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Trabajo presentado en el EpIC - EpiGene3Sys meets INC-Spain to ChromDesign the Genome International Nucleome Consortium, celebrado en Granada (España) del 17 al 20 de octubre de 2022., Epigenetic alterations are one of the main mechanisms driving different human diseases, including many cancer types and neurological diseases. The coordination of histone PTMs requires the coordination of several epigenetic enzymes. H3K4me3 and H3K27ac characterize transcriptionally active chromatin, whereas repressive states are enriched in H3K9me3 and H3K27me3 among others. We need to understand the regulatory mechanisms that coordinate these epigenetic enzymes. VRK1 (Vaccinia-related kinase 1) is a nuclear kinase implicated in gene transcription or DNA damage response. We studied the role of VRK1 depletion on various epigenetic enzymes and the levels of different PTMs (H3K4me3, H3K9ac/me3, H3K27ac/me3, and H4K16ac), in two different human cancer cell lines, A549 and U2OS. We studied the interaction of VRK1 with the histone-modifying enzymes (KAT, HDAC, KMT and KDM). VRK1 directly interacts, phosphorylates and activates Tip60/KAT5 in response to DNA damage. VRK1 depletion promotes a decrease of chromatin active marks (H3K4me3, H3K9ac, H3K27ac, and H4K16ac), and an increase of H3K9me3 and H3K27me3. Treatments with drugs targeting histone-modifying enzymes also remodel the acetylation and methylation pattern. HAT (C646 and MG149) and KDM (JMJD2i and ORY-1001) inhibitors mimic the effect of VRK1 depletion. HDAC (Vorinostat, Entinostat, Panobinostat, Selisistat, Thiomyristoil, AGK2, and AK7) and KMT (Chaetocin and Tazemetostat) inhibitors mimic the effect of VRK1 depletion on histone marks. VRK1 forms a stable protein complex with KDM1a, KDM3a, KDM4a, HDAC1, Sirt1/2, and SETDB1. We conclude that VRK1 dynamically coordinate epigenetic marks on chromatin and can be suitable target for synthetic lethality strategies in cancer.

Resumen del trabajo presentado en el Congreso "Cancer Epigenetics: Principles, applications and single-cell resolution", celebrado en Spetses (Grecia) del 28 de agosto al 03 de septiembre de 2022., Epigenetic regulation of gene expression and DNA damage responses are mediated by chromatin remodeling, which involves histone post-translational modifications (PTMs). These PTMs result in dynamic shifts between different degrees of chromatin compaction. Thus, it is crucial to unveil the molecular mechanisms that modulate these changes and the histone-modifying enzymes involved in such regulation. VRK1 (Vaccinia-related kinase 1) is a nuclear/chromatin kinase implicated in different cellular processes like transcription or DNA damage response. In this context, we studied the potential role of VRK1 in the regulation of these epigenetic modifiers. In our study, we evaluated the levels of different PTMs in two human cancer cell lines, A549 (lung adenocarcinoma) and U2OS (osteosarcoma). We next compared the effect of VRK1 silencing with the effect of inhibitors of several epigenetic enzymes. Finally, we studied the potential interaction of VRK1 with specific epigenetic modifiers. Our results showed that VRK1 knock-down induced an alteration of histone PTMs. We observed that VRK1 depletion promotes a decrease in H3K4me3, H3K9ac, H3K27ac, and H4K16ac levels and an increase in H3K9me3 and H3K27me3. Moreover, VRK1 absence mimics the effect of HATi (C646 and MG149) and KDMi (JMJD2i and ORY-1001), while the presence of VRK1 mimics the effect of HDACi (Vorinostat, Entinostat, Panobinostat, Selisistat, Thiomyristoil, AGK2, and AK7) and KMTi (Chaetocin and Tazemetostat). Furthermore, VRK1 forms a stable protein complex with KDM1a, KDM3a, KDM4a, HDAC1, SIRT1/2, and SETDB1. Our findings revealed that permissive chromatin marks (H3K4me3, H3K9ac, H3K27ac, H4K16ac) decreased while repressive marks (H3K9me3, H3K27me3) increased in VRK1-depleted cells. Therefore, the loss of VRK1 expression had the same effect as the inhibition of the epigenetic enzymes that interact with VRK1, suggesting that VRK1 might be a regulator of them and used as potential target for novel cancer therapies.

Resumen del trabajo presentado en el EACR 2022 Congress - Innovative Cancer Science: Translating Biology to Medicine, celebrado en Sevilla (España) del 20 al 23 de junio de 2022., [Introduction]: Epigenetic alterations are one of the main mechanisms driving different human diseases, including many cancer types and neurological diseases. Histone posttranslational modifications (PTMs) are changes in the amino acid residues of the histone tail which regulate gene expression. The coordination of PTMs requires the strictly regulation of multiple epigenetic enzymes, termed writer and eraser proteins. Chromatin active states are characterized by PTMs such as H3K4me3 and H3K27ac, whereas repressive states are enrichment in H3K9me3 and H3K27me3 among others. Thus, it could be crucial to understand the mechanisms that mediate the coordination of such enzymes. VRK1 (Vaccinia-related kinase 1) is a nuclear kinase implicated in different cellular process like transcription or DNA damage response. In this context, we studied the potential role of VRK1 in the regulation of those epigenetic modifiers. [Material and Methods]: We compared the transient VRK1 silencing with the effect of modulators of various epigenetic enzymes. In our study, we evaluated the expression levels of different PTMs (H3K4me3, H3K9ac/me3, H3K27ac/me3, and H4K16ac), by immunodetection in two different human cancer cell lines, lung adenocarcinoma A549 and osteosarcoma U2OS. We studied the interaction of VRK1 with the histonemodifying enzymes of this PTMs by co-immunoprecipitation. [Results and Discussions]: Our results showed that VRK1 depletion caused an alteration of post-translational histone marks in cells. We observed that VRK1 depletion promotes a decrease of chromatin active marks (H3K4me3, H3K9ac, H3K27ac, and H4K16ac), and an increase of H3K9me3 and H3K27me3. Treatments with drugs targeting histone-modifying enzymes also remodel the acetylation and methylation pattern. HAT (C646 and MG149) and KDM (JMJD2i and ORY-1001) inhibitors mimic the effect of VRK1 depletion. HDAC (Vorinostat, Entinostat, Panobinostat, Selisistat, Thiomyristoil, AGK2, and AK7) and KMT (Chaetocin and Tazemetostat) inhibitors mimic the effect of VRK1 on histone marks. VRK1 forms a stable protein complex with KDM1a, KDM3a, KDM4a, HDAC1, Sirt1/2, and SETDB1. [Conclusion]: Our findings revealed that permissive chromatin marks decreased (H3K4me3, H3K9ac, H3K27ac, H4K16ac) while repressive marks (H3K9me3, H3K27me3) increased in VRK1-depleted cells. Therefore, the loss of VRK1 expression in the cells had the same effect than the inhibition of these enzymes, suggesting that VRK1 might be a regulator of those epigenetic enzymes and used as new target in synthetic lethality strategies

Resumen del trabajo presentado en el EACR 2022 Congress - Innovative Cancer Science: Translating Biology to Medicine, celebrado en Sevilla (España) del 20 al 23 de junio de 2022., [Introduction]: Histone posttranslational modifications (PTMs) and nucleosome composition modulate chromatin dynamic remodeling, which is required for gene expression regulation and for the DNA damage response after oxidative stress in cells. Alteration of PTMs and chromatin structure leads to defective oxidative stress signaling and to an unbalance between oxidants and antioxidants, causing pathologies like cancer and neurodegenerative diseases. VRK1 (Vaccinia-related kinase 1) is a nucleosomal serine-threonine kinase involved in epigenetic chromatin changes and in the DNA damage response (DDR). VRK1 phosphorylates the acetyltransferase Tip60 in response to DNA damage, which carries out the acetylation of H4K16, a PTM necessary for chromatin remodeling prior to DNA repair. In addition, VRK1 phosphorylates γH2AX and 53BP1, both critical for a proper DDR in different pathways. Furthermore, VRK1 is overexpressed in many cancer types such as breast cancer, colon, lung, liver, and gliomas. [Material and Methods]: We used H2O2 200 µM at different times to induce oxidative stress in A549 cells. We studied VRK1 autophosphorylation and activation by performing radioactive kinase assays. For the measurement of reactive oxygen species (ROS) levels, we quantified the intensity of the marker 8-oxo-guanine using immunofluorescence. We used a commercial kit (TUNEL) to detect DNA ends in cells, corresponding with DNA damage levels. Different PTMs (H3K9ac, H4K16ac, H3K27ac, H3K9me3, and H3K4me3), weredetected by immunofluorescence. [Results and Discussions]: In this work, we have demonstrated that VRK1 is autophosphorylated and activated in response to oxidative stress. Furthermore, we observed that the depletion of VRK1 led to the accumulation of ROS assessed by the increase in 8-oxo-guanine levels, causing an increase in DNA damage in cells. VRK1 knock-down completely altered the epigenetic landscape of histone PTMs. We observed that the absence of the kinase led to a decrease in H3K9ac, H4K16ac, and H3K27ac and an increase in H3K9me3 and H3K4me3 after hydrogen peroxide exposure. These results suggest that the depletion of the kinase impairs chromatin remodeling after H2O2 treatment. Therefore, we propose VRK1 as an early modulator of the oxidative stress response. [Conclusion]: VRK1 plays a key role in the oxidative stress response by regulating chromatin remodeling after hydrogen peroxide exposure.

Chromatin is dynamically remodeled to adapt to all DNA-related processes, including DNA damage responses (DDR). This adaptation requires DNA and histone epigenetic modifications, which are mediated by several types of enzymes; among them are lysine methyltransferases (KMTs). Methods: KMT inhibitors, chaetocin and tazemetostat (TZM), were used to study their role in the DDR induced by ionizing radiation or doxorubicin in two human sarcoma cells lines. The effect of these KMT inhibitors was tested by the analysis of chromatin epigenetic modifications, H4K16ac and H4K20me2. DDR was monitored by the formation of γH2AX, MDC1, NBS1 and 53BP1 foci, and the induction of apoptosis. Results: Chaetocin and tazemetostat treatments caused a significant increase of H4K16 acetylation, associated with chromatin relaxation; and increased DNA damage, detected by the labeling of free DNA-ends. These inhibitors significantly reduced H4K20 dimethylation levels in response to DNA damage and impaired the recruitment of 53BP1, but not of MDC1 and NBS1, at DNA damaged sites. This modification of epigenetic marks prevents DNA repair by the NHEJ pathway and leads to cell death. Conclusion: KMT inhibitors can function as sensitizers to DNA damage-based therapies and be used in novel synthetic lethality strategies for sarcoma treatment.

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.

Resumen del trabajo presentado en el 16th Aseica International Congress, celebrado en Valencia (España) del 06 al 08 de noviembre de 2018., [Introduction]: Chromatin undergoes dynamic changes in both normal cellular processes (cellular division, transcription and replication) and in pathological situations (DNA damage, among others). DNA is constantly exposed to exogenous (environmental and chemical factors) and endogenous damage agents (oxidative stress) which cause lesions. In order to repair those lesions, cells have developed several mechanisms that take part in a global process called DNA damage response (DDR), essential to maintain genome integrity and homeostasis. Unrepaired lesions could lead to cancer and neurodegenerative diseases. Vaccinia related kinase 1 (VRK1) is a nucleosomal serine threonine kinase that has been proved to play an important role in DDR by participating in the assembly of 53BP1 and γH2AX foci and by forming a stable complex with NBS1 after gamma radiation exposure. Moreover, VRK1 participates in the epigenetic changes that are necessary for chromatin remodeling after DNA damage. In this study, we have used two drugs that induce different types of DNA damage: temozolomide and actinomycin D. Temozolomide is an alkylating agent that causes DNA methylation and is in use for glioblastoma treatments. Actinomycin D intercalates into the DNA causing transcription inhibition. In addition, we have used olaparib, a PARP inhibitor, in order to study whether it sensitizes temozolomide effect on glioblastoma cells. [Objectives]: Our aim is to determine the role of VRK1 in the DNA damage response when the DNA is exposed to two drugs that cause different types of damage: DNA methylation by alkylating agents and transcription inhibition by actinomycin D. [Methods]: To study VRK1 response, cells were treated with temozolomide 200 μM and actinomycin D 70 nM (separately), and 53BP1, NBS1 and the acetylation of histone H4 were studied using immunofluorescence techniques. VRK1 was depleted to study its effect on these damage markers after DNA damage. Moreover, temozolomide doses was reduced (50 μM) when combinated with olaparib 5 μM. [Results]: In our study, we have observed that the acetylation of the histone H4 increases after 5 minutes of both types of damage (transcription inhibition and DNA methylation), which means that the chromatin is reorganized in order to be repaired. In addition, we have noticed that NBS1 increases after 15 minutes and the number of 53BP1 foci also increases after 30 minutes of both treatments. NBS1 is an early component of the DDR pathway and 53BP1 is part of a specific repairing route. Therefore, these results suggest that VRK1 participates in several DDR steps. Besides, VRK1 depletion reduces the acetylation of histone H4, necessary for chromatin remodeling before DNA damage response, and it affects 53BP1 foci formation, a marker of DNA double-strand breaks. Depletion of VRK1 impairs the DDR to treatment with olaparib (PARP inhibitor) and/or temozolomide, and thus compromise cell viability. Additionally, we have observed that the combination of temozolomide and olaparib allow us to reduce both doses getting the same effect, which can result in a reduction of their toxicity. [Conclusion]: Based on these results, we conclude that VRK1 participates in the DNA repair process after actinomycin D and temozolomide treatments. Furthermore, VRK1 could participate in the early damage response because the acetylation of the histone H4, necessary for chromatin remodeling before repairing, is reduced by VRK1 depletion. Moreover, we suggest that temozolomide, in combination with olaparib, could have a stronger effect at lower doses on glioblastoma cells indicating that the toxicity associated with these drugs can be reduced., [Objectives]: Our aim is to determine the role of VRK1 in the DNA damage response when the DNA is exposed to two drugs that cause different types of damage: DNA methylation by alkylating agents and transcription inhibition by actinomycin D., [Methods]: To study VRK1 response, cells were treated with temozolomide 200 μM and actinomycin D 70 nM (separately), and 53BP1, NBS1 and the acetylation of histone H4 were studied using immunofluorescence techniques. VRK1 was depleted to study its effect on these damage markers after DNA damage. Moreover, temozolomide doses was reduced (50 μM) when combinated with olaparib 5 μM., [Results]: In our study, we have observed that the acetylation of the histone H4 increases after 5 minutes of both types of damage (transcription inhibition and DNA methylation), which means that the chromatin is reorganized in order to be repaired. In addition, we have noticed that NBS1 increases after 15 minutes and the number of 53BP1 foci also increases after 30 minutes of both treatments. NBS1 is an early component of the DDR pathway and 53BP1 is part of a specific repairing route. Therefore, these results suggest that VRK1 participates in several DDR steps. Besides, VRK1 depletion reduces the acetylation of histone H4, necessary for chromatin remodeling before DNA damage response, and it affects 53BP1 foci formation, a marker of DNA double-strand breaks. Depletion of VRK1 impairs the DDR to treatment with olaparib (PARP inhibitor) and/or temozolomide, and thus compromise cell viability. Additionally, we have observed that the combination of temozolomide and olaparib allow us to reduce both doses getting the same effect, which can result in a reduction of their toxicity., [Conclusion]: Based on these results, we conclude that VRK1 participates in the DNA repair process after actinomycin D and temozolomide treatments. Furthermore, VRK1 could participate in the early damage response because the acetylation of the histone H4, necessary for chromatin remodeling before repairing, is reduced by VRK1 depletion. Moreover, we suggest that temozolomide, in combination with olaparib, could have a stronger effect at lower doses on glioblastoma cells indicating that the toxicity associated with these drugs can be reduced.

1 p. (Abtracts of SFRR-International 2021 Virtual Meeting), H2020 grant-675132, “Masstrplan”; RTI2018-097624-B-100 (MCINN, ERDF); RETIC-Aradyal RD16/0006/0021 (ISCIII-ERDF); MICINN BES-2016-076965 (AVP), PRE2019-088194 (PG).

17 p.-10 fig., Cysteine residues can undergo multiple posttranslational modifications with diverse functional consequences, potentially behaving as tunable sensors. The intermediate filament protein vimentin has important implications in pathophysiology, including cancer progression, infection, and fibrosis, and maintains a close interplay with other cytoskeletal structures, such as actin filaments and microtubules. We previously showed that the single vimentin cysteine, C328, is a key target for oxidants and electrophiles. Here, we demonstrate that structurally diverse cysteine-reactive agents, including electrophilic mediators, oxidants and drug-related compounds, disrupt the vimentin network eliciting morphologically distinct reorganizations. As most of these agents display broad reactivity, we pinpointed the importance of C328 by confirming that local perturbations introduced through mutagenesis provoke structure-dependent vimentin rearrangements. Thus, GFP-vimentin wild type (wt) forms squiggles and short filaments in vimentin-deficient cells, the C328F, C328W, and C328H mutants generate diverse filamentous assemblies, and the C328A and C328D constructs fail to elongate yielding dots. Remarkably, vimentin C328H structures resemble the wt, but are strongly resistant to electrophile-elicited disruption. Therefore, the C328H mutant allows elucidating whether cysteine-dependent vimentin reorganization influences other cellular responses to reactive agents. Electrophiles such as 1,4-dinitro-1H-imidazole and 4-hydroxynonenal induce robust actin stress fibers in cells expressing vimentin wt. Strikingly, under these conditions, vimentin C328H expression blunts electrophile-elicited stress fiber formation, apparently acting upstream of RhoA. Analysis of additional vimentin C328 mutants shows that electrophile-sensitive and assembly-defective vimentin variants permit induction of stress fibers by reactive species, whereas electrophile-resistant filamentous vimentin structures prevent it. Together, our results suggest that vimentin acts as a break for actin stress fibers formation, which would be released by C328-aided disruption, thus allowing full actin remodeling in response to oxidants and electrophiles. These observations postulate C328 as a “sensor” transducing structurally diverse modifications into fine-tuned vimentin network rearrangements, and a gatekeeper for certain electrophiles in the interplay with actin., This work was supported by Grants RTI2018-097624-B-I00 and PID2021-126827OB-I00, funded by MCIN/AEI/10.13039/501100011033 and ERDF, “A way of making Europe”; RETIC Aradyal RD16/0006/0021 from ISCIII, cofunded by ERDF; European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant agreement no. 675132 “Masstrplan”; PGJ is the recipient of a predoctoral contract PRE2019-088194, from MCIN/AEI/10.13039/501100011033 and ESF, “Investing in your future”, Spain. AEM is the recipient of a postdoctoral contract from the Juan de la Cierva Program, FJC2021-047028-I, funded by MCIN/AEI/10.13039/501100011033 and by European Union NextGenerationEU/PRTR