Inhibition of the Epigenetic Modifier EZH2 By Mir-138 Upregulates Tumor Suppressor Gene Rbpms and Contributes to Drug Susceptibility in Preclinical Model of Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy driven by numerous genetic and epigenetic alterations including miRNA aberration. Several studies have shown that enhancer of zeste homologue 2 (EZH2) is overexpressed in MM. However, the detailed oncogenic mechanism of EZH2 in drug resistance of MM is not clear. Our previous study showed that miR-138 is an EZH2 target gene, which was epigenetically silenced by histone methylation in its promoter region in drug resistant MM cell lines and primary patient samples. Moreover, luciferase reporter assay confirmed direct targeting of EZH2 by miR-138 in MM cells, suggesting a double negative feedback loop between miR-138 and EZH2 in MM. We also identified that EZH2 and miR-138 expression were negatively correlated in primary patient samples. Here, we investigated how unbalanced miR-138/EZH2 regulatory axis is involved in pathogenesis and drug resistance of MM.

To examine the functional effect of miR-138 on cell proliferation and apoptosis in MM cells, two resistant cell lines (8226-R5 and MM.1R) were transduced with miR-138 precursor or scramble lentiviruses and treated with different anti-myeloma drugs including bortezomib (BTZ) , lenalidomide (Len), dexamethasone (Dex) and doxorubicin (Dox). Overexpression of miR-138 in drug resistant cells could re-sensitize the resistant cells to anti-myeloma drugs. Also, reduction of cell proliferation upon overexpression of miR-138 was associated with apoptosis of the resistant cells. In addition, we explored whether EZH2 could rescue the effect of miR-138 on MM cells growth. To this end, we performed functional rescue assay by overexpressing EZH2 in 8226-R5 and MM.1R cells and co-transfecting with miR-138 mimics or scramble. EZH2 overexpression partially abolished the anti-tumor effect induced by miR-138 and BTZ confirming that combination of miR-138 and BTZ induces an inhibitory effect in MM cells by targeting EZH2.

Furthermore, to investigate the downstream mechanism of EZH2 targeting in MM drug resistance, H3K27me3 ChIP-seq and genome-wide profiling datasets were analyzed and revealed that the EZH2 target gene, RNA-binding protein with multiple splicing (RBPMS), was significantly silenced in MM in comparison to other types of cancers. It was also downregulated in ISS stage III of MM compared to stage I and II. Notably, we identified that RBPMS was downregulated in 8226-R5 and MM1.R resistant cell lines in comparison to 8226 and MM1.S cells, respectively. The ChIP-qPCR also showed that the histone H3K27 was significantly methylated in the promoter region of RBPMS in drug resistant cells and primary patient samples in comparison to parental lines and normal donor samples, respectively. Additionally, functional study on RBPMS silenced cells revealed that knockdown of RBPMS could confer drug resistance to parental MM cells, suggesting a tumor suppressor activity for RBPMS in MM. Remarkably, targeting of EZH2 by miR-138 overexpression could restore the RBPMS expression level in drug resistant cells.

In order to translate our findings to a therapeutic model, we next stablished a mouse xenograft model and investigated the effect of miR-138 on tumorigenesis of MM resistant cells in vivo. Intratumoral injection of synthetic miR-138 significantly suppressed tumorigenesis in combination with BTZ. The miR-138 or BTZ alone moderately prolonged survival compared to control treatment. However, the combination treatment of miR-138 mimics and BTZ significantly extended the overall survival (p= 0.0395). In addition, EZH2 protein and mRNA level were decreased in miR-138 mimics treated groups compared with controls whereas the expression of RBPMS was induced, indicating in vivo targeting of EZH2 by miR-138. IHC analysis of xenograft tumor sections showed that combination treatment of miR-138 mimics and BTZ resulted in a decrease in the proliferation index (Ki67) and an increase in the apoptotic index (Tunnel), compared to either BTZ or miR-138 mimics alone. Collectively, these results support that miR-138 functions as a tumor suppressor miRNA and contributes to the effect of EZH2 on MM cell growth and drug resistance.

Our findings indicate that targeting of EZH2 by miR-138 sensitizes MM cells to anti-myeloma drugs and contributes to induction of apoptosis of MM cells, as well as suppression of MM tumor growth in vivo and could be a potential candidate for clinical application to the treatment of MM.