Imaging VHL-Associated Tumors with Labile O2 Biosensors

描述（由申请人提供）：缺氧诱导因子（HIF）在多种肿瘤中过量产生，是参与血管生成和肿瘤细胞代谢的基因的关键调节因子。von HippeI-Lindau肿瘤抑制蛋白（pVHL）是一种泛素连接酶的组分，在氧气存在下，它靶向HIF进行聚泛素化，从而降解蛋白酶体。最近的研究表明，pVHL与HIF的相互作用是通过HIF中存在的保守肽（下文称为“ODD”肽）的酶促脯氨酰羟基化来调节的。我的初步研究表明，ODD肽与荧光素酶（'ODDLuc'）的融合产生了一种嵌合蛋白，该蛋白在氧气存在下被泛素化并被pVHL破坏。由于荧光素酶活性可以非侵入性地测量，我假设ODDLuc嵌合体将允许我实时测量完整组织和动物中氧气的可用性和pVHL通路的完整性。具体目标1：我将验证ODDLuc荧光素酶在肿瘤异种移植物中的表达反映了氧的可用性和VHL泛素连接酶的活性。具体目标2：我将通过产生ODDLuc敲入小鼠（ROSA 26-ODDLuc）并确定该小鼠中的荧光素酶信号是否可用于监测缺氧来进行体内研究。虽然ROSA 26-ODDLuc小鼠显然在研究缺乏氧合（缺血）的疾病中具有很大的实用性，但我的目标是开发用于成像VHL相关肿瘤的小鼠模型。因此，在特定目标3中：ROSA 26-ODDLuc小鼠将与已被工程化以产生VHL（-/-）肿瘤的小鼠杂交。将验证这些小鼠通过荧光素酶成像检测VHL损失的能力。我设想这些小鼠未来的可能性之一是它们用于小分子的药效学研究，这些小分子可能被预测为预防或治疗VHL相关肿瘤。

DESCRIPTION (provided by applicant): Hypoxia-inducible factor (HIF) is overproduced in a wide variety of tumors and is a key regulator of genes involved in angiogenesis and tumor cell metabolism. The von HippeI-Lindau tumor suppressor protein (pVHL) is a component of a ubiquitin ligase that targets HIF for polyubiquitination, and hence proteasomal degradation, in the presence of oxygen. Recent studies have shown that the interaction of pVHL with HIF is regulated by enzymatic prolyl hydroxylation of a conserved peptide (hereafter called the 'ODD' peptide) present in HIF. My preliminary studies suggest that fusion of the ODD peptide to luciferase ('ODDLuc') gives rise to a chimeric protein that is ubiquitinated and destroyed by pVHL in the presence of oxygen. Since luciferase activity can be measured non-invasively, I hypothesize that the ODDLuc chimera will allow me to measure, in real-time, the availability of oxygen and the integrity of the pVHL pathway in intact tissues and animals. In Specific Aim 1: I will validate that ODDLuc luciferase expression in tumor xenografts reflects the availability of oxygen and the activity of the VHL ubiquitin ligase. In Specific Aim 2: I will proceed to in vivo studies by generating an ODDLuc knock-in mouse (ROSA26-ODDLuc) and determining whether the luciferase signal in this mouse can be used to monitor hypoxia. Although clearly the ROSA26-ODDLuc mouse would have great utility in studying diseases in which there is a lack of oxygenation (ischemia) my goal is to develop a mouse model for imaging VHL-associated neoplasms. Therefore, in Specific Aim 3: the ROSA26-ODDLuc mouse will be crossed to mice that have been engineered to develop VHL (-/-) tumors. These mice will be validated for the ability to detect VHL loss by luciferase imaging. One of the future possibilities I envision for these mice is their use in pharmacodynamic studies of small molecules that might be predicted to prevent or treat VHL-associated neoplasms.