Valproic Acid Upregulates NKG2D Ligand Expression and Potentially Enhances Natural Killer Cell–mediated Cytolysis of Myeloma

Multiple myeloma (MM) is a plasma cell malignancy. Although high-dose chemotherapy supported by autologous hematopoietic stem-cell transplantation can produce higher response rates and longer survival than standard chemotherapy, MM remains largely incurable by current therapeutic strategies. Clinical and preclinical data have been demonstrated that natural killer (NK) cells have an anti-myeloma effect. However, low NK cell activity affected the successful utilization of NK cell adoptive immunotherapy for myeloma. In this study, we investigated the effect of valproic acid (VPA), as a histone deacetylase inhibitor, on the expression of NKG2D ligands on human myeloma cell lines and primary myeloma cells derived from patients. We also evaluated the sensitivity of myeloma cells to NK cell-mediated lysis before and after treatment with VPA, and the possible underlying mechanisms.

ARK, OPM2 myeloma cell lines and primary myeloma cells (n = 4) from patients with myeloma were cultured with 0.5–3mM VPA for 24 to 72 hours. Cells cultured without VPA were as control groups. To evaluate the effect of VPA on NKG2D ligand expression of myeloma cells, mRNA and protein expression of NKG2D ligands (including MICA/B, ULBP1, ULBP2 and ULBP3) of myeloma cells were tested by Real time-PCR and flow cytometry. ⁵¹Cr release assay and NKG2D antibody blocking experiments were combined to examine the susceptibility of myeloma cells to NK cell-mediated lysis in response to VPA and the possible killing mechanisms.

ARK and OPM-2 myeloma cell lines were treated with various concentrations of VPA. The most prominent upregulations of mRNA expressions of MICA/B, ULBP2 and ULBP 3 were observed in the two tested myeloma cell lines. The enhancement of cell surface expression of MICA/B, ULBP2 and ULBP 3 was detected by flow cytometry after cells treatment with VPA. We also found that VPA upregulated MICA/B expression on OPM2 MM cell line in a time- and dose-dependent manner. These data suggest that VPA could upregulate expression of NKG2D ligands in myeloma cell line at both the mRNA and protein levels. A similar increase of surface expression of NKG2D ligands was obtained in 2–3 mM VPA treated primary MM cells (n=4). NK cell function was studied by ⁵¹Cr release assay. VPA treatment of OPM2 showed higher sensitivity to NK cell lysis than untreated cells (specific killing: 72.8±6.2% vs 32.1±3.7% at E/T 10:1, p <0.01). The killing of VPA treatment of patient myeloma cells by NK cells also significantly increased compared with control cells (specific killing: 46.7±5.6% vs 21.6±4.5% at E/T 10:1, n=4, p <0.01). The enhancing effect of VPA was blocked by pretreatment of OPM2 cells with a combination of anti-MICA/B, anti-ULBP1,2,3 mAbs or pretreatment of NK cells with anti-NKG2D mAbs, indicating the contribution of NKG2D-NKG2D ligands interations to VPA-modulated lysis of myeloma. We also found that constitutive phosphorylated extracellular signal-regulated kinase (ERK) but not AKT signaling pathway may be involved in VPA-induced higher NKG2D ligand expression.

Histone deacetylase inhibitor valproic acid could upregulate cell surface expression of NKG2D ligands, rendering myeloma cells more sensitive to NK cells, and thereby enhance NK cell–mediated lysis of myeloma. Our findings imply that regulation of the expression of NKG2D ligands by treatment with histone deacetylase inhibitors may be an attractive strategy for immunotherapy of myeloma.

This work was supported by grants from National Natural Science Foundation of China (81071856 and 30973450 to JS) and funds from Shanghai Tenth People's Hospital (10RD103 and 11SC103 to JS).

No relevant conflicts of interest to declare.