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

项目摘要/摘要 三阴性乳腺癌(TNBC)是一种缺乏传统临床经验的侵袭性乳腺癌亚型 目标；因此，细胞毒性化疗是目前的护理标准。靶向治疗的发展 由于分子的异质性和缺乏治疗靶向性的高频，对TNBC来说是具有挑战性的 “司机”的改动。在TNBC病例中，最统一的特征是~80%的肿瘤含有突变 抑制基因TP53。P53的突变通常是错误的，已被认为会导致- 功能(GOF)活性导致新的致癌表型。尽管这其中的机械论基础 GOF活性尚不清楚，TP53的变化与染色体的增加高度相关 不稳定性(CIN)和非整倍体的发展，并与代谢失调有关。 为了研究P53 GOF突变蛋白，我们实验室建立了两个等基因的细胞系模型(未转化 乳腺上皮细胞和TNBC细胞株)。这些模型包括 表达两种常见“热点突变型”P53蛋白的克隆细胞系(R175H和R273H)，野生型(WT) P53蛋白，或无P53蛋白(空)。这组细胞系允许研究各种形式的P53，所有 由内源基因启动子表达和调控，不受 异位的和不受监管的过度表达。此外，这些模型提供了一个独特的机会来剖析 新的和评估已提出的源于功能性(LOF)p53缺失的GOF机制和表型 和/或伴随突变型P53蛋白表达的增加。我们已经证明了我们的等基因细胞系 突变型P53具有较高的CIN水平、异倍体形成和代谢紊乱。另外， 我们发现突变蛋白的稳定与非整倍体的发展密切相关。 在目标1中，我将部署生化技术并分析一系列基因组数据集，这些数据集由 我们的细胞系模型来评估突变型p53和p73相互作用与CIN的关系。在AIM 2中I 将使用生化技术和靶向代谢组学研究非整倍体的发展是如何奠定基础的 突变型P53的GOF表型稳定和代谢改变。通过这些目标，我将测试 假设发现和剖析突变型P53 LOF和/或GOF机制，从而产生 与肿瘤细胞中非整倍体相关的细胞状态，将导致新的前- TNBC的临床靶点。我预计，对新机制的剖析以及对 建议的突变型p53 GOF表型机制的重复性将改善目前的 了解突变型P53在肿瘤发生中的作用。我们的研究产生的结果有 临床翻译的潜力，不仅在TNBC(对其来说，靶向治疗的需要至关重要)，而且 在其他类型的人类癌症中，存在高频的P53突变。

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.