Nutlin-3a enhances natural killer cell-mediated killing of neuroblastoma by restoring p53-dependent expression of ligands for NKG2D and DNAM-1 receptors

In this study, we explored whether Nutlin-3a, a well-known nontoxic small-molecule compound antagonizing the inhibitory interaction of MDM2 with the tumor suppressor p53, may restore ligands for natural killer (NK) cell-activating receptors (NK-ARs) on neuroblastoma (NB) cells to enhance the NK cell-mediated killing. NB cell lines were treated with Nutlin-3a, and the expression of ligands for NKG2D and DNAM-1 NK-ARs and the NB susceptibility to NK cells were evaluated. Adoptive transfer of human NK cells in a xenograft NB-bearing NSG murine model was assessed. Two datasets of NB patients were explored to correlate p53 expression with ligand expression. Luciferase assays and chromatin immunoprecipitation (ChIP) analysis of p53 functional binding on PVR promoter were performed. Primary NB cells were also treated with Nutlin-3a, and NB spheroids obtained from one high-risk patient were assayed for NK-cell cytotoxicity. We provide evidence showing that the Nutlin-3a-dependent rescue of p53 function in NB cells resulted in: (i) increased surface expression of ligands for NK-ARs, thus rendering NB cell lines significantly more susceptible to NK cell-mediated killing; (ii) shrinkage of human NB tumor masses that correlated with overall survival upon adoptive transfer of NK cells in NB-bearing mice; (iii) increased expression of ligands in primary NB cells and boosting of NK cell-mediated disaggregation of NB spheroids. We also found that p53 was a direct transcription factor regulating the expression of PVR ligand recognized by DNAM-1. Our findings demonstrated an immunomodulatory role of Nutlin-3a, which might be prospectively employed for a novel NK cell-based immunotherapy for NB.