Histone deacetylase inhibitors enhance expression of NKG2D ligands in Ewing sarcoma and sensitize for natural killer cell-mediated cytolysis

Using flow cytometry, ELISA and immunohistochemistry, expression of natural killer cell receptor ligands was assessed in chemotherapy-sensitive/-resistant Ewing sarcoma cell lines, plasma and tumours. Natural killer cell cytotoxicity was evaluated in Chromium release assays. Using ATM/ATR inhibitor caffeine, the contribution of the DNA damage response pathway to histone deacetylase inhibitor-induced ligand expression was assessed.Despite comparable expression of natural killer cell receptor ligands, chemotherapy-resistant Ewing sarcoma exhibited reduced susceptibility to resting natural killer cells. Interleukin-15-activation of natural killer cells overcame this reduced sensitivity. Histone deacetylase inhibitor-pretreatment induced NKG2D-ligand expression in an ATM/ATR-dependent manner and sensitized for NKG2D-dependent cytotoxicity (2/4 cell lines). NKG2D-ligands were expressed in vivo, regardless of chemotherapy-response and disease stage. Soluble NKG2D-ligand plasma concentrations did not differ between patients and controls.Our data provide a rationale for combination immunotherapy involving immune effector and target cell manipulation in first-/second-line treatment regimens for Ewing sarcoma.Ewing sarcoma is an aggressive round cell sarcoma characterized by specific gene fusions most commonly involving TET gene family products, though rarely other activating transcription factors [1-3]. It usually affects bone or soft tissue in children and young adults. Despite multimodal therapy consisting of high-dose chemotherapy, surgery and radiotherapy, survival of patients with Ewing sarcoma has not improved significantly during the past decade. Patients with therapy-resistant or metastatic Ewing sarcoma have the most unfavorable prognosis, with a 5-year overall survival of less than 30% [4-6], which has recently been demonstrated to be independent of Ewing sarcoma-ETS fusion type [4,5].Natural killer (NK) cells are the main cytotoxic effector cells of the innate imm