Functional annotation of the TP53 mutome by CRISPR/Cas9-based saturating mutagenesis

p53编码基因TP 53中的突变是癌细胞中最常见的遗传改变，并与较差的生存预后相关。然而，尽管TP 53突变在癌症患者中的高频率及其预后意义，TP 53突变状态在临床决策中很少被考虑。原因之一当然是TP 53突变的广谱性，这使得单个TP 53突变的临床后果难以预测。我们今天对TP 53突变在肿瘤发生和癌症治疗中的作用的了解大多来自对最普遍的热点突变的研究。这些错义突变消除了作为肿瘤抑制转录因子的生理功能，以显性负性方式抑制野生型p53，有时赋予突变蛋白新的致癌特性。这些活动共同促进肿瘤细胞适应性并增强治疗抗性。然而，70%的TP 53突变不是热点突变，其功能特性仍有待研究。为了全面系统地表征单个TP 53突变的功能，我们建立了利用CRISPR/Cas9介导的基因组编辑对人类肿瘤细胞内源性TP 53基因位点进行定点突变的方法。我们显示了p53残基175至185的初步诱变筛选的初步数据，其覆盖了癌症中所有TP 53错义突变的11.7%。该筛选定量地揭示了个体TP 53突变如何影响用Mdm 2抑制剂治疗后的肿瘤细胞适应性，Mdm 2抑制剂是临床上最先进的针对p53通路的抗癌化合物。我们现在建议将这种全面和系统的筛选扩展到外显子5-8中的> 10，000个TP 53突变，覆盖癌症中所有TP 53错义突变的约95%。使用已建立的方法，我们将研究个体TP 53突变如何影响肿瘤细胞适应性和p53调节的细胞命运决定，以响应癌症治疗，包括化疗和放疗以及野生型或突变型p53的靶向治疗（预）临床开发的不同阶段。这些研究将在结直肠癌模型中进行，并与非小细胞肺癌模型进行比较。总之，这将产生关于人类肿瘤细胞中个体TP 53突变功能的丰富数据资源，预计将使TP 53突变状态为临床决策提供更多信息。

Mutations in the p53 encoding gene TP53 are the most frequent genetic alterations in cancer cells and correlate with a poor survival prognosis. However, despite the high frequency of TP53 mutations in cancer patients and their prognostic implications, TP53 mutation status is only rarely considered in clinical decision making. One reason is certainly the broad spectrum of TP53 mutations that makes the clinical consequences of an individual TP53 mutation difficult to predict. Most of what we know today about the role of TP53 mutations in tumorigenesis and cancer therapy comes from studies on the most prevalent hot-spot mutations. These are missense mutations that ablate the physiological function as a tumor suppressive transcription factor, inhibit wild-type p53 in a dominant-negative manner and sometimes endow the mutant protein with new oncogenic properties. Together these activities promote tumor cell fitness and enhance therapy resistance. However, 70% of all TP53 mutations are not hot-spot mutations and their functional properties still remain to be investigated.To characterize the function of individual TP53 mutations in a comprehensive and systematic manner, we have established a site-directed mutagenesis of the endogenous TP53 gene locus in human tumor cells using CRISPR/Cas9-mediated genome editing. We show preliminary data from a pilot mutagenesis screen of p53 residues 175 to 185, which covered 11.7% of all TP53 missense mutations in cancer. This screen revealed quantitatively how individual TP53 mutations affect tumor cell fitness upon treatment with Mdm2 inhibitors, the clinically most advanced targeted anti-cancer compounds for the p53 pathway. We now propose to extend this comprehensive and systematic screen to >10,000 TP53 mutations in exons 5-8, covering approximately 95% of all TP53 missense mutations in cancer. Using the established approach, we will investigate how individual TP53 mutations influence tumor cell fitness and p53-regulated cell fate decisions in response to cancer treatments including chemo- and radiotherapy as well as targeted therapies for wild-type or mutant p53 in different stages of (pre)clinical development. The studies will be performed in a model of colorectal cancer and compared to a model for non-small cell lung cancer. Together, this will generate a rich data resource on the function of individual TP53 mutations in human tumor cells that is expected to make the TP53 mutation status more informative for clinical decision making.