A systematic p53 mutation library links differential functional impact to cancer mutation pattern and evolutionary conservation

The TP53 gene is frequently mutated in human cancer. Research has focused predominantly on six major “hotspot” codons, accounting for only ~30% of cancer-associated p53 mutations. To comprehensively characterize the consequences of the p53 mutation spectrum, we created a synthetically designed library and measured the functional impact of ~10,000 DNA-binding domain (DBD) p53 variants in human cells in culture and in vivo. Our results highlight the differential outcome of distinct p53 mutations in human patients and elucidate the selective pressure driving p53 conservation throughout evolution. Furthermore, while loss of anti-proliferative functionality largely correlates with the occurrence of cancer-associated p53 mutations, we observe that selective gain-of-function may further favor particular mutants in vivo. Finally, when combined with additional acquired p53 mutations, seemingly neutral TP53 SNPs may modulate phenotypic outcome and presumably tumor progression. A synthetic library of 9,833 TP53 DNA-binding domain (DBD) sequence variants, composed of 4 sub-libraries (DBDA, DBDB, DBDC and DBDD) was expressed in human cells (H1299) and the relative fitness endowed by each variant was measured in a pooled time-course competition assay. For sub-libraries DBDA and DBDB, a biological replicate was performed. A similar experiment was performed with sub-library DBDD in HCT116 cells. In addition, the relative enrichment of these variants (DBDD expressed in HCT116 cells) was further measured in vivo (cells subcutaneously injected into mice) as well as in a 3D culture spheroid setting (using a small-scale library).