CS1 Promotes Multiple Myeloma Cell Adhesion, Clonogenic Growth, and Tumorigenicity Via C-Maf-Mediated Interactions with Bone Marrow Stromal Cells (BMSCs)
Authors: Tai Yu-Tzu , Soydan Ender , Fulciniti Mariateresa , Song Weihua , Li Xian-Feng , Burger Peter , Hong Fangxin , Tonon Giovanni , Kim Kihyun , Rumizen Mathew J. , Nahar Sabikun , Podar Klaus , Chauhan Dharminder , Hideshima Teru , Carrasco Daniel R. , Munshi Nikhil C , Afar Daniel , Anderson Kenneth C. 
Published: October 2019

Abstract
Cell membrane protein CS1 is highly expressed by tumor cells from the majority of multiple myeloma (MM) patients (>95%) regardless of cytogenetic abnormalities and response to current treatments. Furthermore, CS1 is detected in MM patient sera and correlates with active MM. However, its role in MM pathophysiology is undefined. In the present study, we first generated CS1 null OPM2 MM cells using lentiviral CS1 short interfering RNA. Specific CS1 knockdown was confirmed by depletion of CS1 mRNA and membrane protein, whereas CS1 was expressed in parental OPM2 and OPM2 cells infected with control lentiviral vector (cntOPM2). Immunoblotting of phopho-site of multiple kinase screen analysis showed decreased phosphorylation of ERK1/2, AKT, and STAT3 in CS1null OPM2 cells vs. cntOPM2 cells. Serum deprivation markedly blocked survival at earlier time points in CS1null OPM2 cells vs. cntOPM2 cells. Earlier apoptosis in CS1null OPM2 cells correlated with earlier activation of caspases, PARP cleavage, and increased proapoptotic proteins BNIP3, BIK. CS1 knockdown further delayed development of OPM2 tumor and prolonged survival in mice. CS1null OPM2 cells failed to grow tumors in the majority of mice (n=8) at 5 weeks after cell inoculation, whereas cntOPM2 cells formed tumors within 1.5 weeks in all animals (n=8). Interestingly, CS1 was expressed in tumors that developed late in mice injected with CS1null OPM2 cells. Concomitantly, we overexpressed CS1 in CS1-low expressing U266 cells by transfecting an expressing plasmid pflagCS1 or control vector. Enforced CS1 expression enhanced U266 cell growth and survival. In contrast to the majority of U266 cells (>95%) that grow in suspension in standard tissue culture flasks, all U266CS1 cells exhibited adherent growth and homotypic adhesion. Importantly, overexpressed CS1 increased adhesion of U266 and MM1S cells to BMSCs. Furthermore, U266CS1 cells formed more and larger colonies in methylcellulose than U266 cells. Interestingly, tumors that developed in mice injected with U266 cells expressed significantly higher levels of CS1 than injected U266 cells; moreover, exercised tumors grew in an adherent manner in vitro. Overlapping differentially expressed genes in U266CS1 vs. U266 and CS1null OPM2 vs. cntOPM2 was next analyzed by gene expression profiling. Importantly, c-maf pathway was significantly upregulated in U266CS1 vs. U266 cells and downregulated in CS1null OPM2 vs. cntOPM2 cells, as evidenced by differentially expressed c-maf and its target genes, i.e., cyclin D2, integrin αE/β7 at both mRNA and protein levels. Myeloma cell adhesion-induced VEGF secretion by BMSCs was greater with U266CS1 than U266 cells. Finally, immunoblotting showed upregulation of c-maf and cyclin D2 in U266 tumors overexpressing CS1. These studies provide direct evidence of the role of CS1 in myeloma pathogenesis, define molecular mechanisms regulating its effects, and further support novel therapies targeting CS1 in MM.