targetability of mechanistic vulnerabilities corresponding to a range of oncogenotypes present in patient populations lacking effective therapy. Chemically addressable addictions to ciliogenesis in TTC21B mutants and GLUT8-dependent serine biosynthesis in KRAS/KEAP1 double mutants are prominent examples. These observations indicate a wealth of actionable opportunities within the complex molecular etiology of cancer.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular/efectos de los fármacos , Neoplasias Pulmonares/patología , Bibliotecas de Moléculas Pequeñas/farmacología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Familia 4 del Citocromo P450/deficiencia , Familia 4 del Citocromo P450/genética , Descubrimiento de Drogas , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Glucocorticoides/farmacología , Proteínas Facilitadoras del Transporte de la Glucosa/antagonistas & inhibidores , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo
2.
Cell ; 155(3): 552-66, 2013 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-24243015

RESUMEN

Context-specific molecular vulnerabilities that arise during tumor evolution represent an attractive intervention target class. However, the frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets. To confront this challenge, we have applied parallel screening of chemical and genetic perturbations within a panel of molecularly annotated NSCLC lines to identify intervention opportunities tightly linked to molecular response indicators predictive of target sensitivity. Anchoring this analysis on a matched tumor/normal cell model from a lung adenocarcinoma patient identified three distinct target/response-indicator pairings that are represented with significant frequencies (6%-16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occurring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature. Target efficacies were validated in vivo, and mechanism-of-action studies informed generalizable principles underpinning cancer cell biology.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Indoles/farmacología , Neoplasias Pulmonares/metabolismo , Triazinas/farmacología , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Portadoras , Línea Celular Tumoral , Proteína Coatómero/metabolismo , Femenino , Genes ras , Xenoinjertos , Humanos , Neoplasias Pulmonares/patología , Lisosomas/metabolismo , Ratones , Terapia Molecular Dirigida , Proteína con Dominio Pirina 3 de la Familia NLR , Trasplante de Neoplasias , Fosforilación Oxidativa
3.
Nature ; 538(7623): 114-117, 2016 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-27680702

RESUMEN

The common participation of oncogenic KRAS proteins in many of the most lethal human cancers, together with the ease of detecting somatic KRAS mutant alleles in patient samples, has spurred persistent and intensive efforts to develop drugs that inhibit KRAS activity. However, advances have been hindered by the pervasive inter- and intra-lineage diversity in the targetable mechanisms that underlie KRAS-driven cancers, limited pharmacological accessibility of many candidate synthetic-lethal interactions and the swift emergence of unanticipated resistance mechanisms to otherwise effective targeted therapies. Here we demonstrate the acute and specific cell-autonomous addiction of KRAS-mutant non-small-cell lung cancer cells to receptor-dependent nuclear export. A multi-genomic, data-driven approach, utilizing 106 human non-small-cell lung cancer cell lines, was used to interrogate 4,725 biological processes with 39,760 short interfering RNA pools for those selectively required for the survival of KRAS-mutant cells that harbour a broad spectrum of phenotypic variation. Nuclear transport machinery was the sole process-level discriminator of statistical significance. Chemical perturbation of the nuclear export receptor XPO1 (also known as CRM1), with a clinically available drug, revealed a robust synthetic-lethal interaction with native or engineered oncogenic KRAS both in vitro and in vivo. The primary mechanism underpinning XPO1 inhibitor sensitivity was intolerance to the accumulation of nuclear IκBα (also known as NFKBIA), with consequent inhibition of NFκB transcription factor activity. Intrinsic resistance associated with concurrent FSTL5 mutations was detected and determined to be a consequence of YAP1 activation via a previously unappreciated FSTL5-Hippo pathway regulatory axis. This occurs in approximately 17% of KRAS-mutant lung cancers, and can be overcome with the co-administration of a YAP1-TEAD inhibitor. These findings indicate that clinically available XPO1 inhibitors are a promising therapeutic strategy for a considerable cohort of patients with lung cancer when coupled to genomics-guided patient selection and observation.


Asunto(s)
Transporte Activo de Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Carioferinas/antagonistas & inhibidores , Carioferinas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Femenino , Proteínas Relacionadas con la Folistatina/genética , Genes Letales/genética , Vía de Señalización Hippo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Mutación , Inhibidor NF-kappaB alfa/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/metabolismo , Porfirinas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal , Factores de Transcripción de Dominio TEA , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Verteporfina , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Señalizadoras YAP , Proteína Exportina 1
4.
Bioorg Med Chem Lett ; 25(20): 4393-8, 2015 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-26372654

RESUMEN

An efficient total synthesis of the potent V-ATPase inhibitor saliphenylhalamide (SaliPhe), a synthetic variant of the natural product salicylihalamide A (SaliA), has been accomplished aimed at facilitating the development of SaliPhe as an anticancer and antiviral agent. This new approach enabled facile access to derivatives for structure-activity relationship studies, leading to simplified analogs that maintain SaliPhe's biological properties. These studies will provide a solid foundation for the continued evaluation of SaliPhe and analogs as potential anticancer and antiviral agents.


Asunto(s)
Amidas/farmacología , Inhibidores Enzimáticos/farmacología , Salicilatos/farmacología , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Amidas/síntesis química , Amidas/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Salicilatos/síntesis química , Salicilatos/química , Relación Estructura-Actividad , ATPasas de Translocación de Protón Vacuolares/metabolismo
5.
Cancer Lett ; 604: 217219, 2024 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-39244005

RESUMEN

Luminal Androgen Receptor (LAR) triple-negative breast cancers (TNBC) express androgen receptors (AR), exhibit high frequency of PIK3CA mutations and intact RB. Herein, we investigated combined blockade of the CDK4/6 and PI3K signaling with palbociclib, alpelisib, and capivasertib, which inhibit CDK4/6, PI3Kα, and AKT1-3, respectively. The combination of palbociclib/capivasertib, but not palbociclib/alpelisib, synergistically inhibited proliferation of MDA-MB-453 and MFM-223 LAR cells [synergy score 7.34 (p = 5.81x10-11) and 4.78 (p = 0.012), respectively]. The AR antagonist enzalutamide was inactive against MDA-MB-453, MFM-223, and CAL148 cells and did not enhance the efficacy of either combination. Palbociclib/capivasertib inhibited growth of LAR patient-derived xenografts more potently than palbociclib/alpelisib. Treatment of LAR cells with palbociclib suppressed phosphorylated-RB and resulted in adaptive phosphorylation/activation of S473 pAKT and AKT substrates GSK3ß, PRAS40, and FoxO3a. Capivasertib blocked palbociclib-induced phosphorylation of AKT substrates more potently than alpelisib. Treatment with PI3Kß inhibitors did not block phosphorylation of AKT substrates, suggesting that PI3Kß did not mediate the adaptive response to CDK4/6 inhibition. Phosphokinase arrays of MDA-MB-453 cells treated with palbociclib showed time-dependent upregulation of PDGFRß, GSK3ß, STAT3, and STAT6. RNA silencing of PDGFRß in palbociclib-treated MDA-MB-453 and MFM-223 cells blocked the upregulation of S473 pAKT, suggesting that the adaptive response to CDK4/6 blockade involves PDGFRß signaling. Finally, treatment with palbociclib and the PDGFR inhibitor CP637451 arrested growth of MDA-MB-453 and MFM-223 cells to the same degree as palbociclib/capivasertib. These findings support testing the combination of CDK4/6 and AKT inhibitors in patients with LAR TNBC, and further investigation of PDGFR antagonists in this breast cancer subtype.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Proliferación Celular , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Piperazinas , Proteínas Proto-Oncogénicas c-akt , Piridinas , Receptores Androgénicos , Neoplasias de la Mama Triple Negativas , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Piperazinas/farmacología , Animales , Femenino , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/metabolismo , Piridinas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/metabolismo , Receptores Androgénicos/metabolismo , Receptores Androgénicos/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proliferación Celular/efectos de los fármacos , Ratones , Pirimidinas/farmacología , Sinergismo Farmacológico , Transducción de Señal/efectos de los fármacos , Feniltiohidantoína/farmacología , Feniltiohidantoína/análogos & derivados , Benzamidas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Nitrilos/farmacología , Pirroles/farmacología , Tiazoles
6.
Cancer Immunol Res ; 10(7): 829-843, 2022 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-35561311

RESUMEN

The MYC oncogene is frequently amplified in triple-negative breast cancer (TNBC). Here, we show that MYC suppression induces immune-related hallmark gene set expression and tumor-infiltrating T cells in MYC-hyperactivated TNBCs. Mechanistically, MYC repressed stimulator of interferon genes (STING) expression via direct binding to the STING1 enhancer region, resulting in downregulation of the T-cell chemokines CCL5, CXCL10, and CXCL11. In primary and metastatic TNBC cohorts, tumors with high MYC expression or activity exhibited low STING expression. Using a CRISPR-mediated enhancer perturbation approach, we demonstrated that MYC-driven immune evasion is mediated by STING repression. STING repression induced resistance to PD-L1 blockade in mouse models of TNBC. Finally, a small-molecule inhibitor of MYC combined with PD-L1 blockade elicited a durable response in immune-cold TNBC with high MYC expression, suggesting a strategy to restore PD-L1 inhibitor sensitivity in MYC-overexpressing TNBC.


Asunto(s)
Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Neoplasias de la Mama Triple Negativas , Animales , Antígeno B7-H1 , Línea Celular Tumoral , Represión Epigenética , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Evasión Inmune , Ratones , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología
7.
Clin Cancer Res ; 27(15): 4379-4396, 2021 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-34011560

RESUMEN

PURPOSE: FGFR1 overexpression has been associated with endocrine resistance in ER+ breast cancer. We found FGFR1 localized in the nucleus of breast cancer cells in primary tumors resistant to estrogen suppression. We investigated a role of nuclear FGFR1 on gene transcription and antiestrogen resistance. EXPERIMENTAL DESIGN: Tumors from patients treated with letrozole were subjected to Ki67 and FGFR1 IHC. MCF7 cells were transduced with FGFR1(SP-)(NLS) to promote nuclear FGFR1 overexpression. FGFR1 genomic activity in ER+/FGFR1-amplified breast cancer cells ± FOXA1 siRNA or ± the FGFR tyrosine kinase inhibitor (TKI) erdafitinib was examined by chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). The nuclear and chromatin-bound FGFR1 interactome was investigated by mass spectrometry (MS). RESULTS: High nuclear FGFR1 expression in ER+ primary tumors positively correlated with post-letrozole Ki67 values. Nuclear FGFR1 overexpression influenced gene transcription and promoted resistance to estrogen suppression and to fulvestrant in vivo. A gene expression signature induced by nuclear FGFR1 correlated with shorter survival in the METABRIC cohort of patients treated with antiestrogens. ChIP-Seq revealed FGFR1 occupancy at transcription start sites, overlapping with active transcription histone marks. MS analysis of the nuclear FGFR1 interactome identified phosphorylated RNA-Polymerase II and FOXA1, with FOXA1 RNAi impairing FGFR1 recruitment to chromatin. Treatment with erdafitinib did not impair nuclear FGFR1 translocation and genomic activity. CONCLUSIONS: These data suggest nuclear FGFR1 contributes to endocrine resistance by modulating gene transcription in ER+ breast cancer. Nuclear FGFR1 activity was unaffected by FGFR TKIs, thus supporting the development of treatment strategies to inhibit nuclear FGFR1 in ER+/FGFR1 overexpressing breast cancer.


Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/genética , Moduladores de los Receptores de Estrógeno/uso terapéutico , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/fisiología , Transcripción Genética/fisiología , Neoplasias de la Mama/química , Núcleo Celular , Femenino , Humanos , Receptores de Estrógenos/análisis , Células Tumorales Cultivadas
8.
Cell Chem Biol ; 26(10): 1380-1392.e6, 2019 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-31378711

RESUMEN

Gene expression signature-based inference of functional connectivity within and between genetic perturbations, chemical perturbations, and disease status can lead to the development of actionable hypotheses for gene function, chemical modes of action, and disease treatment strategies. Here, we report a FuSiOn-based genome-wide integration of hypomorphic cellular phenotypes that enables functional annotation of gene network topology, assignment of mechanistic hypotheses to genes of unknown function, and detection of cooperativity among cell regulatory systems. Dovetailing genetic perturbation data with chemical perturbation phenotypes allowed simultaneous generation of mechanism of action hypotheses for thousands of uncharacterized natural products fractions (NPFs). The predicted mechanism of actions span a broad spectrum of cellular mechanisms, many of which are not currently recognized as "druggable." To enable use of FuSiOn as a hypothesis generation resource, all associations and analyses are available within an open source web-based GUI (http://fusion.yuhs.ac).


Asunto(s)
Productos Biológicos/farmacología , Descubrimiento de Drogas , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Programas Informáticos , Productos Biológicos/química , Células HCT116 , Células HeLa , Humanos , Fenotipo , Transcriptoma , Células Tumorales Cultivadas
9.
Nat Commun ; 9(1): 2050, 2018 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-29784984

RESUMEN

The originally published version of this Article contained an error in the spelling of the author Nathaniel W. Oswald, which was incorrectly given as Nathaniel W. Olswald. This has now been corrected in both the PDF and HTML versions of the Article.

10.
Nat Commun ; 8(1): 2270, 2017 12 22.
Artículo en Inglés | MEDLINE | ID: mdl-29273768

RESUMEN

Drugs that mirror the cellular effects of starvation mimics are considered promising therapeutics for common metabolic disorders, such as obesity, liver steatosis, and for ageing. Starvation, or caloric restriction, is known to activate the transcription factor EB (TFEB), a master regulator of lipid metabolism and lysosomal biogenesis and function. Here, we report a nanotechnology-enabled high-throughput screen to identify small-molecule agonists of TFEB and discover three novel compounds that promote autophagolysosomal activity. The three lead compounds include the clinically approved drug, digoxin; the marine-derived natural product, ikarugamycin; and the synthetic compound, alexidine dihydrochloride, which is known to act on a mitochondrial target. Mode of action studies reveal that these compounds activate TFEB via three distinct Ca2+-dependent mechanisms. Formulation of these compounds in liver-tropic biodegradable, biocompatible nanoparticles confers hepatoprotection against diet-induced steatosis in murine models and extends lifespan of Caenorhabditis elegans. These results support the therapeutic potential of small-molecule TFEB activators for the treatment of metabolic and age-related disorders.


Asunto(s)
Autofagia/efectos de los fármacos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/agonistas , Biguanidas/farmacología , Caenorhabditis elegans/efectos de los fármacos , Digoxina/farmacología , Inhibidores Enzimáticos/farmacología , Lactamas/farmacología , Longevidad/efectos de los fármacos , Síndrome Metabólico/metabolismo , Animales , Autofagosomas/efectos de los fármacos , Autofagosomas/metabolismo , Caenorhabditis elegans/metabolismo , Calcio/metabolismo , Restricción Calórica , Dieta Alta en Grasa , Hígado Graso/metabolismo , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Inanición
11.
Cancer Discov ; 7(8): 832-851, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28455392

RESUMEN

Genomic diversity among melanoma tumors limits durable control with conventional and targeted therapies. Nevertheless, pathologic activation of the ERK1/2 pathway is a linchpin tumorigenic mechanism associated with the majority of primary and recurrent disease. Therefore, we sought to identify therapeutic targets that are selectively required for tumorigenicity in the presence of pathologic ERK1/2 signaling. By integration of multigenome chemical and genetic screens, recurrent architectural variants in melanoma tumor genomes, and patient outcome data, we identified two mechanistic subtypes of BRAFV600 melanoma that inform new cancer cell biology and offer new therapeutic opportunities. Subtype membership defines sensitivity to clinical MEK inhibitors versus TBK1/IKBKε inhibitors. Importantly, subtype membership can be predicted using a robust quantitative five-feature genetic biomarker. This biomarker, and the mechanistic relationships linked to it, can identify a cohort of best responders to clinical MEK inhibitors and identify a cohort of TBK1/IKBKε inhibitor-sensitive disease among nonresponders to current targeted therapy.Significance: This study identified two mechanistic subtypes of melanoma: (1) the best responders to clinical BRAF/MEK inhibitors (25%) and (2) nonresponders due to primary resistance mechanisms (9.9%). We identified robust biomarkers that can detect these subtypes in patient samples and predict clinical outcome. TBK1/IKBKε inhibitors were selectively toxic to drug-resistant melanoma. Cancer Discov; 7(8); 832-51. ©2017 AACR.See related commentary by Jenkins and Barbie, p. 799This article is highlighted in the In This Issue feature, p. 783.


Asunto(s)
Biomarcadores de Tumor/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Animales , Carcinogénesis/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/clasificación , Melanoma/patología , Ratones , Mutación , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Ensayos Antitumor por Modelo de Xenoinjerto
12.
PLoS One ; 11(1): e0144970, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26788852

RESUMEN

CRISPR/Cas9 is an enabling RNA-guided technology for genome targeting and engineering. An acute DNA binding constraint of the Cas9 protein is the Protospacer Adjacent Motif (PAM). Here we demonstrate that the PAM requirement can be exploited to specifically target single-nucleotide heterozygous mutations while exerting no aberrant effects on the wild-type alleles. Specifically, we target the heterozygous G13A activating mutation of KRAS in colorectal cancer cells and we show reversal of drug resistance to a MEK small-molecule inhibitor. Our study introduces a new paradigm in genome editing and therapeutic targeting via the use of gRNA to guide Cas9 to a desired protospacer adjacent motif.


Asunto(s)
Sistemas CRISPR-Cas , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas p21(ras)/genética , ARN/química , Bencimidazoles/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Células HEK293 , Humanos , ARN/metabolismo , Edición de ARN , ARN Guía de Kinetoplastida/genética
13.
Mol Cell Biol ; 34(12): 2350-8, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24732797

RESUMEN

The RASSF1A gene is one of the most frequently inactivated genes in over 30 different types of cancers (H. Donninger, M. D. Vos, and G. J. Clark, J. Cell Sci. 120:3163-3172, 2007, http://dx.doi.org/10.1242/jcs.010389). Despite the prevalence of RASSF1A silencing in human cancer, the mechanism by which RASSF1A functions as a tumor suppressor is not well understood. Characterization of the consequences of RASSF1A loss on epithelial cell proliferation revealed that RASSF1A expression suppresses both microRNA 21 (miR-21) expression and extracellular signal-regulated kinase 1/2 (ERK1/2) activation. The mechanism of the former is through restraint of SCF(ßTrCP)-dependent destruction of the repressor element 1 silencing transcription factor (REST) tumor suppressor and consequent inhibition of miR-21 promoter activation. The mechanism of the latter is through physical sequestration of MST2, which results in accumulation of inactivating S259 phosphorylation of RAF1. Whether or not inactivation of these RASSF1A regulatory relationships can unleash enhanced proliferative capacity is dependent upon the coupling of SCF(ßTrCP) and miR-21 to suppression of SKP2 protein translation and stability. Airway epithelial cultures retain this coupling and therefore respond to RASSF1A inactivation by p27-dependent cell cycle arrest. In contrast, colonic crypt-derived epithelial cells have uncoupled SCF(ßTrCP) from SKP2 and respond to RASSF1A inactivation by enhanced proliferation rates. These observations help account for context-specific molecular etiology of oncogenic transformation and suggest intervention strategies for recently developed SKP2 inhibitors.


Asunto(s)
Ciclo Celular/genética , Genes Supresores de Tumor , Oncogenes , Transducción de Señal/genética , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Masculino , MicroARNs/metabolismo , Datos de Secuencia Molecular , Proteínas Represoras/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo
14.
Sci Signal ; 6(297): ra90, 2013 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-24129700

RESUMEN

A challenge for biomedical research is the development of pharmaceuticals that appropriately target disease mechanisms. Natural products can be a rich source of bioactive chemicals for medicinal applications but can act through unknown mechanisms and can be difficult to produce or obtain. To address these challenges, we developed a new marine-derived, renewable natural products resource and a method for linking bioactive derivatives of this library to the proteins and biological processes that they target in cells. We used cell-based screening and computational analysis to match gene expression signatures produced by natural products to those produced by small interfering RNA (siRNA) and synthetic microRNA (miRNA) libraries. With this strategy, we matched proteins and miRNAs with diverse biological processes and also identified putative protein targets and mechanisms of action for several previously undescribed marine-derived natural products. We confirmed mechanistic relationships for selected siRNAs, miRNAs, and compounds with functional roles in autophagy, chemotaxis mediated by discoidin domain receptor 2, or activation of the kinase AKT. Thus, this approach may be an effective method for screening new drugs while simultaneously identifying their targets.


Asunto(s)
Productos Biológicos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ontología de Genes , Transcriptoma/efectos de los fármacos , Animales , Autofagia/efectos de los fármacos , Autofagia/genética , Bacterias/química , Bacterias/clasificación , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Línea Celular Tumoral , Células Cultivadas , Análisis por Conglomerados , Biología Computacional/métodos , Evaluación Preclínica de Medicamentos/métodos , Redes Reguladoras de Genes/efectos de los fármacos , Redes Reguladoras de Genes/genética , Células HCT116 , Humanos , Invertebrados/química , Células MCF-7 , Biología Marina , Ratones , Ratones Endogámicos BALB C , MicroARNs/genética , Estructura Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN
15.
PLoS One ; 7(8): e39284, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22876275

RESUMEN

Influenza A virus infects 5-20% of the population annually, resulting in ~35,000 deaths and significant morbidity. Current treatments include vaccines and drugs that target viral proteins. However, both of these approaches have limitations, as vaccines require yearly development and the rapid evolution of viral proteins gives rise to drug resistance. In consequence additional intervention strategies, that target host factors required for the viral life cycle, are under investigation. Here we employed arrayed whole-genome siRNA screening strategies to identify cell-autonomous molecular components that are subverted to support H1N1 influenza A virus infection of human bronchial epithelial cells. Integration across relevant public data sets exposed druggable gene products required for epithelial cell infection or required for viral proteins to deflect host cell suicide checkpoint activation. Pharmacological inhibition of representative targets, RGGT and CHEK1, resulted in significant protection against infection of human epithelial cells by the A/WS/33 virus. In addition, chemical inhibition of RGGT partially protected against H5N1 and the 2009 H1N1 pandemic strain. The observations reported here thus contribute to an expanding body of studies directed at decoding vulnerabilities in the command and control networks specified by influenza virulence factors.


Asunto(s)
Interacciones Huésped-Patógeno/genética , Subtipo H1N1 del Virus de la Influenza A/fisiología , Mucosa Respiratoria/virología , Animales , Línea Celular , Análisis por Conglomerados , Efecto Citopatogénico Viral , Células Epiteliales/virología , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Lactatos/farmacología , Anotación de Secuencia Molecular , Organofosfonatos/farmacología , Interferencia de ARN , Replicación Viral
16.
Summit Transl Bioinform ; 2009: 32-7, 2009 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-21347167

RESUMEN

MOTIVATION: With the increasing volume of scientific papers and heterogeneous nomenclature in the biomedical literature, it is apparent that an improvement over standard pattern matching available in existing search engines is required. Cognition Search Information Retrieval (CSIR) is a natural language processing (NLP) technology that possesses a large dictionary (lexicon) and large semantic databases, such that search can be based on meaning. Encoded synonymy, ontological relationships, phrases, and seeds for word sense disambiguation offer significant improvement over pattern matching. Thus, the CSIR has the right architecture to form the basis for a scientific search engine. RESULT: Here we have augmented CSIR to improve access to the MEDLINE database of scientific abstracts. New biochemical, molecular biological and medical language and acronyms were introduced from curated web-based sources. The resulting system was used to interpret MEDLINE abstracts. Meaning-based search of MEDLINE abstracts yields high precision (estimated at >90%), and high recall (estimated at >90%), where synonym, ontology, phrases and sense seeds have been encoded. The present implementation can be found at http://MEDLINE.cognition.com. CONTACT: Elizabeth.goldsmith@UTsouthwestern.edu Kathleen.dahlgren@cognition.com.

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