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

表征 FMS 样酪氨酸激酶 (FLT3) 的新型相互作用伙伴

• 批准号: 8636833
• 负责人: Amy Susan Duffield
• 金额: $ 21.14万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8636833
• 关键词: Acute; Acute Myelocytic Leukemia; Affect; Autocrine Communication; Binding; Biological Assay; Biology; Cell Proliferation; Cell physiology; Cells; Clinical; Clinical Trials; Co-Immunoprecipitations; Coupled; Cytokinesis; Cytoskeletal Modeling; Development; Dose; Drug Targeting; FLT3 ligand; Goals; Growth; Hematopoietic; Hematopoietic stem cells; Homing; Leukemic Cell; Ligands; Marrow; Mass Spectrum Analysis; Monomeric GTP-Binding Proteins; Mutation; Myelogenous; Paracrine Communication; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphotransferases; Point Mutation; Protein Tyrosine Kinase; Proteins; Receptor Protein-Tyrosine Kinases; Resistance; Signal Pathway; Signal Transduction; Stem cells; Stromal Neoplasm; Tyrosine Kinase Inhibitor; Work; cell motility; clinically relevant; cohort; design; fetal liver kinase-2; in vitro activity; inhibitor/antagonist; leukemia; mutant; neoplastic cell; novel; outcome forecast; public health relevance; research study; response; rho; stem; stem cell differentiation; therapeutic target; tumor

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.

描述（由申请人提供）：fms样酪氨酸激酶3 （FLT3）在骨髓造血干细胞中表达。FLT3的激活启动下游信号级联反应，这对干细胞的存活、增殖和分化非常重要。虽然这种酪氨酸激酶的表达通常在造血细胞分化时丢失，但许多急性髓性白血病异常表达FLT3。此外，多达三分之一的AML病例携带FLT3激活突变，包括近膜结构域（FLT3/ITD）的内部串联重复，以及较少出现的激酶结构域点突变，如FLT3/D835Y。AML中FLT3/ITD突变的存在预示着预后不良。FLT3是一个很有前景的治疗靶点，几种针对FLT3的酪氨酸激酶抑制剂（TKIs）目前正处于临床试验阶段。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FLT3生物学有更全面的了解。尽管FLT/ITD突变的临床意义已被详细描述，至少一些相关的下游信号通路已被表征，但对与FLT3相互作用的蛋白群知之甚少。为了鉴定一组与FLT3/ITD相互作用的蛋白质，我们使用联合免疫沉淀法和质谱法进行了筛选。这些实验产生了一组潜在的相互作用物，包括细胞分裂2奉献体（DOCK2）。DOCK2激活小g蛋白，包括rho亚家族小gtpase Rac1和Rac2，并影响细胞功能的各个方面，包括干细胞在骨髓中的归巢和保留、细胞运动、细胞骨架组织和细胞增殖。在本提案中，我们的目标是确定DOCK2和野生型FLT3之间相互作用的功能后果，以及DOCK2和FLT3与激活突变之间的相互作用。接下来，我们将评估DOCK2与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DOCK2等相互作用物的相互作用将有助于更深入地了解这种酪氨酸激酶的功能，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。