Evaluation of BRAF-inhibitor induced alterations in glucose metabolism

BRAF 抑制剂诱导的葡萄糖代谢改变的评估

• 批准号: 8595089
• 负责人: Nicholas Theodosakis
• 金额: $ 4.72万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8595089
• 关键词: American; BRAF gene; Behavior; Benign; Binding; Biological Assay; Biology; Catabolism; Cell Death; Cell Line; Cell Proliferation; Cell Survival; Cells; Cessation of life; Characteristics; Citric Acid Cycle; Clinic; Clinical; Combined Modality Therapy; Confocal Microscopy; Cultured Cells; Data; Deoxyglucose; Development; Diagnosis; Drug resistance; Evaluation; FDA approved; Flow Cytometry; Generations; Genes; Glucose; Glucose Transporter; Glutamine; Glycogen Synthase Kinases; Glycolysis; Growth; Human; Image; Immunoprecipitation; In Vitro; Individual; Ion Channel; Label; Lead; MAP Kinase Gene; Mass Spectrum Analysis; Measurement; Mediating; Melanoma Cell; Metabolic; Minor; Mitochondrial Membrane Protein; Mutate; Mutation; Normal tissue morphology; Pathway interactions; Patients; Pharmaceutical Preparations; Phase III Clinical Trials; Phosphotransferases; Play; Positron-Emission Tomography; Post-Translational Protein Processing; Process; Production; Progression-Free Survivals; Protein Binding; Protein Isoforms; Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins c-akt; Proxy; RNA Interference; Radioactive; Recurrence; Reducing Agents; Relative (related person); Research; Resistance; Resistance development; Role; SLC2A1 gene; Signal Pathway; Signal Transduction; Signaling Protein; Skin Cancer; Small Interfering RNA; Staging; Testing; Tumor Tissue; VDAC1 gene; Western Blotting; aerobic glycolysis; analog; base; cancer cell; cytotoxicity; deprivation; glucose analog; glucose metabolism; glucose transport; glucose uptake; hexokinase; improved; inhibitor/antagonist; kinase inhibitor; macromolecule; melanoma; mitochondrial membrane; mutant; novel; novel strategies; public health relevance; research study; response; tumor; uptake

DESCRIPTION (provided by applicant): Approximately 80,000 Americans are diagnosed with melanoma each year, leading to over 9,000 deaths. Of these, approximately 80% harbor activating mutations in the MAPK pathway signaling protein BRAF; most commonly a V600E substitution. In 2011, the FDA approved the drug vemurafenib, a selective BRAF Ser/Thr kinase inhibitor, based on phase III trials showing a median progression-free survival benefit of 5.3 months in patients with late-stage BRAF-mutated melanoma. FDG-PET, which uses uptake of a radioactive glucose analog as a proxy for metabolic activity, is used to image melanoma in the clinic due to the tumors' highly elevated glucose metabolism relative to normal tissue. Though the majority of patients show a dramatic decrease in PET positivity within two weeks of beginning therapy, in 40% of these patients, less than 30% maximal tumor shrinkage is observed in spite of the survival benefit. Furthermore, in the majority of these patients, tumor recurrence occurs in between 2 and 18 months, with a corresponding increase in PET positivity, signifying tumor resistance to the drug. Our preliminary data suggest that melanoma cells grown in the presence of vemurafenib show a decrease in glucose uptake and proliferation, associated with a significant decrease in hexokinase activity. Based on these observations, we hypothesize that the effects of vemurafenib on PET positivity and progression-free survival are partly due to vemurafenib-induced decreases in glucose metabolism. Specific aims: This proposal is comprised of two specific aims. The first aim is focused on quantifying the vemurafenib-induced decrease in glucose uptake in a large number of BRAF-mutant sensitive and resistant melanoma cell lines. We will use fluorescent glucose analog-based flow cytometry, as well as analysis of changes in transmembrane glucose transport using a 3H-O-methyl-D-glucose uptake assay, changes in hexokinase activity and localization, and RNAi to functionally evaluate the importance of individual hexokinase and glucose transporter genes. We will also evaluate glutamine as an alternative metabolic substrate through isotopic labeling and flux analysis, as well as glucose and glutamine deprivation experiments. The second aim will focus on further exploring preliminary data identifying altered hexokinase biology as a possible mechanism for the observed decrease in glucose uptake. We will use immunoprecipitation, mass spectrometry, Western blotting, RNAi, and confocal microscopy to evaluate changes in the MAPK, AKT, and GSK3¿ signaling pathways, changes in post-translational modification, and altered binding of isoforms to VDAC mitochondrial membrane channels, as well as other proteins, as potential causes of decreased activity. Relevance: This project will not only evaluate the currently-unexplored metabolic effects of vemurafenib, but may also yield valuable information on the translational correlates of FDG-PET imaging in the setting of BRAF inhibition. New discoveries in the mechanisms underpinning metabolic changes may also suggest new metabolically-based combination therapies aimed at overcoming vemurafenib resistance.

描述（由适用提供）：每年大约有80,000名美国人被诊断出患有黑色素瘤，导致9,000多人死亡。其中，MAPK途径信号传导蛋白BRAF中约有80％的携带激活突变；最常见的是V600E替代。 2011年，FDA基于III期试验，批准了一种选择性BRAF SER/THR激酶抑制剂的药物vemurafenib，该试验表明，晚期BRAF突破性黑色素瘤患者的中位无进展生存率为5.3个月。由于肿瘤相对于正常组织的高度升高的葡萄糖代谢，因此使用放射性葡萄糖类似物作为代谢活性的代理的FDG-PET用于临床中的黑色素瘤。尽管大多数患者在开始治疗后的两周内显示出宠物积极性的急剧下降，但在40％的患者中，尽管生存益处，但观察到最大肿瘤收缩率不到30％。此外，在大多数患者中，肿瘤复发发生在2到18个月之间，宠物电位相应增加，表明肿瘤对药物的耐药性。我们的初步数据表明，黑色素瘤细胞在维美富尼存在下生长显示葡萄糖摄取和增殖的降低，与己糖激酶活性的显着降低有关。基于这些观察结果，我们假设vemurafenib对宠物势和无进展生存的影响部分是由于葡萄糖代谢中武马替尼引起的下降。具体目的：该提案由两个具体目的组成。第一个目的是量化大量BRAF突变敏感和抗性黑色素瘤细胞系中葡萄糖诱导的葡萄糖摄取减少的降低。我们将使用3H-O-甲基-D-葡萄糖摄取测定法，己糖激酶活性和定位的变化以及RNAI在功能上评估单个己糖酶和葡萄糖转运蛋白的重要性。我们还将通过同位素标记和通量分析以及葡萄糖和葡萄糖剥夺实验评估谷氨酰胺作为替代代谢底物。第二个目标将集中于进一步探索初步数据，以识别改变的己糖酶生物学，作为观察到的葡萄糖摄取减少的可能机制。我们将使用免疫沉淀，质谱法，蛋白质印迹，RNAi和共聚焦显微镜来评估MAPK，AKT和GSK3…信号通路，翻译后修饰的变化，以及等质组合型与VDAC MITochranial Membrane Membrane niments portives and pottions的变化，以及其他prote蛋白以及其他protee蛋白以及其他partistion。相关性：该项目不仅将评估vemurafenib的当前未塑造代谢作用，而且还可以在BRAF抑制作用的环境中就FDG-PET成像的转化相关性产生有价值的信息。基于代谢变化的机制中的新发现也可能表明旨在克服vemurafenib抗性的新型组合疗法。