Nuclear functions regulated by the VRK1 kinase

ABSTRACT

In the nucleus, the VRK1 Ser-Thr kinase is distributed in nucleoplasm and chromatin, where it has different roles. VRK1 expression increases in response to mitogenic signals. VRK1 regulates cyclin D1 expression at G0 exit and facilitates chromosome condensation at the end of G2 and G2/M progression to mitosis. These effects are mediated by the phosphorylation of histone H3 at Thr3 by VRK1, and later in mitosis by haspin. VRK1 regulates the apigenetic patterns of histones in processes requiring chromating remodeling, such as transcription, replication and DNA repair. VRK1 is overexpressed in tumors, facilitating tumor progression and resistance to genotoxic treatments. VRK1 also regulates the organization of Cajal bodies assembled on coilin, which are necessary for the assembly of different types of RNP complexes. VRK1 pathogenic variants cuase defects in Cajal bodies, functionally altering neurons with long axons and leading to neurological diseases, such as amyotrophic laterla sclerosis, spinal muscular atrophy, distal hereditay motor neuropathies and Charcot-Marie-Tooth.

KEYWORDS:

• Cajal bodies
• cell cycle
• chromatin
• DNA damage
• histones
• VRK1

Introduction

Dynamic remodeling of chromatin is required for its adaptation to various normal and pathological functions. These functions include gene transcription and silencing, replication, recombination, and DNA damage responses, for which there are different types of DNA repair pathways. Chromatin is organized by DNA-wrapping nucleosomes formed by histones, which can be modulated by several types of posttranslational modifications. These modifications permit multiple locally different conformations to adapt to specific functions regulated by different epigenetic enzyme families. These chromatin remodeling processes require functional and temporal coordination. VRK1 (vaccinia-related kinase 1), also known in Drosophila melanogaster as NHK1 (nucleosomal histone kinase 1) [1]. In pluricellular eukaryotes, this Ser-Thr kinase family appeared later in evolution, constituting an isolated branch that diverged from casein kinases, after which, each evolved independently [2]. The VRK protein family is composed of three members, but VRK2 and VRK3 are mostly cytosolic [3]. Nuclear VRK1 regulates the dynamic reorganization of chromatin required for several types of functions, either physiological such as transcription, replication and recombination, or pathological, such as the response to different types of DNA damage [4,5], as well as to other functions such as chromatin interaction with components of the nuclear envelope [6,7], and Cajal bodies [8,9].

VRK1, chromatin and epigenetic modifications of histones

Specific patterns of histone epigenetic modifications are associated with different biological functions [10,11], including transcription, replication, recombination, and DNA damage responses. Alterations of these patterns can lead to pathological situations such as cancer [12–14] and neurological diseases [15]. Chromatin organization, in which nucleosomes are the basic unit, underlies all biological processes implicated in its functions and requires a complex coordination in which kinases play a relevant role [11,16,17]. VRK1 is a Ser-Thr kinase, mostly located in the nucleus and chromatin, where it plays different roles, but there is also a cytosolic subpopulation at lower concentrations [4,5]. VRK1 [18] and NHK-1 [19] facilitate chromosome condensation in human and Drosophila melanogaster cells, respectively. The VRK1 protein has a low-complexity C-terminal flexible region that folds over the catalytic site, and has several alternative conformations permitting different protein interactions, which are critical for the structural stability, regulation of the kinase activity [20], and selection of interacting proteins [4]. At the distal end of the VRK1 C-terminal flexible region, there is a basic Arg-rich motif that directly interacts with the nucleosome acidic patch [21]. In this context, VRK1 directly interacts with histones H2A [22], H3 [23,24], H4 [25–27] and H2AX [28]. VRK1 directly interacts with histones H2A and H3, and phosphorylates histone H2A in Thr120 [22] and H3 in Thr3 [24,28], two modifications associated with local chromatin remodeling. VRK1 also phosphorylates histone H2AX at Ser139 (γH2AX) in the response to DNA damage [28], which is considered the guardian of the genome [29]. In centromeres, defects in H2A-Thr120 phosphorylation by BUB1 alter chromosome segregation, generating multinucleated cells [30].

The different patterns of histone epigenetic modifications are associated with several types of biological functions [10,11], including transcription, replication, recombination, and DNA damage responses to pathological situations such as cancer [12–14] and neurological diseases [31]. Epigenetic modifications alter nucleosomes, which can have different conformations and functional effects on chromosomal dynamics [32,33]. In addition to kinases, chromatin organization is also regulated by other enzymes that perform additional types of histone epigenetic modifications, such as acetylation, methylation, and ubiquitination [17]. These epigenetic posttranslational modifications of histones are indirectly regulated by VRK1, and these epigenetic modifications are altered by either VRK1 depletion or by VRK-IN-1 [34], a specific inhibitor of this kinase [35]. VRK1 can only play an additional role in the regulation of other histone epigenetic modifications through indirect mechanisms, in which these epigenetic enzymes are likely targets to be regulated and coordinated by VRK1 [27,34]. VRK1, in addition to histone phosphorylation, indirectly regulates histone epigenetic patterns through either phosphorylation or direct interaction with different types of epigenetic enzymes (Figure 1). VRK1 stably interacts with several epigenetic enzymes, such as HDAC1, PCAF, SETDB1, KDM3A, and KDM4A [27], which belong to different families, but their effects are mostly unknown (Figure 1). VRK1 directly phosphorylates Tip60/KAT5 at Thr158 and Ser199 in response to DNA damage [25,26] promoting Tip60 translocation from the nucleoplasm to chromatin, and the specific acetylation of H4K16 [25,26]. The interaction between VRK1 and SIRT2 inhibits the kinase activity of VRK1 [27], and facilitates the deacetylation of H4K16ac by SIRT2 [36,37]. Deacetylation of H4K16ac by SIRT1 impairs the recruitment of SMARCAD1 to DNA double-strand breaks [38]. The regulation of histone PTMs by VRK1 indicates that this kinase is a master regulator of chromatin organization and is associated with several nuclear functions [27,34].

Figure 1. Effect of VRK1 on different histones in response to different types of stimulation and its interactions with different types of enzymes performing histone posttranslational modifications. VRK-IN-1: VRK inhibitor 1. MG149: Tip60 inhibitor. HDAC1: histone deacetylase 1. PCAF: (P300/CBP-Associated factor or KAT2B), SETDB1: SET domain bifurcated histone lysine methyltransferase 1 or KMT1E. KDM3A: Lysine demethylase 3A or JHMD2A. KDM4A: Lysine demethylase 4A. SIRT1: NAD-Dependent protein deacetylase sirtuin-1. SIRT 2: NAD-Dependent protein deacetylase sirtuin-2.

VRK1 regulates several epigenetic enzymes that control histone posttranslational modifications.

Epigenetic modifications alter nucleosomes, which can have different conformations and functional effects on chromosomal dynamics [32,33]. In addition to phosphorylation, chromatin organization is also regulated by other enzymes that perform additional types of histone epigenetic modifications, such as acetylation, methylation, and ubiquitination [17]. These epigenetic posttranslational modifications of histones can be indirectly regulated by VRK1, and consequently histone epigenetic modifications can be altered by either VRK1 depletion or by VRK-IN-1 [34], a specific inhibitor of this kinase [35]. Loss of VRK1 by depletion, or its inhibition, functionally mimics the effect of different types of epigenetic inhibitors targeting histone acetyl (KAT) and methyl transferases (KMT), as well as histone deacetylases (HDAC) and demethylases (KDM) [34]. Depletion of VRK1, or its inhibition with VRK-IN-1, causes a switch in the patterns of H3K4, H3K9, and H3K27 post-translational modifications [34]. VRK1 depletion/inhibition causes the loss of H3K9/H3K27 acetylation, and permits their methylation. This effect is similar to that of the p300 inhibitor C646 and KDM inhibitors, such as iadademstat (ORY-1001) or JMJD2 inhibitor [34]. Alternatively, HDAC inhibitors (selisistat, panobinostat, and vorinostat) and KMT inhibitors (tazemetostast and chaetocin) have the opposite effect of VRK1 depletion or inhibition, and cause an increase in H3K9ac and a decrease in H3K9me3 [34]. VRK1 depletion also impairs the H4K20me2 modification, which is required for the recruitment of 53BP1 in DNA repair by the non-homologous end-joining (NHEJ) pathway [39], functioning in a manner similar to KDM inhibitors such as chaetocin or tazemetostat [40]. Alternatively, the kinase activity of VRK1 can be inhibited by its interaction with another epigenetic enzyme, as is the case of a direct protein interaction between VRK1 and SIRT2, which inhibits the kinase and alters the pattern epigenetic posttranslational modifications of histones [27].

VRK1 gene transcription and cell cycle progression

Mitogenic signals, such as growth factors induce VRK1 gene expression, which expression parallels that of MYC, FOS, CCND1 (cyclin D1) and Ki67 in proliferative/mitogenic responses [41,42]. However, VRK1 is not active unless it is phosphorylated by autophosphorylation [43], or indirectly by the BRAF, MEK, and ERK, a pathway that is also activated in response to mitogenic signaling. Phosphorylation of VRK1 kinase is blocked by MAPK signaling inhibitors. The activated VRK1 directly phosphorylates several transcription factors implicated in cell cycle progression, which includes c-Jun [44], ATF2 [45] and CREB [46] and p53 [43,44,47] (Figure 2). VRK1 depletion blocks cell cycle progression [42]. In stratified epithelia, the highest VRK1 expression was detected in a few individual cells in the basal layer, the one that has started to divide; in the next layer, all cells are positive and have high levels. As epithelial cell terminal differentiation progresses in the epithelium, the level of VRK1 is progressively reduced [41,48].

Figure 2. VRK1 in proliferation and cell cycle progression. Basal VRK1 expression is increased in response to mitogenic signals. Arrows: indicate direct phosphorylations (green) or dephosphorylation (red). Direct protein interactions (blue lines).

VRK1 regulates cell cycle progression, from G0 exit to mitosis.

VRK1 plays different roles in the cell cycle (Figure 2), from entry into the cell cycle to the end of mitosis. Initially, the expression of VRK1 is upregulated in response to mitogenic signals, such as the addition of growth factors to serum-deprived cells for entry into cell proliferation and the G0/G1 transition [42,49]. In response to mitogenic signals, VRK1 overexpression correlates with c-Myc, FOS, and Ki67 expression and their increased levels [24,41,42,50–52], phosphorylation of retinoblastoma (RB), and the expression of cyclin D1 [22,42,46]. The expression of VRK1 is enhanced by mitogenic signals, and VRK1 is required for G0/G1 and [42,49] and G2/M progression [23,41]. Depletion of VRK1 [42] or targeting with specific aptamers [53] blocks CCND1 gene expression, G0 exit, and cell proliferation. Upstream activation of VRK1 gene expression by mitogenic signals is blocked by inhibitors targeting sequential steps in the MAPK signaling pathway, such as BRAF, MEK, and ERK. VRK1 activation is associated with G0 exit [42], as well as with the levels of cytosolic Vaccinia-related Kinase 2 (VRK2), which regulates the Erb2 pathway [54,55]. In chromatin, activated VRK1 phosphorylates H3 at Thr3, which is required for G0 exit [41,42] and transcription activation [41,44,45,56] in response to mitogenic signals. This phosphorylation of H3 at Thr3 is also necessary for the G1/S transition [41] and G2/M progression [23,24,57]. The translational efficiency of VRK1 mRNA is increased by its binding to the 3’-untranslated region of hnRNPA1 [58], and thus potentiates the expression of CCND1 (cyclin D1) mediated by phosphorylation of CREB [46], which promotes lung cancer tumor cell proliferation [58]. Furthermore, hnRNPA1 directly binds to 3’-untranslated VRK1 mRNA, which contributes to its translation and tumor cell proliferation [58]. VRK1 phosphorylates hnRNPA1 and facilitates its binding of hnRNPA1 to telomeric single-stranded DNA and telomerase RNA, which stimulates telomerase activity [59].

The hnRNP A1 protein is phosphorylated by VRK1, promoting its binding to telomerase RNA and telomeric ssDNA and enhancing the telomerase reaction [59]. Thus, VRK1 deficiency induces shortening of telomeres and aberrant organization in mouse male germ cells [60], which is consistent with the male sterility detected in VRK1 deficient mice [61].

During the G2/M transition [23] and meiotic progression [62], H3Thr3ph is read by survivin and interacts with Aurora B (AURKB) [63]. In mitosis, this transient interaction between VRK1 and AURKB cross inhibits their kinase activities [24]. Active VRK1 is dephosphorylated by the mitogen-activated protein kinase phosphatase 2 (MKP2) phosphatase, preventing the rephosphorylation of H3 [24,57,64], which is later rephosphorylated by haspin during mitotic progression [65], which facilitates the recruitment of the chromosomal passenger complex (CPC) to centromeres, of which AURKB is a catalytic component [66] that phosphorylates H3Ser10 [67]. In Drosophila melanogaster, NHK-1 is required for mitotic [1] and meiotic [68,69] progression. In a murine gene-trap model of VRK1, deficiency in levels of VRK1 causes male sterility by impairing the formation of spermatogonia [61], and female sterility by causing defective folliculogenesis [70] and interfering with the progression of oogenesis [62,71].

For a correct cell division, the fragmentation of some intracellular membranes is necessary for its redistribution into daughter cells, and in this context VRK1 also plays a role. In G2/M, Golgi fragmentation, which is regulated by serine-threonine kinases, is required for its redistribution into daughter cells during mitosis. Golgi fragmentation is activated by MEK1 [72], ERK1 [73] and Plk3 [74]. Golgi fragmentation is induced by mitogenic signals that are blocked downstream of Plk3 by a kinase-dead VRK1(K179E) as well as by the VRK1 knockdown, kinase-dead Plk3, or PD98059 MEK1 inhibitor [75]. Plk3 directly interacts with VRK1 to form a stable complex, and both kinases colocalize with giantin in granules, resulting from Golgi fragmentation in the cytoplasm [75]. Plk3 is upstream and phosphorylates the C-terminal region of VRK1 at Ser342, but VRK1 does not phosphorylate Plk3 [75]. This specific phosphorylation of VRK1 is necessary for Golgi fragmentation, mimicked by the mutation to aspartic acid (S342D), but its mutation to alanine (S342A) blocks Golgi fragmentation. These data indicate that VRK1 phosphorylation at Ser342 is required for cell cycle progression. MEK1-Plk3-VRK1 constitutes a sequential module in the regulation of Golgi fragmentation during cell cycle and mitotic progression [75].

In cells with low VRK1 levels, some VRK1 functions are compensated by the expression of a splice variant of VRK2, which is cytosolic and anchored to the endoplasmic reticulum membranes [76]. This VRK2 variant is generated by alternative splicing and lacks the VRK2 C-terminal region, which contains an endoplasmic reticulum signal. This shorter VRK2 variant is located in the nucleus [76], and partially shares several interaction and phosphorylation partners with VRK1 [77].

Moreover, deficiency in VRK1 levels causes telomere shortening and abnormal arrangement, which triggers the activation of DNA damage responses in male murine germ cells [59]. In mice, deficiency of VRK1 causes male and female infertility due to a progressive loss of spermatogonia [61] and impaired oogenesis by defects in folliculogenesis [70] and meiotic recombination [62].

VRK1 in DNA damage responses

Local DNA damage alters chromatin organization [78,79]. The implication of VRK1 in DNA damage response (DDR) is associated with several functions, chromatin and epigenetic modifications [27,34], and DDR pathways. The organization of chromatin conditions accessibility and sensitivity to different types of DNA damage. The induction of DNA damage by doxorubicin or oxidative stress alters the nuclear phosphoproteome, which is enriched in histone modification proteins and chromatin-associated proteins that are impaired by VRK1 depletion [80,81]. These observations are consistent with the role of VRK1 in biological processes that require chromatin remodeling (Figure 3).

Figure 3. VRK1 and DNA damage responses. Diagram illustrating the main pathways triggered by DNA damage in the response to different types of DNA damage, and the implication of VRK1 in different steps of the process, from altered chromatin to repair pathways.

The response to different types of DNA damage is regulated by VRK1.

VRK1 kinase activity is triggered by DNA damage independent of its type, including oxidative stress, alkylating drugs, ionizing radiation, doxorubicin, olaparib, temozolomide and replication block [28,82–85]. Moreover, VRK1 functional downregulation by mutation or inhibition sensitizes tumor cells to genotoxic treatments [4,31,39,82,86]. All of these types of DNA damage, which locally alter chromatin, induce the activating autophosphorylation of VRK1, which phosphorylates different types of targets, including epigenetic enzymes, DDR proteins, and transcription factors. These proteins are regulated by VRK1 and their functions impaired by VRK1 depletion or inhibition [5,28,82,85].

In the DNA damage response (DDR), VRK1 directly phosphorylates histone H2AX (γH2AX) [28], NBS1 in Ser343 [85], 53BP1 in Ser25/29, and is independent of ATM [82]. VRK1 depletion impairs the activation of ATM, CHEK2, and DNA-PK in response to radiation [82], doxorubicin [84,87] and olaparib [88] treatments. The role of VRK1 in DDR is upstream of ATM and DNA-PK [82]. These proteins form part of the sequential steps in the DNA damage response, which is functional in both proliferating and cell-cycle arrested cells [28,82,84,85].

Alterations in DDR pathways have been implicated in cancer and neurological diseases. High levels of VRK1 confer resistance to different treatments based on DNA damage; thus, VRK1 overexpression has been associated with poorer prognosis in some types of cancer, including multiple myeloma and lung cancer [4]. Furthermore, several neurological, neurodegenerative, and motor neuron diseases (MND) are associated with alterations in DNA damage repair (DDR) pathways [89–91], as well as alterations in Cajal bodies and its associated proteins [92–94]. Among the proteins involved in the pathogenesis of neurological diseases are ATM, NBS1, and CHEK2, all of which are involved in DDR [95–97].

VRK1 and p53 mediated responses

The p53 protein regulates the cell cycle, DNA damage responses, and apoptosis [98,99]. The immediate cell response to DNA damage requires the phosphorylation of p53. VRK1 participates in the early responses to DNA damage. This mechanism detects and reacts to local alterations in the chromatin structure induced by DNA damage. The VRK1 protein directly interacts to form a complex with p53 in the basal state [56,100]. This interaction occurs through the p53 N-terminal regulatory domain, and frequent DNA-contact mutants of p53, such as R273H, R248H or R280K, do not disrupt the VRK1-p53 complex [43,101]. Therefore, the basal VRK1-p53 complex functions as an early warning system for immediate cellular responses to cellular stress and DNA damage. In response to different types of DNA damage, VRK1 directly phosphorylates p53 in Thr18 [43,56,102]. This specific phosphorylation triggers a p53 functional switch from binding to ubiquitin ligases such as mdm2 to transcription factors [103,104]. This specific phosphorylation at Thr18 distorts the p53 hydrophobic alpha helix, preventing the p53 interaction with the mdm2 hydrophobic pocket; thus, p53 degradation by ubiquitination is impaired, which facilitates p53 protein stabilization and accumulation [56], and permits its interaction with DNA and gene transcription [104]. This specific p53 phosphorylation functions as a switch between protein degradation and gene transcription [98,104,105]. The activation and stabilization of p53 permits the induction of cell cycle arrest and facilitates DNA repair or cell death when DNA damage is excessive. In case, the stress or damage is solved in the initial response, the p53 accumulation is reversed by a novel p53-dependent activation of autophagy that removes its activating VRK1 [106], thus facilitating p53 dephosphorylation and its downregulation by mdm2 to bring p53 back to basal levels [100,102,106,107]. In many types of cancers with p53 mutations, this autoregulatory loop is defective, and facilitates cancer cell progression and resistance to treatments [48,102].

VRK1, BAF (barrier-to-autointegration) and nuclear envelope

The BAF (BANF1, barrier-to-autointegration factor 1) protein interacts with LEM domain proteins (LAP2, emerin, and MAN1) in C. elegans [108] and humans [109] (Figure 4). The BAF protein bridges double-stranded DNA in a highly ordered nucleoprotein complex, but its interaction with the DNA sequence is nonspecific [110]. BAF is required to segregate and enclose chromosomes within the nuclear envelope, and also assembles the nuclear lamina [111]. BAF participates in the formation of the nuclear envelope (NE), and NE defects occur prior to chromatin alterations [108] and interact with core histones [112]. The phosphorylation of BAF by VRK1 [113] regulates its interaction with the nuclear envelope [114], and facilitates chromosome attachment and chromatin organization, two processes that are altered by VRK1 depletion [108]. In addition, a fraction of BAF is associated with centromeres and the control of mitotic progression [115], which is also impaired by VRK1 depletion [24] or VRK1 pathogenic variants [31].

Figure 4. Regulation of BAF by VRK1 in mitotic progression. Red and green arrows indicate repression or activation respectively. Black arrows indicate the activity of VRK1 kinase and the PP2A and PP4C phosphatases.

VRK1 regulates the attachment of BAF and chromosomes to the nuclear envelope.

In mammals, non-phosphorylated BAF mediates the interaction of chromatin with the inner nuclear membrane in interphase and facilitates the attachment of chromosomes to the nuclear envelope [116], the binding of emerin to the inner nuclear envelope [117], and assembly of the nuclear lamina [111] (Figure 4). BAF compacts chromatin via a looping mechanism [118]. VRK1 is necessary for the expression of BAF in cell cycle progression [119]. The VRK1 protein directly interacts with and phosphorylates BAF in Ser4 and Thr3 [71,113,114,120,121], preventing the interaction of emerin with the nuclear envelope and DNA, and facilitating BAF detachment from the nuclear envelope [113]. Therefore, the dephosphorylation of BAF is necessary for the reassembly of the nuclear envelope at the end of mitosis, which requires LEM4/ANKLE2, an inner nuclear membrane protein in mammals, and implicates a coordination of PP2A phosphatase and VRK1, a function that is conserved from worms to humans [122]. LEM4/ANKLE2 inhibits VRK1 and controls the dephosphorylation of BAF by protein phosphatase 2A (PP2A), thereby facilitating nuclear envelope assembly [122]. In chromatin, mitogen-activated protein kinase phosphatase 2 (MKP2) interacts with VRK1 and suppresses histone H3 [64]. In the nuclear envelope, LEM4 promotes the dephosphorylation of BAF during mitotic exit [123]. Furthermore, SIRT2 interacts with LEM4 to inhibit its acetylation [124] and prevents BAF phosphorylation, facilitating its detachment from the nuclear envelope. In a zebrafish experimental model, ANKLE2 deficiency caused microcephaly, spermatogenic defects [125] and motor dysfunction [126], symptoms that have also been reported in patients with rare VRK1 mutations [127].

The extensive flexibility of the BAF N-terminal helix α1 and loop α1α2 is strongly reduced in diphosphorylated BAF due to interactions between the phosphorylated residues and the positively charged C-terminal helix α6. These regions are involved in DNA and lamin A/C-binding. BAF phosphorylation causes a major loss of its affinity for dsDNA, but does not impair its binding to the lamin A/C Ig fold domain and emerin nucleoplasmic region, which leaves open the question of the coordination of these protein interactions and their functions [120].

BAF bridges DNA using two pairs of helix-hairpin-helix motifs, which are located on opposite surfaces of the BAF dimers [128]. BAF interaction with DNA is also involved in the regulation of DNA double-strand break (DSB) repair by the inhibition of DNA-PK, which is required for the choice of DSB repair pathway [129]. BAF relocalizes from the nuclear envelope to sites of double-strand breaks [129]. This effect is similar to that of VRK1 depletion, which impairs DNA-PK activity in response to DNA damage [82]. Oxidative stress induces the binding of BAF to Poly-(ADP-Ribose) polymerase 1 (PARP1) causing the inhibition of PARP1 auto-ADP-ribosylation and thus a defective repair of oxidative lesions, particularly in cells with high levels of BAF [129,130]. VRK1 downregulation reduces the expression of BAF and inhibited the proliferation and migration of esophageal squamous cell carcinoma (ESCC) [121]. Depletion of human VRK1 results in aberrant nuclear architecture, in which nuclear BAF is elevated and sustained interaction with its partners is likely to account for aberrant NE organization [114]. This disruption of the nuclear envelope (NE) by inactivation of BAF causes an accumulation of tau protein and is a likely initiating event in the pathogenesis of tauopathies [131].

VRK1, coilin and Cajal bodies

Cajal bodies are nuclear structures identified by Ramón y Cajal in 1903 [132]. These structures are assembled on coilin as its scaffold protein [133] on which different RNP complexes are assembled [134,135] with different types of RNA, including spliceosomal nuclear RNA (snRNA) and small nucleolar RNAs (snoRNAs), by forming small nuclear ribonucleoprotein particles (snRNPs) [135] (Figure 5). These RNP particles are involved in different functions such as splicing, ribosome biogenesis, and telomere maintenance [136].

Figure 5. VRK1, Cajal bodies and neurological diseases altered by VRK1 variants. VRK1 phosphorylates coilin that is translocated to the Cajal bodies, where it interacts with SMN. Alterations in the process by pathogenic variants of VRK1 impair different nuclear proteins and lead to very severe neurological diseases. SMA: spinal muscular atrophy. CMT: Charcot-Marie-tooth. HSP: hereditary spastic paraplegia. DHMN: distal hereditary motor neuropathies. FTD: front- temporal degeneration. ALS: amyotrophic lateral sclerosis. VCP (valosin containing protein). Ku70/80 (ATP dependent helicases). VRK1 and VCP mutant proteins are associated with amyotrophic lateral sclerosis (ALS). Red lines: disease. Green lines: interactions.

VRK1 regulates the organization of Cajal bodies. Pathogenic VRK1 variants impair Cajal bodies and cause neurological diseases.

Coilin regulates the formation and organization of Cajal bodies and functions as a scaffold protein for the assembly of RNP complexes [92,135]. VRK1 directly phosphorylates coilin at Ser184 and facilitates its assembly and stability in CBs organization [9] (Figure 5). This specific phosphorylation of coilin at Ser-184 prevents Cajal body disassembly [8,137]. Non-phosphorylated coilin leads to CBs disintegration, its ubiquitination within nuclei, and its transport to the cytosol, where coilin is degraded in the proteasome [9]. This effect of non-phosphorylated coilin is prevented by either blocking its nuclear export with leptomycin or by inhibiting its proteasomal degradation after its export to the cytosol [9]. Another potential role of coilin is due to its involvement on the organization of the Histone locus bodies (HLBs), which are biomolecular condensates that assemble replication-dependent histone genes in animal cells [138]. HLBs share components with Cajal bodies [136,139,140], and contain factors that are required for the processing of histone pre-mRNAs [141]. However, the role of coilin and its regulation by VRK1 in HLB remains to be characterized.

Rare homozygous or compound heterozygous human VRK1 variants were associated with distal hereditary neuropathies and motor neuron diseases [31] (Figure 6). These VRK1 variants alter Cajal body assembly, and the assembly and stability of RNPs complexes, which are the main factors underlying Cajalopathies [31]. Coilin interacts with multiple proteins [142]. Thus, VRK1 variants might interact differently with protein complexes assembled on coilin and consequently display some differences in their pathological effects. These pathogenic VRK1 variants associated with neurological diseases cause an altered assembly of Cajal bodies [31,86,143,144], and some also have an altered action potential [145]. VRK1 pathogenic variants mainly affect neurons with long axons, such as motor neurons. Among the diseases associated with these VRK1 variants are spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), distal hereditary motor neuropathies (dHMN), and Charcot-Marie-Tooth (CMT) [31]. In addition, mutations in hnRNPA1 have also been reported in patients with amyotrophic lateral sclerosis [146] and myopathies [147].

Figure 6. Targeting of epigenetic modifiers in cancer treatment. The combined drug targeting facilitates a switch from repair mechanisms toward damage accumulation. Different drugs combination in synthetic lethality strategies might facilitate a reduction of individual drug dose, and thus their toxicity, while promoting damage accumulation leading to tumor cell death.

Combined drug targeting of epigenetic modifiers facilitates a switch from repair mechanisms towards tumor cell damage and death.

VRK1 in cancer, targeting VRK1 for novel cancer therapies

High levels of VRK1 facilitate tumor cell proliferation [42,58], resistance to treatments based on DNA damage [4,5,85], and epithelial-mesenchymal transition in breast cancer cells [148,149]. High levels of wild-type VRK1 expression are associated with poorer prognosis in many types of cancer [4] including multiple myeloma [150], lung cancer [4,41,58,151], head and neck squamous cell carcinoma [48], esophageal cancers [152], gastric cancer [153], liver [154], colon [155], breast [84,149,156], hepatocellular carcinomas [154], Wilms tumors [157] and gliomas [158–160].

The identification of druggable targets in chromatin regulation could potentially be exploited in synthetic lethality strategies. In this context, kinases and other epigenetic modifiers are likely targets. The structure of the VRK1 catalytic site is atypical and is thus insensitive to current types of kinase inhibitors [161–164]. The mechanism of the VRK1-ATP complex is not well understood [165]. Thus, most information regarding its loss of function has been obtained by experiments based on the depletion of VRK1, which has been shown to impair cell proliferation [42] and response to DNA damage [4,5], and lately by the use of specific VRK1 inhibitors [27,34,35,166]. Depletion of VRK1 sensitizes cells to genotoxic cancer treatments. Initially, VRK1 depletion sensitized cells to DNA damage induced by ionizing radiation or doxorubicin. DNA damage detected by the induction of ionizing radiation (IR) or doxorubicin caused double-strand breaks detected by the formation of yH2AX [28,84] and 53BP1. VRK1 depletion in combination with either ionizing radiation (IR) [88] or doxorubicin [84] resulted in a reduction in the dose required to achieve a similar effect.

VRK1 depletion or inhibition has an important effect on epigenetic posttranslational modifications of histones, and as an indirect epigenetic modifier, is also a potential target for the development of novel therapeutic combinations in cancer [13,167,168]. VRK1 depletion promotes the loss of H3K9ac and H3K27ac, which are associated with gene transcription and cell proliferation, and facilitates trimethylation of H3K9 and K27 [34]. VRK1 depletion also impairs the accumulation of H4K20m2 [39], similar to KMT inhibitors [40], which is necessary for the recruitment of 53BP1 in the non-homologous end joining (NHEJ) DNA damage repair pathway [169,170], thus sensitizing tumor cells to genotoxic treatments. VRK1 depletion also impairs the DNA damage response triggered by treatment with olaparib [39], a PARP inhibitor, and ionizing radiation [39,88]. Depletion of VRK1 sensitizes glioblastoma (GBM) cells to PARP inhibitors, such as olaparib, by facilitating DNA damage in tumor cells and reducing their viability [39]. In GBM, VRK1 depletion impaired the formation of H4K20me2 that is required for the recruitment of 53BP1 to damaged DNA sites in glioblastoma cells treated with temozolomide and olaparib [39].

The structure of VRK1 indicates that its kinase domain has characteristics that make it a candidate for the development of specific inhibitors [162,163], consistent with the lack of effect of inhibitors targeting different kinase families [164]. Recently, a specific VRK1/VRK2 inhibitor (VRK-IN-1) was developed [35]. This inhibitor impairs the phosphorylation of VRK1 targets, such as histone H3 and Tip60/KAT5 [27,34], altering epigenetic posttranslational modifications of chromatin in both basal and DNA damage responses [27,34]. VRK1 depletion or its inhibition with VRK-IN-1 also facilitate interferon-mediated immune responses [171,172]. Thus, it is a starting point for the development of better inhibitors targeting VRK1, which can be used in combination with additional inhibitors directed to proteins in other signaling pathways, and which in cooperation can facilitate tumor cell death at lower drug doses in synthetic lethality strategies [39]. Targeting VRK1 in glioblastoma cells, neuroblastomas, and pediatric gliomas, in which VRK2 has been eliminated or silenced by VRK2-promoter methylation cells [159,173], is a potential synthetic lethality strategy in these tumors. The combination of drugs targeting different families of epigenetic post-translational modification enzymes can be a useful strategy for cancer treatment (Figure 6).

Perspective

The roles played by VRK1 in the context of several different nuclear functions can have important implications for the understanding of major diseases such as cancer and neurological diseases. High levels of VRK1 promote tumor cell expansion by enhancing cell proliferation and resistance to treatment. In the context of cancer, the inhibition of VRK1 activity has the potential to be a vulnerability in tumor cells and thus become a target for use in novel synthetic lethality strategies in cancer treatment. Regarding neurological diseases, the implication of rare pathogenic VRK1 variants associated with very severe motor neuron diseases can be useful for a better characterization of these diseases and contribute to improved diagnosis and the design of specific new approaches for their clinical management.

Abbreviations

VRK1	=	vaccinia related kinase 1
NHK-1	=	nucleosomal histone kinase-1
CB	=	Cajal body
ALS	=	amyotrophic lateral sclerosis
SMA	=	Spinal muscular atrophy
CMT	=	Charcot-Marie-Tooth
dHMN	=	distal hereditary motor neuropathy.
RNP	=	Ribonucleoprotein.
DDR	=	DNA damage response

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Additional information

Funding

This research was funded by grants from Agencia Estatal de Investigación (doi: 10.13039/501100011033)-Ministerio de Ciencia, Innovación y Universidades-FEDER “Una manera de hacer Europa” (PID2019-105610RB-I00, PID2022-139598OB-I00).