Abstract P5-09-01: Using isogenic model systems to determine mechanisms regulating mutant p53 protein stability in breast cancer cells

Abstract

Abstract Two of the most common events in triple-negative breast cancers (TNBC) are mutation of the tumor suppressor gene TP53 and development of aneuploidy. This is often the case in aggressive and molecularly heterogeneous cancer subtypes such as TNBC, where ~80% of cases harbor a mutation in TP53. In addition to losing wild-type (WT) tumor-suppressive function, mutant p53 proteins are proposed to acquire gain-of-function (GOF) activity, leading to novel oncogenic phenotypes. Mutant p53 proteins can accumulate to high levels in tumors, and this stabilization is thought to be a pre-requisite for mutant p53 to exert its GOF effects. Mechanisms underlying the accumulation of mutant p53 are not well understood. To study mutant p53 GOF mechanisms and phenotypes, we used CRISPR/Cas9-mediated genome editing and developed two isogenic TNBC cell line models (one non-transformed and one tumor-derived) that express the most frequently occurring p53 missense mutations (R175H and R273H), are deficient for functional p53 protein (null), or retain the wild-type (WT) protein. In these engineered models, endogenous p53 expression is regulated by the native p53 promoter, thus providing a controlled system for rigorous functional experimentation across different p53 states. Through functional genomics analyses comparing isogenic TNBC cell lines, which initially differed only by the TP53 genotype, we have evaluated the relationship between mutant p53 and development of aneuploidy and determined that development of aneuploidy and not TP53 genotype contributed to several previously reported mutant p53 GOF phenotypes in vitro and in vivo1. Immunoblotting, immunofluorescence, and immunohistochemistry experiments revealed that mutant p53-expressing cell lines display a range of p53 protein levels, with several maintaining low levels of p53, similar to the cell lines which contain WT p53. In addition, mutant p53 levels correlate with aneuploidy, but not mean nuclear area or whole-genome doubling status, and protein levels increase over time in cell lines that become aneuploid. Using our various clonal cell lines that exhibit differing p53 protein stability, we are currently determining if p53 stability is dysregulated in aneuploid cell lines after copy number gain or loss of genes encoding critical p53 regulators, leading to the accumulation of the mutant protein. Analysis of cancer cell line genetic dependencies and human tumor data from The Cancer Genome Atlas (TCGA) has identified genetic and clinical correlates with increased mutant p53 protein levels. Utilizing biochemical and bioinformatic approaches, we aim to uncover mechanisms for increased mutant p53 stability and identify novel regulators of both WT and mutant p53 proteins. The dissection of mechanisms that contribute to stabilization of p53 protein will not only give insight into the regulation and tumor suppressive function of WT p53, but it has the potential for clinical translation in human cancers that have high-frequency p53 mutation and high levels of mutant p53 protein, as targeting mutant p53 through pharmacologic inhibition or antibody-based therapies is an active area of investigation. 1Article in press in Nature Communications Citation Format: Lindsay Redman-Rivera, Hailing Jin, Brian D Lehmann, Jennifer A Pietenpol. Using isogenic model systems to determine mechanisms regulating mutant p53 protein stability in breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-09-01.