Antileukemic properties of DYRK2 stabilization with VK3. (A) Cell viability of KU-812, K562, and KCL-22 CML cells that were incubated for 48 hours in the presence of VK3 expressed as a percentage of the vehicle control (mean ± standard deviation [SD]). (B) Immunoblot analysis of DYRK2, c-Myc, SIAH1, SIAH2, and PHD3, as well as cleavage of PARP (cPARP) and caspase 3 (cCASP3), in protein lysates of CML cell lines treated with vehicle or 20 μM VK3. (C) Levels of DYRK2, c-Myc, and cleaved PARP (cPARP) in CRISPR/CAS9-mediated DYRK2-knockout K562 cells treated with vehicle or 12 μM VK3 for 48 hours. (D) Effect of VK3 on DYRK2 ubiquitination was analyzed in K562 cells treated with MG-132 and VK3 by immunoprecipitation with anti-DYRK2, and immunoblots were revealed with anti-ubiquitin. DYRK2 level is shown in protein lysates. (E) VK3 cytotoxicity in K562 cells with the DYRK2 gene knocked out by CRISPR/CAS9 compared with that in the parental cell line (n = 3, mean ± SD) (left panel) and immunoblotting of DYRK2 gene deletion (right panel). (F) Dose-dependent induction of apoptosis in K562 cells with p53 overexpression. (G) Effect of the combination of VK3 and IM on the viability of K562 cells (mean ± SD). (H) Isobologram analysis of VK3 and IM combination. (I) Cytotoxicity of VK3 in K562 cells resistant to IM (IM R), generated by culture in the presence of 2.5 μM IM, compared with that in parental cells (mean ± SD). (J) Cell viability (left panel) and immunoblots (right panel) of K562 cells incubated with menadione (VK3) or VK3-BS (mean ± SD). DYRK2 upregulation is shown (right panel). The data are representative of 2 or 3 independent experiments. *P < .05, ***P < .001, ****P < .0001, 2-tailed Student t test. Fa, fraction affected.