Mis-splicing Drives Loss of Function of p53E224DPoint Mutation

Abstract

AbstractBackgroundTp53 is the most commonly mutated gene in cancer. Canonical Tp53 DNA damage response pathways are well characterized and classically thought to underlie the tumor suppressive effect of Tp53. Challenging this dogma, mouse models have revealed that p53 driven apoptosis and cell cycle arrest are dispensable for tumor suppression. Here, we investigated the inverse context of a p53 mutation predicted to drive expression of canonical targets, but is detected in human cancer.MethodsWe established a novel mouse model with a single base pair mutation (GAG>GAC, p53E221D) in the DNA-Binding domain that has wild-type function in screening assays, but is paradoxically found in human cancer in Li-Fraumeni syndrome. Using mouse p53E221Dand the analogous human p53E224Dmutant, we evaluated expression, transcriptional activation, and tumor suppression in vitro and in vivo.ResultsExpression of human p53E224Dfrom cDNA translated to a fully functional p53 protein. However, p53E221D/E221DRNA transcribed from the endogenous locus is mis-spliced resulting in nonsense mediated decay. Moreover, fibroblasts derived from p53E221D/E221Dmice do not express a detectable protein product. Mice homozygous for p53E221Dexhibited increased tumor penetrance and decreased life expectancy compared to p53 WT animals.ConclusionsMouse p53E221Dand human p53E224Dmutations lead to splice variation and a biologically relevant p53 loss of function in vitro and in vivo.