IL-3 protects CP-CML LSPCs from the cytocidal effect of dasatinib, which is overcome by CSL362. CP-CML CD34⁺ cells were cultured with IL-3 (1 ng/mL), dasatinib (10 nM), and/or CSL362 or BM4 isotype-matched control antibody (both 1 µg/mL), as indicated, for 3 days. Cell viability was assessed using AnnexinV/7-Aminoactinomycin D flow cytometry assays (A) n = 7, whereas effects on cell proliferation were determined in CFSE-labeled CP-CML CD34⁺ cells; (B) n = 4, using flow cytometry, where the percentage of undivided cells was estimated using the proliferation tool in FCS Express. (C) CFU (n = 7) and (D) LTC-IC (n = 3) potential were monitored in parallel assays. (E) Combination of CSL362 and dasatinib effectively blocks STAT5 phosphorylation in IL-3–stimulated CP-CML progenitors. CD34⁺ cells of CP-CML patients were pretreated for 20 minutes with dasatinib (100 nM) and/or CSL362 or BM4 control antibody, respectively (both 10 µg/mL), prior to IL-3 (20 ng/mL) stimulation for 10 minutes (as indicated). Fixed and permeabilized cells were subsequently stained with conjugated antibodies against pY694-STAT5, CD34, and CD38 and analyzed by flow cytometry. (F) Quantitation of pSTAT5 flow cytometry data for CD34⁺ and CD34⁺/CD38^– subpopulations as described in panel E from 3 independent experiments is shown. *P < .05; **P < .01 (by unpaired Student t test).