The role of emergency granulopoiesis in the pathogenesis of Fanconi Anemia

描述(申请人提供)：范可尼途径修复崩溃和停滞的脱氧核糖核酸复制分叉通过同源重组修复(HRR)和转移合成(TLS)；从而在细胞周期的S阶段保持基因组完整性。先天性缺乏任何Fanconi蛋白会导致Fanconi贫血(FA)；这种疾病的早期特征是骨髓衰竭(BMF)。据推测，BMF的发生是因为未修复的DNA损伤触发了FA造血干细胞(HSC)和祖细胞的凋亡。在FA的BMF阶段存活的患者有发生骨髓异常增殖症的趋势，并呈克隆性进展。这被认为是由累积的突变引起的，这些突变诱导了对细胞周期检查点的抵抗和/或对DNA损伤的凋亡反应。FA患者表现出稳定的粒细胞减少和感染易感性。我们的研究表明，感染时受损的“紧急粒细胞生成”(EG)也是FA免疫缺陷的原因之一。在正常情况下，EG相关的细胞因子会刺激骨髓立即释放粒细胞，然后是HSC和粒/单核细胞祖细胞(GMP)的扩张。这一增殖期涉及S期缩短。我们发现，经IL1和其他介导EG反应的细胞因子处理后，原代小鼠GMP中Fanconi C和F的表达增加。而且，我们确定FancC缺陷的小鼠不能在体内进行EG反应。相反，我们发现，在大多数FancC-/-小鼠中，重复使用EG刺激会导致全血细胞减少、BMF和死亡。我们还发现，电刺激FancC-/-小鼠的不良反应可被IL1-R拮抗剂阻断。我们假设反复的、失败的紧急粒系造血会加速FA患者的骨髓衰竭。而且，不成功的EG发作为导致克隆进展的突变提供了机会。这一假说将通过三个目标来实现：目标1：明确EG过程中Fanconi通路激活和DNA修复的机制。WT、FancC缺陷或FancA缺陷的小鼠骨髓细胞将被EG相关细胞因子处理，并分析Fanconi途径的激活和DNA修复。体内研究将在不同的EG刺激下进行。目的2：探讨乙二醇致FA骨髓衰竭的分子机制。我们还将使用上面描述的模型来确定EG相关的细胞因子是否与细胞周期检查点激活相关地诱导FANC缺陷的骨髓细胞凋亡。目的3：确定多次失败的EG是否促进FA的克隆进展：我们将使用这些模型来确定在重复的EG发作期间，阻断特定的细胞因子是否阻止克隆进展。这些研究的目的是确定无效的EG发作在BMF中的作用以及在FA中的克隆性进展。这些研究可能会提出可以迅速转化为临床的治疗方法。

DESCRIPTION (provided by applicant): The Fanconi pathway repairs collapsed and stalled DNA-replication forks through homologous recombination repair (HRR) and translesional synthesis (TLS); thereby maintaining genomic integrity during S phase of the cell cycle. Congenital absence of any Fanconi protein results in Fanconi Anemia (FA); a disorder that is characterized during early stages by bone marrow failure (BMF). BMF is hypothesized to occur as unrepaired DNA damage triggers apoptosis in FA hematopoietic stem cells (HSC) and progenitor cells. Patients who survive the BMF stage of FA have a tendency to develop bone marrow dysplasia with clonal progression. This is hypothesized to be caused by accumulating mutations that induce resistance to cell cycle checkpoints and/or the apoptotic response to DNA-damage. FA patients exhibit steady state granulocytopenia and susceptibility to infection. Our studies suggest that impaired "emergency granulopoiesis" (EG) during infectious challenge also contributes to immuno-deficiency in FA. Under normal circumstances, EG-related cytokines stimulate immediate granulocyte release from the bone marrow, followed by expansion of HSC and granulocyte/monocyte progenitors (GMP). This proliferative phase involves S phase-shortening. We found that expression of Fanconi C and F increased in primary murine GMP treated with IL1¿ and other cytokines that mediate the EG response. And, we determined that FancC deficient mice are unable to mount an in vivo EG-response. Instead, we found that repeated episodes of EG-stimulation result in pancytopenia, BMF, and death in the majority of FancC-/- mice. We also found that the adverse effects of EG stimulation in FancC-/- mice are blocked by an IL1-R antagonist. We hypothesize that repeated, failed episodes of emergency granulopoiesis accelerate bone marrow failure in FA. And, that unsuccessful EG episodes provide opportunity for mutations that result in clonal progression. This hypothesis will be pursued through three aims: AIM 1: Define mechanisms of Fanconi pathway activation and DNA-repair during EG. Wt, FancC- deficient, or FancA-deficient murine bone marrow cells will be treated with EG-related cytokines and analyzed for Fanconi pathway activation and DNA-repair. In vivo studies will be performed with various EG stimuli. AIM 2: Identify molecular mechanisms involved in EG-related bone marrow failure in FA. We will also use the models described above to determine if EG-related cytokines induce apoptosis in Fanc-deficient bone marrow in association with cell cycle checkpoint activation. AIM 3: Determine if multiple failed episodes of EG facilitate clonal progression in FA: We will use these models to determine if blocking specific cytokines prevents clonal progression during repeated EG episodes. The goal of these studies is to define the role of ineffective EG episodes in BMF and clonal progression in FA. These studies may suggest therapeutic approaches that could be rapidly translated to the clinic.