NRF2信号通路调控高危MDS进展及耐药的分子机制研究

Myelodysplastic syndromes (MDS) are a group of clonal stem cell disorders manifesting as cytopenias, mainly due to ineffective hematopoiesis. International Prognosis Scoring System (IPSS) provides a clinical model for the risk stratification of MDS. Patients with high-risk MDS are prone to progress to acute myeloid leukemia (AML) and resistant to treatment, the precise mechanism remains to be determined. Oxidative stress (OS), may lead to oxidant of proteins and DNA, is involved in MDS development. The NF-E2 related factor (NRF2), the Aryl Hydrocarbon Receptor (AHR) and Hypoxia-inducible transcription factor-1 alpha (HIF-1α) are transcription factors that are implicated in activating an antioxidant response. The latest research indicated that NRF2 plays a crucial role in the process of OS. And in some cancer cells the AHR competes with HIF-1α for the molecular chaperone HIF-1β, which is also known as Aryl hydrocarbon receptor nuclear translocator (ARNT). However, AHR/ARNT pathways protect cancer cells from apoptosis by up regulating NRF2. NRF2 over expression confers drug resistance in AML by augmenting antioxidant defense. Former findings show that HIF-1α signal is necessary and sufficient to induce dysplastic and cytopenic MDS phenotypes. Our previous studies in immunohistochemistry indicated that NRF2 expression in high-risk MDS was higher than that in low-risk MDS, and was associated with poor survival, whereas HIF-1α expression was opposite. In the further research, we will conduct a series of new methods including magnetic activated cell sorting, western blot, retrovirus transfection technique, RNA interference, animal models to assess OS levels and the connection among NRF2, AHR and HIF-1A in high-risk MDS cells. Investigating the important roles of NRF2 in ROS signaling pathway and chemo resistance in high-risk MDS will be significant for providing the theoretical basis of a new and clinical promising target in the treatment of MDS.

高危骨髓增生异常综合征（MDS）向急性髓系白血病（AML）转化率高，易发生耐药。氧化应激（OS）的激活是MDS发病及进展的重要因素。研究表明NRF2通路是调节OS的重要途径，在实体瘤中AHR激活使NRF2表达增加，HIF-1α/β参与其调节；在AML中NRF2还与耐药密切相关。前期研究发现，MDS患者HIF-1α广泛激活并与不良预后相关；高危MDS患者中NRF2表达增加，而HIF-1α表达却较低危组低下。推想在高危MDS细胞中，HIF-1α表达相对下调，使AHR与HIF-1β结合增强，致NRF2信号通路过度激活，这可能是细胞恶性转化及耐药的重要机制。本研究将应用基因转染、RNA干扰和小鼠模型等方法，研究高危MDS细胞OS状态及NRF2信号通路在疾病进展和耐药中的作用，阐明AHR促进NRF2信号通路调控高危MDS进展及耐药的分子机制，为以NRF2为靶点的MDS治疗提供新思路。

项目研究背景.骨髓异常增生综合征（Myelodysplastic syndrome，MDS）是造血干细胞的恶性克隆性疾病，其中高危患者易于向白血病转化，化疗效果差，易发生耐药。阿糖胞苷（Ara-C）是治疗高危 MDS 病人的传统药物。然而，MDS 病人对阿糖胞苷的治疗反应率不高。文献报道NRF2 与白血病耐药有关，但NRF2是否与MDS 患者的临床预后以及治疗耐药相关目前尚无研究。 .主要研究内容.1）对已发表的基因表达数据进行基因富集分析，免疫组化检测MDS 患者骨髓NRF2的表达情况，分析其对预后的影响。2）体外利用慢病毒转染构建NRF2 以及DUSP1 shRNA的MDS细胞株，利用 Western blot 和Q-PCR 验证敲低是否成功；利用 MTS检测 MDS 细胞活性和增殖情况，通过流式细胞术检测细胞凋亡和周期；通过尾静脉注射干扰表达的细胞至NOD/SCID-IL2Rγnull-SGM3 (NSGS)小鼠构建MDS 移植小鼠模型，进行后续体内实验。染色质免疫沉淀技术及 Q-PCR 验证NRF2 及DUSP1 的关系。 .重要研究结果 .1) NRF2下游靶基因在较高危MDS 患者中显著富集(P=0.020)。高危MDS患者骨髓NRF2 的表达高于低危患者(P=0.010)，NRF2高表达患者总生存期(OS) 比 低表达患者短 (391 天vs. 554 天, P=0.011)。 .2）药物激动或抑制NRF2后，MDS 细胞对阿糖胞苷耐药或增敏。NRF2 shRNA 慢病毒转染MDS 细胞后用阿糖胞苷处理时，细胞的凋亡显著增加并更倾向于S期阻滞。.3）基因对比发现AML耐药与高危MDS中共表达基因37个，选取DUSP1进行研究，染色质免疫沉淀技术及q-PCR 进一步验证 DUSP1是NRF2 的下游靶基因。DUSP1 的敲低能够逆转高NRF2 引起的阿糖胞苷耐药。.4）NRF2以及DUSP1敲低的MDS小鼠，经阿糖胞苷处理后肝脏肿瘤显著缩小(P=0.001)。.科学意义 .本研究发现NRF2 表达水平在高危MDS 患者中升高并与不良预后相关。NRF2 抑制剂能使 MDS 细胞对阿糖胞苷反应性增加， NRF2 是通过下游基因DUSP1在MDS中耐药作用。NRF2-DUSP1 靶向治疗与传统化疗的结合为将来高危 MDS 的治疗带来新希望。