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.

NOTE: All procedures in this protocol were conducted according to the National Institute of Health guidelines for the ethical treatment and care of animals, and according to an approved IACUC animal use protocol.
1. Cell Line Maintenance
<ol>
	<li>Culture BAF3 cells in RPMI-1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 units/ml and 100 &#181;g/ml) and spent culture medium of Wehi Cells. Grow HEK293T cells in DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 units/ml and 100 &#956;g/ml). Maintain cells at 37 &#176;C in a humidified atmosphere containing 5% CO2.</li>
</ol>
2. Plasmid Construction
<ol>
	<li>Clone the mouse JAK2 and its oncogenic isoform JAK2-V617F in pMSCV-puro-GW by LR clonase using standard recombination cloning procedure.</li>
	<li>For the construction of luciferase expression vector (pMSCV-Luc-Cherry-GW) used in in-vivo imaging, amplify luciferase from pLVX-Tet-ON vector by PCR using primers (LUC-SD/RP TTACAATTTGGACTTTCCG and LUC-SD/FP-CACCATGGAAGACGCCAAAAAC) followed with cloning in pENTR-SD-TOPO to generate pENTR-Luc, which is used to transfer the Luciferase gene in retroviral vector, pMSCV-Cherry-GW by recombination mediated cloning using LR clonase.</li>
</ol>
3. Preparation of Random Mutation Library, Screening and Identifying the Mutations
3.1) Random Mutagenesis
<ol>
	<li>Clone full length of JAK2 wild type and the V617F mutation into pMSCV-puro-GW retroviral vector using a commercial recombination cloning technology.</li>
	<li>Use 1 &#181;g of JAK2-V617F plasmid DNA to transform, XL-1 Red, E. coli cells, which is defective in DNA repair mechanisms thus allowing them to incorporate random mutations during replication. More specifically, mix 50 - 100 ng of DNA (more than 100 ng of DNA decreases transformation efficiency) with 100 &#181;l of competent cells in a pre-chilled polypropylene tube and incubated on ice for 30 min, gently swirling every 5 min. For a good library coverage, use four to six tubes of competent cells. 
	NOTE: More than 100 ng of DNA decreases transformation efficiency</li>
	<li>Immerse the tube in a water bath at 42 &#176;C for a heat shock of 45 sec and incubate on ice for 2 min. Next, add 1 ml of SOC medium (2.0% tryptone, 0.5% yeast extract, 0.05% NaCl, 10 mM MgCl2, 10 mM MgSO4, and 0.4% D-glucose). Incubate the tube at 37 &#176;C while shaking at 225-250 rpm for 90 min.</li>
	<li>Plate cells from each tube on four 10 cm LB-agar plates containing 100 &#181;g/ml ampicillin. Incubate the plates for 16 - 24 hr at 37 &#176;C.</li>
	<li>Following visible colonies, collect them by scraping the plates with a sterile plate scraper. Pool cells from each plate and isolate plasmid DNA using a commercial plasmid extraction kit. 
	NOTE: Usually colonies are smaller and take 18-24 hr to be visible because these are slow growing bacterial strains. At this stage, assess the heterogeneity of mutations in the library by restriction digestion with a frequent cutter such as Sau3A1 or Taq1 or sequencing.</li>
</ol>
3.2) Production of Retroviral Supernatants and Transduction
<ol>
	<li>One day before transfection, plate 4 x 106 HEK 293T cells onto 100 cm dishes in DMEM containing 10% FCS, penicillin/streptomycin and 2 mM L-glutamine.</li>
	<li>Next day, replace the medium and transfect the cells with mutagenized pMSCV-JAK2-V617F library. For each 10 cm plate, mix 10 &#181;g of DNA (5 &#181;g of the plasmid library and 5 &#181;g of retroviral packaging plasmid, pCLEco) with 40 &#181;l of FuGENE to a total volume of 400 &#181;l using serum free DMEM media. Incubate the DNA and lipid mix for 20 min at room temperature. After 20 min add the DNA-lipid complex drop wise on top of HEK293T cells.</li>
	<li>After six hr, change the transfection media with fresh media and incubate the plates for 48 hr at 37 &#176;C for virus production. After 48 hr of incubation, collect viral supernatants filtered through a 0.45 &#181;m acrodisc filter.</li>
</ol>
3.3) Selection of Resistant Clones In Vitro
<ol>
	<li>For drug resistant JAK2-V617F mutants, transduce 100 million BAF3 cell with 100 ml of virus supernatants having a viral titer of 0.5-1 x106. More specifically, mix 108 cells with 100 ml of virus supernatant to a total volume of 300 ml using RPMI media, containing 10% serum and 24 &#181;g/ml of polyberene (final concentration of ployberene is 8 &#181;g/ml). Distribute these cell mixers in multiple six well plates (4-5 ml per well).</li>
	<li>Centrifuge the plates at 1,250 x g for 90 min at 25 &#176;C. After centrifugation, incubate the plates at 37 &#176;C for 24 hr.</li>
	<li>Next day, pool the cells together and mix with Ruxolitinib and medium containing soft agar and plated in six well plates. For each drug concentration, mix 10 ml of transduced BAF3 cells (10-20 million cells) with the appropriate amount of Ruxolitinib in a 50 ml tube. Make up the volume to 40 ml using RPMI containing 20% serum. Add 10 ml of 1.2 % agar to the cells, mixcarefully and plate in six well plates.</li>
	<li>Incubate the plates at 37 &#176;C for two weeks. Pick the resistant colonies using 0.2 ml pipette tips and sub-culture them individually in 24 well plates for 3-4 days.</li>
	<li>Harvest the resistant BAF3 cells by centrifugation at 450 x g for five min at room temperature. Isolate the genomic DNA using a commercial genomic DNA isolation kit. PCR amplify full length JAK2 cDNA from 100 ng of genomic DNA using primers (mJAK2FP-ATGGCCTGCCTTACAATGACAG and mJAK2RP-GGTTCTGGAAGTCTGCTTGG) and long-template expand high fidelity PCR systems.</li>
	<li>Separate the PCR products by agarose gel electrophoresis. Excise the 3.4 kb of DNA band representing JAK2 coding frame and isolate the DNA using a commercial gel extraction kit. Sequence the full length of cDNA using 8 internal primers (P1 - P8) spanning the coding sequence and analyze sequences using commercial software.</li>
</ol>
4. In Vitro Validation of Resistant Mutations
Many cell clones carry more than one mutation, To test the contribution of each mutation in resistance phenotype, generate selected variants by site-directed mutagenesis using pMSCV-JAK2V-617F plasmid as template.
<ol>
	<li>Perform site directed mutagenesis on pMSCV-JAK2-V617F plasmid using a commercial mutagenesis kit and oligonucleotides designed to create point mutations. Confirm the identities of mutant clones by sequencing.</li>
	<li>Transduce BAF3 cells with retroviruses made using mutant plasmids followed with selection using puromycin at 1&#181;g/ml.</li>
	<li>Plate 104 BAF3-JAK2-V617F cells (50 &#181;l of RPMI with 10% FCS) into each well of a 96 well plate. Separately add 50 &#181;l of the ruxolitinib containing media to the wells across the plate (final concentration: 0, 1, 3, 5, 10, and 20 &#181;M), and incubate the plates for 60 hr at 37 &#176;C.</li>
	<li>Assess cell viability by adding 10 &#181;l of WST-1 reagent to each well followed with incubation at 37&#176;C for 2-4 hr. Record absorbance at A450 using a plate reader. Perform all assays in triplicate and plot averaged absorbance against INCB018424 concentrations. Perform a best-fit sigmoidal curve using a nonlinear curve fitting algorithm. Score the drug concentration resulting in 50% cell viability as the cellular IC50.</li>
	<li>Next, plate six million BAF3 cells expressing JAK2 variants in six well plates in RPMI medium containing 10% serum with increasing concentrations of ruxolitinib and incubated for 4&#8211;6 hr at 37 &#176;C.</li>
	<li>Collect the cells by centrifugation at 450 x g for five min at 4 &#176;C. Wash the cells one time with PBS, and lyse in 20 mM Tris-Cl (pH 7.5), 50 mM NaCl, 1% NP-40, 0.1% SDS, 5 mM EDTA, 1mM EGTA, 1 mM sodium fluoride, 2 mM Sodium Vanadate, and 5% Glycerol supplemented with protease inhibitor cocktail and phosphatase inhibitors. Sonicate the cell suspension for 1 min followed by addition of 5x gel loading buffer (350 mM Tris-HCl [pH 6.8], 500 mM DTT, 15% SDS, 10 mM Benzamidine, 5 mM EDTA, 5 mM EGTA, 5 mM Sodium Vanadate, Protease cocktail, 50% Glycerol, and 0.001% Bromophenol blue) and denaturation at 70 &#176;C for 5&#8211;10 min.</li>
	<li>Resolve the proteins on a 8% SDS polyacrylamide gel under denaturing conditions. Perform immunoblotting with mouse monoclonal anti-phopsho STAT5. Strip the blots and reprobed with a rabbit polyclonal anti-SATA5 antibody. Visualize the bands using ECL reagents according to the supplier&#8217;s recommendation.</li>
</ol>
5. In Vivo Validation
<ol>
	<li>Transduce BAF3 cells expressing luciferase and cherry (transduced with retroviruses made from pMSCV-Luc-cherry GW) with viruses expressing JAK2-V617F and resistant variants. Select cells that survive puromycin selection for the expression of JAK2 and its resistance variants.</li>
	<li>Inject two million actively growing BAF3-Luc/cherry cells carrying JAK2-V617F and its resistant variants in 200 &#181;l of PBS to 6 Balb/C mice via tail-vein injection.</li>
	<li>After three days of cell transplantations, inject the mice with Ruxolitinib (100 mg/kg) twice daily for two weeks.</li>
	<li>Perform luciferase-based bioluminescence imaging with an imaging system. .
	<ol>
		<li>In brief, prepare luciferin solution by dissolving 300 mg to 25 ml of sterile deionized water, aliquot in smaller volumes and store. 
		NOTE: Store luciferin at -80 &#176;C and protect from light until use.</li>
		<li>Inject 250 &#181;l to each mouse by IP to achieve 125 mg/kg in each mouse. Anesthetize the mice from same group together using inhaled isoflurane (1.5%-2.0%) in a sealed box. Apply vet ointment on the eye to prevent dryness. 
		NOTE: The time taken for mice to sleep (10-15 min) allowed luciferin activity to peak. Keep the time consistent between the groups to avoid variation.</li>
	</ol>
	</li>
	<li>Transfer the mice immediately to imaging chamber stage and maintain anesthesia at 1.5-2% isofluorane during imaging procedures. Image mice with sequential 0.1, 0.5, 1, 5, 30, 60, and 300 sec exposure. Capture images and quantitate bioluminescence intensity using image acquisition and analysis software. Following imaging return the mice back to its cage.</li>
	<li>For, in vivo chimerism harvest total bonemarrow cells. Euthanize the mice with CO2 for 5 min followed with cervical dislocation. Harvest the bones and crush in 4 ml cold PBS to collect the bone marrow. Analyze the percent cherry positive cells under fluorescence microscope and quantify using FACS. Collect twenty thousand events from each sample.</li>
</ol>

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<li>Gorre, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+resistance+to+STI-571+cancer+therapy+caused+by+BCR-ABL+gene+mutation+or+amplification%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification.</a> Science. 293, 876-880 (2001).</li>
<li>Shah, N. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiple+BCR-ABL+kinase+domain+mutations+confer+polyclonal+resistance+to+the+tyrosine+kinase+inhibitor+imatinib+%28STI571%29+in+chronic+phase+and+blast+crisis+chronic+myeloid+leukemia%5BTitle%5D)+AND+%22Cancer+Cell%22%5BJournal%5D)">Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia.</a> Cancer Cell. 2, 117-125 (2002).</li>
<li>Branford, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+frequency+of+point+mutations+clustered+within+the+adenosine+triphosphate-binding+region+of+BCR%2FABL+in+patients+with+chronic+myeloid+leukemia+or+Ph-positive+acute+lymphoblastic+leukemia+who+develop+imatinib+%28STI571%29+resistance%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance.</a> Blood. 99, 3472-3475 (2002).</li>
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No conflicts of interest declared.

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 mutagenized plasmid library built in E. coli, to express the mutant proteins. The library is then introduced in BAF3 cells (susceptible to transformation by an oncogene), followed with selection of cell clones in the presence of chemotherapeutic agents. Sequencing of targeted gene from resistant clones identifies mutations conferring resistance. Finally, identified mutations are recreated by site directed mutagenesis to validate them for drug resistance.
Using this strategy we defined the spectrum of mutations in JAK2 and BCR/ABL conferring resistance to Ruxolitinib and Imatinib, respectively. We identified more than hundred mutations in BCR/ABL. Besides, identifying all major mutations detected in patients, our screening method allowed us to identify novel substitutions of residues both within and beyond the kinase domain. Interestingly, all mutations from our screen have been identified in patient samples but this process took almost more than 10 years 15, thus demonstrating the ability of this screen to anticipate mutations that will pose clinically problematic drug resistance. While resistant screening using random mutagenesis provides many advantages in comparison to other methods, there are potential pitfalls to be aware of when following a screening procedure. For any screening, it is strongly recommended for BAF3 cells to be fully dependent on the target against which resistance is sought. A failure or partial dependence on the target gene may not develop true resistant clones and most likely develop false positives. For instance, four different studies have performed resistant screening against JAK2 inhibitors using BAF3 cells transduced with either EPO or MPL receptors to facilitate JAK2-V617F dependence 16-19. Only eight resistant mutations limited to kinase domain were identified from these screens, although numerous resistant clones developed that lacked mutations, presumably due to emergence of false positives, which was attributed to heterodimerization of JAK1 and JAK3 with cytokine receptors. Therefore, to avoid loss of JAK2 dependence we carried out resistant screening in plain BAF3 that showed robust selection of resistant clones against Ruxolitinib and all of these clones showed presence of resistant mutations. Based on these observations, in order to harness the full spectrum of resistant mutations, we recommend that the BaF3 cells should be fully dependent on the targeted kinase to avoid the emergence of false positives, as has been a norm in previous resistant screenings performed against JAK2 inhibitors16,17. Additionally, it is critical to avoid bulk culture conditions in which cells harboring different mutations are pooled together and allowed to expand in liquid culture, since this can lead to clonal dominance of a few highly drug resistant variants. For instance, during initial selection for imatinib-resistance of mutagenized BCR-ABL in bulk liquid culture, we found only 4 mutant forms that were represented multiple times within the first 100 isolates we sequenced. Therefore, screening using soft agar allows slow growing clones with more modest degrees of drug resistance to be recovered that would not be identified using bulk culture. For this reason, it is recommended to grow bulk culture prior to selection only for 14 to 16 hr, followed with drug selection without IL3 in soft agar. In our experience, growing the cultures beyond 36 hr after viral transduction tend to be dominated by highly proliferative clones, which poses a risk to lose slow growing clones (weakly transformative). Likewise, using cells from an earlier time points such as 6-8 hr after viral transduction are prone to select for highly transformative or overexpressing clones, thus causing a bias and misrepresentation in the identity and frequency of resistance clones. Therefore, we recommend using cells grown for 14-16 hr post viral transduction for screening, which provides tight selection of individual clones and prevents clonal dominance typically observed in bulk liquid cultures.
It is essential to confirm a candidate mutation&#8217;s ability to confer resistance in isolation, since some clones were identified to have multiple mutations. For the JAK2-V617F screen, we recreated a majority of the mutations identified from our random mutagenesis library using site directed mutagenesis. After introducing the isolated mutants into fresh cells, they were then grown in the presence of various drugs to determine IC50s for each mutant. We also tested two different resistant variants for in vivo resistance by monitoring the bioluminescence to further confirm the ability of these single mutations to cause drug resistance in vivo.
Given the success of anti-kinase therapy in treating cancers that galvanized a surge of kinase inhibitors in clinics, it is crucial to identify the clinically problematic resistance conferring mutations for better patient management and developing drugs that can target them. This screening strategy has been successfully used to identify mutations in the BCR/ABL, JAK2 and FLT3 20; however, we believe that it will be generally applicable to a broad range of anti-neoplastic agents.

Protein kinases are key regulatory enzymes of intracellular signal transduction pathways that seemingly modulate every cellular function. A proper control of kinase mediated signaling is crucial to homeostasis and development, which mostly relies on proper regulation of kinases, phosphatases and its degradation by UPS (ubiquitin proteasome system). Deregulated kinases are at the center stage of many cancers and implicated in host of human diseases 1. Human genome encodes more than 500 protein kinases that have been linked, directly or indirectly, to ~400 human diseases 2. These observations supported the concept for therapeutic targeting of kinases by small molecule inhibitors 3-5.
The demonstration of ABL kinase inhibitors, such as Imatinib, in the treatment of chronic myeloid leukemia (CML) provided the proof of concept for this approach6,7. This observation not only revolutionized the anti-kinase therapy but also enforced the idea to identify the genetic lesions in other neoplastic diseases for therapeutic targeting, which lead to discovery of oncogenic mutations in the JAK2 from polycythemia vera (PV) and patients with myeloproliferative neoplasms (MPN). This discovery generated great interest in treating MPNs by targeting JAK2 with small molecule kinase inhibitors. Now, almost a dozen of JAK2 inhibitors are in clinical trials and one of them has been approved recently for the treatment of myelofibrosis. While specific targeting of oncogenic kinases by small molecule inhibitors in cancers bring promising outcome, it also suffers from developing resistance to the treatment. In fact, so far, patients treated with kinase inhibitors, such as Imatinib, Gefitinib, Erlotinib and Dasatinib developed resistance mutations mostly by acquiring mutations in the kinase domain to which drug targets 8-10. Resistance as a result of gene mutation highlights the limitations of targeted monotherapy against the oncogenic kinases, and represents the next challenge in the development of ever more successful cancer chemotherapy. Mechanistic and functional consequences of drug resistance should provide a rationale for selection and design of complimentary compounds for drug development. Mutations identified via in vitro screens, have shown a high degree of correlation with those found in patients. Therefore, in vitro screening for mutations that confer drug-resistance for a given drug target pairs in clinical or preclinical development assists in identifying the resistance patterns that are likely to cause clinical relapse. The identification of these mutant forms is not only helpful in monitoring the patients for drug response and relapse but also essential for the design of more robust next generation inhibitors. For instance, development of next generation BCR/ABL inhibitors, Nilotinib and Ponatinib, were made possible because of greater mechanistic understandings gained from mutagenesis, structural, and functional studies.
Earlier, we have reported the results of our screen using random mutagenesis of BCR/ABL to reveal the spectrum of mutations conferring resistance to inhibitors such as Imatinib11,12, PD16632612, and AP2416313. The results not only identified the mutations conferring clinical resistance and disease relapse, but also provided the mechanistic understanding of drug resistance and principles governing the kinase function11,14. Here we provide additional methodological detail, using Ruxolitinib and JAK2 as a drug target pair, to enable a broader application of this screening strategy.

<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

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 potential of targeting the kinase in cancers by effectively treating the CML patients. This observation revolutionized drug development to target the oncogenic kinases implicated in various other malignancies, such as, EGFR, B-RAF, KIT and PDGFRs. However, one major drawback of anti-kinase therapies is the emergence of drug resistance mutations rendering the target to have reduced or lost affinity for the drug. Understanding the mechanisms employed by resistant variants not only helps in developing the next generation inhibitors but also gives impetus to clinical management using personalized medicine. We reported a retroviral vector based screening strategy to identify the spectrum of resistance conferring mutations in BCR/ABL, which has helped in developing the next generation BCR/ABL inhibitors. Using Ruxolitinib and JAK2 as a drug target pair, here we describe in vitro screening methods that utilizes the mouse BAF3 cells expressing the random mutation library of JAK2 kinase.

Emergence of genetic mutations poses great challenge for the targeted anti kinase therapy. Mutational studies, besides providing mechanistic and functional insights that are instrumental in selection and design of next-generation drug development, also allows better clinical management and may in future be more helpful for personalized treatment. In this experiment, we show screening for ruxolitinib resistance mutations in JAK2-V617F kinase (Figure 1). We constructed pMSCV-JAK2-V617F-cherry.gateway vector by introducing the full-length mouse JAK2-V617FcDNA into pMSCV-Cherry-GW. It is recommended to use bacterial host (XL-1 red strain of E. coli) to develop random mutations over the most popular choice of method, which is PCR, due to its limitations associated with sequence bias and larger gene fragments are difficult to amplify. Randomly mutagenized DNA library was transfected to HEK293T cells for retrovirus production. These mutant viruses were used to, transduce the BAF3 cells that were selected for colony growth in soft-agar in the absence of IL-3 with either 1 or 5 &#181;M of Ruxolitinib. Under these conditions, colonies arise only from cells carrying JAK2V-617F cDNAs that expresses functional and resistant variant of the kinase. After 10 - 14 days, well-separated individual colonies were picked, which varied in size, and expanded them in liquid culture. Genomic DNA was isolated from these cells. The provirus was recovered by direct rescue or by PCR. The recovered proviruses were sequenced to identify mutations (Figure 1). Sequence analyses were performed using DNASTAR package of Lasergene.
Because many cell clones carried more than one mutation, we strongly recommend validating these mutations in isolation by both in vitro and in vivo assays (Figures 2 and 3). To verify the activity of variants in isolation, selected variants were generated by site directed mutagenesis of the JAK2V-617F sequence. These variants were reintroduced into BAF3 cells, and measured for cell proliferation ability at different dosage of Ruxolitinib to evaluate IC50 (IC50 values, Figure 2A). Biochemical assays are performed for phosphotyrosine or phospho-STAT5 by immunoblot analysis on protein lysates of BAF3 cells that were incubated with increasing doses of Ruxolitinib to rule out any off target mediated resistance (Figure 2B). Mutants exhibiting enhanced IC50 values and persistent autophosphorylation at higher Ruxolitinib concentrations are thus confirmed to be drug resistant variants. Because many resistant mutations show variable dose response, therefore it is imperative to test whether these mutations will confer in vivo resistance as well. Usually, we recommend to test only 2 -3 different variants for in vivo experiments, as they are expensive and laborious. To validate in vivo resistance, BAF3 cells were engineered to express JAK2-V617F variants and Luciferase/cherry to enable in vivo tracking. Mice were injected with 1-2 million cherry positive cells (expressing JAK2 variants and luciferase), followed by Ruxolitinib injection for two weeks. After two weeks, mice were imaged for luciferase-catalyzed bioluminescence (Figure 3), and also for BAF3 chimerism in bonemarrow and peripheral blood by measuring the fluorescence of cherry positive cells (Figure 3). Mice expressing JAK2-V617F are sensitive to Ruxolitinib, while resistant variants show progressive increment in bioluminescence over the period of treatments, thus suggesting that BAF3 cells expressing JAK2-V617F variant is resistant to Ruxolitinib treatment in vivo.
<img alt="Figure 1" src="/files/ftp_upload/51984/51984fig1highres.jpg" /> 
Figure 1: A scheme for screening the resistance mutations against kinase inhibitor Ruxolitinib.
<img alt="Figure 2" src="/files/ftp_upload/51984/51984fig2highres.jpg" /> 
Figure 2: A scheme showing in vitro validation using dose dependent cell proliferation assays (A) and western blotting (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51984/51984fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 3" src="/files/ftp_upload/51984/51984fig3highres.jpg" /> 
Figure 3: A schematic representation of in vivo validation using luciferase catalyzed bioluminescence measurement. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51984/51984fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

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

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Biology

14.8K Views.  Cincinnati Children's Hospital Medical Center. Emergence of genetic resistance against kinase inhibitor therapy poses significant challenge for effective cancer therapy. Identification and characterization of resistant mutations against a newly developed drug helps in better clinical management and next generation drug design. Here, we describe our protocol for in vitro screening and validation of resistant mutations.

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.

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 ...

Biochemistry

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.

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

Cancer Research

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.

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.

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.

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.

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.

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

Medicine

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.

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.

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 ...

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.

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

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

Summary

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