Targeting ANP32B impairs human CML development and synergizes with imatinib therapy to eradicate CML (A-B) Relative ANP32B protein expression levels in bone marrow mononuclear cells (BMMCs) of patients with CML and control patients were analyzed (n = 10). All samples were collected at diagnosis before therapy. All patients with CML were diagnosed in chronic phase and were BCR-ABL1 positive. (C) Colony-forming assay for human CML CD34⁺ cells infected with shNC or shANP32B. Numbers of colonies were evaluated at day 10 (n = 3). All samples were collected at diagnosis before therapy. All patients with CML were diagnosed in chronic phase and were BCR-ABL1 positive. (D) shNC- or shANP32B-infected human CML CD34⁺ cells were stably transfected with EV or Flag-tagged ANP32B with the shRNA target sequence mutation, and Western blots of indicated proteins are shown. (E) Colony-forming assay for shNC/EV, shANP32B/EV, and shANP32B/Flag-ANP32B CML CD34⁺ cells. Colony numbers were counted at day 10 after plating (n = 3). (F) Schematic strategy of evaluation of the in vivo effect of ANP32B knockdown in human CML CD34⁺ cells. (G-H) Percentages of human CD34⁺, CD45⁺, and CD45⁺CD11b⁺ cells engrafted in PB (G) and BM (H) 6 months after transplantation (n = 5). Human CD34⁺ cells sorted from CML patient 10 were used in this experiment. (I) Human CML CD34⁺ cells (CML patient 10) infected with shNC or shANP32B were seeded into methylcellulose medium with 5μM imatinib or dimethyl sulfoxide as vehicle. Colony numbers were counted at day 10 after plating (n = 3). (J-K) Survival curves for recipients transplanted with BCR-ABL1–transduced Anp32b^+/+ and Anp32b^−/− Lin⁻ BM cells followed by the treatment with either vehicle or imatinib at day 8 after transplantation (J, n = 5 for Anp32b^+/+/vehicle and Anp32b^−/−/vehicle group, n = 8 for Anp32b^+/+/imatinib, and n = 7 for Anp32b^−/−/imatinib group). Percentages of GFP⁺ cells and GFP⁺LSK cells in BM were analyzed at day 14 after transplantation (n = 5) (K) Error bars denote mean ± SEM. Statistical significance was determined by a 2-tailed, unpaired Student t test (B-C,E,G-I) or log-rank test (J). The experiments in panels J-K were repeated twice with similar results, and the results of 1 representative experiment are shown. The experiments in panels A-H are presented from an independent experiment.