Severe congenital neutropenia (SCN) is frequently associated with dominant point mutations in ELANE, the gene encoding neutrophil elastase (NE). Chronic administration of granulocyte colony-stimulating factor (G-CSF) is a first-line treatment of ELANE-mutant (ELANEmut) SCN. However, some ELANEmut patients including patients with ELANE start codon mutations do not respond to G-CSF. Here, through directed granulopoiesis of gene-edited isogenic normal and patient-derived iPSCs, we demonstrate that ELANE start codon mutations suffice to induce G-CSF resistant granulocytic precursor cell death and refractory SCN. ELANE start codon mutated neutrophil precursors express predominantly nuclear N-terminal truncated alternate NE. Unlike G-CSF sensitive ELANE mutations that induce endoplasmic reticulum and unfolded protein response stress, we found that the mutation of the ELANE translation initiation codon resulted in NE aggregates and activated pro-apoptotic aggrephagy as determined by downregulated BAG1 expression, decreased BAG1/BAG3 ratio, NE co-localization with BAG3, and localized expression of autophagic LC3B. We found that SERF1, an RNA-chaperone protein, known to localize in misfolded protein aggregates in neurodegenerative diseases, was highly upregulated and interacted with cytoplasmic NE of mutant neutrophil precursors. Silencing of SERF1 enhanced survival and differentiation of iPSC-derived neutrophil precursors, restoring their responsiveness to G-CSF. These observations provide a mechanistic insight of G-CSF-resistant ELANEmut SCN, revealing targets for therapeutic intervention.

中文翻译：

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ELANE 突变型中性粒细胞减少症的 G-CSF 耐药性取决于含有截短的中性粒细胞弹性蛋白酶聚集体的 SERF1。


严重的先天性中性粒细胞减少症 （SCN） 通常与编码中性粒细胞弹性蛋白酶 （NE） 的基因 ELANE 的显性点突变有关。粒细胞集落刺激因子 （G-CSF） 的长期给药是 ELANE 突变体 （ELANEmut） SCN 的一线治疗。然而，一些 ELANEmut 患者（包括 ELANE 起始密码子突变患者）对 G-CSF 没有反应。在这里，通过基因编辑的同基因正常和患者来源的 iPSC 的定向粒细胞生成，我们证明 ELANE 起始密码子突变足以诱导 G-CSF 耐药粒细胞前体细胞死亡和难治性 SCN。ELANE 起始密码子突变的中性粒细胞前体主要表达核 N 末端截短的替代 NE。与诱导内质网和展开蛋白反应应激的 G-CSF 敏感 ELANE 突变不同，我们发现 ELANE 翻译起始密码子的突变导致 NE 聚集体和激活的促凋亡聚集性，这是通过下调 BAG1 表达、BAG1/BAG3 比率降低、NE 与 BAG3 共定位和自噬 LC3B 的定位表达来确定的。我们发现 SERF1 是一种 RNA 伴侣蛋白，已知定位于神经退行性疾病中错误折叠的蛋白质聚集体，高度上调并与突变中性粒细胞前体的细胞质 NE 相互作用。沉默 SERF1 增强了 iPSC 衍生的中性粒细胞前体的存活和分化，恢复了它们对 G-CSF 的反应性。这些观察结果提供了 G-CSF 耐药 ELANEmut SCN 的机制见解，揭示了治疗干预的靶点。