Disabled Gene Is Involved in CML Progression and Its Expression Level at Diagnosis Can Predict Major Molecular Response (MMR) to Imatinib Therapy

Despite the role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, the mechanisms leading to CML progression remain unknown. By using our model of Drosophila melanogaster (Dm) transgenic for human Bcr-Abl we have identified Dab1 and Dab2 as genes involved in CML progression. In Dm the loss of function of these genes induced a worsening of the phenotype generated by hBcr-Abl expression. Moreover, an even stronger phenotype was obtained by silencing Disabled using RNAi fly strains.By contrast, Dab gain of function rescued Bcr-Abl phenotype. Disabled is a non receptor tyrosine kinase, identified in Dm as an adaptor protein acting downstream of many tyrosine kinases receptors (RTK). One of the human homolog of Disabled, Dab1 is a large common fragile site gene, involved in neural migration and the other homolog Dab2 codes is an adaptor protein implicated in RTK signalling, endocytosis, cell adhesion and differentiation. The downregulation of both genes is described in many types of cancer suggesting their possible role in oncogenesis but their involvement in haematological malignancies has never been described. The aim of the study was to investigate the role of Dab1/2 in CML progression. Dab1 and Dab2 mRNA was analyzed by Real Time PCR in 94 samples from 82 CML patients (34 PB and 60 BM). Among those patients 55 were collected at diagnosis (19 enrolled in TOPS study), 9 patients in CP collected at the time of imatinib resistance, 7 in accelerated phase and 11 in BC, In addition 21 healthy donors (10 PB and 11 BM). In 18 patients, genes expression was analyzed during remission as well. Protein expression was investigated by Western Blot and Immunofluorescence. In addition, K562 cells were transfected with Dab plasmids to gain insight about the effects of Dab1/2 on cell proliferation and apoptosis. We found that in CML patients Dab1/2 expression levels were significantly decreased in either BM or PB (p<0.002 and p<0.0004) as compared to healthy subjects. Furthermore, in blast crisis Dab1/2 transcript levels are further decreased. In contrast, at the time of remission, the transcript levels were comparable to normal values. Data analysis of patients included in TOPS studies shows that Dab1 values are lower among those achieving MMR by 12 months (median value 2) compared to those without MMR (median 4,59) although this difference does not appear significant (p=0.15). By analyzing all together the CP patients enrolled in different protocols based on imatinib as front line therapy, this difference became significant (p=0,01). Western Blot and immunofluorescence confirmed the absence of Dab1/2 proteins in course of active CML samples while it reappeared during remission. Dab1 and Dab2 transfection of K562 significantly reduced proliferation (p=0,002). In conclusion, our results show a significant decrease of Dab1/2 expression in blast crisis samples, when compared to CP CML and healthy volunteers, which suggest a role of Dab1/2 in slowing down or suppressing a progression. Among CP CML patients the responders to TKI therapy have been detected to express a lower Dab levels of than non responders. We find those data intriguing and suggesting that molecular factors involved in the regulation of CML progression could be uncoupled from the mechanisms regulating response to TKI therapy. Supported by Novartis Oncology, Clinical Development, TOPS Clinical Correlative Studies Network