Role of the IL-6 Pathway in Multiple Myeloma Cell Growth and Its Implications in Target Gene Hypermethylation.

Aberrant methylation of promoter regions of tumor suppressor genes (TSG) has recently become recognized as an important epigenetic event in cancer and tumor progression including multiple myeloma. Interleukin-6 (IL-6), which is known to play a significant role in the pathophysiology of myeloma, regulates DNA methylation by inducing expression of FLI-1, a transcription factor of DNA methyltransferase-1 (DNMT-1), resulting in over-expression of DNMT-1 and thus hypermethylation of TSG. Despite this understanding, attempts to bring IL-6 blockade to the clinic have had limited success. We hypothesize that IL-6 regulation of epigenetic events (hypermethylation) may be an important pathway that could eventually allow rational chemotherapeutic/anit-IL-6 combinations. We have studied the correlation of IL-6 expression and dependence in myeloma cell lines and correlated that to the methylation profile of TSG, DcR1 and CDH1. Using three well-established multiple myeloma cell lines: U266B1 (U266), RPMI 8226 (RPMI), and KAS6/1 (KAS), we have confirmed that KAS is IL-6-dependent (exogenous IL-6 is needed) whereas the U266 and RPMI are IL-6 independent. To determine if blockade of the IL-6 pathway would inhibit the growth of the myeloma cell lines, these cells were grown in the presence of an anti-IL-6 antibody (B-E8; Cell Sciences, Canton, MA) that is known to block IL-6 signaling in the cells. We found that blocking IL-6 (by B-E8, 200 ng/ml) inhibited the growth of U266 (36% inhibition; n≥3, p<0.01) and KAS (68% inhibition; n≥3, p<0.001) cells, but not RPMI cells. We examined IL-6 expression in these three cell lines by RT-PCR and found that U266 expresses IL-6 mRNA, but RPMI and KAS cells do not. This IL-6 mRNA expression pattern correlates well with the anti-IL-6 cellular proliferation findings. Since RPMI is not dependent on the addition of exogenous IL-6 and blocking the IL-6 pathway had no effect on cell growth; therefore, we would not expect the cells to express IL-6. The U266 cells are not dependent on exogenous IL-6 but cellular proliferation can be blocked with an anti-IL-6 antibody; therefore, we expected that the cells would endogenously express IL-6. Furthermore, KAS cells are dependent on exogenous IL-6 and cell growth can be inhibited by anti-IL-6 antibody; therefore, we would not expect the cells to express IL-6. To determine if IL-6 sensitivity correlated with hypermethylation of TSG, we investigated the methylation status of the DcR1(tumor necrosis factor-related apoptosis inducing ligand decoy receptor 1) and CDH1 (Cadherin 1) loci. We have shown that CDH1 is methylated in U266 cells and un-methylated in RPMI cells. Experiments are underway to determine the methylation status in KAS cells and gene expression in all cell-lines for CDH1. Finally, we found that the RPMI and KAS cells are un-methylated and U266 cells are methylated at the DcR1 locus. We have found that in the RPMI and U266 cell lines that methylation of target TSG correlates with anti-IL-6 sensitivity. These data support our hypothesis that an IL-6-dependent pathway may regulate hypermethylation of important TSG in multiple myeloma. Newer chemotherapeutic agents that may affect methylation pathways are being studied in combination with IL-6 blockade.