Role of BCR/ABL Gene Expression Levels in Determining the Phenotype and Imatinib Sensitivity of Transformed Human Hematopoietic Cells.

Increased levels of BCR/ABL expression in chronic myelogenous leukemia (CML) cells is associated with disease progression from chronic phase (CP) to blast crisis (BC), and resistance to imatinib mesylate (IM) treatment. However, it is not clear if these associations directly result from elevated BCR/ABL expression levels or reflect coincidental occurrence of additional abnormalities. Here we used an experimental model of human CML based on ectopic expression of the BCR/ABL gene in human CD34+ cells to directly investigate the role of increased BCR/ABL expression levels in determining the phenotype of transformed hematopoietic cells and their IM response. CD34+ cells were transduced with retroviral vectors expressing BCR/ABL and GFP. Cells with low and high levels of GFP and BCR/ABL expression (BA^lo and BA^hi) were selected by flow cytometry. We confirmed increased expression of BCR/ABL in GFP high cells by quantitative RT-PCR and Western blot analysis. The average BCR/ABL expression levels in BA^lo cells (0.51 ± 0.19, n=3) were consistent with those observed in CD34+ cells from CML CP (0.36 ± 0.11, n=4) and accelerated phase (AP) patients (0.62 ± 0.08, n=3), and in BA^hi (0.95 ± 0.09, n=3) cells with those observed in CML BC patients (0.98 ± 0.09, n=3). BA^hi cells demonstrated significantly higher expansion after growth factor (GF) culture compared with BA^lo cells (expansion of control cells: 14.3 ± 3.7, BA^lo: 33.7 ± 9.3 and BA^hi: 65.4 ± 16.4, n=4). Increased expansion was related to both increased cell division and reduced apoptosis. The proliferation rate of BA^hi cells, measured by labeling cells with the fluorescent dye SNARF-1, was significantly increased compared with BA^lo cells (proliferation index for controls: 5.3 ± 1.0, BA^lo: 24.3 ± 8.1, and BA^hi: 41.8 ± 11.9, n=4). BA^hi cells demonstrated increased resistance to apoptosis upon GF deprivation, (28 ± 6.7%, n=5) compared with BA^lo (45 ± 8%, n=5) or control cells (56 ± 9.3%, n=5). Interestingly, increased BCR/ABL expression levels in human hematopoietic cells was associated with enhanced IM sensitivity rather than with IM resistance (IC⁵⁰ for BA^hi = 0.025 μM; BA^lo = 0.2 μM), related to both increased inhibition of proliferation (% inhibition of 55 ± 4.3% for BA^hi, vs. 35.5 ± 4.4% for BA^lo cells with 0.1 μM IM, n=4) as well as increased induction of apoptosis (% increase in apoptosis with 0.1μM IM for BA^hi 26.4 ± 4% vs. BA^lo 8.4 ± 3 %, n=5). We did not observe a significant difference in phosphorylation of AKT and MAPK between BA^hi and BA^lo cells, but did observe significantly increased STAT5 phosphorylation in BA^hi CD34+ cells compared to BA^lo cells (pSTAT5:STAT5 for BA^lo = 0.41 ± 0.21, BA^hi = 1.01 ± 0.1). Furthermore, electromobility shift assays (EMSA) confirmed increased STAT5 promoter activity in BA^hi cells compare to BA^lo cells. In conclusion, increased levels of BCR/ABL expression in human CD34+ cells results in increased expansion in hematopoietic cell numbers related to enhanced proliferation and increased resistance to apoptosis. However expression of high levels of BCR/ABL was associated with enhanced rather than reduced sensitivity to IM. These observations suggest that BCR/ABL overexpression may contribute to disease progression in CML but may not play a direct role in IM resistance in cells overexpressing BCR/ABL. Our results suggest a role of increased STAT5 activity in abnormal growth of BCR/ABL overexpressing cells.