Epigenetic regulation of the non-canonical Wnt pathway in acute myeloid leukemia

Abstract

Wnt5a is a member of the Wnt family of proteins that signals through the non-canonical Wnt/Ca(2+)pathway to suppress cyclin D1. Deregulation of this pathway has been found in animal models suggesting that it acts as tumour suppressor in acute myeloid leukemia (AML). Although DNA methylation is the main mechanism of regulation of the canonical Wnt pathway in AML, the role of WNT5A abnormalities has never been evaluated in this clinical setting. The methylation status of WNT5A promoter-exon 1 was analyzed by methylation-specific PCR and sequencing in eleven AML-derived cell lines and 252 AML patients. We observed WNT5A hypermethylation in seven cell lines and in 43% (107/252) of AML patients. WNT5A methylation was associated with decreased WNT5A expression (P < 0.001) that was restored after exposure to 5-Aza-2'-deoxycytidine. Moreover, WNT5A hypermethylation correlated with upregulation of CYCLIN D1 expression (P < 0.001). Relapse (15%vs 37%, P < 0.001) and mortality (61%vs 79%, P = 0.004) rates were lower for patients in the non-methylated group. Disease-free survival and overall survival at 6 and 7 years, respectively, were 60% and 27% for unmethylated patients and 20% and 0% for hypermethylated patients (P = 0.0001 and P = 0.04, respectively). Interestingly, significant differences were also observed when the analysis was carried out according to cytogenetic risk groups. We demonstrate that WNT5A, a putative tumor suppressor gene in AML, is silenced by methylation in this disease and that this epigenetic event is associated with upregulation of CYCLIN D1 expression and confers poor prognosis in patients with AML.

Figure 1

Expression and methylation analysis of the WNT5A gene in acute myeloid leukemia (AML)‐derived cell lines. (A) Quantitative real‐time PCR expression of WNT5A in AML‐derived cell lines demonstrating downregulation of gene expression in seven of the 11 cell lines studied. Gene expression was normalized using the expression in bone marrow mononuclear cells (BM‐MNC) from 30 healthy controls (normalized ratio = 100%). The figure represents the mean of three different studies in triplicate. (B) Methylation‐specific PCR (MSP) analysis of WNT5A in the same AML‐derived cell lines. Region 2 of WNT5A was methylated in all of the cell lines that had downregulated WNT5A expression. C+, positive methylated control; C–, BM‐MNC cells from healthy donors. (C) Schematic description of the WNT5A CpG island region 2. Each vertical bar represents a CpG dinucleotide. The gray arrows show the location of the MSP primers and the black arrows the location of bisulfite sequencing primers. The figure also represents the analysis of WNT5A CpG island methylation status by bisulfite sequencing in AML‐derived cell lines. Each box indicates a CpG dinucleotide (white box, unmethylated; black box, methylated) and each line represents the analysis of 21 CpG dinucleotides of a single clone of the WNT5A analysed region. (D) Expression analysis of the Wnt5a gene in the methylated KASUMI‐1 cell line and in the unmethylated HEL cell line, before and after treatment with the demethylating agent 5‐Aza‐2′‐deoxycytidine (DAC) demostrating upregulation of the gene after treatment in AML‐derived cell line that was methylated. Gene expression was normalized with expression in BM‐MNC from healthy controls (normalized ratio = 100%). The figure represents the mean of three different studies in triplicate.

Figure 2

Expression of WNT5A and CYCLIN D1 genes in samples from acute myeloid leukemia (AML) patients. (A) Expression analysis of the WNT5A gene in AML patients. Methylated patients showed downregulation of WNT5A expression. Bars represent the mean expression (95% CI) in AML patients in comparison with the expression in Bone marrow mononuclear cells (BM‐MNC) from 30 healthy controls (normalized ratio = 100%). (B) Hypermethylation of the WNT5A gene is associated with upregulation of CYCLIN D1 in AML patients. Bars represent the mean expression (95% CI) in AML patients in comparison with the expression in BM‐MNC from 30 healthy controls (normalized ratio = 100%).

Figure 3

Kaplan–Meier disease‐free survival (DFS) function for acute myeloid leukemia (AML) patients. DFS curves for (A) all of the patients included in this study, and for patients classified into cytogenetic (B) low‐risk, (C) intermediate‐risk, and (D) high‐risk groups according to the WNT5A methylation status. Solid lines, WNT5A unmethylated patients. Dashed lines, WNT5A methylated patients.

Figure 4

Kaplan–Meier overall survival (OS) function for acute myeloid leukemia (AML) patients. OS curves for (A) all of the patients included in this study, and for patients classified into cytogenetic (B) low‐risk, (C) intermediate‐risk, and (D) high‐risk groups according to the WNT5A methylation status. Solid lines, WNT5A unmethylated patients. Dashed lines, WNT5A methylated patients.