ARTICLE ABSTRACTNonsteroidal anti-inflammatory drugs such as sulindac inhibit Wnt signaling, which is critical to maintain cancer stem cell–like cells (CSC), but they also suppress the activity of 5-lipoxygenase (5-LO) at clinically feasible concentrations. Recently, 5-LO was shown to be critical to maintain CSC in a model of chronic myeloid leukemia. For these reasons, we hypothesized that 5-LO may offer a therapeutic target to improve the management of acute myeloid leukemia (AML), an aggressive disease driven by CSCs. Pharmacologic and genetic approaches were used to evaluate the effects of 5-LO blockade in a PML/RARα-positive model of AML. As CSC models, we used Sca-1+/lin− murine hematopoietic stem and progenitor cells (HSPC), which were retrovirally transduced with PML/RARα. We found that pharmacologic inhibition of 5-LO interfered strongly with the aberrant stem cell capacity of PML/RARα-expressing HSPCs. Through small-molecule inhibitor studies and genetic disruption of 5-LO, we also found that Wnt and CSC inhibition is mediated by the enzymatically inactive form of 5-LO, which hinders nuclear translocation of β-catenin. Overall, our findings revealed that 5-LO inhibitors also inhibit Wnt signaling, not due to the interruption of 5-LO–mediated lipid signaling but rather due to the generation of a catalytically inactive form of 5-LO, which assumes a new function. Given the evidence that CSCs mediate AML relapse after remission, eradication of CSCs in this setting by 5-LO inhibition may offer a new clinical approach for immediate evaluation in patients with AML. Cancer Res; 74(18); 5244–55. ©2014 AACR.