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A. Al Outa et al.
outshined by the emergence of resistance caused by point mutations in the ABL1 kinase domain which necessitated the development of second-generation TKI.14,15 Dasatinib16- 18 and nilotinib19-21 revealed faster and deeper molecular responses compared to imatinib in patients with newly diagnosed chronic phase CML. In vitro, dasatinib is more potent than imatinib22-24 and inhibits a wider spectrum of kinases including the Src family.25 Nilotinib has a greater affinity than imatinib to the ATP binding site in BCR- ABL1 and its spectrum of kinase inhibition involves platelet-derived growth factor receptor (PDGFR) and c-Kit receptors.26 Although nilotinib and dasatinib tackled the majority of imatinib-resistant mutations, neither of them targeted the T315I mutation (threonine to isoleucine sub- stitution at position 315 in the ABL1 kinase domain) (BCR- ABL1T315I).23 Ponatinib, a third generation TKI, remains the only clinically available drug that is designed to overcome the T315I gatekeeper mutation.27,28 However, post-market- ing safety issues with ponatinib involved serious cardio- vascular events which led to its temporary suspension and then reintroduction with special patient recommenda- tions.29,30
In addition to the burden of resistance, therapy with TKI is hindered by their inability to eradicate leukemic stem cells and hence relapse often accompanies discontin- uation of therapy.31 This fact imparts lifelong therapy with TKI despite accompanying side effects which result in ever-expanding costs for remission sustainment. Therefore, it seems evident that despite the breakthrough with TKI, CML remains a pathology that requires vigilant assessment of curative therapeutic interventions.
One simple, multicellular and genetically tractable ani- mal model that has been exploited in recent years for modelling human diseases, including cancer, is Drosophila melanogaster.32 A myriad of advantages is held by this 3 mm long fruit fly as an in vivo model for dissecting the con- tribution of cellular mechanisms to human cancers and therapeutic screening. Fogerty et al. utilized Drosophila to decipher functional analogies between fly ABL1 and human BCR-ABL1 via neural-specific expression of p185 or p210 BCR-ABL1 transgenes. In these transgenes, BCR and the N-terminal sequences are derived from human oncogenes while the C-terminal ABL1 tail is from Drosophila. Both transgenes were capable to substitute the fruit fly ABL1 during axon genesis and flies expressing BCR-ABL1 revealed an increase in the phosphorylation of Enabled (Ena), a substrate for Drosophila Abl (dAbl). Expression of chimeric BCR-ABL1 in Drosophila eyes and CNS resulted in a rough eye phenotype and CNS develop- mental defects.33 Furthermore, a recent study showed that the expression of human BCR-ABL1p210 in Drosophila acti- vates the dAbl pathway and its upstream regulators Ena and Disabled (Dab).34
In this study, we have overexpressed human BCR- ABL1p210 and mutated BCR-ABL1p210/T315I in Drosophila com- pound eyes. BCR-ABL1p210/T315I expression induced a signif- icantly more severe rough eye phenotype compared to BCR-ABL1p210 pointing towards more aggressive tumori- genic capacities of the gatekeeper mutation. We have fur- ther assessed the efficiency of the current TKI used in clin- ics in modifying the characteristic eye phenotypes of transgenic flies. Dasatinib and ponatinib rescued the eye defects observed upon expression of BCR-ABL1p210 making this model a valuable screening platform to pre-clinically evaluate the efficacy of potential novel therapies for CML.
Methods
Fly stocks
Fly stocks were maintained at 25oC on standard agar-based medium. GMR-GAL4 (BDSC 1104) were obtained from Bloomington Stock Center. Treatment was performed at 18oC. Fly work was done following the institutional guide for the care and use of laboratory animals.
Generation of transgenic flies
Transgenic flies, harboring human BCR-ABL1p210 and BCR- ABL1p210/T315I were generated using Phi C31 integrase system and were inserted in the 3rd chromosome for GAL4-UAS expression. BCR-ABL1p210 and BCR-ABL1p210/T315I were inserted into pUAST-attB Drosophila expression vector (Custom DNA cloning). pUAST-attB- myc BCR-ABL1p210 and pUAST-attB-myc BCR-ABL1p210/T315I were injected into y1 w67c23; P {CaryP} ABLattP2 (8622 BDSC) embryos to generate transgenic flies (BestGene Inc, Chino Hills, CA).
TKI administration
Imatinib (I-5577), nilotinib (N-8207), dasatinib (D-3307) and ponatinib (P-7022) were obtained from LC laboratories, MA, USA. Stock solutions were dissolved in DMSO and the required amount of TKI was added to instant Drosophila medium (Carolina Biological Supply Company). Since DMSO is known to be toxic to Drosophila,40 0.03% DMSO was used for low TKI concentra- tions and 0.3% for high concentrations.
Scoring of eye phenotypes and measurement of eye defect area
A grading score, that was modified from the score previously published,35 was used for scoring and is based on the number of ommatidial fusions, the extent of bristle organization and omma- tidial loss (Online Supplementary Table S1). For the measurement of the posterior eye defect area, Image J36 was used. Scanning elec- tron microscopy images were coded by one researcher and analy- sis was blindly performed by another researcher. N=20 flies from each genotype at each temperature were scored and the experi- ment was done in triplicate. For the measurement of posterior eye defect area an average of n=20-30 flies from each group was quan- tified and the experiment was done at least twice.
Scanning electron microscopy
Adult flies were fixed in 2% glutaraldehyde and 2% formalde- hyde in phosphate buffered saline (PBS) (1x), washed, dehydrated with a series of increasing ethanol concentrations, dried with a critical point dryer (k850, Quorum Technologies), mounted on standard aluminum heads and coated with 20 nm layer of gold. Analysis was performed using Tescan, Mira III LMU, Field Emission Gun (FEG) scanning electron microscopy (SEM) with a secondary electron detector.
Western blot analysis
Fly heads were homogenized in Laemmli buffer and samples were loaded in 8% SDS-PAGE. Anti-ABL1 (SC-23, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA) and phospho-ABL1 (#2868, 1:500, Cell Signaling Technology) primary antibodies and anti- mouse (SC-2318, 1:5000, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-rabbit (NA934, 1:5000, GE Healthcare) secondary antibodies were used for protein detection. An extract (150 μg) from 20-30 flies was used.
Statistical analysis
The statistical significance of the difference between the aver- age scores of the rough eye phenotype and the average scores of
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