Fig. 2: c-MYC is downregulated by YPN-005 and FLT3-TKI combination treatments.

Decreased expression of FLT3 signaling molecules and RNA polymerase by the combined treatment of FLT3-ITD-mutated AML cell lines with YPN-005 and FLT3-TKIs. Immunoblot analysis of the expression of FLT3 signaling pathway proteins and RNA polymerase in MV4-11, MOLM-13, and MOLM-14 cells treated with 5 nM quizaritinib, 20 nM YPN-005, or a combination of both for 6 h (a). Induction of mitochondrial damage by combination treatment with YPN-005 and FLT3-TKIs. Electron micrographs of MV4-11 cells either (a) untreated or treated with (b) 5 nM quizartinib (c) 20 nM YPN-005, or (d) a combination of both agents for 72 h. Scale bars = 0.2 µm (b). Changes in mitochondrial membrane potential (MMP, ΔΨm) in MV4-11 cells stained with tetramethylrhodamine ethyl ester were determined by flow cytometry. Cells treated with 50 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were used as positive controls. Depolarization of the MMP was observed in cells treated with 10 nM YPN-005, 20 nM midostaurin, 1 nM quizartinib, 5 nM gilteritinib, or a combination of YPN-005 and an FLT3-TKI for 72 h. MOCK, negative control cells treated with Dimethyl Sulfoxide (DMSO) only (c). The bar graph is illustrating the percent of MMP depolarization in MV4-11 cells treated YPN-005, FLT3-TKIs or both agents as compare to control group. *p < 0.05, ***p < 0.001 (d). Protein expression in FLT3-ITD-mutated AML cells was evaluated using immunoblotting after combination treatment with 10 nM YPN-005 and 1 nM quizartinib (MV4-11 and MOLM-13 cell lines), or 12 nM YPN-005 and 2 nM quizartinib (MOLM-14 cell line) for 6 h (e). Immunoblot detection of c-MYC protein in mouse bone marrow mononuclear cells after a 21 d treatment with either 2 mg/kg YPN-005, 2 mg/kg quizartinib, 30 mg/kg gilteritinib, or a combination of YPN-005 and an FLT3-TKI (n = 3 per group) (f). Immunoblot band density was measured using CSAnalyzer4 and normalized to that of β-actin (g).