CS1, a New Surface Target on Multiple Myeloma (MM) Cells, Protects Myeloma Cells from Apoptosis Via Regulation of ERK1/2, AKT and STAT3 Signaling Cascades.
Authors: Tai Yu-Tzu , Tonon Giovanni , Leiba Merav , Li Xian-Feng , Burger Peter , Song Weihua , Podar Klaus , Chauhan Dharminder , Munshi Nihkil , Anderson Kenneth C. 
Published: October 2019

Abstract
Cell surface glycoprotein CS1 is universally expressed in the majority of MM patients. CS1 gene is localized on chromosome 1q which amplified in many MM patients and in MM cell lines (i.e., H929 and OPM2). We recently further detected CS1 protein in MM patient sera, but not in individuals with monoclonal gammopathy of undetermined significance (MGUS) or in healthy donors (Abstract # 950059). Since circulating CS1 levels correlate with active MM (Abstract # 950059), we postulated that CS1 may regulate MM cell growth and survival. We here defined a role of CS1 in MM cell growth and survival by generating CS1-null OPM2 MM cells using lentiviral CS1 short interfering RNA (CS1 siRNA). Four CS1-null OPM2 transfectants were derived using 4 CS1 siRNA targeting different regions in CS1 gene. Specific knockdown was confirmed by complete abrogation of CS1 mRNA and protein expression. In contrast, CS1 was expressed in parental OPM2 and OPM2 cells infected with control lentiviral vector (cnt-OPM2). Significantly, immunoblotting analysis showed decreased phosphorylation of ERK1/2, AKT, and STAT3 in all 4 CS1-null OPM2 cells, compared with OPM2 and cnt-OPM2 cells. We next examined growth and survival of these cells using MTT and Alamar Blue colorimetric/fluorescence assays. Serum deprivation markedly induced apoptosis at earlier time points in CS1-null OPM2 cells, but not in cnt-OPM2 cells. Earlier apoptosis in CS1-null OPM2 cells was mediated by earlier activation and cleavage of caspases, i.e. caspase 3 (> 7-fold) and caspase 8 (> 10-fold). Microarray gene expression profiling of cells under normal cultures showed altered cell cycle regulators (i.e., decreased CDK1, cyclin B1, cdc25b, cyclin D2, and increased CDK inhibitor p18), reduced survival/anti-apoptotic proteins (i.e., Mcl-1), and increased proapoptotic molecules (i.e., BINP3, BIM) in CS1-null OPM2 vs. cnt-OPM2 cells (>4-fold alteration, p<0.001). Additionally, p-21-activated kinase 1 (PAK1) that regulates cell motility, invasiveness, and survival in breast cancer cells, was completely blocked in CS1-null OPM2 transfectants. These transcriptional changes were further confirmed by immunoblotting using specific Abs. Moreover, CS1-null OPM2 transfectants had defective IL-6/IL-6R signaling, since IL-6-induced pERK1/2, pAKT, and pSTAT3 was completely blocked in CS1-null cells, but not in cnt-OPM2 cells. FGFR3 was overexpressed in cnt-OPM2 cells but was significantly downregulated in CS1-null OPM2 cells. MIP-1alpha (CCL3, an osteoclasts-activating factor), a downstream target of FGFR3 and ERK1/2 signaling, was concomitantly and specifically downregulated in CS1-null OPM2 transfectants. Ingenuity analysis further revealed altered transcriptional factors (i.e., XBP-1 and its downstream target genes, IRF4, RUNX2, SMAD1), immunoproteasome components (i.e., NMI, PSMB9, PSME2), and cell surface receptors (i.e., CCR1, Syndecan 1, IL-6R, gp130) in CS1-null vs cnt-OPM2 cells. Finally, MM1S cells transfected with a CS1-overexpressing plasmid had a moderate growth increase and increased pERK1/2, pAKT, and pSTAT3, compared with parental MM1S cells. Together, these results suggest a role of CS1 in MM cell survival by cross-talking major growth and survival pathways, strongly supporting novel therapies targeting CS1 function in MM.