The role of the SCF/FBW7 ubiquitin ligase complex in hematopiesis and leukemia

描述(申请人提供)：T细胞急性淋巴细胞白血病(T-ALL)是一种由造血干细胞/祖细胞转化而引起的疾病。它主要困扰着儿童和青少年。尽管T-ALL的治疗结果近年来有所改善，但复发患者的预后仍然很差。因此，识别和研究在这种特殊类型的白血病中控制转化诱导和治疗耐药的分子途径是非常重要的。最近的证据表明，在大多数T-ALL患者中，Notch1癌基因的激活突变是细胞转化的触发因素。大多数T-ALL Notch1突变都截断了Notch1蛋白的一部分，称为PEST结构域。尽管该结构域在Notch1蛋白酶体介导的降解过程中起重要作用，但Notch1稳定性的确切调控机制及其在T细胞转化和人类T-ALL中的作用目前尚不清楚。我们在这里提供了大量的实验证据，证明E3泛素连接酶Fbw7是Notch1蛋白稳定性的重要调节因子，它与位于Notch1 PEST结构域的以苏氨酸为中心的降解子序列相互作用。我们还证明了T-ALL诱导的Notch1突变针对这个Fbw7退化子，并且Fbw7基因本身在人类T-ALL的很大一部分中发生突变和失活。这些和我们在应用中提出的其他初步观察结果使我们假设Fbw7是一种重要的新的T细胞白血病的肿瘤抑制因子，它的失活可以由于基本的Fbw7底物的稳定而触发T细胞转化。在这项应用中，我们最初测试了Notch1：Fbw7相互作用在白血病中的重要性，方法是产生在Notch1 PEST结构域上缺乏必要降解的“敲入”小鼠。此外，为了直接证明Fbw7在T-ALL中是一种肿瘤抑制因子，我们建立了条件性的T细胞特异性Fbw7基因敲除小鼠，并研究了这种缺陷对T细胞转化和发育的影响。此外，利用这些基因工具，我们确定了Fbw7在T细胞转化中的重要下游靶点。最后，我们在体外和体内研究了Fbw7突变对T-ALL伽玛分泌酶抑制剂治疗的影响。公共卫生意义：T细胞急性淋巴细胞性白血病(T-ALL)是一种由T淋巴细胞恶性转化引起的疾病。尽管近年来T-ALL的治疗结果有所改善，但复发患者的预后仍然很差。为了鉴定和研究控制T细胞转化的分子途径，我们在这里研究了Notch1和Fbw7之间的相互作用，Notch1是大多数T-ALL患者中突变的癌基因，Fbw7是泛素连接酶，可以泛化和降解核，致癌的Notch1。

DESCRIPTION (provided by applicant): T cell acute lymphoblastic leukemia (T-ALL) is a disease induced by transformation of hematopoietic stem cells/progenitors. It afflicts mainly children and adolescents. Although treatment outcome in T-ALL has improved in recent years, patients with relapsed disease continue to have dismal prognosis. It is thus very important to identify and study the molecular pathways that control both induction of transformation and treatment resistance in this particular type of leukemia. Recent evidence demonstrated that activating mutations in the Notch1 oncogene are the trigger for cell transformation in the majority of T-ALL patients. The majority of the T-ALL Notch1 mutations are truncating a portion of the Notch1 protein called the PEST domain. Although this domain has been previously suggested to be important for Notch1 proteasome-mediated degradation, the exact mechanism of regulation of Notch1 stability and its role in T cell transformation and human T-ALL is currently unknown. We present here a large amount of experimental evidence that identifies the E3 ubiquitin ligase Fbw7 as an important regulator of Notch1 protein stability, through its interaction with a Threonine-centered degron sequence situated in the Notch1 PEST domain. We also demonstrate that T-ALL- inducing Notch1 mutations target this Fbw7 degron and that the Fbw7 gene itself is mutated and inactivated in a significant portion of human T-ALL. These and other preliminary observations presented in our application make us hypothesize that Fbw7 is an important novel tumor suppressor in T cell leukemia and its inactivation can trigger T cell transformation due to the stabilization of essential Fbw7 substrates. In this application we initially test the importance of the Notch1: Fbw7 interaction in leukemia by generating "knock-in" mice that lack the essential degron on the Notch1 PEST domain. Moreover, to directly prove that Fbw7 is a tumor suppressor in T-ALL we generate conditional, T-cell specific Fbw7 knock-out mice and study the effect of the deficiency in T cell transformation and development. Moreover, using these genetic tools we identify the essential downstream targets of Fbw7 in T cell transformation. Finally, we study both in vitro and in vivo the effect of Fbw7 mutations on gamma-secretase inhibitor treatment of T-ALL. PUBLIC HEALTH RELEVANCE: T cell acute lymphoblastic leukemia (T-ALL) is a disease induced by malignant transformation of T lymphocytes. Although treatment outcome of T-ALL has improved in recent years, patients with relapsed disease continue to have dismal prognosis. In an attempt to identify and study the molecular pathways that control T cell transformation we study here the interplay between Notch1, an oncogene mutated in the majority of T- ALL patients, and Fbw7, a ubiquitin ligase that can ubiquitinate and degrade nuclear, oncogenic Notch1.