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：该项目资助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降低儿童和成人该病发病率和死亡率的策略。项目3主要关注造血表型，寻求确定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）自从上次审查该项目以来，我们已经开发了一种纳米免疫测定法，这将使我们能够测试我们的FA干细胞中STAT 5功能障碍模型的有效性。我们还开发并验证了两种适用于我们共同的小分子筛选工作的短期体外试验，其中一种来自我们 已经发表了（l），其中第二篇已提交血液杂志审查 （附录）。事实上，使用后一种测定法，我们已经使用初级筛选方法筛选了50个小 分子，并具有增强FANCC造血干细胞（HSC）存活和减少用toll样受体8（TLRS）激动剂刺激的FANCC缺陷型单核吞噬细胞释放TNF α的纯化剂。因为这些测定法是稳健的和定量的，它们已经取代了我们在上一次申请中提出的测定法（基于有缺陷的STAT 5活化）。所有项目和核心都有两个共同目标：识别可能在临床上有价值的小分子，以减少FA的并发症，并识别具有良好体外特征的药物对FA细胞发挥作用的机制。 本项目（项目3）的每个目标都集成了适用于Fancd 2小鼠的新型试验。项目3将根据项目1和项目2的新发现进行相关的分析。例如，项目2已经证实，Faned 2缺陷型小鼠的HSC比野生型小鼠的HSC显著更少静止。项目2将使用项目3中研究的小鼠HSC进行基于流式细胞术的试验，从而加深项目3中对原代HSC进行的功能和生化研究。同样，项目2的负责人将根据其中一位评审员的建议创建Fancc突变基因敲入小鼠。 项目3的目标：骨髓衰竭和急性髓性白血病是FA发病率和死亡率的主要原因。为此，我们的项目集中于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蛋白的三种关键信号传导功能，这些功能与它们的核核心复合物功能不同（18）（也参见初步研究和进展报告），并且在过去的一年中已经证明这些机制中的至少两种（HSC和造血祖细胞（HPC）中的TNF α超敏性）增加了肿瘤性干细胞克隆的选择系数。(1)基于预防FA中的骨髓衰竭将降低AML的风险，我们的长期目标是：（A）阐明FA蛋白的存活信号传导功能的精确分子机制。（B）证明骨髓衰竭由缺陷存活信号传导和HSC衰老引起，但不是异常DNA损伤应答的结果。（C）鉴定来自所有最常见的FA互补组的细胞中的存活信号传导缺陷，和（D）鉴定将减少干细胞凋亡和衰老并减少TNF α过度产生的治疗剂，一种我们已经证明在骨髓衰竭和急性白血病表型中具有重要意义的细胞因子。（1;21）以下三个目标中的每一个都集中在我们在FA造血细胞中鉴定的三个信号传导缺陷中的一个。