The role of emergency granulopoiesis in the pathogenesis of Fanconi Anemia

Fanconi途径修复崩溃和停滞的DNA复制分叉通过同源 重组修复和转位合成；从而保持S期间基因组的完整性 细胞周期的阶段。先天性缺乏任何Fanconi蛋白导致Fanconi贫血(FA)；一种疾病 这在早期以骨髓衰竭(BMF)为特征。假设bmf发生在 未修复的DNA损伤会引发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克隆性骨髓发育不良的倾向 进步。据推测，这是由累积的突变引起的，这些突变诱导了对细胞周期的抵抗 检查点和/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)也是导致免疫缺陷的原因之一 FA.在正常情况下，EG相关的细胞因子刺激粒细胞立即从 骨髓，随后是HSC和粒细胞/单核细胞祖细胞(GMP)的扩增。这种增殖剂 阶段涉及S阶段的缩短。我们发现Fanconi C和F在原发肿瘤中的表达增加 用IL1和其他细胞因子处理小鼠GMP，以介导EG反应。而且，我们决定 FancC基因缺陷的小鼠不能在体内进行EG反应。相反，我们发现反复出现的情节 在大多数FancC-/-小鼠中，EG刺激导致全血细胞减少、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加速了骨髓。 FA失败。而且，不成功的EG发作为导致克隆的突变提供了机会 进步。这一假设将通过三个目标来实现： 目的1：明确脑震荡时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用EG相关细胞因子处理并分析 用于Fanconi通路的激活和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 使用上述模型来确定EG相关细胞因子是否诱导FANC缺陷骨的细胞凋亡 骨髓与细胞周期检查点激活相关。 目标3：确定多次失败的EG是否促进FA的克隆性进展：我们将使用这些 模型以确定在重复的EG发作期间，阻断特定的细胞因子是否阻止克隆进展。 这些研究的目标是确定无效的EG发作在BMF和克隆进展中的作用。 FA.这些研究可能会提出可以迅速转化为临床的治疗方法。

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.