CEP-701 inhibits engraftment of FLT3/ITD cells in NOD-SCID mice. (A) Immunoblot of leukemia blasts (from patient sample 1) incubated for 1 hour in culture medium with or without 50 nM CEP-701. The blots were probed with antiphosphotyrosine antibody (top blot), then stripped and reprobed with anti-FLT3 antibody (bottom blot). (B) CFU-L assay. NOD-SCID mice injected with 5 × 10³ CD34⁺/CD38^- cells obtained using immunomagnetic selection of the sample shown in panel A were treated with vehicle only or with CEP-701. At 14 weeks, the mice were killed. Bone marrow cells harvested from engrafted mice were cultured in methylcellulose medium for CFU-L formation. Each column on the graph represents the average number of colonies obtained from all marrows within a treatment group. The number of colonies is expressed per 1 × 10³ human CD45 cells plated. Error bar represents SD. (C) Genomic DNA was prepared from bone marrow cells and PCR was performed to amplify the juxtamembrane region of FLT3, yielding the characteristic doublet of the mutant gene. Following electrophoresis in 2.5% agarose, the ethidium bromide-stained gels were visualized under ultraviolet light. HL indicates control DNA obtained from HL60 cells (which harbor only FLT3 wild type); C, control DNA from the unsorted leukemia sample prior to injection into mice.