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是人类癌症中最常突变的原癌基因，并编码一种小的GT3， 调节多种细胞过程，如细胞增殖、代谢、迁移和存活。点 氨基酸G12、G13和Q61的突变通过调节性GTP酶激活蛋白阻止KRAS失活 并促进肿瘤发生。尽管特定KRAS突变变体的频率因癌症类型而异，但 这种观察的机制基础是未知的。据推测，特定的突变体诱导了一种“甜味”。 点”的信号转导改变，以诱导细胞状态优化肿瘤的发展和维持，在特定的 组织中为了支持这一假设，KRAS突变体在GTP水解中表现出不同的生物化学性质 速率和对下游效应物的结合亲和力，支持它们激活信号网络的分歧。 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， 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。等位基因特异性信号传导的系统和综合评价 网络将是有价值的，以更好地了解KRAS的多样性和揭示变异的具体依赖性。的 本提案的首要目标是了解KRAS music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 和典型的KRAS突变体。因此，我们的核心假设是， KRAS变体之间的差异导致差异信号传导途径参与，影响细胞行为， 致瘤特性和对治疗的反应。为了验证这一假设，我将重新表达一个大的KRAS面板， 在我们最近产生的同基因KRAS缺陷PDAC细胞系中观察到的人类癌症突变体， 使用数据独立采集质谱法研究信号网络中的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 人类肿瘤生物标本。目的二是结合体内外实验研究这些突变体的细胞适应性 竞争性测定。总的来说，这些数据将为KRAS突变体的生物学提供基本的见解 这可能解释了临床患病率和治疗反应的差异。更广泛地说，这项工作 可以为PDAC和其他KRAS突变癌症提供新的等位基因特异性治疗策略。最后 高度跨学科和协作的努力将训练我在应用尖端分子， 生物化学和计算技术，以促进我的职业目标，成为一个独立的科学家， 癌症生物学