WNT974 in combination with nilotinib inhibits the long-term engraftment of human CML stem cells in immunodeficient mice without affecting normal stem cells. Normal (1 × 10⁵ cells per mouse) and CML 34⁺ cells (2 × 10⁶ cells per mouse) were cultured in the presence of MSC and treated with nilotinib (Nil) (1 µM), WNT974 (1 µM), or both, or were untreated for 4 days; they were then transplanted into NSG mice (6-8 mice per group). Mice were killed after 16 weeks, and bone marrow (BM) content of femurs was obtained. Graph showing normal human CD45⁺ cell engraftment in BM (A); human CML CD45⁺ cell engraftment in BM (B); BCR-ABL mRNA levels in CML CD45⁺ cells engrafted in BM (C); lineage marker expression of normal CD45⁺ hematopoietic cells engrafted in BM (D); and lineage marker expression of CML CD45⁺ hematopoietic cells engrafted in BM (E). In a second experiment, CML CD34⁺ cells were transplanted into sublethally irradiated NSG mice, and 3 weeks posttransplantation, mice were treated with NIL (50 mg/kg), WNT974 (5 mg/kg), or the combination of these for 3 weeks. Representative results of fluorescence-activated cell sorter analysis of BM cells (F), frequency and absolute number of total human CD45⁺ cells (G), and aggregate results of the percentage (H) and absolute number (I) of human myeloid progenitors and lineage marker expression in the BM from treated mice are shown. (J) Human CD45⁺ cells were selected and plated in CFC assays. (K) FISH analysis was performed on interphase nuclei of 200 CD45⁺ cells from each treatment arm. Error bars represent mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001.