Role of Dok-1 in oncogenic tyrosine kinase signaling

描述(申请人提供)：这项研究的长期目标是确定DOK-1抑制p210Bcr-Abl诱导的导致肿瘤转化的信号事件的分子机制。P210Bcr-Abl是一种具有结构性活性的酪氨酸激酶，是慢性粒细胞白血病(CML)的病原体，CML是一种造血细胞疾病，每年发生的频率约为10万人中有1人。已经证实，酪氨酸激酶活性对p210Bcr-Abl的转化能力是至关重要的。事实上，用特定的Abl激酶抑制剂，如甲磺酸伊马替尼抑制p210Bcr-Abl激酶，可导致疾病缓解，使CML治疗成为靶向癌症治疗的成功范例。然而，大多数接受这些药物治疗的患者保留了残留的白血病细胞，一些患者由于Abl激酶域的突变而产生耐药性，最终导致CML复发。因此，需要开发新的治疗策略来治疗对Abl激酶抑制剂耐药的CML病例。在这种背景下，阐明调节p210Bcr-Abl驱动的白血病发生的关键信号通路和成分是至关重要的。我们发现，在慢性粒细胞白血病患者的慢性期祖细胞中，由p210Bcr-Abl组成的酪氨酸结合蛋白DOK-1抑制有丝分裂信号，并反对p210Bcr-Abl引发的致癌转化。DOK-1在小鼠体内的失活加速了p210Bcr-Abl诱导的CML样疾病的发生，而DOK-1的表达则降低了p210Bcr-Abl表达细胞的增殖活性。这些发现为获得对p210Bcr-Abl介导的致癌信号的分子基础和抵消它的机制的新见解提供了一个独特的框架。本研究旨在阐明DOK-1干扰p210Bcr-Abl介导的信号转导和肿瘤发生的分子机制(S)。为了实现这些目标，第一个具体目标将定义DOK-1中对其抑制p210Bcr-Abl驱动的转化和白血病发生的作用至关重要的决定因素。具体目标2将描绘和表征DOK-1作用于抑制p210Bcr-Abl诱导的致癌转化的信号通路。将使用分子、生化和细胞生物学方法以及小鼠模型来实现这些目标。第三个具体目标是通过生化纯化技术和多维蛋白质鉴定技术相结合，鉴定在p210Bcr-Abl介导的致癌信号中起重要作用的DOK-1相互作用蛋白。从这些研究中获得的信息不仅将为DOK-1的S功能和作用模式提供新的见解，而且还将有助于更好地理解p210Bcr-Abl信号级联中的关键下游事件，这些下游事件导致慢性粒细胞白血病前体细胞的异常扩张。了解DOK-1是如何抑制p210Bcr-Abl致癌信号的，可能会导致改进策略的概念，以控制CML和可能的其他肿瘤疾病。 公共卫生相关性：DOK-1接头蛋白已成为由致癌酪氨酸激酶驱动的肿瘤转化的关键调节因子，包括酪氨酸激酶融合蛋白p210Bcr-Abl，这是慢性粒细胞白血病(CML)的致病事件。这些研究旨在了解DOK-1抑制p210Bcr-Abl诱导的信号事件和致癌转化的分子机制，并可能为开发更有效的策略和药物来控制CML和可能的其他肿瘤疾病提供基础。

DESCRIPTION (provided by applicant): The long-term objective of this study is to define the molecular mechanisms by which Dok-1 inhibits p210Bcr-Abl-induced signaling events that lead to neoplastic transformation. p210Bcr-Abl, a constitutively active tyrosine kinase, is the causative agent of chronic myelogenous leukemia (CML), a disorder of hematopoietic cells that occurs with a frequency of about 1 in 100,000 people a year. It is well established that the tyrosine kinase activity is crucial to the transforming capacity of p210Bcr-Abl. Indeed, inhibition of the p210Bcr-Abl kinase with specific Abl kinase inhibitors, such as imatinib mesylate, leads to disease remission - making treatment of CML a successful paradigm of targeted cancer therapy. However, most patients receiving these drugs retain residual leukemic cells, and some develop drug resistance due to the acquisition of mutations in the Abl kinase domain, which eventually leads to relapse of CML. Therefore, new therapeutic strategies need to be developed to treat CML cases resistant to Abl kinase inhibitors. In this context, it is of utmost importance to elucidate the critical signaling pathways and components that moderate p210Bcr-Abl-driven leukemogenesis. We have found that Dok-1, an adaptor protein constitutively tyrosine phosphorylated by p210Bcr-Abl in chronic phase progenitor cells of CML patients, inhibits mitogenic signaling and opposes oncogenic transformation triggered by p210Bcr- Abl. Inactivation of Dok-1 in mice accelerates the onset of the CML-like disease induced by p210Bcr-Abl, whereas expression of Dok-1 diminishes the proliferative activity of p210Bcr-Abl expressing cells. These findings provide a unique framework for the acquisition of novel insights into the molecular underpinnings of p210Bcr-Abl- mediated oncogenic signaling and mechanisms that counteract it. This application aims to delineate the molecular mechanism(s) by which Dok-1 interferes with p210Bcr-Abl-mediated signaling and oncogenesis. Towards these goals, the first specific aim will define the determinants in Dok-1 that are critical for its inhibitory effect on p210Bcr-Abl-driven transformation and leukemogenesis. Specific aim 2 will delineate and characterize signaling pathways Dok-1 acts upon to inhibit p210Bcr-Abl-induced oncogenic transformation. Molecular, biochemical, and cell biological approaches, as well as mouse models, will be used to achieve these objectives. The third specific aim will identify Dok-1 interacting proteins important for its function in p210Bcr-Abl- mediated oncogenic signaling, by using biochemical purification techniques combined with Multidimensional Protein Identification Technology. Information gained from these studies will not only provide novel insights into Dok-1's function and mode of action, but will also contribute to a better understanding of the critical downstream events in the p210Bcr-Abl signaling cascades that drive aberrant expansion of CML progenitor cells. Understanding how Dok-1 inhibits p210Bcr-Abl oncogenic signaling could lead to the conception of improved strategies to control CML and possibly other neoplastic diseases as well. PUBLIC HEALTH RELEVANCE: The Dok-1 adaptor protein has emerged as a critical regulator of neoplastic transformation driven by oncogenic tyrosine kinases, including the tyrosine kinase fusion protein p210Bcr-Abl, which is the causative event of chronic myelogenous leukemia (CML). The proposed studies are aimed at understanding the molecular mechanisms by which Dok-1 inhibits p210Bcr-Abl- induced signaling events and oncogenic transformation, and may provide the basis for developing more effective strategies and drugs to control CML and possibly other neoplastic diseases as well.