SPAN-Xb Expression in Myeloma Cells Can Be Upregulated by Pharmacologics.

Abstract

SPAN-Xb is a spermatid-specific protein. We recently identified SPAN-Xb as a novel Cancer-Testis (CT) antigen in multiple myeloma (MM) (Blood 2003; 101: 955–960) and that it frequently elicited immune responses in vivo in these patients, suggesting that SPAN-Xb may be a suitable target for immunotherapy of MM. SPAN-Xb, however, was only detected in the tumor cells from 20% of MM patients. To increase the applicability of a SPAN-Xb tumor vaccine for MM, we have set out to determine if SPAN-Xb expression could be upregulated by pharmacologics in 8 MM cell lines.Since most CT antigen expression occurs due to promoter demethylation, we first determined the ability of a hypomethylating agent, 5-azacytidine, to upregulate SPAN-Xb expression. Prior to exposure to 5-azacytidine, SPAN-Xb transcripts were only detected by RT-PCR in ARP-1 and RPMI 8226 cells. Following incubation in 5-azacytidine (2 uM) for 96 hours, the levels of SPAN-Xb transcripts were upregulated, as determined by RT-PCR and real time PCR, in all MM cell lines (ARK-B, ARP-1, H929, IM9, MM1-R, RPMI 8226 and U266) except KMS-11 (Table 1). This was associated with a corresponding increase in the level of SPAN-Xb protein expression, as shown by immunocytochemistry using a murine monoclonal antibody directed at SPAN-Xb.We next determined if SPAN-Xb gene expression could be increased by cytokine pre-incubation of these cells. IL-2 (100 IU/ml) and IL-4 (1000 IU/ml) did not affect SPAN-Xb gene expression. On their own, IL-7 (0.1 ng/ml) and GM-CSF (10 U/ml) did not affect SPAN-Xb gene expression significantly. However, when combined, especially with 5-azacytidine, a summative effect of these pharmacologics on SPAN-Xb gene expression, as determined by real time PCR, was observed in IM9 cells (Table 2). Our results indicate the potential to upregulate SPAN-Xb expression in MM for therapeutic purpose using 5-azacytidine, GM-CSF and IL-7 and provide insight into the possible mechanisms of regulation of SPAN-Xb gene expression.Table 1Cellspre-5-azacytidinepost-5-azacytidineARK-B0456ARP-12601376H9290116IM90532KMS-1100MM1-R0428RPMI 82262721348U2660984SPAN-Xb mRNA copy numbers before and after 5-azacytidine exposureTable 2PharmacologicsSPAN-Xb mRNA5-azacytidine532IL-74GM-CSF9IL-7 + GM-CSF365-azacytidine + IL-710885-azacytidine + GM-CSF9525-azacytidine + IL-7 + GM-CSF1632medium only0SPAN-Xb mRNA copy numbers after exposure to combination pharmacologics