Myeloablative chemoradiotherapy supported by autologous bone marrow transplantation (ABMT) and followed by 13-cis-retinoic acid improves survival in high-risk neuroblastoma, yet over 50% of such patients eventually die from disease progression after ABMT. Neuroblastoma therapy relies heavily upon alkylating agents, suggesting that alkylator resistance plays a key role in neuroblastoma relapse. Alkylator resistance involves tumor glutathione (GSH), a ubiquitous tri-peptide antioxidant that aids in detoxifying alkylating agents, and tumor cell hypoxia may also contribute to alkylator resistance. Buthionine Sulfoximine (BSO), a selective inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme in GSH synthesis, can decrease intracellular levels of GSH and enhance the cytotoxic effect of alkylating agents. We have established neuroblastoma cell lines at time of relapse after ABMT and we have shown that they have stable resistance to alkylating agents, and that one mechanism of resistance is a loss of p53 function, often via mutation. We will detect TP53 mutations (by Affymetrix GeneChip) and measure expression of p53/MDM2 protein in tumor and marrow metastases (by flow cytometry) from recurrent neuroblastomas (treated on COG and NANT protocols) to identify the frequency of p53 loss-of-function (p53 LOF) in recurrent neuroblastoma and to determine if response to salvage therapy correlates with p53 status. We propose to determine the degree of cross-resistance in a panel of 25 neuroblastoma cell lines of cyclophosphamide, carboplatin, melphalan (L-PAM) alone and in combination with topoisomerase inhibitors or BSO under standard culture conditions (20% O2) and physiological hypoxia (2% O2). We will identify the alkylating agents (+/- a topoisomerase inhibitor) with the greatest activity (+/- BSO in hypoxia) against multi-drug resistant, p53 LOF neuroblastomas in pre-clinical models. We will correlate enhanced response to alkylating agents when combined with topoisomerase inhibitors or BSO with the degree of tumor cell DNA damage and will explore down-stream mechanisms of tumor cell death occurring in the absence of p53 function. We will complete ongoing phase I clinical trials of BSO/L-PAM or pyrazoloacridine (PZA) + stem cell support. Based on laboratory and clinical data from this project we will develop additional phase I trials to define the tolerability of new combinations of agents that show promise against recurrent neuroblastoma, and also phase II trials to define the activity of agents and combinations of agents that show promise in phase I studies. This project will lead to greater understanding of drug resistance in neuroblastoma, will define a marker (p53 LOF) to identify drug-resistant neuroblastomas, and will test novel approaches to overcome alkylating agent resistance, especially resistance associated with a loss of p53 function.
Showing the most recent 10 out of 37 publications
