Evaluation of BRAF-inhibitor induced alterations in glucose metabolism

描述(申请人提供)：每年约有80,000名美国人被诊断出患有黑色素瘤，导致超过9,000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替换。2011年，FDA批准了药物vemurafenib，一种选择性的BRAF丝氨酸/苏氨酸激酶抑制剂，基于III期试验，显示BRAF突变的晚期黑色素瘤患者的中位无进展生存益处为5.3个月。FDG-PET使用摄取放射性葡萄糖类似物作为代谢活动的代用品，由于肿瘤相对于正常组织葡萄糖代谢高度升高，因此被用于临床对黑色素瘤进行成像。虽然大多数患者在开始治疗的两周内PET阳性率显著下降，但在40%的患者中，尽管生存受益，但观察到的最大肿瘤缩小率不到30%。此外，在这些患者中的大多数，肿瘤复发发生在2到18个月之间，伴随着PET阳性的相应增加，意味着肿瘤对该药物的耐药性。我们的初步数据表明，在维莫拉非尼存在下生长的黑色素瘤细胞表现出葡萄糖摄取和增殖减少，与己糖激酶活性显著降低相关。基于这些观察，我们假设维莫拉非尼对PET阳性和无进展生存率的影响部分是由于维莫拉非尼诱导的葡萄糖代谢下降。具体目标：该提案由两个具体目标组成。第一个目标是量化维莫拉非尼在大量BRAF突变的敏感和耐药黑色素瘤细胞系中引起的葡萄糖摄取减少。我们将使用基于葡萄糖类似物的荧光流式细胞术，以及通过~3H-O-甲基-D-葡萄糖摄取实验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来从功能上评估单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。第二个目标将集中于进一步探索初步数据，确定己糖激酶生物学变化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、质谱学、Western blotting、RNA干扰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、异构体与VDAC线粒体膜通道以及其他蛋白质结合的变化，作为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拉非尼的代谢效应，还可能提供关于FDG-PET成像在BRAF抑制背景下的翻译相关性的有价值的信息。在支持代谢变化的机制方面的新发现也可能提出旨在克服维莫拉非尼耐药性的新的基于代谢的联合疗法。

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.