## 3 Results
### Effects of Setanaxib Are Independent of FLT3-Signaling, Presence of NOX4, or ROS Quenching  
Next, we sought to assess the mechanism of Setanaxib-mediated decrease in cell viability. Setanaxib has initially been described as a NOX1/4 inhibitor. However, recent data provided first evidence that Setanaxib may not directly inhibit any of the NOX family members [35; 36]. To investigate the involvement of NOX4 inhibition in the observed antiproliferative effects on AML cells, we tested Setanaxib on the human AML cell lines MV4-11 and MOLM13, either expressing NOX4 or after genetic inactivation of NOX4 by CRISPR/Cas9 technology. No difference in antiproliferative activity of the drug was observed regardless of whether the cells contained NOX4 or not, rendering inhibition by affecting enzymatic activity of NOX4 unlikely (Figure 4A). We further analyzed cells in which the common subunit of NOX1, 2, 3, and 4, p22-phox was either present or deleted using CRISPR/Cas9. Elimination of p22-phox is expected to abolish the activity of all four NOX enzymes [37]. Again, Setanaxib was equally inhibitory for cells lacking p22-phox, confirming that its effect is not mediated through the inhibition of enzymatic activity of either NOX1 or NOX4 (Figure 4B). Its inhibitory capacity, independent from the presence of NOX enzyme activity, was specifically also observed in combination with  
Figure 3: Effect of single and combined drug treatments with Setanaxib and doxorubicin in vivo. (**A**,**B**) 2 \(\times\) 10\({}^{6}\) GFP-expressing 32D FLT3-ITD cells were injected in C3H/HeJ mice via the tail vein, and respective drug treatments were applied. Setanaxib was given at 40 mg/kg body weight by daily gavage for 9 days, and doxorubicin at 3 mg/kg body weight by i.p. injection at day 3, 4, and 5 post-transplantation. Mice in independent experiments (two cohorts) were sacrificed 9- or 10-days post-transplantation. The percentage of GFP-positive cells was determined by flow cytometry in (**A**) BM and (**B**) spleen. (**B**,**C**) 5 \(\times\) 10\({}^{5}\) GFP-expressing 32D FLT3-ITD cells were injected in C3H/HeJ mice and respective drug treatments were applied as in (**A**,**B**). Mice in independent experiments (two cohorts) were sacrificed 10 days post-transplantation. The percentage of GFP-positive cells was determined by flow cytometry in (**C**) BM and (**D**) spleen (\(n\) = 4–8 per group, mean \(\pm\) SD). Statistical analyses were carried out using one-way ANOVA with Tukey’s post-test. (n.s.—not significant, \({}^{*}\)\(p\) < 0.05, \({}^{**}\)\(p\) < 0.01, and \({}^{***}\)\(p\) < 0.001). daunorubicin, when using FLT3-ITD-transduced Ba/F3 cells (Figure 4C). Taken together, neither antiproliferative activity of Setanaxib nor synergy with daunorubicin in AML cell lines appears to be causally related to the inhibition of NOX1/4 enzymatic activity. Figure 4: Assessment of a potential mechanism for Setanaxib effects. (**A,B**) Effect of Setanaxib on the proliferation of human AML cells with _NOX4_ or _p22-phax_ knockout. KO human AML cells were obtained using CRISPR/Cas9 technology. The cell lines stably express Cas9 and were either transduced with control sgRNA(sgLuci, designated WT) or sgRNA targeting _NOX4_ or _p22-phax_,  
respectively (designated KO). Absence of the targeted gene was detected as described in Materials and Methods. The experimental setup for the drug treatments and assay of proliferation were as in Figure 1A,B. Cells were counted using a hemocytometer at day 8. Mean \(\pm\) SD of cell treatments of two to three independently sgRNA-transduced cell batches is presented. **(C)** Comparison of synergy of Setanaxib with daanorubicin in Ba/F3-FLT3-ITD cells harboring Cas9 and transduced with sgLuci (WT) or sg_Nox4_ (KO). Treatments were performed as in Figure 2 and proliferation/viability was assessed by Cell Titer Blue assay. The individual experiments were normalized to DMSO controls. Mean \(\pm\) SD (with technical triplicates) for cells from three independent transductions with sgRNA is shown.