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1.
Mol Cancer Res ; 15(10): 1431-1444, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28655712

RESUMEN

Alterations in MEK1/2 occur in cancers, both in the treatment-naïve state and following targeted therapies, most notably BRAF and MEK inhibitors in BRAF-V600E-mutant melanoma and colorectal cancer. Efforts were undertaken to understand the effects of these mutations, based upon protein structural location, and MEK1/2 activity. Two categories of MEK1/2 alterations were evaluated, those associated with either the allosteric pocket or helix-A. Clinically, MEK1/2 alterations of the allosteric pocket are rare and we demonstrate that they confer resistance to MEK inhibitors, while retaining sensitivity to BRAF inhibition. Most mutations described in patients fall within, or are associated with, helix-A. Mutations in this region reduce sensitivity to both BRAF and MEK inhibition and display elevated phospho-ERK1/2 levels, independent from increases in phospho-MEK1/2. Biochemical experiments with a representative helix-A variant, MEK1-Q56P, reveal both increased catalytic efficiency of the activated enzyme, and phosphorylation-independent activity relative to wild-type MEK1. Consistent with these findings, MEK1/2 alterations in helix A retain sensitivity to downstream antagonism via pharmacologic inhibition of ERK1/2. This work highlights the importance of classifying mutations based on structural and phenotypic consequences, both in terms of pathway signaling output and response to pharmacologic inhibition.Implications: This study suggests that alternate modes of target inhibition, such as ERK inhibition, will be required to effectively treat tumors harboring these MEK1/2-resistant alleles. Mol Cancer Res; 15(10); 1431-44. ©2017 AACR.


Asunto(s)
Neoplasias Colorrectales/genética , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 2/genética , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Quinasas raf/metabolismo , Sitio Alostérico , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , MAP Quinasa Quinasa 1/química , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/química , MAP Quinasa Quinasa 2/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Modelos Moleculares , Fosforilación , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas B-raf/genética
2.
Nat Med ; 21(5): 440-8, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25849130

RESUMEN

Resistance to cancer therapies presents a significant clinical challenge. Recent studies have revealed intratumoral heterogeneity as a source of therapeutic resistance. However, it is unclear whether resistance is driven predominantly by pre-existing or de novo alterations, in part because of the resolution limits of next-generation sequencing. To address this, we developed a high-complexity barcode library, ClonTracer, which enables the high-resolution tracking of more than 1 million cancer cells under drug treatment. In two clinically relevant models, ClonTracer studies showed that the majority of resistant clones were part of small, pre-existing subpopulations that selectively escaped under therapeutic challenge. Moreover, the ClonTracer approach enabled quantitative assessment of the ability of combination treatments to suppress resistant clones. These findings suggest that resistant clones are present before treatment, which would make up-front therapeutic combinations that target non-overlapping resistance a preferred approach. Thus, ClonTracer barcoding may be a valuable tool for optimizing therapeutic regimens with the goal of curative combination therapies for cancer.


Asunto(s)
Código de Barras del ADN Taxonómico/métodos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Diferenciación Celular , Línea Celular Tumoral , Crizotinib , ADN/química , ADN Complementario/metabolismo , Transición Epitelial-Mesenquimal , Clorhidrato de Erlotinib , Proteínas de Fusión bcr-abl/genética , Dosificación de Gen , Biblioteca de Genes , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Modelos Teóricos , Oligonucleótidos/genética , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , Pirazoles/administración & dosificación , Piridinas/administración & dosificación , Quinazolinas/administración & dosificación , Análisis de Secuencia de ARN
3.
Mol Cancer Ther ; 14(5): 1224-35, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25724664

RESUMEN

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide and hyperactivation of mTOR signaling plays a pivotal role in HCC tumorigenesis. Tuberous sclerosis complex (TSC), a heterodimer of TSC1 and TSC2, functions as a negative regulator of mTOR signaling. In the current study, we discovered that TSC2 loss-of-function is common in HCC. TSC2 loss was found in 4 of 8 HCC cell lines and 8 of 28 (28.6%) patient-derived HCC xenografts. TSC2 mutations and deletions are likely to be the underlying cause of TSC2 loss in HCC cell lines, xenografts, and primary tumors for most cases. We further demonstrated that TSC2-null HCC cell lines and xenografts had elevated mTOR signaling and, more importantly, were significantly more sensitive to RAD001/everolimus, an mTORC1 inhibitor. These preclinical findings led to the analysis of TSC2 status in HCC samples collected in the EVOLVE-1 clinical trial of everolimus using an optimized immunohistochemistry assay and identified 15 of 139 (10.8%) samples with low to undetectable levels of TSC2. Although the sample size is too small for formal statistical analysis, TSC2-null/low tumor patients who received everolimus tended to have longer overall survival than those who received placebo. Finally, we performed an epidemiology survey of more than 239 Asian HCC tumors and found the frequency of TSC2 loss to be approximately 20% in Asian HBV(+) HCC. Taken together, our data strongly argue that TSC2 loss is a predictive biomarker for the response to everolimus in HCC patients.


Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/genética , Everolimus/uso terapéutico , Hepatitis B/epidemiología , Neoplasias Hepáticas/genética , Proteínas Supresoras de Tumor/genética , Animales , Pueblo Asiatico/genética , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Hepatitis B/genética , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/virología , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos/antagonistas & inhibidores , Mutación , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Resultado del Tratamiento , Proteína 2 del Complejo de la Esclerosis Tuberosa
4.
PLoS One ; 4(8): e6862, 2009 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-19718437

RESUMEN

Weight-bearing stepping, without supraspinal re-connectivity, can be attained by treadmill training in an animal whose spinal cord has been completely transected at the lower thoracic level. Repair of damaged tissue and of supraspinal connectivity/circuitry following spinal cord injury in rat can be achieved by specific cell elimination with radiation therapy of the lesion site delivered within a critical time window, 2-3 weeks postinjury. Here we examined the effects of training in the repaired spinal cord following clinical radiation therapy. Studies were performed in a severe rat spinal cord contusion injury model, one similar to fracture/crush injuries in humans; the injury was at the lower thoracic level and the training was a combined hindlimb standing and stepping protocol. Radiotherapy, in a similar manner to that reported previously, resulted in a significant level of tissue repair/preservation at the lesion site. Training in the irradiated group, as determined by limb kinematics tests, resulted in functional improvements that were significant for standing and stepping capacity, and yielded a significant direct correlation between standing and stepping performance. In contrast, the training in the unirradiated group resulted in no apparent beneficial effects, and yielded an inverse correlation between standing and stepping performance, e.g., subject with good standing showed poor stepping capacity. Further, without any training, a differential functional change was observed in the irradiated group; standing capacity was significantly inhibited while stepping showed a slight trend of improvement compared with the unirradiated group. These data suggest that following repair by radiation therapy the spinal circuitries which control posture and locomotor were modified, and that the beneficial functional modulation of these circuitries is use dependent. Further, for restoring beneficial motor function following radiotherapy, training seems to be crucial.


Asunto(s)
Actividad Motora , Radioterapia/efectos adversos , Traumatismos de la Médula Espinal/fisiopatología , Animales , Femenino , Locomoción , Ratas , Ratas Sprague-Dawley
5.
Clin Cancer Res ; 14(22): 7519-25, 2008 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-19010870

RESUMEN

PURPOSE: Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene are associated with sensitivity of lung adenocarcinomas to the EGFR tyrosine kinase inhibitors, gefitinib and erlotinib. Acquired drug resistance is frequently associated with a secondary somatic mutation that leads to the substitution of methionine for threonine at position 790 (T790M). We aimed to identify additional second-site alterations associated with acquired resistance. EXPERIMENTAL DESIGN: Tumor samples were obtained from 48 patients with acquired resistance. Tumor cell DNA was analyzed for EGFR kinase domain mutations. Molecular analyses were then done to characterize the biological properties of a novel mutant EGFR allele. RESULTS: A previously unreported mutation in exon 21 of EGFR, which leads to substitution of alanine for threonine at position 854 (T854A), was identified in one patient with a drug-sensitive EGFR L858R-mutant lung adenocarcinoma after long-term treatment with tyrosine kinase inhibitors. The T854A mutation was not detected in a pretreatment tumor sample. The crystal structure analyses of EGFR suggest that the T854 side chain is within contact distance of gefitinib and erlotinib. Surrogate kinase assays show that the EGFR T854A mutation abrogates the inhibition of tyrosine phosphorylation by erlotinib. Such resistance seems to be overcome by a new irreversible dual EGFR/HER2 inhibitor, BIBW 2992. CONCLUSIONS: The T854A mutation is the second reported second-site acquired resistance mutation that is within contact distance of gefitinib and erlotinib. These data suggest that acquired resistance to ATP-mimetic EGFR kinase inhibitors may often be associated with amino acid substitutions that alter drug contact residues in the EGFR ATP-binding pocket.


Asunto(s)
Adenocarcinoma/genética , Resistencia a Antineoplásicos/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Adenocarcinoma/tratamiento farmacológico , Afatinib , Anciano , Secuencia de Aminoácidos , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Secuencia de Bases , Línea Celular Tumoral , Clorhidrato de Erlotinib , Femenino , Gefitinib , Humanos , Immunoblotting , Neoplasias Pulmonares/tratamiento farmacológico , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Inhibidores de Proteínas Quinasas/química , Estructura Cuaternaria de Proteína , Quinazolinas/química , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , Alineación de Secuencia
6.
Proc Natl Acad Sci U S A ; 104(52): 20932-7, 2007 Dec 26.
Artículo en Inglés | MEDLINE | ID: mdl-18093943

RESUMEN

In human lung adenocarcinomas harboring EGFR mutations, a second-site point mutation that substitutes methionine for threonine at position 790 (T790M) is associated with approximately half of cases of acquired resistance to the EGFR kinase inhibitors, gefitinib and erlotinib. To identify other potential mechanisms that contribute to disease progression, we used array-based comparative genomic hybridization (aCGH) to compare genomic profiles of EGFR mutant tumors from untreated patients with those from patients with acquired resistance. Among three loci demonstrating recurrent copy number alterations (CNAs) specific to the acquired resistance set, one contained the MET proto-oncogene. Collectively, analysis of tumor samples from multiple independent patient cohorts revealed that MET was amplified in tumors from 9 of 43 (21%) patients with acquired resistance but in only two tumors from 62 untreated patients (3%) (P = 0.007, Fisher's Exact test). Among 10 resistant tumors from the nine patients with MET amplification, 4 also harbored the EGFR(T790M) mutation. We also found that an existing EGFR mutant lung adenocarcinoma cell line, NCI-H820, harbors MET amplification in addition to a drug-sensitive EGFR mutation and the T790M change. Growth inhibition studies demonstrate that these cells are resistant to both erlotinib and an irreversible EGFR inhibitor (CL-387,785) but sensitive to a multikinase inhibitor (XL880) with potent activity against MET. Taken together, these data suggest that MET amplification occurs independently of EGFR(T790M) mutations and that MET may be a clinically relevant therapeutic target for some patients with acquired resistance to gefitinib or erlotinib.


Asunto(s)
Resistencia a Antineoplásicos , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Quinazolinas/farmacología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Antineoplásicos/farmacología , Cromosomas Humanos Par 7 , Estudios de Cohortes , Análisis Mutacional de ADN , Clorhidrato de Erlotinib , Gefitinib , Humanos , Hibridación Fluorescente in Situ , Neoplasias Pulmonares/tratamiento farmacológico , Proto-Oncogenes Mas
7.
PLoS Med ; 4(10): e294, 2007 Oct 09.
Artículo en Inglés | MEDLINE | ID: mdl-17927446

RESUMEN

BACKGROUND: Mutations in the epidermal growth factor receptor (EGFR) gene are associated with increased sensitivity of lung cancers to kinase inhibitors like erlotinib. Mechanisms of cell death that occur after kinase inhibition in these oncogene-dependent tumors have not been well delineated. We sought to improve understanding of this process in order to provide insight into mechanisms of sensitivity and/or resistance to tyrosine kinase inhibitors and to uncover new targets for therapy. METHODS AND FINDINGS: Using a panel of human lung cancer cell lines that harbor EGFR mutations and a variety of biochemical, molecular, and cellular techniques, we show that EGFR kinase inhibition in drug-sensitive cells provokes apoptosis via the intrinsic pathway of caspase activation. The process requires induction of the proapoptotic BH3-only BCL2 family member BIM (i.e., BCL2-like 11, or BCL2L11); erlotinib dramatically induces BIM levels in sensitive but not in resistant cell lines, and knockdown of BIM expression by RNA interference virtually eliminates drug-induced cell killing in vitro. BIM status is regulated at both transcriptional and posttranscriptional levels and is influenced by the extracellular signal-regulated kinase (ERK) signaling cascade downstream of EGFR. Consistent with these findings, lung tumors and xenografts from mice bearing mutant EGFR-dependent lung adenocarcinomas display increased concentrations of Bim after erlotinib treatment. Moreover, an inhibitor of antiapoptotic proteins, ABT-737, enhances erlotinib-induced cell death in vitro. CONCLUSIONS: In drug-sensitive EGFR mutant lung cancer cells, induction of BIM is essential for apoptosis triggered by EGFR kinase inhibitors. This finding implies that the intrinsic pathway of caspase activation may influence sensitivity and/or resistance of EGFR mutant lung tumor cells to EGFR kinase inhibition. Manipulation of the intrinsic pathway could be a therapeutic strategy to enhance further the clinical outcomes of patients with EGFR mutant lung tumors.


Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Receptores ErbB/genética , Neoplasias Pulmonares/tratamiento farmacológico , Quinazolinas/farmacología , Proteína Letal Asociada a bcl/efectos de los fármacos , Proteína Letal Asociada a bcl/genética , Adenocarcinoma/genética , Animales , Apoptosis/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Tumoral/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Clorhidrato de Erlotinib , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Neoplasias Pulmonares/genética , Ratones , Mutación , Sensibilidad y Especificidad
8.
PLoS One ; 2(8): e810, 2007 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-17726540

RESUMEN

BACKGROUND: The EGFR T790M mutation confers acquired resistance to kinase inhibitors in human EGFR mutant lung adenocarcinoma, is occasionally detected before treatment, and may confer genetic susceptibility to lung cancer. METHODOLOGY/PRINCIPAL FINDINGS: To study further its role in lung tumorigenesis, we developed mice with inducible expression in type II pneumocytes of EGFR(T790M) alone or together with a drug-sensitive L858R mutation. Both transgenic lines develop lung adenocarcinomas that require mutant EGFR for tumor maintenance but are resistant to an EGFR kinase inhibitor. EGFR(L858R+T790M)-driven tumors are transiently targeted by hsp90 inhibition. Notably, EGFR(T790M)-expressing animals develop tumors with longer latency than EGFR(L858R+T790M)-bearing mice and in the absence of additional kinase domain mutations. CONCLUSIONS/SIGNIFICANCE: These new mouse models of mutant EGFR-dependent lung adenocarcinomas provide insight into clinical observations. The models should also be useful for developing improved therapies for patients with lung cancers harboring EGFR(T790M) alone or in conjunction with drug-sensitive EGFR kinase domain mutations.


Asunto(s)
Antineoplásicos/uso terapéutico , Receptores ErbB/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Benzoquinonas/farmacología , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Receptores ErbB/metabolismo , Genotipo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Lactamas Macrocíclicas/farmacología , Ratones , Ratones Transgénicos
9.
Clin Cancer Res ; 12(21): 6494-501, 2006 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-17085664

RESUMEN

PURPOSE: In patients whose lung adenocarcinomas harbor epidermal growth factor receptor (EGFR) tyrosine kinase domain mutations, acquired resistance to the tyrosine kinase inhibitors (TKI) gefitinib (Iressa) and erlotinib (Tarceva) has been associated with a second-site EGFR mutation, which leads to substitution of methionine for threonine at position 790 (T790M). We aimed to elucidate the frequency and nature of secondary EGFR mutations in patients with acquired resistance to TKI monotherapy. EXPERIMENTAL DESIGN: Tumor cells from patients with acquired resistance were examined for secondary EGFR kinase domain mutations by molecular analyses. RESULTS: Eight of 16 patients (50% observed rate; 95% confidence interval, 25-75%) had tumor cells with second-site EGFR mutations. Seven mutations were T790M and one was a novel D761Y mutation found in a brain metastasis. When combined with a drug-sensitive L858R mutation, the D761Y mutation modestly reduced the sensitivity of mutant EGFR to TKIs in both surrogate kinase and cell viability assays. In an autopsy case, the T790M mutation was found in multiple visceral metastases but not in a brain lesion. CONCLUSIONS: The T790M mutation is common in patients with acquired resistance. The limited spectrum of TKI-resistant mutations in EGFR, which binds to erlotinib in the active conformation, contrasts with a wider range of second-site mutations seen with acquired resistance to imatinib, which binds to ABL and KIT, respectively, in closed conformations. Collectively, our data suggest that the type and nature of kinase inhibitor resistance mutations may be influenced by both anatomic site and mode of binding to the kinase target.


Asunto(s)
Adenocarcinoma/genética , Resistencia a Antineoplásicos/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Adenocarcinoma/tratamiento farmacológico , Secuencia de Aminoácidos , Antineoplásicos/uso terapéutico , Análisis Mutacional de ADN , Receptores ErbB/química , Receptores ErbB/efectos de los fármacos , Clorhidrato de Erlotinib , Gefitinib , Dosificación de Gen , Humanos , Hibridación in Situ , Neoplasias Pulmonares/tratamiento farmacológico , Datos de Secuencia Molecular , Mutación , Metástasis de la Neoplasia/genética , Estructura Terciaria de Proteína , Quinazolinas/uso terapéutico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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