Abstract
The Mixed Lineage Leukemia Partial Tandem Duplication (MLL PTD) defect, present in 4–10% of adults with acute myeloid leukemia (AML) and normal cytogenetics (NC), predicts for shorter complete remission duration. Functionally, the MLL PTD is associated with aberrant histone methylation. Since posttranslational histone modifications are also frequently associated with changes in the methylation status of gene promoters, we evaluated whether AML with the MLL PTD is associated with a pattern of DNA methylation distinct from AML with NC and wild type MLL (MLL WT). Significant differences in DNA methylation patterns of over 2,000 CpG island fragments, most of them located in the 5′-region of genes, were observed between NC AML with the MLL PTD (n=9) and NC AML with MLL WT (n=23) using Restriction Landmark Genomic Scanning (RLGS) (P<0.001). This analysis revealed 201 RLGS spots were lost, corresponding to DNA methylation events, at a higher frequency in MLL PTD+ AML compared with MLL WT AML. Among the non-polymorphic, methylated spots, spot 3D41 corresponding to the SLC5A8 gene was found methylated in 78% of MLL PTD+ AMLs compared with only 17% of MLL WT AMLs (P<0.003). The SMCT1 protein, encoded by SLC5A8, is a known tumor suppressor reported to be silenced by DNA methylation in colorectal, glioma, thyroid and gastric cancers. This plasma membrane protein mediates intracellular transport of substrates such as butyrate, a histone deacetylase inhibitor (HDACi), and when SLC5A8 is underexpressed, this predicts worse prognosis in colon cancer patients. In MLL PTD+ AML samples exhibiting 3D41 loss by RLGS, bisulfite-PCR sequencing confirmed CpG hypermethylation within the SLC5A8 proximal promoter (range: 24–78% promoter methylation) compared with those AML samples (0%–12% promoter methylation) exhibiting no 3D41 loss. Furthermore, SLC5A8 transcript levels were significantly reduced in MLL PTD+ AML patient samples compared to MLL WT+ AML patient samples (P<0.002). The demethylating agent, decitabine (Dacogen®, MGI Pharma), alone or in sequential combination with one of the following HDACis- depsipeptide, valproic acid or MS275, induced expression of SLC5A8 and cell death in the MLL PTD+ EOL−1 cell line. These results suggest that aberrant promoter DNA methylation and the reduced expression of the tumor suppressor gene SLC5A8 may be associated with the short remission duration observed in MLL PTD+ AML patients. These data also support further investigation of DNA demethylating agents, such as recently FDA-approved Dacogen® and the appropriate timing of administration of HDACi, such as butyrate analogs, as a therapeutic approach for this poor prognostic AML subgroup.
DNA hypermethylation and epigenetic silencing of the tumor suppressor gene, SLC5A8, in acute myeloid leukemia with the MLL partial tandem duplication