Targeting the PHD/HIF Pathway to Mitigate Radiation Induced Gl Toxicity

正常组织毒性胃肠道（Gl）经常发生与放化疗，代表一个主要的临床挑战。不幸的是，目前还没有有效的治疗方法来对抗这一重大的临床问题。胃肠道管腔上皮存在生理性缺氧，缺氧信号对其生存和正常功能至关重要。缺氧诱导因子-1 （HlF-1）是细胞缺氧反应的核心转录因子，其水平与细胞氧张力呈负相关波动。稳定的HIF-1表达已被证明对正常肠道稳态以及炎症挑战期间的生存至关重要。HIF-1是维持肠的几种基本功能所必需的，如屏障和吸收功能。在肠道中增加HIF-1表达可改善上皮屏障功能，增加营养吸收并减少细胞凋亡，以应对感染和炎症应激。相反，在小鼠结肠炎模型中，肠道特异性敲除HIF-1会导致上皮细胞凋亡增加。HIF的氧依赖性调节主要通过脯氨酸羟化酶结构域（dr）蛋白介导，该蛋白将HIF上的脯氨酸部分羟基化，并作为von Hippel Lindau （VHL）蛋白的识别位点，该蛋白靶向HIF进行蛋白酶体破坏。迄今为止，已经确定了三种氧依赖性脯氨酸羟化酶（PHD1-3），然而，每种异构体在肠道内稳态中的作用尚不清楚。由于PHD蛋白调节HIF水平，而HIF反过来保护肠道免受炎症应激，因此我们的假设是PHD蛋白调节胃肠道的放射敏感性，因此特定PHD亚型的删除将通过HIF介导的对上皮完整性和隐窝再生的影响，为肠道提供放射保护。在本研究中，我们将：1)研究PHD 1-3与项目2和核心B在保护Gl束免受低分割和分散辐射方面的具体贡献，2)确定PHD抑制项目3的辐射保护机制；3)利用Project 2探索PHD - 1在肠道干细胞辐射应答中的作用；4)利用Project 2确定一种小分子PHD抑制剂对Gl道的保护效果；5)与Project 4的正常组织放射防护相比，确定PHD 1-3敲除和PHD活性小分子抑制剂对肿瘤生长的治疗效果。该项目代表了放射防护剂发展的新范式变化，因为我们正在通过改变上皮屏障功能来研究组织放射防护的生理基础。

Normal tissue toxicity to the gastrointestinal (Gl) tract occurs frequently with chemoradiotherapy and represents a major clinical challenge. Unfortunately, no effective treatments exist to combat this significant clinical problem. The luminal epithelia of the Gl tract exist in physiologic hypoxia, and thus hypoxic signaling is crucial to the their survival and normal functions. Hypoxia-inducible factor-1 (HlF-1) is a transcription factor at the heart of the cellular hypoxia response whose levels fluctuate inversely with cellular oxygen tension. Stabilized HIF-1 expression has been shown to be critical for normal intestinal homeostasis as well as survival during inflammatory challenges. HIF-1 is required to maintain several basic functions of the intestine, such as barrier and absorptive functions. Augmenting HIF-1 expression in the intestine improves epithelial barrier functions, increases nutrient absorption and reduces apoptosis in response to infection and inflammatory stress. Conversely, intestinal specific knockouts of HIF-1 cause increased epithelial apoptosis in murine models of colitis. The oxygen dependent regulation of HIF is chiefly mediated through the prolyl hydroxylase domain (PHD)-containing proteins which hydroxylate proline moieties on HIF, and serve as a recognition site for the von Hippel Lindau (VHL) protein, which targets HIF for proteasomal destruction. To date, three oxygen-dependent prolyl hydroxylases have been identified (PHD1-3), however, the roles of each isoform in intestinal homeostasis is not clear. Because PHD proteins regulate HIF levels, and HIF, in turn protects the gut from inflammatory stress, it is our hypothesis that the PHD proteins regulates radiosensitivity in the gastrointestinal tract, such that deletion of specific PHD isoforms will afford radioprotection of the gut through HIF-medlated effects on epithelial integrity and crypt regeneration. In this proposal, we will: 1) investigate the specific contributions of PHD 1-3 on radioprotecting the Gl tract from hypofractionated and fractionated radiation with Project 2 and Core B, 2) determine the mechanisms of radioprotection by PHD inhibition with Project 3; 3) explore the role of PHD 1-3 in the radiation response ofthe intestinal stem cell with Project 2, 4) determine the efficacy of a small molecule PHD inhibitor to protect the Gl tract with Project 2; and 5) determine the therapeutic efficacy of PHD 1-3 knockouts and small molecule inhibitors of PHD activity on tumor growth in response to radiation relative to normal tissue radioprotection with Project 4. This project represents a new paradigm change in the development of radioprotectors in that we are investigating the physiological basis of tissue radioprotection through alterations in epithelial barrier function.