Characterizing Novel Interaction Partners of FMS-Like Tyrosine Kinase ( FLT3)

描述（由申请人提供）：FMS样酪氨酸激酶3（FLT3）在骨髓中的造血干细胞中表达。 FLT3的激活启动下游信号级联，这对干细胞的存活、增殖和分化很重要。 这种酪氨酸激酶的表达通常随着造血细胞分化而丧失，而许多急性髓性（AML）白血病异常表达FLT3。 此外，高达三分之一的AML病例在FLT3中携带激活突变，包括在质膜结构域中的内部串联重复（FLT3/ITD），以及较不常见的激酶结构域中的点突变，如FLT3/D835Y。 AML中FLT3/ITD突变的存在预示着预后不良。 FLT3是一个很有前途的治疗靶点，目前有几种针对FLT3的酪氨酸激酶抑制剂（TKI）正在进行临床试验。 这些药物具有显著的体外活性;然而，临床试验的结果在很大程度上令人失望。 虽然已经提出了许多解释这些结果的假设，但我们认为，在我们能够有效地靶向这一途径之前，需要对正常和突变型FLT3生物学有更全面的了解。 尽管已经详细描述了FLT/ITD突变的临床意义，并且已经表征了至少一些相关的下游信号传导途径，但对与FLT3相互作用的蛋白质组知之甚少。 为了鉴定与FLT3/ITD相互作用的一组蛋白质，我们使用与质谱联用的共免疫沉淀测定进行了筛选。 这些实验产生了一组潜在的相互作用物，包括胞质分裂专用因子2（DOCK 2）。 DOCK 2激活小G蛋白，包括Rho亚家族小GTP酶Rac 1和Rac 2，并影响细胞功能的各个方面，包括干细胞归巢和在骨髓中的滞留、细胞运动性、细胞骨架组织和细胞增殖。 在这个提议中，我们的目标是确定DOCK 2和野生型FLT3之间的相互作用以及DOCK 2和FLT3与激活突变之间的相互作用的功能后果。 接下来，我们将评估DOCK 2和FLT3、FLT3/ITD和FLT3/D835Y之间的相互作用如何影响酪氨酸激酶受体对临床相关抑制剂的反应。 我们假设，表征FLT3和FLT3/ITD与DOCK 2等相互作用物的相互作用将提供对这种酪氨酸激酶功能的更深入理解，提出可以增强TKI治疗疗效的机制，并确定新的药物靶点，以便为FLT3/ITD + AML患者开发更有效的治疗方法。

DESCRIPTION (provided by applicant): FMS-Like tyrosine kinase 3 (FLT3) is expressed in hematopoietic stem cells in the marrow. Activation of FLT3 initiates downstream signaling cascades that are important for the survival, proliferation and differentiation of stem cells. Whie expression of this tyrosine kinase is normally lost as hematopoietic cells differentiate, many acute myeloid (AML) leukemias aberrantly express FLT3. Additionally, up to one-third of AML cases harbor activating mutations in FLT3 including internal tandem duplications in the juxtamembrane domain (FLT3/ITD) and, less frequently, point mutations in the kinase domain such as FLT3/D835Y. The presence of a FLT3/ITD mutation in AML portends a poor prognosis. FLT3 is a promising therapeutic target, and several tyrosine kinase inhibitors (TKIs) directed against FLT3 are currently in clinical trials. These agents have dramatic in vitro activity; however, the results of clinical trials have been largely disappointing. While many hypotheses to explain these results have been suggested, we believe that a more complete understanding of normal and mutant FLT3 biology will be required before we are able to effectively target this pathway. Although the clinical implications of FLT/ITD mutations have been described in detail and at least some of the relevant downstream signaling pathways have been characterized, relatively little is known about the cohort of proteins that interacts with FLT3. In order to idenify a panel of proteins that interact with FLT3/ITD, we performed a screen using co- immunoprecipitation assays coupled with mass spectroscopy. These experiments generated a panel of potential interactors, including dedicator of cytokinesis 2 (DOCK2). DOCK2 activates small G-proteins including the Rho-subfamily small GTPases Rac1 and Rac2, and affects various aspects of cellular function including stem cell homing and retention in the marrow, cell motility, cytoskeletal organization and cell proliferation. In this proposal our goal is to determne the functional consequences of the interaction between DOCK2 and wild type FLT3 as well as the interaction between DOCK2 and FLT3 with activating mutations. Next, we will assess how the interaction between DOCK2 and FLT3, FLT3/ITD and FLT3/D835Y affects the response of the tyrosine kinase receptors to clinically relevant inhibitors. We hypothesize that characterizin the interaction of FLT3 and FLT3/ITD with interactors such as DOCK2 will provide a deeper understanding of this tyrosine kinase's function, suggest mechanisms that could enhance the efficacy of TKI therapy, and identify new drug targets in order to develop more efficacious treatments for patients with FLT3/ITD+ AML.