Inhibition of Autophagy Enhances Anti-Angiogenic Efficacy in Intracranial Glioma

描述(申请人提供)：恶性胶质瘤是一种高度侵袭性、侵袭性和血管的肿瘤，其临床预后仍然非常差。胶质瘤是高度血管性的，但目前的抗血管生成治疗(AAT)只能产生短暂的临床益处。有证据表明，胶质瘤通过激活其他血管生成途径和增加侵袭力来适应AAT。我们假设AAT可以与分子靶向(AAT MT)相结合，以破坏使肿瘤能够单独存活的适应性机制。一个关键的靶点是自噬，这是一种降解途径，可能使肿瘤细胞在低氧/营养应激期间存活。自噬可以防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相联系，自噬抑制可能导致细胞死亡。我们已经开发出一种全面的双推注核磁共振成像方法，使我们能够在体内可视化肿瘤的灌注和生长，在患有脑胶质瘤的小鼠中。这种方法首先利用动态对比增强(DCE)MRI来提供KTRANS的地图，这是血管通透性的指标。然后进行动态磁化率对比磁共振(DSC-MRI)测量脑血流量(CBF)。这种双重推注的方法表明，AAT MT与抗血管生成受体酪氨酸激酶抑制剂头孢拉尼(AstraZeneca)和自噬抑制剂奎纳克林一起治疗可以产生协同的细胞毒效应。奎纳克林是一种阻断自噬功能的抗疟疾药物，具有良好的血脑屏障通透性。初步结果表明，体内有抗血管生成/抗肿瘤的效果，从而延长了生存期。具体目标1将表征在正常和缺氧葡萄糖缺乏条件下，小鼠(4C8)和人(U87)胶质瘤细胞对头孢拉尼和奎纳克林单独和联合使用的剂量依赖性反应。这些实验将检验如下假设：a)低氧/营养应激在头孢拉尼和奎纳克林之间的协同作用中发挥作用；b)在低氧/营养应激条件下，头孢拉尼诱导自噬；以及c)通过奎纳克林联合晚期自噬抑制有效地诱导自噬小体积累和肿瘤细胞死亡。具体目的2采用小鼠脑内4C8和U87恶性胶质瘤模型，研究头孢拉尼和奎纳克林单独及联合治疗的体内效应。这些实验验证了这样的假设，即联合使用这两种药物会产生协同的抗血管/抗肿瘤治疗效果。DCE/DSC双推注MRI将用于评估肿瘤生长和血管生成。免疫组织化学将用于体外，以量化肿瘤细胞的凋亡、坏死、自噬、缺氧和微血管密度。这两个特定的目标都将使用细胞和组织裂解物的免疫印迹来量化细胞标记并研究潜在的机制。这些初步研究需要证明对AAT MT治疗恶性胶质瘤的药效和机制进行更详细的研究是必要的，这一点在R01型应用中定义。 公共卫生相关性：恶性胶质瘤是一种高度侵袭性、侵袭性和血管性的肿瘤，其预后仍然非常差，迫切需要新的治疗方法。我们将评估一种新的联合治疗策略在脑内恶性胶质瘤小鼠模型中的疗效，同时也将探讨潜在的机制。这可能会导致对恶性胶质瘤的更好治疗和理解。

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.