Abstract 4512: Identifying novel mechanisms of p53-mediated tumor suppression

TP53 is the most frequently mutated gene in human cancer. Classically, p53-mediated tumor suppression is thought to occur via transactivation of canonical targets that induce cell cycle arrest and apoptosis. However, this model has been challenged by studies using mouse p53 mutants. The mouse transactivation domain mutant, p5325,26, is unable to activate most canonical p53 targets and fails to induce cell cycle arrest or apoptosis in response to DNA damage. However, it retains the ability to induce cellular senescence and suppresses tumor development in mouse models. Indeed, some human p53 mutants found in Li-Fraumeni syndrome as well as cancer retain transactivation, while some p53 mutants are not found in cancer despite loss of transactivation. The goal of our study is to identify novel p53 functions that drive tumor suppression by evaluating p53 mutations where transactivation is uncoupled from tumor suppression activity. To identify human TP53 missense mutants with tumor suppressive function dissociated from the ability to activate canonical p53 pathways, we first utilized the results of a 2003 saturation mutagenesis screen performed in yeast which stratified human p53 mutants by their ability to activate 8 canonical p53 targets. We then cross-referenced these results with tumor sequencing data from the IARC, the TCGA PanCancer Atlas, and the MSK-IMPACT Sequencing Cohort. We identified panels of human p53 missense mutants which either 1) retained transactivation ability in yeast, yet had been identified in sequenced human tumors or 2) were unable to transactivate p53 targets in yeast, but had not been identified in sequenced human tumors. To study these p53 mutants compared to wt p53, we cloned their sequences into doxycycline-inducible pLVX-TetOne-Puro plasmids and puromycin-selected for cell lines stably expressing the construct in p53 null H1299 and HCT116 cells. Using qPCR and western blots we validated the expression of mutant or wt p53 and assessed for transactivation of canonical targets p21, mdm2, puma, gadd45α, and 14-3-3. The IncuCyte Live Cell Analysis system and XTT Cell Proliferation assays were used to evaluate p53-mediated growth arrest of human cancer cells following induction of each mutant. From these experiments, we selected three mutants for further study. Missense p53 mutants E224D and R290H are found in human cancers and in the germline of Li-Fraumeni syndrome patients yet retain the capacity to transactivate p53 targets and arres