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

项目摘要 KRAS是人类癌症中突变最频繁的原癌基因，它编码一个小的GTP酶， 调节多种细胞过程，如细胞增殖、新陈代谢、迁移和存活。点 氨基酸G12、G13和Q61的突变通过调节GTP酶激活蛋白防止KRAS失活 并促进肿瘤的发生。虽然特定的KRAS突变变种的频率因癌症类型而异，但 这一观察的机制基础尚不清楚。据推测，特定的突变体会产生一种“甜味” 信号改变的“斑点”，以诱导特定的肿瘤发生和维持的最佳细胞状态 纸巾。支持这一假说的是，KRAS突变体在GTP水解过程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，支持它们激活信号网络的差异。 此外，临床前和临床数据显示，肿瘤起始能力和肿瘤的等位基因特异性差异。 胰腺导管腺癌(PDAC)的患者预后，提示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转导的系统和综合评价 网络对于更好地理解KRAS多样性和揭示特定变种的依赖关系将是有价值的。这个 这项建议的首要目标是了解KRAS突变体特有的信号差异以及如何 这些改变了细胞的健康状况。我们的初步数据显示了全球信令网络的不同参与 在KRAS突变体中具有典范意义。因此，我们的中心假设是生化差异 在KRAS变异体之间导致不同的信号通路参与，影响细胞行为， 致癌特性和对治疗的反应。为了验证这一假设，我将重新表达一大组KRAS 在我们最近产生的等基因KRAS缺陷型PDAC细胞系中观察到的人类癌症突变的解剖 使用独立于数据的获取质谱学在信令网络中的KRAS变体特定差异 (DIA-MS)。目标1中提出的实验将调查信号通路激活和 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。目标2将探索这些突变体在体外和体内的细胞适合性。 竞争性化验。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突变癌症的等位基因特异性治疗策略提供新的信息。最后，这一点 高度跨学科和协作的努力将培训我在尖端分子应用方面的能力， 生物化学和计算技术，以促进我成为一名独立科学家的职业目标 癌症生物学。

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.