Re-setting the Endothelial Ceramide Rheostat

描述（由申请人提供）：我们最近的研究探索了一种生物化学机制，通过该机制，抗血管生成增强了微血管功能障碍的鞘脂信号传导，以介导通过抗肿瘤治疗（包括一些抗癌药物和高单剂量放疗（SDRT））治愈肿瘤。本申请中提出的研究是基于内皮比任何哺乳动物细胞产生更多的酸性鞘磷脂酶（ASMase）并使用它来发出细胞凋亡信号的观察。我们发表的数据显示，ASMase快速易位至内皮细胞质膜的外部小叶诱导鞘磷脂水解为其中的促凋亡第二信使神经酰胺，从而触发凋亡。我们的初步数据表明，微血管功能障碍的级联增强，其中包括急性灌注缺陷（通过动态MRI和Hoechst 33342染料外渗测量）和缺氧（通过EPR血氧测定法测量），并且这种血管功能障碍抑制肿瘤干细胞中的DNA损伤修复，以增强SDRT的治愈。本申请的假设是，神经酰胺介导的细胞凋亡的强度、血管功能障碍的程度和肿瘤治愈的可能性之间存在直接关系。我们建议验证这一机制，并尝试通过药理学或遗传学上调ASMase信号转导来增强肿瘤治愈。具体而言，我们将检查抗血管生成药物的放射增敏是否通过拨高ASMase活化而发生（Aim 1），通过腺病毒asmase基因治疗直接靶向内皮神经酰胺信号传导以特异性地在肿瘤血管系统中过表达ASMase是否会比抗血管生成药物更有效地放射增敏（Aim 2），以及ASMase定向治疗的肿瘤可治愈性是否通过急性血管损伤发生（Aim 3）。因此，我们提出了一种新的病理生理模型抗血管生成放射增敏，其中的神经酰胺信号的强度是不是一个静态的内皮生物学功能，而是动态调节的血管生成因子分泌的肿瘤细胞，是易处理的。认识到内皮神经酰胺变阻器对于SDRT的肿瘤治愈是必需的，表明了提高内皮神经酰胺信号传导强度的治疗潜力。实际上，在重置ASMase活化的条件下组合抗VEGF策略几乎可以立即用于临床。此外，拟议的研究探索了通过使用腺病毒基因疗法直接访问神经酰胺生物学以仅在新生血管生成血管中过表达asmase来对肿瘤治愈产生更大影响的潜力。 公共卫生相关性：内皮神经酰胺变阻器在微血管功能障碍级联的顶部起作用，其通过抗肿瘤治疗介导肿瘤治愈，包括一些抗癌药物和高单剂量放射治疗（SDRT）。酸性鞘磷脂酶（ASMase）介导的第二信使神经酰胺产生的强度决定了急性内皮细胞凋亡的程度，这沉淀了肿瘤内的短暂灌注缺陷。急性缺氧（或再灌注）增强，其与肿瘤干细胞DNA损伤偶联，协调决定肿瘤治愈。本申请的目的是探索使用抗血管生成药物和腺病毒基因治疗载体（称为Ad 5 H2 E-PPE 1（3x）-ASMase）对这种神经酰胺变阻器的药理学和遗传学上调，该载体设计用于在修饰的前内皮素原启动子下游的肿瘤内皮细胞中特异性表达人ASMase。初步临床前数据表明，这种方法对改善SDRT诱导的肿瘤治愈有很大影响。目前使用抗血管生成药物或ASMase基因治疗上调内皮神经酰胺变阻器的临床预测设想其作为SDRT和寡转移性癌症化疗管理的辅助治疗，估计美国每年的人群规模约为900，00例患者。

DESCRIPTION (provided by applicant): Our recent studies explore a biochemical mechanism by which anti-angiogenesis enhances sphingolipid signaling of microvascular dysfunction to mediate tumor cure by anti-neoplastic therapies, including some anti- cancer drugs and high single dose radiotherapy (SDRT). The studies proposed in this application are predicated on the observation that endothelium make more acidic sphingomyelinase (ASMase) than any mammalian cell and uses it to signal apoptosis. Our published data show that rapid ASMase translocation to the external leaflet of the endothelial cell plasma membrane induces sphingomyelin hydrolysis to the pro- apoptotic second messenger ceramide therein, triggering apoptosis. Our preliminary data indicate a cascade of microvascular dysfunction ensues, which includes acute perfusion defects (measured by Dynamic MRI and Hoechst 33342 dye extravasation) and oxygen deprivation (measured by EPR oximetry), and that this vascular dysfunction represses DNA damage repair in tumor stem cells to enhance cure by SDRT. It is the hypothesis of this application that there is a direct relationship between the intensity of ceramide-mediated apoptosis, the extent of vascular dysfunction, and the probability of tumor cure. We propose to validate this mechanism and attempt to enhance tumor cure by pharmacologic or genetic up-regulation of ASMase signaling. Specifically, we will examine whether radiosensitization by anti-angiogenic drugs occurs by dialing up ASMase activation (Aim 1), whether directly targeting endothelial ceramide signaling via adenoviral asmase gene therapy to overexpress ASMase specifically in tumor vasculature will radiosensitize more effectively than anti-angiogenic drugs (Aim 2), and whether tumor curability with ASMase-directed therapies occurs via acute vascular compromise (Aim 3). As such, we propose a new pathophysiologic model for anti-angiogenic radiosensitization, in which intensity of the ceramide signal is not a static function of endothelial biology, but rather is dynamically regulated by angiogenic factors secreted by tumor cells and is pharmacologically tractable. Recognition that an endothelial Ceramide Rheostat is obligate for tumor cure for SDRT suggests the therapeutic potential of turning up the intensity of endothelial ceramide signaling. Practically, combining ant- VEGF strategies under conditions that re-set ASMase activation can be taken to the clinic almost immediately. Furthermore, the proposed studies explore the potential for greater impact on tumor cure by directly accessing ceramide biology using adenoviral gene therapy to overexpress asmase exclusively in neo-angiogenic vasculature. PUBLIC HEALTH RELEVANCE: An endothelial Ceramide Rheostat functions atop of a cascade of microvascular dysfunction that mediates tumor cure by anti-neoplastic therapies, including some anti-cancer drugs and high single dose radiotherapy (SDRT). The intensity of acid sphingomyelinase (ASMase)-mediated generation of the second messenger ceramide determines the extent of acute endothelial cell apoptosis, which precipitates transient perfusion defects within tumors. Acute hypoxia (or reperfusion) ensues, which couples to tumor stem cell DNA damage, coordinately determining tumor cure. The purpose of this application is to explore pharmacologic and genetic up-regulation of this Ceramide Rheostat using anti-angiogenic drugs and an adenoviral gene therapy vector, termed Ad5H2E-PPE1(3x)-ASMase, designed to express human ASMase specifically in tumor endothelium downstream of a modified pre-proendothelin promoter. Preliminary pre-clinical data indicate a large impact of this approach on improving SDRT-induced tumor cure. The current clinical forecast for use of anti-angiogenic drugs or ASMase gene therapy to up-regulate the endothelial Ceramide Rheostat envisions its application as adjunct to SDRT and chemotherapeutic management of oligometastatic cancer, with an estimated annual population size in the United States of ~900,00 patients.