Implicación de la quinasa humana VRK1 en la respuesta al daño génico inducido por estrés oxidativo

Trabajo Fin de Máster presentado por el licenciado en Biotecnología Ignacio Campillo Marcos para optar al título de Máster en Biología y Clínica del Cáncer por la Universidad de Salamanca, que ha sido realizado en el Instituto de Biología Molecular y Celular del Cáncer (Curso 2013/2014).[ES]: La célula está continuamente expuesta a agentes tanto exógenos como endógenos causantes de daños en el material genético que pueden afectar a la integridad genómica. Para evitar que esto ocurra, cuenta con mecanismos de reparación específicos para cada tipo de lesión. En este trabajo demostramos la implicación de la serina – treonina quinasa VRK1 en la respuesta al daño oxidativo, especialmente importante en órganos con alta tasa metabólica como el cerebro. Esta quinasa se activa en respuesta a las roturas de doble cadena generadas por la exposición al agua oxigenada y participa en la formación de focos de 53BP1 y γ H2AX en presencia y ausencia de suero. Además, al silenciar VRK1, el número de focos de 53BP1 disminuye significativamente. En conjunto, estos resultados muestran que VRK1 juega un papel importante en las etapas tempranas de la respuesta al daño en el ADN y ponen de manifiesto su implicación en la reparación de las lesiones asociadas al estrés oxidativo.[EN]: Cells are continuously exposed to both exogenous and endogenous agents responsible for DNA damage that could affect genomic integrity. They have developed specific DNA repair mechanisms so as to avoid each of these lesions. In this work we demonstrate serine - threonine kinase VRK1 implication in oxidative damage response, which is essential in organs with high metabolic rates such as brain. This kinase is activated in response to double-strand breaks induced by hydrogen peroxide and it participates in 53BP1 and γ H2AX foci formation, both in presence or absence of serum. Furthermore, the number of 53BP1 foci is significantly reduced after VRK1 knockdown. Therefore, all these results show that VRK1 plays a crucial role in early steps of DNA damage response and exhibit its implication in DNA lesions related to oxidative stress.Peer Reviewe

Nuevas funciones de la quinasa humana VRK1 en la respuesta al daño en el ADN y la estructura de la cromatina

[ES] Los cambios en la estructura de la cromatina, esenciales en distintos contextos celulares, están estrictamente regulados tanto espacial como temporalmente. Esta regulación depende de gran cantidad de proteínas, entre las que se encuentran las distintas familias de quinasas. Durante esta tesis se ha caracterizado, en primer lugar, la implicación de VRK1, una quinasa humana muy abundante y preferentemente nuclear, en la respuesta al daño génico inducido por olaparib, un inhibidor selectivo de PARP ampliamente utilizado como agente quimioterapéutico, y/o radiación ionizante. Se ha visto que la combinación de ambos tratamientos permite reducir las dosis empleadas de cada uno de ellos por separado generando la misma cantidad de daño en el ADN y que VRK1 participa en distintas etapas de la respuesta a este daño independientemente de factores clave en la reparación como p53 o ATM. A continuación, se ha estudiado el efecto de la inhibición específica de modificadores de cromatina sobre la regulación del ciclo celular y el reclutamiento de proteínas implicadas en la respuesta al daño en el ADN. Más concretamente, se ha demostrado, por una parte, que los cambios en la metilación asociados al tratamiento con inhibidores de metiltransferasas ("chaetocin") y demetilasas ("JMJD2 inhibitor") interfieren en la correcta progresión del ciclo celular y, por otra, que la inhibición de metiltransferasas mediada por "chaetocin" altera la estructura de la cromatina e impide que la proteína mediadora 53BP1 se una a las regiones dañadas del ADN y forme focos de reparación. Por último, se ha puesto de manifiesto que la presencia o ausencia de quinasa humana VRK1 altera el patrón de modificaciones covalentes de las histonas, lo que estaría relacionado directamente con un papel regulador de esta quinasa sobre modificadores de cromatina concretos (acetilasas, deacetilasas, metilasas y/o demetilasas). En su conjunto, los resultados obtenidos en este trabajo refuerzan la importancia que tienen los cambios en la cromatina para el mantenimiento de la homeostasis celular y el papel de VRK1 en la regulación de estos cambios tanto a nivel basal como en respuesta al daño génico

VRK1 phosphorylates and protects NBS1 from ubiquitination and proteasomal degradation in response to DNA damage

AbstractNBS1 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

Effectiveness of an intervention for improving drug prescription in primary care patients with multimorbidity and polypharmacy:Study protocol of a cluster randomized clinical trial (Multi-PAP project)

This study was funded by the Fondo de Investigaciones Sanitarias ISCIII (Grant Numbers PI15/00276, PI15/00572, PI15/00996), REDISSEC (Project Numbers RD12/0001/0012, RD16/0001/0005), and the European Regional Development Fund ("A way to build Europe").Background: Multimorbidity is associated with negative effects both on people's health and on healthcare systems. A key problem linked to multimorbidity is polypharmacy, which in turn is associated with increased risk of partly preventable adverse effects, including mortality. The Ariadne principles describe a model of care based on a thorough assessment of diseases, treatments (and potential interactions), clinical status, context and preferences of patients with multimorbidity, with the aim of prioritizing and sharing realistic treatment goals that guide an individualized management. The aim of this study is to evaluate the effectiveness of a complex intervention that implements the Ariadne principles in a population of young-old patients with multimorbidity and polypharmacy. The intervention seeks to improve the appropriateness of prescribing in primary care (PC), as measured by the medication appropriateness index (MAI) score at 6 and 12months, as compared with usual care. Methods/Design: Design:pragmatic cluster randomized clinical trial. Unit of randomization: family physician (FP). Unit of analysis: patient. Scope: PC health centres in three autonomous communities: Aragon, Madrid, and Andalusia (Spain). Population: patients aged 65-74years with multimorbidity (≥3 chronic diseases) and polypharmacy (≥5 drugs prescribed in ≥3months). Sample size: n=400 (200 per study arm). Intervention: complex intervention based on the implementation of the Ariadne principles with two components: (1) FP training and (2) FP-patient interview. Outcomes: MAI score, health services use, quality of life (Euroqol 5D-5L), pharmacotherapy and adherence to treatment (Morisky-Green, Haynes-Sackett), and clinical and socio-demographic variables. Statistical analysis: primary outcome is the difference in MAI score between T0 and T1 and corresponding 95% confidence interval. Adjustment for confounding factors will be performed by multilevel analysis. All analyses will be carried out in accordance with the intention-to-treat principle. Discussion: It is essential to provide evidence concerning interventions on PC patients with polypharmacy and multimorbidity, conducted in the context of routine clinical practice, and involving young-old patients with significant potential for preventing negative health outcomes. Trial registration: Clinicaltrials.gov, NCT02866799Publisher PDFPeer reviewe

Olaparib and ionizing radiation trigger a cooperative DNA-damage repair response that is impaired by depletion of the VRK1 chromatin kinase

[Background] 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.[Methods] 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.[Results] 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.[Conclusion] 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.I.C-M was supported by FPI-MINECO-FSE contract (BES-2014-06772). The laboratory was supported by grants from Agencia Estatal de Investigación- Ministerio de Ciencia, Innovacion y Universidades-FEDER (SAF2016–75744-R), and Consejería de Educación de la Junta de Castilla y León (CLC-2017-01, and UIC-258) to P. A. L.We acknowledge support of the publication fee by the CSIC Open Access Support Initiative through its Unit of Information Resources for Research (URICI

Implication of the VRK1 chromatin kinase in the signaling responses to DNA damage: a therapeutic target?

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 ¿H2AX, 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.I.C-M was supported by FPI-MINECO-Fondo Social Europeo predoctoral contract (BES-2014-06772). The laboratory was supported by grants from Agencia Estatal de Investigación-MINECO (SAF2016-75744-R) and Consejería de Educación de la Junta de Castilla y León (CSI001U16, UIC-017) to P.A.L.We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer Reviewe

VRK1 phosphorylates Tip60/KAT5 and is required for H4K16 acetylation in response to DNA damage

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).This work was supported by grants from Agencia Estatal de Investigación-Ministerio de Economía y Competitividad-FEDER [SAF2016-75744-R; RED2018-102801-T, PID2019-105610RB-I00] Consejería de Educación de la Junta de Castilla y León-ERDF [CLC-2017-01, and UIC-258] to P.A.L., R.G.-G, P.M.-G. and I.C.-M. were supported by Consejería de Educación-Junta de Castilla y León-Fondo Social Europeo (ESF), Ministerio de Educación y Universidades [FPU 16-01883] and MINECO-FPI [BES-2014-067729] predoctoral fellowships respectively

Novel role of the human kinase VRK1 in response to DNA Damage induced by oxidative stress

Resumen del trabajo presentado al 15th ASEICA International Congress, celebrado en Sevilla (España) del 21 al 23 de octubre de 2015.[Introduction]: In basal conditions, cells are exposed to exogenous and endogenous agents, such as ionizing radiation (IR) or oxidative stress, among others, which are responsible for double- and single-strand breaks (DSBs and SSBs, respectively) in the DNA. As a consequence of this, cells can activate different DNA repair mechanisms based on the type of DNA lesions. All these mechanisms take part in a global process called DNA Damage Response (DDR), essential to maintain the integrity and stability of genetic information. It is widely known that oxidative stress induces both SSBs and DSBs, particularly in cells with high metabolic rates, like cancer or neural cells. For this reason, it is particularly relevant to know the proteins which detect and repair the damage, returning to homeostatic equilibrium afterwards. Recently, VRK1 has been described to be required for the assembly of 53BP1 foci and participates in the recruitment and formation of γ H2AX foci in response to ionizing radiation. Furthermore, VRK1 interacts with p53 and is activated by UV-induced DNA damage. Overall, these processes could be an indicator of the crucial role this kinase may play in several steps of DDR. [Objectives]: Our aim is to determine whether VRK1 is activated and participates in the assembly of 53BP1 foci after inducing oxidative stress in human cancer cell lines.[Methods]: To study VRK1 activation by oxidative stress, cell lines were starved and treated with 10 mM of hydrogen peroxide (H2O2). Next, endogenous p53 was immunoprecipitated at different points in time and the level of threonine 18 phosphorylation, which depends on VRK1 activation, was determined with a phosphospecific antibody. Moreover, in vitro assays were performed, using purified protein GST-p53 (1-85) as substrate and analyzing its phosphorylation in threonine 18. Along this same line, the assembly of 53BP1 foci was assessed by immunofluorescence in the presence or absence of VRK1. [Results]: In this work analysis, we can observe that VRK1 is activated by oxidative stress in the absence of serum, which reduces VRK1 activation mediated by growth factors. Exclusively under H2O2 exposition, this kinase is able to activate and phosphorylate p53. In turn, what it is shown is that VRK1 plays an essential role in DNA repair after oxidative stress induction. When cell lines were treated with H2O2, the number of 53BP1 foci increased considerably for the following 30-60 minutes, beginning to decrease at that point. On the contrary, VRK1 depletion was accompanied by a huge reduction of the number of 53BP1 foci, despite the fact that cells had been previously treated with hydrogen peroxide. [Conclusions]: Based on these results, we conclude that VRK1 participates in DNA repair process after H2O2 exposure, because it is activated and required for the assembly of 53BP1 foci under these conditions. Furthermore, it could also be possible that this kinase plays a crucial role in the early steps of DDR, mainly indicated by its hypothetical involvement in the recruitment and formation of γ H2AX foc.Peer Reviewe

The Vaccinia-Related Kinase I (VRK1) in epigenetic regulation of chromatin

Resumen del trabajo presentado al 15th ASEICA International Congress, celebrado en Sevilla (España) del 21 al 23 de octubre de 2015.[Introduction]: The differences in chromatin compaction are determined by the interaction of DNA with histones and other non histone chromosomal proteins. Chromatin needs to relax or condense to regulate gene transcription or DNA damage response, among other processes. These key reversible mechanisms depend on histone post translational modifications, such as lysine acetylation or methylation, which are regulated by ATP-dependent enzymes: histone acetyltransferases (HAT) and methyltransferases (KMT), called “writers”, and histone deacetylases (HDACs) and demethylases (KDMT), called “erasers”. Epigenetics deregulation is gradually gaining importance as potential player in aging and neurodegeneration. VRK1 is a nuclear Ser-Thr kinase implicated in many cellular processes, such as proliferation, cell cycle or DNA Damage Repair (DDR). Previously we observed that VRK1 is present in the chromatin fraction and is able to interact and phosphorylate H2AX in Ser139 after DNA Damage induced by irradiation. Besides, it is necessary for H4 acetylation in an ATM-independent manner. [Objectives]: We aim to study if VRK1regulates epigenetic mechanisms and has a role in chromatin condensation/relaxation by regulating histone writers or erasers. [Methods]: We used pharmacological inhibitors of HATs (C646 and MG149); HDACs (Entinostat and SAHA); KMTs (Chaetocin); and KDMTs (JMJD2 inhibitor). Some of them are advancing in preclinical regulatory studies. Serum-deprived U2OS cells were silenced for VRK1 and immunofluorescence experiments for lysine-specific H3 methylation and H4 acetylation were performed. [Results]: First, as we knew that VRK1 was necessary for 53BP1 and γ H2AX foci formation after irradiation and to confirm that VRK1 has a role in relaxing chromatin after DNA damage, we tested the effect of histone acetylation/deacetylation and methylation/demethylation inhibition on foci formation. The depletion of VRK1 by siRNA in U2OS, A549 and ATM-/- cells affected the acetylation of H3 in Lys14 and Lys9 and also the H4 acetylation in Lys16, important markers of transcriptional activation. In contrast, siVRK1 enhanced the H3 methylation in Lys9 (a gene repressor marker) but not in Lys4 (transcriptional activator residue), confirming the role of VRK1 in positively mediating gene transcription. As expected, the pharmacological treatment of C646 and of MG149 reduced H3K9 and H4K16 acetylation, while the inhibition of HATs and VRK1 depletion didn’t rescue both acetylations, showing that siVRK1 works as a HATs inhibitor. Moreover, the H3K9 trimethylation enhanced by siVRK1 or JMJD2 inhibitor had a further increase with the presence of the both inhibitions, suggesting that siVRK1 is also a strong KDMT inhibitor. [Conclusions]: In conclusion, we suppose that VRK1 regulates epigenetic mechanisms and supports the chromatin relaxation through two ways: activating HATs and also activating KDMTs. These results could be useful to further studies that will expand the roles of VRK1 in chromatin remodeling and also in the number of target proteins regulated by VRK1. Futurperspectives could be focus on the activation of HATs p300 and Tip60 or some of the members of the Jumonji family by VRK1.Peer Reviewe

VRK1 depletion is associated to a defective DNA damage response after combining ionizing radiation and PARP inhibitors

Resumen del trabajo presentado en el 16th Aseica International Congress, celebrado en Valencia (España) del 06 al 08 de noviembre.[Introduction]: Eukaryotic cells are continuously exposed to exogenous and endogenous agents, which are responsible for inducing DNA damage. In order to prevent the adverse consequences of these DNA lesions in the integrity and stability of genetic information, higher organisms have developed DNA damage signaling and repair machineries adapted specifically to the type of damage. Defects in DNA repair, along with the effect of the DNA damage itself and the failure to stall or stop the cell cycle before damaged DNA is passed on to daughter cells, can lead to genomic instability, a typical feature of cancer. Because of that, inhibitors of DNA damage response (DDR) pathways and agents that induce DNA breaks have become an effective anticancer therapy. PARP-1 (Poly (ADP-ribose) polymerase 1) is an important protein involved in DNA repair and transcriptional regulation and is recognized as a key regulator of cell survival and cell death. In turn, it is considered a master component of a number of transcription factors implicated in tumor development and inflammation. As a DNAbinding and chromatin-associating enzyme, it mediates single-strand DNA break repair, alternative end-joining of DNA double-strand breaks, and also aspects of homologous recombination (HR). For this reason, pharmacologic inhibition of PARP-1/2 is synthetically lethal in association with genetic or functional defects in BRCA1/2 and other genes involved in HR. Furthermore, this inhibition sensitizes malignant cells and tumors to ionizing radiation (IR), although it is poorly understood the mechanisms through which PARP inhibition causes radiosensitization. Recently, VRK1 (the most abundant kinase in chromatin) has been described to be required for the assembly of 53BP1 foci and participates in the recruitment and formation of γH2AX foci in response to ionizing radiation. This kinase also phosphorylates and protects Nbs1 to proteosomal degradation after inducing DNA damage, and, moreover, it interacts with p53 and is activated by UV-induced DNA lesions. Therefore, it is possible that VRK1 plays a critical role in several steps of DDR after combining IR and PARP inhibitors.[Objectives]: Our first aim is to determine how Olaparib (PARP inhibitor widely employed in cancer treatment) and IR dosages might be reduced after their combination in order to decrease the side effects on patients, and, secondly, whether VRK1 depletion interferes with DDR in response to DNA damage induced by these two anticancer treatments.[Methods]: To study the effect of combining Olaparib and IR on foci formation, cells were treated with increasing dosages of this PARP inhibitor and irradiated using rising doses afterwards. Then, these cells were fixed and the assembly of DNA repair foci was assessed by immunofluorescence. On the other hand, VRK1 knockdown was performed using different siRNAs and VRK1 levels were tested by immunofluorescence and western blot.[Results]: Based on the assembly of γH2AX and 53BP1 foci, we can conclude that 5 μM Olaparib and IR 1 Gy is the most effective combination of both anticancer treatments, since the number of these foci is comparable to higher dosages of Olaparib or IR separately in three different tumor cell lines. Later, VRK1-depletion experiments show a drastic reduction of DNA repair foci (γH2AX, Nbs1 and 53BP1) after inducing DNA damage by Olaparib, IR or their combination, both in presence or in absence of serum. Furthermore, the consequences associated to VRK1 knockdown are reproducible in p53- and ATM-null cells. Finally, H4K16 acetylation levels, which are directly related to local relaxation of chromatin in response to DNA lesions, are also decreased significatively in absence of this kinase.[Conclusions]: Overall, these results indicate, first of all, that the combination of PARP inhibitors like Olaparib and IR allows the reduction of dosages for each treatment, with a positive impact on cancer patients, and, in second place, VRK1 depletion is correlated with a defective DNA damage response after inducing DNA damage with IR and/or Olaparib, which is independent of p53 or ATM, essential in other DNA repair pathways. As a consequence of this, VRK1 chromatin kinase could become a new therapeutic target in current approaches against cancer