Nilotinib Inhibits Bcr-Abl Kinase Activity in CML Progenitor Cells More Effectively Than Imatinib but Is Equipotent in Inducing Growth Inhibition.

The therapeutic success of imatinib mesylate (IM) in chronic myeloid leukemia (CML) is impaired by persistence of malignant stem cells. Mechanisms contributing to incomplete elimination of CML stem cells include resistance to proapoptotic effects of IM, persistent signaling through growth stimulating pathways and Abl kinase mutations resulting in IM resistance. We investigated whether nilotinib, a more potent Bcr-Abl tyrosine kinase (TK) inhibitor than IM, could more effectively target CML progenitors. CD34+ progenitors from CML patients and healthy donors were cultured with nilotinib (0–10μM) and IM (0–5μM) in growth factor (GF) containing medium. After 96h incubation, cells were harvested and assayed for colony-forming-(CFC) and long-term culture initiating cells (LTCIC). CD34+38- primitive progenitor cells (PPC) and CD34+38+ committed progenitor cells (CPC) labeled with CFSE were incubated under similar conditions and evaluated for proliferation and apoptosis assays by annexin-V staining and FACS analysis. The IC50 for CML LTCIC-suppression was 0.9μM for nilotinib and 0.8μM for IM. Normal LTCIC were not significantly suppressed at doses below 10μM nilotinib and 5μM IM. The IC50 for CML CFC suppression for nilotinib was 6.4μM and 1.0μM for IM. Normal CFC showed less growth inhibition and the IC50 was not reached at the dose range tested. CFSE assays indicated dose-dependent antiproliferative activity of both compounds with IC50-values of 3.0μM nilotinib and 1.8μM IM for CML PPC, and 8.2μM nilotinib and 4.0μM IM for CML CPC. In CML PPC, apoptosis significantly increased following nilotinib treatment (8±2.1% [control] to 22±2.7% [10μM], n=4, p=.015). IM treatment also increased apoptosis in CML PPC (to 35±3.8% [5μM], n=4, p=.0008). In CML CPC, apoptosis increased from 33±2.3% [control] to 50±1.7% (n=4, p=.002) with 10μM nilotinib, and to 53±2.1% (n=4, p=.001) with 5μM IM exposure. The effects on IM and nilotinib on Bcr-Abl-TK activity were investigated by Western blotting with anti-CrkL antibodies after overnight drug exposure of CML CD34+ cells in GF containing media. Importantly, 0.25μM nilotinib significantly reduced P-CrkL levels (from 85.7±4.1% [control] to 13.9±4.5%, n=4, p=.000002), whereas a higher concentration of IM (1–5μM) was needed to achieve similar inhibition. Nilotinib resulted in increased MAPK activity (to 13.3-fold [5μM]) in CML CD34+ cells. MAPK kinase activity was also increased following treatment with IM (to 22.7-fold [5μM]). P-AKT and P-STAT5 levels were not significantly changed in response to either drug. In contrast incubation with either drug in the absence of GF resulted in inhibition of MAPK, Akt and STAT5 activity in CML CD34+ cells. In conclusion, nilotinib is significantly more potent than IM in inhibiting Bcr-Abl TK in CML progenitors but does not induce greater suppression of progenitor growth. As with IM, inhibition of cell division represents the predominant mode of inhibition of progenitor growth. Nilotinib or IM treatment inhibits Bcr-Abl dependent MAPK, STAT5 and Akt activity, but does not inhibit these signaling mechanisms in CML progenitors in the presence of GF. Our results suggest that combined inhibition of Bcr-Abl TK and additional signaling mechanisms may be required to achieve elimination of CML progenitors by targeted therapies.