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Letters to the Editor
Targeting Wnt signaling in acute myeloid leukemia stem cells
In this work we tested whether targeting Wingless and Int-1 (Wnt) signaling in acute myeloid leukemia (AML) leukemic stem cells (LSC) by using a porcupine inhibitor (WNT974) is a good strategy to eradicate LSC. Wnt/b-catenin is an evolutionarily conserved pathway that is involved in embryonic development and stem cells by regulating cell fate and differentiation decisions.1 In normal conditions, the Wnt/b-catenin pathway mediates normal hematopoietic stem cell self-renewal, prolifera- tion and differentiation and is tightly controlled.2,3 While b-catenin is highly expressed in normal hematopoietic stem cells, it is downregulated during myeloid differenti- ation.2,4 Aberrant activation of this pathway gives rise to the accumulation of b-catenin in the nucleus, and pro- motes the transcription of many oncogenes such as c-Myc (MYC). b-catenin is highly expressed in AML patients and overexpression of b-catenin in normal CD34+ hematopoietic precursors in vitro leads to a myeloprolifer- ative disorder of immature cells.5,6 Further work identi- fied a crucial role of the Wnt/b-catenin pathway in the development of LSC,7 and different authors proposed tar- geting the Wnt pathway in AML blasts.8-11 Thus, we
hypothesize that targeting Wnt in LSC, using a novel Wnt inhibitor called WNT974, could be an efficient strat- egy to eradicate LSC, prevent relapse and improve overall outcomes in AML.
WNT974 is an inhibitor of the enzyme porcupine, which is a membrane-bound O-acyltransferase located in the endoplasmic reticulum, and it is required for the palmitoylation of Wnt ligands. Inhibition of porcupine prevents the secretion and activity of Wnt ligands outside the cell, leading to a decrease in Wnt ligand cell surface receptor phosphorylation and a reduction in the expres- sion of Wnt target genes.12,13 Several studies documented that WNT974 is able to target the Wnt pathway in solid tumors and the LSC in chronic myeloid leukemia.14 Based on this information, we hypothesized that Wnt inhibi- tion by using WNT974 may block the aberrant Wnt acti- vation in AML LSC and prevent leukemogenesis. To ver- ify this hypothesis, we first evaluated whether WNT974 treatment can inhibit the Wnt signaling pathway using the K562 cell line, which is the cell line used initially to report the activity of this drug in myeloid leukemias.
Treatment of K562 cells with dimethylsulfoxide (DMSO), as a control, or WNT974 at different concentra- tions (0.5, 2.5 and 10 mM) for 24 and 48 h showed a reduction of Wnt pathway targets such as ROR2, LRP6 and GSK3B protein (Figure 1A), and AXIN2 mRNA
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Figure 1. Validation of WNT974 activity in cell lines. (A) Western blot assay of ROR2, LRP6 and GSK3B after WNT974 treatment or control (dimethylsulfoxide) for 24 and 48 h. Densitometry analysis was conducted using ImageJ software. Band intensity is reported relative to b-actin (ACTB). (B) AXIN2 mRNA fold change expression of K562 cells treated with WNT974 at different concentrations, or control (DMSO), for 24 h. *P<0.05, **P<0.01 (t-test). (C) Luciferase Renilla assay of HCT116 cells transfected with a wild-type or mutant b-catenin activity reporter vector. Thirty hours after transfection, the cells were treated with Wnt3a ligand (200 ng/mL), WNT974 5 μM, or a combination for 24 h. Data are normalized to the control wild-type. *P<0.05, **P<0.01, ***P<0.001 (t-test). (D) MYC mRNA relative expression of bone marrow CD34+ selected primary acute myeloid leukemia cells treated with WNT974 1 mM for 24 h (*P<0.05, **P<0.01 by the t- test). GAPDH was used as a normalizer.
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