Downregulation of MDM2 Leads to Anti-Proliferative Effects through Activation of p53-Associated Pathway Mediated By Both Dual Inhibitor MX69 and Mir-548c-3p in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by abnormal proliferation of clonal plasma cells in the bone marrow. MM remains an incurable disease with a high rate of relapse and development of drug resistance. Mouse double minute 2 homolog (MDM2) has been characterized as an oncogene that is associated with cancer development and radio/chemotherapy resistance in cancer. However, the mechanism(s) underlying MDM2 overexpression and its association with drug resistance in MM have not been fully explored. In addition, the effect of the newly discovered dual inhibitor MX69, which targets MDM2 and XIAP, is unknown in MM.

To assess the effects of MDM2 overexpression in MM patients, we performed GEO data (GSE6477, GSE31161) differential expression analysis on bone marrow samples from three patient groups- normal donors, newly diagnosed MM and relapsed MM. We found that relapsed MM patients expressed high MDM2 compared to newly diagnosed MM patients, and both groups expressed higher MDM2 compared to normal donors (p<0.001). Furthermore, immunoblotting and qRT-PCR showed upregulated MDM2 expression in drug resistant MM cell lines (MM1R, 8226R5) as compared to their parental drug sensitive cells (MM1S, 8226S).

XIAP is an important inhibitor of apoptotic proteins caspases 3, 7 and 9. Elevated expression of XIAP is involved in chemotherapy resistance in cancer. To determine whether MDM2 and XIAP downregulation by dual inhibitor MX69 could induce apoptosis in MM cells, we treated two drug resistant MM cell lines with MX69. Cell viability assay showed that MX69 reduced viability of MM cells in a time- and dose-dependent manner. Since MDM2 is an antagonist of tumor suppressor p53, we measured the expression of p53 following MX69 treatment and found that MX69-mediated downregulation of MDM2 significantly enhanced p53, PUMA and p21 expression in MM cell lines harboring wild-type p53. In MM p53 null cells, we found that p53 family pro-apoptotic factors p63 and p73 were increased upon MX69 treatment.

To investigate the downstream mechanism of MDM2 targeting in MM drug resistance, we knocked down MDM2 in MM cells via siRNA-mediated silencing. Cell viability assay indicated that siMDM2 reduced cell proliferation compared to negative control (P=0.009). Following MDM2 silencing, XIAP expression was decreased and p53 expression was increased in p53 positive MM cell lines, whereas p63, p73 and p21 were increased in both p53 positive and p53 null MM cells. Furthermore, combined treatment of MX69 with dexamethasone (Dex), doxorubicin (Dox) or lenalidomide (Len) displayed synergistic toxic effects (combination index ranging from 0.437 to 0.8) in drug resistant MM cell lines. MX69 alone or in combination with Dex, Dox and Len significantly increased apoptosis and decreased proliferation of MM cells.

To explore whether miRNAs play a role in regulating MDM2 overexpression and MM drug resistance, several miRNA target prediction algorithms were exploited to generate a selective miRNA library for subsequent screening. Four miRNAs (miR-890, miR-211-5p, miR-429 and miR-548c-3p) were common among GSE49261, GSE39571, GSE16558 and GSE17498 GEO datasets. These four miRNAs were downregulated in MM patients and negatively correlated with MDM2. Furthermore, analyzing prediction score revealed that miR-548c-3p had the highest binding score with MDM2 3'UTR. Moreover, MiR-548c-3p showed a significant downregulation in drug resistant cells in comparison with their parental sensitive cells (p<0.0001). To determine whether MDM2 expression is selectively regulated by miR-548c-3p, we transfected two MM drug resistant cells with miR-548c-3p mimics and found that MDM2 mRNA and protein levels were suppressed and the cell viability was significantly reduced compared to the scrambled controls.

In conclusion, our results indicate that MDM2 overexpression is associated with disease progression and drug resistance in MM. Treatment of drug resistant MM cells with MDM2/XIAP dual inhibitor MX69 sensitizes the cells to anti-myeloma drugs and induces apoptosis. Our finding of the regulatory interaction between miR-548c-3p and MDM2 reveals key insights into the dysregulation of MDM2 in MM and provides a new therapeutic approach to target MDM2 by restoration of miR-548c-3p.