新的逆转录病毒致癌基因Np9调控白血病干细胞自我更新信号通路分子机制的研究

It is well known that leukemia stem cell (LSC) plays a critical role in the initiation, drug resistance and relapse of leukemia, but it is unknown what factors determine the survival and self-renewal of LSC and how they regulate corresponding actions. Recently, we demonstrated that the regulatory gene Np9 of retrovirus is a novel viral oncogene. The viral protein Np9 encoded by this viral gene was detected in 56% of leukemia patients, but rare in normal hematopoietic stem cell samples. Moreover, the abundance of viral Np9 protein was positively correlated with the level of LSC in leukemia patients. Further studies showed that the viral Np9 was a critical molecule that simultaneously up-regulated multi-signaling molecules, such as β-catenin, involved in the survival and self-renewal of leukemia stem cells. However, its mechanism remains unclear. In this proposal, we will try to identify up-stream and down-stream partners interacting with the viral Np9 protein and corresponding pathways using human genome array,CoIP, Protein MS, Western blot, shRNA, etc. We will focus on the identification of signaling pathways essential for the survival and self-renewal of LSC and expect to reveal the molecular mechanism by which the viral Np9 regulates the self-renewal of LSC. These data will lay a foundation for the viral Np9 as therapeutic target of leukemia stem cells, and will be of scientific significance and potential application for clinic.

已知白血病干细胞(LSC)在白血病起始,耐药和复发中起着十分重要的作用,但调控LSC存活和自我更新的关键因子及相应分子机制尚未阐明。新近,我们在国际上率先证实逆转录病毒调节基因Np9是一种新的病毒致癌基因,其编码的Np9蛋白在56%的白血病人群中高表达,但在正常造血干细胞样本中未见表达,且其表达量与LSC呈显著正相关。进一步发现Np9是一种能同时上调β-catenin等多个与LSC存活和自我更新相关信号分子的关键分子之一,能强有力促进白血病细胞生长,但其作用机制不详。本项目拟应用全基因表达芯片,免疫共沉淀,蛋白质质谱,Western blot,shRNA等技术分离和鉴定Np9蛋白上下游信号分子及相应信号通路,重点关注与LSC存活和自我更新相关的信号通路,以期阐明Np9调控LSC自我更新信号通路的分子机制,为下一步以Np9作为新的白血病治疗性靶标奠定基础,具有重要科学理论意义和潜在应用价值。

背景:我们前期研究发现人内源性逆转录病毒HERV-K调节基因Np9是一种能同时上调β-Catenin等多个与白血病干细胞(LSC)存活和自我更新相关信号分子的病毒致癌基因，但其作用机制不详。主要研究内容：应用基因表达芯片、免疫共沉淀、蛋白质质谱、Western blot、shRNA等技术分离和鉴定Np9蛋白上下游信号分子及相应信号通路，重点关注与LSC存活和自我更新相关的信号通路，以期阐明Np9调控LSC自我更新信号通路的分子机制。重要结果和关键数据：（1）基因表达芯片和染色质免疫共沉淀-测序技术(ChIP-Seq)结果显示：Np9蛋白高表达广泛影响白血病干/祖细胞增殖相关基因及转录因子，其中c-Myc, HOXA3, MEIS1, STAT1等多个基因与LSC自我更新相关。（2）发现Np9蛋白与β-Catenin、c-Myc、MDM2等多个LSC自我复制相关的信号分子形成蛋白复合物。Np9蛋白可能通过劫持核β- catenin蛋白促进LSC增殖和自我更新（3）发现CaMKIIγ是Np9蛋白重要下游效应分子。应用CaMKIIγ基因敲除小鼠白血病模型证实，CaMKIIγ是髓系白血病干细胞存活和自我更新重要因子。与野生型CaMKⅡγ小鼠白血病模型相比，CaMKⅡγ缺失组小鼠骨髓和脾脏中的白血病干细胞水平降低了21.5倍 和5.1倍。机制研究发现，CAMKIIγ可能通过磷酸化和随后蛋白酶体依赖降解p27Kip1这一分子刹车来唤醒休眠期白血病干细胞，从而促进髓系白血病干细胞增殖与自我更新。(4) 我们发现CaMKIIγ是维持c-myc蛋白稳定性关键激酶之一。CaMKIIγ通过直接磷酸化c-myc蛋白方式稳定c-myc蛋白，促进淋巴瘤的发生。此外，我们还发现CaMKⅡγ通过磷酸化FOXO3a 促进T淋巴细胞白血病发生。 (5) 我们实验研究还发现，Np9过表达可诱导细胞基因组不稳定，产生新的融合基因，基因突变等。(6) 发现天然化合物雷公藤甲素是Np9小分子转录抑制剂。雷公藤甲素通过抑制逆转录病毒HERV-K Np9基因转录诱导人白血病细胞凋亡和生长抑制。科学意义：研究成果对于阐明病毒Np9及其效应分子CaMKIIγ在白血病干细胞中的作用及机制提供了新的思路，为下一步以Np9和CaMKIIγ激酶作为白血病治疗潜在靶标奠定基础。