RANKL expression in myeloma cells is regulated by a network involving RANKL promoter methylation, DNMT1, microRNA and TNFα in the microenvironment.

We studied the regulation of RANKL expression in myeloma by promoter DNA methylation. Methylation-specific polymerase chain reaction showed complete methylation of RANKL promoter in WL-2 myeloma cells but partial methylation in eight other lines. 5-AzadC treatment of WL-2 cells led to demethylation and re-expression of RANKL. Transwell and contact co-culture of WL-2 cells with normal bone marrow-derived mesenchymal stromal cells (BMSCs) resulted in comparable repression of DNA methyltransferase-1 (DNMT1) and re-expression of RANKL in WL-2 cells. Moreover, treatment of WL-2 cells with TNFα led to repression of DNMT1 and re-expression of RANKL in association with upregulation of miR-140-3p and miR-126, which are partially offset by addition of anti-TNFα antibody to transwell-coculture of WL2 with BMSC. Taken together, our results showed that TNFα in the marrow microenvironment led to RANKL demethylation and re-expression in myeloma cells through DNMT1 repression and upregulation of miR-126-3p and miR-140, both known to repress DNMT1 translation.