VRK1 is a nuclear Ser-Thr chromatin kinase that does not mutate in cancer, and is overexpressed in many types of tumors and associated with a poor prognosis. Chromatin VRK1 phosphorylates several transcription factors, including p53, histones and proteins implicated in DNA damage response pathways. In the context of cell proliferation, VRK1 regulates entry in cell cycle, chromatin condensation in G2/M, Golgi fragmentation, Cajal body dynamics and nuclear envelope assembly in mitosis. This kinase also controls the initial chromatin relaxation associated with histone acetylation, and the non-homologous-end joining (NHEJ) DNA repair pathway, which involves sequential steps such as γH2AX, NBS1 and 53BP1 foci formation, all phosphorylated by VRK1, in response to ionizing radiation or chemotherapy. In addition, VRK1 can be an alternative target for therapies based on synthetic lethality strategies. Therefore, VRK1 roles on proliferation have a pro-tumorigenic effect. Functions regulating chromatin stability and DNA damage responses have a protective anti-tumor role in normal cells, but in tumor cells can also facilitate resistance to genotoxic treatments.

Regulation of cell division requires the integration of signals implicated in chromatin reorganization and coordination of its sequential changes in mitosis. Vaccinia-related kinase 1 (VRK1) and Aurora B (AURKB) are two nuclear kinases involved in different steps of cell division. We have studied whether there is any functional connection between these two nuclear kinases, which phosphorylate histone H3 in Thr3 and Ser10, respectively. VRK1 and AURKB are able to form a stable protein complex, which represents only a minor subpopulation of each kinase within the cell and is detected following nocodazole release. Each kinase is able to inhibit the kinase activity of the other kinase, as well as inhibit their specific phosphorylation of histone H3. In locations where the two kinases interact, there is a different pattern of histone modifications, indicating that there is a local difference in chromatin during mitosis because of the local complexes formed by these kinases and their asymmetric intracellular distribution. Depletion of VRK1 downregulates the gene expression of BIRC5 (survivin) that recognizes H3-T3ph, both are dependent on the activity of VRK1, and is recovered with kinase active murine VRK1, but not with a kinase-dead protein. The H3–Thr3ph–survivin complex is required for AURB recruitment, and their loss prevents the localization of ACA and AURKB in centromeres. The cross inhibition of the kinases at the end of mitosis might facilitate the formation of daughter cells. A sequential role for VRK1, AURKB, and haspin in the progression of mitosis is proposed.

DNA damage causes a local distortion of chromatin that triggers the sequential processes that participate in specific DNA repair mechanisms. This initiation of the repair response requires the involvement of a protein whose activity can be regulated by histones. Kinases are candidates to regulate and coordinate the connection between a locally altered chromatin and the response initiating signals that lead to identification of the type of lesion and the sequential steps required in specific DNA damage responses (DDR). This initiating kinase must be located in chromatin, and be activated independently of the type of DNA damage. We review the contribution of the Ser-Thr vaccinia-related kinase 1 (VRK1) chromatin kinase as a new player in the signaling of DNA damage responses, at chromatin and cellular levels, and its potential as a new therapeutic target in oncology. VRK1 is involved in the regulation of histone modifications, such as histone phosphorylation and acetylation, and in the formation of γH2AX, NBS1 and 53BP1 foci induced in DDR. Induction of DNA damage by chemotherapy or radiation is a mainstay of cancer treatment. Therefore, novel treatments can be targeted to proteins implicated in the regulation of DDR, rather than by directly causing DNA damage.

NBS1 is an early component in DNA-Damage Response (DDR) that participates in the initiation of the responses aiming to repair double-strand breaks caused by different mechanisms. Early steps in DDR have to react to local alterations in chromatin that are induced by DNA damage. NBS1 participates in the early detection of DNA damage and functions as a platform for the recruitment and assembly of components that are sequentially required for the repair process. In this work we have studied whether the VRK1 chromatin kinase can affect the activation of NBS1 in response to DNA damage induced by ionizing radiation. VRK1 is forming a basal preassembled complex with NBS1 in non-damaged cells. Knockdown of VRK1 resulted in the loss of NBS1 foci induced by ionizing radiation, an effect that was also detected in cell-cycle arrested cells and in ATM (-/-) cells. The phosphorylation of NBS1 in Ser343 by VRK1 is induced by either doxorubicin or IR in ATM (-/-) cells. Phosphorylated NBS1 is also complexed with VRK1. NBS1 phosphorylation by VRK1 cooperates with ATM. This phosphorylation of NBS1 by VRK1 contributes to the stability of NBS1 in ATM (-/-) cells, and the consequence of its loss can be prevented by treatment with the MG132 proteasome inhibitor of RNF8. We conclude that VRK1 regulation of NBS1 contributes to the stability of the repair complex and permits the sequential steps in DDR.

The VRK1 chromatin kinase regulates the organization of locally altered chromatin induced by DNA damage. The combination of ionizing radiation with inhibitors of DNA damage responses increases the accumulation of DNA damage in cancer cells, which facilitates their antitumor effect, a process regulated by VRK1. Tumor cell lines with different genetic backgrounds were treated with olaparib to determine their effect on the activation of DNA repair pathways induced by ionizing radiation. The effect of combining olaparib with depletion of the chromatin kinase VRK1 was studied in the context of double-strand breaks repair pathway after treatment with ionizing radiation. The initiation and progression of DDR were studied by specific histone acetylation, as a marker of local chromatin relaxation, and formation of γH2AX and 53BP1 foci. In this work, we have studied the effect that VRK1 by itself or in collaboration with olaparib, an inhibitor of PARP, has on the DNA oxidative damage induced by irradiation in order to identify its potential as a new drug target. The combination of olaparib and ionizing radiation increases DNA damage permitting a significant reduction of their respective doses to achieve a similar amount of DNA damage detected by γH2AX and 53BP1 foci. Different treatment combinations of olaparib and ionizing radiation permitted to reach the maximum level of DNA damage at lower doses of both treatments. Furthermore, we have studied the effect that depletion of the VRK1 chromatin kinase, a regulator of DDR, has on this response. VRK1 knockdown impaired all steps in the DDR induced by these treatments, which were detected by a reduction of sequential markers such as H4K16 ac, γH2AX, NBS1 and 53BP1. Moreover, this effect of VRK1 is independent of TP53 or ATM, two genes frequently mutated in cancer. The protective DNA damage response induced by ionizing radiation is impaired by the combination of olaparib with depletion of VRK1, and can be used to reduce doses of radiation and their associated toxicity. Proteins implicated in DNA damage responses are suitable targets for development of new therapeutic strategies and their combination can be an alternative form of synthetic lethality.

Dynamic remodeling of chromatin requires acetylation and methylation of histones, frequently affecting the same lysine residue. These alternative epigenetic modifications require the coordination of enzymes, writers and erasers, mediating them such as acetylases and deacetylases. In cells in G0/G1, DNA damage induced by doxorubicin causes an increase in histone H4K16ac, a marker of chromatin relaxation. In this context, we studied the role that VRK1, a chromatin kinase activated by DNA damage, plays in this early step. VRK1 depletion or MG149, a Tip60/KAT5 inhibitor, cause a loss of H4K16ac. DNA damage induces the phosphorylation of Tip60 mediated by VRK1 in the chromatin fraction. VRK1 directly interacts with and phosphorylates Tip60. Furthermore, the phosphorylation of Tip60 induced by doxorubicin is lost by depletion of VRK1 in both ATM +/+ and ATM−/− cells. Kinase-active VRK1, but not kinase-dead VRK1, rescues Tip60 phosphorylation induced by DNA damage independently of ATM. The Tip60 phosphorylation by VRK1 is necessary for the activating acetylation of ATM, and subsequent ATM autophosphorylation, and both are lost by VRK1 depletion. These results support that the VRK1 chromatin kinase is an upstream regulator of the initial acetylation of histones, and an early step in DNA damage responses (DDR).