## 3 Results
### Setanaxib Is Synergistic with Daunorubicin in FLT3-ITD-Positive Cells  
We subsequently tested the synergy of Setanaxib in combinations with the cytotoxic and targeted drugs commonly used to treat AML, daunorubicin, cytarabin, and midostaurin. For simplicity, we used here the metabolic assay CellTiter Blue, which gave identical results with cell counting in side-by-side comparison using Setanaxib as a single drug (data not shown). To adjust suitable doses, effective dose ranges of the cytotoxic drugs were assessed with human MV4-11 cells and murine FLT3-ITD-expressing 32D cells and found to be in the previously reported range of active concentrations (Supplementary Figure S2, Online Supplement). We observed additive inhibitory effects in particular for the combination of daunorubicin with Setanaxib as shown for FLT3-ITD-positive murine cells in Figure 2A,C-E, but also in murine 32D cells harboring wildtype FLT3. Side-by-side comparison revealed similar sensitivity of wildtype FLT3-expressing 32D cells and FLT3-ITD-expressing 32D cells, with a slightly stronger response to the combination of both drugs in the latter (Figure 2C). A limitation of these experiments is, however, that wildtype FLT3-expressing cells require the presence of IL-3 for survival, and this cytokine activates similar pathways as FLT3-ITD. While Setanaxib alone had minor effects on the induction of apoptosis, high levels of apoptosis were induced by the drug combination (Figure 2B). These effects were synergistic according to data analysis using the method of Chou [33]. Notably, we observed synergistic inhibition of Setanaxib with daunorubicin also in a FLT3-ITD/NPM1-mutated primary human AML cell sample, while a FLT3-wildtype/NPM1-mutated cell sample appeared less sensitive (Figure 2F). While this finding shows susceptibility of primary human cells to the drug combination, the assessment of larger numbers of AML cell samples will be required to establish a potential difference in the sensitivity of primary human AML cells with different FLT3 status. An overview of results regarding the observed synergism of Setanaxib containing combinations with different cell lines is presented in Table 1. Taken together, Setanaxib enhances the efficiency of classical AML therapeutics in vitro. Figure 2: Effect of the single and combined drug treatments on proliferation/viability and induction of apoptosis in AML cells. (**A**) 32D-FLT3-ITD cells were seeded in 96-well plates, single compounds or combinations of compounds (daunorubicin, Setanaxib, or combinations) were added, and the proliferation/viability was assessed by Cell Titer Blue assay after 72 h. The fluorescent signal, which was directly proportional to the number of viable cells, was measured in a plate reader at 540/610 nm (excitation/emission, respectively, RFU-relative fluorescence units). (**B**) Apoptosis rate was measured by FACS analysis using Annexin V/7-AAD staining after 48 h of single or combined treatments with daunorubicin and Setanaxib. The combination index was determined using the software Calcusyn. The obtained ‘combination index (CI)’ numbers are shown above the bars. Three independent experiments were conducted; error bars represent mean \(\pm\) SD. (**C**) Comparison of synergistic effects in wildtype (WT) FLT3-expressing 32D-cells, FLT3-ITD-expressing 32D- cells, and Ba/F3-FLT3-ITD cells. Note that the cultivation of WT-FLT3 32D cells requires addition of IL-3 to the medium. (**D**) Synergy of Setanaxib and daunorubicin in a murine leukemic cell line transformed with FLT3-ITD and MLL-AP9 obtained as indicated in the legend to Figure 1. (**E**) Synergy of Setaxanib with doxorubicin in 32D-FLT3-ITD cells. The individual experiments were normalized to DMSO controls. Three independent experiments (with technical triplicates) were conducted; error bars represent mean \(\pm\) SD. (*** \(p\) < 0.0001 by two-tailed _t_-test).