靶向蛋白磷酸酶2A的新型预激方法对高危骨髓增生异常综合征恶性克隆细胞的作用研究

The treatments of patients with high risk myelodysplastic syndromes （MDS）still present a difficult problem. Up to now, the disease is regarded as a kind of refractory malignant clonal disease. The incurability is due to a subpopulation of cell cycle quiescent cancer cells which is insensitive to drugs targeting cell growth and replication. The important mechanisms are related to cell cycle arrest and DNA damage defense of tumor cells. The traditional cell cycle stimulation strategy by using the granulocyte colony-stimulating factor (G-CSF) with low dose chemotherapy proves to be effective partially in patients with high risk MDS with excess blasts, yet it still has limitation and needs to be improved. In previous our studies, we demonstrated that a small molecule compound named LB1, an inhibitor of protein phosphatase 2A (PP2A) can drive quiescent cancer cells into cell cycle and simultaneously inhibits cycle arrest despite the presence of chemotherapy induced by DNA-damage. In combination with other chemotherapeutic agents it substantially enhances the anti-tumor effect by inhibiting cell-cycle arrest and inducing cell cycle progression. The effect of inhibiting cell-cycle arrest and inducing cell cycle progression has also been confirmed in MDS clonal cells. This project is try to study the potential current treatment effect on MDS clonal cells by adding the new cell-cycle stimulator, LB1 or in combination with G-CSF in chemotherapy in vivo and in vitro. We will use a series of new methods including molecular biology technique, gene chip, confocal laser scanning technique and microPET et al to determine the efficacy and superiority of the novel treatment strategy with LB1. The molecular mechanism involved in the new combination will also be investigated to provide the theory basis of clinical application in the treatment of high risk MDS.

骨髓增生异常综合征(MDS)是恶性血液病治疗领域的难题, 高危MDS难治与肿瘤细胞休眠及启动DNA损伤防御机制密切相关。传统基于G-CSF的预激方法治疗MDS有局限性。我们前期合成的低毒性小分子斑蝥素衍生物LB1，具有靶向抑制蛋白磷酸酶2A的作用，在实体瘤中证实通过调节G2/M周期节点、促使G0期细胞活化、阻断DNA损伤修复等机制，增强难治性肿瘤细胞的化疗敏感性；我们发现LB1对MDS恶性克隆细胞也有同样作用。本研究拟将作用于G2/M的LB1与作用于G1/S的G-CSF联合预激方法，从多个周期节点对MDS细胞进行预激，用基因芯片、分子生物学、激光共聚焦、MicroPET等技术，对MDS肿瘤细胞及荷瘤小鼠进行体内、外研究，探讨新型预激方法对MDS细胞的作用，研究新型预激与传统预激的区别，阐明其增强化疗敏感性的分子机制，证实新型预激较传统预激的优越性。为高危MDS的临床治疗提供新的方法和策略。

蛋白磷酸酶2A（PP2A）是一个高度保守的特异性磷酸酶，通过和丝氨酸/苏氨酸相互作用在调节细胞周期蛋白活性和细胞凋亡中发挥着重要的调控作用。本课题组合成了一种PP2A特异性的小分子药物——LB1，通过MTT、流式细胞学、ELISA、Western Blot、PCR、克隆形成以及小鼠荷瘤模型构建等一系列实验方法，探索了其在高危MDS中的作用。结果发现LB1可以有效的抑制MDS细胞的活性（IC50为5.35μM），诱导G2/M细胞周期阻滞，促进细胞发生凋亡，抑制MDS细胞克隆形成能力。低剂量的LB1不仅在体外细胞株和MDS患者原代细胞中可以明显地增敏柔红霉素的细胞毒作用（P<0.001），且在小鼠体内荷瘤模型中也可以显著抑制肿瘤负荷（P<0.001），延长小鼠总体生存时间（P=0.002）。进一步的研究发现，LB1是通过诱导miR-181b-1的表达，从而抑制其下游靶基因BCL-2，继而达到增强柔红霉素细胞杀伤作用的。我们的数据表明LB1联合柔红霉素是高危MDS患者治疗的新方案。此外，我们发现在MDS中，地西他滨序贯联合去甲氧柔红霉素的治疗方案可以通过上调Wnt信号通路上游抑制因子，抑制该通路活性，最终实现体内和体外良好的抑癌作用。该结果为今后临床MDS的治疗提供了很好的理论基础。而结合本实验室数据以及国内外有关IDH基因突变和MDS患者临床预后的文章进行Meta分析，我们还发现IDH基因突变是MDS患者预后不佳的重要因素。这一结果可以很好的完善现有MDS的预后评价系统。