Characterization of the PKM2-RAS Interaction as a Novel Metabolic Vulnerability of RAS-driven Tumors

PKM2-RAS 相互作用作为 RAS 驱动肿瘤的新型代谢脆弱性的表征

基本信息

批准号：
10361189
负责人：
Juan Andres Kochen Rossi
金额：
$ 3.85万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
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：
2021-04-01 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10361189
关键词：
Affinity Affinity Chromatography Amino Acids Anabolism Binding Biochemical Biological Assay Cancer Etiology Carbon Cell Line Cell Proliferation Cell membrane Cells Cellular Metabolic Process Centrifugation Cessation of life Characteristics Citric Acid Cycle Clinical Trials Co-Immunoprecipitations Colon Adenocarcinoma Colorectal Adenocarcinoma Coupled Coupling Cytotoxic Chemotherapy Data Dependence Engineering Fellowship Flow Cytometry Gel Chromatography Glucose Glycolysis Guanosine Triphosphate Human Impairment KRAS2 gene Lung Adenocarcinoma Malignant - descriptor Malignant Neoplasms Mass Spectrum Analysis Measures Mediating Membrane Proteins Metabolic Metabolism Methods Mitochondria Molecular Monomeric GTP-Binding Proteins Mutate Mutation Analysis Nucleotide Biosynthesis Oncogenes Oncogenic Oxidative Phosphorylation Pancreatic Adenocarcinoma Pathway interactions Pentosephosphate Pathway Pharmacology Phosphoenolpyruvate Pre-Clinical Model Prevalence Process Program Sustainability Protein Isoforms Pyruvate Pyruvate Kinase RAS genes RAS inhibition Radiolabeled Reaction Receptor Signaling Recombinants Role Serine Structure Sucrose System Testing Therapeutic Work base cancer cell cell growth cellular engineering dimer experimental study extracellular in vivo inorganic phosphate liquid chromatography mass spectrometry live cell imaging macromolecule mutant neoplastic cell novel programs protein crosslink ras Proteins response shunt pathway stability testing stable isotope standard of care targeted treatment therapeutic evaluation tumor

项目摘要

Project Summary/Abstract RAS is the most prevalent oncogene in human cancer and is disproportionately present in aggressive cancers, such as lung, pancreas, and colon adenocarcinomas, that are leading causes of cancer-related deaths in the US. Unfortunately, attempts to target RAS have largely failed such that cytotoxic chemotherapy remains the standard of care for RAS-driven tumors. Hence, there exists a need to identify vulnerabilities of RAS-driven tumors that can be targeted by novel directed therapies. RAS-mutant tumors rewire glycolysis in order to divert glucose-carbons away from mitochondrial oxidative phosphorylation and into glycolytic shunt pathways for the biosynthesis of nucleotides, amino acids, and reducing equivalents needed to sustain proliferation. One mechanism by which tumors achieve shunting of glucose-carbons into biosynthetic processes is through expression of the M2 isoform of pyruvate kinase (PKM2), as its dynamic enzymatic activity allows malignant cells to regulate glycolytic flux. Strikingly, our lab recently identified PKM2 as a potential metabolic effector of RAS proteins. Preliminary data suggests that RAS directly binds PKM2 in a GTP-dependent manner that diminishes the stability of PKM2 multimers and inhibits its enzymatic function. The proposed work in this fellowship will determine the precise molecular mechanisms employed by oncogenic RAS to undermine PKM2 tetramer stability and test whether inhibition of PKM2 by RAS alters central carbon metabolism in RAS-mutant tumor cells. To understand the molecular mechanism of inhibition, PKM2 domains and critical residues that mediate the interaction with RAS will be identified by mutational analysis and binding assays. Given the essential role of multimerization for PKM2 function, the effects of oncogenic RAS proteins on PKM2 multimer stability and formation will be measured utilizing sucrose velocity gradients. The effects of RAS on PKM2 multimerization will be validated in vivo by analysis of PKM2 multimer profiles across a panel of oncogenic and wildtype RAS-bearing colorectal adenocarcinoma cell lines. We hypothesize that inhibition of PKM2 by oncogenic RAS rewires glycolysis to divert glucose-carbons towards the biosynthesis of macromolecules. To test this hypothesis, the central carbon metabolism of cell lines engineered to express wildtype PKM2 or PKM2 mutants with diminished affinity to RAS will be measured by coupling stable isotope tracing with liquid-chromatography and mass spectrometry. We hypothesize that RAS-driven tumor cells rely on inhibition of PKM2 to sustain the biosynthetic requirements of sustained malignant proliferation and, therefore, represents a metabolic vulnerability that can be targeted therapeutically. To test this, cellular viability and proliferation of a panel of oncogenic and wildtype RAS-bearing colorectal adenocarcinoma cell lines will be measured in response to pharmacological stimulation of PKM2. Collectively, this work will determine the molecular mechanisms of PKM2 inhibition by RAS, elucidate the metabolic consequences of the interaction, and evaluate a novel directed therapy for RAS-driven tumors.

项目概要/摘要 RAS 是人类癌症中最常见的癌基因，并且不成比例地存在于侵袭性癌症中，例如肺癌、胰腺癌和结肠腺癌，它们是导致癌症相关死亡的主要原因我们。不幸的是，针对 RAS 的尝试基本上失败了，因此细胞毒性化疗仍然是最有效的治疗方法。 RAS 驱动肿瘤的护理标准。因此，需要识别 RAS 驱动的漏洞可以通过新型定向疗法靶向的肿瘤。 RAS突变肿瘤重新连接糖酵解以转移葡萄糖-碳远离线粒体氧化磷酸化并进入糖酵解分流途径维持增殖所需的核苷酸、氨基酸和还原当量的生物合成。一肿瘤将葡萄糖-碳分流至生物合成过程的机制是通过丙酮酸激酶 (PKM2) 的 M2 亚型的表达，因为其动态酶活性允许恶性细胞调节糖酵解通量。引人注目的是，我们的实验室最近发现 PKM2 是 RAS 的潜在代谢效应子蛋白质。初步数据表明 RAS 以 GTP 依赖性方式直接结合 PKM2，从而减少 PKM2 多聚体的稳定性并抑制其酶功能。这项研究金的拟议工作将确定致癌 RAS 破坏 PKM2 四聚体稳定性的精确分子机制并测试 RAS 抑制 PKM2 是否会改变 RAS 突变肿瘤细胞的中心碳代谢。到了解抑制的分子机制、PKM2 结构域和介导的关键残基与 RAS 的相互作用将通过突变分析和结合测定来鉴定。鉴于其重要作用 PKM2 功能的多聚化、致癌 RAS 蛋白对 PKM2 多聚体稳定性的影响以及将利用蔗糖速度梯度来测量形成。 RAS 对 PKM2 多聚化的影响通过分析一组致癌性和野生型 RAS 的 PKM2 多聚体概况进行体内验证结直肠腺癌细胞系。我们假设致癌 RAS 重新布线对 PKM2 的抑制糖酵解将葡萄糖碳转向大分子的生物合成。为了检验这个假设，经改造表达野生型 PKM2 或 PKM2 突变体的细胞系的中心碳代谢减少与 RAS 的亲和力将通过稳定同位素示踪与液相色谱和质量联用来测量光谱测定法。我们假设 RAS 驱动的肿瘤细胞依靠抑制 PKM2 来维持生物合成持续恶性增殖的需要，因此代表了一种代谢脆弱性，可以成为治疗的目标。为了测试这一点，一组致癌和野生型的细胞活力和增殖将测量携带 RAS 的结直肠腺癌细胞系对药物刺激的反应 PKM2 的。总的来说，这项工作将确定 RAS 抑制 PKM2 的分子机制，阐明相互作用的代谢后果，并评估针对 RAS 驱动肿瘤的新型定向疗法。