Functional genomics approach to evaluate mechanisms of mutant p53 gain-of-function phenotypes

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Project Summary/Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that lacks traditional clinical targets; as a result, cytotoxic chemotherapy is the current standard of care. Development of targeted therapies for TNBC is challenging due to molecular heterogeneity and a lack of therapeutically targetable, high-frequency “driver” alterations. The most unifying feature across TNBC cases is that ~80% harbor a mutation in the tumor suppressor gene TP53. Mutations in p53 are commonly missense and have been proposed to result in gain-of- function (GOF) activity leading to novel oncogenic phenotypes. Although the mechanistic underpinnings of this GOF activity are not understood, alterations in TP53 are highly correlated with increased chromosomal instability (CIN) and the development of aneuploidy, and have been associated with dysregulated metabolism. To study p53 GOF mutant proteins, our lab developed two isogenic cell line models (non-transformed mammary epithelial and TNBC cell lines) using CRISPR/Cas-mediated genome editing. The models include clonal cell lines expressing two common “hotspot mutant” p53 proteins (R175H and R273H), wild-type (WT) protein, or no p53 protein (Null). This panel of cell lines allows for the study of various forms of p53, all expressed and regulated by the endogenous gene promoter and without the confounding effects caused by ectopic and unregulated overexpression. Additionally, these models afford a unique opportunity to both dissect novel and evaluate proposed GOF mechanisms and phenotypes that stem from loss of functional (LOF) p53 and/or concomitant gain of mutant p53 protein expression. We have shown that our isogenic cell lines with mutant p53 have higher levels of CIN, development of aneuploidy and dysregulated metabolism. Additionally, we have found that stabilization of mutant protein significantly correlates with the development of aneuploidy. In Aim 1 I will deploy biochemical techniques and analysis of an array of genomics data sets generated from our cell line models to evaluate the relationship between mutant p53 and p73 interactions and CIN. In Aim 2 I will use biochemical techniques and targeted metabolomics to study how development of aneuploidy underlies the GOF phenotypes of mutant p53 stabilization and altered metabolism. Through these aims I will test the hypothesis that discovery and dissection of mutant p53 LOF and/or GOF mechanisms, which generate cellular states associated with aneuploidy in tumor cells, will lead to the identification of novel pre- clinical targets for TNBC. I anticipate that the dissection of novel mechanisms as well as the evaluation of the reproducibility of proposed mechanisms for mutant p53 GOF phenotypes will improve the current understanding of the role mutant p53 in tumorigenesis. The results generated from our studies have the potential for clinical translation, not only in TNBC (for which the need for a targeted therapy is critical), but also in other types of human cancer that have high-frequency p53 mutation.