Splice-altering TP53 missense mutations as drivers of dysfunction and targets for RNA-based therapy.

Abstract

TP53 missense mutations introduce single amino acid substitutions in the p53 protein, which can lead to diverse functional consequences. However, their post-transcriptional impact, particularly on RNA splicing, remains underexplored. Herein, we analyzed cancer genome databases and identified 34 TP53 missense and synonymous mutations capable of generating de novo-splice sites. Using minigene assays, we confirmed the splicing-altering potential of several mutations, including c.178 C > A, c.182 A > G, c.318 C > G, c.356 C > G, c.362 C > A, c.551 A > G, and c.922 C > G. To assess the physiological relevance of these splicing changes, we developed mutant TP53 knock-in cell models using bacterial artificial chromosome DNA-mediated homologous recombination. These missense mutations can function as frameshift or hypomorphic mutations due to aberrant splicing, significantly compromising TP53 mRNA integrity. Meanwhile, severe impairment of constitutive splicing suggests that it correlates with an increased likelihood of nonsense-mediated mRNA decay for mutation-driven alternative transcripts carrying a frameshifted premature stop codon. By designing antisense morpholino oligomers targeting de-novo splice site, we were able to restore csV1 expression and enhance p53 function. This reclassification of select p53 mutations from simple missense to splicing-disruptive mutations reveals their underlying loss-of-function mechanisms and highlights their therapeutic reversibility. Our findings present a novel framework for RNA-based therapeutic strategies aimed at correcting splicing defects in TP53-mutant cancers.