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

Summary

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.

Abstract

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.

Introduction

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 approach^6,7. 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 Imatinib^11,12, 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 function^11,14. Here we provide additional methodological detail, using Ruxolitinib and JAK2 as a drug target pair, to enable a broader application of this screening strategy.

Protocol

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

1. Culture BAF3 cells in RPMI-1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 units/ml and 100 µg/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 μg/ml). Maintain cells at 37 °C in a humidified atmosphere containing 5% CO2.

2. Plasmid Construction

1. Clone the mouse JAK2 and its oncogenic isoform JAK2-V617F in pMSCV-puro-GW by LR clonase using standard recombination cloning procedure.
2. 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.

3. Preparation of Random Mutation Library, Screening and Identifying the Mutations

3.1) Random Mutagenesis

1. Clone full length of JAK2 wild type and the V617F mutation into pMSCV-puro-GW retroviral vector using a commercial recombination cloning technology.
2. Use 1 µg 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 µl 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
3. Immerse the tube in a water bath at 42 °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 °C while shaking at 225-250 rpm for 90 min.
4. Plate cells from each tube on four 10 cm LB-agar plates containing 100 µg/ml ampicillin. Incubate the plates for 16 - 24 hr at 37 °C.
5. 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.

3.2) Production of Retroviral Supernatants and Transduction

1. 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.
2. Next day, replace the medium and transfect the cells with mutagenized pMSCV-JAK2-V617F library. For each 10 cm plate, mix 10 µg of DNA (5 µg of the plasmid library and 5 µg of retroviral packaging plasmid, pCLEco) with 40 µl of FuGENE to a total volume of 400 µl 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.
3. After six hr, change the transfection media with fresh media and incubate the plates for 48 hr at 37 °C for virus production. After 48 hr of incubation, collect viral supernatants filtered through a 0.45 µm acrodisc filter.

3.3) Selection of Resistant Clones In Vitro

1. 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 µg/ml of polyberene (final concentration of ployberene is 8 µg/ml). Distribute these cell mixers in multiple six well plates (4-5 ml per well).
2. Centrifuge the plates at 1,250 x g for 90 min at 25 °C. After centrifugation, incubate the plates at 37 °C for 24 hr.
3. 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.
4. Incubate the plates at 37 °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.
5. 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.
6. 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.

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.

1. 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.
2. Transduce BAF3 cells with retroviruses made using mutant plasmids followed with selection using puromycin at 1µg/ml.
3. Plate 10⁴ BAF3-JAK2-V617F cells (50 µl of RPMI with 10% FCS) into each well of a 96 well plate. Separately add 50 µl of the ruxolitinib containing media to the wells across the plate (final concentration: 0, 1, 3, 5, 10, and 20 µM), and incubate the plates for 60 hr at 37 °C.
4. Assess cell viability by adding 10 µl of WST-1 reagent to each well followed with incubation at 37°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.
5. 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 4–6 hr at 37 °C.
6. Collect the cells by centrifugation at 450 x g for five min at 4 °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 °C for 5–10 min.
7. 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’s recommendation.

5. In Vivo Validation

1. 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.
2. Inject two million actively growing BAF3-Luc/cherry cells carrying JAK2-V617F and its resistant variants in 200 µl of PBS to 6 Balb/C mice via tail-vein injection.
3. After three days of cell transplantations, inject the mice with Ruxolitinib (100 mg/kg) twice daily for two weeks.
4. Perform luciferase-based bioluminescence imaging with an imaging system. .
   1. 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 °C and protect from light until use.
   2. Inject 250 µl 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.
5. 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.
6. 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.

Results

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 vecto...

Discussion

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...

Materials

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