Proliferation, cell survival, and CrkL phosphorylation of Ph⁺ and Ph⁻ cells after 72 hours of culture with nilotinib with or without IM. (A) The reportedly improved potency of the novel agent was confirmed in our study by ³H-thymidine uptake proliferation assays to determine IC50 for each cell type. In the Ph⁺ cell line K562, IC50 was calculated as 30 nM ± 10 nM for nilotinib as compared with 600 nM ± 60 nM for IM. The IC50 for the Ph⁻ cell line HL60 was 1 μM for nilotinib and 10 μM for IM. (B) Nilotinib reduces P-Crkl expression in K562 in a concentration-dependent manner. Nilotinib (0.05 μM; dark blue line) and IM (1 μM; red line) are approximately equipotent in this regard, confirming the reported 20-fold increased potency of the new agent in cell lines. Baseline P-Crkl for Ph^neg HL60 cells is shown by the dark gray–filled peak; isotype control antibody staining is shown by the black-filled peak; and maximal P-Crkl expression is shown by the no drug control in the light gray–filled peak. (C) It was evident that in the majority of cases the total primary CML cell number surviving 72 hours of exposure to nilotinib, even at 5 μM, was greater than input, although there was a concentration-dependent restriction of the amplification of this output with respect to no drug 5GF-only control. Output cells were found to be Ph⁺ by fluorescence in situ hybridization (FISH), hence the observed cellular proliferation in the presence of drug was not owing to normal (Ph⁻) cells. Representative data from 1 of 5 individual CML CD34⁺ cell samples is shown. (D) In total CD34⁺ cells from individual patients with CML, nilotinib either equally inhibited Crkl phosphorylation with imatinib at equal concentration (Figure 1D, left panel) or failed to inhibit Crkl phosphorylation (Figure 1D, right panel) at 16 hours. Black fill indicates isotype control; dark gray fill, baseline Crkl-P in Ph^neg cells; light gray fill, 5GF-only–treated CML cells; dark blue line, 5 μM-nilotinib–treated CML cells; red line, 5 μM-imatinib–treated CML cells.