Development of prodrugs targeting DNA repair as novel anti-cancer therapeutics for neuroblastoma

背景神经母细胞瘤是最常见的儿童颅外肿瘤，超过一半的高危患者在初次治疗后复发。迫切需要新的精确药物。DNA损伤反应（DDR）在神经母细胞瘤的发生发展中起着重要作用。靶向DDR蛋白如ATR提供了显著的治疗潜力，但对非癌组织有毒性风险。为了减轻这一点，一种新的前药的有效ATR抑制剂激活的肿瘤特异性蛋白酶氨基肽酶-N已开发的二级主管。本项目的重点是评价和进一步开发这种先导化合物。目的评价一系列新型ATR抑制剂前药在神经母细胞瘤细胞中的作用，解决以下问题：（1）前药在神经母细胞瘤细胞/异种移植物中相对于正常细胞/组织离体活化的定量评估（2）确定前药在神经母细胞瘤细胞/异种移植物模型中的功效（3）检查前药与（i）神经母细胞瘤前线（ii）电离辐射（4）测试前药与（i）靶向疗法组合的功效（即ALK、TrKB抑制剂），（ii）微小残留疾病治疗（dinutuximab，isotretinoin）新颖性该项目的重点是评估和开发一种全新的神经母细胞瘤治疗药物-一种无毒的肿瘤特异性前药。靶向DDR的药物已经在成人中试验了几年，但它们在已知DDR关键缺陷的儿科肿瘤中的潜力直到最近才得到研究。ATR是神经母细胞瘤有吸引力的治疗靶点。及时性神经母细胞瘤迫切需要新的治疗方法。靶向ATR可能会增加生存率，但脱靶毒性是一个重大风险。靶向肿瘤的前药将使这种毒性最小化。我们已经成功地使用这种方法使用其他靶点（包括计划进行临床试验的药物），据我们所知，这是第一次应用于靶向DDR的治疗。这将为神经母细胞瘤患者试验的无毒治疗铺平道路。实验方法神经母细胞瘤细胞系将使用qPCR、蛋白质印迹和已建立的活性测定来表征氨基肽酶-N的表达/活性。将使用克隆形成存活测定法评估前药细胞毒性与活性药物。使用彗星试验和DDR标记物的免疫染色将结果与DNA损伤程度相关联。将通过询问预测的活性DDR途径（例如双链/单链断裂修复）来确定作用机制。将使用CsCl密度梯度分级分离，然后进行免疫印迹，定量蛋白质连接的DNA断裂的蓄积。将使用LCMS分析在离体肿瘤/正常组织中释放的前药/活性药物。

BackgroundNeuroblastoma is the most common extra-cranial childhood cancer, with more than half of high-risk patients suffering relapse following initial treatment. Novel precision medicines are desperately required. The DNA Damage Response (DDR) plays an important role in neuroblastoma development. Targeting DDR proteins such as ATR offers significant therapeutic potential, but risks toxicity to non-cancerous tissues. To mitigate this, a novel prodrug of a potent ATR inhibitor activated by tumour-specific protease aminopeptidase-N has been developed by the secondary supervisor. This project focuses on evaluation and further development of this lead compound. ObjectivesEvaluation of a series of novel ATR inhibitor prodrugs in neuroblastoma cells, addressing the following:(1) Quantitative assessment of prodrug activation in neuroblastoma cells/xenografts vs normal cells/tissues ex vivo (2) Determine efficacy of prodrug in neuroblastoma cells/xenograft models (3) Examine efficacy of combinations of prodrug with (i) neuroblastoma frontline (induction) chemotherapies, (ii) ionising radiation (4) Test efficacy of prodrug in combinations with (i) targeted therapies (i.e. ALK, TrKB inhibitors), (ii) minimal residual disease therapies (dinutuximab, isotretinoin)NoveltyThis project is focused on evaluation and development of an entirely new therapeutic class for neuroblastoma - a non-toxic tumour-specific prodrug. Drugs targeting DDR have been trialled in adults for several years, but their potential in paediatric tumours where key defects in DDR are known has only recently been investigated. ATR is an attractive therapeutic target for neuroblastoma. TimelinessThere is a desperate need for novel therapeutics for neuroblastoma. Targeting ATR could potentially increase survival, but off-target toxicity is a significant risk. A tumour-targeted prodrug will minimise such toxicity. We have had success with this approach using other targets (including a drug scheduled for clinical trial), and to the best of our knowledge this is the first application to therapeutics targeting DDR. This will pave the way towards a non-toxic therapeutic for trials in neuroblastoma patients.Experimental ApproachNeuroblastoma cell lines will be characterised for expression/activity of aminopeptidase-N using qPCR, Western blotting and established activity assays. Prodrug cytotoxicity versus active drug will be assessed using clonogenic survival assays. Results will be correlated with the extent of DNA damage using comet assays and immunostaining for DDR markers. The mechanism of action will be determined by interrogating predicted active DDR pathways e.g. double-/single-strand break repair. Accumulation of protein-linked DNA breaks will be quantified using CsCl density gradient fractionation followed by immunoblotting. Prodrug/active drug released in tumour/normal tissues ex vivo will be analysed using LCMS.