Neuroblastoma (NB), the most common malignant sympathetic nervous system tumor of childhood, arises from the neural crest. Despite current aggressive treatment strategies employing intensive myeloablative chemotherapy with autologous bone marrow transplantation, most patients with high risk (Stage 4) NB die of their disease. In order to improve the outcome for patients with this disease, the basis for treatment failures as well as mechanisms that underlie successful responses to chemotherapy in NB cells must be better understood. The primary objective of our work has been to study the regulation of cell death in NB. Two hypotheses are central to our investigation. The first is that defects in apoptosis are essential for the pathogenesis of this disease. The second is that treatments can be developed based on understanding how the death machinery in NB is effectively engaged. In the current proposal we outline studies to extend our understanding of the signaling pathways controlling the therapeutic regulation of apoptosis in NB. A particular emphasis is placed on signaling activated by Doxorubicin (Dox) and etoposide (VP16). Dox and VP16 are presently used in all active treatment protocols for NB. The direction for this work is guided by recent results showing both that NF-kappa:B activation is required for Dox to kill certain N-type NB cells while in representative S-type cell lines NF-KappaB activation is constitutive, supporting cell survival. Proposed experiments will determine whether these differences in NF-kappa B activity are fundamentally linked with S- or N-phenotypic differentiation. Moreover, we will study primary NB tumors to characterize NF-KappaB activity directly in human tumor tissue. We will define the cellular mechanisms that mediate the Dox and VP16-induced NF- kappaB response and verify the prediction that NF-KB governs the response to Dox therapy using xenograft models. The results from these objectives will lead to an understanding of how additional agents could be selected to gain therapeutic advantage (e.g. molecules that more proximally target the pathway causing NFKappaB activity) depending on NF-KappaB function in the diseased cells. Lastly, we will apply specific knowledge acquired during the last funding period pertaining to the susceptibility of NB cells to pro-apoptotic BH3 containing proteins in proof-of-principle experiments leading towards the development of a novel disease selective peptide-based treatment.