WNT974 in combination with nilotinib inhibits the proliferation and clonogenic potential of human CML primitive and committed progenitor cells. Normal and CML 34⁺ cells (n = 4-5) were labeled with CFSE. CFSE⁺ primitive cells (34⁺38⁻) and committed cells (34⁺38⁺) were sorted by flow cytometry and cultured in the presence or absence of MSC for 6 days with WNT974, Nil (1 µM), or both, or left untreated. A proliferation index was calculated on the basis of reduction in CFSE levels for CML 34⁺38⁻ cells (A) and for normal 34⁺38⁻ cells (B). Cells were also plated in methylcellulose progenitor assays, and colony-forming cell (CFC) frequencies were determined after 14 days for CML 34⁺38⁻ cells (C) and for normal 34⁺38⁻ cells (D). CML 34⁺ cells (n = 3) were labeled with CFSE, CD34, CD38, and CD26, and CD34⁺CD38⁻CD26⁺ and CD34⁺CD38⁻CD26⁻ cells were selected by fluorescence-activated cell sorting (E) and cultured in the presence of MSC for 6 days with WNT974, NIL, or both, or left untreated. (F) Example CFSE plots of one CML sample are shown. Proliferation index was measured on the basis of reduction of CFSE levels and both actual values (G), and fold change in relation to untreated controls (H) are shown. Error bars represent mean ± SEM. FSC, forward scatter; Nil, nilotinib; w, with; w/o, without. *P < .05; **P < .01.