Re-setting the Endothelial Ceramide Rheostat

描述(申请人提供)：我们最近的研究探索了一种生化机制，通过这种机制，抗血管生成增强了微血管功能障碍的鞘磷脂信号，从而通过抗肿瘤治疗，包括一些抗癌药物和高单剂量放射治疗(SDRT)，介导了肿瘤的治愈。在这项申请中提出的研究是基于观察到内皮细胞比任何哺乳动物细胞产生更多的酸性鞘磷脂酶(ASMase)并使用它来发出凋亡信号。我们发表的数据显示，ASMase快速移位到内皮细胞质膜的外叶，诱导鞘磷脂水解酶中促凋亡的第二信使神经酰胺，引发细胞凋亡。我们的初步数据表明，随之而来的是一连串的微血管功能障碍，包括急性灌注缺陷(通过动态核磁共振和Hoechst 33342染料外渗测量)和缺氧(通过EPR血氧仪测量)，这种血管功能障碍抑制了肿瘤干细胞的DNA损伤修复，从而加强了立体定向放射治疗的治愈。这一应用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，并试图通过药物或遗传上调ASMase信号来加强肿瘤治疗。具体地说，我们将研究抗血管生成药物是否通过上调ASMase激活而发生放射增敏(Aim 1)，通过腺病毒Asmase基因治疗直接靶向内皮神经酰胺信号转导在肿瘤血管中过度表达ASMase是否会比抗血管生成药物更有效地辐射增敏(Aim 2)，以及ASMase定向治疗的肿瘤治愈是否通过急性血管损害发生(Aim 3)。因此，我们提出了一种新的抗血管生成放射增敏的病理生理学模型，在该模型中神经酰胺信号的强度不是内皮生物学的静态函数，而是由肿瘤细胞分泌的血管生成因子动态调节的，并且在药理上是容易处理的。认识到内皮神经酰胺变阻器对于SDRT的肿瘤治愈是必要的，这表明提高内皮神经酰胺信号强度的治疗潜力。实际上，在重置ASMase激活的条件下结合ANT-VEGF策略几乎可以立即应用于临床。此外，拟议的研究探索了通过使用腺病毒基因疗法直接进入神经酰胺生物学以在新生血管中过度表达asmase来对肿瘤治疗产生更大影响的可能性。 公共卫生相关性：内皮神经酰胺风变仪的功能位于一系列微血管功能障碍的顶部，这些功能通过抗肿瘤治疗(包括一些抗癌药物和高单次剂量放射治疗(SDRT))介导肿瘤治愈。酸性鞘磷脂酶(ASMase)介导的第二信使神经酰胺的产生强度决定了急性内皮细胞凋亡的程度，这导致了肿瘤内的一过性灌注缺陷。急性缺氧(或再灌注)接踵而至，这与肿瘤干细胞DNA损伤相结合，共同决定了肿瘤的治愈。本应用的目的是利用抗血管生成药物和腺病毒基因治疗载体Ad5H2E-PPE1(3x)-ASMase，探索这种神经酰胺变色剂的药理和遗传上调作用。Ad5H2E-PPE1(3x)-ASMase设计用于在修饰的前内皮素原启动子下游的肿瘤内皮中特异性表达人ASMase。初步的临床前数据表明，这种方法在提高SDRT诱导的肿瘤治愈方面有很大影响。目前的临床预测是使用抗血管生成药物或ASMase基因疗法上调内皮细胞神经酰胺Rheostat，预计它将作为SDRT和化疗治疗少转移癌的辅助手段，估计美国每年的人口规模约为90万人。

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.