Effect of Dasatinib on BCR-ABL and Src Mediated Growth Signaling in Primary CML Hematopoietic Progenitors.

Resistance of malignant progenitors to elimination remains a problem in chronic myeloid leukemia (CML) patients in remission with Imatinib (IM) treatment. Potential mechanisms by which CML progenitors may resist elimination include incomplete inhibition of BCR-ABL tyrosine kinase (TK) activity and persistent activation of growth factor (GF) mediated signaling in IM treated cells. We investigated whether Dasatinib (DAS), a potent BCR-ABL and Src TK inhibitor, demonstrates increased efficacy in inhibiting BCR-ABL and Src dependent growth signaling in CML progenitors. We observed P-Src levels to be increased in CML compared with normal CD34+ and CD34+38– cells. In addition P-Src levels were increased in CD34+ cells from advanced phase compared with chronic phase (CP) CML patients. Culture of CD34+ progenitors from CP CML patients with DAS (0.05–0.15μM) resulted in effective suppression of P-Src levels (to 1.9±1.9% of controls with 0.15μM DAS, p<0.001, n=5) compared to only modest suppression with 5μM IM (64±24%, n=5). Treatment with DAS reduced P-CrkL levels, reflecting BCR-ABL TK activity, to 13±3% of control (n=5, p<0.001), with similar levels of suppression achieved using 5μM IM (12±5%, n=5, p<.001). Incubation of CML CD34+ cells with DAS or IM without GF resulted in inhibition of P-MAPK and P-STAT5, likely reflecting inhibition of BCR-ABL mediated activation of these pathways, but did not significantly affect P-AKT levels. Treatment with IM in the presence of exogenous GF induced increased P-MAPK levels in CML CD34+ cells (11±4 fold increase vs. controls, n=6). However DAS treatment did not induce a significant increase in P-MAPK (2±1 fold change, n=6), suggesting a role for Src signaling in increased MAPK activity in IM-treated cells. P-AKT and P-STAT5 levels were not significantly altered by DAS or IM in the presence of GF. Incubation of CML CD34+ cells with DAS for 96 hours resulted in 63±7% suppression of LTCIC (p<0.001), which was similar to 5μM IM (67±9%, p=0.001). CML CFC growth was similarly suppressed by DAS (70±8.%, n=3) and IM (73±10%). DAS resulted in increased suppression of normal LTCIC (45±5%, p<0.001, n=3) compared to IM (19±11%, NS). CD34+38– and CD34+38+ cells were labeled with CFSE, incubated with DAS or IM for 96 hours and evaluated for proliferation using FACS analysis. Both compounds had significant antiproliferative activity on CML CD34+38– cells (45±11% inhibition with DAS, p=0.005 vs. 70±2% with IM, p<0.001, n=4) and CD34+38+ cells. Annexin V labeling indicated that DAS treatment did not increase apoptosis in CML or normal CD34+38– and CD34+38+ cells at the tested dose range, whereas IM modestly increased apoptosis in CML CD34+38+ cells (to 9±1%, p=0.01, n=5) but not CML CD34+38– cells or normal progenitors. In summary, we show that Src activity is increased in CML CD34+ and CD34+38– cells. DAS is significantly more potent than IM in inhibiting Src activity in CML cells. Unlike IM, DAS does induce increased MAPK levels in CML cells. Despite effective Src and BCR-ABL TK inhibition, AKT signaling remains active in DAS treated CML cells even in the absence of exogenous GF, and MAPK, STAT5 and AKT signaling all remain active in the presence of exogenous GF. Therefore effective Src inhibition does not translate into increased suppression or apoptosis of CML progenitors by DAS, indicating a need to target additional signaling mechanisms to achieve elimination of CML progenitors.