The critical mechanisms underlying human hematopoietic stem cell (HSC) transformation by the BCR-ABL gene in chronic myeloid leukemia (CML) are still not well understood. Since treatment with imatinib fails to eliminate primitive CML hematopoietic cells, there is a pressing need to identify additional mechanisms that can be targeted to enhance elimination of CML stem cells. A tyrosine residue at position 177 (Y177) in the BCR-ABL protein binds the adapter protein Grb2 and appears to play an important role in BCR-ABL induced myeloid leukemogenesis in murine CML models. We have recently shown that a tyrosine to phenylalanine mutation of Y177 (Y177F) also results in significant reduction of abnormalities in proliferation and differentiation in BCR-ABL expressing human CD34+ cells and in BCR-ABL induced activation of Ras, Akt and STAT5 signaling (
). Since Grb2 signaling is implicated in several other human malignancies and likely plays an important role in signaling downstream of BCR-ABL, we were interested in investigating the role of Grb2 in BCR-ABL-mediated transformation of primary human hematopoietic cells. Cord blood (CB) CD34+ cells were transduced with bicistronic retrovirus vectors coexpressing wild type or Y177F mutated BCR-ABL genes with the GFP gene. Coimmunoprecipitation studies confirmed the association of Grb2 with BCR-ABL and was abrogated by the Y177F mutation. We next investigated the effect of inhibition of Grb2 expression in BCR-ABL transduced human CD34+ cells. CB CD34+ cells were co-transduced with retroviral vectors coexpressing the BCR-ABL and GFP genes or control vectors expressing the GFP gene alone together with lentivirus vectors coexpressing Grb2 shRNA constructs and the dsRed gene or the dsRed gene alone. Cells expressing CD34+, GFP and dsRed were selected using flow cytometry. Western blot analysis indicated that Grb2 levels were reduced by 80.8±14.5% (n=3, P < 0.014); in CD34+ cells transduced with BCR-ABL and Grb2 shRNA compared with cells expressing BCR-ABL alone. Expression of Grb2 shRNA resulted in significant reduction in expansion of BCR-ABL expressing CD34+ cells compared with cells expressing BCR-ABL alone after 7 days of culture in serum free medium (SFM) with low concentrations of growth factor similar to those present in bone marrow stroma conditioned medium (BA alone 48.5±7.7; BA+Grb2 shRNA, 16.7±4.7, n=3). Co-expression of Grb2 shRNA also resulted in significant reduction in the total number of colonies generated by BCR-ABL expressing CD34+ cells in methylcellulose progenitor culture (BA, 123±31; BA+Grb2, 40±9; n=3, p<0.03), with reduction being seen mainly for erythroid colonies. Expression of Grb2 shRNA also reduced cell expansion from control CD34+ expressing GFP alone but the difference was not statistically significant. Similarly Grb2 inhibition did not result in a significant difference in the number of colonies generated from control CD34+ cells. Grb2 inhibition was associated with reduced levels of P-MAPK, but not P-AKT in BCR-ABL expressing CD34+ cells. Interestingly Grb2 inhibition also results in decreased levels of P-STAT5 consistent with the observed reduction in erythroid colonies. In conclusion RNAi mediated inhibition of Grb2 expression results in significant inhibition of BCR-ABL induced proliferation of human CD34+ cells proliferation, indicating an important role for Grb2 in BCR-ABL mediated transformation of CML cells. These observations support further evaluation of inhibition of Grb2 signaling in targeting of CML stem cells.
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