急性B淋巴细胞白血病中CASP8AP2通过LEF1/β-catenin调控细胞干性及化疗敏感性的研究

CASP8AP2 was significantly associated with relapse of childhood B-ALL. We have found that CASP8AP2 could be co-precipitated with the transcription repressor CtBP2 and ZEB2. All the three proteins could bind to the LEF1 promoter. Meanwhile, LEF1 and some important factors associated with cell stemness including JAG1, CD44, and SALL4 were up-regulated after knock-down of CASP8AP2. Furthermore, CASP8AP2 could possibly promote the biogenesis of miR-27b. Based on these results, we propose a hypothesis that CASP8AP2 makes effect on the stemness and chemoresistance of leukemic cells through its regulation on the expression of miR-27b and LEF1. To prove this hypothesis, we plan to carry out studies at molecular, cellular, and experimental animal’s levels to show the main points as follows: (1) Low expression of CASP8AP2 results in up-regulation of LEF1 and down-regulation of miR-27b in B-ALL. (2) Down-regulation of miR-27b leads to up-regulation of STYK1 which is its potential target. STYK1 could activate Akt and inactivate GSK-3β successively. This makes stabilization and translocation into nucleus of β-catenin. (3) Transcription of stemness related genes, including JAG1, CD44, SALL4, could be activated by the complex of β-catenin and LEF1 in nucleus. (4) The activation of these three genes results in increased stemness and decreased chemosensitivity of leukemic cells. The innovations of this study are to prove that CASP8AP2 is able to make effects on histone modifications in promotor region through interaction with CtBP2, and influences leukemic cell stemness. This study will be of help to clarify the mechanism of leukemia relapse in childhood B-ALL, and provide important basis for new therapeutic targets and methods for these patients.

CASP8AP2与儿童B-ALL复发密切相关。我们发现CASP8AP2与转录抑制因子CtBP2、ZEB2共沉淀，三者均与LEF1启动子结合；敲低CASP8AP2后LEF1及重要干性相关因子上调；CASP8AP2可能促进miRNA-27b生成。我们推测CASP8AP2通过调控miRNA、LEF1表达，影响白血病细胞的干性与耐药性。拟从分子、细胞及动物水平，证明在B-ALL中CASP8AP2低表达导致LEF1上调与miR-27b下调；后者通过靶基因STYK1激活Akt、使GSK-3β失活，造成β-catenin入核增多，与LEF1形成复合体，激活干性相关基因JAG1、CD44、SALL4转录，导致白血病细胞干性增强，化疗敏感性降低。创新点是首次证明CASP8AP2能与CtBP2互作调控启动子区组蛋白修饰，并影响细胞干性。本项目将为阐明儿童ALL复发机制，寻找新的治疗靶点和治疗方法提供重要依据。

本项目主要探讨了CASP8AP2与CtBP2/ZEB2的相互作用，研究CASP8AP2在CtBP2-ZEB2复合体结合靶基因启动子、募集组蛋白与DNA修饰酶、影响细胞耐药性中作用，分析相关分子的临床预后价值，以及CASP8AP2对STYK1-AKT1-GSK3β通路及β-catenin的亚细胞定位的影响。研究结果显示，儿童ALL初诊时CASP8AP2、CtBP2基因低表达与复发密切相关，联合分析两个基因的表达水平能更准确地预测患儿预后。CASP8AP2可能对ZEB2蛋白具有保护作用，影响CtBP2与ZEB2的相互作用及其与LEF1及CD44启动子的结合，从而共同抑制LEF1、CD44的转录及表达。CASP8AP2影响CtBP2募集组蛋白去甲基化酶LSD1和去乙酰化酶HDAC1，敲减CASP8AP2使CD44增强子区ZEB2结合位点附近LSD1、HDAC1的结合明显减少。敲减CASP8AP2导致的697细胞耐药性能被LSD1抑制剂ORY-1001和HDAC1抑制剂TSA逆转。DRD功能域参与CASP8AP2结合CtBP2，该功能域丢失明显增强白血病细胞耐药性。DNMT3A基因低表达与儿童ALL预后较差、白血病细胞耐药相关，CASP8AP2、CtBP2、DNMT3A存在相互作用并影响部分基因转录起始点DNA甲基化水平。敲减CASP8AP2不影响697细胞STYK1、AKT1、GSK3β表达和磷酸化水平，以及β-catenin核浆分布。本项目揭示了CASP8AP2通过影响CtBP2/ZEB2互作，调控组蛋白、DNA修饰酶的募集及下游基因的表达，进而在白血病细胞化疗耐受中发挥重要作用，采用组蛋白去甲基化酶和去乙酰化酶抑制剂有可能克服细胞耐药性。因此，对于CASP8AP2低表达的儿童ALL，可以探讨将上述组蛋白修饰酶抑制剂整合入化疗方案，有望提高白血病疗效，改善患儿临床预后。