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A CML Drosophila model for treatment screening
the posterior eye defect area was evaluated using two-way analy- sis of variance (ANOVA) followed by the Tukey’s multiple com- parisons test. One–way ANOVA was used when comparing the averages of the posterior eye defect area for dose response and was followed by Tukey’s multiple comparisons test. Associations with p < 0.05 were considered significant. Statistical tests were done using GraphPad Prism 6.0 software.
Results
Expression of human BCR-ABL1 in Drosophila eyes induces transformation
To assess the transformative potential of human BCR-ABL1p210 and BCR-ABL1p210/T315I in Drosophila, we expressed the transgenes in the adult eye using the glass- multimer reporter promoter GMR-GAL4 which drives the expression in all differentiating photoreceptor cells poste- rior to the morphogenetic furrow.37 GMR-GAL4>w1118 flies were used as a control. The temperature sensitivity of the GAL4-UAS system allowed us to the control BCR-ABL1 expression levels.38 Therefore crosses were performed at 18oC, 25oC, and 29oC allowing for a reciprocal increase in transgene expression upon increased temperatures. Enclosed flies were imaged using light microscopy and SEM and evaluations of phenotypes were performed using a grading score (Online Supplementary Table S1) which graded the severity of the phenotype based on the extent of mechano-sensory bristles alignment, misplace- ment and duplication as well as the extent of ommatidial facets loss indicating the disruption in cellular proliferation and differentiation collectively defining interrupted nor- mal development.39 BCR-ABL1p210 and BCR-ABL1p210/T315I showed a rough eye phenotype with increased severity at a higher temperature compared to control flies. At 18oC BCR-ABL1p210 and BCR-ABL1p210/T315I flies exhibited a rough eye phenotype characterized by ommatidial fusions and areas of lost ommatidial facets, particularly at the posteri- or end of the eye, as well as multiple ectopic mechano- sensory bristles which are duplicated at some instances (Figure 1 B, F, J; Figure 2 B, F, J). At 25oC, a more severe rough eye was observed in both BCR-ABL1p210 and BCR- ABL1p210/T315I with loss of the majority of ommatidial facets (Figure 1 D, H, L; Figure 2 D, H, L). At 29oC, the severity increased to involve the loss of the majority of mechano- sensory bristles in addition to the total loss of ommatidial facets in both BCR-ABL1p210 and BCR-ABL1p210/T315I express- ing flies (Figure 1 N, P, R; Figure 2 N, P, R). The average roughness of BCR-ABL1p210 significantly increased from 6.2 at 18oC to 8.2 (P<0.0001) at 25oC and to 9.5 (P<0.0001) at 29oC (Figure 1). As for BCR-ABL1p210/T315I, the average roughness significantly increased from 6.6 at 18oC to 8.9 (P<0.0001) at 25oC and to 10 (P<0.0001) at 29oC (Figure 2). Western blot analysis confirmed the expression and phos- phorylation of BCR-ABL1p210 and BCR-ABL1p210/T315I in Drosophila eyes (Figure 3).
Dasatinib and ponatinib rescue human BCR-ABL1p210 mediated defects in Drosophila
Since expression of BCR-ABL1 at high temperature induced severe eye defects in adult flies, we opted to use the lowest temperature (18°C) which produced milder phenotypes for TKI screening efficiency allowing the easy visualization of any rescue due to drug activity. Four TKI were tested which included imatinib, nilotinib, dasatinib
and ponatinib. BCR-ABL1p210 flies were crossed to GMR- Gal4 flies and progeny were fed on multiple concentra- tions of the TKI (treated) or on DMSO alone (untreated). Untreated BCR-ABL1p210 and BCR-ABL1p210/T315I flies showed the same defects described previously at 18oC focusing particularly on the posterior end of the eye with a characteristic defective area characterized by loss of ommatidial facets (Figures 4-6). The posterior eye defect area in untreated BCR-ABL1p210 flies showed an average of 4580 μm2 and 4044 μm2 on 0.03% DMSO and 0.3% DMSO respectively (Figures 4-6). On the other hand, untreated BCR-ABL1p210/T315I expressing flies showed a wider area of defect at the posterior end with an average significant increase in the defect area to 11148 μm2 (P<0.0001) and 8728 μm2 (P<0.0001) on 0.03% DMSO and 0.3% DMSO respectively as compared to untreated BCR-ABL1p210 expressing flies (Figures 4-6).
Feeding 150 μM or 1500 μM imatinib to BCR-ABL1p210 expressing flies did not eliminate the posterior eye defect. However, the average posterior eye defect area showed a tendency to decrease with 1500 μM imatinib (3047 μm2) as compared to that of 150 μM imatinib (4142 μm2) and untreated flies (4044 μm2) (Figure 4). Interestingly, the per- centage of flies with total rescue (total disappearance of the posterior eye defect) with 150 μM and 1500 μM ima- tinib was 4% and 21% respectively. Similarly, feeding 28 μM ((Online Supplementary Figure S1 E, K) or 280 μM (Online Supplementary Figure S1 F, L) nilotinib to BCR- ABL1p210 expressing flies did not eliminate the posterior eye defect. However, the average posterior eye defect area showed a tendency to decrease with 280 μM nilotinib (2480 μm2) compared to that of 28 μM nilotinib (3871 μm2) and untreated flies (4044 μm2) (Online Supplementary Figure S1). The percentage of flies with total rescue with 28 μM and 280 μM nilotinib was 7% and 13% respective- ly.
Testing the potent TKI (dasatinib and ponatinib) showed more efficient rescue. Feeding 20 μM dasatinib or 280 μM ponatinib to BCR-ABL1p210 expressing flies improves the overall eye ommatidial arrangement and more specifically eliminates the characteristic posterior eye defect by restoring its normal ommatidial develop- ment (Figure 5 D, H; Figure 6 D, H). The average posterior eye defect area significantly decreased from 4580 μm2 (in untreated flies) to 0 μm2 (P<0.0001) with 20 μM dasatinib (Figure 5) and from 4044 μm2 (in untreated flies) to 267 μm2 (P<0.0001) with 280 μM ponatinib (Figure 6). The percentage of flies with total rescue was 100% with dasa- tinib and 86% with ponatinib.
A dose-response analysis for BCR-ABL1p210 expressing flies treated with dasatinib showed a significant decrease in the average posterior eye defect area from 4580 μm2 in untreated flies to 2372 μm2 (P<0.0001) with 1 μM dasa- tinib, to 131 μm2 (P<0.0001) with 10 μM and to 0 μm2 (P<0.0001) with 20 μM dasatinib. The percentage of flies with total rescue increased from 25% to 92% and to 100% with 1 μM, 10 μm and 20 μM dasatinib respectively (Figure 7). Similarly, ponatinib also showed a dose- response whereby the average posterior eye defect area decreased significantly from 4044 μm2 in untreated flies to 1684 μm2 (P<0.0001) with 28 μM and to 267 μm2 (P<0.0001) with 280 μM ponatinib (Figure 7). The percent- age of flies with total rescue increased from 48% to 86% with 28 μM and 280 μM ponatinib respectively.
The BCR-ABL1p210/T315I mutation is known to exhibit
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