Restoration of Mir-138 Expression By Epigenetic Modulation Overcomes Drug Resistance in Multiple Myeloma Cells

Multiple myeloma (MM) is a plasma cell malignancy characterized by highly heterogeneous genomic alteration including frequent changes in chromatin remodeling enzymes. The recent studies showed that deregulation of epigenetics mechanisms play an essential role in MM development. In particular, overexpression of enhancer of zeste homologue 2 (EZH2), the catalytic subunit of Polycomb Complex 2 (PRC2), has been shown in several cancers including MM. However, the detailed function of EZH2 histone methyltransferase in drug resistance of MM has not yet been elucidated. This study sought to investigate the role of histone modification changes in drug resistance of MM. First, we checked if the expression level of the epigenetic modulating enzyme, EZH2, changed in drug resistant cells (8226-R5 and MM1R) in comparison to parental sensitive cells (8226 and MM1S). We found that EZH2 expression was upregulated in 8226-R5 and MM1R cells. In addition, the global H3K27me3 level, a silencing histone modification mark induced by EZH2, was dramatically increased in the resistant MM cells. To support our finding, we analyzed some publically available gene expression datasets (GSE 6477 and GSE26760) and found that the EZH2 expression level was significantly upregulated in relapsed patients in comparison to newly diagnosed MM patients. It also revealed that overexpression of EZH2 correlated with the progression of the disease and poor survival of the MM patients.

We next examined the functional effect of EZH2 inhibition on resistant cells using a novel selective EZH2 inhibitor (EPZ-6438) which turned out to significantly inhibit proliferation of MM cells by MTT assay. Furthermore, combination of EPZ-6438 with bortezomib revealed synergistic cytotoxicity effects on 8226-R5 and MM1R cells. In parallel, global H3K27me3 level in MM cells was reduced by EPZ-6438 further confirming its specific functionality. To explore whether miRNAs are associated with EZH2 overexpression in drug resistance, we used several target scan algorithms and identified mir-138, a tumor suppressor miRNA, to potentially target EZH2. We found that mir-138 expression level was downregulated in 8226-R5 and MM1R relative to 8226 and MM1S, respectively. To investigate if the reduction is due to overexpression of EZH2, we performed H3k27me3 chip on the promoter region of mir-138 gene in MM cell lysates. The chip qPCR showed that occupancy of H3K27me3 in the promoter region of mir-138 was increased in 8226-R5 and MM1R cells compared to 8226 and MM1s cells leading to repression of mir-138 gene transcription in the resistant cells.As a result, we concluded that reduction of mir-138 expression level in the resistant MM cells is due to chromatin remodeling changes in its promoter region and identified mir-138 gene as a target of EZH2.

To further demonstrate the role of EZH2 in mir-138 expression, 8226-R5 and MM1R were treated with EZH2 inhibitor and mir-138 expression level was compared to untreated cells. We found a significant upregulation of mir-138 expression in EPZ-6438 treated cells compared with untreated controls. The chip qPCR results also confirmed that the inhibition of EZH2 decreased H3K27me3 occupancy on the mir-138 promoter region which indicated that epigenetic silencing is mechanism underlying miR-138 suppression in drug resistant cells. Furthermore, we overexpressed mir-138 in 8226-R5 and MM1R cells and treated them with bortezomib to assess cell viability using MTT assay. Overexpression of mir-138 sensitized 8226-R5 and MM1R cells to bortezomib. We also observed that overexpression of mir-138 reduced EZH2 expression level. These results suggest a negative feedback loop between miR-138 and EZH2 in our model system.

Altogether, these findings indicate that downregulation of miR-138, likely due to EZH2 overexpression, contributes to drug resistance of MM and the restoration of mir-138 expression by epigenetics modulation re-sensitizes drug resistant MM cells to anti myeloma drugs. We report, for the first time, that miR-138 is involved in a negative feedback loop with EZH2 in MM cells and provide a framework for targeting EZH2 or restoration of mir-138 as novel strategies for the treatment of MM.