Universal Technology for Profiling the Dynamics of Normal & Oncogenic Signaling

描述(申请人提供)：受体酪氨酸激酶(RTK)，如表皮生长因子受体(EGFR)，在许多蛋白质信号通路的启动中是必不可少的。因此，动态控制单个RTK的多个磷酸化修饰可以显示对多个信号转导途径的关键控制。生长因子网络中这种敏感动态的改变和RTK的异常活性通常会产生严重的生物学后果，并与许多人类癌症的致癌过程有关。事实上，Iressa(Tm)和Gleevec(Tm)等酪氨酸激酶抑制剂的成功预示着最近一种精确靶向癌症治疗的策略。了解这些早期动态事件的分子机制可能是理解和预测致癌行为的本质以及预测RTK靶向治疗效果的关键。对早期时间调节状态的深入了解将使选择性靶向下游通路的更精确的策略成为可能，并可能为基于动态而不是静态干预的癌症治疗提供一种独特的方法。需要一个新技术和方法的集成平台，以便在体外和细胞培养中在分子水平上对这些快速、早期的事件提供时间分辨率。这项R21/R33的联合提案将创建一套经过验证的创新和成熟技术的通用集，包括快速反应方法、新的时间分辨电喷雾电离质谱仪(ESI-MS)技术、纳米级ESI-TOF、使用ESI-MS LC/MS/MS绘制的磷酸肽图谱以及适用于分析多种RTK、其自动磷酸化模式和关键的亚秒到多秒时间域中的下游信号事件的位点特异性磷酸酪氨酸抗体检测。我们选择了一个原型RTK，表皮生长因子受体酪氨酸激酶(EGFR)来开发这个平台。表皮生长因子受体(EGFR)酪氨酸激酶通路与许多癌症有关，是靶向癌症治疗的重要分子靶点，例如最近被FDA批准的小分子ATP模拟易瑞沙(Gefinitib)，它强调了特异性激酶靶向的产生作为癌症治疗的新范式。探索自动磷酸化最早事件的分子和时间细节的能力将揭示签名模式，这将提供对正常和致癌形式之间的差异的新理解，并扩展对新兴靶向激酶抑制剂治疗类别的功能理解。我们认为，信号中磷酸化的时间动态的轮廓为区分正常细胞和癌细胞以及对靶向抑制剂的反应性提供了独特的分子指纹或标记。需要新的实验工具和技术在分子水平上区分癌细胞和正常细胞，并评估新的癌症治疗方法的有效性。这个R21/R33项目描述了一种开发新技术的战略，这些技术将使我们能够了解癌细胞中如何在分子水平上发生变化，并更详细地了解新的选择性靶向癌症疗法是如何工作的。

DESCRIPTION (provided by applicant): Receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR) are essential in the initiation of many protein signaling pathways. Dynamic control of multiple phosphorylation modifications of a single RTK thus can manifest critical control on multiple signal transduction pathways. Alterations of this sensitive dynamic in growth factor networks and aberrant activities of RTKs often have severe biological consequences and are linked to oncogenic processes in many human cancers. Indeed the success of tyrosine kinase inhibitors such as Iressa(tm) and Gleevec(tm) heralds a recent strategy of precisely targeted cancer therapy. An understanding of the molecular mechanisms of these early dynamic events may hold the key to understanding and predicting the nature of oncogenic behavior and for predicting the affects of RTK targeted therapy. An increased understanding of the early temporally regulated states will enable more precise strategies for selectively targeting downstream pathways and may offer a unique approach to cancer therapy based on dynamic rather than static intervention. An integrated platform of novel technologies and approaches are needed to provide temporal resolution of these rapid, early events at a molecular level both in vitro as well as in cell culture. This R21/R33 combined proposal will create a validated general set of innovative and established technologies including rapid reaction methodologies, a novel time-resolved electrospray ionization mass spectrometry (ESI-MS) technique, nanospray ESI-TOF, phosphopeptide mapping using ESI-MS LC/MS/MS and site-specific phosphotyrosine antibody detection that will be applicable for the analysis of a wide range of RTKs, their autophosphorylation patterns and downstream signaling events in the critical subsecond to multisecond time domain. We have chosen a prototypical RTK, epidermal growth factor receptor tyrosine kinase (EGFR) to develop this platform. The epidermal growth factor receptor (EGFR) tyrosine kinase pathway is linked to a large number of cancers and an important molecular target for targeted cancer therapeutics such as the small molecule ATP mimetic Iressa (gefinitib) that has recently been approved by the FDA and underscores the generation of specific kinase targeting as a new paradigm for cancer therapy. The ability to probe the molecular and temporal details of the earliest events of autophosphorylation will reveal signature patterns that will provide a new understanding of the differences between normal and oncogenic forms of EGFR and an expanded functional understanding of the emerging therapeutic class of targeted kinase inhibitors. We believe that a profile of the temporal dynamics of phosphorylation in signaling provides a unique molecular fingerprint or signature for distinguishing normal and cancer cells and the responsiveness to targeted inhibitors. New experimental tools and technologies are needed to distinguish cancer cells from normal cells at a molecular level and evaluate the effectiveness of new cancer therapies. This R21/R33 project describes a strategy to develop novel technologies that will allow us to understand how changes occur at a molecular level in a cancerous cell and a more detailed understanding of how new selectively targeted cancer therapies work.