研究背景与目的

骨髓增生异常综合征（Myelodysplastic syndrome, MDS）是一组异质性的起源于造血干祖细胞的恶性克隆性疾病，其特征包括外周血细胞减少、一系或多系血细胞发育异常、无效造血并且向白血病转化风险高。剪接因子基因突变为MDS最常见的基因突变，发生于约50%的MDS患者中，其中U2AF1突变频率约10~20%，提示U2AF1突变导致的前体mRNA（pre-mRNA）异常剪接在MDS疾病发生发展中起到了关键作用。既往研究报道U2AF1突变损害成熟红细胞的生成，引起粒单系细胞向粒细胞分化偏倚，但它对巨核谱系细胞发育影响尚不清楚，基于此，本研究分析了就诊于我中心的MDS患者U2AF1突变的临床特征，并利用患者CD34⁺细胞与U2AF1突变细胞系建立了体外巨核细胞分化体系，旨在探究U2AF1突变在MDS巨核细胞发育异常（dys-megakaryopoiesis）中的作用及可能的机制。

方法

纳入初诊同时具备完善二代测序资料的MDS患者，比较U2AF1突变及野生型MDS患者临床和实验室参数特征差异；

利用免疫组织化学染色方法对伴有U2AF1突变的MDS患者（MDS^MT）与不伴有剪接因子突变的MDS患者（MDS^WT）的骨髓病理切片及涂片进行CD41染色，统计分析骨髓巨核细胞数目、胞体直径大小和发育异常巨核细胞比例；

采用BSA速度梯度分离方法富集MDS患者原代巨核细胞，使用流式细胞术检测U2AF1^MT MDS与U2AF1^WT MDS患者不同倍体巨核细胞分布；

构建U2AF1^S34F逆转录病毒小鼠模型，通过流式与病理细胞形态学分析观察小鼠体内巨核细胞发育情况；

收集并分离健康供者(n=5)、伴有U2AF1突变的MDS患者（MDS^MT, n=15）和不伴有剪接因子突变的MDS患者（MDS^WT，n=16）的骨髓单个核细胞进行转录组测序，使用rMATS分析获得U2AF1突变导致的异常剪接基因，通过GO富集分析获得可能影响功能的靶基因；

利用qPCR及RT-PCR在患者骨髓单个核细胞中进行靶基因验证。

构建患者CD34⁺细胞体外巨核分化体系，流式分选CD41⁺单细胞，利用单细胞PCR靶向测序技术检测U2AF1突变的CD41⁺单细胞mRNA水平是否存在靶基因异常剪接。

构建诱导型过表达U2AF1野生型蛋白与突变型蛋白的HEL与K562细胞系，建立细胞体外巨核分化体系，检测伴有U2AF1突变的HEL与K562细胞倍体化程度。

采用shRNA技术在HEL与K562细胞系中敲除筛选获取的靶基因，观察靶基因异常剪接对巨核细胞倍体化过程的影响；

构建过表达靶基因剪接异构体的质粒并转染细胞系，检测剪接异构体蛋白表达水平，进行体外分化实验，检测剪接异构体对于细胞倍体化过程的影响；

通过Western Blot检测靶基因及其下游通路蛋白磷酸化水平；

探索不同药物是否可以促进U2AF1^S34F突变巨核细胞成熟与倍体化。

实验结果

MDS患者中U2AF1突变与男性、年轻、外周血更低的血红蛋白水平、骨髓涂片更高的巨核细胞计数、血清高TNF-α水平以及骨髓高纤维化等级相关；

MDS^MT患者骨髓病理切片单位面积巨核细胞计数更多，巨核细胞胞体直径更小，骨髓细胞涂片发育异常巨核细胞比例更高，且以微小巨核细胞（胞体直径≤40μm）比例升高为主；

流式分析结果显示MDS^MT患者低倍体（2N及4N）巨核细胞比例高于MDS^WT患者，高倍体（≥8N）巨核细胞比例显著降低；

U2AF1^S34F小鼠相比于U2AF1^WT小鼠体内的巨核系祖细胞比例显著升高，骨髓病理显示巨核细胞数目增多且成簇分布；

转录组测序分析结果显示与健康供者及MDS^WT患者相比，MDS^MT患者基因异常剪接类型主要以外显子跳跃为主，异常剪接基因主要富集在DNA合成与细胞周期调控通路;

骨髓单个核细胞中U2AF1突变导致了CHEK1两种剪接异构体CHEK1-L和CHEK1-S表达改变，mRNA水平CHEK1-S显著增多，CHEK-L减少；

通过体外分化实验证实伴有U2AF1^S34突变的CD41⁺单细胞中存在CHEK1 mRNA的异常剪接，CHEK1-S/L比例更高；

与野生型相比，U2AF1^S34F突变的HEL与K562细胞体外向巨核细胞分化过程中，2N比例显著增高，4N及8N细胞比例显著降低，伴有U2AF1^S34F突变的巨核细胞相对不成熟且TNF-α表达水平高于野生型细胞；

CHEK1异常剪接导致CHEK1-L蛋白表达降低，在HEL与K562细胞中敲低CHEK1可抑制巨核细胞倍体化；

过表达CHEK1-S剪接异构体影响巨核细胞倍体化；

CHEK1自身及其下游通路CDC25A与CDC25C蛋白的磷酸化水平升高；

选择性CHEK1抑制剂prexasertib可在体外促进U2AF1^S34F突变巨核细胞倍体化；小分子化合物tiliroside与U2AF1蛋白具有良好亲和力，体外实验可促进U2AF1^S34F突变巨核细胞倍体化；去甲基化药物地西他滨和阿扎胞苷可在体外促进U2AF1^S34F突变巨核细胞倍体化。

结论

    伴有U2AF1突变的MDS患者具有独特的临床特征，U2AF1突变在患者与小鼠体内均抑制巨核细胞成熟与倍体化过程，导致低倍体巨核细胞增多，高倍体巨核细胞减少，伴有U2AF1突变的巨核细胞相对不成熟且产生更多的TNF-α。机制研究表明CHEK1的选择性剪接导致CHEK1蛋白表达改变，进而引起下游通路异常，导致异常巨核细胞生成，靶向该通路可能成为MDS治疗新思路。

Background and Aim

Myelodysplastic syndrome（MDS）is a group of heterogeneous malignant clonal diseases,  originating from hematopoietic stem/ progenitor cells, characterized by cytopenia, dysplasia of one or more blood cell lineages, ineffective hematopoiesis, and a high risk of transformation to leukemia. Spliceosomal gene mutations are among the most common genetic changes observed in MDS, and occur at a high frequency of about 50%. Mutations in U2AF1 are observed in about 10-20% patients with MDS. U2AF1 is the common target of somatic point mutations in MDS, indicating that abnormal alternative splicing of pre-mRNA driven by mutant U2AF1 plays important role in MDS pathogenesis. Previous studies demonstrated that U2AF1^S34F impairs erythroid differentiation and skews granulomonocytic differentiation toward granulocytes, but its influence in megakaryocytic lineage remains unclear. To investigate the role of U2AF1^S34F mutation in dys-megakaryopoiesis in MDS, we retrospectively analyzed the clinical characteristics of MDS patients with U2AF1 mutation in our center, and established an in vitro megakaryocytic differentiation system using patient CD34⁺ cells and U2AF1 mutant cell lines.

Methods

Newly diagnosed MDS patients with complete next generation sequencing data were recruited to compare the clinical characteristics between those with U2AF1 mutant and wildtype MDS patients.

Immunohistochemical staining was utilized to perform CD41 staining on bone marrow pathology sections and smears from patients with U2AF1 mutations (MDS^MT) and those without splicing factor mutations (MDS^WT), followed by statistical analysis of the number and diameters of megakaryocytes (MKs) and the proportion of abnormal MKs in the bone marrow.

Bone marrow (BM) MKs were separated by BSA velocity sedimentation method, and flow cytometry was employed to detect the polyploid of MKs in U2AF1^MT and U2AF1^WT MDS patients.

U2AF1^S34F retroviral mice model was established to evaluated MKs maturation and polyploidization through flow cytometry and pathological morphology analysis.

Alternative splicing (AS) events analysis was based on the bone marrow mononuclear cells (BMMNC) RNA-Seq file from healthy donor (HD, n=5), MDS without splicing mutation (MDS^WT, n=16) and MDS with U2AF1^S34 mutation (MDS^MT, n=16). rMATS analysis identified aberrantly spliced genes caused by U2AF1 mutations, followed by Gene Ontology (GO) enrichment analysis to identify target genes.

Validation of target genes was carried out in patients’ BMMNC using qPCR and RT-PCR.

An in vitro MK differentiation system using patients’ CD34⁺ cells was established, and CD41⁺ single cell was sorted by flow cytometry, then single-cell PCR targeted sequencing technology was utilized to detect the aberrant splicing of target genes in CD41⁺ single cells with U2AF1 mutations.

We established inducible overexpression stable HEL and K562 cell lines, specifically overexpressed U2AF1 wildtype or mutant proteins. MKs differentiation system was established to assess the degree of polyploidization in HEL and K562 cells harboring U2AF1 mutations.

We used shRNA technology to knock down target gene in HEL and K562 cell lines, with the goal of testing the impact of target gene on megakaryocytic polyploidization.

Plasmid overexpressing isoforms was constructed and transfected into cell lines to assess the expression levels of isoform. In vitro MKs differentiation experiments were conducted to examine the effect of isoforms on the process of polyploidization.

The phosphorylation levels of CHEK1 and its downstream pathway proteins were assessed using Western Blot.

Exploring whether different drugs can promote the maturation and polyploidization of U2AF1 mutant MKs.

Results

U2AF1 mutation in MDS patients was associated with prevalence of male, younger age, lower peripheral blood hemoglobin levels, increased MKs numbers on peripheral blood smears, elevated serum TNF-α levels, and higher fibrosis grades.

In MDS^MT patients, there was a higher count of MKs per unit area, smaller cell diameters on sections, and higher proportion of abnormal megakaryocytes on smears, with a predominant increase in micro-megakaryocytes (cell diameter ≤40μm, micro-MKs).

In agreement with morphological findings, we also found that in CD41⁺CD42⁺ populations, MDS^MT have more low-ploidy MKs (2N and 4N micro-MKs) compared to MDS^WT (Figure C), accompanied by less high-ploidy MKs, which denoted that U2AF1^S34F induced a marked defect in MK polyploidization.

U2AF1^S34F mutation caused MK progenitor cells significantly increased in mice, and bone marrow pathology of these mice showed increased number of MK and MK clustering.

RNA-sequencing showed that the aberrant splicing of genes related to the DNA synthesis and cell cycle checkpoint pathways were predominantly affected by U2AF1^S34F.

Isoform expression of CHEK1 was markedly altered, with a short isoform of CHEK1 (CHEK1-S) highly expressing in BMMNC samples with U2AF1^S34F mutation.

RT-PCR and quantitative real-time also confirmed the significant CHEK1 isoform imbalance (ratio of S/L from 0.27 to 4.28, P<0.001) in single CD41+ cell with U2AF1^S34F, compared to those without U2AF1^S34F.

Notable increase of 2N cells and decrease of 4N and 8N cells were seen in HEL and K562 cells with U2AF1^S34F mutation. Megakaryocytes with U2AF1^S34F mutation were relatively immature and had higher levels of TNF-α.

Aberrant splicing of CHEK1 led to reduced expression of the CHEK1-L protein, and knocking down of CHEK1-L in HEL and K562 cells can inhibit MKs polyploidization.

Overexpression of the CHEK1-S affected MKs polyploidization.

Phosphorylation levels of CHEK1 and its downstream pathways CDC25A and CDC25C were elevated in U2AF1^S34F HEL cells.

CHEK1 inhibitor prexasertib can promote polyploidization of U2AF1^S34F mutant MKs in vitro; the small molecule compound tiliroside exhibits high affinity with U2AF1 protein and promotes polyploidization of U2AF1^S34F mutant MKs in vitro; decitabine and azacitidine can promote polyploidization of U2AF1^S34F mutant MKs in vitro.

Conclusions

MDS patients with U2AF1 mutation exhibit unique clinical characteristics. U2AF1 mutation inhibited the maturation and polyploidization of megakaryocytes in both patients and mice, resulting in an increase of low-ploidy megakaryocytes and a decrease of high-ploidy megakaryocytes. MKs with U2AF1 mutation are relatively immature and produce more TNF-α. Aberrant splicing of CHEK1 led to isoform switching, subsequently causing abnormalities in downstream pathways and thus resulting in the generation of abnormal MKs，especially micro-MKs in MDS. This study explained the mechanism of dys-megakaryopoiesis in MDS and may serve as a novel therapeutic target in MDS.