Figure 5

Mutated p53-dependent induction of CBFB contributes to the resistance to RUNX1 inhibition therapy. (a) Schematic diagram of the procedure to establish Chb-M’-resistant MV4-11 clones (MV4-11M’R) and subsequent mutation analysis by next generation sequencing (NGS). (b) Calculation of IC50 of Chb-M’ in Chb-M’-naïve and Chb-M’-resistant MV4-11 cells (MV4-11M’R) (n = 3). (c) Induction of p53 and CBFB in MV4-11 M’R cells. Cell lysates prepared from the parental MV4-11 and MV4-11M’R cells were analyzed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control. (d) Chb-M’ treatment further stimulates the expression levels of p53 and CBFB in MV4-11M’R cells. MV4-11M’R cells were exposed to the indicated concentrations of Chb-M’. Twenty-four hours after treatment, cell lysates were prepared and subjected to immunoblotting with the indicated antibodies. GAPDH was used as a loading control. (e) Frequent p53 mutation at codon R248W in MV4-11 M’R cells. Genomic DNA was prepared from MV4-11 M’R cells according to the standard procedure, and analyzed for p53 mutations by next generation sequencing. (f) Schematic drawing of the transplantation assay in NOG mice. Chb-M’ treatment (twice/week) was continued until the recipient mice show the sign of leukemia development. AML cells were then extracted from sacrificed mice with leukemia. (g) Chb-M’-resistant AML cells highly express p53 and CBFB. Cell lysates prepared from parental, control and resistant cells were analyzed by immunoblotting with the indicated antibodies. GAPDH was used as a loading control.