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New Drosophila model for chronic myeloid leukemia
Moreover, ena-RNAi weakly suppressed the BCR-ABL1 phenotype (Figure 7G), decreasing the phenotype pene- trance. As a control, we did not observe any phenotype due to Dab or ena downregulation or Dab overexpression in prohemocytes (Figure 7F,G).
Discussion
In order to identify candidate genes and pathways involved in the onset and progression of CML we devel- oped and validated a CML genetic model based on trans-
AC
genic Drosophila expressing BCR-ABL1. In order to build and characterize a human functional model that could be sensitive to pharmacological inhibition and suitable for studying the effects of BCR-ABL1 mutations identified in patients with CML, we chose to express a completely human p210-BCR-ABL1 protein, in contrast what has been done previously.7 The expression of the oncopro- tein in all eye cells committed to differentiation as pho- toreceptors or accessory cells (gmrGal4 driver) induces a strong phenotype characterized by altered differentia- tion of the ommatidia cells.44 The lack of phenotype in flies expressing a BCR-ABL1 kinase-dead mutant sup-
B
D
E
Figure 7. BCR-ABL1 expression in the hematopoietic precursor cells of the lymph gland impairs Drosophila blood cell homeosta- sis increasing the number of circulating blood cells. (A) A w1118 mid-L3 instar larva used as the wild-type control. (B) A mid-L3 larva conditionally expressing BCR-ABL1 in the hematopoietic pre- cursors of the lymph gland medullary zone under the control of the domelessGal4 driver construct (dome:GFP/+;BCR-ABL1_3M,tub80TS/+). BCR- ABL1 expression was induced in stage L2 or early-L3 larvae by exposing the animals to 29°C during the indicated larval instars to disrupt the ability of the temperature-sensitive Gal80 mutant to inhibit Gal4 transactivation activity. The black arrows in (B) point to melanotic nod- ules. Anterior is on the left. (C) Penetrance of the melanotic nodule phenotype in mid-L3 control larvae expressing GFP under the control of the domelessGal4 driver (domeGFP), in larvae con- stitutively expressing a kinase-dead BCR-ABL1 mutant protein (dome:GFP/+;BCR-ABL1 KD/+)
FG
after bleeding of
ABL1_3M,tub80TS/+ and dome:GFP/+ larvae. BCR-ABL1 expression induces the appearance of melanotic nodules and this correlates with an increase of circulating hemocytes. (E) Penetrance of the melanotic nodule phenotype in mid-L3 control larvae (dome:GFP), in larvae expressing Abl-RNAi (dome/Abl-RNAi), in larvae in which BCR-ABL1 alone (dome:GFP/+;BCR- ABL1_3M,tub80TS/+) or together with Abl-RNAi (dome/Abl-RNAi;BCR-ABL1_3M,tub80TS/+) is expressed from the L2 instar. (F) Penetrance of the melanotic nodule phenotype in mid-L3 con- trol larvae (dome:GFP), in larvae expressing Dab- RNAi (dome/+;Dab-RNAi/+), in larvae condition-
and in larvae (dome:GFP/+;BCR-ABL1_3M,tub80TS/+) expression was induced starting from the L2 (L2) or from the early-L3 (eL3) instars. (D) Evaluation of the average number of hemocytes per field
in which BCR-ABL1
ally expressing the (dome:GFP/+;tub80TS/+;UAS-Dab/+), and in lar- vae in which BCR-ABL1 alone (dome:GFP/+;BCR- ABL1_3M,tub80TS/+) or together with either Dab-RNAi (dome/+;BCR- ABL1_3M,tub80TS/+;Dab-RNAi/+) or UAS-Dab (dome/+;BCR-ABL1_3M,tub80TS/+;UAS-Dab/+) is expressed from the L2 instar. (G) Penetrance of the melanotic nodule phenotype in mid-L3 control larvae (dome:GFP), in larvae expressing ena-RNAi (dome/+;ena-RNAi/+), and in larvae in which BCR-ABL1 alone (dome:GFP/+;BCR- ABL1_3M,tub80TS/+) or together with ena-RNAi (dome/+;BCR-ABL1_3M,tub80TS/ena-RNAi) is expressed from the L2 instar. The average phe- notype penetrance is calculated from three inde- pendent experiments, each involving 15-95 lar- vae. The statistical comparisons were conducted using a Student t test (*P<0.05, **P<0.01, ***P<0.001, ns=not significant). Bars indicate the standard error.
dome:GFP/+;BCR-
Dab protein
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