摘要
[背景与目的]：白血病干细胞(Leukemia stem Cells, LSC)和正常造血干细胞(Hematopoietic Stem Cells, HSC)间的竞争与失衡伴随着白血病病程发展的全程,是决定治疗转归的关键。最新研究提示PI3K/PTEN/AKT/mTOR通路可能是纠正两者间失衡的理想干预靶点。尽管目前己发现两代mTOR复合物抑制剂,但其在临床应用中的效果并不理想。本研究应用细胞印迹高通量筛选技术,寻找mTOR复合物2特异性抑制剂。并初步探讨化合物Z1选择性降解部分混合谱系白血病(Mixed Lineage Leukemia,MLL)细胞中Rictor蛋白的分子机制,为MLL白血病的基因靶向治疗提供一定的理论基础。然而Rictor在MLL白血病中究竟扮演着什么样的角色呢?于是我们一方面探讨Rictor分子在MLL白血病细胞集落形成能力、增殖和凋亡等方面所起的作用；一方面体内研究Rictor分子在MLL白血病发病和维持中所起的作用。
[方法]：
1.高通量筛选技术寻找mTOR复合物2特异性抑制剂：采用细胞印迹高通量筛选(Cytoblot High Throughput Assay)技术,从华北制药集团新药研发中心小分子单体化合物文库中筛选mTOR复合物2特异性抑制剂,并进一步运用Western免疫印记法,初步验证筛选得到的小分子化合物对各种白血病细胞系中的mTOR复合物关键组分蛋白的抑制作用。
2.化合物Z1降解Rictor蛋白分子机制的初步探讨：以THP-1细胞作为Rictor降解机制研究的模型细胞,Western免疫印迹技术检测蛋白合成抑制剂放线菌酮(Cycloheximide, CHX)和Z1联合蛋白酶体抑制剂MG-132分别作用该细胞后Rictor蛋白表达水平的变化；荧光素酶-蛋白酶体活性分析技术(Luciferase-Proteasome-Glo Cell-Based Assay)检测Z1处理后细胞蛋白酶体活性变化；构建体外泛素化模型,检测Rictor蛋白泛素化修饰情况。
3. Rictor分子对MLL白血病细胞作用的体外实验研究：首先运用Rictor-shRNA慢病毒反义封闭技术对MLL白血病细胞、非MLL白血病细胞及正常细胞中的Rictor分子加以反义封闭；然后利用该系统所带GFP经流式分选获得GFP-Rictor-RNAi细胞；经流式细胞术、细胞免疫荧光术及Western免疫印迹法鉴定封闭效应后,通过甲基纤维素集落形成实验检测Rictor反义封闭对各种细胞集落形成能力的差别,并同时运用流式细胞术检测相应细胞增殖和凋亡情况的差别。
4. Rictor分子对MLL白血病发病和维持影响的初步研究：建立白血病人源化NOD/SCID免疫缺陷小鼠模型；动态监测小鼠外周血hCD45+细胞及hCD45/GFP双阳性细胞比例,对异种移植是否成功加以评估,同时也对Rictor-RNAi组与其他组间抑制率进行比较；在实验的终点,比较各组小鼠的整体观以及肝脏、脾脏等器官；流式细胞仪分析各组骨髓、肝脏和脾脏hCD45/GFP双阳性细胞比例,并比较其差别；免疫组织化学染色技术检测各组肝脏、脾脏、肾脏和肺hCD45阳性细胞表达情况；RT-PCR技术检测移植小鼠外周血单个核细胞MLL融合基因表达；绘制生存曲线,评估各组小鼠生存期差别。
[结果]：
1.Z1化合物对部分MLL白血病细胞系中的mTOR复合物2保守组分Rictor蛋白有选择性抑制作用,而其他白血病细胞系经Z1化合物作用后仅出现mTOR复合物2下游分子磷酸化水平(如p-AKT ser473)的下降。
2.用Western免疫印迹技术检测到,经10nmol/L CHX处理后不同时间点的THP-1细胞中Rictor蛋白均无降解现象；MG-132可以逆转Z1引起的THP-1细胞中Rictor蛋白的降解；THP-1细胞中蛋白酶体活性被Z1所抑制；GST标签的Rictor片段蛋白可以在体外被经Z1作用后的THP-1细胞粗提液(S100)泛素化。
3.经流式细胞术鉴定,分选后各组GFP阳性细胞均在90%以上,同时细胞免疫荧光术及Western免疫印迹法均证实Rictor蛋白封闭效应明显；MLL白血病细胞Rictor-RNAi组集落形成能力明显低于NC-RNAi组(P<0.001),而非MLL白血病细胞和正常细胞中两组间差别均没有统计学意义(P>0.05)；同时,MLL白血病细胞Rictor-RNAi组增殖能力也明显低于NC-RNAi组(P<0.01),在MLL白血病细胞Rictor-RNAi组中MG-132失去逆转Z1引起的细胞凋亡的作用,这些现象在其他类型的细胞中均没有出现。
4.动态监测THP-1异种移植模型小鼠外周血hCD45+细胞随着时间的延长而比例增加,说明建模成功；THP-1感染Rictor-RNAi组模型鼠外周血hCD45+细胞比例与THP-1未感染慢病毒组以及THP-1感染NC-GFP组模型鼠外周血hCD45+细胞比例在为期9周的动态检测期均无明显差别(P>0.05),但THP-1感染Rictor-RNAi组模型鼠外周血hCD45/GFP双阳性细胞比例从第7周开始就显著低于NC-GFP对照组(P<0.01);实验监测期的终点,可见Rictor-RNAi组模型鼠整体状况好于NC-GFP对照组,且肝脏瘤结节数目体积均小于对照组；Rictor-RNAi组模型鼠肝脏、脾脏和骨髓中hCD45/GFP双阳性细胞比例均显著低于NC-GFP对照组(P<0.01)；Kaplan-Meier曲线生存分析显示Rictor-RNAi组模型鼠生存时间长于对照组(P=0.033)。
[结论]
1.运用细胞印迹高通量筛选技术,从小分子单体化合物文库初步筛选并得到一种能够选择性降解部分MLL白血病细胞系中mTOR复合物2保守组分Rictor蛋白的特异性小分子化合物,为研究MLL白血病的靶向治疗提供了新的分子靶点。
2. Rictor作为一种高度保守蛋白在一般情况下是很稳定的,但Z1可引起THP-1细胞中Rictor蛋白通过泛素-蛋白酶体途径(ubiquitin-proteasome pathway, UPP)降解,这种降解作用很可能是通过Z1活化该途径的E3泛素连接酶而实现的,与蛋白酶体自身活性无关。
3. Rictor分子在MLL白血病细胞集落形成能力、增殖和凋亡调控等方面起着重要的作用,提示它可能是MLL白血病靶向治疗中的一个关键靶基因。
4. Rictor分子是MLL白血病发病和维持所必须的。
[Background and objective]:The competition and imbalance growing state between leukemia stem cells and normal hematopoietic stem cells is the crux of therapeutical approaches and outcome of leukemia as accompanying the whole process of generation and development of leukemia. The latest research reveals that PI3K/PTEN/AKT/mTOR signaling pathway may be a new target for correcting the imbalance between leukemia stem cells and normal hematopoietic stem cells. Two generations of mTOR complex inhibitor have been developed, however, the practical clincal applications have received unfavorable outputs. In present study, we use cytoblot high throughput assay to identify cell-specific inhibitor of mTOR complex 2. Then to investigate the degradation effect of protein Rictor in parts of MLL cell lines induced by lead compound Z1, and to supply some theories for gene targeted therapy to MLL. This study was designed to investigate the role of Rictor in the colony forming, proliferation and apoptosis-regulation of MLL leukemic cells in vitro. To study the effect of Rictor in the pathogenesis and maintain of leukemia in vivo.
[Methods]:
1. Searching for specific inhibitor of mTOR complex 2 by developing a high-throughput cytoblot assay:specific inhibitor of mTOR complex 2 was screened from small molecule monomeric compound library of NCPC new drug research center by using cytoblot high throughput assay. Western blot was then used to determine the inhibiting effect of compound against the key component protein in mTOR complex.
2. An approach to the mechanism of lead compound Zl in degradation protein Rictor: THP-1 cell line was taked as the cell model, Western blot was employed to detect the expressions of protein rictor after the treatment of CHX and Z1-MG-132-combined treatment, respectively; Luciferase-Proteasome-Glo Cell-Based Assay was used to measure the poteasome activity changes of THP-1 after treated by Zl; ubiquitylation in vitro model was constructed, and the situation of ubiquitylation in protein rictor was also detected.
3. Study on the role of Rictor to MLL Ieukemic cells in vitro:Rictor-RNA interference (RNAi) by transcription of short hairpin RNAs (shRNAs) from a lentiviral system was applied to silence Rictor gene expression in MLL leukemic cells, non-MLL leukemic cells and normal cells; and then GFP-Rictor-RNAi cells were isolated by Flow cytometry using the targeting-GFP in this lentivirus RNAi system; after established by flow cytometry, cellular immunofluorescence and Western blot, methyl cellulose colony forming assay was used to detect the differences among these Rictor-RNAi groups. Meanwhile, the proliferation dynamics of DDAO-SE labeled cells was detected by flow cytometry and analyzed with ModFit software, and flow cytometry was also used to observe apoptosis of each cell group.
4. Preliminary Study on the influence of Rictor in the pathogenesis and maintain of MLL leukemia in vivo:Mouse model of hukemia was established in NOD/SCID mice. To evaluate the effect of xenograft by dynamic monitoring the percentages of hCD45 positive cells and hCD45/GFP double positive cells in mice peripheral blood, meanwhile, it could be used as a means for evaluating transplant rates of group Rictor-RNAi distinguishing from the other two control groups. At the end point of observation post transplantation, the aspects, the sizes of livers and spleens, also with the number of tumor nodes in liver and spleen were compared among each group. Flow cytometry was used to analyze the percentages of hCD45/GFP double positive cells in bone marrow, liver and spleen. The expression of the hCD45 in the livers, spleens, kidneys and lungs of the mice post transplantation was detected by immunocytochemistry. The rearrangements of MLL gene in mice peripheral blood was detected by RT-PCR. The survival curve of THP-1 transplantation mice was draw, and used to evaluate the differences of survival time among each group by Kaplan-Meier method.
[Results]:
1. The screen results indicated that compound Z1 had selectively block activation on Rictor protein which was a conserved component of mTOR complex 2, this phenomenon appeared only in parts of MLL leukemia cell lines. Meanwhile, only decreased level of phosphorylated mTOR complex 2 downstream molecules (such as p-AKT ser473) was appeared in other leukemia cell lines treated by compound Z1.
2. No degradation effect of rictor in THP-1 was detected after the treatment of 10μmol/L cycloheximide in different time points by western blot; MG-132 could reverse the degradation effect of rictor in THP-1 cell line by Z1; the activity of poteasome in THP-1 was inhibted by Z1; GST targeting Rictor fragment could be ubiquitylated by THP-1 S-100 fraction which was treated by Z1.
3. After certified by flow cytometry, the positive rate of each sorted cell group was over 90%, meanwhile, both of cellular immunofluorescence and Western blot confirmed that protein Rictor was blocked obviously; colony forming ability of group Rictor-RNAi in MLL leukemic cells was found to significantly lower than group NC-RNAi (P< 0.001) There was no significant difference in colony forming ability between Rictor-RNAi and NC-RNAi groups in non-MLL leukemic cells and normal cells (P>0.05). Furthermore, proliferation of group Rictor-RNAi in MLL leukemic cells was also found to significantly lower than group NC-RNAi (P< 0.01).The effects of Z1 in combination with proteasome inhibitor MG-132 reversing the induced apoptosis by Z1 disappeared in group Rictor-RNAi of MLL leukemic cells, not in non-MLL leukemic cells or normal cells.
4. The results of dynamic monitoring showed that the percentages of hCD45 positive cells in THP-1 xenograft mice peripheral blood increased with the increase of storage time, and it illustrated the success of this transplantation model. The percentages of hCD45 positive cells in THP-1 infected Rictor-RNAi group was not evidently different from those in the THP-1 blank group or THP-1 infected NC-GFP group during the nine-week dynamic monitoring period (P>0.05). However, the percentages of hCD45 positive cells in THP-1 infected Rictor-RNAi group had became significantly lower than those in the THP-1 blank or NC-GFP group from the seventh week (P<0.01). At the end point of observation post transplantation, the aspect of Rictor-RNAi group mice was better than that of THP-1 blank or NC-GFP group. Meanwhile, the sizes of the livers in Rictor-RNAi group were smaller than those in other two groups, and the numbers of liver tumor nodes in Rictor-RNAi group were less than those in other two groups. The percentages of hCD45/GFP double positive cells in mice liver nodes, spleens and bone marrow of Rictor-RNAi group were significantly lower than those of NC-GFP group(P<0.01).The analysis by Kaplan-Meier method showed that survival time of Rictor-RNAi group was longer than those of control groups (P=0.033).
[Conclusion]:
1. Taking cytoblot high throughput assay, a specific small molecule monomeric compound was found out which could degrade a conserved component of mTOR complex 2—Rictor selectively, and it will provide new target molecules for targeted gene therapy of MLL leukemia.
2. A highly conserved protein Rictor is stable in normal situation, and the protein rictor could be degradated by ubiquitin-proteasome pathway(UPP), the degradation is likely to be achieved through the activation of E3 ubiquitin ligase by Z1, and has no relationship with the activation of proteasome.
3. Rictor could play a critical role in the colony forming, proliferation and apoptosis regulation of MLL leukemia. It revealed that Rictor might be a potential key target gene in target therapy for MLL leukemia.
4. Rictor was necessary for the pathogenesis and maintain of MLL leukemia in vivo.
引文
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