Universal Technology for Profiling the Dynamics of Normal & Oncogenic Signaling

描述（由申请人提供）：受体酪氨酸激酶（RTK），如表皮生长因子受体（EGFR），在许多蛋白质信号传导途径的启动中至关重要。因此，对单个RTK的多个磷酸化修饰的动态控制可以表现出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的异常活性通常具有严重的生物学后果，并且与许多人类癌症中的致癌过程有关。事实上，酪氨酸激酶抑制剂如易瑞沙和格列卫的成功预示着最近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性质以及预测RTK靶向治疗效果的关键。对早期时间调节状态的更多理解将使选择性靶向下游通路的策略更加精确，并可能提供基于动态而非静态干预的独特癌症治疗方法。需要一个新技术和方法的综合平台，以在体外和细胞培养中在分子水平上提供这些快速、早期事件的时间分辨率。该R21/R33组合提案将创建一套经过验证的通用创新和成熟技术，包括快速反应方法、新型时间分辨电喷雾电离质谱（ESI-MS）技术、纳米喷雾ESI-TOF、使用ESI-MS LC/MS/MS的磷酸肽图谱和位点特异性磷酸酪氨酸抗体检测，这些技术将适用于分析各种RTK，它们的自磷酸化模式和下游信号事件在关键的亚秒到多秒的时间域。我们选择了一个典型的RTK，表皮生长因子受体酪氨酸激酶（EGFR）开发这个平台。表皮生长因子受体（EGFR）酪氨酸激酶途径与大量癌症相关，并且是靶向癌症治疗剂的重要分子靶标，例如最近已被FDA批准的小分子ATP模拟物易瑞沙（gefinitib），并且强调了特异性激酶靶向的产生作为癌症治疗的新范例。探测最早期自身磷酸化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EGFR的正常和致癌形式之间的差异的新理解，以及对新兴治疗类靶向激酶抑制剂的扩展功能理解。我们相信，信号传导中磷酸化的时间动力学特征提供了独特的分子指纹或特征，用于区分正常细胞和癌细胞以及对靶向抑制剂的反应性。需要新的实验工具和技术来在分子水平上区分癌细胞和正常细胞，并评估新癌症疗法的有效性。这个R21/R33项目描述了一种开发新技术的策略，使我们能够了解癌细胞在分子水平上如何发生变化，并更详细地了解新的选择性靶向癌症疗法如何工作。

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.