title: Nek1 - developmental involvement in DNA repair and role as a target in radiotherapy
Creators: name: Freund, Isabel
Publisher: name: UNSPECIFIED
Year: 2022

description: Organisms are inevitably exposed to ionizing radiation (IR) which is emitted by various natural sources such as decaying radionuclides. Since its discovery in the 19th century, IR has become a highly relevant tool, especially in the field of medicine where it is used for diagnostic procedures and the treatment of tumors. However, its property to alter the structure of the exposed matter by breaking chemical bonds threatens the integrity of an important molecule that presents the fundamental prerequisite of life, namely DNA. Exposing DNA to IR results in different types of lesions of which the DNA double-strand break (DSB) represents the most detrimental. Since DSBs can also result from several endogenous processes, cells evolved certain mechanisms to minimize the harmful impact of this lesion on their genomic integrity, collectively termed DNA damage response (DDR). The DDR consists of highly coordinated signaling pathways that allow for damage detection, cell cycle arrest, and damage repair. While originally studied for their involvement in ciliogenesis, centrosome organization, and mitosis, the members of the never-in mitosis-gene A (NIMA) related kinase (Nek) family increasingly move into the focus of DDR research as nearly all have functions in related processes. However, one member, Nek1, stands out in this context due to its multifunctional role in the DDR, including the regulation of cell cycle checkpoints, apoptosis, and DNA repair. In the following work, the results of two projects are presented, each highlighting a different aspect of Nek1 as an important kinase of the DNA damage response. The first project is based on research conducted by the Löbrich lab, which identified Nek1 as a regulator of a factor required for the successful execution of the DSB repair pathway "Homologous Recombination" (HR), namely Rad54. Subsequent in vivo studies surprisingly revealed that Nek1 is important for HR in adult mice but not in embryos, which exhibit a normal repair behavior despite a Knock-Out mutation in the Nek1 gene. The objective of this project was therefore to further characterize the apparent differential regulation of HR during development. To this end, embryonic and adult fibroblast lines were isolated from different mice strains and analyzed for DSB repair and differences in gene expression. The collected data suggest that the kinases required for the activation of Rad54 indeed change in response to an organism’s developmental stage: Nek1 exclusively controls Rad54’s activation in adult cells whereas Nek3 and Nek5 can redundantly activate Rad54 in embryonic cells. Thus, this study not only consolidates previous findings of the Löbrich lab but is also the first to report a developmental change in the regulation of HR and to associate Nek3 and Nek5 with DNA repair. Considering its multifunctionality in the DDR, pharmacological inactivation of Nek1 has the potential to significantly improve the treatment of cancer by e.g. radiotherapy. The Rödel lab confirmed this assumption by demonstrating that depleting Nek1 significantly sensitizes two different cancer cell lines to single-dose irradiation as shown by their reduced ability to form colonies. Since further experiments revealed that Nek1-depleted cancer cells are still capable of inducing a functional G2/M checkpoint, the second project of this work investigated the extent to which fractionated irradiation can enhance the radiosensitizing effect of Nek1 depletion. Cancer cells were therefore subjected to three fractionation regimes, in which a total radiation dose of 6 Gy was applied in three small fractions either every 2 h, 6 h, or 24 h, and evaluated for cell cycle behavior as well as colony-forming ability. Indeed, the 6 h interval tremendously increases the radiosensitivity of Nek1-depleted cells beyond the level observed for single-dose irradiations while Nek1-proficient cells are less affected. This finding has been additionally strengthened in in vivo xenograft studies. Taken together, this work strengthens Nek1 as a promising target in cancer therapy. It further demonstrates that the efficacy of fractionated radiotherapy can be significantly increased if the employed regime is adapted to the cycle rate of cancer cells and takes the cell cycle-dependent function of Nek1 as an HR factor into account.