Effect of Growth Factor Stimulation on Imatinib-Mediated Proliferation Inhibition and Apoptosis of CML CD34+ Cells.

Imatinib mesylate (Gleevec) is a highly effective treatment for CML, but the long-term durability of response is unclear. We have identified residual BCR/ABL+ progenitors in patients achieving CCR on imatinib (Blood 2003, 101:4701). We have shown that imatinib inhibits CML progenitors through suppression of abnormally enhanced proliferation (Blood 2002, 99:3792) and that apoptosis is restricted to dividing cells with quiescent progenitors being resistant to apoptosis (Blood 2003, 102:652a). Here we have investigated whether growth factor (GF) stimulation of imatinib-treated CML progenitors could enhance proliferation and sensitivity to apoptosis and reduce the number of viable quiescent cells. CML and normal CD34+ cells were analyzed for apoptosis following culture in the presence or absence of imatinib (1μM) for 72 hours with either low (1μg/ml SCF and FL3, 0.2μg/ml IL6, G-CSF and IL3) or high (100μg/ml SCF and FL3, 20μg/ml IL6, G-CSF and IL3) GF concentrations. Cells were labeled with CFSE prior to culture to track cell division and allow separate examination of apoptosis in quiescent and proliferating cells. High GF conditions increased proliferation of both normal and CML CD34+ cells. Suppression of CML progenitor proliferation by imatinib was reduced in high GF conditions, compared to low GFs, when IL-3 was present (p=0.009), but not when IL3 was absent (p=0.065). Interestingly, normal CD34+ cells stimulated with high GFs demonstrated increased sensitivity to imatinib-mediated inhibition of proliferation, both with (p=0.005) and without IL3 (p=0.013). High GF conditions also resulted in reduced apoptosis of CML CD34+ cells, both with (p=0.003) and without IL-3 (p=0.001). In contrast, normal CD34+ cells demonstrated increased sensitivity to imatinib-induced apoptosis in high GFs. Imatinib did not induce apoptosis in undivided cells under either low or high GF conditions. For proliferating CML CD34+ cells we observed a reduction in apoptosis induced by imatinib in high GF conditions (+IL3: p=0.012; −IL3: p=0.019). The percent of cells used to initiate culture that remained as viable, undivided cells following exposure to imatinib was calculated. There was a trend towards a reduction in the number of quiescent, viable CML CD34+ cells after imatinib treatment in high GF conditions (p=0.071). However, high GF conditions also led to a reduction in the number of quiescent, viable normal cells. In conclusion, we show that high GF conditions counter imatinib-mediated proliferation suppression in CML CD34+ cells but also protect cells from apoptosis. GF stimulation also enhances the sensitivity of normal CD34+ cells to imatinib. A trend towards a reduction in viable, quiescent CML progenitors was seen, even with short-term GF exposure, supporting further evaluation of the use of GF stimulation to reduce residual CML progenitors during imatinib treatment. Effect of Imatinib on CD34+ Cells | | | Low Growth Factors | High Growth Factors | |:---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------:| -------------- | ------------------ | ------------------- | ---------- | | ¹relative to cells not exposed to imatinib. ²CFSE compared to starting population: less=divided; same=undivided. ³% of input cells that remain undivided & non-apoptotic with imatinib. | | | | +IL3 | −IL3 | +IL3 | −IL3 | | Proliferation Suppression¹ | CML | 54 ± 2 | 56 ± 2 | 45 ± 2 | 52 ± 1 | | | Normal | 25 ± 4 | 30 ± 4 | 51 ± 1 | 53 ± 2 | | Apoptosis Increase¹ | CML | 23 ± 2 | 29 ± 3 | 17 ± 3 | 20 ± 3 | | | Normal | 3.4 ± 3.6 | 4.4 ± 3.9 | 13 ± 4 | 13 ± 2 | | | CML-Divided² | 27 ± 3 | 41 ± 8 | 9.9 ± 1.8 | 8.6 ± 2.5 | | | CML-Undivided² | −4.1 ± 3.7 | −2.8 ± 4.1 | −1.4 ± 5.5 | 0.6 ± 5.0 | | % remaining undivided and viable³ | CML | 25 ± 7 | 22 ± 5 | 20 ± 8 | 22 ± 8 | | | Normal | 46 ± 11 | 46 ± 12 | 25 ± 2 | 37 ± 7 |