Pre-Stimulation of CML CD34+ Cells with High Concentrations of Growth Factors Counters Imatinib-Mediated Proliferation Suppression and Enhances Apoptosis of Non-Dividing Cells.
Authors: Holtz Melissa S. , Forman Stephen J. , Bhatia Ravi 
Published: April 2020

Abstract
While imatinib mesylate is a highly effective treatment for CML, there is accumulating evidence that it may not adequately target quiescent malignant HSCs. In vitro exposure to imatinib inhibits CML progenitor growth primarily through suppression of abnormally enhanced proliferation. Apoptosis appears to be restricted to dividing cells while quiescent progenitors are resistant to apoptosis. One approach to more effectively enhance the sensitivity of HSCs to imatinib may be to induce them to cycle using hematopoietic growth factors (GF). We have shown that exposure of CML CD34+ progenitors to imatinib (1μM) in high GF conditions (100ng/ml SCF and FL3, 20ng/ml IL6, G-CSF and IL3) reduced the total number of viable, undivided cells compared to control cells cultured in 100-fold lower GF conditions (low GF). High GF treated cells were more proliferative but less sensitive to imatinib-mediated apoptosis (Blood2004, 104:2967). We hypothesized that pre-stimulation with high GF prior to imatinib exposure would further reduce viable, non-dividing CML progenitors. CML CD34+ cells were cultured in high GF for 48 hours and then exposed to imatinib (1μM) for 48 hours in either high or low GF conditions. Compared to cells exposed to imatinib without any pre-stimulation, high GF pre-stimulation significantly reduced imatinib-mediated inhibition of proliferation in both low GF (22±5%, p=0.0009) and high GF (18±3%; p=0.0003). Pre-stimulation decreased imatinib-mediated apoptosis when compared to the same conditions with no pre-stimulation [19±2% for imatinib treatment in low GF (p&lt;0.0001) and 7±3% in high GF (p=0.064)]. However, although overall apoptosis decreased, pre-stimulation resulted in increased apoptosis of undivided cells exposed to imatinib in either low GF (14±5%; p=0.022) or high GF (13±6%, p=0.065). These results are notable since increased apoptosis of undivided cells was not previously observed in any other condition. Importantly, the percent of input cells remaining viable and undivided decreased significantly for pre-stimulated cells exposed to imatinib in low GF (19±3%; p&lt;0.0001) or high GF (7±2%; p=0.016). These results highlight the potential use of GF stimulation to enhance targeting of CML HSC. Additional studies examined whether GF readily available for clinical use (G-CSF and/or GM-CSF) could also enhance imatinib targeting of quiescent CML progenitors. CML CD34+ cells were exposed to 1mM imatinib for 96 hours in a basal low GF cocktail (250pg/ml G-CSF, 10pg/ml GM-CSF, 200pg/ml SCF, 1ng/ml IL6, 200pg/ml MIP1α, 50pg/ml LIF) alone or with the addition of G-CSF (50ng/ml) and/or GM-CSF (10ng/ml). While overall apoptosis decreased, apoptosis of undivided cells significantly increased for cells exposed to imatinib in high concentrations of G-CSF + GM-CSF compared to those in basal GF alone (8.7±1.3%; p=0.007). The percent of input cells remaining viable and undivided in the presence of imatinib significantly decreased with high G-CSF + GM-CSF compared to basal GF alone (7.2%±1.1; p=0.007). In conclusion, pre-stimulation with high concentrations of GF can lead to increased proliferation and enhance reduction of non-dividing CML CD34+ cells by imatinib. These results are of significance because non-dividing primitive cells have previously proven highly resistant to elimination by imatinib and support translational clinical studies to investigate whether intermittent GF administration can enhance elimination of residual CML stem and progenitor cells in patients in remission on imatinib treatment.