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崩溃的复制叉所必需的。范科尼 贫血（FA）是由范可尼基因的遗传突变引起的。 FA 患者出现骨髓衰竭 (BMF) 在童年时期，幸存者经常出现克隆进展。我们确定了紧急情况（压力）的角色 BMF 中的粒细胞生成 (EG) 和 FA 中的克隆进展。 EG 是粒细胞产生的间歇过程 应对传染性挑战。在 EG 期间，S 期缩短，FancC 和 F 表达增加。 与野生型小鼠不同，Fancc-/-小鼠在EG刺激后不会出现粒细胞增多症。重复EG Fancc-/- 小鼠中的攻击诱导 BMF（伴随 HSC 和祖细胞凋亡）或克隆进展。 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是 EG 的重要细胞因子；诱导骨髓谱系定型和 G-CSF 表达。在S期期间， Atr 激活 p53 并促进复制叉损伤未修复的细胞凋亡。在 Fancc-/- 小鼠中，Tp53-haplo- EG 期间粒细胞不足可挽救粒细胞增多症；延迟 BMF 但加速克隆进展。在 Fancc-/- 在小鼠中，每次不成功的 EG 发作都会增加 Atr/p53 的活性；与 BMF 相关。在 相比之下，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 EG 期间 FA 中不成功的 PMN 产生可防止未知阴性的激活 监管途径；维持细胞周期检查点活动和 HSC/GMP 扩展信号。这导致 BMF 和导致克隆进展的突变积累。我们将通过三个目标来实现这一目标： 目标 1：定义终止紧急粒细胞生成的分子触发因素及其作用 FA 中 BMF 的流程。我们将研究 PMN 密度对细胞凋亡和 BMF 的贡献 Fancc-/- 小鼠中的 EG 不成功。 PMN 骨髓密度对已知 EG 相关途径的影响将 在 Wt 与 Fancc-/- 小鼠中确定，并且在无偏见研究中确定了新的途径。 目标 2：识别 FA 中与紧急粒细胞生成诱导的克隆进展相关的事件。 我们将通过研究白血病抑制途径来定义 Fancc-/- 小鼠克隆进展中涉及的事件 通过无偏见的方法介导 EG 终止。结果将与基因表达进行比较 来自人类范可尼贫血的 CD34 骨髓细胞的概况，以确定潜在的转化目标。 目标 3：研究延迟 BMF 或 FA 克隆进展的潜在翻译靶点。我们会 确定 EG 期间激活的新途径对小鼠 BMF 和/或克隆进展的影响 遗传模型。相关小分子抑制剂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。 在 FA 中反复进行 EG 尝试但不成功。这可能表明降低发病率的治疗方法 贫血和感染，或为患者提供明确的治疗，例如干细胞/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.