DYRK2 inhibits self-renewal in leukemia stem/progenitor cells by inducing c-Myc degradation. (A) Immunoblot analysis of DYRK2, c-MYC, phosphorylated c-MYC (Ser 62) [P-c-Myc (S62)] and phosphorylated p53 (Ser 46) [P-p53 (S46)] expression in GFP⁺ LSK cells purified from Klf4^fl/fl and Klf4^Δ/Δ CML mice. Actin was used as a loading control. (B) Immunoblot analysis of KLF4, DYRK2, c-MYC, and GSK3α expression in the 32D-BCR-ABL1 cell line transduced with short hairpin RNA (shRNA) retrovirus specific for luciferase (Luc) or KLF4. Two different KLF4 shRNAs are shown. (C) Effect of proteasome inhibition with MG-132 in the 32D-BCR-ABL1 cell line transduced with luciferase or KLF4 shRNA retrovirus (sh2) on the expression of c-MYC (total and phosphorylated at Ser 62 or at Thr 58). (D) Analysis of endogenous c-MYC levels in leukemia stem/progenitor cells from CML mice 14 days after transplantation of Klf4^Δ/Δc-Myc^gfp/gfp or Klf4^fl/flc-Myc^gfp/gfp bone marrow cells transduced with BCR-ABL1-RFP retrovirus. c-Myc^gfp/gfp mice express the c-Myc–GFP fusion protein knocked in the c-Myc locus to monitor the expression of c-MYC (GFP) by flow cytometry in RFP(BCR-ABL1)⁺ LSK cells. The percentage of RFP⁺ LSK cells with high c-Myc–GFP is also shown (right panel; n = 5, mean). (E) Survival of mice transplanted with Klf4^Δ/Δ Lin⁻ Sca-l⁺ cells cotransduced with BCR-ABL1-GFP and empty vector–RFP (n = 5) or BCR-ABL1-GFP and c-MYC^S62A mutant–RFP (n = 10). (F) Gr1 expression in leukemic cells (GFP⁺) was determined in peripheral blood at 24 dpt. (G) Upregulation of DYRK2 and c-Myc depletion in K562 cells lacking KLF4. (H) Diagram shows the regulation of p53 and c-Myc by DYRK2 downstream of KLF4 and the effect of loss of KLF4. The data are representative of 2 or 3 independent experiments. **P < .01, 2-tailed Student t test.