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1.
Genes Chromosomes Cancer ; 54(2): 99-109, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25251827

RESUMO

Anaplastic lymphoma kinase (ALK) has been demonstrated to be deregulated in sporadic as well as in familiar cases of neuroblastoma (NB). Whereas ALK-fusion proteins are common in lymphoma and lung cancer, there are few reports of ALK rearrangements in NB indicating that ALK mainly exerts its oncogenic capacity via activating mutations and/or overexpression in this tumor type. In this study, 332 NB tumors and 13 cell lines were screened by high resolution single nucleotide polymorphism microarray. Gain of 2p was detected in 23% (60/332) of primary tumors and 46% (6/13) of cell lines, while breakpoints at the ALK locus were detected in four primary tumors and two cell lines. These were further analyzed by next generation sequencing and a targeted enrichment approach. Samples with both ALK and MYCN amplification displayed complex genomic rearrangements with multiple breakpoints within the amplicon. None of the translocations characterized in primary NB tumors are likely to result in a chimeric protein. However, immunohistochemical analysis reveals high levels of phosphorylated ALK in these samples despite lack of initial exons, possibly due to alternative transcription initiation sites. Both ALK proteins predicted to arise from such alterations and from the abnormal ALK exon 4-11 deletion observed in the CLB-BAR cell line show strong activation of downstream targets STAT3 and extracellular signal-regulated kinase (ERK) when expressed in PC12 cells. Taken together, our data indicate a novel, although rare, mechanism of ALK activation with implications for NB tumorigenesis.


Assuntos
Rearranjo Gênico , Neuroblastoma/genética , Receptores Proteína Tirosina Quinases/genética , Translocação Genética , Quinase do Linfoma Anaplásico , Animais , Linhagem Celular Tumoral , Pontos de Quebra do Cromossomo , Éxons , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neuroblastoma/metabolismo , Células PC12 , Polimorfismo de Nucleotídeo Único , Ratos , Receptores Proteína Tirosina Quinases/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo
2.
Biochem J ; 440(3): 405-13, 2011 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21838707

RESUMO

Mutations in the kinase domain of ALK (anaplastic lymphoma kinase) have recently been shown to be important for the progression of the childhood tumour neuroblastoma. In the present study we investigate six of the putative reported constitutively active ALK mutations, in positions G1128A, I1171N, F1174L, R1192P, F1245C and R1275Q. Our analyses were performed in cell-culture-based systems with both mouse and human ALK mutant variants and subsequently in a Drosophila melanogaster model system. Our investigation addressed the transforming potential of the putative gain-of-function ALK mutations as well as their signalling potential and the ability of two ATP-competitive inhibitors, Crizotinib (PF-02341066) and NVP-TAE684, to abrogate the activity of ALK. The results of the present study indicate that all mutations tested are of an activating nature and thus are implicated in tumour initiation or progression of neuroblastoma. Importantly for neuroblastoma patients, all ALK mutations used in the present study can be blocked by the inhibitors, although some mutants exhibited higher levels of drug sensitivity than others.


Assuntos
Antineoplásicos/farmacologia , Mutação de Sentido Incorreto , Neuroblastoma/genética , Pirazóis/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/genética , Quinase do Linfoma Anaplásico , Animais , Animais Geneticamente Modificados , Proliferação de Células , Transformação Celular Neoplásica , Olho Composto de Artrópodes/anormalidades , Olho Composto de Artrópodes/efeitos dos fármacos , Crizotinibe , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Camundongos , Neuritos/efeitos dos fármacos , Neuroblastoma/tratamento farmacológico , Células PC12 , Fenótipo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Fator de Transcrição STAT3/metabolismo
3.
Apoptosis ; 16(8): 783-94, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21562857

RESUMO

Type I interferons constitute a family of pleiotropic cytokines that have a key role in both adaptive and innate immunity. The interferon signalling pathways mediate transcriptional regulation of hundreds of genes, which result in mRNA degradation, decreased protein synthesis, cell cycle inhibition and induction of apoptosis. To elucidate regulatory networks important for interferon induced cell death, we generated interferon resistant U937 cells by selection in progressively increasing concentrations of interferon-α (IFN-α). The results show that IFN-α activates the death receptor signalling pathway and that IFN resistance was associated with cross-resistance to several death receptor ligands in a manner similar to previously described Fas resistant U937 cell lines. Increased expression of the long splice variant of the cellular FLICE-like inhibitor protein (cFLIP-L) was associated with the resistance to death receptor and IFN-α stimulation. Accordingly, inhibition of cFLIP-L expression with cycloheximide or through cFLIP short harpin RNA interference restored sensitivity to Fas and/or IFN-α. Thus, we now show that selection for interferon resistance can generate cells with increased expression of cFLIP, which protects the cells from both IFN-α and death receptor mediated apoptosis.


Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/antagonistas & inibidores , Interferon-alfa/farmacologia , Receptores de Morte Celular/metabolismo , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Cicloeximida/farmacologia , Resistência a Medicamentos , Ativação Enzimática , Ensaios Enzimáticos , Regulação da Expressão Gênica , Humanos , Interferon-alfa/fisiologia , Interfase , Interferência de RNA , Células U937 , Regulação para Cima
4.
Virulence ; 10(1): 37-57, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30518290

RESUMO

The Gram-negative enteropathogen Yersinia pseudotuberculosis possesses a number of regulatory systems that detect cell envelope damage caused by noxious extracytoplasmic stresses. The CpxA sensor kinase and CpxR response regulator two-component regulatory system is one such pathway. Active Cpx signalling upregulates various factors designed to repair and restore cell envelope integrity. Concomitantly, this pathway also down-regulates key determinants of virulence. In Yersinia, cpxA deletion accumulates high levels of phosphorylated CpxR (CpxR~P). Accumulated CpxR~P directly repressed rovA expression and this limited expression of virulence-associated processes. A second transcriptional regulator, RovM, also negatively regulates rovA expression in response to nutrient stress. Hence, this study aimed to determine if CpxR~P can influence rovA expression through control of RovM levels. We determined that the active CpxR~P isoform bound to the promoter of rovM and directly induced its expression, which naturally associated with a concurrent reduction in rovA expression. Site-directed mutagenesis of the CpxR~P binding sequence in the rovM promoter region desensitised rovM expression to CpxR~P. These data suggest that accumulated CpxR~P inversely manipulates the levels of two global transcriptional regulators, RovA and RovM, and this would be expected to have considerable influence on Yersinia pathophysiology and metabolism.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/genética , Ativação Transcricional , Yersinia pseudotuberculosis/genética , Fosforilação , Estresse Fisiológico , Virulência
5.
Anticancer Res ; 28(2A): 593-9, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18506997

RESUMO

BACKGROUND: Acquired resistance to apoptosis is a critical event in tumour development and in insensitivity toward therapy. To investigate resistance mechanisms to Fas/CD95/Apo-1-induced apoptosis, a Fas ligand-resistant variant of the U937 cell line was generated. RESULTS: Selection for Fas resistance resulted in a partial cross-resistance to TRAIL and TNF-alpha. Activation of caspase-8 was found to be impaired and the expression of Fas was reduced. However, FADD expression and ligand-induced aggregation of Fas was intact. Inhibition of various signalling pathways with pharmacological inhibitors revealed that resistance to death receptor-mediated apoptosis was dependent on altered tyrosine phosphatase/kinase activities and de novo protein synthesis. Moreover, FLIP, an anti-apoptotic protein, was expressed to a higher extent in the resistant cells. CONCLUSION: We provide evidence that acquired resistance to Fas-induced apoptosis in U937 cells involves a discrete set of molecular mechanisms which also render the cells cross-resistant to other death ligands.


Assuntos
Caspase 8/metabolismo , Resistencia a Medicamentos Antineoplásicos , Receptor fas/farmacologia , Apoptose/efeitos dos fármacos , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Ativação Enzimática , Proteína Ligante Fas/metabolismo , Proteína de Domínio de Morte Associada a Fas/farmacologia , Humanos , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Células U937
6.
Sci Signal ; 11(557)2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30459281

RESUMO

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that is a clinical target of major interest in cancer. Mutations and rearrangements in ALK trigger the activation of the encoded receptor and its downstream signaling pathways. ALK mutations have been identified in both familial and sporadic neuroblastoma cases as well as in 30 to 40% of relapses, which makes ALK a bona fide target in neuroblastoma therapy. Tyrosine kinase inhibitors (TKIs) that target ALK are currently in clinical use for the treatment of patients with ALK-positive non-small cell lung cancer. However, monotherapy with the ALK inhibitor crizotinib has been less encouraging in neuroblastoma patients with ALK alterations, raising the question of whether combinatorial therapy would be more effective. In this study, we established both phosphoproteomic and gene expression profiles of ALK activity in neuroblastoma cells exposed to first- and third-generation ALK TKIs, to identify the underlying molecular mechanisms and identify relevant biomarkers, signaling networks, and new therapeutic targets. This analysis has unveiled various important leads for novel combinatorial treatment strategies for patients with neuroblastoma and an increased understanding of ALK signaling involved in this disease.


Assuntos
Quinase do Linfoma Anaplásico/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Neuroblastoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteoma , Quinase do Linfoma Anaplásico/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Crizotinibe/farmacologia , Fosfatases de Especificidade Dupla/metabolismo , Perfilação da Expressão Gênica , Humanos , Espectrometria de Massas , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Mapeamento de Interação de Proteínas , Proteômica , RNA Interferente Pequeno/metabolismo , Receptor de Insulina/metabolismo , Análise de Sequência de RNA , Transdução de Sinais
7.
Artigo em Inglês | MEDLINE | ID: mdl-29907598

RESUMO

Tumors with anaplastic lymphoma kinase (ALK) fusion rearrangements, including non-small-cell lung cancer and anaplastic large cell lymphoma, are highly sensitive to ALK tyrosine kinase inhibitors (TKIs), underscoring the notion that such cancers are addicted to ALK activity. Although mutations in ALK are heavily implicated in childhood neuroblastoma, response to the ALK TKI crizotinib has been disappointing. Embryonal tumors in patients with DNA repair defects such as Fanconi anemia (FA) often have a poor prognosis, because of lack of therapeutic options. Here we report a child with underlying FA and ALK mutant high-risk neuroblastoma responding strongly to precision therapy with the ALK TKI ceritinib. Conventional chemotherapy treatment caused severe, life-threatening toxicity. Genomic analysis of the initial biopsy identified germline FANCA mutations as well as a novel ALK-I1171T variant. ALK-I1171T generates a potent gain-of-function mutant, as measured in PC12 cell neurite outgrowth and NIH3T3 transformation. Pharmacological inhibition profiling of ALK-I1171T in response to various ALK TKIs identified an 11-fold improved inhibition of ALK-I1171T with ceritinib when compared with crizotinib. Immunoaffinity-coupled LC-MS/MS phosphoproteomics analysis indicated a decrease in ALK signaling in response to ceritinib. Ceritinib was therefore selected for treatment in this child. Monotherapy with ceritinib was well tolerated and resulted in normalized catecholamine markers and tumor shrinkage. After 7.5 mo treatment, the residual primary tumor shrunk, was surgically removed, and exhibited hallmarks of differentiation together with reduced Ki67 levels. Clinical follow-up after 21 mo treatment revealed complete clinical remission including all metastatic sites. Therefore, ceritinib presents a viable therapeutic option for ALK-positive neuroblastoma.


Assuntos
Quinase do Linfoma Anaplásico/antagonistas & inibidores , Neoplasias Encefálicas/tratamento farmacológico , Neuroblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Sulfonas/uso terapêutico , Células 3T3 , Adolescente , Quinase do Linfoma Anaplásico/genética , Animais , Neoplasias Encefálicas/genética , Anemia de Fanconi/complicações , Anemia de Fanconi/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Humanos , Masculino , Camundongos , Mutação de Sentido Incorreto , Neuroblastoma/complicações , Neuroblastoma/genética , Células PC12 , Ratos
8.
Anticancer Res ; 27(4B): 2109-14, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17695493

RESUMO

BACKGROUND: Interferon-alpha (IFN-alpha) subtypes bind to the same receptor and are expected to have the same biological functions. Whether or not leukocyte IFN, containing six major IFN-alpha proteins had the same anti-tumor effect as one subtype, recombinant IFN-alpha2b, was investigated. MATERIALS AND METHODS: Three melanoma lines were treated with both types of IFN, and the effect on proliferation and survival was estimated both after short-term and prolonged treatment. RESULTS: All the melanoma cell lines were sensitive to the antiproliferative effects of both IFN species during short-term treatment. However, upon prolonged culture, the frequency of resistant colony formation was significantly higher in cultures treated with IFN-alpha2b compared to those treated with leukocyte IFN. There was a qualitative difference between the resistant colonies selected by the two IFN species with respect to morphology, growth rate and sensitivity to apoptosis. CONCLUSION: The development of resistant clones occurred at a lower rate during long-term treatment with leukocyte IFN containing six major subtypes of IFN-alpha as compared to IFN-alpha2b.


Assuntos
Antineoplásicos/farmacologia , Interferon-alfa/farmacologia , Melanoma/tratamento farmacológico , Processos de Crescimento Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Interferon alfa-2 , Melanoma/patologia , Proteínas Recombinantes , Células Tumorais Cultivadas
9.
Cancers (Basel) ; 9(11)2017 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-29084134

RESUMO

Numerous mutations have been observed in the Anaplastic Lymphoma Kinase (ALK) receptor tyrosine kinase (RTK) in both germline and sporadic neuroblastoma. Here, we have investigated the Y1278S mutation, observed in four patient cases, and its potential importance in the activation of the full length ALK receptor. Y1278S is located in the 1278-YRASYY-1283 motif of the ALK activation loop, which has previously been reported to be important in the activation of the ALK kinase domain. In this study, we have characterized activation loop mutations within the context of the full length ALK employing cell culture and Drosophila melanogaster model systems. Our results show that the Y1278S mutant observed in patients with neuroblastoma harbors gain-of-function activity. Secondly, we show that the suggested interaction between Y1278 and other amino acids might be of less importance in the activation process of the ALK kinase than previously proposed. Thirdly, of the three individual tyrosines in the 1278-YRASYY-1283 activation loop, we find that Y1283 is the critical tyrosine in the activation process. Taken together, our observations employing different model systems reveal new mechanistic insights on how the full length ALK receptor is activated and highlight differences with earlier described activation mechanisms observed in the NPM-ALK fusion protein, supporting a mechanism of activation more in line with those observed for the Insulin Receptor (InR).

10.
Artigo em Inglês | MEDLINE | ID: mdl-27446813

RESUMO

Yersinia bacteria target Yop effector toxins to the interior of host immune cells by the Ysc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controlling Ysc-Yop targeting activity. A + 1 frameshift event in the 3-prime end of yopN can also produce a singular secreted YopN-TyeA polypeptide that retains some regulatory function even though the C-terminal coding sequence of this YopN differs greatly from wild type. Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control. Bacteria producing YopN truncated after residue 278, or with altered sequence between residues 279 and 287, had lost type III secretion control and function. In contrast, YopN variants with manipulated sequence beyond residue 287 maintained full control and function. Scrutiny of the YopN-TyeA complex structure revealed that residue W279 functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopN W279G mutant lost all ability to bind TyeA. The TyeA residue F8 was also critical for reciprocal YopN binding. Thus, we conclude that specific hydrophobic contacts between opposing YopN and TyeA termini establishes a complex needed for regulating Ysc-Yop activity.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias/química , Proteínas de Transporte/química , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Domínios e Motivos de Interação entre Proteínas , Sistemas de Secreção Tipo III/metabolismo , Yersinia pseudotuberculosis/metabolismo , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Cálcio/química , Proteínas de Transporte/genética , Linhagem Celular , DNA Bacteriano , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos/microbiologia , Proteínas de Membrana/genética , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estabilidade Proteica , Sistemas de Translocação de Proteínas , Análise de Sequência , Deleção de Sequência , Temperatura , Técnicas do Sistema de Duplo-Híbrido , Sistemas de Secreção Tipo III/genética
11.
Sci Signal ; 7(349): ra102, 2014 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-25351247

RESUMO

Anaplastic lymphoma kinase (ALK) is an important molecular target in neuroblastoma. Although tyrosine kinase inhibitors abrogating ALK activity are currently in clinical use for the treatment of ALK-positive (ALK(+)) disease, monotherapy with ALK tyrosine kinase inhibitors may not be an adequate solution for ALK(+) neuroblastoma patients. Increased expression of the gene encoding the transcription factor MYCN is common in neuroblastomas and correlates with poor prognosis. We found that the kinase ERK5 [also known as big mitogen-activated protein kinase (MAPK) 1 (BMK1)] is activated by ALK through a pathway mediated by phosphoinositide 3-kinase (PI3K), AKT, MAPK kinase kinase 3 (MEKK3), and MAPK kinase 5 (MEK5). ALK-induced transcription of MYCN and stimulation of cell proliferation required ERK5. Pharmacological or RNA interference-mediated inhibition of ERK5 suppressed the proliferation of neuroblastoma cells in culture and enhanced the antitumor efficacy of the ALK inhibitor crizotinib in both cells and xenograft models. Together, our results indicate that ERK5 mediates ALK-induced transcription of MYCN and proliferation of neuroblastoma, suggesting that targeting both ERK5 and ALK may be beneficial in neuroblastoma patients.


Assuntos
Regulação Neoplásica da Expressão Gênica/fisiologia , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Neuroblastoma/fisiopatologia , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Quinase do Linfoma Anaplásico , Animais , Linhagem Celular Tumoral , Primers do DNA/genética , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Fluorescência Verde , Humanos , Processamento de Imagem Assistida por Computador , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Imageamento por Ressonância Magnética , Proteína Quinase 7 Ativada por Mitógeno/genética , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/metabolismo , Células PC12 , Interferência de RNA , Ratos , Reação em Cadeia da Polimerase em Tempo Real
12.
FEBS J ; 280(21): 5269-82, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23889739

RESUMO

Activation of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is a key oncogenic mechanism in a growing number of tumor types. In the majority of cases, ALK is activated by fusion with a dimerizing partner protein as a result of chromosomal translocation events, most studied in the case of the nucleophosmin-ALK and echinoderm microtubule-associated protein-like 4-ALK oncoproteins. It is now also appreciated that the full-length ALK receptor can be activated by point mutations and by deletions within the extracellular domain, such as those observed in neuroblastoma. Several studies have employed phosphoproteomics approaches to find substrates of ALK fusion proteins. In this study, we used MS-based phosphotyrosine profiling to characterize phosphotyrosine signaling events associated with the full-length ALK receptor. A number of previously identified and novel targets were identified. One of these, signal transducer and activator of transcription 3 (STAT3), has previously been observed to be activated in response to oncogenic ALK signaling, but the significance of this in signaling from the full-length ALK receptor has not been explored further. We show here that activated ALK robustly activates STAT3 on Tyr705 in a number of independent neuroblastoma cell lines. Furthermore, knockdown of STAT3 by RNA interference resulted in a reduction in myelocytomatosis neuroblastom (MYCN) protein levels downstream of ALK signaling. These observations, together with a decreased level of MYCN and inhibition of neuroblastoma cell growth in the presence of STAT3 inhibitors, suggest that activation of STAT3 is important for ALK signaling activity in neuroblastoma.


Assuntos
Neuroblastoma/metabolismo , Fosfoproteínas/metabolismo , Proteoma/análise , Receptores Proteína Tirosina Quinases/metabolismo , Fator de Transcrição STAT3/metabolismo , Quinase do Linfoma Anaplásico , Animais , Apoptose , Western Blotting , Proliferação de Células , Humanos , Imunoprecipitação , Luciferases , Neuroblastoma/genética , Neuroblastoma/patologia , Células PC12 , Fosforilação , Fosfotirosina/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptores Proteína Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Transdução de Sinais
13.
Dis Model Mech ; 6(2): 373-82, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23104988

RESUMO

Neuroblastoma is a childhood extracranial solid tumour that is associated with a number of genetic changes. Included in these genetic alterations are mutations in the kinase domain of the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), which have been found in both somatic and familial neuroblastoma. In order to treat patients accordingly requires characterisation of these mutations in terms of their response to ALK tyrosine kinase inhibitors (TKIs). Here, we report the identification and characterisation of two novel neuroblastoma ALK mutations (A1099T and R1464STOP), which we have investigated together with several previously reported but uncharacterised ALK mutations (T1087I, D1091N, T1151M, M1166R, F1174I and A1234T). In order to understand the potential role of these ALK mutations in neuroblastoma progression, we have employed cell culture-based systems together with the model organism Drosophila as a readout for ligand-independent activity. Mutation of ALK at position 1174 (F1174I) generates a gain-of-function receptor capable of activating intracellular targets such as ERK (extracellular signal regulated kinase) and STAT3 (signal transducer and activator of transcription 3) in a ligand-independent manner. Analysis of these previously uncharacterised ALK mutants and comparison with ALK(F1174) mutants suggests that ALK mutations observed in neuroblastoma fall into three classes. These classes are: (i) gain-of-function ligand-independent mutations such as ALK(F1174l), (ii) kinase-dead ALK mutants, e.g. ALK(I1250T) (Schönherr et al., 2011a) and (iii) ALK mutations that are ligand-dependent in nature. Irrespective of the nature of the observed ALK mutants, in every case the activity of the mutant ALK receptors could be abrogated by the ALK inhibitor crizotinib (Xalkori/PF-02341066), albeit with differing levels of sensitivity.


Assuntos
Drosophila melanogaster/enzimologia , Mutação/genética , Neuroblastoma/enzimologia , Neuroblastoma/genética , Receptores Proteína Tirosina Quinases/genética , Quinase do Linfoma Anaplásico , Animais , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Crizotinibe , Modelos Animais de Doenças , Humanos , Concentração Inibidora 50 , Proteínas Mutantes/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neuritos/patologia , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Células PC12 , Fenótipo , Fosforilação/efeitos dos fármacos , Estrutura Terciária de Proteína , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Ratos , Receptores Proteína Tirosina Quinases/química
14.
Transl Oncol ; 4(4): 258-65, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21804922

RESUMO

Activating mutations in the kinase domain of anaplastic lymphoma kinase (ALK) have recently been shown to be an important determinant in the genetics of the childhood tumor neuroblastoma. Here we discuss an in-depth analysis of one of the reported gain-of-function ALK mutations-ALK(I1250T)-identified in the germ line DNA of one patient. Our analyses were performed in cell culture-based systems and subsequently confirmed in a Drosophila model. The results presented here indicate that the germ line ALK(I1250T) mutation is most probably not a determinant for tumor initiation or progression and, in contrast, seems to generate a kinase-dead mutation in the ALK receptor tyrosine kinase (RTK). Consistent with this, stimulation with agonist ALK antibodies fails to lead to stimulation of ALK(I1250T) and we were unable to detect tyrosine phosphorylation under any circumstances. In agreement, ALK(I1250T) is unable to activate downstream signaling pathways or to mediate neurite outgrowth, in contrast to the activated wild-type ALK receptor or the activating ALK(F1174S) mutant. Identical results were obtained when the ALK(I1250T) mutant was expressed in a Drosophila model, confirming the lack of activity of this mutant ALK RTK. We suggest that the ALK(I1250T) mutation leads to a kinase-dead ALK RTK, in stark contrast to assumed gain-of-function status, with significant implications for patients reported to carry this particular ALK mutation.

15.
Cancer Res ; 71(1): 98-105, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21059859

RESUMO

Mutations in the kinase domain of the ALK kinase have emerged recently as important players in the genetics of the childhood tumor neuroblastoma. Here, we report the appearance of a novel ALK mutation in neuroblastoma, correlating with aggressive tumor behavior. Analyses of genomic DNA from biopsy samples initially showed ALK sequence to be wild type. However, during disease progression, mutation of amino acid F1174 to a serine within the ALK kinase domain was observed, which correlated with aggressive neuroblastoma progression in the patient. We show that mutation of F1174 to serine generates a potent gain-of-function mutant, as observed in 2 independent systems. First, PC12 cell lines expressing ALK(F1174S) display ligand-independent activation of ALK and further downstream signaling activation. Second, analysis of ALK(F1174S) in Drosophila models confirms that the mutation mediates a strong, rough eye phenotype upon expression in the developing eye. Thus, we report a novel ALK(F1174S) mutation that displays ligand-independent activity in vivo, correlating with rapid and treatment-resistant tumor growth. The study also shows that initial screening in the first tumor biopsy of a patient may not be sufficient and that further molecular analysis, in particular in tumor progression and/or tumor relapse, is warranted for better understanding of the treatment of neuroblastoma patients.


Assuntos
Mutação , Neuroblastoma/genética , Neuroblastoma/patologia , Proteínas Tirosina Quinases/genética , Quinase do Linfoma Anaplásico , Animais , Sequência de Bases , Primers do DNA , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Lactente , Masculino , Camundongos , Células NIH 3T3 , Neuroblastoma/terapia , Células PC12 , Polimorfismo de Nucleotídeo Único , Ratos , Receptores Proteína Tirosina Quinases , Transdução de Sinais
16.
J Neuroimmunol ; 215(1-2): 102-7, 2009 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-19733917

RESUMO

Epitope reactivity of multiple sclerosis (MS) plasma antibodies against the Epstein-Barr virus protein EBNA-1 and its association with HLA DRB1*1501 status was investigated in a case-referent study. Based on EBNA-1 fragment reactivity and the effect of peptide blocking, four 29-36 amino acid long EBNA-1 fragments were selected for detailed studies. MS cases had increased antibody reactivity against several EBNA-1 domains, of which antibodies against EBNA-1 (amino acid 385-420) in HLA DRB1*1501 positive individuals were associated with a 24-fold risk increase for MS. The data need confirmation in a larger sample but suggest a role for this epitope in the autoimmune pathogenesis of MS.


Assuntos
Anticorpos Antivirais/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Antígenos HLA-DR/imunologia , Antígenos HLA-DR/metabolismo , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Adulto , Idoso , Sequência de Aminoácidos , Sequência de Bases , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Cadeias HLA-DRB1 , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/imunologia , Estrutura Terciária de Proteína , Fatores de Risco
17.
FEBS J ; 276(2): 497-508, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19076218

RESUMO

Susceptibility to cell death is a prerequisite for the elimination of tumour cells by cytotoxic immune cells, chemotherapy or irradiation. Activation of the death receptor Fas is critical for the regulation of immune cell homeostasis and efficient killing of tumour cells by apoptosis. To define the molecular changes that occur during selection for insensitivity to Fas-induced apoptosis, a resistant variant of the U937 cell line was established. Individual resistant clones were isolated and characterized. The most frequently observed defect in the resistant cells was reduced Fas expression, which correlated with decreased FAS transcription. Clones with such reduced Fas expression also displayed partial cross-resistance to tumour necrosis factor-alpha stimulation, but the mRNA expression of tumour necrosis factor receptors was not decreased. Reintroduction of Fas conferred susceptibility to Fas but not to tumour necrosis factor-alpha stimulation, suggesting that several alterations could be present in the clones. The reduced Fas expression could not be explained by mutations in the FAS coding sequence or promoter region, or by silencing through methylations. Protein kinase B and extracellular signal-regulated kinase, components of signalling pathways downstream of Ras, were shown to be activated in some of the resistant clones, but none of the three RAS genes was mutated, and experiments using chemical inhibitors could not establish that the activation of these proteins was the cause of Fas resistance as described in other systems. Taken together, the data illustrate that Fas resistance can be caused by reduced Fas expression, which is a result of an unidentified mode of regulation.


Assuntos
Apoptose , Separação Celular/métodos , Transcrição Gênica/genética , Receptor fas/metabolismo , Apoptose/efeitos dos fármacos , Regulação para Baixo , Humanos , Sistema de Sinalização das MAP Quinases , Metilação , RNA Mensageiro/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Células U937 , Receptor fas/genética
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