Fig. 4: Treatment with FHD-286 significantly depleted FLT3 expression in bulk AML cells and phenotypically defined AML stem cells with mtNPM1 and FLT3-ITD and FHD-286-based combinations exerted synergistic in vitro lethality in patient-derived AML cells expressing mtNPM1 with FLT3-ITD and/or additional FLT3 mutation.

A Immunoblot analysis of FLT3 expression in three patient-derived AML samples with FLT3-ITD and one non-FLT3-ITD AML sample showing variation in FLT3 expression. The expression levels of GAPDH in the cell lysates served as the loading control. B PD mtNPM1 + FLT3-ITD AML cells were treated with 100 nM of FHD-286 for 48 h. Cells were harvested and pellets were processed for total proteome profiling by tandem mass tag mass spectrometry. Volcano plot (log2 fold-change versus -log10 p-value) of mass spectrometry-determined protein expression changes in FLT3 and FLT3 target genes in FHD-286-treated compared to control cells (fold change >0 or <0 and p < 0.05). C Patient-derived mtNPM1 + FLT3-ITD AML cells were treated with 100 nM of FHD-286 for 48 h. CyTOF analysis was conducted utilizing cocktails of rare metal element-tagged antibodies. The heat map shows the log2 fold-change (FHD-286-treated over Control) of depleted and induced proteins in phenotypically defined AML stem/progenitor cells (CLEC12A hi, CD123 hi, CD99 hi, CD33 hi and CD11b low). D Four patient-derived AML samples with mtNPM1 + FLT3-ITD and/or additional FLT3 mutation were treated with the indicated concentrations of gilteritinib or FHD-286 (in duplicate) for 72 h. At the end of treatment, cells were stained with TO-PRO-3 iodide and the % non-viable cells were determined by flow cytometry. E Patient-derived AML cells with mtNPM1 + FLT3-ITD with or without FLT3-TKD or gatekeeper (F691L) mutation were treated with FHD-286 (dose range: 20 nM–50 nM) and quizartinib (dose range: 0.5–2.0 nM), or gilteritinib (dose range: 5 nM–20 nM) for 72 h. At the end of treatment, the % non-viable cells were determined by staining with TO-PRO-3 iodide and flow cytometry analysis. Delta synergy scores were determined by the ZIP method within the SynergyFinder web application. Synergy scores >1.0 indicate a synergistic interaction of the two agents in the combination. Delta Synergy scores for each combination are shown.