Epigenetic silencing of miR-708 enhances NF-B signaling in chronic lymphocytic leukemia

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and their deregulation is involved in tumor development. Epigenetic gene silencing in cancer by DNA methylation contributes to the silencing of tumor-suppressor genes, including miRNAs. We have recently shown that the promoter of miR-708 is aberrantly methylated in chronic lymphocytic leukemia (CLL). To characterize the molecular signaling networks that are influenced by miR-708, we performed a luciferase-based screen evaluating the effects of ectopic miR-708 expression on leukemia-relevant signaling pathways. We found that miR-708 strongly repressed NF-B signaling, a pathway known to be deregulated in CLL. Among the predicted miR-708 targets was IKK (inhibitor of kappa light polypeptide gene enhancer in B cells, kinase-/IKBKB), a key kinase facilitating NF-B signaling. We validated the interaction of miR-708 with the 3-untranslated region of IKK and found that miR-708 overexpression represses endogenous IKK. Phosphorylation of the IKK target IB and expression of known NF-B target genes were impaired by miR-708. Furthermore, we identified an enhancer region downstream of the miR-708 promoter that displays a distinct DNA methylation status in CLL. High enhancer methylation is significantly correlated with lower miR-708 expression and is predominantly found in patients with poor prognosis and shorter time to treatment. These results demonstrate that miR-708 regulates the NF-B pathway by targeting IKK, and that methylation of a key enhancer region contributes to its suppression in CLL. What's new? Mechanisms by which microRNAs (miRNAs) become deregulated in chronic lymphocytic leukemia (CLL) are largely unknown, but for miR-708, a potential tumor suppressor, aberrant promoter methylation may be at fault. Here, miR-708 overexpression was associated with IKK repression and impaired expression of genes targeted by NF-B. Regulation of miR-708 in CLL occurred at a distal enhancer element, where elevated enhancer methylation was correlated with reduced miR-708 expression. Increased miR-708 methylation was found primarily in CLL patients with poor prognosis. The findings shed light on a possible functional connection between an epigenetic mark and regulation of a highly disease-relevant pathway.