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: Tutte le procedure di questo protocollo sono stati condotti secondo l&#39;Istituto Nazionale di Salute linee guida per il trattamento etico e la cura degli animali, e secondo un protocollo di uso animale IACUC approvato. 1. Cella Linea Manutenzione <ol><li> Culture cellule BAF3 in mezzo RPMI-1640 supplementato con 10% siero bovino fetale e penicillina / streptomicina (100 unità / ml e 100 mg / ml) e terreno di coltura di cellule WEHI trascorso. Grow HEK293T cellule in DMEM supplementato con 10% siero bovino fetale e penicillina / streptomicina (100 unità / ml e 100 mg / ml). Mantenere cellule a 37 ° C in atmosfera umidificata contenente il 5% di CO2. </li></ol> 2. plasmidi Construction <ol><li> Clonare il mouse JAK2 e la sua oncogenica isoforma JAK2-V617F in pMSCV-puro-GW da LR clonase utilizzando procedura di clonazione ricombinazione standard. </li><li> Per la costruzione di espressione luciferasi vettoriale (pMSCV-Luc-Cherry-GW) utilizzato nella-viimmagini vo, amplificare luciferasi da pLVX-Tet-ON vettore mediante PCR utilizzando primer (LUC-SD / RP TTACAATTTGGACTTTCCG e LUC-SD / FP-CACCATGGAAGACGCCAAAAAC) seguita con la clonazione in pENTR-SD-TOPO per generare pENTR-Luc, che viene utilizzato per trasferire il gene della luciferasi in vettore retrovirale, pMSCV-Cherry-GW da ricombinazione clonazione mediata con LR clonase. </li></ol> 3. Preparazione di Random Mutazione Biblioteca, riciclaggio e identificare le mutazioni 3.1) casuale Mutagenesi <ol><li> Clone lunghezza della JAK2 wild type e la mutazione V617F in pMSCV-puro-GW vettore retrovirale utilizzando una tecnologia di clonazione ricombinazione commerciale. </li><li> Utilizzare 1 mg di JAK2-V617F plasmide DNA per trasformare, XL-1 Red, E. coli, che è difettoso in meccanismi di riparazione del DNA permettendo loro di integrare le mutazioni casuali durante la replica. Più specificamente, mix 50 - 100 ng di DNA (più di 100 ng di DNA diminuisce l&#39;efficienza della trasformazione) con 100 ml di cellule competenti in un tubo di polipropilene pre-raffreddata e incubate in ghiaccio per 30 minuti, rimestando delicatamente ogni 5 min. Per una buona copertura libreria, utilizzare 5:56 tubi di cellule competenti. NOTA: Più di 100 ng di DNA diminuisce l&#39;efficienza di trasformazione </li><li> Immergere la provetta in un bagno d&#39;acqua a 42 ° C per uno shock termico di 45 sec e incubare su ghiaccio per 2 min. Avanti, aggiungere 1 ml di mezzo SOC (2,0% triptone, estratto di lievito 0,5%, 0,05% di NaCl, MgCl2 10 mM, MgSO4 10 mm, e il 0,4% D-glucosio). Incubare il tubo a 37 ° C agitando a 225-250 rpm per 90 min. </li><li> Cellule piastra di ciascun tubo su quattro 10 centimetri piatti LB-agar contenenti 100 mg / ml di ampicillina. Incubare le piastre per 16-24 ore a 37 ° C. </li><li> A seguito di colonie visibili, raccogliendoli raschiando le piastre con un piatto raschietto sterile. Cellule Pool di ogni piatto e plasmide isolare il DNA utilizzando un kit di estrazione plasmide commerciale. NOTA: Di solito colonie sono più piccoli e prendere 18-24 ore per esserevisibile perché questi sono lenti ceppi batterici crescita. In questa fase, di valutare l&#39;eterogeneità di mutazioni nella libreria da digestione di restrizione con un cutter frequente come Sau3A1 o Taq1 o sequenziamento. </li></ol> 3.2) Produzione di retrovirali surnatante e trasduzione <ol><li> Un giorno prima trasfezione, piastra 4 x 10 6 cellule HEK 293T su 100 centimetri piatti in DMEM contenente 10% FCS, penicillina / streptomicina e 2 mM L-glutammina. </li><li> Il giorno dopo, sostituire il medio e trasfettare le cellule con mutagenizzato biblioteca pMSCV-JAK2-V617F. Per ogni piastra 10 cm miscela 10 mg di DNA (5 ug del plasmide libreria e 5 ug del plasmide retrovirale imballaggi, pCLEco) con 40 ml di FuGENE ad un volume totale di 400 microlitri di siero utilizzando supporti DMEM libera. Incubare la miscela DNA e lipidi per 20 min a temperatura ambiente. Dopo 20 minuti aggiungere il DNA-lipide complesso goccia a goccia in cima HEK293T cellule. </li><li> Dopo sei ore, cambiare il supporto trasfezione wesimo mezzi freschi e incubare le piastre per 48 ore a 37 ° C per la produzione di virus. Dopo 48 ore di incubazione, raccogliere surnatanti virali filtrati attraverso un filtro da 0,45 micron Acrodisc. </li></ol> 3.3) Selezione dei cloni resistenti in vitro <ol><li> Per resistenti ai farmaci mutanti JAK2-V617F, trasduzione 100 milioni BAF3 cella con 100 ml di surnatanti virus hanno un titolo virale di 0,5-1 x10 6. Più specificamente, mescolare 10 8 cellule con 100 ml di surnatante virus ad un volume totale di 300 ml con mezzi RPMI contenente siero, 10% e 24 ug / ml di polyberene (concentrazione finale di ployberene è 8 mg / ml). Distribuire questi mixer cellulari in più sei pozzetti (4-5 ml per pozzetto). </li><li> Centrifugare le piastre a 1.250 xg per 90 min a 25 ° C. Dopo centrifugazione, incubare le piastre a 37 ° C per 24 ore. </li><li> Il giorno dopo, in comune le cellule insieme e mescolare con Ruxolitinib e terreno contenente agar morbido e placcato in six piatti pure. Per ogni concentrazione del farmaco, miscelare 10 ml di cellule trasdotte BAF3 (10-20 milioni di cellule) con la quantità appropriata di Ruxolitinib in un tubo da 50 ml. Portare al volume di 40 ml con RPMI contenente siero del 20%. Aggiungere 10 ml di 1,2% agar alle cellule, mixcarefully e piastra in sei pozzetti. </li><li> Incubare le piastre a 37 ° C per due settimane. Scegli le colonie resistenti con 0,2 ml puntali e sub-cultura singolarmente in 24 tavole e per 3-4 giorni. </li><li> Raccogliere le cellule resistenti BAF3 mediante centrifugazione a 450 xg per cinque minuti a temperatura ambiente. Isolare il DNA genomico utilizzando un kit di isolamento DNA genomico commerciale. PCR amplifica full length JAK2 cDNA da 100 ng di DNA genomico utilizzando primer (mJAK2FP-ATGGCCTGCCTTACAATGACAG e mJAK2RP-GGTTCTGGAAGTCTGCTTGG) e lungo template espandere sistemi PCR ad alta fedeltà. </li><li> Separare i prodotti di PCR mediante elettroforesi su gel di agarosio. Accise 3,4 kb di banda DNA rappresentano JAK2 telaio codifica e isolare il DNAutilizzando un kit di estrazione gel commerciale. Sequenza tutta la lunghezza del cDNA usando 8 primer interni (P1 - P8) che attraversa la sequenza codificante e analizzare sequenze utilizzando software commerciale. </li></ol> 4. In Vitro convalida di mutazioni resistenti Molti cloni cellulari trasportare più di una mutazione, per verificare il contributo di ogni mutazione in resistenza fenotipo, generare varianti selezionati da mutagenesi sito-specifica utilizzando pMSCV-JAK2V-617F plasmide come modello. <ol><li> Eseguire mutagenesi sito-specifica in pMSCV-JAK2-V617F plasmide utilizzando un kit commerciale mutagenesi e oligonucleotidi progettati per creare mutazioni puntiformi. Confermare l&#39;identità dei cloni mutanti mediante sequenziamento. </li><li> Trasdurre cellule BAF3 con retrovirus realizzati con plasmidi mutanti seguita con selezione con puromicina a 1 ug / ml. </li><li> Tavola 10 4 cellule BAF3-JAK2-V617F (50 ml di RPMI con 10% FCS) in tutti i pozzetti di una piastra ben 96. Separatoly aggiungere 50 ml di ruxolitinib contenente supporti ai pozzetti attraverso la piastra (concentrazione finale: 0, 1, 3, 5, 10, e 20 mM), e incubare le piastre per 60 ore a 37 ° C. </li><li> Valutare vitalità cellulare aggiungendo 10 ml di reagente WST-1 ad ogni pozzetto seguito con incubazione a 37 ° C per 2-4 ore. Record assorbanza a A450 con un lettore di piastre. Eseguire tutti i test in triplice copia e trama in media assorbanza contro INCB018424 concentrazioni. Eseguire una curva sigmoidale best-fit utilizzando un algoritmo di curve fitting non lineare. Nota la concentrazione del farmaco con conseguente vitalità cellulare del 50% come IC50 cellulare. </li><li> Successivamente, piastra sei milioni BAF3 cellule esprimenti varianti JAK2 in sei pozzetti in terreno RPMI contenente 10% di siero con concentrazioni crescenti di ruxolitinib ed incubate per 4-6 ore a 37 ° C. </li><li> Raccogliere le cellule per centrifugazione a 450 g per cinque minuti a 4 ° C. Lavare le cellule una volta con PBS, e lisare in 20 mM Tris-Cl (pH 7,5), 50mM NaCl, 1% NP-40, 0.1% SDS, EDTA 5 mM, 1mM EGTA, 1 mM fluoruro di sodio, 2 mM sodio Vanadate, e il 5% glicerina integrato con inibitori della proteasi cocktail e fosfatasi. Sonicare la sospensione cellulare per 1 min seguita da aggiunta di tampone 5x gel loading (350 mM Tris-HCl [pH 6,8], 500 mM DTT, 15% SDS, 10 mM Benzamidine, 5 mM EDTA, EGTA 5 mM, 5 mM Sodio vanadato , proteasi cocktail, 50% glicerolo, e 0,001% blu di bromofenolo) e denaturazione a 70 ° C per 5-10 min. </li><li> Risolvere le proteine ​​su un gel di poliacrilammide SDS dell&#39;8% in condizioni denaturanti. Eseguire immunoblotting con il mouse monoclonale anti-phopsho STAT5. Spellare le macchie e reprobed con un anticorpo anti-SATA5 policlonale di coniglio. Visualizzare le bande utilizzando reagenti ECL secondo le raccomandazioni del fornitore. </li></ol> 5. In Vivo Validation <ol><li> Trasdurre cellule BAF3 esprimono luciferasi e ciliegia (trasdotte con retrovirus realizzati pMSCV-Luc-cherry GW) con i virus che esprimono JAK2-V617F e varianti resistenti. Selezionare cellule che sopravvivono selezione puromicina per l&#39;espressione di JAK2 e le sue varianti resistenza. </li><li> Iniettare due milioni di crescita attiva BAF3-Luc cellule / ciliegia che trasportano JAK2-V617F e le sue varianti resistenti in 200 ml di PBS a 6 Balb / C di topo mediante iniezione-coda vena. </li><li> Dopo tre giorni di trapianti di cellule, iniettare i topi con Ruxolitinib (100 mg / kg) due volte al giorno per due settimane. </li><li> Eseguire base luciferasi imaging bioluminescenza con un sistema di imaging. . <ol><li> In breve, preparare la soluzione luciferina sciogliendo 300 mg per 25 ml di acqua deionizzata sterile, un&#39;aliquota di minori volumi e negozio. NOTA: Conservare luciferina a -80 ° C e proteggere dalla luce fino al momento dell&#39;uso. </li><li> Iniettare 250 microlitri per ogni mouse IP per ottenere 125 mg / kg in ciascun topo. Anestetizzare i topi dal gruppo stesso insieme con isoflurano inalato (1,5% -2,0%), in una scatola sigillata. Applicare pomata veterinario su the occhio per prevenire la secchezza. NOTA: Il tempo necessario per i topi a dormire (10-15 minuti) ha permesso l&#39;attività luciferina a picco. Tenere il tempo coerente tra i gruppi per evitare variazioni. </li></ol></li><li> Trasferire il mouse immediatamente fase camera di imaging e mantenere l&#39;anestesia al 1,5-2% isofluorane durante le procedure di imaging. Topi fotografia con sequenziale 0,1, 0,5, 1, 5, 30, 60, e 300 sec esposizione. Cattura immagini e quantificare l&#39;intensità bioluminescenza utilizzando l&#39;acquisizione delle immagini e software di analisi. A seguito di imaging restituire i topi alla sua gabbia. </li><li> Per, in vivo chimerismo raccolto cellule bonemarrow totali. Eutanasia i topi con CO 2 per 5 minuti, seguito con dislocazione cervicale. Raccogliere le ossa e schiacciare in 4 ml PBS freddo per raccogliere il midollo osseo. Analizzare i cento ciliegi cellule positive al microscopio a fluorescenza e quantificare con FACS. Raccogliere ventimila eventi da ogni campione. </li></ol>

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No conflicts of interest declared.

Il successo clinico di Imatinib nel trattamento CML ha dimostrato non solo il potenziale di colpire chinasi rouge da piccole molecole inibitrici, ma anche le limitazioni allo scoperto di terapia mirata: ricaduta clinica e comparsa di mutazioni di resistenza ai farmaci nel gene bersaglio. L&#39;identificazione di mutazioni di resistenza aiuta a migliorare la gestione clinica e lo sviluppo di inibitori di nuova generazione. Questo protocollo descrive un metodo per identificare mutazioni farmaco-resistenti nel gene bersagl...

Proteina chinasi sono enzimi chiave di regolamentazione degli intracellulari di trasduzione del segnale che apparentemente modulano ogni funzione cellulare. Un adeguato controllo della chinasi mediata segnalazione è fondamentale per l&#39;omeostasi e sviluppo, che si basa principalmente su una corretta regolamentazione delle chinasi, fosfatasi e la sua degradazione da UPS (sistema ubiquitina proteasoma). Chinasi deregolamentati sono al centro della scena di molti tumori e coinvolti in serie di malattie umane 1. Genoma umano codifica più di 500 proteine ​​chinasi che sono stati collegati, direttamente o indirettamente, a ~ 400 malattie umane 2. Queste osservazioni hanno sostenuto il concetto per terapeutico mirati chinasi da piccole molecole inibitrici 3-5. La dimostrazione di inibitori della chinasi ABL, come Imatinib nel trattamento della leucemia mieloide cronica (LMC) ha fornito la prova del concetto di questo approccio 6,7. Questa osservazione ha rivoluzionato non solo la formicai-chinasi terapia, ma anche applicata l&#39;idea di identificare le lesioni genetiche in altre malattie neoplastiche per il targeting terapeutico, che portano alla scoperta di mutazioni oncogeniche in JAK2 da policitemia vera (PV) e nei pazienti con neoplasie mieloproliferative (MPN). Questa scoperta ha generato un grande interesse nel trattamento MPNs di mira JAK2 con piccole molecole inibitori della chinasi. Ora, quasi una dozzina di inibitori della JAK2 sono in studi clinici e uno di loro è stato recentemente approvato per il trattamento della mielofibrosi. Mentre mirata delle chinasi oncogeniche da piccole molecole inibitrici nei tumori portare risultati promettenti, ma soffre anche di sviluppare resistenza al trattamento. In realtà, fino ad ora, i pazienti trattati con inibitori delle chinasi, come l&#39;Imatinib, Gefitinib, Erlotinib e Dasatinib sviluppate mutazioni di resistenza per lo più con l&#39;acquisizione di mutazioni nel dominio chinasi a cui bersagli farmacologici 8-10. Resistenza a seguito di mutazione genica evidenzia i limiti of mirate monoterapia contro le chinasi oncogeniche, e rappresenta la prossima sfida per lo sviluppo di sempre più successo chemioterapia. Conseguenze meccanicistici e funzionali di resistenza ai farmaci dovrebbero fornire un razionale per la selezione e la progettazione di composti gratuiti per lo sviluppo di farmaci. Le mutazioni identificate tramite schermi in vitro, hanno mostrato un alto grado di correlazione con quelli riscontrati in pazienti. Pertanto, lo screening in vitro per le mutazioni che conferiscono resistenza ai farmaci per un dato farmaco coppie bersaglio assist di sviluppo clinico o preclinico per identificare i modelli di resistenza che possono causare una ricaduta clinica. L&#39;identificazione di queste forme mutanti non solo è utile nel monitoraggio dei pazienti per risposta ai farmaci e la recidiva ma anche essenziale per la progettazione di inibitori più robusti prossima generazione. Ad esempio, lo sviluppo della prossima generazione di inibitori BCR / ABL, Nilotinib e Ponatinib, sono stati resi possibili a causa di una maggiore mecintese hanistic acquisita mutagenesi, strutturale, e studi funzionali. In precedenza, abbiamo riportato i risultati del nostro schermo utilizzando mutagenesi casuale di BCR / ABL a rivelare lo spettro di mutazioni che conferiscono resistenza agli inibitori come Imatinib 11,12, PD166326 12, e AP24163 13. I risultati non solo identificare le mutazioni che conferiscono resistenza e malattia clinica ricaduta, ma anche fornito la comprensione meccanicistica della resistenza e dei principi che regolano la funzione chinasi 11,14 droga. Qui forniamo ulteriori dettagli metodologico, utilizzando Ruxolitinib e JAK2 come una coppia di destinazione della droga, per consentire una più ampia applicazione di questa strategia di screening.

<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

La scoperta di BCR / ABL come oncogene conducente leucemia mieloide cronica (CML) ha portato allo sviluppo di Imatinib, che, di fatto, ha dimostrato il potenziale di targeting chinasi in tumori trattando efficacemente i pazienti CML. Questa osservazione ha rivoluzionato lo sviluppo di farmaci a bersaglio le chinasi oncogeni implicati in varie altre neoplasie, come, EGFR, B-RAF, KIT e PDGFRs. Tuttavia, uno svantaggio principale di terapie anti-chinasi è l&#39;emergere di mutazioni di resistenza ai farmaci che rendono l&#39;obiettivo di aver ridotto o perso affinità per la droga. La comprensione dei meccanismi impiegati da varianti resistenti non solo aiuta a sviluppare gli inibitori di nuova generazione, ma dà anche impulso alla gestione clinica utilizzando la medicina personalizzata. Abbiamo riportato una strategia di vettore retrovirale basato di screening per identificare lo spettro di resistenza conferire mutazioni BCR / ABL, che ha contribuito a sviluppare la prossima generazione di BCR / ABL inibitori. Utilizzando Ruxolitinib e JAK2 come una coppia di destinazione della droga, qui si descrivono in metodi di screening in vitro che utilizza le cellule del mouse BAF3 esprimono la biblioteca mutazione casuale del JAK2 chinasi.

L&#39;emergere di mutazioni genetiche pone grande sfida per la terapia chinasi contro mirata. Studi mutazionali, oltre a fornire approfondimenti meccanicistici e funzionali che sono strumentali nella selezione e progettazione di sviluppo di farmaci di nuova generazione, permette anche una migliore gestione clinica e possano in futuro essere più utile per il trattamento personalizzato. In questo esperimento, mostriamo lo screening per le mutazioni di resistenza ruxolitinib in JAK2-V617F chinasi (Figura 1).</stro...

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

Emersione di resistenza genetica contro terapia con inibitori della chinasi pone sfida importante per la terapia del cancro efficace. Identificazione e caratterizzazione di mutazioni resistenti contro un farmaco di nuova concezione contribuisce a una migliore gestione clinica e di progettazione di farmaci di nuova generazione. Qui, descriviamo il nostro protocollo per lo screening e la validazione di mutazioni resistenti in vitro.

Un metodo per lo screening e validazione di mutazioni resistenti contro Kinase Inhibitors

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 ...

a-method-for-screening-validation-resistant-mutations-against-kinase

Research

JoVE Journal

Biology

14.8K Views.  Cincinnati Children's Hospital Medical Center.  Emersione di resistenza genetica contro terapia con inibitori della chinasi pone sfida importante per la terapia del cancro efficace. Identificazione e caratterizzazione di mutazioni resistenti contro un farmaco di nuova concezione contribuisce a una migliore gestione clinica e di progettazione di farmaci di nuova generazione. Qui, descriviamo il nostro protocollo per lo screening e la validazione di mutazioni resistenti in vitro. 

Video: 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.

Analizzare l&#39;attività della proteina con l&#39;ATP radioattivato

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 ...

Ricerca

Biochimica

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.

Valutazione della resistenza agli inibitori della tirosina chinasi di un interrogatorio di trasduzione del segnale da matrici di anticorpo

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

Ricerca sul cancro

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.

Metodi per valutare il ruolo di c-Fos e Dusp1 nella dipendenza dell'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.

Caratterizzare i mutanti correlati alla malattia di RAF Family Kinases utilizzando una serie di metodi pratici e fattibili

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.

Caratterizzazione a livello molecolare utilizzando robusti approcci biochimici di una nuova proteina Kinase

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.

Screening dell'inibitore della kinasi in microarray proteici umane auto-assemblati

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.

Valutazione pre-clinica della tirosina chinasi inibitori per il trattamento di leucemia acuta

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.

Implementazione di<em&gt; In Vitro</em&gt; Drug Resistance saggi: massimizzare il potenziale per scoprire clinicamente rilevanti Resistenza Meccanismi

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.

Saggiare l&#39;attività chinasi di 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.

Identificazione dei mediatori del T-cell Receptor segnalazione tramite lo Screening di librerie chimiche inibitore

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

Un metodo per lo screening e validazione di mutazioni resistenti contro Kinase Inhibitors

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