The authors wish to thank Dr. Veronica Zanoni for editing the manuscript.

NOTE: This protocol has been specifically adjusted for the analysis of the mechanisms underlying acquired resistance to trastuzumab of HER-positive gastric cancer cell lines. All laboratory instruments needed are reported in the Table of Materials.
1. Generation of Targeted Therapy Resistant Subclones
NOTE: This is the most critical and difficult to standardize step in the protocol due to the fact that different cell lines may exhibit different sensitivities to the targeted therapeutic inhibitor independently from their tumor histotype or drug concentration used.
<ol>
	<li>Culture NCI-N87 cell line in RPMI medium supplemented with fetal calf serum (10%) and glutamine (2 mM, 1%), as a monolayer at 37 &#176;C, and seed it onto 25 cm2 culture flasks.</li>
	<li>Add to the culture medium in each flask 10 &#181;g/mL of trastuzumab as the starting dose. Expose the cells to the starting dose until they resume growing. 
	NOTE: The starting dose of the targeted therapy inhibitor should be 10-fold lower than its plasma peak concentration. Plasma peak concentration is the highest level of drug concentration detected in patient blood usually following multiple doses of standard therapy. This value can be retrieved from studies on pharmacokinetics.</li>
	<li>Monitor cell growth with the trypan blue exclusion method; when cell growth is resumed (typically after a period of two to three weeks), expose cells to a 10-fold higher dose concentration of trastuzumab.</li>
	<li>Expose cells to a gradually increasing dose of trastuzumab (from 100 &#181;g/mL to 400 &#181;g/mL) until cells keep growing (after about 2 weeks) despite the presence of a high drug concentration (at least 2.5 -&#160;4-fold higher than the plasma peak).</li>
	<li>Perform a clonogenic assay to evaluate the level of resistance to targeted therapy at every change in the concentration of the target therapy inhibitor in the culture medium, and define relative resistance IC50 index (RR IC50) as follows.
	<ol>
		<li>Seed 500 cells in 24 multi-well culture plates. 
		NOTE: 5 wells must be prepared for each condition set.</li>
		<li>Expose cells to increasing target therapy inhibitor (trastuzumab) concentrations from 100 &#181;g/mL up to 400 &#181;g/mL.</li>
		<li>Incubate cells in a CO2 incubator at 37 &#176;C for 15 days.</li>
		<li>After incubation, fix and stain samples as follows.
		<ol>
			<li>Remove medium and rinse cells with 10 mL 1x PBS. Remove&#160;1x PBS&#160;and add 2 - 3 mL of fixation solution (acetic acid:methanol, 1:7, (vol/vol)). Remove the fixation solution and add 0.5% crystal violet solution. Incubate at room temperature for 2 h</li>
			<li>Remove the crystal violet solution carefully by aspirating with a pipette and immerse plates in tap water to rinse off any crystal violet solution residues. Air-dry at room temperature for up to 2 days.</li>
		</ol>
		</li>
		<li>Count colonies of more than 50 cells using an inverted microscope (4X magnification). 
		NOTE: Two independent observers are needed for this.</li>
		<li>Calculate RR IC50 as the ratio between the IC50 value of target-therapy-resistant subclone cells and the value of the original/na&#239;ve cells.</li>
	</ol>
	</li>
</ol>
2. Validation Protocol for the Candidate Targeted Therapy Resistance Gene (R-gene)
NOTE: After the characterization of gene expression, signatures associated with the acquired resistance to target therapy and any candidate gene as driver of resistance will be investigated through: a) RT-PCR, and b) Western blot analysis.
<ol>
	<li>Real-time RT-PCR
	<ol>
		<li>Extract total RNA from cell lines using acid guanidinium thiocyanate-phenol-chloroform mixture (see Table of Materials).</li>
		<li>Perform reverse transcription (RT) reactions using random hexamer primers and set the thermal cycler as follows: priming at 25 &#176;C for 5 min, RT at 46 &#176;C for 20 min, and RT inactivation at 95 &#176;C for 1 min.</li>
		<li>Use 400 ng of total RNA for a 20 &#181;L reaction. Prepare the reaction mixture for sample as follows: 7 &#181;L RNase free H2O, 2 &#181;L cDNA (10 ng/&#181;L), 1 &#181;L 20x fluorescent-labeled target-specific probe, and 10 &#181;L 2x Mix containing buffer, dNTPs, and DNA polymerase (see Table of Materials).</li>
		<li>Add 20 &#181;L of the reaction mixture to each well plate and run the following program: holding stage at 50 &#176;C for 2 min, at 95 &#176;C for 10 min (1 cycle); then 40 cycles at 95 &#176;C for 15 s and at 60 &#176;C for 1 min.</li>
	</ol>
	</li>
	<li>Western blot
	<ol>
		<li>Recover cell suspensions by adding 2 mL of 0.05% trypsin/EDTA solution and allowing 2 - 3 min for trypsin to work.</li>
		<li>Add 2 volumes of medium containing 10% fetal bovine serum to neutralize trypsin (e.g., 2 mL of medium for each 1 mL of trypsin) and centrifuge at 1,200 x g for 5 min. Discard supernatant and wash cells with cold 1x PBS.</li>
		<li>Centrifuge at 1,200 x g for 5 min and discard supernatant. Resuspend the cell pellet with lysis buffer and incubate on a rocker at 4 &#176;C for 30 min.
		<ol>
			<li>The amount of lysis buffer depends on the number of cells (e.g., 100 &#181;L for 1 x 106 cells); make sure to prepare fresh lysis buffer each time. For 1 mL of lysis buffer preparation (leave on ice): use 975 &#181;L M-PER Mammalian lysate buffer, 15 &#181;L protease and phosphatase inhibitors, and 10 &#181;L 100 &#181;M PMSF inhibitors.</li>
		</ol>
		</li>
		<li>Recover the supernatant protein by cell lysate centrifugation at 13,000 x g at 4 &#176;C for 15 min.</li>
		<li>Determine the protein concentrations by using a BCA protein assay.</li>
		<li>Add 4x LaemmLi Sample Buffer to protein samples (at least 20 - 30 &#181;g of protein) and heat at 100 &#176;C for 3 min.</li>
		<li>Use precast 4 - 20% gels and load 20 - 30 &#181;g of LaemmLi protein solution and 5 - 10 &#181;L of protein standard per sample.</li>
		<li>Fill the Western blot ring with TRIS/GLICYNE /SDS 1x buffer and run gel at 110 V for 30 - 60 min (when time stops check that the dye has reached the end of the gel, otherwise run for a few additional minutes)</li>
		<li>Electroblot to transfer protein into a PVDF membrane (see Table of Materials) by using a semi-dry system.</li>
		<li>Block the membrane by soaking in the appropriate protein solution (milk/BSA 5%) on the shaker at room temperature for 1 h.</li>
		<li>Make a 1:400 solution with anti-IQGAP1 primary antibody in a 5% milk solution (see Table of Materials).</li>
		<li>Place the membrane in primary antibody solution on a shaker in a cold room overnight.</li>
		<li>The following day, discard the antibody solution and soak in T-TBS 1x&#160;solution (1x&#160;T-TBS: 1x&#160;Tris Saline Buffer added by Tween20 0.1%) on a shaker at room temperature for 7 min.</li>
		<li>Discard T-TBS 1x&#160;solution and replace. Repeat 3 times.</li>
		<li>Make a 1:10,000 secondary mouse-antibody solution by adding antibody for standard Western blot detection (see Table of Materials) and place the membrane on a shaker; leave at room temperature for 2 h.</li>
		<li>Discard the antibody solution and soak in T-TBS 1x&#160;solution on a shaker at room temperature for 7 min.</li>
		<li>Discard T-TBS 1x&#160;solution and replace. Repeat 3 times.</li>
		<li>Soak membrane for 5 min in a chemiluminescent solution, and image the membrane on a chemiluminescence imager.</li>
	</ol>
	</li>
</ol>
3. Restoring Target-therapy Inhibitor Sensitivity by Candidate R-gene Knock-down
<ol>
	<li>Cell preparation
	<ol>
		<li>Prepare single cell suspension by trypsinization. Perform transfection on the day that the cells are plated.
		<ol>
			<li>Wash NCI-N87 cells with PBS and incubate at 37 &#176;C with 0.05% trypsin/EDTA solution for 5 - 10 min. Add 2 volume of RPMI medium containing 10% fetal bovine serum to neutralize trypsin (e.g., 2 mL of medium for each 1 mL of trypsin).</li>
			<li>Count cells with a hemocytometer using trypan blue staining. Seed 2.5 x 105 cells (60% confluence) onto a T25 cm2 flask for each condition. 
			NOTE: The appropriate number of cells for seeding is determined by the doubling time of the cell line and the duration of the experiment. The aim is to achieve the target gene knock-down for the entire duration of the experiment. Nine T25 cm2 flasks are ideal for transfection conditions and the clonogenic assay (3 flasks for controls, 3 flasks for negative transfection controls, 3 flasks for gene-target transfection).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Reverse transfection
	<ol>
		<li>Prepare gene-target and negative control transfection mix for each T25 cm2 flask as follows.
		<ol>
			<li>Dilute 12.5 &#181;L of each gene-targeting siRNA oligonucleotides or Negative Control siRNA oligonucleotides in minimal essential transfection medium (ratio 1:55; see Table of Materials).</li>
			<li>Add a cationic liposome transfection reagent (1:1 ratio with gene-targeting siRNA oligonucleotides; see Table of Materials). Incubate at room temperature for 20 min. 
			NOTE: The total volume of the transfection mix is 700 &#181;L.</li>
			<li>Add 700 &#181;L of the transfection mix to each T25 cm2 flask. Incubate cells at 37 &#176;C for 1 - 3 days.</li>
		</ol>
		</li>
		<li>Verify gene knock-down by RT-PCR and Western blot analysis. Double-check the impact of the putative R-gene knock-down on the sensitivity restoration to the targeted therapeutic agent through colony formation experiments (repeat Step 1.5 ).</li>
	</ol>
	</li>
</ol>

<ol><li>Gerber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+therapies%3A+a+new+generation+of+cancer+treatments%5BTitle%5D)+AND+%22Am+Fam+Physician%22%5BJournal%5D)">Targeted therapies: a new generation of cancer treatments.</a> Am Fam Physician. 77 (3), 311-319 (2008).</li>
<li>Zhang, J., Yang, P. L., Gray, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+cancer+with+small+molecule+kinase+inhibitors%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">Targeting cancer with small molecule kinase inhibitors.</a> Nat Rev Cancer. 9 (1), 28-39 (2009).</li>
<li>Nelson, A. L., Dhimolea, E., Reichert, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+trends+for+human+monoclonal+antibody+therapeutics%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">Development trends for human monoclonal antibody therapeutics.</a> Nat Rev Drug Discov. 9 (10), 767-774 (2010).</li>
<li>Chabner, B. A., Roberts, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Timeline%3A+Chemotherapy+and+the+war+on+cancer%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">Timeline: Chemotherapy and the war on cancer.</a> Nat Rev Cancer. 5 (1), 65-72 (2005).</li>
<li>Gilbert, L. A., Hemann, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemotherapeutic+resistance%3A+surviving+stressful+situations%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Chemotherapeutic resistance: surviving stressful situations.</a> Cancer Res. 71 (15), 5062-5066 (2011).</li>
<li>Izar, B., Rotow, J., Gainor, J., Clark, J., Chabner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacokinetics%2C+clinical+indications%2C+and+resistance+mechanisms+in+molecular+targeted+therapies+in+cancer%5BTitle%5D)+AND+%22Pharmacol+Rev%22%5BJournal%5D)">Pharmacokinetics, clinical indications, and resistance mechanisms in molecular targeted therapies in cancer.</a> Pharmacol Rev. 65, 1351-1395 (2013).</li>
<li>Ellis, L. M., Hicklin, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resistance+to+Targeted+Therapies%3A+Refining+Anticancer+Therapy+in+the+Era+of+Molecular+Oncology%5BTitle%5D)+AND+%22Clin+Cancer+Res%22%5BJournal%5D)">Resistance to Targeted Therapies: Refining Anticancer Therapy in the Era of Molecular Oncology.</a> Clin Cancer Res. 15 (24), 7471-7478 (2009).</li>
<li>Asić, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dominant+mechanisms+of+primary+resistance+differ+from+dominant+mechanisms+of+secondary+resistance+to+targeted+therapies%5BTitle%5D)+AND+%22Crit+Rev+Oncol+Hematol%22%5BJournal%5D)">Dominant mechanisms of primary resistance differ from dominant mechanisms of secondary resistance to targeted therapies.</a> Crit Rev Oncol Hematol. 97, 178-196 (2016).</li>
<li>Arienti, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preclinical+evidence+of+multiple+mechanisms+underlying+trastuzumab+resistance+in+gastric+cancer%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">Preclinical evidence of multiple mechanisms underlying trastuzumab resistance in gastric cancer.</a> Oncotarget. 7 (14), 18424-18439 (2016).</li>
<li>Tanner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amplification+of+HER-2+in+gastric+carcinoma%3A+association+with+Topoisomerase+IIalpha+gene+amplification%2C+intestinal+type%2C+poor+prognosis+and+sensitivity+to+trastuzumab%5BTitle%5D)+AND+%22Ann+Oncol%22%5BJournal%5D)">Amplification of HER-2 in gastric carcinoma: association with Topoisomerase IIalpha gene amplification, intestinal type, poor prognosis and sensitivity to trastuzumab.</a> Ann Oncol. 16 (2), 273-278 (2005).</li>
<li>Bang, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trastuzumab+in+combination+with+chemotherapy+versus+chemotherapy+alone+for+treatment+of+HER2-positive+advanced+gastric+or+gastro-oesophageal+junction+cancer+%28ToGA%29%3A+a+phase+3%2C+open-label%2C+randomised+controlled+trial%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial.</a> Lancet. 376 (9742), 687-697 (2010).</li>
<li>ClinicalTrials.gov. Identifier NCT01130337. A Study of Capecitabine [Xeloda] in Combination With Trastuzumab [Herceptin] and Oxaliplatine in Patients With Resectable Gastric Cancer. , Available from:
 <a target="_blank" href="http://clinicaltrials.gov/show/NCT01130337">http://clinicaltrials.gov/show/NCT01130337</a> (2017).</li>
<li>ClinicalTrials.gov. Identifier NCT01748773. A Study of the Combination of Oxaliplatin, Capecitabine and Herceptin (Trastuzumab) and Chemoradiotherapy in The Adjuvant Setting in Operated Patients With HER2+ Gastric or Gastro-Esophageal Junction Cancer (TOXAG Study). , Available from:
 <a target="_blank" href="http://www.clinicaltrials.gov/show/NCT01748773">http://www.clinicaltrials.gov/show/NCT01748773</a> (2017).</li>
<li>ClinicalTrials.gov. Identifier NCT01472029. Explorative Phase II Study of Perioperative Treatment in Patients With Adenocarcinoma of the Gastroesophageal Junction or Stomach (HerFLOT). , Available from:
 <a target="_blank" href="http://www.clinicaltrials.gov/ct2/show/NCT01472029">http://www.clinicaltrials.gov/ct2/show/NCT01472029</a> (2017).</li>
<li>ClinicalTrials.gov. Identifier NCT01196390. Radiation Therapy, Paclitaxel, and Carboplatin With or Without Trastuzumab in Treating Patients With Esophageal Cancer. , Available from:
 <a target="_blank" href="http://clinicaltrials.gov/show/NCT01196390">http://clinicaltrials.gov/show/NCT01196390</a> (2017).</li>
<li>Shimoyama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unraveling+trastuzumab+and+lapatinib+inefficiency+in+gastric+cancer%3A+Future+steps+%28Review%29%5BTitle%5D)+AND+%22Mol+Clin+Oncol%22%5BJournal%5D)">Unraveling trastuzumab and lapatinib inefficiency in gastric cancer: Future steps (Review).</a> Mol Clin Oncol. 2 (2), 175-181 (2014).</li>
<li>Kelly, C. M., Janjigian, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+genomics+and+therapeutics+of+HER2-positive+gastric+cancer-from+trastuzumab+and+beyond%5BTitle%5D)+AND+%22J+Gastrointest+Oncol%22%5BJournal%5D)">The genomics and therapeutics of HER2-positive gastric cancer-from trastuzumab and beyond.</a> J Gastrointest Oncol. 7 (5), 750-762 (2016).</li>
<li>Wong, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genomic+landscape+and+genetic+heterogeneity+in+gastric+adenocarcinoma+revealed+by+whole-genome+sequencing%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Genomic landscape and genetic heterogeneity in gastric adenocarcinoma revealed by whole-genome sequencing.</a> Nat Commun. 5, 5477(2014).</li>
<li>Oshima, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lapatinib+sensitivities+of+two+novel+trastuzumab-resistant+HER2+gene-amplified+gastric+cancer+cell+lines%5BTitle%5D)+AND+%22Gastric+Cancer%22%5BJournal%5D)">Lapatinib sensitivities of two novel trastuzumab-resistant HER2 gene-amplified gastric cancer cell lines.</a> Gastric Cancer. 17 (3), 450-462 (2014).</li>
<li>Pignatta, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prolonged+exposure+to+%28R%29-bicalutamide+generates+a+LNCaP+subclone+with+alteration+of+mitochondrial+genome%5BTitle%5D)+AND+%22Mol+Cell+Endocrinol%22%5BJournal%5D)">Prolonged exposure to (R)-bicalutamide generates a LNCaP subclone with alteration of mitochondrial genome.</a> Mol Cell Endocrinol. 382 (1), 314-324 (2014).</li>
</ol>

The authors have nothing to disclose.

While personalized and targeted therapies have elicited growing enthusiasm, these so-called &#39;smart&#39; therapies face the same major hurdle as traditional chemotherapy drugs, i.e., the rapid and unpredictable acquisition of drug resistance. To improve the results of targeted therapy, drug-resistance problems must be solved through a better understanding of the mechanisms that cause them. Here, we presented a widely accessible in vitro methodology to investigate the mechanisms of acquired resistance...

Following up on the crucial discoveries in molecular and cellular biology, a number of novel synthesized anticancer molecules have been developed to selectively target oncogenic signaling pathways in a broad array of tumor types. In particular, two categories of targeted therapeutic agents with unique properties in oncology, namely synthetic chemical compounds and recombinant monoclonal antibodies, are known to have achieved clinical successes and dramatically altered cancer care in recent decades1,2,3.
Nevertheless, despite their impressive initial response to treatment, the majority of cancer patients have developed drug resistance to all targeted therapeutic agents, both monoclonal antibodies and kinase inhibitors. As a consequence, drug resistance, the major hurdle for classic anti-cancer medicines4, is still a great challenge for emerging targeted therapies5,6.
Resistance to targeted therapy may be intrinsic (i.e., primary) or acquired (i.e., secondary). Intrinsic resistance describes a de novo lack of response to therapy, whereas secondary resistance occurs after a period of response to drug treatment7. The latter is caused by outbreaks of small cohorts of tumor cells within the bulk of the primitive tumor or nestled in distal cryptic anatomical niches, exhibiting up to 90% resistance to one or more targeted therapeutic agents. Molecular mechanisms underlying a resistance development to targeted therapy mainly result from target gene mutations and redundant activation of other pro-survival signaling pathways, whose understanding is still far from complete8.
In this work, we proposed a time- and cost-effective approach to investigate in vitro mechanisms of acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor, a crucial tool used for testing and validating the working hypotheses. In particular, our approach was used to investigate, in gastric cancer, the resistance mechanisms to trastuzumab (e.g., Herceptin), a humanized monoclonal antibody targeting the extracellular domain of HER2 protein9. Trastuzumab + chemotherapy is widely accepted as the standard first-line treatment for HER2-positive metastatic breast cancer. Thanks to recent preclinical studies showing that anti-HER2 therapies have significant activity in both in vitro and in vivo HER2-positive gastric cancer models10,11, molecular drugs targeting HER2 have been extensively examined in clinical trials, some of which are still ongoing, on patients with gastroesophageal cancer12,13,14,15.
These studies have emphasized the rising number of patients experiencing resistance to trastuzumab16, similarly to what is observed for other targeted therapeutic inhibitors. In particular, the response rate of trastuzumab was 47%, and the median overall survival for patients on trastuzumab + chemotherapy was only 2.7 months longer than for patients on chemotherapy alone17. This suggested that while primary trastuzumab resistance is prevalent, secondary trastuzumab resistance is inevitable. For this reason, there is urgent need to clarify the mechanisms causing HER2-targeted therapy resistance in gastric cancer, which is even more problematic given the intra-tumoral genetic heterogeneity of this neoplasia18.
In addition, mechanisms of resistance to trastuzumab in gastric cancer have proven challenging to explain, partially due to the difficulty in obtaining reliable preclinical models representative of this condition. Oshima et al. reported an in vivo selection method of trastuzumab-resistant gastric cancer cell lines, consisting of a repeated culture of a small residual peritoneal metastasis after treatment with trastuzumab19. However, the need of an enclosure, of specific animal handling expertise, and of the approval of the Animal Ethics Committee contributed to making it a time- and cost-consuming method. The approach we describe in this work is simple and widely applicable, and could be easily extended to investigate the resistance mechanisms to other therapeutic drugs in different tumor histotypes20.

<table><tbody><tr><td>Trizol Reagent</td><td>Invitrogen</td><td>15596026</td><td>TRIzol Reagent is a reagent for isolating high-quality total RNA or simultaneously isolating RNA, DNA, and protein from a variety of biological samples</td></tr><tr><td>ISCRIPT CDNA SYNTHESIS KIT</td><td>Bio-Rad</td><td>1708891</td><td>The iScript cDNA synthesis kit is a sensitive and easy-to-use first-strand cDNA synthesis kit for gene expression analysis using real-time qPCR.</td></tr><tr><td>TaqMan gene expression assay</td><td>Life Technologies</td><td>4331182</td><td>Applied Biosystems TaqMan Gene Expression Assays consist of a pair of unlabeled PCR primers and a TaqMan probe with an Applied Biosystems FAM or VIC dye label on the 5&amp;rsquo; end and minor groove binder (MGB) and nonfluorescent quencher (NFQ) on the 3&amp;rsquo; end.</td></tr><tr><td>M-PER Mammalian Protein Extraction Reagent</td><td>Thermo Scientific</td><td>78501</td><td>Thermo Scientific M-PER Mammalian Protein Extraction Reagent is designed to provide highly efficient total soluble protein extraction from cultured mammalian cells.</td></tr><tr><td>Halt Protease and Phosphatase Inhibitor Cocktail (100X)</td><td>Thermo Scientific</td><td>78440</td><td>Thermo Scientific Halt Protease and Phosphatase Inhibitor Cocktail (100X) provides the convenience of a single solution with full protein sample protection for cell and tissue lysates.</td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermo Scientific</td><td>23225</td><td>The Thermo Scientific Pierce BCA Protein Assay Kit is a two-component, high-precision, detergent-compatible assay reagent set to measure (A562nm) total protein concentration compared to a protein standard.</td></tr><tr><td>4x Laemmli Sample Buffer</td><td>Bio-Rad</td><td>1610747</td><td>Use 4x Laemmli Sample Buffer for preparation of samples for SDS PAGE. For reduction of samples, add a reducing agent such as 2-mercaptoethanol to the buffer prior to mixing with the sample.</td></tr><tr><td>4&amp;ndash;20% Mini-PROTEAN TGX Precast Protein Gels</td><td>Bio-Rad</td><td>456-1094</td><td>Long-life TGX (Tris-Glycine eXtended) Gels have a novel formulation and can be used for both standard denaturing protein separations as well as native electrophoresis.</td></tr><tr><td>Precision Plus Protein WesternC Blotting Standards</td><td>Bio-Rad</td><td>1610376</td><td>Precision Plus Protein Prestained Standards are available in Dual Color, All Blue, Kaleidoscope, and Dual Xtra formats. All four have the same gel migration patterns, with 3 high-intensity reference bands (25, 50, and 75 kD).</td></tr><tr><td>10x Tris/Glycine/SDS</td><td>Bio-Rad</td><td>1610732</td><td>10x Tris/glycine/SDS is a premixed running buffer for separating protein samples by SDS-PAGE.</td></tr><tr><td>Trans-Blot Turbo Mini PVDF Transfer Packs</td><td>Bio-Rad</td><td>1704156</td><td>Trans-Blot Turbo Mini PVDF Transfer Packs for fast, efficient transfer of proteins from mini gels using the Trans-Blot Turbo Transfer System. Each vacuum sealed, ready-to-use transfer pack contains two buffer-saturated ion reservoir stacks and a prewetted PVDF membrane.</td></tr><tr><td>Precision Protein StrepTactin-HRP Conjugate</td><td>Bio-Rad</td><td>1610381</td><td>StrepTactin-Horseradish Peroxidase (HRP) Conjugate for chemiluminescent or colorimetric detection of Precision Plus Protein Unstained Protein Standards or Precision Plus Protein WesternC Protein Standards on western blots.</td></tr><tr><td>Immun-Star WesternC solution</td><td>Bio-Rad</td><td>5572</td><td>Immun-Star WesternC solution, a method of protein detection , detects mid-femtogram amounts of protein.</td></tr><tr><td>Universal Negative Control</td><td>Invitrogen</td><td>12935300</td><td>Negative Control siRNA has no significant sequence similarity to mouse, rat, or human gene sequences. The control has also been tested in cell-based screens and proven to have no significant effect on cell proliferation, viability, or morphology.</td></tr><tr><td>opti-MEM Glutamax Medium</td><td>Thermo Fisher Scientific</td><td>51985026</td><td>Opti-MEM Medium is an improved Minimal Essential Medium (MEM) that allows for a reduction of Fetal Bovine Serum supplementation by at least 50% with no change in growth rate or morphology.</td></tr><tr><td>Silencer Select Pre-Designed &amp;amp; Validated siRNA</td><td>Ambion</td><td>4390824</td><td>RNA interference (RNAi) is the best way to effectively knock down gene expression to study protein function in a wide range of cell types.</td></tr><tr><td>Lipofectamine RNAiMAX Transfection Reagent</td><td>Invitrogen</td><td>13778075</td><td>Lipofectamine RNAiMAX Transfection Reagent offers an advanced, efficient solution for siRNA delivery. No other siRNA specific transfection reagent provides such easy and efficient siRNA delivery in a wide variety of cell lines including common cell types, stem cells and primary cells, as well as traditionally hard-to-transfect cell types.</td></tr><tr><td>Mouse anti-IQGAP1</td><td>Invitrigen</td><td>33-8900</td><td>working concentration: 1:400 in 5% milk solution</td></tr><tr><td>goat anti-mouse IgG-HRP: sc-2005</td><td>Santa Cruz</td><td>sc-2005</td><td>working concentration: 1:10000 in 5% milk solution</td></tr><tr><td>PMSF</td><td>Sigma Aldrich</td><td>P 7626</td><td>this is an inhibitor of serine proteases and acetylcholinesterase</td></tr><tr><td>Thermocycler for RT-PCR</td><td>MJ Research</td><td>MJ Research PTC-200 Thermal Cycler</td><td>it allows temperature homogeneity and a ramping speed of up to 3&amp;deg;C/sec. The protocol can be performed also with other thermalcyclers</td></tr><tr><td>Real Time RT-PCR instrument</td><td>Applied Biosystems</td><td>7500 RT-PCR system</td><td>This is a five-color platform that uses fluorescence-based polymerase chain reaction (PCR) reagents to provide a relative quantification using comparative C&lt;sub&gt;T&lt;/sub&gt; assay type. The protocol can be performed also with other thermalcyclers</td></tr><tr><td>Microvolume Spectrophotometers</td><td>Thermo Scientific</td><td>Nanodrop</td><td>It provides an accurate and fast acid nucleid measurement using little volume of sample</td></tr><tr><td>Blotting system</td><td>Bio-Rad</td><td>Trans-Blot Turbo system</td><td>It perfoms a semy-dry transfer in a few minutes</td></tr><tr><td>Image acquisition system and gel documentation</td><td>Bio-Rad</td><td>Chemidoc image system</td><td>It is easy to use for chemiluminescent detection</td></tr></tbody></table>

looking for driver pathways of acquired resistance to targeted therapy: drug resistant subclone generation and sensitivity restoring by gene knock-down

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more impressive clinical efficacy and minimized adverse effects than traditional treatments, drug resistance has become the main limitation to their benefits. Several preclinical in vitro/in vivo models of acquired resistance to targeted agents in clinical practice have been developed mainly by using two strategies: i) genetic manipulation for modeling genotypes of acquired resistance, and ii) in vitro/in vivo selection of resistant models. In the present work, we propose a unifying framework, for investigating the underlying mechanisms responsible for acquired resistance to targeted therapeutic agents, starting from the generation of drug-resistant cellular subclones to the description of silencing procedures used for restoring the sensitivity to the inhibitor. This simple time- and cost-effective approach is widely applicable, and could be easily extended to investigate resistance mechanisms to other targeted therapeutic drugs in different tumor histotypes.

Figure 1 outlines the framework of the experimental procedure. Three gastric cancer cell-lines expressing a high level of HER2 and sensitive to trastuzumab (IC50 &#60; peak plasma level of drug, Figure 2A, left graph) were grown in culture medium containing the targeted therapeutic agent. A dose 10-fold lower than the peak plasma concentration of trastuzumab (10 &#181;g/mL) was set as the starting dose, and gradually i...

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Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

This is a time- and cost-effective in vitro protocol investigating the mechanisms of acquired resistance to targeted therapeutic agents, which is a highly unmet medical need in cancer management.

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

looking-for-driver-pathways-acquired-resistance-to-targeted-therapy

Research

JoVE Journal

Cancer Research

7.2K Views.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. Dit is een tijd - en goedkoop cost-effective in vitro protocol van het onderzoek naar de mechanismen van verworven resistentie aan gerichte therapeutische agenten, dat is een zeer onvervulde medische noodzaak in kanker beheer.

Video: Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to overcome this challenge. Here, a 2-step dose-escalation method for establishing dual-resistance to an epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), gefitinib, and a MET-TKI, PHA665752, is described. This method is based on simple stepwise dose-escalation of inhibitors for inducing acquired resistance in cell lines. The alternate method for generating resistant sublines involves exposing the cells to high concentrations of the inhibitor in one step. The stepwise dose-escalation method has a higher possibility of successfully inducing acquired resistance than this method. Activating EGFR mutations are biomarkers of a response to treatment with EGFR-TKI, which is an applied first-line treatment for non-small cell lung cancers (NSCLC) that harbor these mutations. However, despite reports of effective responses, the use of EGFR-TKI is limited because tumors inevitably acquire resistance. The major mechanisms behind EGFR-TKI resistance include a secondary mutation at the gatekeeper site, T790M in exon 20 of EGFR, and a bypass signal of MET. Thus, a potential solution for this issue would be a combination of EGFR-TKI and MET-TKI. This combined treatment has been shown to be effective in an in vitro study model. Acquired gefitinib-resistance was established through MET-amplification by stepwise dose-escalation of gefitinib for 12 months, and a cell line named PC-9MET1000 was generated in a previous study. To further investigate the mechanisms of acquired MET-TKI and EGFR-TKI resistance, a MET-TKI, PHA665752, was administered to these cells with stepwise dose-escalation in the presence of gefitinib for 12 months. This protocol has also been successfully applied for a number of combination therapies to establish acquired resistance to other inhibitor molecules.

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

An in vitro method for establishing dual resistance to an EGFR-TKI and a MET-TKI in cancer cells is described. This method is useful for developing treatments for patients with EGFR-mutations, who exhibit disease progression despite EGFR-TKI treatment with MET-amplification. It can also be modified for inhibitors targeting other molecules.

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to ...

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, ...

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell lines or increasing its expression with overexpression cell lines. Transient and stable transfection are two methods that are often used for exogenous gene expression. Transient transfection is only useful for short-term expression, whereas stable transfection allows exogenous genes to be integrated into the host cell genome where it will be continuously expressed. As a result, stable transfection is usually employed for research into long-term genetic regulation. Here we describe a simple protocol to generate a stable cell line overexpressing tagged death receptor 3 (DR3) to explore DR3 function. We picked single clones after a retroviral infection in order to maintain the homogeneity and purity of the stable cell lines. The stable cell lines generated using this protocol render DR3-deficient HT29 cells sensitive to antimitotic drugs, thus reconstituting the apoptotic response in HT29 cells. Moreover, the FLAG tag on DR3 compensates for the unavailability of good DR3 antibody and facilitates the biochemical study of the molecular mechanism by which antimitotic agents induce apoptosis.

Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell ...

Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.

A method for establishing afatinib-resistance cell lines from lung adenocarcinoma PC-9 cells was developed, and resistant cells were characterized. The resistant cells can be used to investigate epidermal growth factor receptor tyrosine kinase inhibitor-resistance mechanisms, applicable for patients with non-small cell lung cancer.

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

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 ...

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 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 ...

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

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