1.目的：在费城染色体阴性骨髓增殖性肿瘤（myeloproliferative neoplasm，MPN）中，JAK2^V617F是最主要的驱动基因突变。此外，MPN患者还往往伴随多种表观遗传基因突变，其中ASXL1是最常见的协同突变基因之一，其存在与患者总存活率降低和继发性急性髓系白血病转化风险的增加密切相关。本研究以携带JAK2^V617F纯合突变的人红白血病细胞HEL为基础，建立ASXL1和JAK2^V617F双突变模型，为研究JAK2^V617F与ASXL1协同参与MPN疾病进展提供模型。

方法：利用CRISPR/ Cas9介导的基因编辑技术建立ASXL1基因敲除的HEL细胞系，并进行一系列细胞功能实验来验证ASXL1敲除对HEL细胞增殖、周期以及克隆形成能力的影响。我们利用多种髓系肿瘤治疗药物对HEL-AKO细胞进行了处理，通过加药后HEL-AKO细胞系的克隆形成实验、IC50测定等明确了ASXL1敲除后HEL细胞对多种药物的敏感性变化。我们利用实时定量PCR的方法检测了HEL-AKO细胞中原癌基因HOXA的mRNA表达，进一步通过ChIP-qPCR验证初步探索了ASXL1对HEL细胞产生影响的内在分子机制。

结果：成功构建ASXL1基因完全敲除的HEL细胞系（HEL-AKO），模拟JAK2^V617F 突变与ASXL1基因功能缺失性突变并存。细胞功能实验发现，HEL-AKO细胞较HEL细胞增殖能力变慢，细胞周期G2/M期的进程延长，且HEL-AKO细胞的克隆形成能力减弱，药物敏感实验表明，与HEL细胞相比，HEL-AKO细胞对芦可替尼（ruxolitinib）、地西他滨（decitabine）和伏立诺他（vorinostat）产生了显著的耐受性。实时定量PCR的结果显示，促白血病转化的HOXA基因表达呈显著上调。

结论：运用CRISPR-Cas9基因编辑技术能够实现对JAK2^V617F突变的人红白血病细胞系HEL细胞系的特定编辑，ASXL1基因敲除之后对HEL细胞的生物学功能产生影响，原癌基因HOXA表达上调，并且对髓系肿瘤常用药物地西他滨和伏立诺他产生耐受性。该模型可以用于进一步探讨JAK2^V617F背景下ASXL1发生功能缺失性突变后对骨髓增殖性肿瘤进展的作用机制研究。

目的：在费城染色体阴性骨髓增殖性肿瘤（myeloproliferative neoplasm，MPN）中，JAK2^V617F是最主要的驱动基因突变。此外，MPN患者还往往伴随多种表观遗传基因突变，其中ASXL1是最常见的协同突变基因之一，其存在与患者总存活率降低和继发性急性髓系白血病转化风险的增加密切相关。本研究以携带JAK2^V617F纯合突变的人红白血病细胞HEL为基础，建立ASXL1和JAK2^V617F双突变模型，为研究JAK2^V617F与ASXL1协同参与MPN疾病进展提供模型。

方法：利用CRISPR/ Cas9介导的基因编辑技术建立ASXL1基因敲除的HEL细胞系，并进行一系列细胞功能实验来验证ASXL1敲除对HEL细胞增殖、周期以及克隆形成能力的影响。我们利用多种髓系肿瘤治疗药物对HEL-AKO细胞进行了处理，通过加药后HEL-AKO细胞系的克隆形成实验、IC50测定等明确了ASXL1敲除后HEL细胞对多种药物的敏感性变化。我们利用实时定量PCR的方法检测了HEL-AKO细胞中原癌基因HOXA的mRNA表达，进一步通过ChIP-qPCR验证初步探索了ASXL1对HEL细胞产生影响的内在分子机制。

结果：成功构建ASXL1基因完全敲除的HEL细胞系（HEL-AKO），模拟JAK2^V617F 突变与ASXL1基因功能缺失性突变并存。细胞功能实验发现，HEL-AKO细胞较HEL细胞增殖能力变慢，细胞周期G2/M期的进程延长，且HEL-AKO细胞的克隆形成能力减弱，药物敏感实验表明，与HEL细胞相比，HEL-AKO细胞对芦可替尼（ruxolitinib）、地西他滨（decitabine）和伏立诺他（vorinostat）产生了显著的耐受性。实时定量PCR的结果显示，促白血病转化的HOXA基因表达呈显著上调。

结论：运用CRISPR-Cas9基因编辑技术能够实现对JAK2^V617F突变的人红白血病细胞系HEL细胞系的特定编辑，ASXL1基因敲除之后对HEL细胞的生物学功能产生影响，原癌基因HOXA表达上调，并且对髓系肿瘤常用药物地西他滨和伏立诺他产生耐受性。该模型可以用于进一步探讨JAK2^V617F背景下ASXL1发生功能缺失性突变后对骨髓增殖性肿瘤进展的作用机制研究。

2.目的：ASXL1(Additional sex combs-like 1 )在表观遗传调控中起着重要作用，其突变通常与髓系肿瘤相关，例如骨髓增生异常综合征(myelodysplastic, MDS)、急性髓系白血病(acute myeloid leukemia, AML)和骨髓增殖性肿瘤(myeloproliferative neoplasm, MPNs)等。最近的研究报道，ASXL1突变可以通过改变组蛋白修饰导致造血功能受损。而且也有研究表明，与正常人相比，急性髓细胞白血病(AML)患者中大约29%的全基因组基因发生了异常剪接。剪接是在真核基因表达中不可或缺的过程，成熟mRNA的产生需要转录与剪接相互协调，共同完成。剪接过程中，剪接位点的选择也受RNA聚合酶II延伸率、染色质重塑和组蛋白修饰的影响。本研究旨在确定选择性剪接(Alternative splicing, AS)的异常是否与ASXL1突变有关以及探索ASXL1参与髓系恶性肿瘤发生的其它可能机制。

方法:利用rMATS分析Asxl^-/-和WT小鼠的造血干祖细胞(Lineage negative and c-Kit positive, LK) RNA-seq结果中的差异选择性剪接事件，并使用RMATs2SASHimiplot软件将结果进行可视化。随后利用CRISPR/ Cas9介导的基因编辑技术，在32D细胞中敲除Asxl1基因，建立32D-AKO的细胞系，并利用蛋白免疫印迹试验(Western Blot)来检测ASXL1缺失后组蛋白修饰的改变情况，利用实时定量PCR(real-time PCR)在细胞系中进一步检测异常的mRNA剪接事件，进一步利用ChIP-qPCR验证剪接因子上组蛋白修饰的结合情况，初步探索Asxl1影响选择性剪接的内在机制。

结果：从RNA-Seq的分析结果中我们发现，与正常WT小鼠的LK细胞相比，Asxl1^-/- 小鼠的LK细胞中可变剪接事件显著增加，这些剪接事件包括选择性3’剪接位点、选择性5’剪接位点、内含子保留、外显子跳跃，其中显著增加的选择性剪接事件是外显子跳跃。此外，ASXL1敲除后多种剪接因子的表达降低。在32D-AKO细胞中，与造血细胞增殖、分化相关的多种基因短转录本形式(short-form transcripts)表达水平的增加也得到进一步验证，ChIP-qPCR实验证明，H3K4me4在剪接因子U2AF1、SRSF3启动子区结合显著减少，导致其表达降低，我们的研究表明与ASXL1突变相关的异常RNA剪接增加可能是ASXL1参与髓系恶性肿瘤发生发展的内在机制之一。

结论：ASXL1的缺失可以引起选择性剪接改变，并且主要以外显子跳跃改变为主，并且可以通过影响组蛋白修饰水平引起剪接因子表达异常，由此可见，ASXL1突变可能通过对选择性剪接产生影响来参与髓系肿瘤的进程。

关键词：ASXL1；髓系肿瘤；选择性剪接；剪接因子

1.Purpose       JAK2^V617F is the most frequently found somatic mutation in patients with myeloproliferative neoplasms (MPN). The co-occurrence of ASXL1 and JAK2^V617F mutation is associated with reduced overall survival and increased risk of leukemia transformation. Although previous studies demonstrated that ASXL1 loss cooperated with JAK2V617F mutation, promoted MPN progression, and associated with poor prognosis, but the underlying mechanisms are still unknown. In this study, human leukemia cell line HEL that carried homozygous JAK2^V617F mutation is used to elucidate the role of ASXL1 loss of function mutation on the myeloproliferative neoplasms, which will provide an important model for the study of the role of ASXL1 mutation on the myeloproliferative neoplasms at the cellular level.

Methods: In order to study the pathogenic mechanism of ASXL1 mutation with JAK2^V617F to accelerate myelofibrosis progression, HEL cell line with ASXL1 knockout (HEL-AKO) have been established by using CRISPR/Cas9-mediated gene editing. And a series of experiments based on biological function were preformed to verify the effect of ASXL1 on HEL cell proliferation, clone formation and sensitivity to chemotherapeutic drugs. Colony‑forming cell and cytotoxicity assays were performed to test target drugs induced cell biological phenotypes. The mRNA expression of HOXA genes were also measured in HEL-AKO cells.

Results: HEL-AKO cell line was successfully generated and futehre verified by sequencing results. We found that loss of ASXL1 could inhibit proliferation and induce cell cycle arrest at the G2/M phase. And the colony-forming capacity of HEL-AKO cells is also markedly inhibited. Moreover, the HEL-AKO had higher cloning forming efficiency than HEL CTRL after Ruxolitinib, Decitabine or Vorinostat treatment.

Conclusion: Our study showed that ASXL1 knockout influenced the growth and cell cycle of HEL cells. In addition, loss of ASXL1 could induce resistance to multiple therapeutic drugs. Therefore, HEL-AKO cell line can be used to further explore the underlying mechanisms of concomitant ASXL1 mutation in JAK2^V617F positive MPN.

Key words：ASXL1; JAK2^V617F; Myeloproliferative neoplasms; CRISPR/ Cas9

2.Purpose Additional sex combs-like 1 (ASXL1) plays a critical role in epigenetic regulation, and its mutations are commonly associated with myeloid neoplasms such as myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and myeloproliferative neoplasms (MPNs). Recent studies have suggested that ASXL1 mutation led to impaired hematopoiesis through altered histone modification. A recent study demonstrated that approximately 29% of the genome-wide expressed genes were aberrantly spliced in patients with acute myeloid leukemia (AML) compared with healthy donors. Pre-mRNA splicing occurs largely co-transcriptionally, and the choice of alternative splice site is influenced by RNA polymerase II elongation rate, chromatin remodelers, and histone modifications. It is unknown how aberrant AS would be related to the mutation of ASXL1. In this study, we aim to identify the connection between ASXL1 and malignancies.

Methods Lineage negative and c-Kit positive (LK) cells were collected from the bone marrow of mice with complete knockout of ASXL1(ASXL1^-/-) for RNA sequencing. rMATS was applied to detect differential alternative splicing in LK cells from ASXL1^-/- and WT mice, and RMATs2SASHimiplot was used to visualize the results. We then generated 32D cell line with ASXL1 knockout (32D-AKO) using CRISPR/Cas9-mediated gene editing and examined the expression of ASXL1 in 32D and 32D-AKO cells by Western blot analysis with an antibody against H3K4me3, H3K27me3 and H2AK119Ub et al. The aberrant mRNA splicing events were further determined by real-time PCR. ChIP-qPCR were performed to reveal the underlying mechanisms of ASXL1 loss on splicing.

Results: We found out that the alternative splicing events significantly increased in ASXL1^-/- LK cells compared with WT LK cells, including alternative 3’ splice site, alternative 5’ splice site, intron retention, mutually exclusive exons, and especially exon skipping. The increased expression levels of short-form transcripts of multiple genes associated with hematopoietic cell proliferation and differentiation were further validated in 32D-AKO cells. ChIP-qPCR validated decreased enrichment of H3K4me3 in the transcription start sites of multiple splicing factors. Our study indicated that abnormal RNA splicing associated with ASXL1 mutation might relate to the pathogenesis of myeloid malignancies.

Conclusion: The loss of ASXL1 have an impact on alternative splicing, which can cause abnormal expression of splicing factor by affecting histone modification level. Therefore, the ASXL1 mutation is involved in the progression of myeloid malignancies through influencing alternative splicing

Keywords: ASXL1; myeloid leukemia; alternative splicing; splicing factors