GAS2调控慢性粒细胞白血病干细胞生长的研究

Growth arrest specific 2 (GAS2) could regulate calpain/beta-catenin pathway to maintain the growth of tumour cells including the BCR-ABL expressed murine bone marrow progenitor cells. We found that GAS2 was up-regulated in the CD34+ and CD34+CD38-Ph+ cells from the chronic myeloid leukemia (CML) patients compared to their normal counterparts. Targeting GAS2 with either RNAi or expression of the dominant negative form of GAS2 (GAS2DN) was capable to inhibit the growth of K562, MEG-01 and CD34+ cells from patients, which was partially dependent on the activation of calpain. Unexpectedly, we found that targeting GAS2 could inhibit CML cells through a beta-catenin independent signaling. However, the role of GAS2 and its underlying mechanism in the maintanence and initiation of leukemic stem cells has not been addressed. We propose to study the LTC-IC (long term culture-initiating cell) and xenoengraftment capacityies of CD34+CD38-IL1RAP+ cells from CML patients, when GAS2 in these cells was suppressed. In the other hand, we will deliver Gas2 alone or together with BCR-ABL into the lin-Sca-1+c-Kit+ cells from mice bone marrow to study the effect of these factors on hematopoiesis and the leukemogenesis. Mechanistically, we will utilize proteomic approach to identify key factors regulated by GAS2 to control the growth of CML primitive cells. We believe that the delineation of the role of GAS2 in CML stem cells will obtain novel molecular insights of these cells and provide opportunities to pursue novel therapies.

生长抑制特异蛋白GAS2可通过calpain/beta-catenin途径维持肿瘤细胞生长。我们发现慢性粒细胞白血病（CML）的干、祖细胞中GAS2较正常对照细胞表达显著增高；CML细胞株和原代CD34+细胞的生长需要GAS2；靶向GAS2导致的细胞生长减缓依赖calpain的活化但无需beta-catenin。然而，Gas2能否与BCR-ABL协同恶性转化正常干细胞、GAS2能否维持CML干细胞的生长和GAS2/calpain的关键下游分子是什么等核心问题尚未得到阐述。本项目将在上述预研基础上研究Gas2对BCR-ABL恶性转化能力的影响；检测干扰CML干细胞中GAS2表达对其长期培养起始细胞（LTC-IC）和小鼠移植能力的影响；使用蛋白质组策略揭示GAS2的作用机制。本项目的完成将首次阐述GAS2在CML干细胞中的功能和机制、完善对CML致病分子机制的认知，为改善CML治疗提供新策略。

慢性髓细胞白血病（chronic myeloid leukemia, CML）是源自造血干细胞的恶性血液病，BCR-ABL是该疾病的重要诱发因素，靶向BCR-ABL的酪氨酸激酶抑制剂已极大地改善了该疾病的治疗。然而，部分患者仍会发生药物耐受和疾病复发，这与酪氨酸激酶抑制剂无法清除CML干细胞密切相关。因此，深入研究CML干/祖细胞的生长机制有可能为改善疾病治疗提供新的理论依据和实验基础。本研究显示生长抑制特异蛋白GAS2（growth-arrest specific 2）在CML干/组细胞中较正常对照细胞显著高表达，GAS2沉默或表达GAS2的显性负性突变体（GAS2DN）能抑制CML干/祖细胞的生长。以小鼠BaF3/BCR-ABL细胞为模型，发现GAS2虽不能增强其体外生长能力，但显著增强其诱发小鼠白血病能力。GAS2DN抑制其体外生长和体内诱发白血病的能力。基因表达谱分析提示GAS2DN的表达能抑制核不均一核糖核蛋白HNRPDL（heterogeneous nuclear ribonucleoriboprotein D-like）的表达，qRT-PCR分析显示HNRPDL在CML患者CD34+细胞中表达显著高于正常造血CD34+细胞。HNRPDL能恶性转化BaF3细胞，表明它是一个新型癌蛋白；HNRPDL显著促进CML细胞生长。通过iTRAQ分析，发现异柠檬酸脱氢酶IDH2(Isocitrate dehydrogenase, mitochondrial)的表达受到GAS2DN的抑制，且与Calpain活性相关；而IDH2沉默抑制CML细胞生长。综上，本研究证实GAS2具有增强BCR-ABL恶性转化血液细胞的能力，而CML细胞的生长部分依赖GAS2高表达。GAS2在CML细胞中以多种方式发挥其功能，包括维持新型癌蛋白HNRPDL和代谢相关酶IDH2的表达。这些实验结果表明GAS2高表达在CML干/祖细胞的生长中具有重要作用，对GAS2功能和作用机制的深入研究有望为改善该疾病治疗提供新思路。