EphA3 Kinase Is Constitutively Activated in Chronic Myeloid Leukaemia during Progression to Accelerated and Blast Crisis and It Could Represent a New Molecular Target.

Abstract

Eph receptors tyrosine kinases are involved in many key developmental processes. Ephs are overexpressed or mutated in solid tumours suggesting their possible role in oncogenesis. The majority of the genes and processes involved in CML progression are still unknown. The aim of this study was to investigate the role of EphA3 in CML progression and to explore the possibility to selectively target EphA3 with a monoclonal antibody. The expression of EphA3 and ligands were analyzed by RQ-PCR in 92 samples from 76 CML patients and in 38 healthy controls. 56 patients were in chronic phase (CP) enrolled in TOPS (Cortes, EHA, 2008) studies, 10 in accelerated phase (AP) and 10 in blast crisis (BC). In 5 patients and 10 healthy subjects EphA3 was studied also in enriched CD34+ population. Protein expression and localization were examined using Western Blot and immunofluorescence using specific antibodies. EphA3 expression in CD34+ cells were analyzed by FACS. The effects of EphA3 overexpression were studied by transfecting EphA3 plasmid in 293T e COS cells. In addition, sequencing of EphA3 TK domain was performed in 45 EphA3+ patients. BM, PB and CD34+ cells from CP CML patients expressed very low levels of EphA3, similar to normal controls with a median of 2−Δ ΔCt of 0,0018. By contrast, during the advanced phases of disease (AP and BC) we found high transcript levels (mean of 2−Δ ΔCt 43 and 67 respectively). Moreover, purified CD34+ CML cells presented significantly higher levels as compared to the unfractioned sample (p=0,001). RT-PCR showed no increase of the expression of the ligands Ephrin A2,A3,A4,A5 and B2, all of them able to bind to the receptor EphA3. WB and immunofluorescence confirmed the presence of phosphorylated protein in EphA3 in AP and BC CML cells. Western Blot and immunofluorescence confirmed the presence and the high level of phophorylation of EphA3 protein in AP and BC, but not in CP cells. EphA3 transfection into normal cells results in loss of adhesion, cell rounding and increased proliferation. Incubation of CML cells or transfected cells with a specific antibody able to block the EphA3 receptor induced a significant reduction of proliferation (31±4% of reduction in primary cells and 45±9% in transfected cells), it reduced colony growth (median value of 34,2 vs 76,5) and mainly, it changes the adhesion properties. The antibody did not induced any significant change of proliferation or colony growth in CP cells and normal controls. Finally, no kinase domain mutations were found in EphA3 overexpressing cells. In conclusion, our experimental data have shown that EphA3 was abnormally expressed in cells derived from advanced stage CML. The ectopic expression of EphA3, as demonstrated in our experimental system is responsible for increased proliferation and loss of cell adhesion. The inhibition of EphA3 induced by a specific antibody results in proliferation and colony growth inhibition. Although preliminary, our findings suggest that EphA3 may represent a potential candidate for a molecular therapy in advanced phase of chronic myeloid leukaemia. This investigation was conducted by CML Correlative Studies Network (CCSN), TOPS, which is sponsored by Novartis Oncology