ASXL1突变对伴有JAK2V617F突变的原发性骨髓纤维化干细胞功能影响的研究

Mutations in ASXL1, an epigenetic modifier, are most common in patients with primary myelofibrosis (PMF). ASXL1 mutations change the transcription of some oncogenes and result in dysfunction of cancer stem cells. We found that PMF patients with ASXL1 mutations were more likely to present obvious splenomegaly. The upregulation of CXCR4 was found in ASXL1 knockdown-UKE1 cells with JAK2V617F mutation. Therefore, we speculate that ASXL1 mutations may directly or indirectly change the transcription of CXCR4 and result in abnormal activation of CXCL12-CXCR4/7 axis in PMF stem cell and that PMF stem cells with activation of CXCL12-CXCR4/7 axis have abnormal biological function and lead to splenomegaly and formation of tumor microenviroment. We will identify that how ASXL1 mutations regulate the histone modification through PRC1 and/or PRC2 and transcription of CXCR4 by chromatin immunoprecipitation assay and how abnormal activation of CXCL12-CXCR4/7 axis change the migration, proliferation and differentiation in cell lines with JAK2V617F mutation. According to the findings in vitro, we will confirm these results and identify that how the PMF stem cells with both JAK2V617F and ASXL1 mutations change the hematopoiesis microenviroment in bone marrow and spleen through CXCL12-CXCR4/7 axis in the mouse model. We hope to demonstrate the molecular mechanisms of ASXL1 mutations which associate with dysfuntion of PMF stem cells and formation of tumor microenviroment and find new therapeutic target on spleen reduction and fibrosis reversion.

ASXL1突变是原发性骨髓纤维化（PMF）中最常见的表观遗传相关基因突变。有证据表明ASXL1突变影响了原癌基因的转录，与肿瘤干细胞功能异常密切相关。我们发现伴有ASXL1突变的PMF患者脾肿大更为明显，敲降了ASXL1的JAK2V617F阳性的UKE1细胞CXCR4表达增高。据此推测ASXL1突变可能直接或间接影响了CXCR4的转录，PMF干细胞CXCL12-CXCR4/7通路异常激活导致干细胞生物学功能异常，参与了脾肿大和肿瘤微环境的形成。我们将利用JAK2V617F阳性细胞系和小鼠模型，通过染色质免疫共沉淀技术研究ASXL1突变如何通过PRC1和/或PRC2改变组蛋白修饰进而上调CXCR4表达和异常激活CXCL12-CXCR4/7通路对PMF干细胞迁移、自我复制、增殖、分化和肿瘤微环境的影响。通过对上述分子机制的研究，寻找新的可用于缓解PMF患者脾肿大和逆转骨髓纤维化的治疗靶标。

ASXL1突变是原发性骨髓纤维化（PMF）中最常见的表观遗传相关基因突变。有证据表明ASXL1突变影响了原癌基因的转录，与肿瘤干细胞功能异常密切相关。我们的前期研究发现伴有ASXL1突变的PMF患者脾肿大更为明显，敲降了ASXL1的JAK2V617F+的UKE1细胞CXCR4表达增高。据此推测ASXL1突变可能直接或间接影响了CXCR4的转录，PMF干细胞CXCL12-CXCR4/7通路异常激活导致干细胞生物学功能异常，参与了脾肿大和肿瘤微环境的形成。为了进一步探索在JAK2突变的背景下，ASXL1突变如何通过异常激活CXCL12-CXCR4/7通路对PMF干细胞迁移、自我复制、增殖、分化和肿瘤微环境产生影响，寻找新的可用于缓解PMF患者脾肿大和逆转骨髓纤维化的治疗靶标， 我们以患者临床数据、小鼠模型和肿瘤细胞系为研究平台进行了一系列表型及功能研究。我们分析了75例PMF患者外周血CD34+细胞比例，发现伴ASXL1突变的JAK2V617F+病人脾脏较无ASXL1突变的JAK2V617F+病人其外周血CD34+细胞比例显著增高，同时脾脏显著增大。在小鼠模型中，我们发现虽然ASXL1 fl/fl JAK2V617F小鼠和JAK2V617F 小鼠的脾脏肿大程度并无明显差别，但是ASXL1 fl/fl JAK2V617F小鼠的髓外造血更加明显，其脾脏中造血祖细胞比例，外周血c-kit+细胞比例均显著高于JAK2V617F小鼠。随后，我们进一步以细胞系及小鼠细胞为研究平台，探索JAK2突变背景下，ASXL1突变如何影响CXCR4的表达。我们发现ASXL1敲降后的JAK2V617F+SET2细胞系CXCR4总蛋白及膜表面表达水平均显著下降。ASXL1 fl/fl JAK2V617F小鼠脾脏c-kit+细胞CXCR4 mRNA表达水平亦较JAK2V617F小鼠低，而骨髓中c-kit+细胞CXCR4 mRNA表达水平无明显差异。我们推测可能与CXCR4低表达的c-kit+细胞已从骨髓迁出有关。综上，本研究阐述了伴ASXL1缺失的JAK2V617F+MPN相较JAK2V617F+MPN骨髓中HSPC迁出能力增强，脾脏髓外造血更为显著，其机制可能与ASXL1缺失使得HSPC的CXCR4表达水平降低从而易从骨髓中迁出并在脾脏中定植相关。