SELECTIVE THERAPY OF NEUROBLASTOMA

晚期N-myc扩增神经母细胞瘤患者的3年无进展生存率仅为2- 30%。由于肿瘤经常在疾病原发部位复发或骨髓未完全清除的结果，手术、化疗和清除后的自体移植治疗往往不能治愈。该提案描述了一种基于利用N-MYC作为转录因子的功能，旨在产生肿瘤细胞特异性细胞毒性的神经母细胞瘤。待验证的假设是，当肿瘤细胞暴露于拓扑异构酶I抑制剂CPT-11时，羧酸酯酶基因N-MYC选择性诱导转录将产生选择性细胞毒性。这一假设的基本原理是基于羧酸酯酶将前药CPT-11转化为其活性代谢物SN-38的观察。具体来说，我们打算测试肿瘤细胞中N-MYC的内源性过表达是否可以选择性地反激活N-MYC应答鸟氨酸脱羧酶启动子，从而允许羧酸酯酶的转录。当过表达N-MYC的肿瘤细胞暴露于CPT-11时，该药物将转化为SN-38并产生肿瘤选择性细胞杀伤。我们将研究这种方法的可行性，作为一种潜在的治疗最小残留疾病和清除骨髓自体移植。本提案的目标是确定：1)激活CPT-11的兔肝羧酸酯酶的最合适形式；2) A)从N-MYC反应性启动子可在过表达N-MYC的神经母细胞瘤细胞中实现羧酸酯酶的特异性表达，B)过表达N-MYC的肿瘤细胞选择性表达羧酸酯酶可将CPT-11转化为SN-38并产生肿瘤细胞特异性细胞毒性；3)该方法能否根除异种移植模型中的残留疾病；4) A)所提出的方法是否可以用于使用小鼠异种移植模型从骨髓中清除神经母细胞瘤细胞，以及B)从人外周干细胞（CD34+）细胞中清除原发性神经母细胞瘤细胞，并且对造血细胞的毒性最小。

Patients with advanced stage, N-myc amplified neuroblastoma have only a 2-30 percent 3-year progression-free survival. Therapy with surgery, chemotherapy, and purged autologous transplant is often not curative since tumor frequently recurs at the original site of disease or as a consequence of an incompletely purged bone marrow. This proposal describes an approach designed to produce tumor cell-specific cytotoxicity of neuroblastomas overexpressing N-MYC, based on exploiting the function of N-MYC as a transcription factor. The hypothesis to be tested is that the selective induction of transcription by N-MYC of a carboxylesterase gene will produce selective cytotoxicity when tumor cells are exposed to the topoisomerase I inhibitor CPT-11. The rationale for this hypothesis is based upon the observation that carboxylesterases convert the prodrug CPT-11 to its active metabolite SN-38. Specifically, we propose to test whether endogenous overexpression of N-MYC in tumor cells can selectively transactivate the N-MYC-responsive ornithine decarboxylase promoter allowing transcription of a carboxylesterase. When tumor cells overexpressing N-MYC, and therefore the carboxylesterase, are exposed to CPT-11, the drug will be converted to SN-38 and produce tumor-selective cell kill. We will examine the feasibility of this approach as a potential treatment for minimum residual disease and in the purging of marrow for autologous transplant. The goals of this proposal are to determine: 1) the most appropriate form of rabbit liver carboxylesterase for CPT-11 activation; 2) A) that specific expression of carboxylesterase can be achieved in neuroblastoma cells overexpressing N-MYC from N-MYC responsive promoters, and B) that selective expression of carboxylesterase by tumor cells overexpressing N-MYC can convert CPT-11 to SN-38 and produce tumor cell-specific cytotoxicity; 3) whether this approach eradicates residual disease in a xenograft model; 4) A) whether the proposed approach can be used to purge neuroblastoma cells from marrow using a mouse xenograft model, and B) to purge primary neuroblastoma cells from human peripheral stem (CD34+) cells, with minimal toxicity to hematopoietic cells.