Rational of targeting WNT/beta-catenin signaling in acute myeloid leukemia (AML)

Background and aim: Although recent evidence suggests that aber- rant Wnt signaling can be involved in the neoplastic myeloid cell growth, the role of the Wnt/β-catenin pathway during the disease development and its contribution to AML chemoresistance are still unclear. Methods: AML primary blast cells(25 samples) and AML cell lines were cultured or co-cultured with human bone marrow mes- enchymal stromal cells (hBM-MSCs),in presence or absence of Wnt modulators, including ligands (Wnt3a, Wnt5a/5b), Porcupine inhibitors (IWP-2), LRP6 inhibitors (Niclosamide), or antagonists of TCF/β-catenin (PKF118-310, PNU-74654). Results:In silico analysis showed the enrichment of Wnt signaling components in AML samples. Western Blot and flow cytometry showed the presence of total β-catenin only in 65% of primary sam- ples analyzed. β-catenin positive samples had different degree of activation of the pathway, as revealed by the expression of active forms of β-catenin, including (Ser675)β-catenin and non-phospho- (Ser33/37/Thr41) β-catenin. Notably, we found that active forms of β-catenin increased in AML samples in co-culture with hBM-MSCs, thus suggesting that Wnt signalling could be involved in the crosstalk between bone marrow stroma and AML cells. The addi- tion of Wnt pharmacological inhibitors,such as IWP-2, PNU-74654 and Niclosamide,to the culture medium of β-catenin-positive AML samples, either cultured alone or in co-culture with hBM-MSCs, reduced AML cell proliferation with slight effect on cell death. When associated to Idarubicin,all Wnt inhibitors except IWP-2 syn- ergycally induced a dramatic cell death in AML cells in both culture conditions. However, when Idarubicin was replaced by Ara-C the synergism was observed only with Niclosamide and PKF. Cell death was mainly due to apoptosis, as shown by Annexin-V staining. Conclusion: Overall our data show that chemoterapeutic agents in combination with Wnt inhibitors reduce proliferation and chemoresistance of AML cellsin culture or co-culture with bone marrow stroma, thus highlighting the role of microenvironmental Wnt/β-catenin pathway in supporting AML cell survival. Conse- quently, Wnt/β-catenin signalling may represent a therapeutic target