Romedepsin (RM), A Hdaci, Significantly Increases The Expression Of NKG2D Ligands, MIC A/B, In Leukemia/Lymphoma Cells (LL), In Part Through The Glycogen Synthase Kinase-3 (GSK-3) Pathway, Resulting In Enhanced NK Cytotoxicity: Translational Approach For Adoptive NK Cell Immunotherapy

Abstract

Natural killer (NK) cells recognize malignant cells through the tumor-associated expression of NKG2D-ligands, including MIC A/B. However, tumor cells may shed MIC A/B and escape immuno- surveillance. Glycogen synthase kinase-3 (GSK-3), a constitutively active serine-threonine kinase with numerous functions including regulation of cellular differentiation, stress and apoptosis, also an important regulatory enzyme in the expression of MIC A/B in response to RM (Skov et al, Cancer Res, 2005). To determine the expression of MIC A/B in response to RM in various leukemia and lymphoma cells (LL), its influence on NK cell mediated cytotoxicity and to investigate the role of the GSK-3 pathway in the regulation of expression of MIC A/B in response to RM. LL cells (106/ml, RS 4:11 [MLL-ALL], REH [pre-B cell ALL], Jurkat [T-cell ALL], Toledo [DLBCL], Ramos [Burkitt's Lymphoma]) were exposed to RM (10 ng/mL) for 24 hours, followed by FACS staining with PE-conjugated anti-MIC A/B. Peripheral blood NK cells were isolated via magnetic separation followed by IL-2 activation. Cytotoxicity assays (europium assay) were performed at effector:target (E:T) ratio of 5-10:1. LL cells were also pre-treated for 1 hour with 100 mM lithium chloride (LiCl), a potent inhibitor of GSK-3 activity. Blocking studies were also performed with anti-NKG2D antibodies. MIC A/B expression significantly increased in LL cells in response to RM ([RS4:11 0.2% vs 82%, p< 0.0001], [REH 0.2% vs 46%, p= 0.0003], [Jurkat 1.12% vs 85%, p< 0.0001], [Toledo 0.5% vs 15.8%, p=0.0001], [Ramos 0.57% vs 67%, p=0.0003]). Enhanced expression of MIC A/B in response to RM was inhibited when LL cells are pre-treated with LiCl (Jurkat [RM vs RM+LiCl] 85% vs 18%, p<0.0001; RS 4:11 [RM vs RM+LiCl] 82% vs 5%, p<0.0001; Ramos [RM vs RM+LiCl] 67% vs 35%, p<0.0001). Cytotoxicity assays revealed significant increases in-vitro cytotoxicty in RS 4:11, Ramos and REH cells at E:T ratio of 5-10:1 (Table-1). NKG2D receptor-blocking resulted in significant decrease in NK cell mediated cytotoxicity in REH (p<0.03) and Ramos cells (p<0.001). In-vivo experiments are underway. Expression of MICA/B in LL cells is significantly increased by RM leading to enhanced susceptibility for NKG2D- MIC A/B mediated cytotoxicity by NK cells. Furthermore, up-regulation of MICA/B in LL cells secondary to RM exposure is in part regulated by the GSK-3 signal transduction pathway.Tabled 1Cell LineCondition RM - 10 ng/mlMean % Specific Release±SEM-E:T-5:1Mean % Specific Release±SEM-E:T-10:1P-valueRS4;11+NK(A)3.2%±2%10%±3%5:1-AvsC; BvsD - P<0.01; 10:1-AvsC; BvsD-p<0.05+NK+IL2(B)11%±3.3%12±4%+NK+IRM(C)78±42%108%±47%+NK_IL2+RM(D)123±48%145%±Ramos+NK(A)0.3%±0.3%7.6%±7.6%10:1-AvsC; BvsD - p<0.05+NK+IL2(B)16.3%±8.6%35.3%±13%+NK+IRM(C)62.6%±14%79.6%±15%+NK_IL2+RM(D)78.3%±26%90%±22%Jurkat+NK(A)22.2%±8%63.7%±22%5:1-AvsC; BvsD - p<0.01+NK+IL2(B)51%±11%102%±21%+NK+IRM(C)130%±14%174%±39%+NK_IL2+RM(D)174%±27%208%±34% Open table in a new tab