Name: a method for screening and validation of resistant mutations against kinase inhibitors
Uploaded: 2014-12-07T15:00:00
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.

BEMÆRK: Alle procedurer denne protokol blev udført ifølge National Institute of retningslinjer for etisk behandling og pleje af dyr Sundhed, og ifølge en godkendt IACUC anvendelsen af ​​dyr protokol. 1. cellelinie Vedligeholdelse <ol><li> Kultur BaF3-celler i RPMI-1640 medium suppleret med 10% føtalt bovint serum og penicillin / streptomycin (100 enheder / ml og 100 ug / ml) og brugt dyrkningsmedium WEHI Cells. Grow HEK293T celler i DMEM suppleret med 10% føtalt bovint serum og p...

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The clinical success of Imatinib in treating CML demonstrated not only the potential of targeting the rouge kinases by small molecule inhibitors, but also uncovered limitations of targeted therapy: clinical relapse and emergence of drug resistance mutations in the target gene. Identification of resistance mutations helps in better clinical management and development of next-generation inhibitors. This protocol describes a methodology to identify drug-resistant mutations in the targeted gene. This method uses a randomly m...

Proteinkinaser er centrale regulatoriske enzymer af intracellulære signaltransduktionsveje, som tilsyneladende modulerer hver cellulær funktion. En ordentlig kontrol med kinasemedieret signalering er afgørende for homeostase og udvikling, som for det meste er afhængig af en ordentlig regulering af kinaser, phosphataser og dens nedbrydning af UPS (ubiquitin proteasom system). Dereguleret kinaser er i centrum af mange kræftformer og impliceret i væld af sygdomme hos mennesker 1. Humane genom koder mere end 500 proteinkinaser, der er blevet knyttet direkte eller indirekte, til ~ 400 humane sygdomme 2. Disse observationer støttes konceptet for terapeutiske målretning af kinaser af små molekyle inhibitorer 3-5. Demonstrationen af ABL-kinase inhibitorer, såsom Imatinib, i behandlingen af kronisk myeloid leukæmi (CML), forudsat at proof of concept for denne tilgang 6,7. Denne observation ikke kun revolutionerede myrenI-kinase behandling, men også håndhæves ideen til at identificere de genetiske læsioner i andre neoplastiske sygdomme til terapeutisk målretning, som fører til opdagelsen af ​​onkogene mutationer i JAK2 fra polycytæmi vera (PV), og patienter med myeloproliferative neoplasmer (MPN). Denne opdagelse genereret stor interesse i at behandle MPNs ved at målrette JAK2 med småmolekyle-kinaseinhibitorer. Nu, næsten et dusin af JAK2 inhibitorer er i kliniske forsøg, og en af ​​dem er blevet godkendt for nylig til behandling af myelofibrose. Mens specifik målretning af onkogene kinaser af små molekyler inhibitorer i kræft bringe lovende resultat, men også lider udvikle resistens over for behandlingen. Faktisk hidtil Patienter behandlet med kinase inhibitorer, såsom Imatinib, Gefitinib, Erlotinib og Dasatinib udviklet resistens mutationer meste ved at erhverve mutationer i kinase domæne, som lægemiddelmål 8-10. Modstand som følge af genmutation fremhæver begrænsningerne of målrettet monoterapi mod onkogene kinaser, og repræsenterer den næste udfordring i udviklingen af ​​stadigt mere vellykket cancerkemoterapi. Mekanistiske og funktionelle konsekvenser af resistens bør give en begrundelse for valg og design af gratis forbindelser til lægemiddeludvikling. Mutationer identificeret via in vitro skærme, har vist en høj grad af korrelation med dem, der findes i patienter. Derfor in vitro screening for mutationer, som giver resistens over for medicinen for en given lægemiddelmål par i kliniske eller prækliniske udviklingsprogrammer hjælper med at identificere resistensmønstre, som sandsynligvis vil forårsage klinisk tilbagefald. Identifikationen af ​​disse muterede former er ikke kun nyttige i overvågningen af ​​patienter lægemiddelrespons og tilbagefald, men også afgørende for udformningen af ​​mere robuste næste generations hæmmere. For eksempel udvikling af næste generation BCR / ABL inhibitorer, Nilotinib og Ponatinib blev muliggjort på grund af større mechanistic forståelser gøres med mutagenese, strukturelle, og funktionelle studier. Tidligere har vi rapporteret resultaterne af vores skærm ved hjælp af tilfældig mutagenese af BCR / ABL at afsløre spektret af mutationer giver resistens mod inhibitorer, såsom Imatinib 11,12, PD166326 12, og AP24163 13. Resultaterne ikke blot identificeret mutationer giver klinisk resistens og sygdom tilbagefald, men også den mekanistisk forståelse af modstand og principper for kinase funktion 11,14 stof. Her giver vi yderligere metodologisk detalje, hjælp Ruxolitinib og JAK2 som et lægemiddel målpar, for at muliggøre en bredere anvendelse af denne screening strategi.

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			<td style="width:453px;">Comments/ Description</td>
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			<td height="21" style="height:21px;">Cell and Tissue culture&#160;</td>
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			<td height="21" style="height:21px;">BaF3 Cells</td>
			<td>ATCC</td>
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			<td height="21" style="height:21px;">HEK293T cells</td>
			<td>ATCC</td>
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			<td height="21" style="height:21px;">pMSCV-JAK2-V617F-puro.GW</td>
			<td>A gift from Ross Levine</td>
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			<td>Made in house</td>
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			<td height="21" style="height:21px;">pMSCV-JAK2-V617F/P58A.GW</td>
			<td>Made in house</td>
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			<td>Made in house</td>
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			<td height="21" style="height:21px;">pMSCV-JAK2-V617F/Y931C-cherry.GW</td>
			<td>Made in house</td>
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			<td height="21" style="height:21px;">pMSCV-JAK2-V617F/L983F-cherry.GW</td>
			<td>Made in house</td>
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			<td>Made in house</td>
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			<td height="21" style="height:21px;">RPMI</td>
			<td>Cellgro (corning)</td>
			<td>15-040-CV</td>
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			<td>Cellgro (corning)</td>
			<td>15-013-CV</td>
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			<td>Cellgro (corning)</td>
			<td>30-002-CI</td>
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			<td>Atlanta biological</td>
			<td>S11150</td>
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			<td height="21" style="height:21px;">Trypsin EDTA 1X</td>
			<td>Cellgro (corning)</td>
			<td>25-052-CI</td>
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			<td height="21" style="height:21px;">1XPBS</td>
			<td>Cellgro (corning)</td>
			<td>21-040-CV</td>
			<td></td>
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			<td height="21" style="height:21px;">L-Glutamine</td>
			<td>Cellgro (corning)</td>
			<td>25-005-CL</td>
			<td></td>
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			<td height="21" style="height:21px;">Puromycin</td>
			<td>Gibco (life technologies)</td>
			<td>A11138-03</td>
			<td></td>
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			<td height="21" style="height:21px;">Protamine sulfate</td>
			<td>Sigma</td>
			<td>P3369</td>
			<td>5mg/ml stock in water</td>
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			<td height="21" style="height:21px;">Trapan Blue solution (0.4%)</td>
			<td>Sigma</td>
			<td>T8154</td>
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			<td height="21" style="height:21px;">DMSO</td>
			<td>Cellgro (corning)</td>
			<td>25-950-CQC</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">INCB018424 (Ruxolitinib)</td>
			<td>ChemieTeK</td>
			<td>941678-49-5</td>
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			<td height="21" style="height:21px;">WST-1</td>
			<td>Roche</td>
			<td>11644807001</td>
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			<td height="21" style="height:21px;">0.45uM acro disc filter</td>
			<td>PALL</td>
			<td>2016-10</td>
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			<td height="21" style="height:21px;">70um nylon cell stariner</td>
			<td>Becton Dickinson</td>
			<td>352350</td>
			<td></td>
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			<td height="21" style="height:21px;">Bacterial Culture</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">XL-1 red E.Coli cells</td>
			<td>Agilent Tech</td>
			<td>200129</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">SOC</td>
			<td>New England Biolabs</td>
			<td>B90920s</td>
			<td></td>
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			<td height="21" style="height:21px;">Ampicillin</td>
			<td>Sigma</td>
			<td>A0166</td>
			<td>100mg/ml stock solution&#160;</td>
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			<td height="21" style="height:21px;">Bacto agar</td>
			<td>Difco</td>
			<td>214050</td>
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			<td height="21" style="height:21px;">Terrific broth</td>
			<td>Becton Dickinson</td>
			<td>243820</td>
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			<td height="21" style="height:21px;">Agarose</td>
			<td>Genemate</td>
			<td>E-3119-500</td>
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			<td height="21" style="height:21px;">Kits</td>
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			<td></td>
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			<td height="21" style="height:21px;">Dneasy Blood&#38; tissue kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Expand long template PCR system</td>
			<td>Roche</td>
			<td>1168134001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wizard Sv gel and PCR clean up system</td>
			<td>Promega</td>
			<td>A9282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pure Yield plasmid mini prep system</td>
			<td>Promega</td>
			<td>A1222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR Cloning System with Gateway Technology with pDONR 221 &#38; OmniMAX 2 Competent Cells</td>
			<td>Invitrogen</td>
			<td>12535029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gateway&#160; LR Clonase Enzyme mix&#160;</td>
			<td>Invitrogen</td>
			<td>11791019</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vivo-Glo Luciferin in-vivo Grade</td>
			<td>Promega</td>
			<td>P1043</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">1/2cc Lo-Dose u-100 insulin syringe 28 G1/2</td>
			<td>Becton Dickinson</td>
			<td>329461</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Mortor pestle</td>
			<td>Coor tek&#160;</td>
			<td colspan="2">60316 and 60317</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflorane (Isothesia TM)</td>
			<td>Butler Schien</td>
			<td>29405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NAPCO series 8000 WJ CO2 incubator</td>
			<td>Thermo scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Swing bucket rotor centrifuge 5810R</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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

Opdagelsen af ​​BCR / ABL som drivkraft onkogen i kronisk myeloid leukæmi (CML) resulterede i udviklingen af ​​Imatinib, som i virkeligheden vist potentiale målrette kinasen i kræft ved effektivt at behandle CML-patienter. Denne observation revolutioneret lægemiddeludvikling at målrette onkogene kinaser impliceret i forskellige andre maligniteter, såsom EGFR, B-RAF, KIT og PDGFRs. En stor ulempe ved anti-kinase behandlinger er imidlertid fremkomsten af ​​lægemiddel-resistente mutationer der gør målet at have reduceret eller mistet affinitet for lægemidlet. Forståelse af de mekanismer, der er ansat af resistente varianter ikke kun hjælper med at udvikle den næste generation af inhibitorer, men giver også skub i den kliniske behandling ved hjælp af skræddersyet medicin. Vi rapporteret en retroviral vektor baseret screening strategi for at identificere spektret af resistens tillægger mutationer i BCR / ABL, som har hjulpet med at udvikle den næste generation BCR / ABL inhibitorer. Brug Ruxolitinib og JAK2 som et mål lægemiddel par, her beskriver vi in vitro screeningsmetoder, der udnytter muse BaF3 celler, der udtrykker tilfældig mutation bibliotek af JAK2 kinase.

Fremkomsten af ​​genetiske mutationer udgør stor udfordring for den målrettede anti kinase terapi. Mutations undersøgelser, foruden at give mekanistiske og funktionelle indsigter, der er medvirkende til udvælgelse og design af næste generation af lægemiddeludvikling, også giver en bedre klinisk ledelse og kan i fremtiden være mere nyttigt for personlig behandling. I dette forsøg viser vi screening for ruxolitinib resistens mutationer i JAK2-V617F kinase (Figur 1). Vi konstruerede pMSCV-JAK2...

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

Fremkomsten af ​​genetisk resistens over for kinase hæmmere udgør en betydelig udfordring for effektiv kræftbehandling. Identifikation og karakterisering af resistente mutationer mod en nyudviklet lægemiddel hjælper med bedre kliniske håndtering og næste generation drug design. Her beskriver vi vores protokol for in vitro screening og validering af resistente mutationer.

Fremgangsmåde til screening og validering af resistente mutationer Against kinaseinhibitorer

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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We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

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