The involvement of DNA damage response pathway in nuclear reorganization during netotic initiation

Abstract

The netotic type of cell death plays an important role in innate immunity and host defense. It is characterized by epigenetic changes in chromatin modifications, nuclear envelope disassembly, chromatin expulsion and formation of extracellular chromatin nets loaded with anti-bactericidal granules containing aggressive proteinase and pro-oxidant enzymes. Recently, it was shown by several groups that this process has a negative impact on tumor progression. Methods: To study an involvement of DNA damage response pathway in nuclear reorganization during netotic initiation we selected an HL-60 based model. HL-60 is an acute myeloid leukemia cell line that preserved the capability of differentiation into granulocyte cell lineages. At first step, we performed differentiation of HL-60 cells into neutrophils with DMSO or ATRA. On the fifth day of differentiation, we have applied phorbol myristate to induce netotic cell death. Alternatively, we have treated granulocyte fraction isolated from bone marrow and blood of wild-type mice or from human donor blood with phorbol myristate. To study involvement of DNA damage response pathway we used an inhibitor of Wip1 phosphatase. Wip1 is a DNA damage induced nuclear phosphatase and a major negative switch of several DNA damage response proteins such as ATM, Chk1, Chk2, p53 and others. Results: Recently, several studies have suggested that nuclear structure of neutrophils plays an important role in their survival, their ability to migrate and control the release of NETs. First, we observed that Wip1 phosphatase expressed at high levels in the nucleus of differentiated HL-60 myeloid cells and in neutrophils. We observed that the inhibition of nuclear phosphatase Wip1 activity could extend mouse and human neutrophils and differentiated HL-60 cells survival through dephosphorylation and inhibition of its target proteins. Additionally, neutrophils treated with Wip1 inhibitors tends to show more segmented nuclei which have been correlated before with better anti-tumoral immune functions. Conclusions: In this context, we discovered that the inhibition of nuclear phosphatase Wip1 activity promotes neutrophil migration and control critical functions associated with nuclear reorganization during netotic type of cell death. The targeting of nuclear phosphatase Wip1 in neutrophils could be a new therapeutic strategy in the treatment of several diseases that trigger the inflammatory response and netotic type of death. The reported study was funded by La Ligue contre le Cancer and RSF (grant # 19-75-20128).