Analysis of how the epigenetic modifiers HDAC1, HDAC2, and HDAC3 control cytotoxicity and the induction of DNA damage in cancer cells upon DNA replication stress

分析表观遗传修饰剂 HDAC1、HDAC2 和 HDAC3 在 DNA 复制应激下如何控制细胞毒性和诱导癌细胞中的 DNA 损伤

• 批准号: 496927074
• 负责人: Professor Dr. Oliver Holger Krämer
• 金额: --
• 依托单位: Institut für Toxikologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/496927074?language=en
• 关键词: Analysis; how; epigenetic; modifiers; HDAC1

Over 3 billion base pairs of a mammalian cell are replicated with every cell division. Drugs that disturb the deoxynucleotide pool, such as the ribonucleotide reductase inhibitor hydroxyurea, slow down DNA replication forks. Upon prolonged stalling of DNA replication forks, they collapse and become double-strand DNA breaks. These are repaired by homologous recombination. If such DNA lesions are not repaired, they are cytotoxic. There is an intense search for mechanisms that mediate the complex cellular responses to replication stress and how this determines cell fate. Increasing evidence shows that epigenetic modifiers of the histone deacetylase family control cellular stress responses. However, it is often unknown how individual members of the 18 mammalian histone deacetylases affect replication stress signaling and its consequences for cells. Moreover, there is limited evidence on how histone deacetylases regulate subsets of gene expression patterns and phosphorylation events that shape replication stress responses. We demonstrate that an inhibition of the histone deacetylases HDAC1, HDAC2, and HDAC3 in leukemic cells with replication stress induces programmed cell death (apoptosis). This process relies on the transcription factor p73 and a subsequent induction of the pro-apoptotic protein NOXA. Furthermore, we reveal that the catalytic activity of the tyrosine kinase ABL is required for the accumulation of p73 and NOXA and that p73 attaches to the NOXA gene promoter in cells with replication stress and histone deacetylase inhibition. Histone deacetylase inhibitors reduce proteins that promote homologous recombination and protect replication forks. This mechanism could be the reason why these drugs cause DNA damage and the activation of the ABL-p73-NOXA signaling node upon replication stress. In the proposed project we want to define how HDAC1, HDAC2, and HDAC3 control the fate of leukemic cells and pancreatic ductal adenocarcinoma cells with pharmacologically induced replication stress. We speculate that an HDAC-regulated, p73-dependent expression of NOXA is a newly identified and decisive rheostat for apoptosis induction response to replication stress. We consider a direct binding of HDAC2 on the NOXA promoter. Moreover, we hypothesize that an inhibition of at least one of HDAC1, HDAC2, and HDAC3 suppresses homologous recombination and thereby causes DNA damage and the phosphorylation-dependent activation of ABL and p73. We want to use state-of-the art genetic models (CRISPR-Cas9 knockout and activation), pharmacological tools, protein analysis, DNA damage assays, and RNA-sequencing to test these hypotheses. Such knowledge will give deep insights into molecular mechanisms that control the signaling pathways of the DNA replication stress cascade. We will be able to precisely delineate how shared and individual functions of HDAC1, HDAC2, and HDAC3 determine whether replication stress leads to toxic DNA damage and apoptosis.

哺乳动物细胞的每一次分裂都要复制超过30亿个碱基对。干扰脱氧核苷酸库的药物，如核糖核苷酸还原酶抑制剂羟基脲，可以减缓DNA复制分叉。当DNA复制叉长时间停滞时，它们会崩溃并成为双链DNA断裂。这些是通过同源重组修复的。如果这些DNA损伤没有得到修复，它们就具有细胞毒性。有一个激烈的研究机制，介导复杂的细胞反应复制压力和如何决定细胞的命运。越来越多的证据表明，组蛋白去乙酰化酶家族的表观遗传修饰因子控制细胞应激反应。然而，通常不知道18种哺乳动物组蛋白去乙酰化酶的个体成员如何影响复制应激信号及其对细胞的影响。此外，关于组蛋白去乙酰化酶如何调节基因表达模式亚群和形成复制应激反应的磷酸化事件的证据有限。我们证明，在复制应激的白血病细胞中，组蛋白去乙酰化酶HDAC1、HDAC2和HDAC3的抑制可诱导程序性细胞死亡（凋亡）。这一过程依赖于转录因子p73和随后诱导的促凋亡蛋白NOXA。此外，我们发现酪氨酸激酶ABL的催化活性对于p73和NOXA的积累是必需的，并且p73在复制应激和组蛋白去乙酰化酶抑制的细胞中附着在NOXA基因启动子上。组蛋白去乙酰化酶抑制剂减少促进同源重组和保护复制叉的蛋白质。这一机制可能是这些药物在复制应激时引起DNA损伤和ABL-p73-NOXA信号节点激活的原因。在这个项目中，我们想要确定HDAC1、HDAC2和HDAC3如何通过药理学诱导的复制应激来控制白血病细胞和胰腺导管腺癌细胞的命运。我们推测，hdac调控的、p73依赖性的NOXA表达是一种新发现的、对细胞凋亡诱导对复制应激反应起决定性作用的变阻器。我们认为HDAC2直接结合在NOXA启动子上。此外，我们假设抑制HDAC1、HDAC2和HDAC3中至少一种会抑制同源重组，从而导致DNA损伤和磷酸化依赖性的ABL和p73激活。我们希望使用最先进的遗传模型（CRISPR-Cas9敲除和激活）、药理学工具、蛋白质分析、DNA损伤分析和rna测序来测试这些假设。这些知识将深入了解控制DNA复制应激级联信号通路的分子机制。我们将能够精确地描述HDAC1、HDAC2和HDAC3的共享和个体功能如何决定复制应激是否导致毒性DNA损伤和细胞凋亡。