A Critical Role for the Grb2-Binding Tyrosine 177 in BCR/ABL Mediated Activation of Ras and Akt and Abnormal Expansion of Hematopoietic Cells in CML.

Chronic myelogenous leukemia (CML) results from transformation of a primitive hematopoietic cell by the BCR/ABL fusion protein. However the critical BCR/ABL-activated signaling mechanisms, besides abnormal tyrosine kinase activity, responsible for the transformed phenotype of hematopoietic progenitors in CML remain poorly understood. In this study, we used a human progenitor model of CML based on ectopic expression of BCR/ABL genes in human primary hematopoietic cells to investigate the effect of Y177 in CML progenitor transformation. Cord blood CD34+ cells were transduced with MSCV vectors coexpressing GFP and wild type BCR/ABL gene (BA) or BCR/ABL gene with a tyrosine to phenylalanine mutation at 177 position (Y177F) or control vector expressing GFP alone (R1). CD34+GFP+ cells were selected by flow cytometry sorting. Our results showed that BCR/ABL expressing CD34+ cells resulted in significant expansion of hematopoietic cells of both myeloid (CD45+CD71-; CD14+) and erythroid (CD45-71+; Gly A+) lineage as well as abnormal expansion of immature erythroid progenitor cells (CD33+ Gly A+). BCR/ABL-Y177F expressing cells demonstrated similar myeloid cell expression as control (GFP alone) but significantly reduced erythroid cell expression compared with wild type BCR/ABL. The Y177F mutation reduced enhanced CD34+ cell division induced by BCR/ABL and reduced BCR-ABL mediated inhibition of apoptosis related to GF deprivation. These results indicate that BCR/ABL-Y177 plays a critical role in BCR/ABL mediated hematopoietic cell proliferation and apoptosis and abnormal expansion of CML hematopoietic cells. To investigate the effects of Y177F mutation on downstream signaling pathways we compared complex formation between BCR/ABL and various downstream proteins, in BCR/ABL and BCR/ABL-Y177F expressing cells. Immunoprecipitation (IP) studies revealed that Grb2, Gab2, Shp2, SOS and p85 PI-3 kinase (PI-3K) coimmunoprecipitated with wide type BCR/ABL, but demonstrated markedly reduced association with BCR/ABL-Y177F. Immunoprecipitation with anti-phosphotyrosine antibody revealed increased phosphorylation of Grb2, Gab2 and Shp2 in wide type BCR/ABL expressing cells and reduced phosphorylation in BCR/ABL- Y177F expressing cells. We further demonstrate that increased Ras and AKT activation seen in BCR/ABL-transformed hematopoietic cells was not observed in BCR/ABL-Y177F expressing cells. STAT5 association with BCR/ABL, phosphorylation, translocation to the nucleus and binding to target sequences was partially reduced in BCR/ABL-Y177F expressing cells. In contrast MAPK activity was reduced in BCR/ABL expressing cells and comparatively increased in BCR/ABL-Y177F expressing cells. We further investigated whether BCR/ABL tyrosine kinase by imatinib could further inhibit the growth of BCR/ABL-Y177F expressing CD34+ cells. We found that BCR/ABL-Y177F expressing CD34+ cells were highly sensitive to inhibition by Imatinib and that the combination of BCR/ABL kinase inhibition and blockage of BCR/ABL-Y177 activated signals could enhance targeting of CML stem cells. In conclusion the current study shows that BCR/ABL-Y177 plays a crucial role for in CML hematopoietic progenitor proliferation by mediating activation of multiple downstream signaling pathways, in particular the Ras and PI-3K/Akt pathways. Our results support further exploration of the combination of BCR/ABL kinase inhibition and blockage of BCR/ABL-Y177 activated signals to enhance targeting of CML stem cells.