Li-Fraumeni Syndrome tp53 Hotspot Mutant Zebrafish Exhibit Variant-Specific p53 Dysfunction and Tumour Development Which is Accelerated by kmt2d and pik3ca Modifier Mutations

Abstract

Li-Fraumeni syndrome (LFS), a highly penetrant cancer predisposition syndrome, is associated with a nearly 100% lifetime cancer risk. LFS is characterized by a diverse, early onset tumour spectrum and germline mutations in the TP53 tumour suppressor gene. The observed heterogeneity in age of onset and tumour type in LFS may be due to specific p53 variants, as well as acquired mutations in modifier genes, such as KMT2D and PIK3CA.

Zebrafish are a powerful cancer modelling tool due to their high degree of genetic conservation and tumour development that resembles those found in humans. I characterized two tp53 mutant zebrafish, R217H and R242H, (human hotspot residues, R248 and R273H), and demonstrated R242H mutants exhibit no activation of p53 target genes and are resistant to apoptosis, while the R217H variant is hypomorphic as mutants show partial activation of p53 target genes and reduced p53-mediated apoptosis. tp53 mutants develop spontaneous tumours at 6-9 months that histologically resemble human sarcomas. Homozygous R242H mutants exhibited earlier tumour onset and higher life-time incidence compared to R217H and null mutants. Evaluation of pCAP-250, a promising p53 reactivator, in tp53 mutants was unsuccessful, which prompted the generation of humanized TP53 zebrafish as a more clinically relevant drug screening model.

To model the modifiers, I generated transgenic lines that ubiquitously express zebrafish pik3ca WT or H1048R (human H1047R) and a kmt2d deletion line using CRISPR/Cas9 and crossed all three into tp53 mutant backgrounds. Compound kmt2d -/-;tp53 (R217H/R217H or R242H/R242H) and pik3ca-H1048R;tp53 R242H/R242H zebrafish all have significantly decreased tumour-free survival compared to tp53 mutants, validating KMT2D and PIK3CA as modifiers in LFS.

Overall, p53 mutant zebrafish recapitulate human LFS phenotypes and hold tremendous potential to define underlying tumour-driving mechanisms and serve as a drug screening platform, while kmt2d and pik3ca;tp53 mutants represent novel in vivo tools to evaluate LFS tumourigenesis.