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 radia- tion 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. Keywords VRK1 · H2AX · NBS1 · 53BP1 · p53 · Phosphorylation · DNA damage response · Ionizing radiation Abbreviations Introduction VRK1 Vaccinia-related kinase 1 DSB DNA double-strand break Genome stability DDR DNA damage response NBS1 Nijmegen breakage syndrome 1 (nibrin) Genome stability is essential for the maintenance of spe- NHEJ Non-homologous end-joining cies, but, at the same time, genetic variation is necessary for 53BP1 Tumor protein P53 binding protein 1 their evolution. Therefore, in all species, there are several ATM Ataxia-telangiectasia-mutated Ser/Thr kinase mechanisms aiming to protect DNA from genetic damage of endogenous or exogenous origin. Endogenous DNA damage is a consequence of the biological properties of cells, and includes oxidative stress, replication errors, transcriptional errors, or metabolism of DNA, to which cells are continu- ously exposed . Alternatively, exogenous factors such as ultraviolet light, ionizing radiation, or chemicals also cause DNA damage to which exposure is frequently transient. The DNA damage has many different forms, single- or double- * Pedro A. Lazo strand breaks, nucleotide, or base modification [ 1]. To cope firstname.lastname@example.org with all of them, cells have developed several specific DNA repair mechanisms, which increase their complexity in Experimental Therapeutics and Translational Oncology higher organisms because of the chromatin organization. Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC-Universidad de Salamanca, 37007 Salamanca, Double-strand breaks constitute the most serious form of Spain DNA damage that has two alternative repair mechanisms Instituto de Investigación Biomédica de Salamanca (IBSAL), depending of the situation of the cell cycle. During repli- Hospital Universitario de Salamanca, 37007 Salamanca, cation, DNA double-strand breaks (DSBs) are repaired by Spain Vol.:(0123456789) 1 3 2376 I. Campillo-Marcos, P. A. Lazo homologous recombination (HR) using as template the other in the local chromatin organization. Chromatin can function chromatid. In non-dividing-cells or in G0/G1 phases, DSBs as a signaling platform that has effects not only on its remod- are repaired by non-homologous end-joining (NHEJ) . eling, but can also send signals to other processes involved in Independently of the origin of DNA lesions, these lesions nuclear dynamics . When cells encounter a stress such as have to be detected rapidly and efficiently before cells DNA damage, the activation of complex signaling networks divide, to avoid transmitting the damage to their progeny. triggers the detection and repair of the damage in a specific Because nuclear kinases are capable of rapidly and revers- and sequential process, before returning to the homeostatic ibly responding to changes in the cell and its environment, equilibrium. These networks integrate a wide variety of sig- and of integrating diverse stimuli, they are likely to be nals from inside the cell, transduced through protein kinases involved in sensing, triggering, regulating, and organizing [10–12], to ultimately control cell cycle arrest or progression the sequential steps that are needed for a correct and specific in the case of dividing cells . Moreover, the chromatin- DNA damage response. signaling platform regulates DDR, cell cycle checkpoints, Cells are continuously exposed to DNA damage and it cell death, and senescence, among others. All these pro- can occur at any time during the cellular lifetime. The num- cesses are associated with the maintenance of genetic sta- ber of normal cell division is limited to approximately 40 bility and the transmission of a mutation-free genome to because of telomere shortening, which implies that, in the daughter cells. The major pathological consequence of DNA life of the organism, most cells are not dividing at the time of damage is the potential transmission of mutations to their exposure to DNA damage [3, 4]. Furthermore, cells are most progeny , which are implicated in aging and cancer . of their lifetime in the G0/G1 phases, in which homologous In addition to the role of DNA damage in cancer, alterations recombination is not functional , but are exposed to DNA in DNA repair genes are also associated with neurodegenera- damage. Furthermore, stem cells have an enhanced response tive diseases , since neurons are not dividing in most of to DNA damage mediated by the NHEJ pathway . There- the individual lifetime and have to repair these DNA lesions. fore, most of the DNA lesions will occur and have to be Most research into DNA damage responses has been studied repaired in the absence of replication. Very often, there is a in the context of replication and cell division . large time interval between the moment in which DNA dam- In the highly organized eukaryotic chromatin, the most age occurs and the time when an individual cell replicates, vulnerable DNA is the fraction that is transcriptionally active in which most cells are non-dividing, and are thus able to at the time of exposure to damaging agents, particularly in pass the mutation to their daughter cells. Consequently, each resting or non-dividing cells, such as stem cells or neurons. cell has to deal individually with this problem and to respond In these locations, DNA has to relax and open to allow the independently of its particular situation, which is very vari- access of RNA polymerase and permit gene transcription. In able within a tissue. Cells are either resting or dividing, and these transcriptionally active regions, DNA is more exposed their individual position within a tissue implies that cellular and vulnerable, particularly in non-dividing or cells in G0/ interactions are heterogeneous depending on its location. G1. Therefore, in an individual resting cell, the response to DNA repair mechanisms have to function in all these dif- DNA damage does not have to be linked to cell division, ferent cellular contexts. In the particular case of neurons, differentiation state, or the cell location and its interactions by their exposition to oxidative stress, the accumulation of within a tissue. Even in dividing cells, the G1 phase last DNA damage might be a pathogenic mechanism for dete- several hours before entering replication. DNA damage has rioration of neurological functions associated with aging. to be detected, identified, and repaired immediately in all Recent evidence indicates that a significant proportion of the different types of situations. DNA damage is of endogenous origin [7, 8]. Francis Crick predicted that several redundant mechanisms must exist to repair damaged DNA and maintain genome integrity . Cellular response to DNA damage Since then, several pathways have been identified [10– 13]. Induction of DNA damage is a mainstay of cancer treatment, The cellular reaction to DNA damage involves two major aims; one is to protect the DNA, and the other to protect and the specific targeting of regulatory proteins implicated in DDR can lead to the development of new drugs. cells and the organisms from the consequences of unre- paired DNA damage. The cellular protection against DNA Chromatin and DNA damage damage is mediated by arresting cell cycle in proliferating cells, so that damage can be repaired before its transmission The cellular response to DNA damage has to be initiated and to daughter cells. However, if DNA damage is excessive and cannot be repaired, the alternative response is medi- triggered at the site of the DNA lesion, independent of its type. DNA damage causes a local distortion of the double ated by the induction of cell death, and in that way, there is no progeny of mutated cells. These two types of responses helix, and of its associated nucleosomes, which is reflected 1 3 Implication of the VRK1 chromatin kinase in the signaling responses to DNA damage: a therapeutic… 2377 are associated with p53 and activated by different types of Chromatin DNA damage. DNA repair requires a sequential reorganization of chro- Transcription factors matin to allow for the different and consecutive steps in each DNA Damage Response repair pathway, which includes protection of damaged DNA, ac recognition of the type of lesion, recruitment of specific H2A H3 p53 repair mechanisms, ligation of DNA ends, and restoration P KAT5 to its normal chromatin organization. After DNA damage, in Sox2 P addition to the DNA lesion, the initial effect is a local distor - ac ATF2 P ATM tion of chromatin, which is the initiating event to trigger the P VRK1 cascade of DNA repair processes. As organisms increased P CREB NBS1 in their complexity, new regulatory elements are necessary P P Jun not only to coordinate different functions in DDR, but also γH2AX to adjust to their much more complex and dynamic structure FXR of chromatin. Therefore, new regulatory mechanisms that integrate and coordinate basic processes are necessary. In Fig. 1 VRK1 relation with transcription factors and DNA damage this context, new regulatory elements have evolved from pre- response proteins in chromatin existing proteins. A candidate for this role must be a chroma- tin protein with a reversible enzymatic activity. Among the which are organized in nucleosomes and in direct contact 518 kinase of the human kinome, vaccinia-related kinase-1 (VRK1) is a potential candidate for this role because of its with DNA, contributing to chromatin spatial organization. VRK1 is detected in the chromatin fraction forming a stable association to chromatin and its targets, with the exception of chromosomes condensed in mitosis [19, 20]. complex with histone H3 . Moreover, VRK1 phospho- rylates histones H3 [27, 29, 30], H2A [31, 32] and H2AX . Therefore, it is very likely that nucleosome organi- zation can be modified by covalent modifications because VRK1 roles in chromatin of histone phosphorylations by VRK1. This regulation of histone covalent modifications is essential for different func- The VRK1 chromatin kinase tions, normal or pathological, requiring a dynamic chroma- tin reorganization [33, 34]. VRK1 is a Ser-Thr kinase that belongs to the VRK family that diverged early from branch of the human kinome that An additional role of VRK1 as a chromatin kinase is its association with transcriptional complexes, where it inter- led the casein kinase family . Bacteria and yeast have no VRK or p53 members, invertebrates such as D. mela- acts and phosphorylates several transcription factors that include p53 , CREB , ATF2 , c-Jun , Sox2 nogaster or C. elegans have one member, and mammals have three members in their respective families. The complex- , and the farnesoid X receptor (FXR) . ity of VRK family  parallels that of p53  and the autophagic DRAM (death-related autophagic modulator) VRK1 as a sensor of chromatin alterations . This increased complexity during evolution is likely to reflect the need for additional regulatory or coordinating Chromatin in interphase has a very large size and DNA lesions can occur at any place, heterochromatin and euchro- roles as organisms and their functions became more com- plex. In mammals and C. elegans, it is known as Vrk-1 , matin, which are likely to have a different sensitivity to DNA damage. Alterations of DNA by strand breaks or chemical and in D. melanogaster as nucleosomal histone kinase 1 (NHK-1) . modifications, such as oxidation, alkylation, or intercalation among others, will alter the chromatin organization by intro- VRK1 is a Ser-Thr kinase in nuclei  that is located on chromatin in resting cells and in all phases of the cell cycle ducing a local distortion [40, 41], which is a likely initiating event for triggering the complex processes of DNA repair covering all DNA, except when chromosomes are already condensed in mitosis [27, 28], in which VRK1 is ejected [42–44]. However, responding to DNA damage requires the coupling of chromatin distortion to a signal transduction from mitotic chromosomes. When chromosomes segregate, VRK1 returns to chromatin in daughter cells. VRK1 forms system, probably mediated by a nuclear chromatin kinase. A requirement for a sensor kinase is that its activation stable complexes with several different types of chromatin proteins, ranging from histones, transcription factors, and is independent of the type of DNA damage and, therefore, is not associated to any particular type of DNA damage. proteins involved in DNA repair processes (Fig. 1). The pro- teins more closely associated with DNA are histones , In this latter case, the kinase involved will participate in 1 3 2378 I. Campillo-Marcos, P. A. Lazo specific steps of a particular DNA damage, as is the case for . ATM-null cells, such as the HT144 cell line, has a high ATM, ATR, or DNA-PK in the response to double-strand endogenous level of H4K16ac that is also lost by depletion +/+ DNA breaks . In the particular case of VRK1, its kinase of VRK1 . In ATM cells, this acetylation induced activity increases tenfold after induction of DNA damage by IR does not occur in the absence of VRK1 . These independently of its type, which includes pyrimidine dimers results indicate that VRK1 is a good candidate to regulate caused by ultraviolet light, single-strand DNA breaks caused the enzymes involved in epigenetic modifications of chroma- by hydroxyurea treatment, or double-strand DNA breaks tin. DNA damage causes a local distortion of chromatin that induced by either doxorubicin or ionizing radiation . can affect its different covalent modifications. Consequently, Early sensor mechanism of DNA damage must fulfill the regulation and coordination of histone modifiers such some basic requirements, be a nuclear enzyme that inter- as acetylases, deacetylases, methylases, and demethylases acts with basic chromatin components in nucleosomes, and is very poorly understood. Moreover, VRK1 also directly be a capable of an immediate signaling reaction that is also phosphorylates histone H2A in T120 , which is next to reversible. In this context, a kinase, such as VRK1, is a very K119 ubiquitinated, and both modifications are functional suitable candidate for this role [29, 46, 47]. alternatives, being T120 phosphorylation an activator of Other important early proteins at the site of specific types chromatin. Thus, two histones in nucleosomes, H3 and H2A, of DNA damage are Ku70/Ku80 (XRCC6/XRCC5), which are directly regulated by VRK1. Furthermore, histone H4 have to re-localize and interact with free DNA ends at the is not a phosphorylation target of VRK1, but its covalent breakpoints, mainly in double-strand breaks , a subtype modification by acetylation is sensitive to VRK1 in an ATM- of DNA damage, or in telomeres [48, 49]. It is unknown independent manner, since it is detected in ATM-null cells whether these proteins are targets of VRK1, but it is a real . possibility. Ku70 and Artemis have multiple phosphoryla- It is important to remark that the sensor kinase activity tion sites, but the kinases involved in their specific phospho- has to be regulated by protein–protein interactions. In this rylation and their regulation are unknown. Telomeres are context, the C-terminal region of VRK1 has a low complex- naturally occurring DNA ends in chromosomes and there ity structure, which is very flexible and can adopt different is evidence for a role of VRK1 in their maintenance . conformations . This C-terminal region can fold and Moreover, VRK1 phosphorylates hnRNP A1 (heterogeneous block the active site of the kinase  and proteins inter- nuclear ribonucleoprotein A1) and facilitates its binding to acting with this region can modulate the activity of VRK1. telomeric ssDNA and telomeric RNA . Two proteins that inhibit the VRK1 kinase activity have been identified, macrohistone H2A1.2 in interphase [ 59], VRK1, chromatin relaxation, and histone acetylation and Ran-GDP, but not Ran-GTP , which have an asym- metric nuclear distribution . DNA damage and local disruption of chromatin are associ- ated with an increase in histone acetylation, which is medi- ated by KAT (lysine acetyl transferase) proteins. Histone VRK1 in DNA damage responses acetylation extends over an area of several hundred kilobases flanking the damaged DNA site [51, 52], which requires the VRK1 and histone H2AX local activation of KATs by a not yet identified mechanism. Defects in histone acetylation are associated with an increase VRK1 directly and stably interacts with histones H2AX in cellular sensitivity to DNA damage as a consequence of a and H3 in basal conditions, and is able to phosphoryl- defective DNA repair [53, 54]. Furthermore, acetylation of ate them in vitro with purified proteins in Ser139 and histone H4 in Lys16 disrupts the interaction between H4 and Thr3, respectively . The early response to DNA dam- H2A–H2B, and facilitates the relaxation of chromatin [55, age requires the phosphorylation of H2AX in Ser139 56]. Consistently, the inactivation of KAT5/Tip60 blocks (γH2AX). γH2AX covers large areas of DNA surround- the opening of chromatin at DSBs (double-strand breaks) ing the site of DNA damage  and protects DNA from that are required to facilitate the repair process . Induc- exonuclease attack. This γH2AX organization can also tion of DNA damage by UV light or radiation causes an function as a platform for the recruitment of proteins increase in histone acetylation [52, 57]. Depletion of VRK1, that participate in sequential DDR steps, such as NBS1, a nucleosomal chromatin kinase, causes a loss of histones 53BP1, or BRCA1, among others [40, 63]. Phosphoryla- H3 and H4 acetylation, which are necessary for chromatin tion of histone H2AX in Ser139 (γH2AX) is a mark of relaxation, either in basal conditions or after DNA damage, an early reaction to DNA damage that can be detected by independently of ATM and p53 . VRK1 knockdown formation of γH2AX foci [62, 64]. The phosphorylation also causes a loss of specific histone acetylations, including of H2AX and the formation of γH2AX foci induced by H4K16 acetylation (H4K16ac), induced by DNA damage ionizing radiation (IR) are lost by depletion of VRK1 and 1 3 Implication of the VRK1 chromatin kinase in the signaling responses to DNA damage: a therapeutic… 2379 can be rescued by kinase-active, but not by kinase-dead, VRK1 and NBS1 in early DDR VRK1 . This effect of VRK1 is also independent of ATM, suggesting that VRK1 is an upstream participant. Cellular responses to DNA damage require the formation of VRK1 is also necessary for the activation of ATM and protein complexes in a highly organized fashion. In resting CHK2 in response to IR . However, in the absence cells, VRK1 plays an important role in the formation of ion- of ATM, the γH2AX foci induced by IR have a smaller izing radiation-induced foci formed by γH2AX, NBS1, and size, which indicates that both kinases cooperate either in 53BP1 during DDR. The MRE11 complex holds together the formation or stabilization of the foci . This latter the two free ends of the broken DNA. This complex, formed possibility might be a consequence of the effect of VRK1 NBS1–Mre11–Rad50, is highly dynamic and has a very on the stability of NBS1 . In this context, VRK1 is a complex organization . Phosphorylation of NBS1/nibrin novel chromatin component that reacts to its alterations is necessary for the recruitment of ATM to damaged sites and participates very early in DDR by itself and in coop- and for the stabilization of the repair complex . VRK1 is eration with ATM . activated by DNA double-strand breaks induced by ionizing radiation (IR) or doxorubicin, and specifically phosphoryl- ates NBS1 in Ser343  and 53BP1 in serum-starved cells VRK1 and specific DNA damage response proteins and ATM-null and p53-null cells , indicating that they are independent of both p53 and ATM activation , and Because of the physical association of VRK1 with chroma- consistent with VRK1 role as an early step in the response tin, VRK1 has also been implicated in the regulation of DDR to DNA damage. Depletion of VRK1 causes a loss of NBS1 proteins. The VRK1 kinase has also been directly associated stability that is prevented by treatment with the MG132 pro- with different components in DDR pathways, which have teasome inhibitor . This phosphorylation mediated by been studied in the context of the response to DSBs, in both VRK1 protects the NBS1 protein of RNF8-mediated ubiqui- resting and cycling cells as well as in ATM-null and p53- tination . Therefore, it is likely that NBS1 phosphoryla- null cells. VRK1 physically interacts and directly phospho- tion by VRK1 contributes to the stabilization of foci, and rylates specific proteins participating at different sequential facilitates the recruitment of additional participants in the stages of DDR, which include H2AX , NBS1 , and specific DNA repair process, such as kinases of the PI3K 53BP1 [46, 65] in NHEJ [66, 67]; and all of these activating family, ATM, ATR, or DNA-PK, for specific signaling steps phosphorylations are lost by VRK1 depletion. Intermediate or pathways in DDR . steps in DDR signal transmission are well known. The most common pathways in DNA damage response (DDR) impli- VRK1 and 53BP1 in NHEJ cate protein phosphorylation by different kinases such as ATM , ATR , and DNA-PK ; all members of the Double-strand breaks are the most serious form of DNA PI-3K family, which have been mostly studied in the context damage, particularly in cells that are resting or in the early of cell division and cell cycle checkpoints . In response phases of the cell cycle, which includes differentiated rest- to double-strand breaks induced by ionizing radiation (IR), ing cells, as neurons, and stem cells. Under these conditions, the 53BP1 scaffold protein is recruited to IR-induced foci these DSBs are repaired by non-homologous end-joining (IRIF), and is an important marker for monitoring cellular (NHEJ); and one of its main components is 53BP1, a scaf- DDR by NHEJ. 53BP1 foci induced by ionizing radiation or fold protein that forms foci induced by DNA damage . doxorubicin are intermediate steps in DDR activation [68, VRK1 stably interacts with 53BP1 in the region comprised 69] and are known to be regulated by ATM in response to between residues 955-1354, which is implicated in the DSBs , and by ATR in response to replication stress interaction with H2AX, but its phosphorylation site is in . However, it is also known that DNA damage response Ser25/29 within the 53BP1N-terminal region and occurs can be ATM-independent . The effect of VRK1 in DDR even in the absence of ATM (null cells) . VRK1 deple- is insensitive to inhibitors of PI3KK proteins that target tion causes a defective formation of 53BP1 foci induced by ATM and DNA-PK . This suggests that there are alterna- ionizing radiation or doxorubicin, both in number and size, tive kinases participating in DDR induced by ionizing radia- which requires a kinase-active VRK1 protein for their rescue tion. The complete molecular components that sequentially . Moreover, this effect of VRK1 on 53BP1 foci is insen- participate in DDR, particularly regulatory proteins, remain sitive to ATM and DNA-PK inhibitors and is functional in unknown. In this context, VRK1 knockdown also prevents p53-null and ATM-null cells. All these data indicate that the the activating phosphorylations of ATM in Ser1981, CHK2 effect of VRK1 is independent of both ATM and p53 , in Thr68, and DNA-PK in Ser2056, all induced in response and that VRK1 activation in response to DNA damage is a to IR , suggesting that VRK1 is an early and upstream novel participant in the NHEJ mechanism of mammalian component in this DDR process. DNA damage responses [29, 46, 47]. 1 3 2380 I. Campillo-Marcos, P. A. Lazo stress or DNA damage, the basal intracellular level of the Cellular protection mediated by VRK1 and its p53 protein is very low, but it is always present. This basal target p53 low level of p53 is necessary to initiate a fast response to cellular stress by its immediate phosphorylation. The VRK1 forms a complex and phosphorylates p53 p53 phosphorylation in several residues within its TAD1 region (residues 1–46) is the main determinants of the The p53-mediated responses induced by DNA damage stress response . To trigger an immediate reaction to have two major roles in the context of cellular protection DNA damage, the response will be greatly facilitated by (Fig. 2). The first one is preventing the transmission of the formation of a stable and inactive complex between damaged DNA to daughter cells during cell proliferation. p53 and one of its regulatory kinases that are activated by This p53 action is mediated by the induction of a cell cycle DNA damage. In non-damaged cells, the basal low p53 arrest, and forms part of cell cycle checkpoints [75, 76]. level is partially forming a stable complex with VRK1, The other role is the protection of the organism from the which are detected by reciprocal immunoprecipitations, consequences of accumulating cells with damaged DNA, and are detected in resting and cycling cells . This which is mediated by induction of cell death . The basal VRK1-p53 complex forms a basic early warning p53 transcription factor mediates these two main protec- system for detection of cellular stress and its activation is tive responses that are regulated by p53 immediate phos- induced by DNA damage caused by ultraviolet light, ion- phorylation in response to DNA damage. These cellular izing radiation, or doxorubicin treatments. All these types responses have a different temporal order because of the of DNA damage activate the kinase activity of VRK1, a covalent modifications, which are immediate as the stabi- previous step required for the specific phosphorylation of lization of p53, or require hours, such as the induction of p53 at Thr18 [28, 81]. Therefore, the subpopulation of specific gene expression. basal p53 that is forming a complex with VRK1 facili- The stabilization and activation of p53 is performed by tates a readiness state of p53 to initiate an immediate acti- several Ser-Thr kinases that target different residues within vating response in different cellular stress situations [28, the p53N-terminal transactivation domain (Fig. 2), and 81]. Furthermore, the p53 protein also indirectly plays an have different sequential roles [78– 80]. In the absence of important role in epigenetic regulation of chromatin . Fig. 2 Kinase activation DNA damage induced by DNA damage and the regulation of p53 in G0/G1 cells. An enzyme (X) activated by VRK1, and that has not yet Local chromatin been identified, mediates the alteration AT M activation of ATM CHK2 Ub VRK1 Ub Ub Ub P P Ub Ub Ub Ub p53 p53 p53 p53 p53 mdm2 Target genes Proteasomal degradaon BAX CDKN1A MDM2 BBC3 (PUMA) (p21) NOXA Cell cycle Apoptosis arrest 1 3 Implication of the VRK1 chromatin kinase in the signaling responses to DNA damage: a therapeutic… 2381 autophagic proteins . The reversion of activated p53 Phosphorylation of p53 by VRK1 prevents the interaction with MDM2 and regulates the switch also requires downregulation of p53 activating kinases, including VRK1 and ATM, so that dephosphorylated between ubiquitination and transcription p53 is not re-phosphorylated and becomes accessible to MDM2. In this context, p53 induces of the expression of Non-phosphorylated p53 binds to the human MDM2 (HDM2) ubiquitin ligase . VRK1 uniquely and specifi- DUSP6 and WIP1 phosphatases targeting ATM [95–97], and that of the DUSP4 phosphatase targeting VRK1 [98, cally phosphorylates p53 in Thr-18 [28, 84, 85]. This Thr18 residue is critical to maintain the folding of the p53 α-helix 99]. However, downregulation of p53 activation is more complex and also requires additional deactivation of other required for its binding to a hydrophobic pocket in MDM2 . The phosphorylation of p53 in Thr18 alters the align- kinases, which are mediated by phosphatases, and deacety- lation of p53 . ment of hydrophobic residues in this α-helix, and this altered conformation permits the p53 binding to transcriptional The stabilization and accumulation of p53 by VRK1 in response to DNA damage is reverted by a novel p53-depend- cofactors. Moreover, this phosphorylation of Thr18 deter- mines the change in binding mode from ubiquitin ligases to ent activation of autophagy that removes its activating VRK1 , a p53 stabilizer, and thus permits p53 dephos- transcription factors, and additional p53 phosphorylation in Ser15 or Ser20  contributes to the selection of specific phorylation and its downregulation by MDM2 [85, 100, 101]. Among the degradation processes regulated by p53 is transcriptional cofactors . The specific phosphorylation of p53 in Thr18 places VRK1 upstream of additional phos- autophagy. In normal cells, autophagy contributes to regu- late basal levels of cytosolic and particulate proteins , a phorylation in Ser15 and Ser20 mediated by other kinases . The activated ATM-CHK2, ATR-CHK1, or DNA-PK process that is further activated in response to several types of stress, including DNA damage. Autophagy is required pathways mediate the phosphorylation p53 in Ser15 or Ser20 [78, 79], and all of them are necessary to achieve the full for recycling of proteins implicated in negative cell cycle regulation, and can provide a survival strategy to tumor cells transcriptional activation of p53 . These additional p53 phosphorylations, and their combination, select transcrip- . By this process, regulated by p53, cells remove and digest endogenous proteins, particularly those that are very tional cofactors and activate p53-dependent genes such as CDKN1A (p21) expression , which induces a cell cycle stable, functioning as an important mechanism for tissue remodeling  and maintenance of cellular homeostasis arrest and senescence , and BAX that facilitates apopto- sis [91, 92], among others. The role of p53 in transcription , but it can also result in a form of cell death, thus hav- ing a dual role [105, 106]. in these processes has been extensively reviewed . The kinase activity of all these p53 kinases, VRK1, ATM, The downregulation of VRK1 is a late response that is also mediated by the p53-dependent transcription of DRAM ATR, and DNA-PK, are inducible by DNA damage, but their spatial organization, coordination, and sequential activation (death-related autophagic modulator) . DRAM is a small hydrophobic protein located in the membrane of require further studies for its complete understanding. In this context, because of its interactions with histones, VRK1 is a autophagosomes . Expression of DRAM facilitates degradation of VRK1 in the lysosome, and the elimination of new component that participates very early in the response mechanisms to DNA damage, as well as in specific steps DRAM or Beclin1 prevents the downregulation of VRK1 by proteolytic degradation [85, 100] (Fig. 3). This degradation of DDR. of VRK1 takes place in the cytosol and is sensitive to the inhibition of nuclear export with leptomycin B and to lysoso- Activated p53 induces the downregulation of VRK1 mal inhibitors . DRAM expression induced by p53 reg- ulates the degradation of stable proteins. DRAM is a novel Once DNA damage has been repaired, the cell cycle arrest induced by activated p53 has to be reverted. Otherwise, component of the cell autophagic response . Autophagy is partly regulated by p53-induced DRAM expression , p53 will maintain the cell cycle arrest or even induce apop- tosis. This reversal requires the deactivation of p53, which and p53-induced VRK1 degradation requires entry in the endosomal–lysosomal pathway . In this way, DRAM is mediated by its dephosphorylation and subsequent inter- action with MDM2. However, the phosphorylation of p53 downregulates VRK1 forming an autoregulatory loop  (Fig. 3). Moreover, this autophagic downregulation of VRK1 in Thr18 by VRK1 blocks its interaction with MDM2 and other phosphorylations, in Ser15 and Ser20 further is altered in tumors with p53 mutations that affect its DNA- binding domain, including all the most frequent mutations interfere with the interaction . All these phospho- rylations have to be removed, to revert the p53-mediated detected in human cancer [85, 109], because they disrupt this autoregulatory loop. Consequently, tumors harboring responses, such as a cell cycle arrest, in viable cells. This is accomplished by the regulation by p53 of different target p53 mutations also have very high levels of VRK1, as it has been shown in head and neck squamous cell carcinomas genes that range from ubiquitin ligases, phosphatases, to 1 3 2382 I. Campillo-Marcos, P. A. Lazo Fig. 3 Downregulation of ATM VRK1 by DRAM1 in the DNA damage autophagic pathway induced DRAM1 by p53 and deactivation of p53 by phosphatases and ubiquitin WIP1 ligases the proteasome in DNA damage response. Solid black VRK1 lines represent the activation route. Dashed lines represent ATM the downregulatory routes and each color represents a MDM2 different route. Kinases: VRK1 and ATM. Phosphatases: p53 WIP1 (wild-type P53-induced VRK1 DRAM1 phosphatase 1) and DUSP4 (dual specificity phosphatase endosome DUSP4 4). DRAM1: damage-regulated autophagy modulator 1 Beclin1 p53 Ub-p53 lysosome proteasome  and lung cancer , which can also facilitate cell proliferation and cell cycle progression, where it plays sev- proliferation. eral roles . VRK1 is required for G0 exit, behaving like In conclusion, the main mechanism of downregulation of an early gene such as MYC and FOS, which facilitate the p53 is mediated MDM2, but for this to occur, it is necessary progression in G1 and passing the restriction point . to previously dephosphorylate p53 and its activating kinases, In this context, depletion of VRK1 prevents the expression all of which are regulated by p53 . Once p53 is dephos- of CCND1 (cyclin D1), since VRK1 directly binds to the phorylated, it becomes available for its ubiquitination by human CCND1 promoter , and consequently, retinoblas- MDM2 and degradation in the proteasome, which has been toma cannot be phosphorylated . Later, in cell cycle extensively reviewed and has become a target for therapeutic progression, VRK1 is also required for the phosphorylation intervention with drugs that interfere with the p53-MDM2 of histone H3 that facilitates the initiation of chromatin com- interaction, such as nutlins . paction in G2/M  and cooperates with AURKB in the sequential remodeling of chromatin in the progression of mitosis . Implications of VRK1 in cancer biology However, based on the biological actions of VRK1, either in cell proliferation or DNA damage responses indicates that The functions of VRK1 suggest that it is likely to actively depending on the cellular context, VRK1 might function as participate in tumor biology. Knockdown experiments indi- an oncogene or a tumor suppressor or predisposition gene. cate that VRK1 plays a major role in cell cycle progression VRK1 might behave an oncogene because of its roles in and proliferation [27, 112, 113]. Moreover, VRK1 elimina- the promotion of cell cycle progression and proliferation. tion by CRISPR/Cas9 identifies wild-type VRK1 as an over - However, in other contexts, VRK1 might behave as a tumor expressed oncogenic driver gene , consistent with its suppressor or a tumor susceptibility gene represented by the role in lung adenocarcinomas . In most cell types, the effects mediated by p53 and those associated with genome human VRK1 gene is expressed at different levels and is not stability. These properties, in the context of cancer, can mutated in cancer, and it is overexpressed in many cancer contribute to a poorer prognosis of tumors overexpressing types of different origins correlating with a poorer prognosis VRK1 because of its contribution to the promotion of cell in breast [116, 117], lung , liver , glioblastoma proliferation and resistance to treatments based on DNA , head and neck , and esophageal cancer . damage. Some driver genes are oncogenic in situations in which they Due to the essential role played by VRK1, attempts to are overexpressed by different mechanisms, as it occurs with generate knock-out mice have been unsuccessful. However, members of the MYC and EGFR families that promote cell the consequences of VRK1 deficiency in animal models proliferation. In the context of tumor growth, the human have been studied in gene-trap mice with a fifteen percent VRK1 protein has been implicated in the regulation of residual level of VRK1 [123–125]. In this model, deficient 1 3 Implication of the VRK1 chromatin kinase in the signaling responses to DNA damage: a therapeutic… 2383 animals were sterile, both male and female [123–125], pre- high probability of high toxicity and side effects. The elimi- venting additional studies. The role that VRK1 plays in nation of VRK1 causes a defective DDR that facilitates and response to DNA damage in this model was not studied. increases the sensitivity to DNA damaging agents, such as In one of the studies, the problem was identified as lagging ionizing radiation or doxorubicin . Depletion of VRK1 chromosomes during meiosis leading to sterility , an sensitizes cells to these treatments because of defective DNA observation consistent with the role of VRK1 in dynamic repair, and thus permits the use of lower doses of toxic drugs chromatin reorganization. In addition, VRK1 regulates the to achieve the same result. This is important, because this attachment of chromatin to the nuclear envelop that is medi- sensitization also occurs in non-dividing cells, and might be ated by the phosphorylation of BANF1 . The disrup- useful for targeting non-dividing cells within a tumor that tion of this process can also lead to alterations of chromatin later might cause a relapse. Moreover, the treatment with a reorganization in mitosis and affect cell viability . lower dose of commonly used cancer drugs can contribute to a reduction of the toxicity associated with them. Facilitating some degree of DNA damage in tumor cells VRK1 potential as therapeutic target can contribute to the generation of new antigens and facili- in oncology tate the response to new therapies based on manipulation of the immune system, as supported by the evidence that Protein kinases, because of their structural characteristics, tumors with an intrinsic higher genome instability are better are candidates for development of inhibitors. Knockdown responders to these new therapies . screening is a useful approach to identify potential thera- In conclusion, the pharmacological targeting of VRK1 peutic targets. Knockdown of VRK1 sensitizes cells to other will impair p53-mediated responses, prevent cell cycle pro- cancer treatments based on DNA damage such as ionizing gression and proliferation, and sensitize cells to treatments radiation or doxorubicin by impairing the DNA damage based on DNA damage, such as ionizing radiation and some response [46, 65]. Moreover, depletion of VRK1 inhibits chemotherapeutic drugs. The consequence of therapeutically cell proliferation [113, 128]. VRK1 has been identified as exploiting this target will be a better control of the tumor a potential target in a screening of synthetic lethal relation- if the new drugs are selective regarding both its molecular ships in a massive siRNA screening . These observa- target and the specific tumor cell. tions suggests that inhibitors of VRK1 can be of potential Acknowledgements I.C-M was supported by FPI-MINECO-Fondo use in cancer treatments, by themselves or in combinations, Social Europeo predoctoral contract (BES-2014-06772). The labora- by facilitating inhibition of proliferation and at the same tory was supported by grants from Agencia Estatal de Investigación- time sensitizing cells to treatments based on DNA damage. MINECO (SAF2016-75744-R) and Consejería de Educación de la In cancer treatment, many drugs are directed to the main Junta de Castilla y León (CSI001U16, UIC-017) to P.A.L. driver as targets. However, cancer cells can be derailed if alternative pathways that impinge on basic processes of the Compliance with ethical standards tumor phenotype are targeted. These alternative targets will Conflict of interest The authors declare no conflict of interests. open a wide range of possibilities, as well as provide with alternatives to manage individual cases. Open Access This article is distributed under the terms of the Creative Kinases share a common structure in their catalytic Commons Attribution 4.0 International License (http://creativecom- kinase domain and are druggable proteins . Therefore, mons.org/licenses/by/4.0/), which permits unrestricted use, distribu- the likelihood of cross inhibition with other kinases is very tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the high and makes many kinase inhibitors promiscuous. In the Creative Commons license, and indicate if changes were made. human kinome, there are kinases that are isolated from other major branches, among which is VRK1. 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