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Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces selective apoptosis in a variety of tumors, including most cell lines derived from Ewing's sarcoma family of tumors, an aggressive sarcoma that afflicts children and young adults. To determine the in vivo efficacy of TRAIL receptor agonists in Ewing's sarcoma family of tumors, mice with orthotopic xenografts were treated with anti-TRAIL-R2 monoclonal antibody or TRAIL/Apo2L in a model that can identify effects on both primary tumors and metastases. Administration of either agonist slowed tumor growth in 60% of animals and induced durable remissions in 11 to 19% but did not alter the incidence of metastatic disease. Response rates were not improved by concurrent doxorubicin treatment. Cells recovered from both TRAIL receptor agonist-treated and nontreated tumors were found to be resistant to TRAIL-induced death in vitro unless pretreated with interferon (IFN) gamma. This resistance coincided with a selective down-regulation of TRAIL receptor expression on tumor cells. In vivo treatment with IFNgamma increased tumor expression of TRAIL receptors and caspase 8, but did not increase the antitumor effect of TRAIL receptor agonists on primary tumors. However, IFNgamma treatment alone or in combination with a TRAIL receptor agonist significantly decreased the incidence of metastatic disease and the combination of TRAIL receptor agonist therapy with IFNgamma-mediated impressive effects on both primary tumors and metastatic disease. These data demonstrate that in vivo growth favors TRAIL resistance but that TRAIL receptor agonists are active in Ewing's sarcoma family of tumors and that the combination of TRAIL receptor agonists with IFNgamma is a potent regimen in this disease capable of controlling both primary and metastatic tumors.

The resistance of neuroblastoma (NB) cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis has been attributed to a lack of caspase 8 expression. Here we demonstrate a clinically applicable molecular targeting strategy that not only increases caspase 8 expression ex vivo in NB cell lines but also in the tumor tissues of NB patients receiving IFN-gamma treatment. We identify the functional caspase 8 promoter, which is different from the methylated region reported previously, and show promoter activity is up-regulated by IFN-gamma through a IFN-gamma activation site-containing region. IFN-gamma also induces TRAIL expression in NB cell lines. However, the IFN-gamma restoration of caspase 8 in some NB cells revealed persistent TRAIL resistance in most NB cell lines examined. This additional lesion in the TRAIL path is because of a loss of cell membrane TRAIL receptors (TR1/TR2) not only in cell lines but in most of the NB tumor tissues evaluated. Restoration of TR2 expression by transfection enhances IFN-gamma-induced TRAIL sensitivity. Furthermore, we have found that we can improve TRAIL sensitivity in NB by reconstituting caspase 8 with IFN-gamma and TR2 with chemotherapeutic agents.