Acute myeloid leukemia is characterized by Wnt pathway inhibitor promoter hypermethylation

Abstract

Nuclear localization of non-phosphorylated, active beta-catenin is a measure of Wnt pathway activation and is associated with adverse outcome in patients with acute myeloid leukemia (AML). While genetic alterations of the Wnt pathway are infrequent in AML, inhibitors of this pathway are silenced by promoter methylation in other malignanices. Leukemia cell lines were examined for Wnt pathway inhibitor methylation and total beta-catenin levels, and had frequent methylation of Wnt inhibitors and upregulated beta-catenin by Western blot and immunofluorescence. One hundred sixty-nine AML samples were examined for methylation of Wnt inhibitor genes. Diagnostic samples from 72 patients with normal cytogenetics who received standard high-dose induction chemotherapy were evaluated for associations between methylation and event-free or overall survival. Extensive methylation of Wnt pathway inhibitor genes was observed in cell lines, and 89% of primary AML samples had at least one methylated gene: DKK1 (16%), DKK3 (8%), RUNX3 (27%), sFRP1 (34%), sFRP2 (66%), sFRP4 (9%), sFRP5 (54%), SOX17 (29%), and WIF1 (32%). In contrast to epithelial tumors, methylation of APC (2%) and RASSF1A (0%) was rare. In patients with AML with normal cytogenetics, sFRP2 and sFRP5 methylation at the time of diagnosis was associated with an increased risk of relapse, and sFRP2 methylation was associated with an increased risk for death. In patients with AML: (a) there is a high frequency of Wnt pathway inhibitor methylation; (b) Wnt pathway inhibitor methylation is distinct from that observed in epithelial malignancies; and (c) methylation of sFRP2 and sFRP5 may predict adverse clinical outcome in patients with normal karyotype AML.

Figure 1

Westen blot for total β-catenin in three leukemia cell lines (HL60, KG1, and K562) known to be without mutations in APC, Axin, and β-catenin, normal CD34⁺ bone marrow cells, and HCT116 colon cancer cells harboring a constitutively active β-catenin protein. The leukemia cell lines have higher levels of β-catenin protein than are seen in normal CD34⁺ bone marrow cells, but are lower than observed in HCT116 cells.

Figure 2

Immunofluorescence for nuclei (blue) and β-catenin (red) demonstrating predominantly nuclear localization of β-catenin protein in leukemia cell lines. Low levels of β-catenin were observed in CD34⁺ bone marrow samples, with most protein appearing to be cytoplasmic or membrane bound. HCT116 colon cancer cells are shown as a positive control with abundant β-catenin protein present. All images were taken at ×63, scale bar 10 μm.

Figure 3

Methylation frequency in patients with AML (n = 169), AML and normal karyotype (n = 106), and breast cancer (n = 54). Rates of methylation are similar in the AML group as a whole and in those with normal karyotype. Overall, 89% of the AML samples had at least one methylated Wnt inhibitor gene, and 70% had methylation of two or more genes. APC, RASSF1A, and sFRP4 are frequently methylated in epithelial malignancies and rarely methylated in AML (*p < 0.05 for AML versus breast cancer).

Figure 4

Kaplan–Meier curves demonstrating the impact of sFRP2 (A) and sFRP5 (B) methylation on disease-free survival. In univariate analysis, sFRP2 and sFRP5 methylation was significantly associated with adverse prognosis (p < 0.04 and p < 0.02, respectively). ‘U’ designates unmethylated status and ‘M’ designates methylated status.

Figure 5

(A) Multivariate analysis of disease-free survival for each of the Wnt genes in 72 uniformly treated patients with AML with normal cytogenetics. Methylation of sFRP2 and sFRP5 is associated with a significantly increased rate of relapse. (B) Multivariate analysis of the association between Wnt inhibitor methylation and all-cause mortality in this group. sFRP2 methylation is associated with a significantly increased risk of death. The multivariate model includes patient age at diagnosis, history of antecedent cytopenias/MDS, total WBC at diagnosis, and FLT3–ITD and NPM1^mut status.