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

哺乳动物细胞的每一次细胞分裂都会复制超过30亿个碱基对。干扰脱氧核苷酸库的药物，例如核糖核苷酸还原酶抑制剂羟基脲，会减慢DNA复制叉。当DNA复制叉长时间停滞时，它们会崩溃并成为双链DNA断裂。这些通过同源重组修复。如果这样的DNA损伤没有被修复，它们是细胞毒性的。有一个激烈的搜索机制，介导的复杂的细胞反应复制压力，以及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剂控制细胞的应激反应。然而，它往往是未知的18个哺乳动物组蛋白去乙酰化酶的个别成员如何影响复制应激信号及其对细胞的后果。此外，组蛋白去乙酰化酶如何调节基因表达模式和磷酸化事件的子集，形成复制应激反应的证据有限。我们证明，组蛋白脱乙酰酶HDAC 1，HDAC 2和HDAC 3在白血病细胞复制应激的抑制诱导程序性细胞死亡（凋亡）。这个过程依赖于转录因子p73和随后诱导的促凋亡蛋白NOXA。此外，我们揭示了酪氨酸激酶ABL的催化活性是p73和NOXA积累所必需的，并且p73在具有复制应激和组蛋白脱乙酰酶抑制的细胞中附着于NOXA基因启动子。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。这种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。在拟议的项目中，我们希望确定HDAC 1，HDAC 2和HDAC 3如何控制白血病细胞和胰腺导管腺癌细胞的命运与ESTA诱导的复制应激。我们推测，HDAC调节的，p73依赖的NOXA表达是一个新确定的和决定性的变阻器凋亡诱导复制应激反应。我们认为HDAC 2直接结合在NOXA启动子上。此外，我们假设，HDAC 1，HDAC 2和HDAC 3中的至少一种的抑制抑制同源重组，从而导致DNA损伤和ABL和p73的磷酸化依赖性激活。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损伤测定和RNA测序来测试这些假设。这些知识将使我们深入了解控制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HDAC 1、HDAC 2和HDAC 3的共享和个体功能如何决定复制应激是否导致毒性DNA损伤和凋亡。

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.