Effects of Elevated BCR/ABL Expression Level on Cellular Transformation and Sensitivity to Imatinib in Primitive Human Hematopoietic Cells.

Increased levels of BCR/ABL (BA) expression in CML hematopoietic cells have been associated with disease progression and resistance to the tyrosine kinase inhibitor imatinib mesylate (IM). Although cell lines with varying levels of BA expression have been studied, the role of elevated BA expression in cell transformation and drug resistance has not been directly evaluated in the primitive human hematopoietic cells in which the disease arises. Here we have used a human transduction model of CML to determine the effects of varying BA expression levels on cellular transformation (proliferation, apoptosis and differentiation) as well as responsiveness to IM. Cord blood (CB) CD34+ cells were transduced with MSCV vectors expressing BA and GFP, or control vectors expressing GFP alone followed by CD34+GFP+ cells selection by flow cytometry sorting. For BA expressing cells, two separate populations were selected based on low or high GFP expression (BA lo and BA hi ). Quantitative RT-PCR analysis confirmed increased expression of BA in high GFP expressing cells (5.9±1.5 fold increase in BA:B2M levels in BA hi compared with the BA lo cells, n=3). The proliferation rate of BA hi cells, measured by fold expansion after 3 days of growth factor (GF) culture, was 2.0±0.2 fold higher than BA lo cells, and 6.7±0.1 fold higher than control (n=3). Upon GF deprivation, BA hi cells demonstrated increased resistance to apoptosis (24.4±11.4%, n=3) compared with BA lo (42±12%, n=3) or control cells (45±12%, n=3). BA transduced CD34+ cells generated higher numbers of glycophorin A+ cells than control (35.6%) following GF culture for 7 days. This effect was enhanced in BA hi (91.5%) compared with BA lo cells (77.1%). This was accompanied by an increase in CD33+ myeloid cells and a decrease in CD11b+ cells (36.3, 60.1 and 65.2% CD33+ cells and 6.0, 1.8 and 0.5% CD11b+ cells for control, BA lo and BA hi cells respectively). In addition the frequency of CD41a+ megakaryocytic cells was higher in BA hi (7.0%) relative to BA lo (3.1%) and control cells (1.0%). Next, we asked whether elevated expression of BA resulted in altered sensitivity to IM (0.025μM to 1μM) in an MTS assay. We observed that BA hi cells were more sensitive (83% inhibition at 1μM) to the IM compared with BA lo cells (14% inhibition at 1μM). We also investigated whether elevated levels of expression of two BA kinase mutants, M351T and E255K, were associated with altered IM sensitivity in CD34+ cells. The M351T mutation leads to intermediate level of IM resistance in cell lines. As was observed for wild type BA, CD34+ cells expressing higher levels of M351T demonstrated increased sensitivity to IM. On the other hand, cells expressing high and low levels of the E255K mutant, which is associated with high levels of IM resistance, demonstrated similar levels of IM sensitivity. In conclusion, increased levels of BA expression in human CD34+ cells results in enhanced proliferation; increased resistance to apoptosis following GF withdrawal, and altered differentiation with increased expression of erythroid, megakaryocytic and early myeloid markers and reduced expression of mature myeloid markers. Interestingly, expression of high levels of BA was associated with enhanced rather than reduced sensitivity to IM. Taken together these observations suggest that the effects of varying levels of BA expression on imatinib sensitivity in primitive human hematopoietic cells are determined primarily by increased proliferation rather than reduced apoptosis resulting from enhanced BA expression.