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

• 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
• 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

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.

项目摘要 KRAS是人类癌症中最常见的原始癌症，并编码一个小的GTPase，该癌症是 调节多个细胞过程，例如细胞增殖，代谢，迁移和存活。观点 氨基酸G12，G13和Q61中的突变通过调节GTPase激活蛋白来防止KRAS灭活 尽管特异性KRAS突变体变体的频率与癌症类型不同，但 该观察结果的机械基础尚不清楚。有人认为特定的突变体会诱导“甜 信号改变的斑点”，以诱导针对特定肿瘤发育和维持优化的细胞状态 组织。为了支持这一假设，KRAS突变体在GTP水解中暴露了不同的生化特性 速率和对下游效应的结合亲和力，支持发散信号网络的差异。 此外，临床前和临床数据揭示了等位基因特异性肿瘤起始能力的差异和 胰腺导管腺癌（PDAC）的患者预后，表明发散的信号传导输出可以 导致表型特性改变。对等位基因特异性信号的系统和全面评估 网络对于更好地理解KRAS多样性并揭示特定于变异的依赖性将是有价值的。这 该提案的总体目标是了解KRAS突变体特异性的信号传导差异以及如何 这些改变了细胞适应性。我们的初步数据揭示了全球信号网络的不同参与 和Kras突变体中的规范。因此，我们的核心假设是生化差异 KRAS变体之间会导致差异信号通路参与度，影响细胞行为， 肿瘤性特性和对治疗的反应。为了检验这一假设，我将重新表达大型KRAS面板 在我们最近产生的同源性KRAS缺乏PDAC细胞系中，在人类癌症中观察到的突变体以剖析 KRAS变体特异性差异在信号网络中使用数据无关的采集质谱法 （DIA-MS）。 AIM 1中提出的实验将研究信号通路激活和 KRAS突变体的依赖性并在一般设计的鼠标模型，PDX模型和 人类肿瘤生物测量。 AIM 2将探索这些突变体在体外和体内结合的细胞适应性 竞争测定法。总的来说，这些数据将提供对KRAS突变体生物学的基本见解 这可能可以解释临床患病率和对治疗反应的差异。更广泛地说，这项工作 可以为PDAC和其他KRAS突变体癌症提供新的等位基因特异性治疗策略。最后，这个 高度跨学科和协作的努力将训练我在尖端分子的应用中， 生化和计算技术，以促进我成为一名独立科学家的职业目标 癌症生物学。