Evaluation of BRAF-inhibitor induced alterations in glucose metabolism

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，约80%的人在MAPK途径信号蛋白BRAF中含有激活突变;最常见的是V600 E取代。2011年，FDA批准了药物vemurafenib，一种选择性BRAF Ser/Thr激酶抑制剂，基于III期试验显示晚期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性的代表，由于肿瘤相对于正常组织的葡萄糖代谢高度升高，FDG-PET被用于在临床中对黑色素瘤进行成像。尽管大多数患者在开始治疗的两周内显示PET阳性显著降低，但在这些患者中的40%中，尽管有生存益处，但观察到小于30%的最大肿瘤缩小。此外，在大多数这些患者中，肿瘤复发发生在2至18个月之间，PET阳性率相应增加，表明肿瘤对药物具有耐药性。我们的初步数据表明，在维罗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著降低相关。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是由于维罗非尼诱导的葡萄糖代谢降低。具体目标：本提案包括两个具体目标。第一个目的是集中在大量BRAF突变敏感和耐药黑色素瘤细胞系中定量维罗非尼诱导的葡萄糖摄取减少。我们将使用荧光葡萄糖类似物为基础的流式细胞术，以及分析跨膜葡萄糖转运的变化，使用3 H-O-甲基-D-葡萄糖摄取试验，在己糖激酶活性和定位的变化，和RNAi功能评估个别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。第二个目标将集中在进一步探索初步数据，确定改变己糖激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，蛋白质印迹，RNAi和共聚焦显微镜来评估MAPK，AKT和GSK 3？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结合的改变，作为活性降低的潜在原因。相关性：该项目不仅将评估vemurafenib目前尚未探索的代谢作用，还可能产生关于BRAF抑制背景下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提示新的基于代谢的联合疗法，旨在克服vemurafenib耐药性。

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.