靶向GAS2-Calpain轴抑制CML干细胞的研究

GAS2 (growth-arrest specific 2) is one of the growth-arrest specific proteins，which is an endogenous inhibitor of Calpain as well. Recently, we have demonstrated that GAS2 is a novel regulator of the stem/progenitor cells of chronic myeloid leukemia (CML), targeting GAS2 with both RNAi and the expression of GAS2Δ171-313 is sufficient to activate Calpain, inhibit the growth of CML cells and sensitize them to the treatment of Imatinib mesylate (IM), which suggests GAS2 is a potential therapeutic target to improve the management of CML. .However, how to translate this discovery to improve the treatment of CML is still a challenge. We hypothesize that the delivery of a short GAS2 homologous peptide to CML cells would be able to act as GAS2Δ171-313 to activate Calpain, inhibit the growth of these cells and sensitize them to the treatment of IM. In this proposal, the interaction between GAS2 and Calpain will be studied comprehensively to determine the critical region in GAS2 for its interaction with Calpain. We will also synthesize the GAS2 mimicking peptide and assess its ability to activate Calpain. We will investigate the effects of GAS2 mimicking peptide on the long-term culture initiating cell (LTC-IC) capacities and engraftment abilities in NOD/SCID mice of highly purified CML stem cells. We will also study the ability of GAS2 mimicking peptide to sensitize the quiescent CD34+ CML cells in the treatment of IM as well. The performance of this project will provide novel insights for the improvement of CML management by targeting GAS2-Calpain axis.

生长抑制特异蛋白GAS2（Growth arrest specific 2）是钙蛋白酶Calpain的内源性抑制剂。我们发现GAS2是慢性粒细胞白血病（CML）CD34+细胞的新调控因子；靶向GAS2（RNAi或表达GAS2Δ171-313）可通过活化Calpain抑制CML细胞生长、增强其伊马替尼（IM）敏感性。由于不存在已知的GAS2抑制剂，如何转化这项研究进展以抑制CML干细胞和静息期CD34+细胞仍是严峻挑战。在前期研究基础上，本项目将研究GAS2与Calpain结合的分子机制、确定GAS2与Calpain结合的关键性肽段（GAS2模拟肽），研究GAS2模拟肽对CML干细胞长期培养起始细胞（LTC-IC）能力、数量和小鼠移植能力的影响，探讨GAS2模拟肽单独或联合IM抑制静息期CD34+细胞的能力。本项目将为通过靶向GAS2-Calpain轴改善CML的治疗提供理论依据和实验基础。

人类慢性髓细胞白血病（CML）是源自造血干细胞的恶性血液病，靶向疾病特异的融合蛋白BCR-ABL的伊马替尼（Imatinib mesylate，IM）已极大改善了疾病的治疗。然而，药物耐受、疾病复发仍困扰部分患者，因而有必要更为深入地研究CML细胞的生长与生存机制。前期研究显示生长抑制特异蛋白2（Growth-arrest specific 2, GAS2）在人类慢性髓细胞白血病中高表达，通过表达其显性负性突变体（GAS2DN）能相对特异地抑制CML细胞的生长。为转化利用这一科学发现成果，我们假设GAS2DN的生长抑制功能集中在部分肽段。本项研究中，首先通过生化手段证实GAS2DN的生长抑制功能位于其C-端部分。合成GAS2蛋白N-端不具有生长抑制的部分肽段进行修饰，优化其转染K562和CML患者CD34+细胞的条件。在GAS2DN的C-端区域合成3个肽段，以N-端肽段为对照进行比较，发现肽段-B具有最强的生长抑制功能。它不仅能增强K562细胞的化疗药物敏感性，也能相对特异地抑制CML患者细胞的体外生长。最后，数据也显示肽段-B能抑制处于静息期的CML患者CD34+细胞的体外生长。这些结果显示本项目的基本科学假设正确，GAS2DN中的部分肽段也具有生长抑制功能，肽段-B能作为继续深入研究GAS2-Calpain2在CML细胞生长中的作用的新研究工具，有望为针对GAS2-Calpain2相互作用而设计小化合物提供重要基础，提供靶向GAS2-Calpain2轴而改善CML治疗的新策略。