Oncogenic and teratogenic effects of p53^Y217C, a mouse model of the human hotspot mutant p53^Y220C

Reviewer #1 (Public review):

Summary:

This manuscript by Toledo and colleagues describes the generation and characterization of Y220C mice (Y217C in the mouse allele). The authors make notable findings: Y217C mice that have been backcrossed to C57Bl/6 for five generations show decreased female pup births due to exencephaly, a known defect in p53 -/- mice, and they show a correlation with decreased Xist expression, as well increased female neonatal death. They also noted similar tumor formation in Y217C/+ and p53 +/- mice, suggesting that Y217C may not function as a dominant negative. Notably, the authors find that homozygous Y217C mice die faster than p53 -/- mice and that the lymphomas in the Y217C mice were more aggressive and invasive. The authors then perform RNA seq on thymi of Y217C homozygotes compared to p53 -/-, and they suggest that these differentially expressed genes may explain the increased tumorigenesis in Y217C mice.

Strengths:

Overall, the study is well controlled and quite well done and will be of interest to a broad audience, particularly given the high frequency of the Y220C mutation in cancer (1% of all cancers, 4% of ovarian cancer).

Weaknesses:

No weaknesses were noted by this reviewer.

Reviewer #2 (Public review):

Summary:

Jaber et al. describe the generation and characterization of a knock-in mouse strain expressing the p53 Y217C hot-spot mutation. While the homozygous mutant cells and mice reflect the typical loss-of-p53 functions, as expected, the Y217C mutation also appears to display gain-of-function (GOF) properties, exemplified by elevated metastasis in the homozygous context (as noted with several hot-spot mutations). Interestingly, this mutation does not appear to exhibit any dominant-negative effects associated with most hot-spot p53 mutations, as determined by the absence of differences in overall survival and tumor predisposition of the heterozygous mice, as well as target gene activation upon nutlin treatment.

In addition, the authors noted a severe reduction in the female 217/217 homozygous progeny, significantly more than that observed with the p53 null mice, due to exencephaly, leading them to conclude that the Y217C mutation also has additional, non-cancer-related GOFs. Though this property has been well described and attributed to p53 functional impairment, the authors conclude that the Y217C has additional properties in accelerating the phenotype.

Transcriptomic analyses of thymi found additional gene signature differences between the p53 null and the Y217C strains, indicative of novel target gene activation, associated with inflammation.

Strengths:

Overall, the characterisation of the mice highlights the expected typical outcomes associated with most hot-spot p53 mutations published earlier. The quality of the work presented is well done and good, and the conclusions and reasonably well justified.

Weaknesses:

The manuscript would benefit from the provision of additional data to strengthen the claims made, as follows:

(1) Oncogenic GOF - the main data shown for GOF are the survival curve and enhanced metastasis. Often, GOF is exemplified at the cellular level as enhanced migration and invasion, which are standard assays to support the GOF. As such, the authors should perform these assays using either tumor cells derived from the mice or transformed fibroblasts from these mice. This will provide important and confirmatory evidence for GOF for Y217C.

(2) Novel target gene activation - while a set of novel targets appears to be increased in the Y217C cells compared to the p53 null cells, it is unclear how they are induced. The authors should examine if mutant p53 can bind to their promoters through CHIP assays, and, if these targets are specific to Y217C and not the other hot-spot mutations. This will strengthen the validity of the Y217C's ability to promote GOF.

(3) Dominant negative effect - the authors' claim of lack of DN effect needs to be strengthened further, as most p53 hot-spot mutations do exhibit DN effect. At the minimum, the authors should perform additional treatment with nutlin and gamma irradiation (or cytotoxic/damaging agents) and examine a set of canonical p53 target genes by qRT-PCR to strengthen their claim.