Fanconi Anemia: Molecular pathogenesis of Bone Marrow Failure and Clonal Evolutio

范可尼贫血：骨髓衰竭和克隆进化的分子发病机制

• 批准号: 7918661
• 负责人: Grover Carlton Bagby
• 金额: $ 49.63万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7918661
• 关键词: Acute Myelocytic Leukemia; Acute leukemia; Adult; Agonist; Apoptosis; Binding; Binding Proteins; Biochemical; Biological Assay; Blood; Cell Aging; Cell Line; Cell Survival; Cells; Child; Clinic; Complement; Complementary DNA; Complex; Computer Simulation; DNA Damage; Defect; Disease; Epitopes; Exposure to; FANCG gene; Failure; Family member; Fanconi' s Anemia; Flow Cytometry; Functional disorder; GRP94; Genes; Goals; Hematopoietic; Hematopoietic stem cells; Homologous Gene; Human; Hypersensitivity; Immunoassay; In Vitro; Journals; Knock-in Mouse; Knowledge; Lead; Light; Manuscripts; Marrow; Methods; Mitogen-Activated Protein Kinase Inhibitor; Modeling; Molecular; Mono-S; Mononuclear; Morbidity - disease rate; Mus; NF-kappa B; Nuclear; Nuclear Translocation; Pancytopenia; Pathogenesis; Pathway interactions; Phagocytes; Phenotype; Play; Prevention; Production; Program Research Project Grants; Progress Reports; Protein Isoforms; Protein Tyrosine Kinase; Proteins; Publishing; Resveratrol; Risk; Role; STAT5A gene; Screening procedure; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Stem cells; Structure of thyroid parafollicular cell; Testing; Toll-Like Receptor Pathway; Wild Type Mouse; Work; base; cytokine; design; fitness; high throughput screening; human IRAK1 protein; human TLR8 protein; improved; in vitro Assay; inhibitor/antagonist; interest; kinase inhibitor; lymphoblast; macrophage; monocyte; mortality; mutant; nano; novel; peripheral blood; progenitor; protein B; protein complex; protein phosphatase inhibitor-2; research study; response; second transplant; senescence; small hairpin RNA; small molecule; tool

Overall PPG Obiectives and Points of Integration for Project 3: The central theme of this program project grant applicafion is to clarify the molecular basis ofthe Fanconi anemia phenotype and to exploit this knowledge to identify strategies that will reduce morbidity and mortality in children and adults with this disease. Project 3 focuses largely on the hematopoiefic phenotype seeking define roles of FA proteins in three signal transduction pathways and to test (directly in hematopoietic cells) the notion that FA defects lead to dysfuncfion of these pathways consequently to bone marrow failure.(14-16) Since this project was last reviewed, we have developed a nano-immunoassay that will permit us to test the validity of our model of STAT5 dysfunction in FA stem cells. We have also developed and validated two short-term in vitro assays suitable for use in our shared small molecular screening efforts one of which is derived from a method that we had already published(l) and the second of which has been submitted for review to the journal Blood (Appendix). In fact, using the latter assay we have used the primary screening method to screen 50 small molecules and have idenfified agents that both enhance FANCC''' hematopoiefic stem cell (HSC) survival and reduce the release of TNFa by FANCC deficient mononuclear phagocytes sfimulated with toll-like receptor 8 (TLRS) agonists. Because these assays are robust and quanfitafive, they have supplanted the assay (based upon defective STAT5 acfivation) we had proposed in the last application. All ofthe projects and cores share two objecfives; to identify small molecules that might be of value in the clinic to reduce the complicafions of FA and to identify mechanisms by which agents with favorable in vitro profiles exert their effects on FA cells. Each aim of this project (Project 3) has integrated a novel assay applicable to Fancd2 mice. Project 3 will conduct such assays as they become relevant in the light of new findings in Projects 1 and 2. For example, project 2 has confirmed that HSC of Faned2 deficient mice are significantly less quiescent than those of wild type mice. Their flow cytometry based assay will be conducted by Project 2 using HSC from mice studied in project three, adding depth to the funcfional and biochemical studies being conducted on primary HSCs in project 3. Likewise, the leader of project 2 will create Fancc mutant knock-in mice suggested by one of the reviewers. Goals for Project 3: Bone marrow failure and acute myelogenous leukemia are major causes of morbidity and mortality in FA. For this reason our project has focused on the defining the molecular pathogenesis of these cardinal hematopoiefic phenotypes (17-20). We have defined three key signaling functions of some FA proteins that are disfinct from their nuclear core complex funcfions (18) (also see preliminary studies and progress report) and during the past year have demonstrated that at least two of these mechanisms (TNFa hypersensitivity in HSC and hematopoiefic progenitor cells (HPCs) increase the coefficient of selecfion for neoplasfic stem cell clones.(1) Reasoning that prevention of marrow failure in FA will reduce the risk of AML, our long term obiectives are to: (A) Clarify the precise molecular mechanisms that underiie the survival signaling funcfions of FA proteins, (B) demonstrate that marrow failure arises from defecfive survival signaling and HSC senescence but not as a result of the aberrant DNA damage response, (C) to identify survival signaling defects in cells from all of the most common FA complementafion groups, and (D) to identify therapeufic agents that will reduce stem cell apoptosis and senescence and reduce over-production of TNFa, a cytokine we have shown to be of significance in both the bone marrow failure and acute leukemia phenotype.(1;21) Each of the three aims below focuses on one of the three signaling defects we have identified in FA hematopoiefic cells.

项目 3 的总体 PPG 目标和整合点：该计划项目拨款申请的中心主题是阐明范可尼贫血表型的分子基础，并利用这些知识来确定减少患有此病的儿童和成人发病率和死亡率的策略。疾病。项目 3 主要关注造血表型，寻求定义 FA 蛋白在三种信号转导途径中的作用，并测试（直接在造血细胞中）FA 缺陷导致这些途径功能障碍从而导致骨髓衰竭的概念。(14-16)自上次审查该项目以来，我们开发了一种纳米免疫测定法，使我们能够测试 FA 干细胞中 STAT5 功能障碍模型的有效性。我们还开发并验证了两种适合在我们共同的小分子筛选工作中使用的短期体外测定方法，其中一种方法源自我们的方法 已经发表(l)，其中第二篇已提交给《Blood》杂志审阅 （附录）。事实上，使用后一种检测方法，我们已经使用初级筛选方法筛选了 50 个小 分子，并已确定既能增强 FANCC''' 造血干细胞 (HSC) 存活率，又能减少用 Toll 样受体 8 (TLRS) 激动剂刺激的 FANCC 缺陷型单核吞噬细胞释放 TNFa 的药物。由于这些测定方法稳健且定量，因此它们取代了我们在上次申请中提出的测定方法（基于有缺陷的 STAT5 激活）。所有项目和核心都有两个目标：鉴定可能在临床上有价值的小分子，以减少 FA 并发症，并确定具有良好体外特性的药物对 FA 细胞发挥作用的机制。 该项目（项目 3）的每个目标都集成了适用于 Fancd2 小鼠的新颖测定法。项目 3 将根据项目 1 和 2 中的新发现进行相关的此类测定。例如，项目 2 已证实 Faned2 缺陷小鼠的 HSC 的静止状态明显低于野生型小鼠。他们基于流式细胞术的测定将由项目 2 使用来自项目 3 中研究的小鼠的 HSC 进行，从而增加项目 3 中对原代 HSC 进行的功能和生化研究的深度。同样，项目 2 的领导者将创建 Fancc 突变体敲入-在一位审稿人建议的小鼠中。 项目 3 的目标：骨髓衰竭和急性髓性白血病是 FA 发病和死亡的主要原因。因此，我们的项目重点关注定义这些主要造血表型的分子发病机制 (17-20)。我们已经定义了一些 FA 蛋白的三个关键信号传导功能，这些功能与其核核心复合体功能不同 (18)（另见初步研究和进展报告），并且在过去一年中已经证明了至少其中两个机制（TNFa 超敏反应） HSC 和造血祖细胞 (HPC) 增加了肿瘤干细胞克隆的选择系数。(1) FA 中预防骨髓衰竭将降低风险的推理对于 AML，我们的长期目标是：(A) 阐明 FA 蛋白生存信号功能的精确分子机制，(B) 证明骨髓衰竭是由生存信号缺陷和 HSC 衰老引起的，而不是由 FA 蛋白的生存信号功能缺陷引起的。异常的 DNA 损伤反应，(C) 识别来自所有最常见 FA 互补组的细胞中的生存信号缺陷，以及 (D) 识别可减少干细胞凋亡和衰老并减少 TNFa 的过度产生，TNFa 是一种细胞因子，我们已证明它对骨髓衰竭和急性白血病表型都具有重要意义。(1;21) 以下三个目标中的每一个都侧重于我们拥有的三个信号传导缺陷之一在 FA 造血细胞中鉴定。