特异降解AML1-ETO的融合蛋白TAT-MLLRD-NHR2的制备与应用研究

Acute leukemias account for approximately 1/3 of all childhood cancers. Most leukemias begin with a chromosome translocation, which results in the production of an incorrect oncoprotein which functions differently than the correct form. AML1-ETO, which disrupted tumor suppressor gene AML1, is one of the most frequent ones in acute myeloid leukemias. The leukemia cells containing AML1-ETO were absolutely dependent on the presence of this oncoprotein, and their growth and survival were severely impaired with experimental repression of AML1-ETO. This general phenomenon is well known as "oncoprotein addiction". Thus, targeted inhibition or degradation of AML1-ETO will provide a ideal way to kill the leukemia cells, with little or no effect to the normal cells. .In our previous study, we found in the 293T overexpression system that the repression domain of MLL, MLL-RD, is able to degrade AML1-ETO dramatically. Taking advantage of a well-known AML1-ETO binding motif, NHR2, we're going to generate a novel fusion protein in this proposal: TAT-MLLRD-NHR2, which contains the cell penetration peptide TAT, the AML1-ETO targeted domain NHR2, and the AML1-ETO degradation domain MLL-RD. We hope this fusion protein will work as a "silver bullet" which targets and specifically degrade oncoprotein AML1-ETO, leaving the normal blood cells that have no AML1-ETO unaffected. TAT-MLLRD-NHR2 will be expressed in one of the most widely used mammalian cell expression systems-Chinese hamster ovary cell expression system, and Akta purification system will be used for the protein purification procedure. The purified TAT-MLLRD-NHR2 will be tested on in vitro and in vivo models..The AML1-ETO positive human leukemia cell line, Kasumi-1 and SKNO-1 were used as in vitro research models, HL-60 which has no AML1-ETO is taken as control. After adding the fusion protein TAT-MLLRD-NHR2 in culture medium of these cell lines, the AML1-ETO will be detected by western blot, morphology and the growth curve of these cell lines will be determined. We'll also analyze the cell apopotosis by annexin V/PI staining and DNA ladder assay, as well as western blot of apopotosis related molecules such as Bcl-2, Bcl-xL, caspase3. Cell cycle and differentiation will also be detected..We also plan to build up mouse xenograft leukemia models by injection of AML1-ETO positive human leukemia cell lines through tail vein of NOD/SCID mice. TAT-MLLRD-NHR2 will be administrated and the in vivo AML1-ETO protein level will be detected by western blot. The total blood count, as well as the body weight and survival of these mice will be monitored. Spleen and bone marrow cells will be analysed at given time points by flow cytometry to see the difference of LSK and SLAM populations among the groups. .We believe that our study on the preparation and application of fusion protein TAT-MLLRD-NHR2 will inspire leukemia researchers with new approach and useful tool for AML1-ETO related targeted therapy and mechanism study.

AML1-ETO是急性髓性白血病患者中常见的癌蛋白，能够抑制细胞分化并促进其恶性增殖。利用反义核酸、siRNA或冬凌草甲素特异地靶向AML1-ETO可以造成其mRNA下调或蛋白降解，促使白血病细胞分化或凋亡，而不影响正常血细胞的功能，因此，AML1-ETO是理想的白血病靶向治疗靶标。我们在前期研究中发现蛋白结构域MLL-RD对AML1-ETO具有强烈的降解作用；本申请拟利用特异靶向识别AML1-ETO的NHR2结构域，构建融合蛋白TAT-MLLRD-NHR2，并在哺乳动物细胞表达系统中对其进行表达与纯化制备。本申请还将探索该融合蛋白在体外模型（AML1-ETO阳性细胞系Kasumi-1和SKNO-1）与体内模型（注射AML1-ETO阳性细胞系的NOD/SCID小鼠）中对AML1-ETO的降解及作用。本申请将为白血病的靶向治疗与AML1-ETO在白血病发生发展中作用的机理研究提供新思路。

为了特异降解白血病癌蛋白AML1-ETO以实现靶向治疗，本研究设计、优化并构建了能够特异降解AML1-ETO的融合蛋白TAT-MLLRD-NHR2（TAT-CMN）和REV-MLLRD-NHR2（REV-CMN）。首先，将MLL-RD降解AML1-ETO的结构域由原先的一千多个氨基酸缩减至核心的约60个氨基酸（CXXC结构域，1154-1211aa）；其次，通过对比各种细胞渗透肽与EGFP的融合蛋白进入各白血病细胞系的情况，选取REV和TAT构建CMN（REV更佳）。REV-CMN与TAT-CMN连入我室高效真核表达载体，筛选到高表达CHO细胞株，并进行了小规模的扩大培养，经镍柱、Sephadex G25分子排阻层析纯化，获得纯度>95%的足量蛋白质。REV-CMN和TAT-CMN可抑制AML1-ETO阳性的白血病细胞系生长，但并不影响AML1-ETO阴性白血病细胞系的生长。.本课题的顺利开展，证明这种特异降解癌蛋白的思路可能，这种设计融合蛋白的思路还可推广用于特异降解诸如TEL-AML1和CBF-SMMHC等其他白血病相关癌蛋白，并可用作研究白血病相关癌蛋白致病机理的有力工具。

内容获取失败，请点击重试