In Fanconi Anemia, impaired accumulation of bone marrow neutrophils during emergency granulopoiesis induces hematopoietic stem cell stress. In Fanconi Anemia, impaired accumulation of bone marrow neutrophils during emergency granulopoiesis induces hematopoietic stem cell stress

Fanconi Anemia (FA) is an inherited disorder of DNA-repair caused by mutation in one of 20+ interrelated genes that repair intra-strand DNA crosslinks and rescue collapsed or stalled replication forks. The most common hematologic abnormality in FA is anemia, but progression to bone marrow failure (BMF), clonal hematopoiesis, or acute myeloid leukemia (AML) may also occur. In prior studies, we found that Fanconi DNA-repair is required for successful emergency granulopoiesis; the process for rapid neutrophil production during the innate immune response. Specifically, Fancc-/- mice did not develop neutrophilia in response to emergency granulopoiesis stimuli, but instead exhibited apoptosis of bone marrow hematopoietic stem cells (HSCs) and differentiating neutrophils. Repeated emergency granulopoiesis challenges induced BMF in most Fancc-/- mice, with AML in survivors. In contrast, we found equivalent emergency granulopoiesis-induced neutrophilia in Fancc-/-Tp53+/- mice and wild type (WT) mice, without BMF in either. Since bone marrow neutrophil accumulation triggers termination of emergency granulopoiesis, we hypothesize neutrophilia protects Fancc-/-Tp53+/- bone marrow from the stress of sustained inflammatory physiology, as experienced by Fancc-/- mice. Overall design: We find blocked neutrophil accumulation during emergency granulopoiesis in Wt, Fancc-/- and Fancc-/-Tp53+/- mice in vivo with Ly6G antibody injections. In these studies, emergency granulopoiesis was induced by injection of Alum (ovalbumin + aluminium chloride) with saline as a steady state control. Mice were assessed for bone marrow failure and leukemia during these treatments. Bone marrow from the mice was harvested at various time points after treatment. LSK cells were separated by flow cytometry and subjected to total RNA sequencing.

范科尼贫血（Fanconi Anemia, FA）是一种遗传性DNA修复缺陷疾病，由20余种相互关联的基因发生突变所致，这类基因负责修复链内DNA交联并解救坍塌或停滞的复制叉。范科尼贫血最常见的血液学异常为贫血，但病情可进展为骨髓衰竭（bone marrow failure, BMF）、克隆性造血或急性髓系白血病（acute myeloid leukemia, AML）。既往研究表明，范科尼DNA修复通路对于成功启动应急粒细胞生成——即先天免疫应答期间快速生成中性粒细胞的过程——不可或缺。具体而言，Fancc基因敲除（Fancc-/-）小鼠无法在应急粒细胞生成刺激下出现中性粒细胞增多症，反而表现出骨髓造血干细胞（hematopoietic stem cells, HSCs）及分化中中性粒细胞的凋亡。反复的应急粒细胞生成刺激可使大多数Fancc-/-小鼠出现骨髓衰竭，存活小鼠则进展为AML。与之相反，我们发现Fancc-/-Tp53+/-小鼠与野生型（wild type, WT）小鼠的应急粒细胞生成诱导的中性粒细胞增多症程度相当，且二者均未出现骨髓衰竭。鉴于骨髓中性粒细胞聚集可触发应急粒细胞生成的终止，我们提出假说：中性粒细胞增多症可保护Fancc-/-Tp53+/-小鼠骨髓免受持续性炎症生理状态带来的应激，而Fancc-/-小鼠则会经历此类应激。总体实验设计：我们通过Ly6G抗体注射，在体内阻断野生型、Fancc-/-及Fancc-/-Tp53+/-小鼠应急粒细胞生成过程中的中性粒细胞聚集。本研究中，应急粒细胞生成通过注射明矾（卵清蛋白+氯化铝）诱导，以生理盐水注射作为稳态对照。在上述处理过程中，对小鼠的骨髓衰竭及白血病发生情况进行评估。于治疗后的不同时间点采集小鼠骨髓样本，通过流式细胞术分离LSK细胞，并进行总RNA测序。