GNAO1参与TEL-AML1阳性白血病二次打击的分子机制研究

Benefitting from risk-directed combination chemotherapy and improved supportive cares, the event-free survival of childhood acute lymphoblastic leukemia (ALL) is now between 70% and 80% for those receiving protocol-based therapy. However, there are also 20%-30% childhood ALL cannot be cured, and one of the most important reasons is that the etiology of these diseases is uncertain. It is well known that TEL-AML1 resulting from the translocation of chromosome 12 and 21 is the commonest genetic variants of childhood ALL. TEL-AML1 was considered as the first hit of the pathogenesis of TEL-AML1 positive ALL and coming from the embryonic period. According to the Two-hit theory of leukemia, there should have the second-hit of genetic damage which is the basis of tumor cells to be able to maintain its heritage and the malignant phenotype continuously. In this area, the study of monozygotic twin is one of the most direct and convenient strategy. Although the genome of monozygotic twins are identical and one can be seen as the copy of the other, the minor genetic differences between monozygotic health and leukemia twins can happened after the fertilized egg split, which constitute the basis of the pathogenesis of leukemia. Fortunately, we collected one pairs of monozygotic twins who suffered from TEL-AML1 positive leukemia in two months successively. Through comparing the whole genome sequencing data between paired tumor cell and somatic cells, we find the candidate culprit gene (GNAO1 R209C) of leukemia, which coexist between two leukemia samples. By many kinds of mutational site analysis, such as the evolution of GNAO1 in different species, the orthologous analysis of Gα family, and the prediction of the function of mutational protein by SIFT, Mutation Taster、Polyphen2、LRT、LJB_GERP++, etc., the function of GNAO1 R209C is damaged seriously. In this study, based on the results of the expression of GNAO1 can be regulated by TEL, we try to find if TEL can inhibit the expression of GNAO1 as a transcriptional repressor by luciferase assay, EMSA and ChIP, etc. Then we will transfect different lentivirus groups to CD34 positive hematopoietic stem cells and Ba/F3 cell and detect the difference in clonogenic assay and cell cycle, apoptosis, cell growth curve, etc. To study the transformation of GNAO1 R209C in vivo, we transfected different lentivirus groups to the long-term repopulating HSC (LT-HSC) of the day12 embryonic liver cells of mouse (i.e., enrichment of hematopoietic stem cells) and to detect the difference of the development of leukemia. Moreover other genetic variations of GNAO1 and other Gα protein, such as GNA11, GNA13, GNA14, GNAI1, GNAI2, GNAL, GNAT1, GNAT2, GNAT3, GNAQ, GNAS, and GNAZ will also be sequenced in large-scale clinical samples by next-generation sequencing. Through these studies, GNAO1 will be defined as the second hit of TEL-AML1 positive ALL and which will be the classic mode of the pathologies of childhood ALL.

国内外研究普遍认为TEL-AML1是引起TEL-AML1+白血病发生的第一次打击因素。通过全外显子组测序，我们在一对同卵孪生且先后发病的白血病患儿的肿瘤样本中均发现存在GNAO1基因的突变，并初步证实了GNAO1突变是造成TEL-AML1+白血病发生的第二次打击因素，且极有可能是通过TEL的去转录抑制和持续激活Rac1-Src-STAT3-Myc通路发挥作用。本项研究中，通过制备GANO1野生和突变慢病毒以及TEL干扰和TEL-AML1过表达慢病毒分组转染TEL-AML1-白血病细胞株，观察TEL对GNAO1转录抑制和Rac1-Src-STAT3-Myc通路激活变化；通过将上述病毒分组转染人脐带血造血干细胞，观察体外克隆形成差异；通过转染小鼠胎肝造血干细胞，观察白血病发生情况；通过上述研究，将为GNAO1突变在介导白血病发生和转化的分子机制和白血病的二次打击学说提供理论和实验基础

得益于危险度分层的联合化疗和支持治疗，儿童急性淋巴细胞白血病（ALL）的5年无事件生存率介于70%到80%之间。然而，也有20%-30%的儿童都无法治愈，其中最重要的原因之一就是这些复发难治患者可能都具有遗传学上的不利预后因素。众所周知，12号和21号染色体易位导致的TEL-AML1是儿童期ALL最常见的遗传类型，它起源于胚系变异，是造成白血病的第一次打击。根据白血病的二次打击理论，应该有遗传损伤的二次打击，这是肿瘤细胞能够持续维持其遗传和恶性表型的基础。在这一领域，单卵双生子的研究是最直接和方便的策略之一。虽然单卵双生子的基因组是完全相同的，一个可以看作是另一个的拷贝，但是健康和白血病孪生子之间的微小遗传差异在受精卵分裂后就可能发生，这构成了白血病发病的基础。通过全外显子组测序，我们在一对同卵孪生且先后发病的白血病患儿的肿瘤样本中均发现存在GNAO1基因的突变，并初步证实了GNAO1突变是造成TEL-AML1+白血病发生的第二次打击因素。通过本课题进行的深入系统的研究，我们确定了G蛋白亚单位αo1（GNAO1）的R209C突变为一个新的高致病性位点。此外，GNAO1错义突变仅在所有患者中复发，并与TEL-AML1融合相关。GNAO1 R209C突变体的异位表达增加了其GTPase活性并促进了细胞增殖和细胞肿瘤转化。伴随TEL-AML1融合，gnao1r209c突变体通过激活PI3K/Akt/mTOR信号促进白血病的发生。另外，活化的mTORC1磷酸化p300乙酰转移酶，乙酰化TEL-AML1，从而增强了GNAO1 R209C的TEL-AML1的转录活性。综上所述，我们的研究为GNAO1突变体与TEL-AML1融合蛋白之间的相互作用引起白血病转化提供了临床证据，表明GNAO1可能是人类白血病的潜在治疗靶点，同时也为白血病的二次打击学说提供理论依据和实验基础。