Molecular mechanisms for bone marrow failure and clonal progression during the innate immune response in Fanconi Anemia

Fanconi DNA修复途径是拯救停滞或倒塌的复制叉子所必需的。范科尼 贫血(FA)是由Fanconi基因的遗传突变引起的。FA患者发展为骨髓衰竭(BMF) 在儿童时期，幸存者经常出现克隆进展。我们确定了紧急情况(压力)的作用 BMF的粒细胞生成(EG)和FA的克隆性进展。Eg是粒细胞产生的间歇性过程。 以应对传染性的挑战。在EG中，S时相缩短，FancC和F的表达增加。 与野生型小鼠不同，Fancc-/-小鼠在EG刺激下不会出现粒细胞增多症。重复的EG 在Fancc-/-小鼠中的攻击诱导了BMF，伴随着HSC和祖细胞的凋亡，或者克隆性进展。 用IL1-R拮抗剂治疗Fancc-/-小鼠可以保护它们免受这些不良后果的影响。IL1β为 EG的基本细胞因子；诱导髓系分化和G-CSF的表达。在S阶段， ATR可激活未修复复制叉状损伤的细胞的P53和凋亡。在Fancc-/-小鼠中，TP53-单链- 不足挽救了EG期间的粒细胞增多；延缓了BMF，但加速了克隆性进展。在Fancc-/- 小鼠，每一次失败的EG发作都会增加ATR/P53的活性；与BMF相关。在……里面 相比之下，在Fancc-/-TP53/-小鼠中，ATR/P53活性随着连续成功的EG周期而降低。 我们假设在EG期间在FA中不成功地产生PMN可以阻止未知负值的激活 调节通路；维持细胞周期检查点活动和HSC/GMP扩张信号。这导致了 BMF和突变的积累导致克隆性进展。我们将通过三个目标来实现这一目标： 目标1：确定终止紧急粒系造血的分子诱因及其作用 在FA的BMF中进行处理。我们将研究PMN密度在细胞凋亡和BMF中的作用 Fancc-/-小鼠的EG不成功。PMN骨髓密度对已知的EG相关途径的影响将 在Wt与Fancc-/-小鼠中被确定，并在无偏倚研究中发现了新的途径。 目的2：确定与紧急粒系造血诱导的FA克隆进展相关的事件。 我们将通过研究白血病抑制通路来确定Fancc-/-小鼠克隆进展中涉及的事件 通过无偏见的方法调解EG的终止。结果将与基因表达进行比较 人类Fanconi贫血患者CD34骨髓细胞的图谱以确定潜在的翻译靶点。 目的3：研究潜在的翻译靶点以延缓FA的BMF或克隆性进展。我们会 确定在EG期间激活的新通路对小鼠BMF和/或克隆性进展的影响 遗传模型。使用小分子抑制剂的相关中间体将在临床前研究中进行测试。 这些研究的目标是确定BMF和/或克隆进展的分子机制。 FA中反复出现的、不成功的EG尝试。这可能会提出治疗方法，以减少因 对于贫血和感染，或为患者提供明确的治疗，如干细胞/骨髓移植。

The Fanconi DNA repair pathway is required for rescue of stalled or collapsed replication forks. Fanconi Anemia (FA) is caused by inherited mutation of Fanconi genes. FA patients develop bone marrow failure (BMF) in childhood, with survivors frequently developing clonal progression. We identified a role for emergency (stress) granulopoiesis (EG) in BMF and clonal progression in FA. EG is an episodic process for granulocyte production in response to infectious challenge. During EG, S phase is shortened and FancC and F expression increase. Unlike wild type mice, Fancc-/- mice did not develop granulocytosis upon stimulation of EG. Repeated EG challenge in Fancc-/- mice induced either BMF, with apoptosis of HSC and progenitors, or clonal progression. Treatment of Fancc-/- mice with an IL1-R antagonist protected them from these adverse consequences. IL1β is an essential cytokine for EG; inducing myeloid lineage commitment, and G-CSF expression. During the S phase, Atr activates p53 and apoptosis of cells with unrepaired replication fork damage. In Fancc-/- mice, Tp53-haplo- insufficiency rescued granulocytosis during EG; delaying BMF but accelerating clonal progression. In Fancc-/- mice, increasing activity of Atr/p53 occurred with each unsuccessful EG episode; associated with BMF. In contrast, Atr/p53 activity decreased with consecutive, successful EG cycles in Fancc-/-Tp53+/- mice. We hypothesize unsuccessful PMN production in FA during EG prevents activation of unknown negative regulatory pathways; sustaining cell cycle checkpoint activity and HSC/GMP expansion signals. This induces BMF and accumulation of mutations that lead to clonal progression. We will pursue this through three aims: Aim 1: Define molecular triggers for termination of emergency granulopoiesis and the role of this process in BMF in FA. We will investigate contribution of PMN density to apoptosis and BMF during unsuccessful EG in Fancc-/- mice. The impact of PMN bone marrow density on known EG-related pathways will be determined in Wt vs Fancc-/- mice, and novel pathways identified in non-biased studies. Aim 2: Identify events associated with emergency granulopoiesis-induced clonal progression in FA. We will define events involved in clonal progression in Fancc-/- mice by studying leukemia suppressor pathways that mediate EG termination and by non-biased approaches. Results will be compared to gene expression profiles in CD34+ bone marrow cells from human Fanconi Anemia to identify potential translational targets. Aim 3: Investigate potential translational targets to delay BMF or clonal progression in FA. We will determine the impact of novel pathways that are activated during EG on BMF and/or clonal progression in murine genetic models. Relevant intermediates with small molecule inhibitors will be tested in pre-clinical studies. The goal of these studies is to define molecular mechanisms for BMF and/or clonal progression during recurrent, unsuccessful EG attempts in FA. This may suggest therapeutic approaches to decrease morbidity due to anemia and infection, or bridge patients to definitive treatments such as stem cell/bone marrow transplant.