SMAD7 expression and radiotoxicity: Clarification of mechanisms for the observed correlation in fibroblasts with clinical toxicity and development of a cell model for evaluation of pharmacological interventions

尽管进行了大量的研究，但迄今为止，还没有建立可用于临床的生物标志物，用于预测放射治疗的更高级别急性毒性。根据目前的文献报道和自己的初步研究，SMAD 7（转化生长因子β信号传导途径的内源性自身抑制剂）的表达似乎是一种有前途的方法。基于我们的观察，即当在患者匹配的成纤维细胞中模拟放化疗时，SMAD 7的早期诱导与较低的临床急性毒性一致，本项目提案应解决以下问题：1）观察到的SMAD 7诱导与模拟放化疗对成纤维细胞中整个转录组的影响有何关系？2)给定由（1）鉴定的最强转录物，它们分别如何受到SMAD 7的特异性靶向过表达和抑制的影响？3)辐射与SMAD 7启动子活性相互作用的机制如何理解可变转录诱导？4)是否存在调节内源性SMAD 7转录的遗传多态性？5)如何在CRISPR/Cas9技术构建的细胞模型中分析放化疗诱导的SMAD 7转录变化的动态剂量-时间效应？在该模型系统中是否存在显示出有效SMAD 7诱导的确定药物？具体的目的是获得详细的信息，以便更好地理解SMAD 7转录在放射治疗期间发生的相关性，并将相关发现应用于临床。在本申请所基于的患者来源的成纤维细胞中检测到的临床毒性和离体转录效应之间的直接关系代表了这种翻译方法的特殊特征。在这方面，这项建议的问题代表了床边-benach-床边三明治的中间部分。应用研究的主要结果应在药物启动试验（IIT）中进行前瞻性测试，以评估临床相关性和可行性。

Despite intensive research efforts no biomarkers predictive for higher grade acute toxicity by radiotherapy could be established for clinical use so far. Following current literature reports and own pilot investigations expression of SMAD7, the endogenous autoinhibitor of the transforming growth factor beta signalling pathway, appears as a promising approach. Based on our observation that early induction of SMAD7 when radiochemotherapy was simulated in patient-matched fibroblasts was coincident with lower clinical acute toxicity the following issues should be addressed by this project proposal: 1) How is the observed SMAD7 induction in relation to the effects of simulated radiochemotherapy on the entire transcriptome in fibroblasts? 2) Given the strongest transcripts indentified by (1) how are they affected by specifically targeted overexpression and suppression of SMAD7, respectively? 3) How are the mechanisms by which radiation interacts with SMAD7 promoter activity to understand variable transcription induction? 4) Are there any inherited genetic polymorphisms modulating endogenous SMAD7 transcription? 5) How are the dynamic dose-time effects for SMAD7 transcription changes induced by radiochemotherapy to be analyzed in a cellular model to be engineered by the CRISPR/Cas9 technology? Are there established pharmaceuticals showing effective SMAD7 induction in this model system? The specific aim is to get detailed information for better understanding of the relevance of SMAD7 transcription occurring during radiotherapy and to apply relevant findings for soon clinical use. The direct relationship detected between clinical toxicity and transcriptional effects ex vivo in patient-derived fibroblasts on which this application is based represent a special feature of this translational approach. In this regard, the issues of this proposal represent the middle part of a bedside-benach-bedside sandwich. The major results of the applied investigations should then be prospectively tested in an investigator-initiated trial (IIT) to evaluate clinical relevance and feasibility.