骨髓微环境SLIT2-ROBO1信号失活促进MDS细胞恶性增殖的作用机制及靶向干预研究

SLIT2-ROBO1 signaling inhibits stem cell expansion, cell migration and angiogenesis, and its abnormalities can lead to tumorigenesis. However, its role in myelodysplastic syndrome (MDS) has not been studied. Our previous study has shown that inactivated ROBO1 mutations are frequent in MDS cells and associated with increased risk of leukemic transformation. Further study showed that ROBO1 ligand SLIT2 expression was significantly reduced in bone marrow supernatant of MDS. Co-culture of SLIT2-depleted normal MSC with MDS cells significantly promoted cell proliferation and induced apoptosis resistance to cytotoxic agents. Based on these data, we speculate that SLIT2-ROBO1 signaling is a tumor suppressor signal, and its suppression promotes malignant proliferation of MDS cells. In this project, we will investigate the mechanism of SLIT2-ROBO1 signal inactivation in MDS malignant proliferation and the potential target intervention by performing a series of functional experiments including virus transfection, gene expression profile, western blotting, ChIP-sequencing, mouse transplantation model, etc. Finally, this project can provide some novel findings in MDS pathogenesis on microenvironment and scientific evidence for novel targeted therapy.

SLIT2-ROBO1/2信号调控干细胞生成、细胞迁移和血管生成，其异常可导致肿瘤发生。然而该信号在骨髓增生异常综合征(MDS)中的作用尚不清楚。我们前期研究显示MDS患者SLIT2受体ROBO1/2频繁发生失活性突变，突变患者转白风险增加。进一步研究显示MDS骨髓上清SLIT2表达明显降低，SLIT2敲除的正常间充质干细胞与MDS细胞共培养后明显促进MDS细胞增殖且对细胞毒药物诱导的凋亡产生抵抗。申请人推测SLIT2-ROBO1/2信号是一个抑癌信号，该信号受抑有助于MDS恶性细胞增殖和凋亡抵抗。本项目在前期基础上，采用病毒转染、基因表达谱、WB、ChIP测序、小鼠移植模型、药物干预等功能学实验，阐述SLIT2-ROBO1/2信号失活在MDS恶性增殖中的作用机制及可能的靶向干预。该项目有助于理解SLIT2-ROBO1/2信号在微环境发病机制中的作用，为靶向骨髓微环境治疗提供依据。

SLIT2-ROBO1/2信号调控干细胞生成、细胞迁移和血管生成，其异常可导致肿瘤发生。然而该信号在骨髓增生异常综合征(MDS)中的作用尚不清楚。在本研究中，定量PCR分析显示高危MDS患者尤其是RAEB-T患者存在明显低的ROBO1表达，ROBO1低表达患者存在缩短的生存时间。ELISA分析显示ROBO1配体SLIT2表达明显降低，且与MDS WHO分型风险相关。体外实验显示外源性SLIT2加入激活SLIT2-ROBO1/2信号，明显抑制MDS/白血病细胞增殖，诱导细胞凋亡；而抑制ROBO1/2则使该信号失活，失去对肿瘤细胞的增殖抑制作用。此外，SLIT2敲除的骨髓间充质细胞与MDS/AML细胞共培养后导致肿瘤细胞增殖增加，且对TNF-α诱导的凋亡耐受。上述提示骨髓间充质依赖的SLIT2-ROBO1/2信号是一个抑癌信号，该信号受抑有助于MDS恶性细胞增殖和凋亡抵抗。我们进一步采用基因表达谱芯片筛选SLIT2敲除MSC或正常MSC与MDS细胞共培养后差异性表达基因，Pathway整合分析显示在PI3K-AKT和TP53信号通路可能作为SLIT2-ROBO1/2信号的下游通路。定量PCR验证结果显示SLIT2-ROBO1/2信号抑制MYC、CCND1和BCL2的基因表达，促进TP53和P21表达。MYC、CCND1和BCL2是PI3K-AKT信号通路的关键核内靶基因。免疫印迹分析显示SLIT2-ROBO1/2信号抑制AKT和GSK3β磷酸化水平，导致GSK3β增加和核内β-catenin减少，从而抑制靶基因转录。这些数据表明SLIT2-ROBO1/2信号失活导致下游PI3K-AKT信号通路的过度激活，从而促进了MDS肿瘤细胞的恶性增殖和凋亡抵抗。该项目实施有助于理解SLIT2-ROBO1/2信号在微环境发病机制中的作用，为靶向骨髓微环境治疗提供依据。