Enzymatically Inactive 5-Lipoxygenase Inhibits Wnt-Signaling Activation Induced by Leukemia Inducing Oncogenes.

Abstract 2424

Acute myeloid leukemia (AML) is characterized by an abnormal accumulation of hematopoietic progenitors in the bone marrow (BM). The AML phenotype is induced and maintained by specific chromosomal translocations, such as t(8;21), t(15;17), t(6;9). These leukemia initiating events lead to an accelerated proliferation due to a differentiation block that prevents progenitors from reaching the post-proliferative stage of blast cells. This is supported by the aberrant stem cell capacity of poorly defined leukemic stem cells (LSC). The related AML associated fusion proteins (FPs) such as PML/RARα-t(15;17) (P/R), AML-1/ETO-t(8;21) or DEK/CAN-t(6;9) (D/C) recapitulate the leukemic phenotype in vitro and in vivo. Deregulated activation of the Wnt-signaling by FPs lead to aberrant self renewal of LSC in AML and is fundamental for maintenance of the LSC population. Wnt-signaling can be inhibited by non steroidal anti inflammatory drugs (NSAID, i.e. Sulindac, Indomethacin), mainly dual COX 1/2 inhibitors that in high concentrations, which are relevant for the Wnt-signaling inhibition also target the 5-Lipoxygenase (5-LO). Both COX1/2 and 5-LO are the key enzymes in the arachidonic acid metabolism. COX1/2 are the key enzymes for the synthesis of prostaglandines whereas 5-LO is responsible for the biosynthesis of leukotrienes, a group of pro-inflammatory lipid mediators. Reportedly the loss of 5-LO abolished the LSC of BCR/ABL induced CML-like disease. Therefore we targeted 5-LO by selective inhibitors (CJ 13,610 or Zileuton) and have recently shown that the inhibition of the 5-LO enzymatic activity interferes with the stem cell capacity of both PML/RARα and DEK/CAN positive LSC. In order to definitively show that the effects of the 5-LO inhibitors are related to the inhibition of 5-LO, we extended our studies on P/R- and D/C-positive LSC in a 5-LO^−/−background. Here we show that loss of 5-LO expression not only failed to inhibit the stem cell activity of P/R or D/C expressing LSC, but significantly increased the colony number and the replating efficiency of D/C-positive LSC as compared to 5-LO+/+ controls. No significant differences were seen between short-term (ST) or long-term (LT) stem cell capacity of P/R or D/C expressing HSC in 5-LO−/− as compared to a 5-LO+/+ background. These data led us to the conclusion that the inhibition, but not the absence of 5-LO is important for the effect of 5-LO inhibitors on the Wnt-signaling. In order to prove the hypothesis that the inactive 5-LO is the inhibitor of the Wnt-signaling, we expressed 5-LO in U937 cells which are 5-LO-negative. We used U937 P/R9 cells, in which the inducible expression of PML/RAR strongly activates the Wnt-signaling as revealed by the Topflash/Fopflash transactivation system. The expression of 5-LO in absence of leukotriene stimulation reduced the Wnt-signaling activation by P/R to the levels seen in controls. These effects of 5-LO on Wnt-signaling can be explained by the direct interaction between 5-LO and β-catenin as revealed by co-immunoprecipitations in U937 cells.

Taken together our data strongly suggest 1.) an important role of 5-LO in the regulation of the Wnt-signaling pathway and thereby for the maintenance of LSC and 2.) that the inhibitory effect of 5-LO inhibitors are not due to the interruption of the 5-LO mediated lipid signaling but to the presence of an inactivated 5-LO protein, which acquires novel functions that are disconnected from the lipid signaling.