Targeting an MDM2/MYC Axis to Overcome Drug Resistance in Multiple Myeloma.

Abstract

Simple SummaryWe show that high expression of MDM2 is associated with poor prognosis and enhanced drug resistance in human myeloma cell lines (HMCLs). Inhibition of MDM2 by RNAi or by the MDM2/XIAP dual inhibitor MX69 significantly increased the sensitivity of resistant HMCLs and primary MM samples to bortezomib and other anti-myeloma drugs, demonstrating that MDM2 can modulate drug response. We uncovered a novel oncogenic regulatory loop between MDM2 and c-Myc that mediated MM drug resistance. MDM2 inhibition resulted in a remarkable induction of apoptosis and suppression of relapsed MM cell growth. Mechanistically, MDM2 stabilized c-Myc mRNA to upregulate its expression, while c-Myc transcriptionally inducedMDM2. Targeting this regulatory loop with MX69 re-sensitized chemo-resistant MM cells to anti-myeloma agents, irrespective of TP53 tumor suppressor status, and prolonged survival in xenograft MM mouse model. These results establish a rationale for therapeutic targeting of the MDM2/c-Myc axis to improve the clinical outcome of patients with refractory/relapsed MM.Background: MDM2 is elevated in multiple myeloma (MM). Although traditionally, MDM2 negatively regulates p53, a growing body of research suggests that MDM2 plays several p53-independent roles in cancer pathogenesis as a regulator of oncogene mRNA stability and translation. Yet, the molecular mechanisms underlying MDM2 overexpression and its role in drug resistance in MM remain undefined. Methods: Both myeloma cell lines and primary MM samples were employed. Cell viability, cell cycle and apoptosis assays, siRNA transfection, quantitative real-time PCR, immunoblotting, co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), soft agar colony formation and migration assay, pulse-chase assay, UV cross-linking, gel-shift assay, RNA-protein binding assays, MEME-analysis for discovering c-Myc DNA binding motifs studies, reporter gene constructs procedure, gene transfection and reporter assay, MM xenograft mouse model studies, and statistical analysis were applied in this study. Results: We show that MDM2 is associated with poor prognosis. Importantly, its upregulation in primary MM samples and human myeloma cell lines (HMCLs) drives drug resistance. Inhibition of MDM2 by RNAi, or by the MDM2/XIAP dual inhibitor MX69, significantly enhanced the sensitivity of resistant HMCLs and primary MM samples to bortezomib and other anti-myeloma drugs, demonstrating that MDM2 can modulate drug response. MDM2 inhibition resulted in a remarkable suppression of relapsed MM cell growth, colony formation, migration and induction of apoptosis through p53-dependent and -independent pathways. Mechanistically, MDM2 was found to reciprocally regulate c-Myc in MM; MDM2 binds to AREs on c-Myc 3′UTR to increase c-Myc mRNA stability and translation, while MDM2 is a direct transcriptional target of c-Myc. MDM2 inhibition rendered c-Myc mRNA unstable, and reduced c-Myc protein expression in MM cells. Importantly, in vivo delivery of MX69 in combination with bortezomib led to significant regression of tumors and prolonged survival in an MM xenograft model. Conclusion: Our findings provide a rationale for the therapeutic targeting of MDM2/c-Myc axis to improve clinical outcome of patients with refractory/relapsed MM.