Acute Myeloid Leukemia Is Characterized by Wnt Pathway Inhibitor Promoter Methylation.

The Wnt pathway contributes to a stem-cell like phenotype in a variety of cancer subtypes. Nuclear localization of non-phosphorylated, active β-catenin is a surrogate marker for Wnt pathway activation and has been associated with adverse outcome in patients with acute myeloid leukemia (AML). Wnt pathway inhibitors including APC, DKK1, DKK3, LKB1/STK11, RASSF1A, RUNX3, SFRP1, SFRP2, SFRP4, SFRP5, SOX17, and WIF1 contain extensive promoter region CpG islands. Wnt pathway inhibitors are silenced by promoter methylation in many malignancies including lung cancer, colon cancer and acute lymphoid leukemias. To determine if methylation of these Wnt pathway inhibitors is present in AML, we evaluated leukemia cell lines (i.e., K562, HNT34, KG1, KG1A, U937, and HL60) for evidence of promoter methylation. Additionally, 188 AML patient (median age 61 years) samples from the Johns Hopkins Hospital leukemia tumor bank were assessed for the presence of Wnt pathway inhibitor methylation. All samples were bisulfite treated and evaluated for promoter methylation using methylation specific PCR. Diagnostic samples from a subgroup of patients with normal cytogenetics (n=73) who received high dose induction therapy were evaluated for potential associations between methylation of individual Wnt pathway inhibitor genes or total number of methylated genes and event free or overall survival.

RESULTS: Extensive promoter methylation of the Wnt pathway inhibitor genes was observed in leukemia cell lines. Of the primary leukemia samples, 85% had at least one methylated gene. Promoter methylation of Wnt inhibitors was common in these samples with the following frequencies: DKK1 (16%;30/188), DKK3 (8%;15/188), RUNX3 (27%;50/188), SFRP1 (34%;63/188), SFRP2 (66%;124/188), SFRP4 (9%;16/188), SFRP5 (54%;102/188), SOX17 (29%;54/188), and WIF1 (32%;61/188). This is among the first comprehensive evaluations of Wnt pathway inhibitor methylation in primary samples from AML patients. The frequency of methylation seen here is comparable to that observed in established tumor suppressor genes in patients with AML, including p15^INK4B, SOCS1 and CDH1. In marked contrast with epithelial tumors, methylation of APC (2%;2/108) and RASSF1A (0%;0/188) was rare. LKB1/STK11 methylation was also uncommon (2%;2/108). Previous reports have associated Wnt pathway activation with poor prognosis. In our treated patients with normal cytogenetics, no correlation was observed between methylation of Wnt pathway inhibitors and event free or overall survival. In conclusion, 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 Wnt pathway genes does not correlate with adverse clinical outcome.