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R. Bernardoni et al.
ports the role of kinase activity in the eye phenotype. Moreover, BCR-ABL1 expression and phosphorylation levels correlate with the severity of the phenotype. Consistently, BCR-ABL1 expression under the control of gmrGal4 induces a decrease of photoreceptors expressing Elav in eye imaginal discs and this correlates with the disruption of the adult eye. Interestingly, partial loss of dAbl function also slightly reduces the number of eye cells expressing Elav at L3, and to a much greater extent at later stages of development. This suggests that dAbl is implicated in the maintenance of neuronal commit- ment45,46 and confirms that loss or gain of function of dAbl/BCR-ABL1 can alter eye cell development.7 We have shown that human BCR-ABL1 interacts and inter- feres with the dAbl signaling pathway. Animals express- ing BCR-ABL1 and heterozygous for the recessive Abl1 allele or coexpressing either Abl-RNAi or a kinase-dead dominant negative Abl (AblK417N) showed a weaker pheno- type, suggesting that BCR-ABL1 and dAbl proteins most likely share binding sites and/or targets of the kinase activity. Consistently, co-expression of human BCR- ABL1 and dAbl synergizes and the phenotype becomes more severe. Notably, dAbl overexpression per se induces a weak “rough” eye phenotype but the differentiation program is not severely disrupted. We cannot exclude that this is due to a level of dAbl expression below a crit- ic threshold but it could also suggest that excessive dAbl might be still, at least partially, negatively regulated. This possible negative regulation seems to be overcome by BCR-ABL1 since all animals co-expressing dAbl and BCR- ABL1 showed a severe class -1 phenotype. Consistently, LOF or downregulation of genes known to interact genet- ically with dAbl LOF mutations interact in the same way with BCR-ABL1 expression. Namely, pros and fax alleles or deletions enhance the phenotype and this is consistent with their roles in neuronogenesis and neuronal differen- tiation. Moreover, ena LOF suppresses and Dab LOF enhances the dAbl LOF phenotype19,20 and we observed that both ena and Dab LOF and downregulation through RNAi also modify the BCR-ABL1 phenotype in the same way. Ena belongs to the ENA/VASP protein family involved in regulation of the actin cytoskeleton.47,48 dAbl regulates Ena by modulating its localization, most likely through its phosphorylation. It is known that both dAbl and the human/Drosophila BCR-ABL chimera phosphory- late Ena7 in vitro and we established that human BCR-ABL1 expression in the eye also increases Ena phosphorylation. This conservation of phosphorylation targets significantly increases the reliability of our model for identifying rele- vant BCR-ABL1 functional interactors. In this view the observation that decreased Ena function suppresses phe- notypes due to both dAbl mutations24 and BCR-ABL1 expression suggests that both phenotypes can be due to Ena mislocalization and consequently actin cytoskeleton alterations can be suppressed if Ena expression decreases. In Drosophila, Abl and Dab are often co-expressed and the phenotype due to Dab mutations mimics the dAbl pheno- type. Epistasis experiments have shown that Dab func- tions upstream of both dAbl and Ena, controlling their localization and thus the actin cytoskeleton, and Dab LOF does indeed enhance the phenotype due to dAbl muta- tions.30 Interestingly, Dab deletion or downregulation has the same effect on the BCR-ABL1 phenotype. These find- ings could be explained if Dab is able to regulate, at least partially, BCR-ABL1 localization. This interaction might
mitigate more severe BCR-ABL1-dependent effects when Dab is expressed at a physiological level but not if Dab is downregulated or its gene dosage is halved. Furthermore, our study showed that Dab human homologs are less expressed in both peripheral blood and bone marrow of CML patients at diagnosis compared to their expression in controls and are re-expressed in patients during molec- ular remission. Moreover, Dab1 expression in transfected K562 cells significantly decreases cell proliferation, con- firming that Dab activity might alleviate the pathogenic effects of BCR-ABL1. We then assessed whether our model could help to fish-out homologs of leukemia-rele- vant genes in an ongoing dosage-sensitive genetic screen of the whole Drosophila genome. To this aim we consid- ered STAT5, a transcription factor phosphorylated and activated by BCR-ABL1. Interestingly, LOF conditions of STAT92E, encoding the fly homolog of various human STAT, led to suppression of the BCR-ABL1 phenotype. In order to discover a tissue that could be a reliable second read-out for identifying BCR-ABL1 interactors relevant for hematopoiesis and leukemia, we moved to the larval hematopoietic organ, the lymph gland. We conditionally expressed human BCR-ABL1 in the lymph gland medullary zone where quiescent prohemocytes reside. Only BCR-ABL1 expression during L2 induces the appearance of melanotic nodules, which correlates with an increase of circulating hemocytes. This phenotype can be suppressed by dAbl downregulation, confirming that dAbl is expressed in the lymph gland medullary zone43 where it contributes to BCR-ABL1 pathway activation and to induction of the hematopoietic phenotype. It is worth noting that both Dab and ena interact functionally with BCR-ABL1 during hematopoiesis. In fact, while Dab downregulation enhances the melanotic nodule pheno- type and Dab overexpression suppresses it, ena downreg- ulation decreases the penetrance of this phenotype, con- firming that ena and Dab are also expressed in the lymph gland medullary zone43 and modulate BCR-ABL1 activity. This phenotype is visible if BCR-ABL1 is expressed from the L2, when prohemocytes become quiescent, but not if it is expressed from the early L3, when the quiescent pro- hemocytes are still present in the medullary zone of the lymph gland. We are tempted to speculate that the dAbl pathway, activated by BCR-ABL1, could be involved in the mechanisms that regulate entry of prohemocytes into the quiescent state rather than maintenance of this state. This seems consistent with the observation that the lymph glands in mid-L3 larvae expressing BCR-ABL1 from L2 are very small compared to those in controls and do not show any clear partition (Giordani and Bernardoni, unpublished data). This suggests that, upon BCR-ABL1 expression, most of the prohemocytes could undertake the differentiation pathway and leave the lymph gland prematurely without becoming quiescent. We did not test all pathways interacting with BCR-ABL1, for example the Tyr-receptor/Ras pathway, which is known to compete with BCR-ABL1 for binding with the Grb2/Drk proteins1 and is likely involved in the eye phe- notype since the Sevenless Tyr-receptor has an estab- lished role in eye differentiation.49,50 Nevertheless, we present here a new and efficient CML model based on Drosophila transgenic for human BCR-ABL1. This model could be a powerful tool for identifying new genes and pathways involved in the pathogenesis and progression of CML.
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haematologica | 2019; 104(4)