Name: a method for screening and validation of resistant mutations against kinase inhibitors
Uploaded: 2014-12-07T15:00:00.000+00:00
Duration: 12 min 40 s
Description: Watch this Scientific Journal Video about A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors at JoVE.com

This study was supported by grants to M.A. from NCI (1RO1CA155091), NHLBI (1R21HL114074) and the Leukemia Research Foundation. M.A. is a recipient of V-Scholar award from the V- Foundation. Authors are thankful to Dr. Sara Rohrabaugh for editing.

NOTA: Todos os procedimentos deste protocolo foram conduzidos de acordo com o Instituto Nacional de diretrizes de saúde para o tratamento ético e tratamento dos animais, e de acordo com um protocolo de uso de animais IACUC aprovado. 1. Linha celular de manutenção <ol><li> Culturas de células BAF3 em meio RPMI-1640 suplementado com 10% de soro bovino fetal e penicilina / estreptomicina (100 unidades / ml e 100 ug / ml) e meio de cultura gasto de células WEHI. Cultivar células HEK293T em DMEM suplementado com 10% de soro bovino fetal e penicilina / estreptomicina (100 unidades / ml e 100 ug / ml). Manter as células a 37 ° C numa atmosfera humidificada contendo 5% de CO2. </li></ol> 2. Construção de plasmídeo <ol><li> Clonar o rato JAK2 e JAK2 sua isoforma oncogénica-V617F em pMSCV-puro-GW por clonase LR, utilizando um processo de recombinação de clonagem padrão. </li><li> Para a construção do vector de expressão de luciferase (Luc-pMSCV-Cherry-GW) utilizado na in-viimagiologia vo, amplificar a luciferase de pLVX-Tet-ON vector por PCR utilizando iniciadores (LUC-SD / RP-LUC TTACAATTTGGACTTTCCG e SD / FP-CACCATGGAAGACGCCAAAAAC) seguido pela clonagem em pENTR-SD-TOPO para gerar pENTR-Luc, que é usada para transferir o gene de luciferase em vector retroviral, pMSCV-Cherry-GW de clonagem por recombinação mediada usando clonase LR. </li></ol> 3. Preparação da Random Mutation Library, Triagem e identificação das Mutações 3.1) A mutagénese aleatória <ol><li> Comprimento total Clone de JAK2 tipo selvagem e a mutação V617F em pMSCV-puro-GW retroviral vector usando uma tecnologia de recombinação clonagem comercial. </li><li> Use 1 ug de ADN plasmídeo de JAK2 V617F-se transformar, XL-1 Vermelho, E. coli, que é defeito nos mecanismos de reparo do DNA permitindo-lhes incorporar mutações aleatórias durante a replicação. Mais especificamente, mistura 50 - 100 ng de ADN (mais do que 100 ng de ADN diminui a eficiência de transformação) com 100 ul de células competentes de um tubo de polipropileno pré-gelada e incubadas em gelo durante 30 min, agitando suavemente a cada 5 min. Para uma boa cobertura a biblioteca, utilizam 4-6 tubos de células competentes. NOTA: mais do que 100 ng de ADN diminui a eficiência de transformação </li><li> Mergulha-se o tubo num banho de água a 42 ° C durante um choque térmico de 45 seg e incubar em gelo durante 2 min. Em seguida, adicionar 1 ml de meio SOC (2,0% de triptona, 0,5% de extrato de levedura, 0,05% de NaCl, 10 mM de MgCl2, 10 mM de MgSO4, e 0,4% de D-glucose). Incubar o tubo a 37 ° C enquanto agitando a 225-250 rpm durante 90 min. </li><li> Placa células de cada tubo em quatro placas de 10 cm de LB-ágar contendo 100 ug / ml de ampicilina. Incubar as placas durante 16-24 horas a 37 ° C. </li><li> Seguindo colónias visíveis, coletá-los raspando as placas com uma placa raspador estéril. Piscina células de cada placa e isolar o ADN de plasmídeo utilizando um kit de extracção de plasmídeo comercial. NOTA: Normalmente, as colônias são menores e tomar 18-24 horas para servisível, porque estes são crescentes estirpes bacterianas lentas. Nesta fase, avaliar a heterogeneidade de mutações na biblioteca por digestão de restrição com um cortador frequente, tais como Sau3A1 ou Taq1 ou sequenciação. </li></ol> 3.2) A produção de retrovirais sobrenadantes e transdução <ol><li> Um dia antes da transfecção, placa de 4 x 10 6 células HEK 293T Onto 100 centímetros pratos em DMEM contendo 10% de FCS, penicilina / estreptomicina e 2 mM de L-glutamina. </li><li> No dia seguinte, substituir o meio e transfecção das células com mutado biblioteca pMSCV-JAK2-V617F. Para cada placa de 10 cm, misturar 10 ug de ADN (5 ug da biblioteca de plasmídeo e 5 ug de plasmídeo de empacotamento retroviral, pCLEco) com 40 ul de FuGENE para um volume total de 400 ul utilizando meio DMEM livre de soro. Incubar a mistura de ADN e de lípido durante 20 minutos à temperatura ambiente. Após 20 min adicionar a queda complexo DNA-lipídico sábio em cima de células HEK293T. </li><li> Após seis horas, mudar o meio de transfecção wom meios frescos e incubar as placas durante 48 horas a 37 ° C para a produção de vírus. Após 48 horas de incubação, recolher os sobrenadantes virais filtradas através de um filtro de 0,45 um Acrodisc. </li></ol> 3.3) Seleção de clones resistentes In Vitro <ol><li> Para mutantes resistentes à JAK2 V617F-droga, a transdução de 100 milhões de células BAF3 com 100 ml de sobrenadantes de vírus com um título virai de 0,5-1 x 10 6. Mais especificamente, misturar 10 8 células com 100 ml de sobrenadante do vírus para um volume total de 300 ml, utilizando meio RPMI, contendo soro a 10% e 24 ug / ml de polyberene (concentração final de ployberene é de 8 ug / ml). Distribuir estes misturadores de células em placas de seis poços múltiplos (4-5 ml por poço). </li><li> Centrifuga-se as placas a 1.250 xg durante 90 min a 25 ° C. Após centrifugação, incubar as placas a 37 ° C durante 24 horas. </li><li> No dia seguinte, reunir as células e misture com Ruxolitinib e meio contendo agar macio e banhado em six placas de poços. Para cada concentração de fármaco, misturar 10 mL de células BAF3 transduzidas (10-20 milhões de células) com a quantidade adequada de Ruxolitinib em um tubo de 50 ml. Completar o volume de 40 ml, utilizando RPMI contendo 20% de soro. Adicionar 10 ml de 1,2% de agar para as células, e mixcarefully placa em placas de seis poços. </li><li> Incubar as placas a 37 ° C durante duas semanas. Escolha as colónias resistentes, utilizando 0,2 ml pontas para pipetas e sub-cultura-los individualmente em placas de 24 poços para 3-4 dias. </li><li> Colher as células BAF3 resistentes por centrifugação a 450 xg durante cinco minutos à temperatura ambiente. Isolar o ADN genómico utilizando um kit comercial de isolamento de ADN genómico. PCR amplificar comprimento total JAK2 cDNA a partir de 100 ng de DNA genômico utilizando primers (mJAK2FP-ATGGCCTGCCTTACAATGACAG e mJAK2RP-GGTTCTGGAAGTCTGCTTGG) e longa-template expandir sistemas de PCR de alta fidelidade. </li><li> Separa-se os produtos de PCR por electroforese em gel de agarose. Extirpar o 3.4 kb de banda DNA representando JAK2 quadro de codificação e isolar o DNAutilizando um kit de extracção de gel comercial. Sequência de todo o comprimento do cDNA utilizando 8 primers internos (P1 - P8) que mede a sequência de codificação e analisar seqüências usando software comercial. </li></ol> 4. Validação in vitro de mutações resistentes Muitos clones de células transportar mais do que uma mutação, Para testar a contribuição de cada mutação no fenótipo de resistência, gerar variantes seleccionadas por mutagénese dirigida a um local utilizando pMSCV-JAK2V-617F plasmídeo como modelo. <ol><li> Realizar mutagénese dirigida em pMSCV-JAK2 V617F-plasmídeo utilizando um kit de mutagénese comercial e oligonucleótidos concebidos para criar mutações pontuais. Confirme as identidades dos clones mutantes por sequenciação. </li><li> Transdução de células BAF3 com retrovírus feita usando plasmídeos mutantes seguiram com a seleção usando ï¿½puromicina em 1 ug / ml. </li><li> Placa 4 10 células BAF3-JAK2 V617F (50-ul de meio RPMI com FCS a 10%) em cada poço de uma placa de 96 poços. Separadoly adicionar 50 ul de meio contendo o ruxolitinib aos poços ao longo da placa (concentração final: 0, 1, 3, 5, 10, e 20 uM), e incuba-se as placas durante 60 horas a 37 ° C. </li><li> Avaliar a viabilidade das células por adição de 10 ul de reagente WST-1 a cada poço, seguido de incubação com a 37 ° C durante 2-4 horas. Ficha absorvância a A450 usando um leitor de placas. Realizar todos os ensaios em triplicado e enredo média absorção contra concentrações INCB018424. Realize uma curva sigmoidal melhor ajuste usando um algoritmo de ajuste de curva não-linear. Conseguir a concentração de droga que resulta em 50% de viabilidade celular como a IC50 celular. </li><li> Em seguida, placa seis milhões de células BAF3 que expressam variantes de JAK2 em placas de seis poços em meio RPMI contendo 10% de soro com concentrações crescentes de ruxolitinib e incubadas durante 4-6 horas a 37 ° C. </li><li> Recolher as células por centrifugação a 450 xg durante cinco minutos a 4 ° C. Lavar as células uma vez com PBS, e lise em 20 mM Tris-Cl (pH 7,5), 50mM de NaCl, 1% de NP-40, 0,1% de SDS, 5 mM de EDTA, 1 mM EGTA, 1 mM de fluoreto de sódio, 2 mM de vanadato de sódio, e 5% de glicerol suplementado com inibidores de cocktail inibidor de protease e fosfatase. Sonicar a suspensão de células durante 1 min, seguido por adição de tampão de carregamento de gel 5x (350 mM Tris-HCl [pH 6,8], 500 mM de DTT, 15% de SDS, 10 mM de benzamidina, 5 mM de EDTA, 5 mM de EGTA, 5 mM de vanadato de sódio , Protease Cocktail, 50% de glicerol, e azul de bromofenol a 0,001%) e desnaturação a 70 ° C durante 5-10 min. </li><li> Resolver as proteínas em um gel de poliacrilamida SDS 8% em condições de desnaturação. Execute immunoblotting com anticorpo monoclonal anti-phopsho STAT5. Tira as manchas e sondado com um anticorpo policlonal de coelho anti-SATA5. Visualize as bandas que utilizam reagentes ECL de acordo com a recomendação do fornecedor. </li></ol> 5. In Vivo de validação <ol><li> Transdução de células que expressam BAF3 luciferase e cereja (transduzidas com retrovírus feitos de pMSCV-Luc-cherry GW) com vírus que expressam JAK2-V617F e variantes resistentes. Seleccionar células que sobrevivem puromicina selecção para a expressão de JAK2 e suas variantes de resistência. </li><li> Injectar dois milhões de células em crescimento activo / cereja BAF3-Luc-JAK2 V617F que transportam e as suas variantes resistentes em 200 ul de PBS a 6 ratinhos Balb / c por via injecção venosa da cauda. </li><li> Depois de três dias de transplantes de células, injectar os ratinhos com Ruxolitinib (100 mg / kg) duas vezes por dia durante duas semanas. </li><li> Execute-base luciferase bioluminescência de imagem com um sistema de imagem. . <ol><li> Em resumo, preparar solução luciferina dissolvendo 300 mg a 25 ml de água deionizada estéril, da alíquota em volumes e loja de menores. NOTA: luciferina Armazenar a -80 ° C e protegido da luz até à utilização. </li><li> Injectar 250 ul a cada rato por IP para conseguir 125 mg / kg em cada rato. Anestesiar os ratos do mesmo grupo em conjunto, utilizando isoflurano inalado (1,5% -2,0%) em uma caixa selada. Aplicar vet pomada em the olho para evitar ressecamento. NOTA: O tempo necessário para que os ratos de dormir (10-15 min) permitiu atividade luciferina a pico. Mantenha o tempo consistente entre os grupos para evitar variação. </li></ol></li><li> Transferir os ratos imediatamente a fase câmara de imagem e manter a anestesia em 1,5-2% de isofluorano durante os procedimentos de imagiologia. Ratinhos imagem sequencial com 0,1, 0,5, 1, 5, 30, 60, e 300 segundos de exposição. Capturar imagens e quantificar a intensidade de bioluminescência usando aquisição de imagem e software de análise. Na sequência de imagens devolver os ratos volta a sua jaula. </li><li> Para, células de medula óssea no total in vivo quimerismo colheita. Eutanásia os ratos com CO 2 para 5 min seguido com luxação cervical. Colher os ossos e esmagar em 4 ml de PBS frio para recolher a medula óssea. Analisar as células positivas por cento de cereja sob microscópio de fluorescência e quantificar utilizando FACS. Recolha vinte mil eventos de cada amostra. </li></ol>

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No conflicts of interest declared.

O sucesso clínico de imatinib no tratamento da CML demonstrado não só o potencial de atacar as quinases rouge por inibidores de moléculas pequenas, mas também descoberto limitações de terapia direcionada: recaída clínica e aparecimento de mutações de resistência a drogas no gene alvo. Identificação de mutações de resistência ajuda na melhor gestão clínica e desenvolvimento de inibidores de próxima geração. Este protocolo descreve uma metodologia para a identificação de mutações resistentes a dr...

As proteína-quinases são enzimas reguladoras-chave de vias intracelulares de transdução de sinal que aparentemente modulam cada função celular. Um controle adequado da quinase mediada sinalização é fundamental para a homeostase e desenvolvimento, que se baseia principalmente na regulamentação adequada de quinases, fosfatases e sua degradação por UPS (sistema ubiquitina proteassoma). Quinases desregulados estão no centro do palco de muitos cânceres e envolvida em série de doenças humanas 1. Genoma humano codifica mais de 500 proteínas cinases que foram ligadas, directa ou indirectamente, para ~ 400 doenças humanas 2. Estas observações suportado o conceito para direccionamento terapêutico de cinases por pequenas moléculas inibidoras 3-5. A demonstração de inibidores da quinase ABL, tais como imatinib, no tratamento de leucemia mielóide crónica (LMC) forneceu a prova de conceito para esta abordagem 6,7. Esta observação não só revolucionou a formigai-quinase terapêutica mas também aplicada a ideia para identificar as lesões genéticas em outras doenças neoplásicas para direccionamento terapêutico, que levam à descoberta de mutações oncogénicas em JAK2 de policitemia vera (PV) e os pacientes com neoplasias mieloproliferativas (NMP). Esta descoberta gerou grande interesse no tratamento MPNs alvejando JAK2 com inibidores da quinase de moléculas pequenas. Agora, quase uma dúzia de inibidores de JAK2 estão em ensaios clínicos, e um deles foi aprovado recentemente para o tratamento de mielofibrose. Enquanto direccionamento específico de quinases oncogénicas por pequenas moléculas inibidoras em cancros trazer resultados promissores, também sofre de desenvolvimento de resistência ao tratamento. De facto, até agora, os pacientes tratados com inibidores de cinase, tais como imatinib, gefitinib, erlotinib e dasatinib mutações de resistência desenvolvida principalmente através da aquisição de mutações no domínio de cinase de droga que tem como alvo 8-10. Resistência como resultado de mutação genética destaca as limitações de of monoterapia alvo contra as quinases oncogénicas, e representa o seguinte desafio para o desenvolvimento de quimioterapia do cancro cada vez mais bem sucedidos. Consequências mecanicistas e funcionais de resistência aos medicamentos deve fornecer uma justificação para a selecção e design de compostos de cortesia para o desenvolvimento de drogas. As mutações identificadas via telas in vitro, mostraram um alto grau de correlação com os encontrados em pacientes. Portanto, rastreio in vitro para mutações que conferem resistência aos fármacos para um dado alvo pares droga auxilia em desenvolvimento clínico ou pré-clínicos em identificar os padrões de resistência que são susceptíveis de causar recaída clínica. A identificação dessas formas mutantes não só é útil no acompanhamento dos pacientes para a resposta à droga e recaída, mas também essencial para a concepção de inibidores mais robustos de próxima geração. Por exemplo, o desenvolvimento de inibidores BCR / ABL próxima geração, o nilotinib e ponatinibe, foram possíveis por causa da maior mecentendimentos hanistic adquirida com mutagênese, estrutural, e estudos funcionais. Anteriormente, relataram os resultados de nossa tela usando mutagênese aleatória de BCR / ABL para revelar o espectro de mutações de resistência aos inibidores tais como Imatinib 11,12, PD166326 12 e 13 AP24163. Os resultados não só identificou as mutações que conferem resistência e recidiva da doença clínica, mas também forneceu a compreensão mecanicista da resistência e dos princípios que regem a função quinase 11,14 drogas. Aqui nós fornecemos detalhes metodológicos adicionais, usando Ruxolitinib e JAK2 como um par alvo da droga, para permitir uma aplicação mais ampla dessa estratégia de triagem.

<table><tbody><tr><td>Cell and Tissue culture&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>BaF3 Cells</td><td>ATCC</td><td></td><td></td></tr><tr><td>HEK293T cells</td><td>ATCC</td><td></td><td></td></tr><tr><td>pMSCV-JAK2-V617F-puro.GW</td><td>A gift from Ross Levine</td><td></td><td></td></tr><tr><td>pMSCV-JAK2-V617F/Y931C.GW</td><td>Made in house</td><td></td><td></td></tr><tr><td>pMSCV-JAK2-V617F/L983F.GW</td><td>Made in house</td><td></td><td></td></tr><tr><td>pMSCV-JAK2-V617F/P58A.GW</td><td>Made in house</td><td></td><td></td></tr><tr><td>pMSCV-V617F-Cherry.GW</td><td>Made in house</td><td></td><td></td></tr><tr><td>pMSCV-JAK2-V617F/Y931C-cherry.GW</td><td>Made in house</td><td></td><td></td></tr><tr><td>pMSCV-JAK2-V617F/L983F-cherry.GW</td><td>Made in house</td><td></td><td></td></tr><tr><td>pMSCV-Luciferase-puro.GW</td><td>Made in house</td><td></td><td></td></tr><tr><td>RPMI</td><td>Cellgro (corning)</td><td>15-040-CV</td><td></td></tr><tr><td>DMEM</td><td>Cellgro (corning)</td><td>15-013-CV</td><td></td></tr><tr><td>Penn/Strep</td><td>Cellgro (corning)</td><td>30-002-CI</td><td></td></tr><tr><td>FBS</td><td>Atlanta biological</td><td>S11150</td><td></td></tr><tr><td>Trypsin EDTA 1X</td><td>Cellgro (corning)</td><td>25-052-CI</td><td></td></tr><tr><td>1x PBS</td><td>Cellgro (corning)</td><td>21-040-CV</td><td></td></tr><tr><td>L-Glutamine</td><td>Cellgro (corning)</td><td>25-005-CL</td><td></td></tr><tr><td>Puromycin</td><td>Gibco (life technologies)</td><td>A11138-03</td><td></td></tr><tr><td>Protamine sulfate</td><td>Sigma</td><td>P3369</td><td>5 mg/ml stock in water</td></tr><tr><td>Trypan Blue solution (0.4%)</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>DMSO</td><td>Cellgro (corning)</td><td>25-950-CQC</td><td></td></tr><tr><td>INCB018424 (Ruxolitinib)</td><td>ChemieTeK</td><td>941678-49-5</td><td></td></tr><tr><td>WST-1</td><td>Roche</td><td>11644807001</td><td></td></tr><tr><td>0.45 micron disc filter</td><td>PALL</td><td>2016-10</td><td></td></tr><tr><td>70 micron nylon cell strainer</td><td>Becton Dickinson</td><td>352350</td><td></td></tr><tr><td>Bacterial Culture</td><td></td><td></td><td></td></tr><tr><td>XL-1 red &lt;em&gt;E. coli&lt;/em&gt; cells</td><td>Agilent Tech</td><td>200129</td><td></td></tr><tr><td>SOC</td><td>New England Biolabs</td><td>B90920s</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A0166</td><td>100 mg/ml stock solution&amp;nbsp;</td></tr><tr><td>Bacto agar</td><td>Difco</td><td>214050</td><td></td></tr><tr><td>Terrific broth</td><td>Becton Dickinson</td><td>243820</td><td></td></tr><tr><td>Agarose</td><td>Genemate</td><td>E-3119-500</td><td></td></tr><tr><td>Kits</td><td></td><td></td><td></td></tr><tr><td>Dneasy Blood&amp;amp; tissue kit</td><td>Qiagen</td><td>69506</td><td></td></tr><tr><td>Expand long template PCR system</td><td>Roche</td><td>1168134001</td><td></td></tr><tr><td>Wizard Sv gel and PCR clean up system</td><td>Promega</td><td>A9282</td><td></td></tr><tr><td>Pure Yield plasmid mini prep system</td><td>Promega</td><td>A1222</td><td></td></tr><tr><td>PCR Cloning System with Gateway Technology with pDONR 221 &amp;amp; OmniMAX 2 Competent Cells</td><td>Invitrogen</td><td>12535029</td><td></td></tr><tr><td>Gateway&amp;nbsp; LR Clonase Enzyme mix&amp;nbsp;</td><td>Invitrogen</td><td>11791019</td><td></td></tr><tr><td>Mouse reagents</td><td></td><td></td><td></td></tr><tr><td>Vivo-Glo Luciferin in-vivo Grade</td><td>Promega</td><td>P1043</td><td></td></tr><tr><td>1/2 cc Lo-Dose u-100 insulin syringe 28 G1/2</td><td>Becton Dickinson</td><td>329461</td><td></td></tr><tr><td>Mortor pestle</td><td>Coor tek&amp;nbsp;</td><td>60316 and 60317</td><td></td></tr><tr><td>Isoflorane (Isothesia TM)</td><td>Butler Schien</td><td>29405</td><td></td></tr><tr><td>Instruments</td><td></td><td></td><td></td></tr><tr><td>NAPCO series 8000 WJ CO2 incubator</td><td>Thermo scientific</td><td></td><td></td></tr><tr><td>Swing bucket rotor centrifuge 5810R</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>TC-10 automated cell counter</td><td>Bio-RAD</td><td></td><td>This is not necessary, one can use standard hemocytomemetr for cell counting</td></tr></tbody></table>

a method for screening and validation of resistant mutations against kinase inhibitors

A descoberta de BCR / ABL como um oncogene condutor na leucemia mielóide crónica (LMC), resultou no desenvolvimento de Imatinib, o qual, na verdade, demonstraram o potencial do alvo a quinase em cancros por tratar eficazmente os pacientes de CML. Esta observação revolucionou o desenvolvimento de drogas para alvejar as quinases oncogénicas implicados em várias outras doenças malignas, tais como, o EGFR, B-RAF, KIT e PDGFRs. No entanto, uma grande desvantagem de terapias anti-quinase é o aparecimento de mutações de resistência a drogas que tornam o alvo ter reduzido ou perdido afinidade para a droga. A compreensão dos mecanismos empregados pela variantes resistentes não só ajuda no desenvolvimento dos inibidores de nova geração, mas também dá impulso à gestão clínica usando a medicina personalizada. Nós relatou uma estratégia de rastreamento retroviral vector base para identificar o espectro de resistência conferindo mutações no BCR / ABL, o que tem ajudado no desenvolvimento da próxima geração BCR / ABL inibidores. Usando Ruxolitinib e JAK2 como um par alvo da droga, aqui descrevemos em métodos de rastreio in vitro que utiliza as células do rato BAF3 expressam a biblioteca mutação aleatória de JAK2 quinase.

Surgimento de mutações genéticas coloca grande desafio para a terapia anti-quinase alvo. Estudos de mutação, além de proporcionar idéias mecanicistas e funcionais, que são fundamentais para a seleção e projeto de desenvolvimento de medicamentos de última geração, também permite uma melhor gestão clínica e pode, no futuro, ser mais útil para o tratamento personalizado. Neste experimento, mostramos triagem para mutações de resistência ruxolitinib em JAK2 V617F-quinase (Figura 1). Const...

Watch this Scientific Journal Video about A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors at JoVE.com

A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

Surgimento de resistência genética a terapia com inibidor da quinase poses desafio significativo para a terapia do cancro eficaz. Identificação e caracterização de mutações resistentes contra uma droga recém-desenvolvido ajuda na melhor gestão clínica e concepção de medicamentos próxima geração. Aqui, descrevemos o nosso protocolo de rastreio in vitro e validação das mutações resistentes.

Um método de triagem e Validação de mutações resistentes aos inibidores da quinase

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

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Research

JoVE Journal

Biology

14.8K visualizações.  Cincinnati Children's Hospital Medical Center.  Surgimento de resistência genética a terapia com inibidor da quinase poses desafio significativo para a terapia do cancro eficaz. Identificação e caracterização de mutações resistentes contra uma droga recém-desenvolvido ajuda na melhor gestão clínica e concepção de medicamentos próxima geração. Aqui, descrevemos o nosso protocolo de rastreio in vitro e validação das mutações resistentes. 

Vídeo: A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

Assaying Protein Kinase Activity with Radiolabeled ATP

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering allosteric details of their regulation. Kinases comprise signaling networks whose defects are often hallmarks of multiple forms of cancer and related diseases, making an assay platform amenable to manipulation of upstream regulatory factors and validation of reaction requirements critical in the search for improved therapeutics. Here, we describe a basic kinase assay that can be easily adapted to suit specific experimental questions including but not limited to testing the effects of biochemical and pharmacological agents, genetic manipulations such as mutation and deletion, as well as cell culture conditions and treatments to probe cell signaling mechanisms. This assay utilizes radiolabeled [&#947;-32P] ATP, which allows for quantitative comparisons and clear visualization of results, and can be modified for use with immunoprecipitated or recombinant kinase, specific or typified substrates, all over a wide range of reaction conditions.

Protein kinases are highly evolved signaling enzymes and scaffolds that are critical for inter- and intracellular signal transduction. We present a protocol for measuring kinase activity through the use of radiolabeled adenosine triphosphate ([&#947;-32P] ATP), a reliable method to aid in elucidation of cellular signaling regulation.

Ensaio de Atividade da Proteína Quinase com ATP Radiomarcado

Protein kinases are able to govern large-scale cellular changes in response to complex arrays of stimuli, and much effort has been directed at uncovering ...

Bioquímica

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates approaching 85% are common. Unfortunately, patients often become refractory to the treatment by altering their signal transduction pathways. An implementation of the expression profiling with microarrays can identify the overall mRNA-level changes, and proteomics can identify the overall changes in protein levels or can identify the proteins involved, but the activity of the signal transduction pathways can only be established by interrogating post-translational modifications of the proteins. As a result, the ability to identify whether a drug treatment is successful or whether resistance arose, or the ability to characterize any alterations in the signaling pathways, is an important clinical challenge. Here, we provide a detailed explanation of antibody arrays as a tool which can identify system-wide alterations in various post-translational modifications (e.g., phosphorylation). One of the advantages of using antibody arrays includes their accessibility (an array does not require either an expert in proteomics or costly equipment) and speed. The availability of arrays targeting a combination of post-translational modifications is the primary limitation. In addition, unbiased approaches (phosphoproteomics) may be more suitable for the novel discovery, whereas antibody arrays are ideal for the most widely characterized targets.

Here, we present a protocol for antibody arrays to identify alterations in signaling pathways in various cellular models. These changes, caused by drugs/hypoxia/ultra-violet light/radiation, or by overexpression/downregulation/knockouts, are important for various disease models and can indicate whether a therapy will be effective or can identify mechanisms of drugs resistance.

Avaliação da resistência aos inibidores de tirosina quinase por um interrogatório das vias de transdução de sinal por matrizes de anticorpo

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates ...

Pesquisa do câncer

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

Métodos para avaliar o papel de c-Fos e Dusp1 em dependência do Oncogene

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

Caracterizar Mutants relacionados à doença da família da RAF kinases usando um conjunto de métodos práticos e viáveis

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important roles for the cell and may unravel new cellular processes. Among proteins, kinases are major actors as they belong to cell signaling pathways and have the ability to switch on or off many processes crucial to the fate of the cell, such as cell growth, division, differentiation, motility, and death.
In this study, we focused on a new potential kinase protein, LIMK2-1. We demonstrated its existence by Western Blot using a specific antibody. We evaluated its interaction with an upstream regulating protein using coimmunoprecipitation experiments. Coimmunoprecipitation is a very powerful technique able to detect the interaction between two target proteins. It may also be used to detect new partners of a bait protein. The bait protein may be purified either via a tag engineered to its sequence or via an antibody specifically targeting it. These protein complexes may then be separated by SDS-PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel) and identified using mass spectrometry. Immunoprecipitated LIMK2-1 was also used to test its kinase activity in vitro by &#947;[32P] ATP labeling. This well-established assay may use many different substrates, and mutated versions of the bait may be used to assess the role of specific residues. The effects of pharmacological agents may also be evaluated since this technique is both highly sensitive and quantitative. Nonetheless, radioactivity handling requires particular caution. Kinase activity may also be assessed with specific antibodies targeting the phospho group of the modified amino acid. These kinds of antibodies are not commercially available for all the phospho modified residues.

We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by &#947;[32P] ATP labeling.

Caracterização no nível molecular usando abordagens bioquímicas robustas de uma nova proteína quinase

Extensive whole genome sequencing has identified many Open Reading Frames (ORFs) providing many potential proteins. These proteins may have important ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

Triagem de inibidores da quinase em Microarrays de proteínas humanas Automontados

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

A avaliação pré-clínica da tirosina quinase inibidores para o tratamento de leucemia aguda

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Medicina

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

Implementação de<em&gt; In Vitro</em&gt; Droga de Resistência Ensaios: maximizar o potencial para Descobrindo clinicamente relevantes mecanismos de resistência

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Doseamento do atividade da quinase de LRRK2 In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Biologia

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

Identificação de mediadores do Receptor de células T através da triagem de bibliotecas de inibidor químico de sinalização

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Um método de triagem e Validação de mutações resistentes aos inibidores da quinase

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