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

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 infusion of IL-2 activated NK cells 24 hrs later. Xenografted NOD-SCID mice were monitored by volumetric measurement of tumor size, bioluminescent imaging (Inoue et al, Exp. Hem, 2007) and survival. The survival distribution for each of the groups of mice was estimated using Kaplan-Meier estimates. The log-rank test was used to compare the survival distributions between treatment groups.

MIC A/B expression significantly increased in response to RM ([RS4:11 0.2% vs. 82%, p< 0.0001] and [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 RS 4:11 [RM vs RM+LiCl] 82% vs. 5%, p<0.0001; Ramos [RM vs. RM+LiCl] 67% vs. 35%, p<0.0001). In vitro cytotoxicity assays revealed significant increases against both RS 4:11 and Ramos cells at E:T ratio of 5–10:1 (p<0.01). The median survival time for NOD-SCID mice with RS4:11 was 24 days and RS4:11 + NK cell therapy was 34 days and RS4:11 +RM + NK cell therapy was: 46.5 days, respectively; Log-rank test p-value = 0.003. Median survival time for mice with Ramos was 16 days, Ramos + NK cells was 29 days and Ramos + RM + NK cell therapy was 32 days, respectively; Log-rank test p-value <0.001

Expression of MIC A/B in LL cells is significantly increased by RM leading to enhanced susceptibility for NKG2D- MIC A/B mediated cytotoxicity by NK cells. NK cells + RM leads to increase in survival of NOD-SCID mice xenograft with LL. Furthermore, up-regulation of MIC A/B in LL cells secondary to RM exposure is in part regulated by the GSK-3 signal transduction pathway.

No relevant conflicts of interest to declare.