Role of the IL-6 Pathway in Multiple Myeloma Cell Growth and Its Implications in Target Gene Hypermethylation.
Authors: Ingersoll Susan B. , Thoni Natalie D. , Ahmed Farhana , Monahan Kimberly A. , Caballero Lizette , Batista Anginett , Ahmad Sarfraz , Edwards John R. 
Published: October 2019

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.