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Abstract 1908 Introduction: MAGE-C1/CT7 encodes for a cancer/testis antigen (CTA) frequently expressed in multiple myeloma (MM) that may be a potential target for immunotherapy in this still incurable disease. The expression of this CTA is restricted to malignant plasma cells and a positive correlation between MAGEC1/CT7 expression and advanced stage has been demonstrated for MM. It has been suggested that MAGE-C1/CT7 might play a pathogenic role in MM; however, the exact function of this protein in the pathophysiology of MM is not yet understood. Objectives: (1) To clarify the role of MAGE-C1/CT7 in the control of cellular proliferation and cell cycle regulation in myeloma cell line SKO-007 and (2) to evaluate the impact of silencing MAGE-C1/CT7 on cells treated with bortezomib. Material and Methods: Short hairpin RNA (shRNA) specific for MAGE-C1/CT7 was inserted in the pRETROSUPER(pRS) retroviral vector. The pRS-shRNA-MAGE-C1/CT7 was co-transfected with pCL-amphotropic packing vector in 293T cells to produce virus particles. Sko-007 myeloma cell line was transduced for stable expression of shRNA-MAGE-C1/CT7. Downregulation of MAGE-C1/CT7 was confirmed by real time PCR (RQ-PCR) and western blot. Functional studies included cell proliferation, cell cycle analysis using propidium iodide, and analysis of apoptosis using annexin V staining. Results: SKO-007 MM cell line was transduced for stable expression of shRNA-MAGE-C1/CT7. SKO-007 cells were divided into three derivatives: empty vector (pRS) and ineffective shRNA (antisense strand deleted – GC bases) [both used as controls for all the experiments] and inhibited (shMAGE-C1/CT7). MAGE-C1/CT7 mRNA expression was ∼5 times lower in inhibited cell line than control cells by RQ-PCR. Western blot showed 70–80% decrease in MAGE-C1/CT7 protein expression in inhibited cells when compared with controls. Functional assays did not indicate a difference in cell proliferation and DNA synthesis when inhibited cells were compared with controls. We used empty vector, ineffective shRNA and inhibited cells to determine whether inhibition of MAGE-C1/CT7 was associated with cell cycle dysregulation. We detected differences between inhibited cells and both controls regarding the proportion of myeloma cells in the G2/M phase (p Conclusions: MAGE-C1/CT7 antigen inhibition did not change cell proliferation and DNA synthesis in SKO-007 cells. However, we found that MAGE-C1/CT7 plays in cell cycle regulation, protecting SKO-007 cells against bortezomib-induced apoptosis. Therefore, MAGE-C1/CT7 silencing by shRNA could be a strategy for future therapies in MM, i.e. in combination with proteasome inhibitors. [Supported by CNPq and LICR] Disclosures: No relevant conflicts of interest to declare.

Abstract 4888 Introduction MAGEC1/CT7 gene encodes for a cancer testis (CT) antigen frequently expressed in multiple myeloma (MM) and may be a potential target for immunotherapy in this still incurable disease. This CT gene expression is restricted to malignant plasma cells and may be related with tumor progression, since it seems to play a role as a “gatekeeper” gene for other CT antigens and can be associated with a more aggressive phenotype. However, the exact function of this protein for tumor biology is not yet understood. Objective The aim of this study is to elucidate the role of MAGEC1/CT7 in the control of cellular proliferation, DNA synthesis and invasive potential of Sko-007 myeloma cell line. Material and Methods We used a short hairpin RNA (shRNA) specific for MAGEC1/CT7 gene that was previously inserted in the pRS (pRETROSUPER) retroviral vector. The pRS-shRNA-MAGEC1/CT7 construct was co-transfected with pCL-amphotropic packing vector in 293T cells to produce virus particles. Sko-007 myeloma cell line was transduced and selected for stable expression of shRNA-MAGEC1/CT7. Expression of MAGEC1/CT7 was analyzed by Western Blot and Real Time PCR (RQ-PCR). Functional studies included cell proliferation, 3H thymidine incorporation and invasion potential, using matrigel-coated transwell filters. Results Sko-007 was chosen to perform the initial functional studies since the basal MAGEC1/CT7 gene expression level was higher in this cell line when compared with the other myeloma cell lines (U266 and SK-MM-2). Sko-007wt (wild type) cell was divided into three derivatives, Sko-007ev (empty vector), Sko-007shMAGEC1/CT7Δ (antisense strand deleted – GC bases) and Sko-007shMAGEC1/CT7. MAGEC1/CT7 expression was approximately five times down regulated in Sko-007shMAGEC1/CT7 when compared with Sko-007wt, Sko-007ev, and Sko-007shMAGEC1/CT7Δ cells by RQ-PCR. Sko-007shMAGE1/CT7 had an 80% decrease in MAGEC1/CT7 protein expression when compared with Sko-007wt, Sko-007ev and Sko-007shMAGEC1/CT7Δ by Western Blot. Our preliminary functional assays did not show any change in cell proliferation and DNA synthesis when Sko-007shMAGEC1/CT7 was compared with Sko-007wt, Sko-007ev and Sko-007shMAGEC1/CT7Δ. Indeed, Sko-007 cell line has invasive potential, but no change was observed in Sko-007shMAGEC1/CT7 or other cells (Sko-007wt, Sko-007ev and Sko-007shMAGEC1/CT7Δ). Conclusions Expression of MAGEC1/CT7 mRNA could be inhibited by the shRNA strategy, but the concomitant reduction of its protein level did not seem to have impact in proliferation, DNA synthesis or in invasive potential of Sko-007 MM cell line (Supported CNPq and LICR São Paulo Branch). Disclosures No relevant conflicts of interest to declare.