Abstract 4729: MAGE-C2 promotes proliferation and enhances resistance to p53 DNA damage-mediated apoptosis in multiple myeloma.

Abstract

Abstract Background: MAGE-C2 is member of the MAGE class I family of genes and is commonly and specifically expressed in Multiple Myeloma (MM). Expression of MAGE genes is associated with an aggressive clinical course of MM and resistance to chemotherapy, suggesting that these genes may confer a survival advantage on myeloma cells. Here, we have analysed on a molecular level the influence of MAGE-C2 on the malignant phenotype of myeloma cells. Methods: The biological function of MAGE-C2 was investigated, by lentiviral shRNA stable gene silencing, in two MM cell lines (MOLP-8, IM-9) expressing wild-type (wt) p53 and two MM cell lines (U-266, SK-007) bearing a missense mutant p53 (A161T) that has partially lost its transcriptional activity. The effects of MAGE-C2 silencing were examined by assessing cell viability, proliferation and colony formation. Finally, we investigated the cell cycle distribution and the expression of key molecules of cell cycle progression and apoptosis using a real-time PCR array and western blot. Results: We found that lentivirus-mediated silencing of MAGE-C2 inhibited the proliferation and anchorage-independent growth of MM cell lines. The anti-proliferative effect of MAGE-C2 silencing was due to a 50-80% decrease of cells in the S phase, a cell cycle arrest at both G0/G1 and G2/M transitions, and an increase in the subG0/G1 diploid population based on an initiation of apoptotic cell death. Loss of MAGE-C2 expression was associated with an increase in the phosphorylation of the histone variant H2A.X on Ser139 which is a sensitive indicator of DNA damage. A constitutive phosphorylation on Ser1981 and Ser428, respectively, of the primary sensors of DNA damage ataxia telangiectasia mutant (ATM) and Rad3-related protein kinase (ATR) was detected before MAGE-C2 silencing suggesting the presence of an ongoing DNA damage in MM cell lines. Downstream of ATM and ATR, MAGE-C2 silencing led to the activation of CHK2 and/or CHK1 through phosphorylation at Ser296 and Thr68, respectively, and an increase in the endogenous level of p53 protein. The stabilization and activation of p53, through phosposphorylation at Ser20 by CHK1 and CHK2, correlated with an up-regulation of two transcriptional target genes, p21WAF1 and GADD45A, known to be essential for p53-induced G1 and G2 arrest, respectively. Accordingly, MAGE-C2 silencing induced a more than two-fold increase in the expression of the pro-apoptotic Bcl-2 genes such as BAX and BAK, being the main down-stream mediators of p53-dependant apoptosis following DNA damage. Conclusions: Our findings support an anti-apoptotic function of MAGE-C2 in MM through the regulation of key molecules involved in DNA damage repair pathway and p53-mediated apoptosis. The central role of MAGE-C2 in the biology of myeloma suggests that this CTA represents a promising target for myeloma-specific immunotherapies or other targeted modes of therapy for MM. Citation Format: Nesrine Lajmi, Sara Yousef, Tim Luetkens, Julia Templin, Carsten Bokemeyer, Nicolaus Kröger, Djordje Atanackovic. MAGE-C2 promotes proliferation and enhances resistance to p53 DNA damage-mediated apoptosis in multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4729. doi:10.1158/1538-7445.AM2013-4729