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.

项目摘要/摘要 RAS是人类癌症中最普遍的癌基因，并且在侵略性癌症中不成比例地存在，例如肺，胰腺和结肠腺癌，它们是导致癌症相关死亡原因的原因我们。不幸的是，靶向RA的尝试在很大程度上失败了，因此细胞毒性化疗仍然是 RAS驱动肿瘤的护理标准。因此，有必要确定RAS驱动的漏洞可以通过新颖的定向疗法靶向的肿瘤。 RAS突变肿瘤重新糖酵解以转移从线粒体氧化磷酸化并进入糖酵解分流途径的葡萄糖 - 碳碳碳碳碳纤维孔核苷酸，氨基酸和减少相同的生物合成以维持增殖。一肿瘤将葡萄糖 - 碳碳的分流到生物合成过程中的机制是通过丙酮酸激酶（PKM2）的M2同工型的表达，因为其动态酶促活性允许恶性细胞调节糖酵解通量。令人惊讶的是，我们的实验室最近将PKM2识别为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，以减少 PKM2多聚体的稳定性并抑制其酶促功能。这项奖学金的拟议工作将确定致癌性RAS使用的精确分子机制，以破坏PKM2四聚体稳定性并测试RAS对PKM2的抑制是否会改变Ras突变肿瘤细胞中的中央碳代谢。到了解介导的抑制，PKM2结构域和关键残基的分子机制与RAS的相互作用将通过突变分析和结合测定确定。鉴于 PKM2功能的多聚化，致癌Ras蛋白对PKM2多聚体稳定性和将使用蔗糖速度梯度来测量形成。 RAS对PKM2多聚化的影响将通过分析pkm2多聚体轮廓在体内验证结直肠腺癌细胞系。我们假设通过致癌RAS重新布线抑制PKM2 糖酵解将葡萄糖 - 碳纤维转向大分子的生物合成。为了检验这一假设，旨在表达野生型PKM2或PKM2突变体的细胞系的中央碳代谢通过将稳定的同位素跟踪与液相色谱和质量耦合，对RAS的亲和力将测量光谱法。我们假设由RAS驱动的肿瘤细胞依赖于PKM2的抑制来维持生物合成持续恶性增生的要求，因此代表了一种代谢脆弱性以治疗为目标。为了测试这一点，细胞活力和一群致癌和野生型的增殖含有RAS的结直肠腺癌细胞系将根据药理刺激进行测量 PKM2。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，并评估了一种新型的针对RAS驱动肿瘤的定向治疗。