## 3 Results
### Effects of Setanaxib Are Independent of FLT3-Signaling, Presence of NOX4, or ROS Quenching
**(D,E)** The 32D-FLT3-ITD cells were subjected to single or combined drug treatments with daanorubicin, diphenyleneiodonium (DPI) or N-acetylcysteine (NAC) as indicated for 72 h, and proliferation/viability was assessed by Cell Titer Blue assay. Three independent experiments (in triplicate) were conducted; error bars represent mean \(\pm\) SD. Statistical analyses were carried out using two-tailed \(t\)-test (n.s.--not significant, * \(p<0.05\), **\(p<0.01\), ***\(p<0.001\), and ***\(p<0.0001\)). Inhibitory activity of Setanaxib and its observed synergy with anthracyclines may, however, be based on general antioxidant activity. We addressed this issue indirectly by testing two compounds with an established general activity on redox metabolism: Diphenyleneiodonium (DPI), a flavoprotein inhibitor and general NOX inhibitor, and N-acetylcysteine (NAC), a general antioxidant. When we used these compounds in concentrations known to exert antioxidant effects, DPI as single compound inhibited cell growth (Figure 4D), while NAC only had a little effect (Figure 4E). Importantly, no synergy with daanorubicin could be observed (Figure 4D,E). Although the activity of Setanaxib did not appear to be restricted to FLT3-ITD-positive AML cells, we also assessed the potential effects of Setanaxib on FLT3-ITD-mediated oncogenic signaling. MV4-11 cells were treated with Setanaxib and lysates were analyzed by immunoblotting for activation of FLT3, STAT5, AKT, and ERK based on the presence of their phosphorylated forms. AC220 (quizartinib), a potent FLT3 kinase inhibitor [38], and DPI were used as controls. As shown in Figure 5A-C, AC220 reduced activation of the assessed signaling molecules strongly. DPI showed a significant inhibitory effect on ERK activation (Figure 5C). Of note, Setanaxib did not significantly inhibit any of these signaling events even at the high concentration of 60 \(\upmu\)M (Figure 5A-C). Anthracyclines such as daanorubicin induce apoptosis in cancer cells by causing DNA damage. However, they also contribute to the elevation of ROS. This ROS induction can contribute to the induction of cancer cell apoptosis [39; 40]. Therefore, we considered that Setanaxib, despite its proposed targets NOX1 and 4, may rather modulate ROS metabolism and affect daanorubicin-induced ROS elevation. Setanaxib treatment resulted in dose-dependent elevation of ROS in MOLM13 cells as indicated by H2 DCFDA fluorescence (Figure 5D). Again, this effect was independent of NOX4 expression. NAC, DPI, and midostaurin caused a reduction in ROS levels, as described before [22; 41]. Importantly, daanorubicin enhanced ROS levels, and the combination of Setanaxib and daanorubicin showed additive effects in enhancing ROS (Figure 5E). Similar observations were made in HEK93 cells, engineered to inductively overexpress NOX4. NOX4 overexpression caused elevated ROS formation, which was further enhanced by Setanaxib. CRISPR/Cas9-mediated knockout of NOX4 in these cells abolished tet-induced ROS elevation; however, a dose-dependent induction of ROS formation by Setanaxib was likewise visible (Figure 5F). These findings support that Setanaxib can promote ROS formation, and that this effect is independent from NOX4. While the exact mechanisms of Setanaxib-induced ROS formation remain so far elusive, synergy in elevated and critical ROS production may play a role for the observed synergy in cytotoxicity. Figure 5: Setanaxib promotes ROS formation elicited by daunorubicin. (**A**–**C**) MV4-11 cells were treated with the indicated concentrations of the FLT3-ITD inhibitor AC220 (quizartinib, positive control), the general NOX inhibitor diphenylenoodium (DPI), or Setanaxib for 4 h in serum-free medium. Thereafter, cells were lysed and lysates subjected to immunoblotting for assessment of pathway activation. Blots were probed with activation-specific antibodies to pFLT3 (pY589/591), pSTAT5 (pY694), pAkt (Ser473), or pErk1/2 (Thr202/Tyr204) antibodies.