糖皮质激素下调DOT1L表达的分子机制及在儿童ALL激素耐药中的作用研究

It is well documented that epigenetic dysregulation contributes to drug resistance. DOT1L (disruptor of telomeric silencing 1 like) is the only histone methyltransferase that produces methylated H3K79. The onco-MLL loses its C-terminal segment and is fused with a partner protein, which can recruit DOT1L, causing abnormal H3K79 methylation in children with acute lymphoblastic leukemia (ALL). This leads to eventually leukemia initiation. Hence DOT1L is a drug target for ALL. However, it has not been reported the roles of DOT1L in glucocorticoid resistance in ALL.We first found that glucocorticoid (GC) can inhibit the expression and transcriptional activity of DOT1L in GC sensitive ALL cells. The expression of DOT1L played an important role in the ALL cells' glucocorticoid resistance. Bioinformatic analysis showed that there were some glucocorticoid receptor(GR) possible binding sites in DOT1L promoter region. On this basis, we will study the molecular mechanism of down-regulation of DOT1L by GC and identify that DOT1L is a new GR target gene. Moreover, the in vitro and in vivo experiments will be performed to observe the effect of overexpression or knockdown of DOT1L in glucocorticoid resistance. Taken altogether,this study provides possibility to find the important role of DOT1L in the ALL cells' glucocorticoid resistance and theoretical basis for reversal of glucocorticoid resistance in ALL.

糖皮质激素GC耐药是困扰儿童急性淋巴细胞性白血病ALL治疗的难题。组蛋白修饰异常是肿瘤耐药的新机制。近年研究表明组蛋白赖氨酸甲基转移酶DOT1L异常修饰H3K79甲基化与ALL发生发展密切相关。那么ALL GC耐药是否与DOT1L相关,目前尚不清楚。鉴于此，本项目在"GC能明显下调GC敏感型ALL细胞DOT1L表达,但不能下调GC耐药型ALL细胞DOT1L表达"及"生物信息学分析DOT1L 5＇调控区有GR结合位点"的基础上，拟开展如下研究：①解析GC下调DOT1L表达的分子机制，明确DOT1L是GR的一个新靶基因；②构建沉默或高表达DOT1L基因的重组慢病毒载体，经体内外实验探讨DOT1L在GC耐药中的作用。旨在阐明"作为GR新的靶基因，DOT1L表达下调是新的GC敏感性生物学标志物，其高表达参与GC耐药形成"这一全新观念，从而为逆转GC耐药，提高儿童ALL治疗效果寻找一个可能的靶点。

糖皮质激素（GC）耐药是困扰儿童急性淋巴细胞白血病（ALL）治疗的难题。组蛋白修饰异常是肿瘤耐药的新机制。近年研究表明，组蛋白赖氨酸甲基转移酶DOT1L（disruptor of telomeric silencing 1 like）异常修饰H3K79甲基化与ALL的发生发展密切相关。那么ALL的GC耐药是否与DOT1L有关呢？目前尚不清楚。在本项目中，我们发现：①在GC敏感的白血病和淋巴瘤细胞中，GC可通过活化GC受体（GR）而降低DOT1L mRNA的稳定性，从而下调DOT1L的水平及其效应蛋白H3K79的甲基化水平，促进瘤细胞凋亡。荷瘤裸鼠实验显示，GC可在体内抑制来源于敏感肿瘤细胞的移植瘤生长，并在移植瘤组织中下调DOT1L的水平和H3K79的甲基化水平。以上结果表明，通过活化GR而下调DOT1L是GC杀伤敏感型白血病和淋巴瘤细胞的重要机制；②在GC耐药的白血病和淋巴瘤细胞中，GC对瘤细胞的杀伤作用明显减弱，对DOT1L的水平无明显影响；沉默DOT1L可显著增加耐药型瘤细胞对GC的敏感性。荷瘤裸鼠实验显示，沉默DOT1L可在体内增敏GC抑制耐药性移植瘤生长的作用。以上结果表明，DOT1L密切参与了白血病和淋巴瘤细胞对GC的耐药过程，其为探索通过下调DOT1L的水平或抑制其活性而增敏GC杀伤耐药型瘤细胞的新措施提供了科学依据。 . 此外，我们还发现：①SARI（suppressor of AP-1 regulated by interferon）是GR的新靶基因，通过活化GR而上调SARI在GC杀伤B淋巴瘤细胞的过程中发挥了重要作用，提示SARI可能是治疗B淋巴瘤的潜在新靶点以及判断GC治疗B淋巴瘤敏感性的潜在新标志；②含有SARI、寡聚精氨酸穿膜肽9R、柔性连接子G4S和可溶性BLyS突变体（mBLyS）的重组蛋白SARI-G4S-9R-G4S-mBLyS可选择性识别并内化进入BLyS受体阳性的淋巴瘤细胞，进而通过抑制AP-1的活性而导致瘤细胞生长抑制和凋亡；这种选择性抗瘤效应由mBLyS和BLyS受体的相互作用所介导，并依赖效应分子SARI和穿膜肽9R。以上结果提示，含有SARI、9R和mBLyS的重组蛋白在靶向治疗Burkitt′s淋巴瘤和其他BLyS受体阳性肿瘤方面具有潜在的应用前景。.就以上有关研究结果已发表SCI论文1篇、会议论文2篇。