In our analysis of 565 newly diagnosed patients, MAGE-A3 expression was observed in 25% of patients. However, MAGE-A3 expression was much higher in relapsed patients (50%) and higher still in patients with highly proliferative disease (80%). Further, there was a correlation of adverse survival (OS and EFS) with MAGE-A3 expression. Our data are supported by other studies in which MAGE-A3 expression has been correlated with advanced MM, MM progression, MM burden, a high plasma cell proliferation index and cytogenetic abnormalities (6
). The increased frequency of MAGE family gene expression in aggressive disease may be because of a functional role in cell-cycle progression and drug resistance (42
). These features make MAGE-A3 an attractive target for MM immuno-therapy. MAGE-A3 is also immunogenic and not expressed by normal tissues surveyed by the immune system. We and others have previously reported that MAGE-A3 protein vaccination induces broad, potent and long-lasting immune responses (31
). A concern is that several studies have reported heterogeneous expression of CT-Ag in tumors, which raises the specter of tumor escape by MAGE-A3-negative MM clones (17
). We therefore hypothesized that epigenetic modulation could induce MAGE-A3 expression, thus permitting recognition and killing of MM by MAGE-A3-specific CTL.
We were indeed able to induce MAGE-A3 expression by exposing the MAGE-A3-negative MM cell lines LP1 and ANBL6 and the colorectal cell line COLO205 to the methyltransferase inhibitor 5AC at an in vitro
concentration of 500 nm
, which is comparable to a clinically achievable dose of 12.5 mg/m2
). Baseline MAGE-A3 expression was correlated with MAGE-A3 promoter methylation status. Untreated cell lines that lacked expression of MAGE-A3 exhibited methylated promoter sequences as opposed to MAGE-A3-positive cell lines that had unmethylated sequences detected. Furthermore, MS-PCR of ANBL6 and LP1 indicated that the MAGE-A3 promoters were hypermethylated prior to treatment and hypomethylated after treatment with 5AC. These results implicate MAGE-A3 promoter demethylation as a mechanism of MAGE-A3 induction that can be modulated by 5AC (15
We also investigated the effect of the HDACi MGC on MAGE-A3 gene expression. MGC alone did not appreciably increase MAGE-A3 expression; however, sequential treatment with 5AC followed by MGC led to enhanced MAGE-A3 gene expression, suggesting that histone deacetylase (HDAC) activity may play a modest role in the transcriptional regulation of MAGE-A3.
MAGE-A3 protein expression was confirmed in ANBL6 cells and LP1 cells transfected with HLA-A*6801 after 5AC treatment by Western blotting and flow cytometry. When ANBL6 was subjected to longer treatment with 5AC, we observed enhanced MAGE-A3 protein expression compared with shorter incubations, suggesting that the maximal effect on MAGE-A3 promoter demethylation had not been reached. Killing of LP1 A68 targets after treatment with 5AC/MGC by MAGE-A3-specific CTL also occurred only after prolonged exposures. Failed target lysis could be the result of insufficient density of MAGE-A3 peptide/HLA complexes on the target cell surface, or differences in MAGE-A3 intracellular processing, that can affect the context in which the processed peptides are displayed on the cell surface (56
). The addition of MGC may have enhanced MAGE-A3 protein expression while longer treatment periods may allow additional time for optimal protein turnover, thus increasing peptide availability enough to cross the threshold needed for recognition by MAGE-A3 CTL. Synergism between MGC and 5AC was observed at the RNA level. Combinatorial or synergistic activity in terms of cytolysis was not formally tested because of limited MAGE-A3-specific CTL availability.
In summary, MAGE-A3 and other CT-Ag have been investigated extensively as a target for immunotherapy. Heterogeneous tumor antigen expression must be addressed when designing immunotherapeutic treatments to prevent escape by selection of tumor antigen-negative cancer cells. Combining vaccination or adoptive transfer of MAGE-specific T cells with an agent that epigenetically ‘turns on’ MAGE-A3 production could lead to immune recognition and eradication of both highly proliferative antigen-positive clones as well as residual, previously antigen-negative, MM cells. A potential disadvantage of the use of hypomethylating agents is the enhancement of genomic instability. Furthermore, up-regulation of MAGE-A3 could potentially have other negative consequences, including anti-apoptotic effects, promotion of the survival of clonogenic precursors and the induction of drug resistance (48
Clinical studies are therefore warranted to determine whether effective up-regulation of MAGE-A3 protein is sufficiently sustained for T-cell expansion and cytolysis of MM cells to occur, which can then negate the possible adverse effects of the induction of MAGE-A3 expression. This study provides proof of the principle that MAGE-A3-expressing MM can be recognized by MAGE-A3-specific CTL generated from CTL precursors isolated from a MAGE-A3-vaccinated MM patient. A combination regimen of hypomethylation, HDAC inhibition and vaccine therapy in patients with MAGE-A3-positive MM, either in remission or with stable disease, after therapy, could be a first clinical trial to test the hypothesis that MAGE-A3-specific immunosurveillance may have therapeutic benefit.