Sensitization to Chemoradiation by Therapeutic Targeting of the DNA Damage Response

摘要 迫切需要提高局部晚期肺癌患者的放化疗疗效 胰腺癌(LAPC)。由于未修复的DNA双链断裂(DSB)是大多数 靶向细胞DNA损伤反应(DDR)通路是细胞对放化疗反应的反应之一 通过防止DNA有效修复来提高放化疗疗效的有希望的方法 损伤，导致肿瘤细胞活力丧失。由于DDR中的缺陷，如p53突变，在 70%的胰腺导管腺癌(PDAC)，靶向抑制DDR提供了一个机会 选择性地增强肿瘤细胞对化疗的敏感性，但不能提高正常细胞的敏感性。我们的总体目标是 拟议的研究是从CTEP收集中识别调节细胞反应的试剂 DNA损伤，并最好地使肿瘤细胞对标准护理放化疗敏感。要做到这一点 在目标1中，我们将在一组PDAC细胞系中进行无偏高通量筛选(HTS) CTEP收集的调节细胞DDR的药物小组，包括靶向ATR的药物， DNAPK、PARP和WEE1(目标1A)。不同浓度单药对细胞活力的初步筛选 将单独进行或与放化疗联合进行。除了集中，最优的 将对具有化疗放射的靶向制剂进行测序。DDR的活细胞分析也是如此 作为凋亡细胞死亡将被用作正交筛选，以协助HIT的优先顺序和验证。在AIM 1B，将通过实施“黄金标准”进一步评估根据定义的标准选择的一组代理 利用一组PDAC细胞系进行克隆存活分析。在AIM 1C中，我们将进行正常的细胞计数器 使用上述试剂对正常细胞进行筛选，以确定哪些药物与 放化疗。这些优先药物的疗效将在AIM 1D中使用一组患者- 衍生异种移植(PDX)球体外植体培养。具有最佳增强比的两种试剂(1B和1D)， 在目标2中，将研究在最佳浓度和时间表(1A和1B)下的安全性(1C)。我们将 用两种PDX比较提名药物联合放化疗的疗效 和具有免疫能力的同基因小鼠PDAC模型(Aim 2A)。由这些引起的正常组织损伤 将对两种药物(目标2B)进行评估，结合肿瘤疗效，将导致选择药物来 在临床试验中追求。目标3将描述的化学辐射增强活性的机制基础 优势药物(目标3A)，目标是选择药效学和成像生物标记物(目标3B 和3C)，在小鼠模型上进行体内研究，用于未来的临床试验。完成这些目标将 提供有关疗效、安全性和生物反应标志物的必要临床前数据(机械和 以影像为基础)，为LAPC患者未来的临床试验设计提供理论基础。

ABSTRACT There is an urgent need to improve the efficacy of chemoradiation therapy for patients with locally advanced pancreatic cancer (LAPC). Since unrepaired DNA double-strand breaks (DSB) are responsible for the majority of cell death in response to chemoradiation, targeting cellular DNA damage response (DDR) pathways is a promising approach to enhance the efficacy of chemoradiation therapy by preventing efficient repair of DNA damage, leading to loss of tumor cell viability. Since defects in the DDR such as P53 mutation occur in up to 70% of pancreatic ductal adenocarcinomas (PDAC), targeted inhibition of the DDR provides an opportunity to selectively enhance sensitivity to chemoradiation in tumor but not normal cells. The overall goal of our proposed study is to identify agents from the CTEP collection that modulate the cellular response to DNA damage and best sensitize tumor cells to standard of care chemoradiation therapy. To achieve this goal, we will in Aim 1 conduct an unbiased high throughput screen (HTS) in a panel of PDAC cell lines using a panel of agents from the CTEP collection that modulate the cellular DDR, including drugs that target ATR, DNAPK, PARP, and WEE1 (Aim 1A). An initial cell viability screen at various concentrations of single agents alone or in combination with chemoradiation will be conducted. In addition to concentration, optimal sequencing of targeted agents with chemoradiation will be investigated. A live cell assay for the DDR as well as apoptotic cell death will be utilized as orthogonal screens to assist in hit prioritization and validation. In Aim 1B, a group of agents selected by defined criteria will be further evaluated by conducting a “gold standard” clonogenic survival assay using a panel of PDAC cell lines. In Aim 1C we will conduct a normal cell counter screen using the above agents against normal cells to identify those which cause toxicity in combination with chemoradiation. The efficacy of these prioritized agents will be validated in Aim 1D using a panel of patient- derived xenograft (PDX) sphere explant cultures. Two agents with the optimal enhancement ratio (1B and 1D), and safety (1C) at the optimal concentration and schedule (1A and 1B) will be studied in Aim 2. We will compare the therapeutic efficacy of the nominated agents in combination with chemoradiation using both PDX and immune competent syngeneic mouse models of PDAC (Aim 2A). Normal tissue injury caused by these two agents (Aim 2B) will be evaluated, which together with tumor efficacy will lead to the choice of the agent to pursue in clinical trials. Aim 3 will delineate the mechanistic basis for the chemoradiation potentiating activity of the superior agent (Aim 3A) with the goal of selecting pharmacodynamic and imaging biomarkers (Aims 3B and 3C), using in vivo studies in mouse models, for the future clinical trial. Completion of these aims will provide the required preclinical data regarding efficacy, safety, and biomarkers of response (mechanistic- and imaging- based) for the rationale design of a future clinical trial for patients with LAPC.