RESUMO
The discovery of novel anti-cancer drugs targeting anaplastic lymphoma kinase (ALK), an oncogenic tyrosine kinase, raises the need for in vitro assays suitable for screening compounds for ALK inhibition. To this aim we have developed and optimized an ALK-specific enzyme-linked immunosorbent assay that employs a novel ALK peptide substrate and purified ALK kinase domain.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Tirosina Quinases/antagonistas & inibidores , Sequência de Aminoácidos , Quinase do Linfoma Anaplásico , Animais , Relação Dose-Resposta a Droga , Humanos , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Fosforilação , Estrutura Terciária de Proteína , Receptores Proteína Tirosina Quinases , Estaurosporina/farmacologiaRESUMO
Deregulated apoptosis is a common finding in tumorigenesis. The oncogenic tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) delivers a strong survival signal in anaplastic large cell lymphomas (ALCLs). Although NPM/ALK activates multiple antiapoptotic pathways, the biologic relevance and therapeutic potential of more downstream apoptotic effectors are mostly unknown. In this report, the NPM/ALK-mediated induction of Bcl-XL (but not of Bcl-2) was identified in human ALCL-derived cells. NPM/ALK kinase activity was required to promote Bcl-XL expression and its protective effect on mitochondrial homeostasis. Down-regulation of Bcl-XL significantly reduced the antiapoptotic potential of NPM/ALK in both transformed murine Ba/F3 pro-B cells and human ALCL-derived KARPAS-299 cells. To elucidate the role of Bcl-XL in vivo, Ba/F3-NPM/ALK+ cells expressing a doxycycline (Dox)-inducible Bcl-XL antisense transgene (pTet-ON) were injected into nude mice. Doxycycline administration prevented a fatal systemic disease in 15 of 15 intravenously injected mice and the appearance of subcutaneous tumor xenografts in 9 of 12 mice; in vivo down-regulation of Bcl-XL was also documented. Our results show a pivotal role for Bcl-XL in ALK-mediated oncogenicity; a single protein placed downstream of a known oncogene can be crucial for the survival of neoplastic cells both in vitro and in vivo. Bcl-XL deserves further investigation as a possible therapeutic target in ALK+ ALCLs.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/genética , Linfoma Difuso de Grandes Células B/patologia , Proteínas Nucleares/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Quinase do Linfoma Anaplásico , Animais , Apoptose/fisiologia , Sequência de Bases , Linhagem Celular Transformada , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Feminino , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/patologia , Membranas Intracelulares/fisiologia , Linfoma Difuso de Grandes Células B/genética , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Mitocôndrias/fisiologia , Proteínas Nucleares/metabolismo , Nucleofosmina , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases , Proteínas Recombinantes/metabolismo , Transfecção , Transplante Heterólogo , Proteína bcl-XRESUMO
BACKGROUND AND OBJECTIVES: Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome that has been associated with the expression of platelet-derived growth factor b receptor (PDGFRbeta) fusion proteins, namely TEL/PDGFRbeta. These fusion proteins possess a constitutive PDGFRbeta tyrosine kinase activity, leading to aberrant PDGFRbeta signaling and cellular transformation. The expression of PDGFRbeta fusions in CMML could have therapeutic relevance, as PDGFRb is inhibited by the selective tyrosine kinase inhibitor, imatinib. Here, we investigated the possibility of employing imatinib to treat CMML. DESIGN AND METHODS: We assessed the effect of imatinib on TEL/PDGFRbeta transformed cells in terms of proliferation, by trypan blue exclusion and 3H-thymidine uptake, and TEL/PDGFRbeta autophosphorylation by anti-phosphotyrosine immunoblotting. TEL/PDGFRbeta expression in mononuclear cells from the peripheral blood of 27 clinically diagnosed CMML patients was determined by reverse transcriptase-polymerase chain reaction. RESULTS: Imatinib potently inhibited the proliferation of TEL/PDGFRbeta transformed cells (IC50=7.5 nM), and TEL/PDGFRbeta kinase activity. However, TEL/PDGFRbeta expression was detected in only 1 of 27 CMML patients (4%, confidence intervals: 0-13%). Additionally, another PDGFRbeta fusion protein, Hip1/PDGFRbeta, had a similarly low incidence in the same samples: 1 of 25 (4%, confidence intervals: 0-14%). INTERPRETATION AND CONCLUSIONS: Although imatinib represents an attractive therapeutic agent for neoplasias associated with abnormal PDGFRbeta signaling, the low frequency of the TEL/PDGFRbeta and Hip1/PDGFRbeta fusion proteins in CMML suggests that its application to this disease maybe limited. Detection of PDGFRbeta fusion genes in individual patients is necessary in order to employ this drug rationally in CMML.