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R. Bernardoni et al.
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Figure 3. Ena loss of function suppresses the eye phenotype due to BCR-ABL1 expression which increases phosphorylation of the dAbl target Ena. (A) Piled his- togram chart showing the frequencies of the three phenotypic classes in flies co-expressing BCR-ABL1 (gmrGal4,4M) and EGFP (UAS-EGFP/+;gmrGal4,4M/+), or one of two independent ena-RNAi lines (VDRC#43056 and VDRC#106484). (B,C) Eye imaginal discs from wild-type late third instar larvae expressing EGFP under the control of the gmrGal4 driver in cells posterior to the morphogenetic furrow (B) and expressing the pan-neuronal marker Elav in cells committed to terminal differen- tiation (C). (D,E) Elav expression in eye imaginal discs from late third instar larvae expressing BCR-ABL1 (D) or larvae co-expressing BCR-ABL1 and ena-RNAi (E) under the control of the gmrGal4 driver construct. BCR-ABL1 expression reduces the number of differentiated photoreceptors, as indicated by a decrease of Elav-expressing cells, and ena downregulation suppresses this phenotypic trait. (F-H) Quantification of Ena expression and tyrosine-phosphorylation in protein extracts (F) or Ena- immunoprecipitated proteins (G) from the heads of adult flies expressing either EGFP (lane 1) or BCR-ABL1 (lane 2). Independent loads of equal amount of protein extracts or Ena-immunoprecipitated proteins were probed with antibodies raised against phosphorylated tyrosine residues (p-Tyr), Ena or α–tubulin as loading control. (H) Average signal intensity from replica of the experiment shown in (F) and (G). Ena immunoprecipitation and probing for tyrosine-phosphorylation confirmed the increase of Ena tyrosine-phosphorylation in animals expressing BCR-ABL1. The statistical comparisons in (A) were conducted using the Mann-Whitney test (*P<0.05, **P<0.01, ***P<0.001).
STAT5, hindering apoptosis in leukemic cells.26 The JAK/STAT pathway is required during Drosophila eye morphogenesis and larval hematopoiesis.27,28 Interestingly, loss of STAT92E function (STAT92E06346), the fly counterpart of STAT5, induced strong suppression of the BCR-ABL1 phenotype (Figure 4, Online Supplementary Figure S3A,B). Flies coexpressing a STAT dominant nega- tive allele (STATDN) and BCR-ABL1 showed an even weaker phenotype (Figure 4A, Online Supplementary Figure S3A,C) confirming that STAT is involved in the BCR-ABL1-activated pathway in the Drosophila eye.
The human homologs of Disabled, Dab1 and Dab2, are altered in patients with chronic myeloid leukemia
To better explore the efficacy of the model we ana- lyzed the Disabled gene that encodes for an adaptor pro- tein acting downstream of many receptor tyrosine kinas- es.17,29 In the embryo Dab LOF disrupts the intracellular localization of dAbl and consequently that of phospho- rylated Ena and F-Actin accumulation.30 In the fly eye we observed an enhancement of the BCR-ABL1 phenotype in animals heterozygous for a Dab deletion. Thus, we further reduced Dab function by gene silencing.
Figure 4. A component of the BCR-ABL1-activated pathway in human leukemia modulates the eye phenotype in Drosophila. Piled histogram chart showing the frequencies of the three phenotypic classes in flies expressing BCR-ABL1
allele or over- expressing a dominant negative allele STATDN. Reduction of the function of STAT, a gene encoding the homolog of the STAT5 protein involved in the BCR-ABL1-acti- vated pathway in human leukemia cells, suppresses the BCR-ABL1-dependent phenotype in the fly eye. The statistical comparisons were conducted using the
(gmrGal4,4M) and heterozygous for a loss of function STAT92E Mann-Whitney test (*P<0.05, **P<0.01, ***P<0.001).
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haematologica | 2019; 104(4)