Figure 3.

WP1130 reduces the viability of cells expressing wild-type and mutant Bcr/Abl. (A) BaF/3 cells (1 × 10⁴) expressing wild-type or mutant Bcr/Abl (T315I, L364I, E355G) were treated with the indicated concentration of WP1130 or imatinib mesylate for 72 hours before analysis of cell viability (MTT staining). IL-3–dependent BaF/3 cells (parental) were also screened for drug sensitivity. The results represent the average ± SEM of 4 assays. (B) BaF/3 (2 × 10⁶) (parental) cells were treated with IL-3 (1 ng/mL) alone or pretreated for 2 hours with 5 μM WP1130 before IL-3 incubation for 15 minutes. Cell lysates were prepared, and Jak2 was immunoprecipitated and immunoblotted with anti-pY (top) followed by anti-Jak2 (bottom). Total cell lysates were also blotted for pStat5 and Stat5. (C) CD34⁺ cells from 2 bone marrow donors (BM) or 3 patients with newly diagnosed CML were cultured with WP1130 at the concentration indicated before analysis of effects on colony formation as described in “Materials and methods.” Results are reported as the percentage of colonies in control samples (vehicle alone). Analysis was performed in triplicate, and the results are presented as mean ± SEM. (D) Fractionated bone marrow cells obtained from patients in CML myeloid and lymphoid blast crisis were cultured in AML and ALL colony culture assays, respectively. Data are reported as the percentage of control of duplicate cultures. Patient 1 was in lymphoid blast crisis and patient 2 was in myeloid blast crisis.