The role of emergency granulopoiesis in the pathogenesis of Fanconi Anemia

描述（由申请人提供）：Fanconi途径通过同源重组修复（HRR）和跨病变合成（TLS）修复塌陷和停滞的DNA复制叉;从而在细胞周期的S期保持基因组完整性。先天性缺乏任何Fanconi蛋白导致Fanconi贫血（FA）;一种在早期阶段以骨髓衰竭（BMF）为特征的病症。BMF被假设为发生未修复的DNA损伤触发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骨髓发育不良伴克隆进展的趋势。这被假设是由诱导对细胞周期检查点的抗性和/或对DNA损伤的凋亡反应的累积突变引起的。FA患者表现出稳态粒细胞减少症和感染易感性。我们的研究表明，受损的“紧急粒细胞生成”（EG）在感染的挑战也有助于在FA的免疫缺陷。在正常情况下，EG相关的细胞因子刺激粒细胞从骨髓中立即释放，随后是HSC和粒细胞/单核细胞祖细胞（GMP）的扩增。该增殖期涉及S期缩短。我们发现，Fanconi C和F的表达增加，在用IL 1？和其他介导EG反应的细胞因子处理的原代鼠GMP中。而且，我们确定FancC缺陷小鼠不能产生体内EG反应。相反，我们发现重复的EG刺激导致大多数FancC-/-小鼠的全血细胞减少、BMF和死亡。我们还发现，EG刺激在FancC-/-小鼠中的不良作用被IL 1-R拮抗剂阻断。我们推测，反复发作的紧急粒细胞生成失败加速FA患者的骨髓衰竭。而且，不成功的EG事件为导致克隆进展的突变提供了机会。这一假设将通过三个目标来实现：目的1：定义EG期间范科尼通路激活和DNA修复的机制。Wt、FancC缺陷型或FancA缺陷型鼠骨髓细胞将用EG相关细胞因子处理，并分析Fanconi途径活化和DNA修复。将使用各种EG刺激进行体内研究。目的2：明确FA中EG相关骨髓衰竭的分子机制。我们还将使用上述模型来确定EG相关的细胞因子是否诱导与细胞周期检查点激活相关的Fan缺陷骨髓中的细胞凋亡。目标3：确定多次失败的EG发作是否促进FA的克隆进展：我们将使用这些模型来确定在重复的EG发作期间阻断特定细胞因子是否阻止克隆进展。这些研究的目的是确定无效EG发作在BMF中的作用和FA中的克隆进展。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.