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R. Bernardoni et al.
analysis demonstrated that the severity of the phenotype correlated with the amount and phosphorylation of BCR-ABL1 protein (Figure 1K-M): indeed the low level of BCR-ABL1 expression observed in line 1M (Figure 1K-M) resulted in a very mild phenotype (Figure 1G). To better understand the origin of the phenotype, we analyzed the expression of the pan-neuronal and eye photoreceptor marker Elav16 in eye imaginal discs expressing BCR- ABL1. The typical Elav+ photoreceptor clusters (Figure 1P) were reduced in number and altered in BCR-ABL1- expressing flies and this correlated with the described defects of the eye’s ordered structure (Figure 1P-T). To assess whether the phenotype depends on BCR-ABL1 kinase activity, we generated transgenic flies to express a kinase-dead mutant BCR-ABL1. gmrGal4-driven expres- sion of the mutant protein did not affect eye develop- ment, indicating that the BCR-ABL1 phenotype requires the enzymatic activity of the oncoprotein (Online Supplementary Figure S1A-C,H).
Expression of human BCR-ABL1 interferes with eye development by altering dAbl signaling
To better understand the consequences of BCR-ABL1 overexpression in the eye, we investigated whether the human oncoprotein could activate the endogenous path- way regulated by the Drosophila Abl kinase (dAbl). To quantify the phenotype we classified BCR-ABL1 eyes (line 4M) into three phenotypic classes. Class 0 repre- sents the most frequent “glazed” phenotype. Class +1 is less severe: the eye is bigger and more prominent, and some ommatidia can be observed. Class -1 is more severe, being characterized by a less differentiated eye with evident lack of pigmentation in the most posterior region (Figure 2A). Interestingly, phenotype expressivity did not change comparing gmrGal4,UAS-BCR-ABL1 4M animals with gmrGal4,UAS-BCR-ABL1 4M;UAS-EGFP (Figure 2B, Online Supplementary Figure S1H,I) indicating that a single gmrGal4 copy does not express a Gal4 limit- ing amount that could be titrated by increasing the num- ber of UAS sequences. Since overexpression of dAbl (UAS-Abl) induces a very mild rough eye phenotype (Online Supplementary Figure S1A,B,G), we investigated whether it could enhance the BCR-ABL1 phenotype. We observed a worsening of the phenotype: all of the eyes belonged to class -1, showing smaller eyes and more evi- dent loss of pigmentation (Figure 2B, Online Supplementary Figure S1G,H,N). We then investigated whether dAbl loss of function (LOF) could suppress the BCR-ABL1 phenotype. gmrGal4,UAS-BCR-ABL1 4M ani- mals heterozygous for a dAbl hypomorphic recessive lethal allele (Abl1/+) showed a very mild phenotypic sup- pression but were not statistically different from controls (Figure 2B, Online Supplementary Figure S1E,H,L). However, dAbl downregulation through RNAi (Abl- RNAi) or expression of a dominant negative kinase- defective dAbl (UAS-AblK417N) induced a significant sup- pression of the BCR-ABL1 phenotype (Figure 2B, Online Supplementary Figure S1D,F,H,I,K,M). Interestingly, we observed that animals expressing either Abl-RNAi or UAS-Abl or UAS-AblK417N showed a similar mild disorgan- ization of the ommatidia (Online Supplementary Figure S1A,B,D,F,G) suggesting that the pathway activated by dAbl is indeed implicated in eye development. Furthermore, the genetic interactions between BCR- ABL1 expression and dAbl loss or gain of function sug-
gest that dAbl, dAblK417N and overexpressed BCR-ABL1 could compete for common binding targets. To confirm that BCR-ABL1 overexpression affects eye development by altering dAbl signaling cascade, we analyzed whether BCR-ABL1 could functionally interact with components of the dAbl pathway. In detail, we focused on four genes whose LOF mutations genetically interact with a dAbl mutant phenotype. Mutations of prospero (pros), a tran- scription factor that regulates neuronal differentiation17, failed axon connections (fax), implicated both in neurogen- esis and axonogenesis18 and Disabled (Dab) that regulates cellular localization of dAbl19, enhance the mutant dAbl phenotype. Moreover, enabled (ena) gene mutations sup- press a dAbl mutant phenotype.20 Interestingly, we found that either a deletion or a mutant allele of pros (Figure 2C, Online Supplementary Figure S2A-C) and fax (Figure 2D, Online Supplementary Figure S2A,D,E) was able to enhance the BCR-ABL1 phenotype. Moreover, although the insertional DabEY10190 allele did not change the BCR- ABL1 phenotype significantly, a deletion uncovering the Dab locus enhanced it (Figure 2E, Online Supplementary Figure S2A,F,G), confirming that BCR-ABL1 expression alters eye development likely by interacting with compo- nents of the dAbl pathway.
BCR-ABL1 expression increases phosphorylation of the dAbl substrate Ena
A genetic screen had previously identified an ena LOF allele as a suppressor of the recessive lethality due to dAbl LOF mutations.20 Ena is a cytoskeletal regulator that facilitates actin polymerization.21 Its cellular localization depends on dAbl5,20,22 and it is phosphorylated by both human and Drosophila Abl.23,24 Heterozygosis of a LOF ena allele or of an ena deletion suppressed the BCR-ABL1 phenotype (Figure 2F, Online Supplementary Figure S2A,J,K). ena silencing with two independent constructs (ena-RNAi), induced a size increase and strong decrease of depigmented tissue in eyes expressing BCR-ABL1 (Figure 3A, Online Supplementary Figure S2A,L,M). Consistently, the analysis of Elav expression highlighted a more correct organization of photoreceptor clusters (Figure 3B-E). Furthermore, we looked at tyrosine-phos- phorylation of the endogenous Ena. Flies expressing BCR-ABL1 showed increased levels of Ena tyrosine- phosphorylation (Figure 3F,H) even after Ena immuno- precipitation (Figure 3G,H) suggesting that Ena might be phosphorylated by BCR-ABL1. Taken together our data indicate that alteration of several components of the dAbl pathway could be important for the mechanism by which BCR-ABL1 overexpression affects eye develop- ment, likely phosphorylating conserved targets in fly eye cells.
A component of the BCR-ABL1-activated pathway in human leukemia modulates the eye phenotype in Drosophila
To further assess the effectiveness of the model, we investigated whether a Drosophila homolog of a gene known to be involved in BCR-ABL1 signaling in human leukemia was also able to modulate the BCR-ABL1 phe- notype. Signal transducer and activator of transcription 5 (STAT5) is a transcription factor activated in response to cytokines and its role in malignant transformation is well established.25 Several studies showed that BCR-ABL1 induces phosphorylation and constitutive activation of
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haematologica | 2019; 104(4)