GM-CSF overcomes NI-induced proliferation inhibition of primary CD34+ progenitors by JAK-2/STAT-5 pathway activation. (A) CD34+-enriched progenitors from patients with first-diagnosis CML (n = 4) were cultured in vitro in IMDM medium (+ 5 growth factors) in the presence of 10 μM NI plus 10 ng/mL of either 1 of the 3 test cytokines (GM-CSF, IL-3, or G-CSF) as indicated. After 72 hours, cells were seeded in soft agar, and 10 to 14 days later, emerging colonies (CFCs) were counted. Bars represent mean fold increase ± SD in CFC counts obtained after NI exposure in the presence of indicated test cytokines versus a standard cytokine cocktail only. *P < .05 (1-way ANOVA Dunnett adjustment for multiple comparisons). (B) BCR/ABL-independent STAT-5 activation by GM-CSF in primary CML progenitors. CD34+-enriched primary CML progenitors of patient no. 7 (CML at diagnosis) were treated for 48 hours with NI, the JAK-2 inhibitor AG490 (100 μM) with or without GM-CSF (10 ng/mL), and were analyzed by FACS for the regulation of ic-p-STAT-5 (top 2 histograms) and ic-p-CrkL (bottom histograms) according to the indicated gating strategy: CD34high/SSClow cells were gated into CD34high/CD116low (gate R1) or CD116high (gate R2) and separately analyzed for R1 and R2. The gray curves in each histogram plot represent baseline expression levels; the colored curves represent different treatments as indicated. (C) CD34+-enriched progenitors from a patient with first-diagnosis CML (patient no. 3) were cultured in vitro in a liquid culture containing a cytokine cocktail of 5 growth factors (± GM-CSF, as indicated) in the presence of NI or not and/or AG490. After 72 hours, cells were seeded in triplicates in soft agar and emerging colonies (CFCs) were counted 10 to 14 days later. Bars represent mean CFC counts ± SD of triplicates.