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

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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.