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

评估突变 p53 功能获得表型机制的功能基因组学方法

• 批准号: 10017661
• 负责人: Lindsay Redman
• 金额: $ 3.02万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-09 至 2022-07-08
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10017661
• 关键词: Affect; African American; Amino Acids; Aneuploidy; Apoptosis; Binding; Biochemical; Breast Cancer cell line; Cell Cycle; Cell Cycle Arrest; Cell Cycle Regulation; Cell Fractionation; Cell Line; Cells; Cessation of life; Chromosomal Instability; Chromosome abnormality; Clinical; Clinical Data; Clone Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Complex; Cytotoxic Chemotherapy; Development; Disease; Dissection; Evaluation; Event; Evolution; Family member; Frequencies; Future; Gene Expression; Genes; Genome; Genomic Instability; Genomic approach; Genomics; Goals; Heat-Shock Proteins 90; Heterogeneity; Human; Immunofluorescence Immunologic; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Metabolic; Metabolism; Missense Mutation; Modeling; Molecular; Mutation; Neoplasm Metastasis; Oncogenic; Outcome; Phenotype; Prevention strategy; Property; Protein Analysis; Protein p53; Proteins; Relapse; Reporting; Reproducibility; Research; Research Proposals; Role; TP53 gene; Techniques; Testing; Therapeutic; Time; Tumor Suppressor Genes; Tumor Suppressor Proteins; Tumor-Derived; Woman; base; clinical translation; experimental study; functional genomics; functional loss; gain of function; genome editing; genomic data; improved; inhibitor/antagonist; loss of function; malignant breast neoplasm; mammary epithelium; metabolomics; mutant; neoplastic cell; new therapeutic target; novel; overexpression; pre-clinical; promoter; protein expression; protein protein interaction; senescence; standard of care; stem; survival outcome; targeted treatment; therapeutic development; therapeutic target; transcription factor; triple-negative invasive breast carcinoma; tumorigenesis; validation studies

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.

项目概要/摘要 三阴性乳腺癌（TNBC）是一种侵袭性乳腺癌亚型，缺乏传统的临床治疗方法。 因此，细胞毒性化疗是目前靶向治疗的标准。 由于分子异质性和缺乏可靶向治疗的高频药物，TNBC 的治疗具有挑战性 TNBC 病例中最统一的特征是约 80% 的肿瘤存在突变。 p53 的抑制基因 TP53 突变通常是错义的，并且已被认为会导致增益。 功能（GOF）活动导致新的致癌表型，尽管其机制基础。 GOF 活性尚不清楚，TP53 的改变与染色体增加高度相关 不稳定性（CIN）和非整倍性的发展，并与代谢失调有关。 为了研究p53 GOF突变蛋白，我们实验室开发了两种同基因细胞系模型（非转化 乳腺上皮细胞和 TNBC 细胞系）使用 CRISPR/Cas 介导的基因组编辑。 表达两种常见“热点突变”p53 蛋白（R175H 和 R273H）的克隆细胞系，野生型 (WT) 蛋白质，或无 p53 蛋白质（空） 该细胞系组允许研究各种形式的 p53。 由内源基因启动子表达和调节，并且没有由内源基因启动子引起的混杂效应 此外，这些模型为解剖提供了独特的机会。 新颖并评估因功能性 (LOF) p53 缺失而提出的 GOF 机制和表型 我们已经证明我们的同基因细胞系具有突变p53蛋白表达。 p53 突变体具有更高水平的 CIN、非整倍体的发展和代谢失调。 我们发现突变蛋白的稳定性与非整倍性的发展显着相关。 在目标 1 中，我将部署生化技术并对一系列基因组数据集进行分析 我们的细胞系模型用于评估突变型 p53 和 p73 相互作用与 CIN 之间的关系。 将使用生化技术和靶向代谢组学来研究非整倍性发展的基础 通过这些目标，我将测试突变 p53 稳定性和代谢改变的 GOF 表型。 假设发现并剖析了突变型 p53 LOF 和/或 GOF 机制，从而产生 与肿瘤细胞非整倍性相关的细胞状态将导致新的预 我预计新机制的剖析以及 TNBC 的评估。 所提出的突变 p53 GOF 表型机制的可重复性将改善目前的 了解突变体 p53 在肿瘤发生中的作用 我们的研究结果表明： 临床转化的潜力，不仅在 TNBC（对于靶向治疗的需求至关重要），而且在 在具有高频 p53 突变的其他类型的人类癌症中。