V(D)J重组脱靶效应促进Ph染色体阳性急性淋巴细胞白血病发生机制

Ph chromosome positive acute lymphoblastic leukaemia (ALL) comprises a heterogeneous group of disorders characterized by additional recurring chromosomal abnormalities including translocations, trisomies and deletions. BCR–ABL1 cooperating with mutations in master regulators of cell development and oncogenic lesions drive lymphomagenesis in BCR-ABL1+ ALL. The variable region genes encoding Ig and TCR polypeptides are assembled during lymphocyte development by V(D)J recombination, a site-specific recombination reaction named for the arrays of V (variable), D (diversity), and J (joining) gene segments that are its substrates. V(D)J recombination is initiated when the proteins encoded by the recombination activating genes RAG1 andRAG2, bind to recombination signal sequences (RSSs) that flank segments. The RAG proteins then introduce DNA double strand breaks between the RSSs and gene segments, and the reaction is completed by DNA end processing and ligation mediated by non-homologous end joining repair factors. Many potential substrates for RAG-mediated cleavage are remarkably abundant in vertebrate genomes. Such sequences called cryptic RSSs (cRSSs), RAG2 specifically interacts with H3K4me3 which spread in genome. RAG has potential abilities to cleavage the sites above, the process named off-target of V(D)J recombination. In order to address RAG-mediated off-target of V(D)J recombination involving in BCR–ABL1+ALL, We will generate wild type and mutated RAG BCR–ABL1+ALL mouse models, confirm that RAG cleavage can directly induce BCR–ABL1+ALL lymphomagenesis. We will select RAG binding sites in BCR–ABL1+ALL genome, and we hope to find potential biomarkers for Ph+ ALL. All the results will help us to find the better therapeutic targets for Ph+ALL.

BCR-ABL1+ALL基因组常出现除Ph染色体以外的其他基因删除等结构异常。BCR-ABL1协同这些与细胞发育和肿瘤发生相关的异常基因共同促进ALL的发生。淋巴细胞特有的RAG蛋白识别和断裂抗原受体基因片段旁的重组信号序列（recombination signal sequence, RSS），通过V(D)J重组形成编码抗原受体可变区的基因。哺乳动物基因组存在类似RSS序列（cRSS）并且RAG2可以结合基因组H3K4Me3修饰的位点，RAG有机会对这些位点产生断裂作用，称这过程为V(D)J重组脱靶效应。为了揭示RAG介导的V(D)J重组脱靶效应参与BCR-ABL1+ALL发生，我们建立了野生型、RAG功能突变和结构突变的BCR-ABL1+ALL小鼠模型，直接证明RAG参与疾病发生；筛选RAG对基因组作用的位点，寻找可以诊断和预测疾病发生发展的生物学标记，为开发更有效的治疗靶点奠定基础。

Ph染色体阳性急性淋巴细胞白血病（acute lymphoblastic leukaemia，ALL）伴有特征性t(9;22)(q34;q11)的染色体（Philadelphia染色体，Ph）。该异常染色体产生BCR-ABL1融合基因，表达高度异常的酪氨酸激酶，促使早期B细胞发育阶段发生恶性转化。因此，该疾病也称为BCR-ABL1+B-ALL。研究BCR-ABL1+B-ALL发生机制认为BCR-ABL1融合基因的形成是疾病发生的始动因素，BCR-ABL1蛋白持续表达酪氨酸激酶活性，改变了基因组转录或表观遗传学，后果表现为白血病细胞基因组对内在或环境的DNA二次损害因素敏感，促进疾病的发生发展。发育的B细胞通过淋巴细胞特有的重组激活基因蛋白（RAG1和RAG2）组成的RAG重组催化断裂Ig位点的基因片段和其旁的重组信号序列，然后通过传统的非同源末端连接途径(non-homologous end joining，NHEJ)把断裂的基因片段组合在一起，形成编码Ig可变区的基因。形成多样性Ig可变区基因的过程称为V(D)J重组。我们围绕RAG在BCR-ABL1+B-ALL发生发展的作用开展了一系列研究。 我们建立了BCR-ABL1+B-ALL小鼠模型，发现该小鼠模型与临床BCR-ABL1+B-ALL特征相似，以中枢神经系统侵润为特征性表现。建立fRAG, cRAG1和cRAG2不同遗传背景的小鼠BCR-ABL1+B-ALL模型，证明RAG的非核心参与BCR-ABL1+B-ALL发生和发展。BCR-ABL1作用或者BCR-ABL1和RAG协调作用，造成NHEJ在BCR-ABL1+B-ALL细胞受到抑制，增加了基因组的不稳定性增加。V(D)J脱靶效应的基因位点增加，促进疾病的发生发展。这些结果为该疾病的诊疗提供新的策略。