Enhancing Activity of Alkylating Agents in Neuroblastoma

描述(申请人提供)：自体骨髓移植(ABMT)支持的清髓放化疗和13-顺式维甲酸可提高高危神经母细胞瘤的存活率，但超过50%的此类患者最终死于ABMT后的疾病进展。神经母细胞瘤的治疗严重依赖烷化剂，提示烷化剂抵抗在神经母细胞瘤复发中起关键作用。烷化剂抵抗与肿瘤谷胱甘肽(GSH)有关，GSH是一种普遍存在的三肽抗氧化剂，有助于解毒烷化剂，肿瘤细胞缺氧也可能导致烷化剂抵抗。丁硫氨酸亚磺胺(BSO)是谷氨酰半胱氨酸合成酶(GCS)的选择性抑制剂，是GSH合成的限速酶，可降低细胞内GSH水平，增强烷化剂的细胞毒作用。我们已经建立了ABMT后复发的神经母细胞瘤细胞系，我们已经证明它们对烷化剂具有稳定的耐药性，并且耐药性的一个机制是P53功能的丧失，通常是通过突变。我们将检测TP53突变(通过Affymetrix基因芯片)和检测肿瘤和复发神经母细胞瘤(经COG和NANT方案治疗)的骨髓转移瘤中P53/MDM2蛋白的表达，以确定复发神经母细胞瘤中P53功能丧失(P53 LOF)的频率，并确定挽救治疗的反应是否与P53状态相关。我们建议在标准培养条件(20%O2)和生理缺氧(2%O2)条件下，测定25株神经母细胞瘤细胞株对环磷酰胺、卡铂、马法兰(L-PAM)以及与拓扑异构酶抑制剂或BSO联合使用的交叉耐药程度。我们将在临床前模型中确定对多药耐药的神经母细胞瘤具有最高活性的烷化剂(+/-拓扑异构酶抑制剂)(+/-BSO)。我们将把与拓扑异构酶抑制剂或BSO结合时对烷化剂的增强反应与肿瘤细胞DNA损伤的程度相关联，并将探索在缺乏p53功能的情况下发生的肿瘤细胞死亡的下游机制。我们将完成正在进行的BSO/L-PAM或吡唑吖啶(PZA)+干细胞支持的I期临床试验。基于这个项目的实验室和临床数据，我们将开发额外的第一阶段试验，以确定对复发神经母细胞瘤有希望的新药物组合的耐受性，也将开发第二阶段试验，以确定在第一阶段研究中显示出希望的药物和药物组合的活性。该项目将有助于更好地了解神经母细胞瘤的耐药性，将定义一个标记物(P53 LOF)来识别耐药神经母细胞瘤，并将测试克服烷化剂耐药性的新方法，特别是与p53功能丧失相关的耐药性。

DESCRIPTION (provided by applicant): 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.