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（由申请人提供）：每年约有 80,000 名美国人被诊断患有黑色素瘤，导致 9,000 多人死亡。其中，大约 80% 的 MAPK 通路信号蛋白 BRAF 存在激活突变；最常见的是 V600E 替代品。 2011 年，FDA 批准了药物维莫非尼（vemurafenib），这是一种选择性 BRAF Ser/Thr 激酶抑制剂，基于 III 期试验，该试验显示晚期 BRAF 突变黑色素瘤患者的中位无进展生存期为 5.3 个月。 FDG-PET 使用放射性葡萄糖类似物的摄取作为代谢活动的代表，由于肿瘤的葡萄糖代谢相对于正常组织高度升高，因此在临床上用于对黑色素瘤进行成像。尽管大多数患者在开始治疗后两周内 PET 阳性率显着下降，但在 40% 的患者中，尽管生存获益，但观察到最大肿瘤缩小不到 30%。此外，大多数患者的肿瘤复发发生在 2 至 18 个月内，PET 阳性率相应增加，表明肿瘤对药物产生耐药性。我们的初步数据表明，在维莫非尼存在下生长的黑色素瘤细胞显示出葡萄糖摄取和增殖的减少，与己糖激酶活性的显着降低相关。基于这些观察结果，我们假设维罗非尼对 PET 阳性和无进展生存期的影响部分归因于维罗非尼诱导的葡萄糖代谢降低。具体目标：该提案由两个具体目标组成。第一个目标是量化大量 BRAF 突变敏感和耐药黑色素瘤细胞系中维莫非尼诱导的葡萄糖摄取减少。我们将使用基于荧光葡萄糖类似物的流式细胞术，以及使用 3H-O-甲基-D-葡萄糖摄取测定分析跨膜葡萄糖转运的变化、己糖激酶活性和定位的变化以及 RNAi，以在功能上评估各个己糖激酶和葡萄糖转运蛋白基因的重要性。我们还将通过同位素标记和通量分析以及葡萄糖和谷氨酰胺剥夺实验来评估谷氨酰胺作为替代代谢底物的作用。第二个目标将侧重于进一步探索初步数据，确定己糖激酶生物学的改变是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、蛋白质印迹、RNAi 和共聚焦显微镜来评估 MAPK、AKT 和 GSK3¿ 信号通路的变化、翻译后修饰的变化以及异构体与 VDAC 线粒体膜通道以及其他蛋白质的结合改变，这些都是活性降低的潜在原因。相关性：该项目不仅将评估目前尚未探索的维莫非尼的代谢效应，而且还可能产生关于 BRAF 抑制情况下 FDG-PET 成像的翻译相关性的有价值的信息。代谢变化机制的新发现也可能表明旨在克服维罗非尼耐药性的新的基于代谢的联合疗法。