Imaging Kinase Activity In Vivo

描述（由申请人提供）： 针对癌症中活性的特异性受体酪氨酸激酶（RTK）的治疗方法的可用性为解决如何使用靶向治疗最佳治疗复杂癌症提供了独特的机会。我们假设，针对肿瘤发生的多个关键途径的药物治疗癌症将减少治疗耐药性的出现，从而增加有效治疗的可能性。在本申请中，我们将获得与神经胶质瘤发病机制有关的受体酪氨酸激酶的嵌合等位基因，并分别与萤火虫或海肾荧光素酶的激活偶联。我们将衍生融合至分裂萤火虫荧光素酶（flue）的EGFR和融合至分裂海肾荧光素酶（rluc）的PDGFRa的嵌合等位基因。我们将验证EGFR的活化重建活性flue，PDGFRa的活化重建活性rluc，并且flue和rluc活性可以独立测量。我们将测试EGFR或PDGFRa的选择性抑制剂以确定它们分别对flu和rluc的影响。我们将使用这些嵌合的EGFR-fluc和PDGFRa-rluc等位基因来产生胶质瘤的异种移植模型，以测试用单独针对受体酪氨酸激酶或一起针对两种酪氨酸激酶的抑制剂治疗肿瘤的影响。我们的长期目标是使用这些模型来开发减少治疗耐药性出现的治疗范例。具体目标是： 目标1：开发融合至裂解萤火虫荧光素酶的EGFR的嵌合活化等位基因，并分别产生融合至裂解海肾荧光素酶（rluc）的PDGFRa的活化等位基因。我们建议将每个荧光素酶的N-和C-末端的一半分别与两个单独的RTK单体融合。由于对接蛋白结合EGFR和PDGFRa的胞质结构域以响应活化，我们还将检测每个RTK-N-末端luc嵌合体与由融合至适当分裂luc的C-末端一半的对接蛋白组成的嵌合体。 目标二。建立胶质瘤细胞和动物模型，其中PDGFR和EGFR通路被激活并可被独立抑制。我们将产生用于胶质瘤的细胞培养物和异种移植物模型，所述胶质瘤由EGFR与分裂的fluc、PDGFRa与分裂的rluc或两者的嵌合体驱动。然后将对受体激活后luc活性的重建进行非侵入性成像。在用适当的抑制剂治疗后，我们假设flu、rluc或两者的生物发光将降低，从而允许我们单独成像EGFR或PDGFRa的阻断，即使当两种RTK在个体肿瘤中都是活性的。

DESCRIPTION (provided by applicant): The availability of therapies directed against specific receptor tyrosine kinases (RTKs) active in cancer offers a unique opportunity to address how to optimally treat complex cancers using targeted therapies. We hypothesize that treatment of cancers with agents targeted against multiple pathways critical to tumorigenesis will diminish the emergence of therapeutic resistance, thereby increasing the likelihood of effective treatment. In this application, we will derive chimeric alleles of receptor tyrosine kinases implicated in the pathogenesis of glioma, and coupled to activation of firefly or Renilla luciferase respectively. We will derive chimeric alleles of EGFR fused to split firefly luciferase (flue) and of PDGFRa fused to split Renilla luciferase (rluc). We will verify that activation of EGFR reconstitutes active flue, that activation of PDGFRa reconstitutes active rluc, and that flue and rluc activities can be measured independently. We will test selective inhibitors of EGFR or PDGFRa to determine their impact on flue and rluc respectively. We will use these chimeric EGFR-fluc and PDGFRa-rluc alleles to generate xenograft models of glioma, to test the impact of treating tumors with inhibitors directed against either receptor tyrosine kinase alone, or against both tyrosine kinases together. Our long-term goal is to use these models to develop treatment paradigms that diminish the emergence of therapeutic resistance. The Specific Aims are: Aim 1: To develop chimeric activated alleles of EGFR fused to split firefly luciferase, and to separately generate activated alleles of PDGFRa fused to split Renilla luciferase (rluc). We propose to fuse the N- and C-terminal halves of each respective luciferase separately to two individual RTK monomers. Since docking proteins bind to the cytoplasmic domains of EGFR and PDGFRa in response to activation, we will also test each RTK-N-terminal luc chimera in conjunction with a chimera consisting of a docking protein fused to the C-terminal half of the appropriate split luc. Aim 2. To create cell and animal models for glioma in which PDGFR and EGFR pathways are activated and can be inhibited independently. We will generate cell culture and xenograft models for glioma driven by chimeras of EGFR with split flue, PDGFRa with split rluc, or both. The reconstitution of luc activity upon receptor activation will then be imaged non-invasively. Upon treatment with an appropriate inhibitor, we hypothesize that the bioluminescence of flue, rluc, or both will decrease, allowing us to image separately the blockade of EGFR or PDGFRa even when both RTKs are active in an individual tumor.