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

项目3的总体PPG目标和集成点：该项目资助申请的中心主题是阐明Fanconi贫血表型的分子基础，并利用这一知识来确定将降低患有这种疾病的儿童和成人的发病率和死亡率的策略。项目3主要集中在造血表型上，寻求确定FA蛋白在三条信号转导途径中的作用，并(直接在造血细胞中)测试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)自从该项目最后一次审查以来，我们开发了一种纳米免疫分析方法，使我们能够在FA干细胞中测试我们的STAT5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分析方法，其中一种来自我们的一种方法 已经发表了(L)，第二篇已提交给《血液》杂志审阅 (附录)。事实上，使用后一种检测方法，我们已经使用了初步筛查方法来筛选50个小的 这些药物既能提高FANCC造血干细胞(HSC)的存活率，又能减少用Toll样受体8(TLRs)激动剂刺激的FANCC缺陷单核吞噬细胞释放TNFa。由于这些检测是可靠的和合格的，它们取代了我们在上一次申请中提出的检测(基于有缺陷的STAT5活性)。所有的项目和核心都有两个共同的目标：确定可能在临床上有价值的小分子，以减少FA的复杂性，并确定具有良好体外特征的药物对FA细胞发挥作用的机制。 该项目(项目3)的每个目标都整合了一种适用于FANCD2小鼠的新的检测方法。项目3将根据项目1和2中的新发现进行相关的检测。例如，项目2已确认Faned2缺陷小鼠的HSC显著低于野生型小鼠。他们的基于流式细胞术的分析将由项目2使用项目3中研究的小鼠的HSC进行，增加了项目3中对初级HSC进行的功能和生化研究的深度。同样，项目2的负责人将根据一位评审者的建议创建Fancc突变敲入小鼠。 项目3的目标：骨髓衰竭和急性髓系白血病是FA发病率和死亡率的主要原因。出于这个原因，我们的项目集中在定义这些主要造血表型的分子发病机制(17-20)。我们定义了一些FA蛋白的三个与其核核心复合体功能不同的关键信号功能(18)(也见初步研究和进展报告)，并在过去一年中证明了至少两种机制(HSC和造血祖细胞(HPC)中的TNFa超敏)增加了肿瘤干细胞克隆的选择系数。(1)我们的长期目标是：(A)阐明FA蛋白生存信号功能下的确切分子机制，(B)证明骨髓衰竭是由缺陷的生存信号和HSC衰老引起的，而不是由于异常的DNA损伤反应，(C)从所有最常见的FA互补组中确定细胞中的生存信号缺陷，以及(D)寻找将减少干细胞凋亡和衰老并减少TNFa过度产生的治疗药物，我们已经证明，TNFa在骨髓衰竭和急性白血病表型中都具有重要意义。21)下面的三个目标中的每一个都集中在我们在FA造血细胞中识别的三个信号缺陷之一。

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.