Blockage of CGEN-928 Expression Down-Regulates AKT Signaling Pathway and Inhibits Multiple Myeloma (MM) Cell Proliferation

Abstract 5102 We have recently reported that CGEN-928 is highly expressed on the cell membrane of cell lines, human xenografts, and primary tumor cells from MM. Anti-CGEN-928 (anti-TM21) polyclonal antibody blocked the expression of CGEN-928 which decreased MM tumor cell proliferation and increased apoptosis in the MM cell lines MM1s, RPMI8226 and U266 as well as primary MM tumor cells. The mechanism through which blocking CGEN-928 decreases MM tumor cell proliferation and enhances apoptosis has not been elucidated clear. In this study, a CGEN-928 shRNA (lentiviral particles) was used to silence this gene's expression, and determine its impact on the AKT signal transduction pathway which has been shown to play an important role in MM tumor cell metabolism proliferation, and survival. Briefly, MM1s or primary MM tumor cells were cultured in a 12-well plate for 24 hours prior to the viral infection. On the following day, a mixture of 5ug/ml Polybrene and fresh medium were added to the cells. The CGEN-928 shRNA lentiviral particles were then added to the culture. While transducing cells, we treated a portion of the cells with a negative control through introduction of control shRNA lentiviral particles. To ensure we achieved a successful transduction, we also treated another portion of the cells with cop GFP control Lentiviral particles. We confirmed that 75% of MM cells were transduced based on GFP+ cell counts after 24 hours treatment. The day following the transduction, the cultured medium was removed and replaced with fresh medium without polybrene. Two days following transduction, we used fresh 10ug/ml puromycin-containing medium to select stable MM cells. We replaced the medium with fresh puromycin-containing medium every three days until resistant MM tumor cells were stable. Proliferation rate of the MM1s tumor cells transduced with CGEN-928 shRNA (85%) 24 hours was much lower than the tumor cells transduced with control lentiviral particles rate (170%). The proportion of MM cells undergoing apoptosis treated with CGEN-928 shRNA (42%) was higher than MM cells transduced with control lentiviral particles (13%). We next examined several protein phosphorylation sites related to AKT signaling pathway by Western blot. The results showed AKT1 phosphorylation in MM tumor cells transduced with CGEN-928 shRNA or anti- CGEN-928 polyclonal antibody was decreased and phosphorylation of c-Raf, GSK-3β, factors downstream of AKT were also down-regulated. PTEN phosphorylation slightly decreased in MM cell treated with anti-CGEN-928 antibody but did not change in MM cells silenced with CGEN-928 shRNA. We further examined downstream gene expression of the AKT pathway when CGEN-928 was silenced using siRNA or the anti-CGEN-928 TM-21 antibody. We found AKT1 gene expression was reduced in the presence of CGEN-928 siRNA or antibody but it did not impact ATK2 and AKT3. mTOR gene expression in MM tumor cells was decreased with exposure to CGEN-928 siRNA but anti-TM21 showed no effect. Cyclin D1 gene expression in MM tumor cell was not affected by CGEN-928 siRNA and antibody. These studies suggest that blockage of CGEN-928 antigen expression inhibits MM tumor cell proliferation and enhance tumor cell apoptosis through AKT signaling pathway. Currently, a monoclonal anti-CGEN-928 antibody is in development that will be used by our group to evaluate its anti-MM effects both in vitro and in vivo using our SCID-hu models of human MM. Disclosures: Berenson: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau; Onyx Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Medtronic: Consultancy, Honoraria, Research Funding, Speakers Bureau; Merck: Research Funding; Genentech: Research Funding.