Purpose The type I Melanoma Antigen GEnes (MAGEs) are commonly expressed in cancers, fueling speculation that they may be therapeutic targets with oncogenic potential. They form complexes with RING domain proteins that have E3 ubiquitin ligase activity and promote p53 degradation. MAGE-A3 was detected in tumor specimens from patients with multiple myeloma and its expression correlated with higher frequencies of Ki-67+ malignant cells. In this report, we examine the mechanistic role of MAGE-A in promoting survival of proliferating multiple myeloma cells. Experimental Design The impact of MAGE-A3 expression on survival and proliferation in vivo was examined by immunohistochemical analysis in an independent set of tumor specimens segregated into two groups; newly diagnosed, untreated patients and patients who had relapsed after chemotherapy. The mechanisms of MAGE-A3 activity were investigated in vitro by silencing its expression by shRNA interference in myeloma cell lines and primary cells and assessing the resultant effects on proliferation and apoptosis. Results MAGE-A3 was detected in a significantly higher percentage of relapsed patients compared to newly diagnosed, establishing a novel correlation with progression of disease. Silencing of MAGE-A demonstrated that it was dispensable for cell cycling, but was required for survival of proliferating myeloma cells. Loss of MAGE-A led to apoptosis mediated by p53-dependent activation of pro-apoptotic Bax expression and by reduction of survivin expression through both p53-dependent and independent mechanisms. Conclusions These data support a role for MAGE-A in the pathogenesis and progression of multiple myeloma by inhibiting apoptosis in proliferating myeloma cells through two novel mechanisms.

1795 Poster Board I-821 The type I Melanoma Antigen GEne (MAGE) proteins MAGE-A3 and CT7 (MAGE-C1) were commonly detected in primary tumor cells from multiple myeloma patients and their expression was correlated with advanced disease and proliferation. They belong to the Cancer-Testis antigen (CTAg) family of tumor-associated proteins. In gene expression analyses of primary myeloma cells, CTAg were associated with proliferative gene signatures and poor clinical outcome. These findings suggest that type I MAGE may play a pathogenic role in proliferation or survival in multiple myeloma cells. To test this hypothesis, we examined MAGE expression, proliferation, and apoptosis in primary myeloma specimens and human myeloma cell lines (HMCL). First, we examined CTAg expression and proliferation in vivo at two critical clinical milestones, in newly diagnosed, untreated patients and patients who relapsed after chemotherapy. MAGE-A3 was detected in a higher percentage of tumor specimens from relapsed patients (77%) compared to those from newly diagnosed patients (36%, p=0.0003), whereas CT7 was detected in about 75% of both patient populations. The percentage of proliferating myeloma cells, as measured by staining for the proliferation marker Ki-67, was significantly higher in relapsed specimens (19.0 ± 3.5%) compared to newly diagnosed (6.9 ± 1.3%, p=0.0002), demonstrating an association between MAGE-A3, progression of disease and proliferation. Second, we investigated the functional role of MAGE-A3 by silencing this gene in HMCL by shRNA interference. Targeted lentiviral shRNA transduction efficiently knocked down MAGE-A3 mRNA (≥90% compared to controls) and protein in MM.1r and Arp-1 HMCL by 48 hours and this effect was maintained up to 96 hours. Pulse labeling of HMCL with bromodeoxyuridine for 30 minutes revealed that silencing of MAGE-A3 led to cell cycle arrest, as evidenced by the complete loss of cells in S phase and accumulation of cells in both G1 and G2. This was accompanied by increased expression of the tumor suppressor p53 and the endogenous cyclin-dependent kinase (CDK) inhibitor p21Cip1, a p53 target that inhibits CDKs in both late G1 and G2. However, CDK4/6-specific phosphorylation of the retinoblastoma gene product (Rb) was unimpaired, indicating that control of the mid-G1 cell cycle checkpoints by Rb remained intact and suggesting that MAGE-A3 acted in part to promote G1-S progression. Within 24 hours of cell cycle arrest, 70-80% of MAGE-A3-silenced cells underwent apoptosis as measured by Annexin V staining, compared to '20% in cells transduced with a non-target control lentivirus or untreated. Furthermore, this apoptosis was caspase-dependent, as it was completely prevented by the pan-caspase inhibitor Quinoline-Val-Asp-CH2-OPh, and was triggered by the loss of mitochondrial outer membrane potential in the activation of the intrinsic apoptosis pathway. Taken together, the in vivo and in vitro results suggest that MAGE-A3 promoted myeloma cell proliferation by inhibiting p53-dependent expression of p21, and loss of this activity leads to growth arrest and cell cycle-coupled apoptosis via activation of the intrinsic apoptosis pathway. Understanding the biochemical mechanism of MAGE-A3 in cell cycle regulation and survival may identify novel therapeutic strategies for multiple myeloma. Proof of principle in this disease may lead to broader application of these strategies in other cancers that express MAGE-A3. Disclosures Niesvizky: Proteolix: Research Funding, data monitoring committee; Seattle Genetics, Inc: Research Funding; Celgene: Research Funding, Speakers Bureau; Millenium: Research Funding, Speakers Bureau.

<div>Abstract<p><b>Purpose:</b> The type I Melanoma Antigen GEnes (MAGEs) are commonly expressed in cancers, fueling speculation that they may be therapeutic targets with oncogenic potential. They form complexes with RING domain proteins that have E3 ubiquitin ligase activity and promote p53 degradation. MAGE-A3 was detected in tumor specimens from patients with multiple myeloma and its expression correlated with higher frequencies of Ki-67⁺ malignant cells. In this report, we examine the mechanistic role of MAGE-A in promoting survival of proliferating multiple myeloma cells.</p><p><b>Experimental Design:</b> The impact of MAGE-A3 expression on survival and proliferation <i>in vivo</i> was examined by immunohistochemical analysis in an independent set of tumor specimens segregated into two groups: newly diagnosed, untreated patients and patients who had relapsed after chemotherapy. The mechanisms of MAGE-A3 activity were investigated <i>in vitro</i> by silencing its expression by short hairpin RNA interference in myeloma cell lines and primary cells and assessing the resultant effects on proliferation and apoptosis.</p><p><b>Results:</b> MAGE-A3 was detected in a significantly higher percentage of relapsed patients compared with newly diagnosed, establishing a novel correlation with progression of disease. Silencing of MAGE-A showed that it was dispensable for cell cycling, but was required for survival of proliferating myeloma cells. Loss of MAGE-A led to apoptosis mediated by p53-dependent activation of proapoptotic Bax expression and by reduction of survivin expression through both p53-dependent and -independent mechanisms.</p><p><b>Conclusions:</b> These data support a role for MAGE-A in the pathogenesis and progression of multiple myeloma by inhibiting apoptosis in proliferating myeloma cells through two novel mechanisms. <i>Clin Cancer Res; 17(13); 4309–19. ©2011 AACR</i>.</p></div>

<div>Abstract<p><b>Purpose:</b> The type I Melanoma Antigen GEnes (MAGEs) are commonly expressed in cancers, fueling speculation that they may be therapeutic targets with oncogenic potential. They form complexes with RING domain proteins that have E3 ubiquitin ligase activity and promote p53 degradation. MAGE-A3 was detected in tumor specimens from patients with multiple myeloma and its expression correlated with higher frequencies of Ki-67⁺ malignant cells. In this report, we examine the mechanistic role of MAGE-A in promoting survival of proliferating multiple myeloma cells.</p><p><b>Experimental Design:</b> The impact of MAGE-A3 expression on survival and proliferation <i>in vivo</i> was examined by immunohistochemical analysis in an independent set of tumor specimens segregated into two groups: newly diagnosed, untreated patients and patients who had relapsed after chemotherapy. The mechanisms of MAGE-A3 activity were investigated <i>in vitro</i> by silencing its expression by short hairpin RNA interference in myeloma cell lines and primary cells and assessing the resultant effects on proliferation and apoptosis.</p><p><b>Results:</b> MAGE-A3 was detected in a significantly higher percentage of relapsed patients compared with newly diagnosed, establishing a novel correlation with progression of disease. Silencing of MAGE-A showed that it was dispensable for cell cycling, but was required for survival of proliferating myeloma cells. Loss of MAGE-A led to apoptosis mediated by p53-dependent activation of proapoptotic Bax expression and by reduction of survivin expression through both p53-dependent and -independent mechanisms.</p><p><b>Conclusions:</b> These data support a role for MAGE-A in the pathogenesis and progression of multiple myeloma by inhibiting apoptosis in proliferating myeloma cells through two novel mechanisms. <i>Clin Cancer Res; 17(13); 4309–19. ©2011 AACR</i>.</p></div>

Supplementary Figures S1-S3 from MAGE-A Inhibits Apoptosis in Proliferating Myeloma Cells through Repression of Bax and Maintenance of Survivin