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

神经母细胞瘤是最常见的儿童颅外癌症，超过一半的高危患者在初始治疗后复发。迫切需要新型精准药物。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）提供了巨大的治疗潜力，但对非癌组织有毒性风险。为了减轻这种情况，二级主管开发了一种由肿瘤特异性蛋白酶氨基肽酶- n激活的强效ATR抑制剂的新型前药。本项目重点对该先导化合物进行评价和进一步开发。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)定量评估前药在神经母细胞瘤细胞/异种移植物与正常细胞/组织体内的激活(2)确定前药在神经母细胞瘤细胞/异种移植物模型中的疗效(3)检查前药与(1)神经母细胞瘤一线（诱导）化疗的联合疗效；（ii）离子辐射(4)前药与(i)靶向治疗（即ALK， TrKB抑制剂），（ii）最小残留疾病治疗（迪努妥昔单抗，异维甲酸）联合的疗效测试新项目的重点是评估和开发一种全新的神经母细胞瘤治疗类别-一种无毒的肿瘤特异性前药。针对DDR的药物已经在成人中进行了数年的试验，但直到最近才对其在儿童肿瘤中的潜力进行了研究，其中DDR的关键缺陷是已知的。ATR是神经母细胞瘤的一个有吸引力的治疗靶点。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。针对肿瘤的前药将使这种毒性最小化。我们在使用其他靶点（包括一种计划进行临床试验的药物）时已经取得了成功，据我们所知，这是首次将这种方法应用于针对DDR的治疗方法。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、Western blotting和已建立的活性测定来表征氨基肽酶n的表达/活性。药物前细胞毒性与活性药物将通过克隆生存试验进行评估。结果将与使用彗星测定和DDR标记的免疫染色的DNA损伤程度相关。作用机制将通过询问预测的活性DDR途径来确定，例如双链/单链断裂修复。蛋白质链DNA断裂的积累将使用CsCl密度梯度分选和免疫印迹法进行量化。体外肿瘤/正常组织释放的前药/活性药物将使用LCMS进行分析。

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.