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R. Bernardoni et al.
Drosophila Abl (dAbl) proteins and the existence of Drosophila homologs for many proteins that interact functionally with BCR-ABL1 in mammals strongly sup- port the idea that dAbl and presumably BCR-ABL1 signal transduction pathways could be highly conserved from fly to human. The dAbl gene is expressed at high levels in differentiating neurons and plays an important role in central nervous system, eye and epithelia development, mainly regulating cytoskeleton remodeling.4-6 Interestingly, Forgerty and colleagues demonstrated that the neural expression of a chimeric BCR-ABL protein car- rying the human BCR fused to dAbl is able to rescue the dAbl mutant phenotype, suggesting that the chimeric BCR-ABL protein can effectively compensate for lack of dAbl.7 To further identify genes and pathways involved in the onset and progression of CML, we developed and validated a genetic model based on transgenic flies that drive inducible human BCR-ABL1 expression under the control of tissue- and stage-specific promoters, providing both an excellent and powerful model to identify novel functional interactors.
Methods
Generation of BCR-ABL1 transgenic flies
The BCR-ABL1 coding sequence was amplified by poly-
merase chain reactions and cloned into the P-element expres- sion vector pKS69. BCR-ABL1 kinase-dead (BCR-ABL1KD) was obtained through site-directed mutagenesis (Online Supplementary Data). Plasmids were prepared using QiafilterTM Plasmid Maxi Kit (Qiagen, Venlo, the Netherlands) and injected in Drosophila embryos (The BestGene, Inc, Chino Hills, CA, USA).
Drosophila stocks
Fly stocks were obtained from Bloomington Drosophila Stock
Center (Department of Biology, Indiana University, Bloomington, IN, USA). RNA interference (RNAi) lines were obtained from the Vienna Drosophila RNAi Center (Vienna, Austria). domelessGal4 and STATDN flies were kindly provided by A. Giangrande (IGBMC, Illkirch, France) (Online Supplementary Data).
Immunoblotting
Adult heads were dissected and homogenized in a protein extraction buffer. For cell lines, 107 cells were lysed in RIPA buffer. The following primary antibodies were used: c-Abl (sc- 23), Dab1 (sc-271136), p-Tyr (sc-7020), GAPDH (sc-137179) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), α-tubulin (CP06; Oncogene Research Products, Merck KGaA, Darmstadt, Germany) mouse monoclonal antibodies, BCR (sc-20707) rabbit polyclonal antibody (Santa Cruz Biotechnology) and mouse 5G2 anti-Enabled supernatant (Developmental Studies Hybridoma Bank - DSHB, University of Iowa, IA, USA). For immunoprecipitation, 1 mg of total protein extract was incubat- ed with anti-Enabled supernatant and subsequently with pro- tein A sepharose (Amersham Bioscience, GE Healthcare, Waukesha, WI, USA) (Online Supplementary Data).
Fluorescent Immunolabeling
Fly eye primordium
Eye imaginal discs were dissected from third instar larvae, fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, labeled with the rat anti-Elav 7E8A10 supernatant
(DSHB), incubated with a Cy3-conjugated anti-rat secondary antibody (Jackson Immunoresearch, Newmarket, UK) and exposed to HOECHST (Sigma-Aldrich Corp., St. Louis, MO, USA) before mounting in Fluormount-G (Electron Microscopy Sciences, Hatfield, PA, USA) (Online Supplementary Data).
Primary cells
The protocol was approved by the local ethics committee (approval n. 212/2015). White blood cells (105) were obtained from peripheral blood. Immunofluorescence was performed as previously described8. Mouse anti-Dab1 and anti-Dab2 primary antibodies (sc-271136 and sc-136963, Santa Cruz BIotechnology) and anti-mouse Alexa Fluor 568 secondary anti- body (Molecular Probes-Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) were used (Online Supplementary Data).
Genetic analysis
Eye
Flies carrying gmrGal4 or sevGal4 driver constructs were crossed to the UAS-BCR-ABL1 transgenic lines. To analyze the phenotype, flies from a recombinant line carrying both gmrGal4 and UAS- BCR-ABL1 on the third chromosome (gmrGal4,UAS-BCR-ABL1 4M/TM3) were crossed to lines carrying single gene mutations, deficiencies or RNAi constructs. Fifteen to 30 F1 flies from three independent crosses were classified into three phenotypic classes described in the Results section.
Melanotic nodules
domelessGal4-driven BCR-ABL1 expression was controlled with the TARGET system9,10 (Online Supplementary Data). We performed conditional expression in the medullary zone of the lymph gland starting at different stages during larvae development by moving the animals from 18°C to 29°C. Analysis of the melanotic nodule phenotype and temperature shift experiments were performed as previously described.11
RNA extraction and quantitative analysis
RNA was extracted using standard procedures. Expression levels of Dab1 and Dab2 were evaluated by real-time poly- merase chain reaction using specific on-demand kits (Hs00245445_m1 for ABL1, Hs00221518_m1 for Dab1, Hs00184598_m1 for Dab2, Applied Biosystems, ThermoFisher Scientific) according to published methods.12
Results
Expression of human BCR-ABL1 affects eye cell differentiation
The aim of this work was to set up a CML Drosophila model based on the expression of a completely human BCR-ABL1 fusion protein. Available Drosophila genetic tools allow expression of proteins of interest in develop- ing eye cells, often inducing viable and visible phenotyp- ic traits that can be used as a bait in genetic screening. The Drosophila eye differentiates during the third instar larva (L3) from the eye imaginal disc, a monolayer epithelium that is accessible to dissection. We generated several stable transgenic fly lines to express BCR-ABL1 protein using the yeast Gal4/UAS (Upstream Activating Sequence) transcriptional regulation system controlled by a gene promoter active in specific tissues and stages (Gal4 drivers).13 BCR-ABL1 expression was first triggered with the sevenlessGal4 (sevGal4) construct that drives
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haematologica | 2019; 104(4)