Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 9 de 9
Filtrar
1.
Nature ; 468(7326): 968-72, 2010 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-21107320

RESUMO

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.


Assuntos
Resistencia a Medicamentos Antineoplásicos , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Regulação Alostérica , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ativação Enzimática/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Biblioteca Gênica , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , MAP Quinase Quinase Quinases/genética , Melanoma/tratamento farmacológico , Melanoma/enzimologia , Melanoma/genética , Melanoma/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fases de Leitura Aberta/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Vemurafenib
2.
Proc Natl Acad Sci U S A ; 106(48): 20411-6, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19915144

RESUMO

Genetic alterations that activate the mitogen-activated protein kinase (MAP kinase) pathway occur commonly in cancer. For example, the majority of melanomas harbor mutations in the BRAF oncogene, which are predicted to confer enhanced sensitivity to pharmacologic MAP kinase inhibition (e.g., RAF or MEK inhibitors). We investigated the clinical relevance of MEK dependency in melanoma by massively parallel sequencing of resistant clones generated from a MEK1 random mutagenesis screen in vitro, as well as tumors obtained from relapsed patients following treatment with AZD6244, an allosteric MEK inhibitor. Most mutations conferring resistance to MEK inhibition in vitro populated the allosteric drug binding pocket or alpha-helix C and showed robust ( approximately 100-fold) resistance to allosteric MEK inhibition. Other mutations affected MEK1 codons located within or abutting the N-terminal negative regulatory helix (helix A), which also undergo gain-of-function germline mutations in cardio-facio-cutaneous (CFC) syndrome. One such mutation, MEK1(P124L), was identified in a resistant metastatic focus that emerged in a melanoma patient treated with AZD6244. Both MEK1(P124L) and MEK1(Q56P), which disrupts helix A, conferred cross-resistance to PLX4720, a selective B-RAF inhibitor. However, exposing BRAF-mutant melanoma cells to AZD6244 and PLX4720 in combination prevented emergence of resistant clones. These results affirm the importance of MEK dependency in BRAF-mutant melanoma and suggest novel mechanisms of resistance to MEK and B-RAF inhibitors that may have important clinical implications.


Assuntos
Benzimidazóis/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , MAP Quinase Quinase 1/genética , Melanoma/genética , Conformação Proteica , Proteínas Proto-Oncogênicas B-raf/genética , Sequência de Bases , Linhagem Celular Tumoral , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Dados de Sequência Molecular , Mutagênese , Mutação de Sentido Incorreto/genética , Ligação Proteica/genética , Análise de Sequência de DNA , Ensaio Tumoral de Célula-Tronco
3.
Cancer Discov ; 8(2): 184-195, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29247021

RESUMO

Ulixertinib (BVD-523) is an ERK1/2 kinase inhibitor with potent preclinical activity in BRAF- and RAS-mutant cell lines. In this multicenter phase I trial (NCT01781429), 135 patients were enrolled to an accelerated 3 + 3 dose-escalation cohort and six distinct dose-expansion cohorts. Dose escalation included 27 patients, dosed from 10 to 900 mg twice daily and established the recommended phase II dose (RP2D) of 600 mg twice daily. Ulixertinib exposure was dose proportional to the RP2D, which provided near-complete inhibition of ERK activity in whole blood. In the 108-patient expansion cohort, 32% of patients required dose reduction. The most common treatment-related adverse events were diarrhea (48%), fatigue (42%), nausea (41%), and dermatitis acneiform (31%). Partial responses were seen in 3 of 18 (17%) patients dosed at or above maximum tolerated dose and in 11 of 81 (14%) evaluable patients in dose expansion. Responses occurred in patients with NRAS-, BRAF V600-, and non-V600 BRAF-mutant solid tumors.Significance: Here, we describe the first-in-human dose-escalation study of an ERK1/2 inhibitor for the treatment of patients with advanced solid tumors. Ulixertinib has an acceptable safety profile with favorable pharmacokinetics and has shown early evidence of clinical activity in NRAS- and BRAF V600- and non-V600-mutant solid-tumor malignancies. Cancer Discov; 8(2); 184-95. ©2017 AACR.See related commentary by Smalley and Smalley, p. 140This article is highlighted in the In This Issue feature, p. 127.


Assuntos
Aminopiridinas/uso terapêutico , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Inibidores de Proteínas Quinases/uso terapêutico , Pirróis/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Aminopiridinas/farmacologia , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Estadiamento de Neoplasias , Neoplasias/diagnóstico , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Adulto Jovem
4.
Mol Cancer Res ; 15(10): 1431-1444, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28655712

RESUMO

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.


Assuntos
Neoplasias Colorretais/genética , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 2/genética , Mutação , Inibidores de Proteínas Quinases/farmacologia , Quinases raf/metabolismo , Sítio Alostérico , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MAP Quinase Quinase 1/química , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/química , MAP Quinase Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Modelos Moleculares , Fosforilação , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/genética
5.
Mol Cancer Ther ; 16(11): 2351-2363, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28939558

RESUMO

Aberrant activation of signaling through the RAS-RAF-MEK-ERK (MAPK) pathway is implicated in numerous cancers, making it an attractive therapeutic target. Although BRAF and MEK-targeted combination therapy has demonstrated significant benefit beyond single-agent options, the majority of patients develop resistance and disease progression after approximately 12 months. Reactivation of ERK signaling is a common driver of resistance in this setting. Here we report the discovery of BVD-523 (ulixertinib), a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and ERK1/2 selectivity. In vitro BVD-523 treatment resulted in reduced proliferation and enhanced caspase activity in sensitive cells. Interestingly, BVD-523 inhibited phosphorylation of target substrates despite increased phosphorylation of ERK1/2. In in vivo xenograft studies, BVD-523 showed dose-dependent growth inhibition and tumor regression. BVD-523 yielded synergistic antiproliferative effects in a BRAFV600E-mutant melanoma cell line xenograft model when used in combination with BRAF inhibition. Antitumor activity was also demonstrated in in vitro and in vivo models of acquired resistance to single-agent and combination BRAF/MEK-targeted therapy. On the basis of these promising results, these studies demonstrate BVD-523 holds promise as a treatment for ERK-dependent cancers, including those whose tumors have acquired resistance to other treatments targeting upstream nodes of the MAPK pathway. Assessment of BVD-523 in clinical trials is underway (NCT01781429, NCT02296242, and NCT02608229). Mol Cancer Ther; 16(11); 2351-63. ©2017 AACR.


Assuntos
Aminopiridinas/administração & dosagem , Melanoma/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas B-raf/genética , Pirróis/administração & dosagem , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/genética , Melanoma/patologia , Camundongos , Mutação , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
6.
PLoS One ; 10(9): e0138486, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26378449

RESUMO

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


Assuntos
Linhagem da Célula/genética , Regulação Neoplásica da Expressão Gênica/genética , Expressão Gênica/genética , Neoplasias Pancreáticas/genética , Biossíntese de Proteínas/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Animais , Apoptose/genética , Caspase 8/genética , Linhagem Celular Tumoral , Humanos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
7.
Cancer Res ; 73(15): 4840-51, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23737487

RESUMO

Melanomas that contain B-RAF(V600E) mutations respond transiently to RAF and MEK inhibitors; however, resistance to these agents remains a formidable challenge. Although B- or C-RAF dysregulation represents prominent resistance mechanisms, resistance-associated point mutations in RAF oncoproteins are surprisingly rare. To gain insights herein, we conducted random mutagenesis screens to identify B- or C-RAF mutations that confer resistance to RAF inhibitors. Whereas bona fide B-RAF(V600E) resistance alleles were rarely observed, we identified multiple C-RAF mutations that produced biochemical and pharmacologic resistance. Potent C-RAF resistance alleles localized to a 14-3-3 consensus binding site or a separate site within the P loop. These mutations elicited paradoxical upregulation of RAF kinase activity in a dimerization-dependent manner following exposure to RAF inhibitors. Knowledge of resistance-associated C-RAF mutations may enhance biochemical understanding of RAF-dependent signaling, anticipate clinical resistance to novel RAF inhibitors, and guide the design of "next-generation" inhibitors for deployment in RAF- or RAS-driven malignancies.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Melanoma/genética , Mutação , Proteínas Proto-Oncogênicas c-raf/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Imunoprecipitação , Inibidores de Proteínas Quinases/farmacologia
9.
PLoS One ; 4(11): e7887, 2009 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-19924296

RESUMO

BACKGROUND: Detection of critical cancer gene mutations in clinical tumor specimens may predict patient outcomes and inform treatment options; however, high-throughput mutation profiling remains underdeveloped as a diagnostic approach. We report the implementation of a genotyping and validation algorithm that enables robust tumor mutation profiling in the clinical setting. METHODOLOGY: We developed and implemented an optimized mutation profiling platform ("OncoMap") to interrogate approximately 400 mutations in 33 known oncogenes and tumor suppressors, many of which are known to predict response or resistance to targeted therapies. The performance of OncoMap was analyzed using DNA derived from both frozen and FFPE clinical material in a diverse set of cancer types. A subsequent in-depth analysis was conducted on histologically and clinically annotated pediatric gliomas. The sensitivity and specificity of OncoMap were 93.8% and 100% in fresh frozen tissue; and 89.3% and 99.4% in FFPE-derived DNA. We detected known mutations at the expected frequencies in common cancers, as well as novel mutations in adult and pediatric cancers that are likely to predict heightened response or resistance to existing or developmental cancer therapies. OncoMap profiles also support a new molecular stratification of pediatric low-grade gliomas based on BRAF mutations that may have immediate clinical impact. CONCLUSIONS: Our results demonstrate the clinical feasibility of high-throughput mutation profiling to query a large panel of "actionable" cancer gene mutations. In the future, this type of approach may be incorporated into both cancer epidemiologic studies and clinical decision making to specify the use of many targeted anticancer agents.


Assuntos
Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Mutação , Neoplasias/metabolismo , Algoritmos , Códon , Análise Mutacional de DNA , Primers do DNA/genética , Genótipo , Glioma/genética , Humanos , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas B-raf/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA