A Systematic p53 Mutation Library Links Differential Functional Impact to Cancer Mutation Pattern and Evolutionary Conservation

Abstract

The TP53 gene is frequently mutated in human cancer. Research has focused predominantly on six major "hotspot" codons, which account 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.

The Emerging Landscape of p53 Isoforms in Physiology, Cancer and Degenerative Diseases

“…Therefore, it is recommended that the p53 isoform expression is reported with respect to the mutational status of TP53, to establish more precise correlations with clinical outcomes [109,110]. However, stating whether p53 is WT or mutant may not suffice as it is becoming apparent that the mutational landscape of p53 is highly heterogeneous and the various p53 mutants are not equal in their cellular and phenotypical effects [111,112]. For example, Li Fraumeni syndrome (LFS) patients with specific p53 DBD mutations (associated with impaired anti-proliferative functions in H1299 cultured cells) have tumour incidences at an earlier age compared to LFS patients harbouring germline TP53 mutations retaining anti-proliferative functions [112].…”

A quantitative model to predict pathogenicity of missense variants in the TP53 gene

“…Results from systematic transactivation assays performed in yeast (Kato et al, 2003) were used for comparison with the classifica- When compared to data from DBD region variants assayed for antiproliferative function using a high through-put mammalian assay (Kotler et al, 2018) In addition, we compared our results to data from mammalian clonogenic survival assays conducted as part of this study, which also included reduced penetrance p.Arg337His variant as a partial activity pathogenic control. As shown in Figure 4 the performance of the clonogenic assay and systematic transactivation assays (Kato et al, 2003) was moderate, with an R 2 of 0.58.…”

“…Variants classified as pathogenic or likely pathogenic by the model were associated with earlier age of cancer onset (including a greater proportion of pediatric cancers), and were enriched in Classic LFS probands and depleted in a cohort of cancer-free women over 70 years. When compared to the output of reported functional assays, the majority of variants classified as pathogenic or likely pathogenic were nonfunctional in transactivation yeast assays (Kato et al, 2003), showed disruption of antiproliferative function/LOF (Giacomelli et al, 2018;Kotler et al, 2018), or reduced activity in the mammalian clonogenic survival assays developed in this study.…”

“…The outputs of the model were also compared to three datasets from human cellular models. In a recent study (Kotler et al, 2018), a massively parallel mutational scan was used to measure the functional impact of~10,000 TP53 variants in the DNA Binding Finally, we used a clonogenic assay to examine the 22 TP53 variants (17 missense, two nonsense, one frameshift, and two inframe indels) previously identified in 23 unrelated sarcoma probands from the International Sarcoma Kindred Study (ISKS) cohort (Ballinger et al, 2016). This analysis used the p53-null human H1299 cell line (Mitsudomi et al, 1992) derived from a non-small-cell lung carcinoma.…”

“…3.3 | Clinical information correlates with variant pathogenicity assigned using the quantitative model (Kato et al, 2003) 54.6% ≤20% activity 1.1% ≤20% activity Antiproliferative function (Kotler et al, 2018) 76.7% disruptive function 3.6% disruptive function LOF (Giacomelli et al, 2018) 74.4% LOF 11.1% LOF Clonogenic survival assays 90.9% disrupted function 0% disrupted function ClinVar 87.1% "P" or "LP" 0% "P" or "LP" than VUS was assigned, were classified as P/LP. The discordant variant was p.Asn235Ser, with functional transactivation class and 55 alleles in gnomAD.…”