tp53 R217H and R242H Mutant Zebrafish Display Dysfunctional p53 Hallmarks and Recapitulate LFS Phenotypes [RNA-seq]

Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome associated with a highly penetrant and diverse tumor spectrum characterized by germline mutations in the TP53 tumor suppressor gene. To better understand how TP53 mutations predispose individuals with LFS to cancer development, we characterized tp53 R217H and R242H zebrafish lines as the first zebrafish p53 hotspot mutants (human R248 and R273H). These mutants have lost several key wildtype p53 functions and recapitulate many LFS phenotypes. Specifically, we have shown that the R217H and R242H alleles result in partial-to-no activation of key p53 target genes, are resistant to apoptosis in a dominant negative manner and exhibit a defective G1 cell-cycle checkpoint in vivo. The loss of these wildtype p53 functions predisposed the fish to develop spontaneous tumors as early as 6 months of age. Tumor histology resembles human sarcomas, a predominant LFS tumor type. tp53 R242H mutants developed tumors earlier with a higher lifetime incidence than tp53 null or R217H mutants, suggesting it is a more aggressive mutation, while the R217H allele may be hypomorphic. Additionally, we observed mutation-specific differences both in the tumor type and sex bias across tp53 null, R217H, and R242H genotypes with associated diverse transcriptomic and DNA methylome profiles, impacting metabolism, cell signalling, and biological macromolecule synthesis and degradation. These tp53 zebrafish mutants demonstrate fidelity to their human counterparts and provide new insights into underlying tumorigenesis mechanisms and kinetics, which may inform more tailored tumor surveillance approaches and novel therapeutic targets in LFS. We generated tp53 zebrafish mutants representing two of the most common mutation residues found in human cancers, tp53 R217H and R242H (human R248 and R273) using CRISPR to knock in the mutations and also obtained a CG1 tp53 null line from the Langenau lab (Ignatius et al., 2018). We also used the transparent casper strain as a comparison to zebrafish with wildtype p53. Of note, the tp53 null fish are in the CG1 background while the casper, tp53 R217H, and tp53 R242H fish are in the AB background. We performed bulk RNA sequencing on homozygous pooled samples with fifty 30 hour post-fertilization (hpf) larvae exposed to 0 or 30 Gy of irradiation at 24 hpf. We had three biological replicate for each genotype and treatment group for a total of 8 different groups with 24 samples total (casper 0 Gy, casper 30 Gy, tp53 null 0 Gy, tp53 null 30 Gy, tp53 R217H 0 Gy, tp53 R217H 30 Gy, tp53 R242H 0 Gy, tp53 R242H 30 Gy).