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. 2021 May 1;81(9):2442-2456.
doi: 10.1158/0008-5472.CAN-20-1750. Epub 2021 Feb 26.

The Common Germline TP53-R337H Mutation Is Hypomorphic and Confers Incomplete Penetrance and Late Tumor Onset in a Mouse Model

John R Jeffers  1 , Emilia M Pinto  1 , Jerold E Rehg  1 , Michael R Clay  1 , Jinling Wang  1 , Geoffrey Neale  2 , Richard J Heath  3 , Guillermina Lozano  4 , Enzo Lalli  5   6 , Bonald C Figueiredo  6   7 , Alberto S Pappo  8 , Carlos Rodriguez-Galindo  9 , Wenan Chen  10 , Stanley Pounds  11 , Raul C Ribeiro  8 , Gerard P Zambetti  12
Affiliations

The Common Germline TP53-R337H Mutation Is Hypomorphic and Confers Incomplete Penetrance and Late Tumor Onset in a Mouse Model

John R Jeffers et al. Cancer Res. .

Abstract

The TP53-R337H founder mutation exists at a high frequency throughout southern Brazil and represents one of the most common germline TP53 mutations reported to date. It was identified in pediatric adrenocortical tumors in families with a low incidence of cancer. The R337H mutation has since been found in association with early-onset breast cancers and Li-Fraumeni syndrome (LFS). To study this variability in tumor susceptibility, we generated a knockin mutant p53 mouse model (R334H). Endogenous murine p53-R334H protein was naturally expressed at high levels in multiple tissues and was functionally compromised in a tissue- and stress-specific manner. Mutant p53-R334H mice developed tumors with long latency and incomplete penetrance, consistent with many human carriers being at a low but elevated risk for cancer. These findings suggest the involvement of additional cooperating genetic alterations when TP53-R337H occurs in the context of LFS, which has important implications for genetic counseling and long-term clinical follow-up. SIGNIFICANCE: A p53-R334H knockin mouse serves as an important model for studying the most common inherited germline TP53 mutation (R337H) that is associated with variable tumor susceptibility.

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Conflict of interest statement

The authors declare no potential conflicts of interest.

Figures

Figure 1.
Figure 1.. Generation of germline p53-R334H mouse model.
A) Targeting strategy. The p53R334H point mutation in exon 10 is marked by an asterisk. B) Identification of targeted ES cell clones by PCR amplification using an outside probe yielding a 3.6 kb wild-type band and a 4.9 kb mutant band. C) Genotyping of animals using allele-specific primers result in a 109 bp wild-type band and a 128 bp mutant p53 band. PCR products were resolved on a 3% LMP agarose gel. D) Sequencing of the p53 missense mutation from genomic DNA. E) Genotype analysis of offspring from p53R334H heterozygous crosses.
Figure 2.
Figure 2.. Mutant p53R334H/R334H mice develop tumors with long latency and incomplete penetrance.
A) Kaplan-Meier tumor free survival curves. B) Histopathology of tumors arising in homozygous p53R334H mouse (20 months of age). Osteosarcoma, vertebra, H&E (2.5X) (upper left panel), osteosarcoma, vertebra, H&E (40X) (upper middle panel), osteosarcoma, p53 IHC (40X) (upper right panel); mammary adenocarcinoma, H&E (40X) (lower left panel) mammary adenocarcinoma, p53 IHC (40X) (lower right panel). C) Histopathology of primary and metastatic sarcoma arising in homozygous p53R334H mouse (21 months of age). Angiosarcoma with inset highlighting microcapillary formation with red blood cells (40X) (left panel), angiosarcoma CD31 staining (40X) (middle panel), metastatic angiosarcoma attached to blood vessel wall (10X) (right panel).
Figure 3.
Figure 3.. Mutant p53-R334H is abnormally expressed and functionally-impaired in thymocytes.
A) FACS analysis of isolated thymocytes in cell culture either untreated (Control) or 16 hrs post-ionizing radiation (5 Gy γ-IR). Results are representative of four independent experiments. B) Western blot analysis of isolated thymocytes in cell culture at 0 (untreated), 2 and 5 hrs post-γ-IR (5 Gy). Proteins were detected using the following primary antibodies: p53 (1C12); p21 (F5); Puma (P4743); β-Actin (AC-15). C) Western blot analysis of tissues from wild-type, p53R334H/R334H, p53R172H/R172H and p53−/− mice either untreated (Control) or 6 hrs post-treatment 5 Gy whole-body IR. Each sample represents an individual animal. Proteins were detected using the following primary antibodies: p53 (1C12); p21 (F5); β-Actin (AC-15). D) Immunohistochemistry (IHC) analysis of p53 in thymus from mice either untreated or 6 hrs post-treatment with 5 Gy whole body IR. p53 was detected using rabbit polyclonal CM5 antibody (Leica).
Figure 4.
Figure 4.. Mutant p53-R334H transcriptional response is attenuated in thymocytes in vivo in response to DNA damage.
Affymetrix gene expression analysis of wild-type and R334H thymocytes either untreated (Control) or 3 hrs post-treatment 5 Gy whole body (IR). Each lane represents an individual mouse. A) Heat map depicting a global perspective of all gene profiles measured by the microarray; B) Heat map display of 48 core enrichment genes in the Hallmark p53 pathway that are activated in WT cells following radiation (GSEA enrichment score 2.66, p<0.001, FDR<0.001); C) Heat map display of 41 core enrichment genes in the Hallmark MYC targets V2 that are activated in WT cells following radiation (FDR<0.001); D) Quantitative real time analysis of wild-type and p53-R334H thymocytes either untreated (Control) or 3 hrs post-treatment 5 Gy whole body IR. (n = 3).
Figure 5.
Figure 5.. Mutant p53-R334H is functional in MEFs in response to DNA damage and in vitro senescence.
A) Mutant p53-R334H confers short term proliferative advantage in low passage MEFs. Curves represent the average of three independent MEF cell lines for each genotype counted daily in duplicate. (P = 0.002) B) Mutant p53-R334H competently induced cellular senescence in serially passaged MEFs. (P = 0.008) C) Mutant p53-R334H induces target gene expression in response to DNA damage induced by 10 μM etoposide at 4 hrs post-treatment. Proteins were detected using the following primary antibodies p53 (PAb7); p21 (C19). D) Mutant p53-R334H induces target gene expression in response to DNA damage induced by 30 J UV radiation at 24 hours. Proteins detected using the following primary antibodies p53 (1C12); p21 (F-5). MEFs at passage 4 were used in experiments presented in panels C and D.
Figure 6.
Figure 6.. Mutant p53-R334H has a prolonged half-life and compromised in forming stable tetramers in primary mouse embryo fibroblasts.
A) Cycloheximide half-life experiment. Early passage MEFs (passage 5) were treated with 10 μg/ml cycloheximide and harvested at indicated time points to determine p53 levels by Western blot analysis using mouse MAb 1C12. B) Protein complex analysis of primary MEFs by size exclusion chromatography and western blot analysis (see Materials and Methods). P53 protein was detected using MAb 1C12. C) SaOS2 cells transfected with CMV Neo vector without an insert, p53 wild-type or p53-R334H cDNA. Co-immunoprecipitation using Mdm2 (2A10) Ab and blotted for p53 using Sheep anti-p53 polyclonal Ab.
Figure 7.
Figure 7.. Mutant p53-R334H functions as a tumor suppressor in Eμ-Myc lymphomagenesis.
A) Kaplan Meier survival of Eμ-Myc transgenic wild-type, p53WT/R334H and p53R334H/R334H mice. B) Western blot analysis of B cell lymphomas arising in Eμ-Myc WT, p53WT/R334H and p53R334H/R334H mice. C) Characterization of p53 and p19Arf status in B cell lymphomas arising Eμ-Myc WT, p53WT/R334H and p53R334H/R334H mice. Proteins were detected by Western blot analysis using the following primary antibodies: p53 (1C12); p19Arf (ab80; ABCAM).
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