Down-regulation of DLX3 expression in MLL-AF4 childhood lymphoblastic leukemias is mediated by promoter region hypermethylation.

Hypermethylation of CpG islands is the most well defined epigenetic change in neoplasia and plays an important role in the inactivation or silencing of cancer related genes. DLX genes (1-7), with large CpG islands in their 5' region, are implicated in a number of processes among which haematopoiesis. They are characterized by highly dynamic spatio-temporal expression and supposed to be involved in resistance to apoptosis of several tumor cell lines. In acute lymphoblastic leukemia (ALL) hypermethylation is a common phenomenon frequently associated with poor prognosis in specific genetic childhood leukemia subgroups. These data together with the presence of large CpG islands in the up-stream regions of the DLX genes make them attractive candidates for methylation regulated gene expression and leukemia related aberrancies. To validate the role of DLX genes in paediatric B-ALL cells, we studied two cell lines and two groups of patients with paediatric chromosomal rearrangements: MLL-AF4 and TEL-AML1, respectively. Analysis of methylation and gene expression patterns of DLX3 in 64 specimens of B-lineage ALL revealed that DLX3 presents aberrant methylation in paediatric B-ALL patients. In vitro experiments with 5-Aza-2'dC on leukemia cell lines, confirmed by Western blot analysis, indicated that the methylation of DLX3 CpG islands has a functional role and interferes with the DLX3 gene and DLX3 protein expression in B-ALL cells. Importantly, hypermethylation of DLX3 significantly reduces its expression in MLL-AF4 rearranged leukemias while methylation is almost absent in TEL-AML1 positive ALL specimens. These results show that differential DLX3 methylation could be a new epigenetic marker for genotypic B-cell leukemia subgroup with high-risk features.