VK3-BS targets murine and human CML stem/progenitor cells through DYRK2. (A) Analysis of HSCs in bone marrow of wild-type mice after 2 weeks of daily administration of VK3-BS. (B) GFP⁺ LSK cells were isolated from mice 8 dpt with BCR-ABL1–transduced HSCs to confirm DYRK2 upregulation upon in vitro incubation with VK3. CML mice were treated daily for 2 weeks with VK3-BS or vehicle starting at 8 days posttransplant (arrows). After treatment, bone marrow cells were analyzed by flow cytometry and transplanted into secondary mice to evaluate residual leukemia stem/progenitor cells with leukemia-initiating capacity. (C) Detection of myeloid leukemia cells and frequency of GFP⁺ LSK cells in whole bone marrow of primary CML at the end of treatment (n = 8, mean ± standard deviation [SD]). (D) Frequency of GFP⁺ Gr1⁺ leukemic cells at 24 dpt and overall survival in secondary CML. (E) Annexin V staining of bone marrow cells from chronic-phase CML patients (Pt) and healthy individuals incubated in vitro with vehicle or VK3 for 48 hours (triplicates, mean ± SD). (F) Stabilization of DYRK2 protein leading to the phosphorylation of p53-S46 and c-Myc reduction in bone marrow from 2 CML patients by treatment with VK3 compared with those in bone marrow cells from a healthy donor. (G) Colony formation in methylcellulose media of CD34⁺ cells from chronic-phase CML patients and normal individuals treated with VK3 for 24 hours (triplicates, mean ± SD) (left panel). Representative images of colonies from CD34⁺ CML cells treated with 5 μM VK3 (right panels) and control. (H) Experimental design for the CML xenograft model: CD34⁺ stem/progenitor cells from CML patients were transplanted into sublethally irradiated NSG mice (5 × 10⁴ cells per mouse); engraftment was confirmed by flow cytometry after 4 weeks, and mice were randomized for daily treatment (arrows) with vehicle or VK3 for 2 weeks. (I) Detection of human CD45 blood cells (hCD45) vs murine CD45 blood cells (mCD45) was determined by flow cytometry in blood and qPCR detection of BCR-ABL in bone marrow at the end of treatment (see also supplemental Figure 17). The data are representative of ≥3 independent experiments. *P < .05,**P < .01, ***P < .001, ****P < .0001, 2-tailed Student t test and the log-rank test. BMT, bone marrow transplantation; n.d., not detected; n.s., not significant.