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

p53编码基因TP53的突变是癌细胞中最常见的遗传改变，并与不良的生存预后相关。然而，尽管TP53突变在癌症患者中频率很高，并具有预后意义，但TP53突变状态在临床决策中很少被考虑。其中一个原因当然是TP53突变的广谱性，这使得个体TP53突变的临床后果难以预测。我们今天所知道的关于TP53突变在肿瘤发生和癌症治疗中的作用的大部分知识来自于对最普遍的热点突变的研究。这些错义突变削弱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和增强治疗抵抗。然而，70%的TP53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TP53突变的功能，我们利用CRISPR/ cas9介导的基因组编辑技术，建立了人类肿瘤细胞内源性TP53基因位点的定点突变。我们展示了p53残基175 - 185的先导诱变筛选的初步数据，其中涵盖了癌症中所有TP53错义突变的11.7%。该筛选定量揭示了个体TP53突变如何影响Mdm2抑制剂治疗后的肿瘤细胞适应性，Mdm2抑制剂是临床上最先进的p53通路靶向抗癌化合物。我们现在建议将这一全面而系统的筛查扩展到5-8外显子的100万个TP53突变，覆盖癌症中约95%的TP53错义突变。利用已建立的方法，我们将研究个体TP53突变如何影响肿瘤细胞适应性和p53调控的细胞命运决定，以响应癌症治疗，包括化疗和放疗，以及在临床发展的不同阶段（前）针对野生型或突变型p53的靶向治疗。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，这将产生关于个体TP53突变在人类肿瘤细胞中的功能的丰富数据资源，有望使TP53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.