Fatty Acid Synthase and lipophagy as therapeutic targets of glioblastoma

自从发现奥利司他介导的作用以来，阻断从头脂肪酸合成被认为是一种抗癌策略。最近的研究表明，包括GBM和脑转移瘤在内的脑肿瘤严重依赖于通过脂肪酸合成酶（FATCH）的从头脂肪酸合成。一种新的Festival抑制剂TVB-2640（Denifanstat）最近进入了胶质母细胞瘤（GBM）的3期临床试验。它现在可用于临床前研究，并克服了以前的药代动力学和毒性限制。但肿瘤细胞可以通过对脂滴的再利用来逃避脂质缺乏。因此，本项目将研究TVB-2640在体外和体内单独使用以及与氯喹联合使用的抗GBM疗效，并确定GBM敏感性的潜在分子决定因素。分子研究解决了高至低敏感性GBM细胞中的脂质组学和代谢组学模式、线粒体呼吸、细胞活力和自噬途径。我们的初步数据显示，TVB-2640和自噬抑制剂氯喹（CQ）具有很强的协同作用。因此，该项目应专门研究肿瘤细胞重新利用脂滴（LD）中脂质的能力，作为假定的逃逸机制。为了解决噬脂性，候选LD靶向蛋白（例如spartin）和自噬蛋白（ATG 5/7）的敲低/敲除细胞系以及与TVB-2640 + CQ的共同治疗应在GBM细胞系和器官型脑切片培养物中进行测试。此外，我们研究了TVB-2640单一疗法和TVB-2640+CQ共同疗法（相对于CQ单药，相对于安慰剂，4组）在小鼠的原位GBM异种移植物和同基因模型中的体内治疗功效。读数应为生存率、肿瘤生长、组织学、认知行为和最终脂质组学和代谢组学模式。抑制肿瘤导致肿瘤饥饿状态。为了揭示脂质缺乏如何影响临床GBM，应从同基因和异种移植小鼠GBM以及参加ERGO 3试验的患者的临床GBM样品中获得脂质组学和代谢组学数据以及自体/脂肪吞噬的组织学（与Dept.神经肿瘤学，FFM），解决了禁食对增强抗GBM治疗的影响。我们计划建立质谱成像（MSI），用于显示GBM切片和周围脑组织的脂质梯度。这些研究将共同揭示FASN抑制作用在GBM治疗中的假定用途。

Blocking de novo fatty acid synthesis has been considered as an anti-cancer strategy since the discovery of orlistat-mediated effects. Recent work shows that brain tumours including GBM and brain metastases heavily rely on de novo fatty acid synthesis via fatty acid synthase (FASN). A novel FASN inhibitor, TVB-2640 (Denifanstat) has recently entered a phase-3 clinical trial for glioblastoma (GBM). It is now commercially available for preclinical research and has overcome previous pharmacokinetic and toxicity limitations. But tumour cells may escape from lipid shortage via reutilization from lipid droplets. Therefore, the present project shall investigate anti-GBM efficacy of TVB-2640 in vitro and in vivo alone and in combination with chloroquine and define the underlying molecular determinants of GBM susceptibilities. The molecular studies address lipidomic and metabolomic patterns, mitochondrial respiration, cell viability and autophagy pathways in high-to-low sensitive GBM cells. Our preliminary data have revealed strong synergistic effects of TVB-2640 and the autophagy inhibitor, cholorquine (CQ). Hence, the project shall specifically address the ability of the tumour cells to reutilize lipids from lipid droplets (LD) as putative escape mechanism. To address lipophagy, knockdown/knockout cell lines of candidate LD targeting proteins (e.g. spartin) and of autophagy proteins (ATG5/7), and co-therapy with TVB-2640 + CQ shall be tested in GBM cell lines and in organotypic brain slice cultures. Furthermore, we investigate the in vivo therapeutic efficacy of TVB-2640 monotherapy and TVB-2640+CQ co-therapy (versus CQ mono, versus placebo, 4 groups) in orthotopic GBM xenograft and syngeneic models in mice. Readouts shall be survival, tumour growth, histology, cognitive behaviour and final lipidomic and metabolomic patterns. FASN inhibition causes a state of tumour starvation. To reveal how lipid shortage affects clinical GBM, lipidomic and metabolomic data and histology of auto/lipophagy shall be obtained from syngeneic and xenotransplant mouse GBM and from clinical GBM samples of patients who participated in the ERGO3 trial (collaboration with Dept. of Neurooncology, FFM) that addresses the impact of fasting to enhance anti-GBM treatments. We plan to establish mass spectrometry imaging (MSI) for visualization of lipid gradients across GBM sections and surrounding brain tissue. Together, the studies shall unravel the putative usage of FASN inhibition for GBM treatment.