Fig. 1: Synergistic effects of combination treatments of YPN-005 and FLT3-TKIs on FLT3-ITD-mutated AML cells.

The combination matrix shows the interactions between YPN-005 and the corresponding FLT3-TKI. Synergy scores, calculated in accordance with the Bliss independence model, are shown in the combination matrix. The largest numeral in each box represents the synergy score (Blue color); negative values indicate antagonism (Red color). Bliss indices > 10 indicate synergism (a). Induction of apoptosis by the combined treatment of FLT3-ITD-mutated AML cell lines with YPN-005 and FLT3-TKIs. Apoptosis was evaluated using Annexin V/PI staining after combination treatment with YPN-005 and FLT3-TKIs. Flow cytometry analysis was performed after incubation with each agent for 72 h. *p < 0.05, **p < 0.01, ***p < 0.001 (b). The protein expression of cleaved PARP and cleaved caspase-3, -8, and -9 was determined by immunoblotting, combination treatment with YPN-005 and quizartinib after 6 h. The arrows indicate the bands formed by each of the subunits of the corresponding cleaved protein (c). In vivo evaluation of the synergistic effects of YPN-005 and FLT3-TKIs. NSG mice were engrafted with MOLM-13 cells expressing GFP and either treated with the vehicle, 2 mg/kg YPN-005, 2 mg/kg quizartinib, 30 mg/kg gilteritinib, or both YPN-005 and quizartinib or gilteritinib (n = 5 per group). The vehicle (saline) group was compared against each treatment group. The in vivo imaging system was used to evaluate the effects of each treatment in the orthotopic xenograft mouse model (d). Survival analysis was estimated using the Kaplan–Meier method. Kaplan–Meier curves with log rank (Mantel-Cox) tests were obtained for survival determination. **p < 0.01 was considered statistically significant (e). Inhibition of the colony-forming ability of primary AML with FLT3-ITD cells by combined treatment with YPN-005 and quizartinib compared to that after single agent treatments. The images are representative micrographs of the resulting colony forming units from control and treated AML cells from three different patients (f).