## 4 Discussion and Conclusions  
In this study, we have found that Setanaxib, an inhibitor with proposed activity against NOX1 and 4 function, shows antiproliferative effects on AML cells. This effect was prominently seen in leukemia cells harboring the oncogenic driver FLT3-ITD, but also in cell lines which do not carry an FLT3 mutation. Importantly, in both the FLT3-ITD-expressing and wildtype FLT3 cells it strongly synergizes with cytotoxic agents, in particular the anthracyclines such as daanorubicin. Surprisingly, these effects are independent of NOX1/4 expression and may instead be mediated by enhancement of anthracyclin-induced ROS production possibly through an unknown pathway of modulating ROS metabolism. Recent studies have questioned the target specificity of Setanaxib for NOX1/4 and suggested that the compound may affect ROS metabolism through other mechanisms such as inhibition of peroxidase [35; 36]. We have used genetic deletion using CRISPR/Cas9 to assess the relevance of NOX4 as a potential target for the antiproliferative activity of Setanaxib in AML cells. Setanaxib was used in many studies and the observed effects considered being via NOX4 or NOX1 inhibition. However, thorough validation using genetic deletion of _NOX_ genes in combination with inhibitor treatment is lacking in these studies. In a recent study showing cytotoxic activity of Setanaxib on liver cancer cells in vitro, a potentially interesting mechanism of action has been proposed: Treatment with Setanaxib fostered mitochondrial ROS formation and caused cell death, which was at least in part mediated by high ROS levels [42]. As we have likewise found elevated ROS formation in AML cells upon Setanaxib treatment, a mitochondrial origin of elevated ROS may be considered. Testing this hypothesis as well as assessing the potential causal link of ROS formation to cytotoxicity require further investigations. While moderate antiproliferative effects of Setanaxib on AML cells were reported earlier [25], the strong synergy with cytotoxic agents was unexpected and novel. Antiproliferative effects of Setanaxib could also be observed in vivo. It attenuated disease as a single compound in mice injected with 5 \(\times\) 10\({}^{5}\) tumor cells. In mice injected with 2 \(\times\) 10\({}^{6}\) tumor cells, only the combination of Setanaxib with doxorubicin attenuated disease compared with solvent-treated mice. Overall, the observed effects using the syngeneic FLT3-ITD 32D/C3H/HeJ mouse model, in which disease progression is very rapid, were however quite limited. Exploring the in vivo effects of the drug and its combination with anthracyclines further, e.g., in experiments using patient-derived xenograft models is clearly warranted. Further assessment of Setanaxib for possible treatment of AML appears interesting and reasonable. Notably, this compound is in the advanced stages of clinical development. Early clinical trials have suggested a low toxicity profile of the compound [43]. Moreover, Setanaxib has good oral bioavailability. To date, five phase I and three phase II clinical studies have been carried out with Setanaxib and no safety signal and no dose limiting toxicity have been observed. Doses up to 800 mg/day were earlier found to be safe compared to placebo in a 24-week phase II trial in PBC patients (NCT03226067). Evendosing as high as 1600 mg/d has recently been tested without identifying any dose-limiting toxicity (phase I, ClinicalTrials.gov identifier: NCT04327089) [44]. In a phase II trial in patients with diabetic nephropathy, the patients in the Setanaxib-treated arm finished the 12-week treatment period with even fewer adverse events compared to the placebo arm but did not reach the primary clinical endpoint [26] (NCT03740217, NCT02010242). In a phase II trial on primary biliary cholangitis, Setanaxib was successful in reaching its primary and secondary efficacy endpoints (NCT03226067). Other phase II clinical trials using Setanaxib are ongoing for kidney disease in type 1 diabetes, and for idiopathic pulmonary fibrosis [45].