MAGE-A inhibit apoptosis and promote proliferation in multiple myeloma through regulation of BIM and p21Cip1

Abstract

The type I Melanoma Antigen Gene (MAGE) A3 is a functional target associated with survival and proliferation in multiple myeloma (MM). To investigate the mechanisms of these oncogenic functions, we performed gene expression profiling (GEP) of p53 wild-type human myeloma cell lines (HMCL) after MAGE-A knockdown, which identified a set of 201 differentially expressed genes (DEG) associated with apoptosis, DNA repair, and cell cycle regulation. MAGE knockdown increased protein levels of pro-apoptotic BIM and of the endogenous cyclin-dependent kinase (CDK) inhibitor p21^Cip1. Depletion of MAGE-A in HMCL increased sensitivity to the alkylating agent melphalan but not to proteasome inhibition. High MAGEA3 was associated with the MYC and Cell Cycling clusters defined by a network model of GEP data from the CoMMpass database of newly diagnosed, untreated MM patients. Comparative analysis of CoMMpass subjects based on high or low MAGEA3 expression revealed a set of 6748 DEG that also had significant functional associations with cell cycle and DNA replication pathways, similar to that observed in HMCL. High MAGEA3 expression correlated with shorter overall survival after melphalan chemotherapy and autologous stem cell transplantation (ASCT). These results demonstrate that MAGE-A3 regulates Bim and p21^Cip1 transcription and protein expression, inhibits apoptosis, and promotes proliferation.

Keywords: DNA repair; MAGE-A3; apoptosis; cell cycle regulation; multiple myeloma.

Figure 1. Gene expression profiling of HMCL after silencing of MAGE-A.

(A) Volcano plot of 201 gene set with minimum 2-fold change in expression enriched from RNA seq data from MM.1r and H929 HMCL after silencing of MAGE-A by RNAi with two distinct shRNA lentiviral constructs compared to non-target control. Several MAGE-A family members are downregulated, likely reflecting direct effect of shRNA construct. Pro-apoptotic Bcl-2 genes such as BAX are among the significantly upregulated genes. P_adj calculated by multiple testing using the Benjamini-Hochberg method. (B) Heat map of top over- and under-expressed genes. Apoptosis-associated genes are depicted in red. DNA-binding and repair genes are depicted in blue. Cell cycle-associated genes are depicted in green. NT, non-target shRNA lenti (SHC002, denoted as 002. shMAGE-A, MAGE-A3-targeted shRNA lenti (TRCN0000128375 and TRCN0000129750, denoted as 375 and 750, respectively).

Figure 2. Silencing of MAGE-A in HMCL results in stabilization of phosphorylated BIM and p21^Cip1.

MM.1r and H929 were transduced with MAGE-A3-specific lentiviral shRNA constructs or controls as described, and heavy membrane preps (A, C) were probed for expression of Bcl-2 family proteins. (A) Western blot of heavy membrane preps for Bcl-2 family proteins. MAGE-A3 knockdown and increased p53 protein were confirmed by western blot of whole cell lysate. COXIV, load control, representative of all blots. (B) Optical densitometry of western blots for BIM el as in (A) demonstrates significantly higher levels of this protein after MAGE-A silencing compared to controls. Data was pooled from five replicates, representing 13–15 data points each. Error bars, standard error of the mean. (C) Western blot of heavy membrane preps for p-ser69 and p-ser77 Bim el demonstrate stabilization of the phosphorylated protein after MAGE-A knockdown. (D) Whole cell lysates were probed by western blot for p21^Cip1. (E) Optical densitometry of western blots for p21^Cip1 as in (D) demonstrates significantly higher levels after MAGE-A silencing. Data pooled from five replicates, representing 15 data points each. Con, Untreated control cells. shNT, non-target shRNA lentiviral construct. shMA, MAGE-A3 targeted lentiviral shRNA construct TRCN0000128375.

Figure 3. Silencing of MAGE-A in HMCL increased sensitivity to melphalan-induced apoptosis.

MM.1r and H929 cells were transduced with MAGE-A3-specific lentiviral shRNA constructs or controls as described and incubated with increasing concentrations of melphalan (top graphs) or bortezomib (bottom graphs) and cell viability was assessed after 24 hrs by Cell TiterGlo assay.

Figure 4. MAGEA3 expression is associated with cell cycling, DNA replication/repair, and poor prognosis.

(A) Comparison of average MAGEA3 expression in patient subsets identified in MMNet analysis of the CoMMpass iA7 release²⁵ demonstrated high MAGEA3 associated with the MYC and CC clusters. (B) Subjects from the CoMMpass iA9 release dataset were stratified by MAGEA3 expression and differential gene expression was assessed between the highest and lowest quartiles. (C) Gene set enrichment analysis of differentially expressed genes demonstrated strong associations with cell cycling, DNA replication and repair pathways associated with high MAGEA3. (D) Kaplan-Meier projection of overall survival in MAGEA3 low and high quartiles of all subjects shows a significant association with shorter overall survival in the MAGEA3 high quartile compared to low. (E) Subset analysis of MAGEA3 high and low quartiles in patients who underwent melphalan conditioning and autologous stem cell transplantation for consolidation showed significantly worse overall survival associated with the MAGEA3 high quartile.

Figure 5. Model of MAGE-A3 activity in MM.

(A) DNA damage response pathways promote BIM expression and p53 transcriptional activity, resulting in increased expression of BAX and p21^Cip1. BIM displaces BAX from anti-apoptotic Bcl-2 family proteins, allowing it to translocate to mitochondrial membranes and initiate apoptosis. P21^Cip1 inhibits cyclin D1/CDK4/6 activity, blocking progression through the early G1 checkpoint. (B) MAGE-A3/RING complex activity, possibly activated through interactions with DNA repair pathways, down-regulates BIM and p53 through transcriptional and post-translational mechanisms, resulting in survival and proliferation.