Role of Dok-1 in oncogenic tyrosine kinase signaling

描述（由申请人提供）：本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是一种组成型活性酪氨酸激酶，是慢性髓性白血病（CML）的病原体，慢性髓性白血病是一种造血细胞疾病，每年发生的频率约为10万分之一。酪氨酸激酶活性对p210Bcr-Abl的转化能力起着至关重要的作用。事实上，使用特异性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的患者保留了残留的白血病细胞，一些患者由于Abl激酶结构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。在这种情况下，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CML患者的慢性期祖细胞中，由p210Bcr-Abl磷酸化的衔接蛋白Dok-1可抑制有丝分裂信号传导，并对抗p210Bcr-Abl引发的致癌转化。小鼠中Dok-1的失活加速了p210Bcr-Abl诱导的cml样疾病的发生，而Dok-1的表达降低了表达p210Bcr-Abl的细胞的增殖活性。这些发现为获取p210Bcr-Abl介导的致癌信号的分子基础及其抵消机制的新见解提供了一个独特的框架。本应用旨在描述Dok-1干扰p210bcr - abl介导的信号传导和肿瘤发生的分子机制。为了实现这些目标，第一个具体目标将确定Dok-1中的决定因素，这些决定因素对p210bcr - abl驱动的转化和白血病发生的抑制作用至关重要。特异性目标2将描述和表征Dok-1抑制p210bcr - abl诱导的致癌转化的信号通路。分子、生物化学和细胞生物学方法，以及小鼠模型，将被用来实现这些目标。第三个具体目标是通过生化纯化技术结合多维蛋白鉴定技术，鉴定在p210Bcr-Abl介导的致癌信号传导中起重要作用的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驱动CML祖细胞异常扩增的p210Bcr-Abl信号级联中的关键下游事件。了解Dok-1如何抑制p210Bcr-Abl的致癌信号传导可能有助于改进控制CML和其他肿瘤疾病的策略。

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.