DLX4 hypermethylation is a prognostically adverse indicator in de novo acute myeloid leukemia

Hypermethylation of distal-less homeobox 4 (DLX4) has been increasingly identified in several cancers. Our study was aimed to determine the role of DLX4 methylation in regulating DLX4 expression and further analyze its clinical significance in de novo acute myeloid leukemia (AML) patients. DLX4 methylation level was detected by real-time quantitative methylation-specific PCR and bisulfite sequencing PCR. Treatment with 5-aza-2'-deoxycytidine (5-aza-dC) was used for demethylation studies. Clinical significance of DLX4 methylation was obtained by the comparison between the patients with and without DLX4 methylation. DLX4 was significantly methylated in AML patients compared with controls (P < 0.001). DLX4 methylation was negatively associated with DLX7 (the shorter DLX4 isoform) (R = -0.202, P = 0.021) but not BP1 (the longer DLX4 isoform) (R = -0.049, P = 0.582) expression in AML patients. DLX7 and BP1 messenger RNA (mRNA) were significantly increased after 5-aza-dC treatment in leukemic cell lines THP1 and Kasumi-1. DLX4 methylated patients showed significantly higher frequency of U2AF1 mutation compared with DLX4 unmethylated patients (P = 0.043). Both all AML and non-M3 patients with DLX4 methylation presented significantly lower complete remission rate than those with DLX4 unmethylation (P = 0.001 and <0.001, respectively). DLX4 methylated cases had significantly shorter overall survival than DLX4 unmethylated cases among both all AML (P = 0.003), non-M3 AML (P = 0.001), and cytogenetically normal AML (P = 0.032). Multivariate analysis confirmed that DLX4 methylation was independent risk factor in both all AML and non-M3 patients. Our study indicates that DLX4 hypermethylation is negatively associated with DLX7 expression and predicts poor clinical outcome in de novo AML patients.