Decoding the TP53 mutome: CRISPR perspectives on functional diversity and therapeutic interventions

The key transcription factor p53 plays a crucial role in executing diverse tumor suppressive functions in human cells, as evidenced by the high prevalence of mutations in its encoding gene, TP53, across various cancer entities. Remarkably, TP53 mutations predominantly manifest as missense mutations, exhibiting an unparalleled diversity with over 2,000 distinct mutations identified in cancer patients. Notably, about 70% of these mutations remain poorly characterized, highlighting the need for comprehensive understanding to incorporate p53 status into clinical evaluation. This dissertation focuses on the detailed characterization of more than 94% of reported cancer-associated mutations within the TP53 gene. Employing CRISPR-mediated mutagenesis at the endogenous TP53 locus in cancer cells, we conducted investigations in a highly physiological context. Our research elucidates the functional implications of numerous mutations, previously of uncertain significance, unveiling factors such as moderate thermodynamic destabilization, loss of specific functions, and splicing defects. This knowledge was further implemented into an interactive database, encompassing all existing analyses on TP53 mutations. This resource facilitates data recapitulation, diverse visualizations, and predictive insights into the tumorigenic potential and therapy response of variants. Exploring clinical targeting of p53, we utilized Designed Ankyrin Repeat Proteins to restore p53 transcriptional activity, exemplified in Human Papilloma Virus-infected cancer cell lines. To incorporate the importance of the tumor microenvironment, we established a highly physiological genetically engineered mouse model for small-cell lung cancer. Therefore, we leveraged adenoviruses for CRISPR-mediated knockout of Trp53 and other cancer-associated genes, resulting in a rapid, flexible, and efficient method for generating cancer mouse models. Moreover, incorporating in vivo labeling with Gaussia princeps luciferase enabled longitudinal and animal-friendly monitoring of tumor development. In conclusion, this dissertation presents a comprehensive annotation of the TP53 mutome, offering valuable insights into its diverse landscape and clinical implications, including genetic risk assessment and therapeutic strategies. Additionally, it introduces an advanced method for generating genetically induced cancer mouse models, paving the way for further investigations into the influence of the TP53 mutome on the tumor microenvironment.