## 3 Results
### Setanaxib Has Efficiency In Vivo  
We used a mouse model of FLT3-ITD-induced myeloproliferation to test the efficiency of the drug combination in vivo. In this model, FLT3-ITD-positive 32D cells are injected into syngeneic C3H/HeJ mice, which develop an early onset and fatal myeloproliferation. This model operates with a relatively high burden of inoculated tumor cells (2 \(\times\) 10\({}^{6}\) per mouse), leading to death within less than two weeks. When used as monotherapy, Setanaxib had beneficial effects with moderately prolonged survival and reduction in tumor burden at death [25]. For reasons of animal welfare, we applied this model in a modified form. Instead of survival, the tumor burden was measured as the abundance of GFP-positive cells in bone marrow and spleen. To assess for potential additive value, we used doxorubicin, an anthracycline that can be applied intraperitoneally in mice [34]. Doxorubicin was titrated to identify a dose with limited efficiency (3 mg/kg body weight by i.p. injection at day 3, 4, and 5 post-transplantation). Setanaxib at 40 mg/kg daily showed minor efficiency when used as oral monotherapy in mice injected with 2 \(\times\) 10\({}^{6}\) tumor cells (Figure 3A,B). Doxorubicin as single compound appeared to reduce tumor burden, but the effect was not significant. Only the combination of doxorubicin and Setanaxib attenuated the disease to a significant extent, as measured by a reduction in GFP-positive cells in bone marrow (BM) and spleen (Figure 3A,B) compared with only solvent-treated animals. When using fewer numbers of inoculated cells (5 \(\times\) 10\({}^{5}\)), Setanaxib had an inhibitory effect as a single drug (Figure 3C,D). In the only Setanaxib group, 3/4 mice showed less than 2% of GFP-positive cells in BM and spleen while mice in the control group showed around 20% of GFP-positive cells in both compartments. Doxorubicin on its own was likewise potent in reducing the tumor burden as a single drug. Under these conditions, no significant difference was detectable when comparing the effects of the drug combination with that of doxorubicin treatment alone. We only observed a trend of improved efficiency using the combination of both drugs when compared with the solvent control. Taken together, the experiments indicate the potential of Setanaxib to attenuate the myeloproliferative disease in this model alone and in combination with anthracycline therapy. \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multicolumn{5}{c}{**Proliferation/Viability (Cell Titer Blue)**} \\ \hline \hline \multirow{5}{*}{Setanaxib} & Chemotherapy drugs & 32D FLT3-ITD & MV4-11 & MOLM13 \\ \cline{2-5}  & Cytarabine & +++ & \(\pm\) & \(\pm\) \\ \cline{2-5}  & Daunorubicin & +++++ & +++ & +++ \\ \cline{2-5}  & Midostaurin & ++++ & +++ & ++ \\ \hline \multicolumn{5}{c}{Apoptosis (Annexin V + 7-AAD)} \\ \hline \hline \multicolumn{5}{c}{Chemotherapy drugs} & 32D FLT3-ITD & MV4-11 & \\ \cline{2-5}  & Cytarabine & +++ & \(\pm\) & \\ \cline{2-5}  & Daunorubicin & +++++ & + & \\ \cline{2-5}  & Midostaurin & ++++ & ++ & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Analysis of the potential synergy of the combined treatments of different AML cell lines with Setanaxib and with different cytotoxic drugs. 32D FLT3-ITD, MV4-11, or MOLM13 cells were subjected to drug treatments. Experimental conditions for cell treatments were as in Figure 2. Setanaxib was applied at 1, 3, 10 or 30 \(\upmu\)M, cytarabine at 100 or 500 nM, daunorubicin at 20 or 30 nM, and midostaurin at 10 or 20 nM. The proliferation/viability of the cells was assessed by Cell Titer Blue assay after 72 h of treatments. Apoptosis rates were measured by FACS analysis after 48 h of treatments. The CI values were calculated with the Chou method using the software Calcusyn. A CI of \(<\)1 indicates synergism (\(<\)0.1 very strong synergism (++++), 0.1 to 0.3 strong synergism (++++), 0.3 to 0.7 synergism (+++), 0.7 to 0.85, moderate synergism (++), 0.85 to 0.90, low synergism (+), and 0.90 to 1.1, nearly additive (\(\pm\)).