Romedepsin (RM), a Histone Deacytelase Inhibitor (HDACi), Significantly Increases the Expression of NKG2D Ligands, MIC A/B, In Pediatric Leukemia/Lymphoma Cells (LL), Resulting In Enhanced In Vitro and In Vivo NK Cytotoxicity In NOD-SCID Mice: Translational Approach for Adoptive NK Cell Targeted Immunotherapy

• Published in: Blood 2010-11, Vol.116 (21), p.4297-4297
• Main Authors: Satwani, Prakash, Saha, Aniket, Bavishi, Sejal, Zhao, Frances, Ayello, Janet, van de Ven, Carmella, Cairo, Mitchell S.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V116.21.4297.4297

Abstract 4297 Natural killer (NK) cells recognize malignant cells through the tumor-associated expression of NKG2D ligands, including MIC A/B (Crewenka et al, Science, 1998). Tumor cells expressing ligands for NKG2D can become susceptible to NK cell killing despite normal MHC class I expression (Lanier LL, Nat Med, 2001). However, tumor cells may shed MIC A/B and escape immuno-surveillance. HDACi increases the expression of NKG2D ligands MIC A/B. Glycogen synthase kinase-3 (GSK-3), a constitutively active serine/threonine kinase with numerous functions including regulation of cellular differentiation, stress and apoptosis, is also an important regulatory enzyme in the expression of MIC A/B in response to romidepsin (RM) (Skov et al, Cancer Res, 2005). To determine the expression of MIC A/B in response to RM in leukemia and lymphoma cells (LL), its influence on NK cell mediated in vitro and in vivo cytotoxicity in NOD-SCID mice 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], Ramos [Burkitt's lymphoma]) were exposed to RM (10 ng/mL) (generously provided by Gloucester Pharmaceuticals) 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 12 hrs incubation with interleukin-2 (IL-2) [3000 IU/ml]. Cytotoxicity assays (europium assay) were performed at effector target (E:T) ratio of 5–10:1. RS4:11 and Ramos cells were also pre-treated for 1 hour with 100mM lithium chloride (LiCl), a potent inhibitor of GSK-3 activity. The mammalian expression construct (ffLucZeo-pcDNA [generously provided by Laurence Cooper, MD, PhD]) was transfected to RS4:11 and Ramos cells using lipofectin. The transfected cells were selected by zeocin to make stable transfection cells using lipofectin. Six week old NOD-SCID mice received 5×106 LL cells subcutaneously. Once LL engraftment was established in NOD-SCID mice, the xenografted animals were divided in various groups, 1) Control NOD-SCID mice were injected with PBS, 2) NOD-SCID mice with leukemia or lymphoma, 3) NOD-SCID mice with leukemia or lymphoma + NK cell therapy and 4) NOD-SCID mice with leukemia or lymphoma + RM + NK cell therapy. NOD-SCID xenografted mice in group 3 received weekly injections of purified IL-2 activated adult NK cells (5×106) for 6 weeks and mice in group 4 received weekly injections of RM (4.4mg/kg) followed by an inf