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

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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.