Multiple Myeloma Cells Modulate ICAM-3 To Evade Natural Killer Cell-Mediated Lysis

Ex vivo activated/expanded natural killer (ENK) cells can induce myeloma cell lysis both in vitro and in murine models and are currently being studied clinically in the setting of high-risk relapsing disease and asymptomatic disease at high risk of progression. This prompted us to study, in myeloma cell lines, whether intrinsic resistance to ENK cell lysis exists, whether repeated challenge with ENK leads to increased resistance, and what the underlying mechanisms of resistance are. Of 11 myeloma cell lines tested in standard 4h chromium release assays, 8 were avidly killed (78-89% lysis, E:T Ratio 10:1) whereas 3 lines were less sensitive (41-65% lysis). Repeated exposure to ENK challenge decreased sensitivity in 4 of 11 lines, that was at least in part due to down-regulation of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand-Receptors on the myeloma cell surface (Garg et al, Blood 2012, 120:4020). In this study we investigated the resistance issue further via metabolomics, gene expression profiling (GEP) and flow cytometry analysis of OPM2, which was intrinsically resistant and developed further resistance after challenge with ENK cells.

Metabolomics was studied using a quantitative proteomic strategy entailing stable isotope labeling with amino acids in cell culture – mass spectrometry (SILAC-MS). Resistant and parental OPM2 cells were grown either in medium with heavy amino acids (¹³C₆ L-Lysine and ¹³C₆ L-Arginine) or with light amino acids (¹²C₆ L-Lysine and ¹²C₆L-Arginine). Reverse labeling with heavy or light amino acids was also done to confirm the results. Cell lysates from heavy and light amino acid labeled cells were pooled, simultaneously resolved on SDS-PAGE, protein bands were excised and analyzed on a mass spectrometer after trypsin digestion. GEP was performed using the Affymetrix U133 Plus 2.0 microarray platform (Santa Clara, CA). The fold change of signal intensity for genes and proteins in resistant vs. parental OPM2 was calculated. The most differentially expressed genes (top 150-fold up or down) and proteins (up or down by 1.3-fold) were compared for commonality. Cell surface protein expression was determined via flow cytometry. The ability of ENK to lyse myeloma cell targets in the presence of isotype control or ICAM-3 blocking antibody was tested in 4h chromium release assays.

Metabolomics identified >3800 proteins and revealed that the abundance of 352 proteins was significantly altered in resistant myeloma cells. These altered proteins were mainly associated with cell cycle, morphology, organization, cellular compromise, immune response, and survival. Further, a comparison of these differentially expressed proteins with GEP data revealed 3 commonly up-regulated molecules: TBC1D8B, HSPA1A and IFI16; and 2 down-regulated molecules: intercellular adhesion molecule (ICAM-3) and BAI3. Of these, ICAM-3, a ligand for leukocyte function-associated antigen-1 (LFA-1) and a potent signaling molecule, was selected for further studies. Flow cytometry confirmed that ICAM-3 cell surface expression was > 8-fold lower on resistant versus parental OPM2 cells. Further, blocking of ICAM-3 in cytotoxicity assays resulted in decreased lysis (43% blocked, E:T ratio 5:1), suggesting that this molecule is functionally important and takes part in ENK cell-mediated killing.

In conclusion, quantitative proteomic analysis demonstrated dynamic changes in the ENK-resistant OPM2 myeloma cells that correlated with GEP and differences in ICAM-3 expression may have functional implications. Studies evaluating the expression of ICAM-3 in myeloma patients at diagnosis and relapse are in progress. Myeloma cells may down-regulate ICAM-3 as a mechanism of escape from immune surveillance and therefore, ICAM-3 may be a useful biomarker to predict sensitivity to ENK cell-mediated killing and aid in the selection of patients most likely to benefit from ENK cell therapy.

Barlogie:Celgene: Consultancy, Honoraria, Research Funding; Myeloma Health, LLC: Patents & Royalties.