MAGE-a Inhibits Apoptosis in Multiple Myeloma through Regulation of Bim and PUMA

Type I Melanoma Antigen Genes of the MAGE-A family (A1-A12) are commonly expressed in multiple myeloma (MM). Their expression is associated with high-risk gene expression profiles (GEP) and correlated with relapse and progression of disease. Type I MAGE proteins partner with RING domain proteins to form ubiquitin ligase complexes, and in laboratory models of solid tumors they ubiquitinylate cancer-associated targets involved in metabolism, autophagy, and metastasis. MAGE-A proteins recruit HDACs to chromatin and p53 transcription complexes, and this activity is associated with chemotherapy resistance. We demonstrated that silencing of MAGE-A in MM cells caused rapid induction of intrinsic apoptosis, characterized by loss of ubiquitinylation and stabilization of p53 protein and activation of a pro-apoptotic p53 transcriptional program including increased RNA and protein expression of Bax. MAGE-A RNA and protein expression in primary myeloma specimens were correlated with resistance to panobinostat (an HDAC inhibitor)-based chemotherapy in a recent clinical trial for relapsed myeloma from our group (NCT01651039) and silencing of MAGE-A sensitized human myeloma cell lines (HMCL) to panobinostat-induced apoptosis. These findings lead to the hypothesis that MAGE-A genes play a critical role in myeloma pathogenesis by inhibiting apoptosis and promoting resistance to chemotherapy, resulting in the survival of clones that spawn relapse. To investigate the mechanisms of these anti-apoptotic functions, we examined the role of MAGE-A in response to different classes of chemotherapy agents and analyzed the impact of MAGE-A on transcription and protein expression of key regulators of apoptosis. We silenced MAGE-A expression in human myeloma cell lines (HMCL) MM.1r and H929 (both are p53 wt) by transduction of a MAGE-A-specific shRNA lentivirus construct, which resulted in nearly complete loss of MAGE-A protein at 24-48 hrs, or a non-target lenti construct and incubated the cells with different classes of anti-myeloma chemotherapy agents; panobinostat, the alkylating agent melphalan, the proteasome inhibitor bortezomib, or the cereblon modifying drug lenalidomide. Treated and control cells were incubated with a range of concentrations of drug for 24 hrs followed by assessment of cell viability. Silencing of MAGE-A significantly increased sensitivity (decreased LD50) to panobinostat and melphalan, but not bortezomib or lenalidomide. We examined the effect of MAGE-A silencing on gene expression by RNAseq. GEP of MAGE-A shRNA transduced HMCL versus controls enriched a set of 201 differentially expressed genes. This set featured increased expression of key initiators of apoptosis, including PUMA, a p53 transcriptional target, and Bim, which is known to play a significant role in hematopoietic development. Functional enrichment analysis of differentially expressed genes demonstrated activation of p53-mediated apoptosis and DNA damage response pathways in response to MAGE-A silencing. We analyzed protein expression of Bcl-2 family members by silencing MAGE in HMCL and western blotting of heavy membrane preparations to concentrate membrane-associated proteins. Loss of MAGE-A resulted in higher levels of Bim and PUMA proteins compared to controls. Of note, the short isoform of Bim (Bim S), which is the most potent activator of apoptosis, was relatively higher in abundance compared to the long and extra long isoforms (Bim L and EL). Bim protein is negatively regulated by phosphorylation at Ser69 and 77 by Erk1/2 and other kinases followed by ubiquitinylation and proteasomal degradation. In the absence of MAGE-A, phosphorylated Bim is stabilized, suggesting that it is not ubiquitinylated. It appears MAGE-A regulates Bim at both the transcriptional and post-translational levels. These results demonstrate that MAGE-A genes inhibit apoptosis and promote chemotherapy resistance in myeloma cells through regulation of Bim and PUMA. The observed effects with HDAC inhibitor and alkylating agent and the pathway enrichment in the GEP studies suggest that MAGE-A interacts with p53-mediated pathways to inhibit apoptosis in the setting of DNA damage and chromatin remodeling. Therefore, MAGE-A is a promising novel therapeutic target, and inhibition of its anti-apoptotic activity may directly induce apoptosis and increase sensitivity to chemotherapy-induced apoptosis.