Differential pathway engagement and the biological consequences of KRAS variants in cancer

癌症中 KRAS 变异的差异通路参与和生物学后果

基本信息

批准号：
10313571
负责人：
Yanixa Quinones Aviles
金额：
$ 4.6万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10313571
关键词：
Ablation Affect Affinity Alleles Amino Acids Bar Codes Base Sequence Binding Biochemical Biological Biological Assay Biology CRISPR/Cas technology Cancer Biology Cancer Model Cell Line Cell Proliferation Cell model Cell physiology Cells Clinical Clinical Data Competence Computational Technique Data Dependence Development Evaluation Excision Exhibits Frequencies GTPase-Activating Proteins Gene Expression Gene Expression Profile Genetic Genetically Engineered Mouse Goals Growth Guanosine Triphosphate Human Hydrolysis Immunohistochemistry In Vitro Individual KRAS2 gene Lead Maintenance Malignant Neoplasms Mass Spectrum Analysis Mediating Metabolism Methods Molecular Monitor Monomeric GTP-Binding Proteins Mus Mutate Mutation Oncogenic Oncoproteins Output Pancreatic Ductal Adenocarcinoma Pathway interactions Patient-Focused Outcomes Patients Pharmacology Phenotype Phosphoproteins Point Mutation Population Prevalence Prognosis Property Proteins Proto-Oncogenes Reporting Research Scientist Series Signal Pathway Signal Transduction Signaling Molecule Spottings Supervision System Testing Therapeutic Therapeutic Intervention Tissues Training Transplantation Treatment outcome Tumor Cell Line Variant Western Blotting Work base cancer cell cancer type career cell behavior comparative experimental study fitness improved in vitro Assay in vivo inhibitor/antagonist insight migration mutant pancreatic ductal adenocarcinoma cell pancreatic ductal adenocarcinoma model patient derived xenograft model patient response phosphoproteomics pre-clinical prevent response subcutaneous therapeutic target transcriptome sequencing treatment response tumor tumor initiation tumorigenesis tumorigenic

项目摘要

Project Summary KRAS is the most frequently mutated proto-oncogene in human cancer and encodes a small GTPase that regulates multiple cellular processes such as cell proliferation, metabolism, migration, and survival. Point mutations in amino acids G12, G13 and Q61 prevent KRAS inactivation by regulatory GTPase-activating proteins and facilitate tumorigenesis. Although the frequency of specific KRAS mutant variants differ by cancer type, the mechanistic basis for this observation is unknown. It has been postulated that specific mutants induce a “sweet spot” of signaling alterations to induce a cell state optimized for tumor development and maintenance in specific tissues. In support of this hypothesis, KRAS mutants exhibit different biochemical properties in GTP hydrolysis rates and binding affinity to downstream effectors, supporting divergence in their activation of signaling networks. Furthermore, preclinical and clinical data revealed allele-specific differences in tumor initiation capacity and patient prognosis in pancreatic ductal adenocarcinoma (PDAC), suggesting that divergent signaling output could lead to altered phenotypic properties. A systematic and comprehensive evaluation of allele-specific signaling networks would be valuable to better understand KRAS diversity and reveal variant-specific dependencies. The overarching objective of this proposal is to understand KRAS mutant-specific differences in signaling and how these alter cellular fitness. Our preliminary data revealed differential engagement of global signaling networks and canonical amongst KRAS mutants. Therefore, our central hypothesis is that the biochemical differences between KRAS variants result in differential signaling pathway engagement, affecting cellular behavior, tumorigenic properties, and response to therapy. To test this hypothesis, I will re-express a large panel of KRAS mutants observed in human cancer in our recently generated isogenic KRAS deficient PDAC cell lines to dissect KRAS variant-specific differences in signaling networks using data-independent acquisition mass spectroscopy (DIA-MS). Experiments proposed in Aim 1 will investigate differences in signaling pathway activation and dependency of KRAS mutants and validate them in genetically engineered mouse models, PDX models, and human tumor biospecimens. Aim 2 will explore the cellular fitness of these mutants combining in vitro and in vivo competitive assays. Collectively, these data will provide fundamental insights into the biology of KRAS mutants that could potentially explain differences in clinical prevalence and response to therapy. More broadly, this work could inform new allele-specific therapeutic strategies for PDAC and other KRAS mutant cancers. Finally, this highly interdisciplinary and collaborative effort will train me in the application of cutting-edge molecular, biochemical, and computational techniques to facilitate my career goal of becoming an independent scientist in cancer biology.

项目概要 KRAS 是人类癌症中最常突变的原癌基因，编码一种小型 GTP 酶，调节多种细胞过程，如细胞增殖、代谢、迁移和存活。氨基酸 G12、G13 和 Q61 的突变可通过调节 GTP 酶激活蛋白防止 KRAS 失活尽管特定 KRAS 突变体的频率因癌症类型而异，但这一观察结果的机制基础尚不清楚，推测特定突变体会诱导“甜味”。信号改变的“点”，以诱导针对特定肿瘤发展和维持而优化的细胞状态为了支持这一假设，KRAS 突变体在 GTP 水解中表现出不同的生化特性。速率和与下游效应器的结合亲和力，支持其信号网络激活的差异。此外，临床前和临床数据揭示了肿瘤起始能力和等位基因特异性差异。胰腺导管腺癌 (PDAC) 患者的预后，表明不同的信号输出可能导致表型特性改变的等位基因特异性信号传导的系统和全面的评估。网络对于更好地理解 KRAS 多样性和揭示特定变体的依赖关系非常有价值。该提案的首要目标是了解 KRAS 突变体在信号传导方面的特异性差异以及如何这些改变了细胞的适应性。我们的初步数据揭示了全球信号网络的差异参与。因此，我们的中心假设是生化差异。 KRAS 变异之间导致不同的信号通路参与，影响细胞行为，为了验证这一假设，我将重新表达一大组 KRAS。在我们最近生成的同基因 KRAS 缺陷 PDAC 细胞系中观察到的人类癌症突变体使用数据无关的采集质谱分析信号网络中 KRAS 变体特异性差异 (DIA-MS) 目标 1 中提出的实验将研究信号通路激活和信号通路的差异。 KRAS 突变体的依赖性，并在基因工程小鼠模型、PDX 模型和中验证它们目标 2 将结合体外和体内研究这些突变体的细胞适应性。总的来说，这些数据将为 KRAS 突变体的生物学提供基本见解。更广泛地说，这可能解释了临床患病率和治疗反应的差异。可以为 PDAC 和其他 KRAS 突变癌症提供新的等位基因特异性治疗策略。高度跨学科和协作的努力将训练我应用尖端分子、生物化学和计算技术促进我成为一名独立科学家的职业目标癌症生物学。