Inhibition of Autophagy Enhances Anti-Angiogenic Efficacy in Intracranial Glioma

描述（申请人提供）：恶性胶质瘤是一种侵袭性强、侵袭性强、临床预后极差的血管性肿瘤。胶质瘤是高度血管化的，但目前的抗血管生成治疗（AAT）只能诱导短暂的临床疗效。有证据表明，胶质瘤通过激活替代血管生成途径和增加侵袭来适应AAT。我们假设AAT可以与分子靶向（AAT+MT）结合，破坏使肿瘤能够在AAT单独生存的适应机制。一个关键的目标是自噬，这是一种降解途径，可能使肿瘤细胞在缺氧/营养胁迫下存活。自噬阻止有毒蛋白质和细胞器的积累，并提供能量和生物合成底物。自噬途径与控制细胞死亡的途径有关，自噬抑制可诱导细胞死亡。我们已经开发了一种全面的双丸灌注MRI方法，使我们能够在颅内胶质瘤小鼠体内可视化肿瘤灌注和生长。该方法首先利用动态对比增强（DCE） MRI提供血管通透性指标Ktrans的地图。然后进行动态敏感性对比MRI （DSC-MRI）测量脑血流量（CBF）。这种双丸治疗方法提示AAT+MT联合抗血管生成受体酪氨酸激酶抑制剂Cediranib（阿斯利康）和自噬抑制剂quinacrine具有协同细胞毒作用。喹啉是一种阻断自噬体功能的抗疟疾药物，具有良好的血脑屏障渗透性。初步结果显示抗血管生成/抗肿瘤在体内有效，从而延长生存期。特异性Aim 1将表征小鼠（4C8）和人（U87）胶质瘤细胞在正常和缺氧葡萄糖条件下对Cediranib和quinacrine单独和联合的剂量依赖性体外反应。这些实验将验证以下假设：a)缺氧/营养胁迫在Cediranib和quinacrine之间的协同作用中起作用；b) Cediranib在缺氧/营养胁迫条件下诱导自噬；c)联合晚期自噬抑制可有效诱导自噬体积累和肿瘤细胞死亡。特异性目的2将通过颅内小鼠4C8和U87恶性胶质瘤模型，研究Cediranib和quinacrine单独和联合治疗在体内的效果。这些实验验证了两种药物联合使用可产生协同抗血管/抗肿瘤治疗效果的假设。双剂量DCE/DSC MRI将用于评估肿瘤生长和血管生成。体外免疫组化，定量肿瘤细胞凋亡、坏死、自噬、缺氧和微血管密度。特异目标将使用细胞和组织裂解物的免疫印迹来量化细胞标记物并研究潜在的机制。需要这些初步研究来证明AAT+MT治疗恶性胶质瘤的药物疗效和机制的更详细研究是正确的，恶性胶质瘤定义为r01型应用。

DESCRIPTION (provided by applicant): Malignant gliomas are highly aggressive, invasive, and vascular tumors for which clinical prognosis remains extremely poor. Gliomas are highly vascular, but current anti-angiogenic therapy (AAT) induces only a transitory clinical benefit. Evidence suggests that gliomas adapt to AAT, by activation of alternative angiogenic pathways and increased invasion. We hypothesize that AAT can be combined with molecular targeting (AAT+MT), to disrupt the adaptive mechanisms that enable tumors to survive AAT alone. A key target is autophagy, a degradative pathway that may enable tumor cell survival during hypoxic/nutrient stress. Autophagy prevents toxic protein and organelle accumulation and provides energy and biosynthetic substrates. Autophagic pathways are linked to pathways controlling cell death, and autophagic inhibition may induce cell death. We have developed a comprehensive dual bolus perfusion MRI method that enables us to visualize tumor perfusion and growth in vivo, in mice with intracranial gliomas. This method first utilizes dynamic contrast enhanced (DCE) MRI to provide maps of Ktrans, an index of vascular permeability. Then dynamic susceptibility contrast MRI (DSC-MRI) is done to measure cerebral blood flow (CBF). This dual bolus method suggests that AAT+MT therapy with the anti-angiogenic receptor tyrosine kinase inhibitor Cediranib (AstraZeneca) and the autophagy inhibitor quinacrine can have synergistic cytotoxic effects. Quinacrine is an anti-malarial agent which interrupts autophagosome function and has excellent blood brain barrier permeability. Preliminary results show anti-angiogenic/anti-tumor efficacy in vivo with resulting extended survival. Specific Aim 1 will characterize the dose-dependent in vitro responses of mouse (4C8) and human (U87) glioma cells to Cediranib and quinacrine, singly and in combination, under normal and oxygen glucose deprived conditions. These experiments will test hypotheses that a) hypoxic/nutrient stress plays a role in the synergism between Cediranib and quinacrine; b) induction of autophagy occurs with Cediranib under conditions of hypoxic/nutrient stress; and c) combined late-stage autophagic inhibition via quinacrine effectively induces autophagosome accumulation and tumor cell death. Specific Aim 2 will investigate the effects of single and combined Cediranib and quinacrine treatment in vivo, employing the intracranial mouse 4C8 and U87 malignant glioma models. These experiments test the hypothesis that combined administration of the two agents results in a synergistic anti-vascular/anti-tumor therapeutic efficacy. Dual bolus DCE/DSC MRI will be used to assess tumor growth and angiogenesis. Immunohistochemistry will be used ex vivo, to quantify tumor cell apoptosis, necrosis, autophagy, hypoxia and microvessel density. Both Specific Aims will employ immunoblot with cell and tissue lysates to quantify cellular markers and investigate underlying mechanisms. These preliminary studies are required to justify more detailed studies of the drug efficacy and the mechanisms of AAT+MT in treating malignant glioma, defined in a R01-type application. PUBLIC HEALTH RELEVANCE: Malignant gliomas are a highly aggressive, invasive, and vascular tumor for which prognosis remains extremely poor and for which new therapeutic modalities are urgently needed. We will evaluate efficacy of a novel combination treatment strategy in intracranial mouse models of malignant glioma, while also investigating underlying mechanisms. It could lead to better treatment for, and understanding of, malignant glioma.