背景:红系和髓系分化障碍通常发生在白血病中。鉴于红细胞和骨髓谱系之间的关系尚不清楚。寻找红系和髓系分化的共调节因子可能有助于寻找髓系白血病治疗的新靶点。在造血方面,据报道,ALA(α硫辛酸)通过剪接因子SF3B1靶向粒细胞-单核细胞祖细胞中的转录因子ELK1来抑制中性粒细胞谱系的确定。然而,需要进一步探索以确定ELK1是否是红系和髓系分化的常见调节因子.
方法:分离的CD34+的体外培养,进行CMPs(常见的骨髓祖细胞)和CD34+CD371-HSPCs(造血干细胞祖细胞)以测定单核细胞的分化潜能,中性粒细胞,和红细胞。将ELK1转导的CD34HSPC的长同工型(L-ELK1)或短同工型(S-ELK1)的过表达慢病毒移植到NSG小鼠中,以测定移植后3个月的人淋巴细胞和骨髓分化差异。敲除在CD371+GMPs(粒细胞-单核细胞祖细胞)中高表达的SRSF11,ALA上调并与ELK1-RNA剪接位点结合,进行分析在源自CD34+CD123midCD38+CD371-HPCs(造血祖细胞)的红系分化中的功能。对L-ELK1和S-ELK1过表达的CD34+CD123midCD38+CD371-HPCs进行RNA测序以测定ELK1改变的信号。
结果:这里,我们提出了新的证据,即ALA通过靶向CD34+CD371-造血干祖细胞(HSPCs)中的转录因子ELK1促进红系分化.ELK1的长同工型(L-ELK1)或短同工型(S-ELK1)的过表达均抑制红系细胞分化,但ELK1的敲除并不影响红细胞分化。RNAseq分析CD34+CD123midCD38+CD371-HPCs显示L-ELK1上调中性粒细胞活性相关基因的表达,磷酸化,和缺氧信号,而S-ELK1主要调节缺氧相关信号。然而,L-ELK1上调的大多数基因仅被S-ELK1中度上调,这可能是由于与L-ELK1相比,S-ELK1中缺乏血清反应因子相互作用和调节结构域。总之,中性粒细胞和红细胞的分化可能需要依赖于L-ELK1和S-ELK1的剂量,才能在早期谱系定型时通过RNA剪接信号实现精确调节.
结论:发现ALA和ELK1通过RNA剪接体调节人的粒细胞生成和红细胞生成,ALA-ELK1信号可能是人类白血病治疗的靶点。
BACKGROUND: Erythroid and myeloid differentiation disorders are commonly occurred in leukemia. Given that the relationship between erythroid and myeloid lineages is still unclear. To find the co-regulators in erythroid and myeloid differentiation might help to find new target for therapy of myeloid leukemia. In hematopoiesis,
ALA (alpha lipoic acid) is reported to inhibit neutrophil lineage determination by targeting transcription factor ELK1 in granulocyte-monocyte progenitors via splicing factor SF3B1. However, further exploration is needed to determine whether ELK1 is a common regulatory factor for erythroid and myeloid differentiation.
METHODS: In vitro culture of isolated CD34+, CMPs (common myeloid progenitors) and CD34+ CD371- HSPCs (hematopoietic stem progenitor cells) were performed to assay the differentiation potential of monocytes, neutrophils, and erythrocytes. Overexpression lentivirus of long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 transduced CD34+ HSPCs were transplanted into NSG mice to assay the human lymphocyte and myeloid differentiation differences 3 months after transplantation. Knocking down of SRSF11, which was high expressed in CD371+GMPs (granulocyte-monocyte progenitors), upregulated by
ALA and binding to ELK1-RNA splicing site, was performed to analyze the function in erythroid differentiation derived from CD34+ CD123mid CD38+ CD371- HPCs (hematopoietic progenitor cells). RNA sequencing of L-ELK1 and S-ELK1 overexpressed CD34+ CD123mid CD38+ CD371- HPCs were performed to assay the signals changed by ELK1.
RESULTS: Here, we presented new evidence that
ALA promoted erythroid differentiation by targeting the transcription factor ELK1 in CD34+ CD371- hematopoietic stem progenitor cells (HSPCs). Overexpression of either the long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 inhibited erythroid-cell differentiation, but knockdown of ELK1 did not affect erythroid-cell differentiation. RNAseq analysis of CD34+ CD123mid CD38+ CD371- HPCs showed that L-ELK1 upregulated the expression of genes related to neutrophil activity, phosphorylation, and hypoxia signals, while S-ELK1 mainly regulated hypoxia-related signals. However, most of the genes that were upregulated by L-ELK1 were only moderately upregulated by S-ELK1, which might be due to a lack of serum response factor interaction and regulation domains in S-ELK1 compared to L-ELK1. In summary, the differentiation of neutrophils and erythrocytes might need to rely on the dose of L-ELK1 and S-ELK1 to achieve precise regulation via RNA splicing signals at early lineage commitment.
CONCLUSIONS: ALA and ELK1 are found to regulate both human granulopoiesis and erythropoiesis via RNA spliceosome, and
ALA-ELK1 signal might be the target of human leukemia therapy.