Mutant p53 mouse cancer models using somatic CRISPR base editing

• Applicant: Professor Dr. Thorsten Stiewe
• Subject Area: Hematology, Oncology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 511189127

TP53 encodes the tumor suppressive transcription factor p53 and is the most frequently mutated gene in cancer cells. Different to other tumor suppressor genes, TP53 is most commonly hit by missense mutations which lead to a (variable) reduction in tumor suppressor activity. In addition, accumulation of mutant p53 proteins may result in dominant-negative and neomorphic (gain-of-function) activities that promote tumor progression and constitute a potential therapeutic target. However, more than 2000 functionally diverse missense mutations have been described, so that an in-depth understanding of single mutants and their consequences is needed to make better use of the TP53 status for personalized treatment decisions. Genetically-engineered p53 mutant knock-in mice have become valuable preclinical tools and have massively advanced our understanding of mutant p53 functions in tumorigenesis and cancer therapy. However, the underlying effort and costs associated with generating germline knock-ins preclude a systematic in vivo comparison of multiple mutations and deeper insight into the functional diversity of p53 mutations.In this project, we plan to make use of recent progress in somatic genome editing with advanced CRISPR tools to compare different p53 mutations in vivo. Most tumors with a p53 missense mutation are caused by C-to-T transitions which can be engineered with cytosine base editors (CBE), i.e. fusion proteins consisting of a Cas9 nickase, a cytosine deaminase and a uracil-N-glycosidase inhibitor. In our preliminary work, we have developed an improved CBE with superior base editing efficiency that allows the precise induction of various p53 point mutations which could not be generated with previously described CBEs. Moreover, we show successful somatic induction of small cell lung cancer (SCLC) using intratracheal infection of adult mice with adenoviral vectors delivering classical CRISPR-Cas9 nucleases. We now plan to combine these technologies and use adenoviral delivery of CBEs to generate SCLC tumors with defined single nucleotide substitutions (missense and nonsense mutations) in p53 and cooperating driver genes. The resulting tumor cell and tumor microenvironmental phenotypes will be profiled comprehensively and systematically. These studies are expected to provide deep and unprecedented insight into the functional diversity associated with distinct p53 mutations in the defined in vivo context of small cell lung cancer.

• DFG Programme: Research Grants