MAGE-A3 Promotes Chemotherapy Resistance and Proliferation in Multiple Myeloma through Regulation of BIM and p21^Cip1

The type I Melanoma Antigen Gene (MAGE) A3 is a promising functional target in multiple myeloma (MM). MAGE-A3 expression in primary MM specimens is associated with proliferation and progression of disease. Previous laboratory studies showed that MAGE-A3 inhibits p53-dependent and independent apoptosis and cell cycle regulation in human myeloma cell lines (HMCL) and primary cells. We investigated the mechanisms of these oncogenic activities in laboratory models and through analysis of gene expression and clinical outcome data. Gene expression profiling (GEP) by RNA sequencing (RNAseq) of p53+/+ HMCL MM.1r and H929 after MAGE-A knockdown identified a set of 201 differentially expressed genes (DEG, p<0.05). Of the top DEGs, eight were BH3-only Bcl-2 family members (BAX, BCL2L11/BIM, BBC3/PUMA, BMF, BCL2L14/BCLG) or apoptotic pathway genes (TNFRSF10B/TRAILR2, CASP3, TRIAP1). Four of the top DEGs were cell cycle regulation genes (TRAF4, CDKN1A/p21^CIP1, TRIM5, MAP2K6/MEK6) and three were DNA binding/damage repair genes (APOBEC3B, DDB2, ARID3A). We interrogated protein expression after MAGE knockdown and demonstrated significantly increased levels of pro-apoptotic BIM, but other Bcl-2 proteins were either not altered or not detected. BIM protein stability is regulated by serine phosphorylation, which leads to subsequent ubiquitinylation and degradation. MAGE knockdown resulted in higher levels of ser69- and ser77-phosphorylated BIM, indicating that MAGE does not affect phosphorylation but likely mediates further post-translational modification and degradation. We also detected increased levels of the endogenous cyclin-dependent kinase (CDK) inhibitor p21^Cip1, which correlated with the GEP results. Depletion of MAGE-A in HMCL increased the sensitivity to induction of apoptosis by the alkylating agent melphalan but not to proteasome inhibition, suggesting that inhibition of BIM expression may contribute to chemotherapy resistance. To assess the clinical significance of these mechanisms, we analyzed RNAseq data from the iA9 release of the Multiple Myeloma Research Foundation CoMMpass database of more than 650 newly diagnosed MM patients based on high or low MAGEA3expression, which revealed a set of significantly DEGs (p<0.05) that included several MAGE family members and related X-linked genes. Gene set enrichment analysis demonstrated associations with cell cycle and DNA replication pathways, similar to that observed in HMCL. We ranked subjects based on MAGEA3 mRNA expression levels and correlated the highest (219 subjects) and lowest quartiles (212 subjects) with clinical outcome, which showed that the MAGEA3high group had worse overall survival (Hazard Ratio = 2.5, p<0.01, data not shown). This survival difference was even more striking in the subgroup of subjects that had undergone high dose melphalan chemotherapy and autologous stem cell transplantation (ASCT, fig.). MAGEA3 high subjects (39 subs) had worse progression-free (p<0.1) and overall survival (p<0.01) compared to the lowest (37 subs). These results demonstrate that MAGE-A3 negatively regulates BIM at both the transcriptional and post-translational levels, which favor survival and resistance to chemotherapy. MAGE-A3 also inhibits p21^Cip1transcription and protein expression, likely through ubiquitinylation and degradation of p53 protein, which promotes passage through the early G1 checkpoint and proliferation. These mechanisms are a biochemical basis for MAGE-A3-mediated resistance to chemotherapy-induced apoptosis and the associations with progression of disease and tumor proliferation. These results provide a rationale to investigate methods to inhibit MAGE-A3 activity in MM.

View largeDownload slide

View largeDownload slide

Close modal