VRK1 Regulates Sensitivity to Oxidative Stress by Altering Histone Epigenetic Modifications and the Nuclear Phosphoproteome in Tumor Cells.

The chromatin organization and its dynamic remodeling determine its accessibility and sensitivity to DNA damage oxidative stress, the main source of endogenous DNA damage. We studied the role of the VRK1 chromatin kinase in the response to oxidative stress. which alters the nuclear pattern of histone epigenetic modifications and phosphoproteome pathways. The early effect of oxidative stress on chromatin was studied by determining the levels of 8-oxoG lesions and the alteration of the epigenetic modification of histones. Oxidative stress caused an accumulation of 8-oxoG DNA lesions that were increased by VRK1 depletion, causing a significant accumulation of DNA strand breaks detected by labeling free 3'-DNA ends. In addition, oxidative stress altered the pattern of chromatin epigenetic marks and the nuclear phosphoproteome pathways that were impaired by VRK1 depletion. Oxidative stress induced the acetylation of H4K16ac and H3K9 and the loss of H3K4me3. The depletion of VRK1 altered all these modifications induced by oxidative stress and resulted in losses of H4K16ac and H3K9ac and increases in the H3K9me3 and H3K4me3 levels. All these changes were induced by the oxidative stress in the epigenetic pattern of histones and impaired by VRK1 depletion, indicating that VRK1 plays a major role in the functional reorganization of chromatin in the response to oxidative stress. The analysis of the nuclear phosphoproteome in response to oxidative stress detected an enrichment of the phosphorylated proteins associated with the chromosome organization and chromatin remodeling pathways, which were significantly decreased by VRK1 depletion. VRK1 depletion alters the histone epigenetic pattern and nuclear phosphoproteome pathways in response to oxidative stress. The enzymes performing post-translational epigenetic modifications are potential targets in synthetic lethality strategies for cancer therapies.

ALS-associated VRK1 R321C mutation causes proteostatic imbalance and mitochondrial defects in iPSC-derived motor neurons.

Vaccinia-related kinase 1 (VRK1) is a gene which has been implicated in the pathological process of a broad range of neurodevelopmental disorders as well as neuropathies, such as Amyotrophic Lateral Sclerosis (ALS). Here we report a family presenting ALS in an autosomal recessive mode of inheritance, segregating with a homozygous missense mutation located in VRK1 gene (p.R321C; Arg321Cys). Proteomic analyses from iPSC-derived motor neurons identified 720 proteins eligible for subsequent investigation, and our exploration of protein profiles revealed significant enrichments in pathways such as mTOR signaling, E2F, MYC targets, DNA repair response, cell proliferation and energetic metabolism. Functional studies further validated such alterations, showing that affected motor neurons presented decreased levels of global protein output, ER stress and downregulation of mTOR signaling. Mitochondrial alterations also pointed to decreased reserve capacity and increased non-mitochondrial oxygen consumption. Taken together, our results present the main pathological alterations associated with VRK1 mutation in ALS.

Nuclear functions regulated by the VRK1 kinase.

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.

Inactivation of VRK1 sensitizes ovarian cancer to PARP inhibition through regulating DNA-PK stability.

Ovarian cancer is the leading cause of gynecologic cancer death. Among the most innovative anti-cancer approaches, the genetic concept of synthetic lethality is that mutations in multiple genes work synergistically to effect cell death. Previous studies found that although vaccinia-related kinase-1 (VRK1) associates with DNA damage repair proteins, its underlying mechanisms remain unclear. Here, we found high VRK1 expression in ovarian tumors, and that VRK1 depletion can significantly promote apoptosis and cell cycle arrest. The effect of VRK1 knockdown on apoptosis was manifested by increased DNA damage, genomic instability, and apoptosis, and also blocked non-homologous end joining (NHEJ) by destabilizing DNA-PK. Further, we verified that VRK1 depletion enhanced sensitivity to a PARP inhibitor (PARPi), olaparib, promoting apoptosis through DNA damage, especially in ovarian cancer cell lines with high VRK1 expression. Proteins implicated in DNA damage responses are suitable targets for the development of new anti-cancer therapeutic strategies, and their combination could represent an alternative form of synthetic lethality. Therefore, normal protective DNA damage responses are impaired by combining olaparib with elimination of VRK1 and could be used to reduce drug dose and its associated toxicity. In summary, VRK1 represents both a potential biomarker for PARPi sensitivity, and a new DDR-associated therapeutic target, in ovarian cancer.

Identification of VRK1 as a Novel Potential Biomarker for Prognosis and Immunotherapy in Hepatocellular Carcinoma.

Background: Research has indicated that VRK1 is essential for the tumor cell cycle. However, its prognostic and immunotherapeutic predictive significance has not been documented in hepatocellular carcinoma (HCC). Methods: The TCGA, ICGC, and GSE14520 datasets were used to investigate VRK1 expression and its predictive significance of survival outcomes. The qRT-PCR and immunohistochemistry (IHC) were used to confirm the findings. The immunotherapeutic response of VRK1 was anticipated by the IMvigor210 cohort. Lastly, the association between immune infiltration, m6A modification, and functional enrichment of differentially expressed genes (DEGs) was investigated in connection to VRK1 expression. Results: VRK1 expression was markedly elevated on both the mRNA and protein levels in HCC. In HCC patients, a high expression of VRK1 was linked to a poor prognosis. Furthermore, there was a substantial positive correlation seen between increased VRK1 expression and the response rate to anti-PD-L1 immunotherapy. Relationships between VRK1 and m6A-related genes as well as different immune cells were shown by correlation studies. Lastly, enrichment analysis revealed a tight relationship between VRK1 and important biological functions, including DNA replication, cell cycle control, and fatty acid metabolism. Conclusion: Our research reveals the potential of VRK1 as a novel biomarker for prognosis and immunotherapy response in HCC patients.

Pathogenic effects of Leu200Pro and Arg387His VRK1 protein variants on phosphorylation targets and H4K16 acetylation in distal hereditary motor neuropathy.

Rare recessive variants in the human VRK1 gene are associated with several motor neuron diseases (MND), such as amyotrophic lateral sclerosis, spinal muscular atrophy, or distal hereditary motor neuropathies (dHMN). A case with dHMN carrying two novel VRK1 gene variants, expressing Leu200Pro (L200P) and Arg387His (R387H) variant proteins, identified that these protein variants are functionally different. The Leu200Pro variant shares with several variants in the catalytic domain the loss of the kinase activity on different substrates, such as histones, p53, or coilin. However, the distal Arg387His variant and the distal Trp375* (W375X) chinese variant, both located at the end of the low complexity C-terminal region and proximal to the termination codon, retain their catalytic activity on some substrates, and mechanistically their functional impairment is different. The L200P variant, as well as most VRK1 pathogenic variants, impairs the phosphorylation of BAF and histone H4K16 acetylation, which are required for DNA attachment to the nuclear envelope and chromatin accessibility to DNA repair mechanisms, respectively. The R387H variant impairs phosphorylation of H2AX, an early step in different types of DNA damage responses. The functional variability of VRK1 protein variants and their different combinations are a likely contributor to the clinical phenotypic heterogeneity of motor neuron and neurological diseases associated with rare VRK1 pathogenic variants. KEY MESSAGES: VRK1 variants implicated in motor neuron diseases are functionally different. The L200P variant is kinase inactive, and the R387H variant is partially active. VRK1 variants alter H4K16 acetylation and loss of coilin and BAF phosphorylation. VRK1 variants alter Cajal bodies and DNA damage responses. VRK1 variant combination determines the neurological phenotype heterogeneity.

VRK1 promotes DNA-induced type I interferon production.

BACKGROUND: Type I interferons (IFNs) are an essential class of cytokines with antitumor, antiviral and immunoregulatory effects. However, over-productive the type I IFNs are tightly associated with autoimmune disorders. Thus, the induction of type I interferons is precisely regulated to maintain immune hemostasis. This study aimed to identify a novel regulator of type I interferon signaling. METHODS AND RESULTS: Primary BMDMs, isolated from mice, and human cell lines (HEK293 cells, Hela cells) and murine cell line (MEF cells) were cultured to generate in vitro models. After knockdown VRK1, real-time PCR and dual-luciferase reporter assay were performed to determine the expression level of the type I IFNs and ISGs following HTDNA and Poly (dA:dT) stimulation. Additionally, cells were treated with the VRK1 inhibitor, and the impact of VRK1 inhibition was detected. Upon HTDNA and Poly (dA:dT) stimulation, knockdown of VRK1 attenuated the induction of the type I IFNs and ISGs. Consistently, VRK-IN-1, a potent and selective VRK1 inhibitor, significantly suppressed the induction of the type I IFNs and ISGs in human and murine cell lines. Further, VRK-IN-1 inhibited induction of the type I IFNs in mouse primary BMDMs. Intriguingly, VRK1 potentiated the cGAS-STING- IFN-I axis response at STING level. CONCLUSIONS: Our study reveals a novel function of VRK1 in regulating the production of type I IFNs. VRK-IN-1 might be a potential lead compound for suppressing aberrant type I IFNs in autoimmune disorders.

Loss of VRK1 alters the nuclear phosphoproteome in the DNA damage response to doxorubicin.

Dynamic chromatin remodeling requires regulatory mechanisms for its adaptation to different nuclear function, which are likely to be mediated by signaling proteins. In this context, VRK1 is a nuclear Ser-Thr kinase that regulates pathways associated with transcription, replication, recombination, and DNA repair. Therefore, VRK1 is a potential regulatory, or coordinator, molecule in these processes. In this work we studied the effect that VRK1 depletion has on the basal nuclear and chromatin phosphoproteome, and their associated pathways. VRK1 depletion caused an alteration in the pattern of the nuclear phosphoproteome, which is mainly associated with nucleoproteins, ribonucleoproteins, RNA splicing and processing. Next, it was determined the changes in proteins associated with DNA damage that was induced by doxorubicin treatment. Doxorubicin alters the nuclear phosphoproteome affecting proteins implicated in DDR, including DSB repair proteins NBN and 53BP1, cellular response to stress and chromatin organization proteins. In VRK1-depleted cells, the effect of doxorubicin on protein phosphorylation was reverted to basal levels. The nuclear phosphoproteome patterns induced by doxorubicin are altered by VRK1 depletion, and is enriched in histone modification proteins and chromatin associated proteins. These results indicate that VRK1 plays a major role in processes requiring chromatin remodeling in its adaptation to different biological contexts.

Comprehensively prognostic and immunological analysis of VRK Serine/Threonine Kinase 1 in pan-cancer and identification in hepatocellular carcinoma.

BACKGROUND: VRK1 is a member of the vaccinia-related kinase (VRK) family of serine/threonine protein kinases, which is related to the occurrence and development of malignant tumors. The expression pattern, predictive value, and biological function of VRK1 in various cancers remain largely elusive and warrant further investigation. METHODS: Public databases, such as TCGA, GTEx, and UCEC, were utilized to comprehensively analyze the expression of VRK1 across multiple cancer types. Prognostic significance was assessed through Univariate Cox regression and Kaplan-Meier analyses. Additionally, Spearman's correlation analysis was employed to explore the potential associations between VRK1 expression and various factors, including tumor microenvironment scores, immune cell infiltration, and immune-related genes. Moreover, to validate the findings, differential expression of VRK1 in HCC tissues and cell lines was further confirmed using qPCR, Western blot, and immunohistochemistry techniques. RESULTS: The upregulation of VRK1 was observed in most cancer types, and was associated with worse prognosis in ACC, KICH, KIRP, LGG, LIHC, LUAD, MESO, and PCPG. In various cancers, VRK1 expression exhibited positive correlations with immune infiltrating cells, immune checkpoint-related genes, TMB, and MSI. Furthermore, the promoter methylation status of VRK1 varied across different tumor types, and this variation was associated with patient prognosis in certain cancers. In our experimental analyses, we observed significantly elevated expression of VRK1 in both HCC tissues and HCC cells. Functionally, we found that the downregulation of VRK1 had a profound impact on HCC cells, leading to a significant decrease in their proliferation, migration, and invasion capabilities. CONCLUSION: The expression of VRK1 exerts a notable influence on the prognosis of several tumors and exhibits a strong correlation with tumor immune infiltration. Moreover, in the context of HCC, VRK1 may act as an oncogene, actively promoting tumor progression.

Behavioral and neurological effects of Vrk1 deficiency in zebrafish.

Vaccinia-related kinase 1 (VRK1) is a serine/threonine kinase, for which mutations have been reported cause to neurodegenerative diseases, including spinal muscular atrophy, characterized by microcephaly, motor dysfunction, and impaired cognitive function, in humans. Partial Vrk1 knockdown in mice has been associated with microcephaly and impaired motor function. However, the pathophysiological relationship between VRK1 and neurodegenerative disorders and the precise mechanism of VRK1-related microcephaly and motor function deficits have not been fully investigated. To address this, in this study, we established vrk1-deficient (vrk1-/-) zebrafish and found that they show mild microcephaly and impaired motor function with a low brain dopamine content. Furthermore, vrk1-/- zebrafish exhibited decreased cell proliferation, defects in nuclear envelope formation, and heterochromatin formation in the brain. To our knowledge, this is the first report demonstrating the important role of VRK1 in microcephaly and motor dysfunction in vivo using vrk1-/- zebrafish. These findings contribute to elucidating the pathophysiological mechanisms underlying VRK1-mediated neurodegenerative diseases associated with microcephaly.

VRK1 variants at the cross road of Cajal body neuropathogenic mechanisms in distal neuropathies and motor neuron diseases.

Distal hereditary neuropathies and neuro motor diseases are complex neurological phenotypes associated with pathogenic variants in a large number of genes, but in some the origin is unknown. Recently, rare pathogenic variants of the human VRK1 gene have been associated with these neurological phenotypes. All VRK1 pathogenic variants are recessive, and their clinical presentation occurs in either homozygous or compound heterozygous patients. The pathogenic VRK1 gene pathogenic variants are located in three clusters within the protein sequence. The main, and initial, shared clinical phenotype among VRK1 pathogenic variants is a distal progressive loss of motor and/or sensory function, which includes diseases such as spinal muscular atrophy, Charcot-Marie-Tooth, amyotrophic lateral sclerosis and hereditary spastic paraplegia. In most cases, symptoms start early in infancy, or in utero, and are slowly progressive. Additional neurological symptoms vary among non-related patients, probably because of their different VRK1 variants and their genetic background. The underlying common pathogenic mechanism, by its functional impairment, is a likely consequence of the roles that the VRK1 protein plays in the regulation on the stability and assembly of Cajal bodies, which affect RNA maturation and processing, neuronal migration of RNPs along axons, and DNA-damage responses. Alterations of these processes are associated with several neuro sensory or motor syndromes. The clinical heterogeneity of the neurological phenotypes associated with VRK1 is a likely consequence of the protein complexes in which VRK1 is integrated, which include several proteins known to be associated with Cajal bodies and DNA damage responses. Several hereditary distal neurological diseases are a consequence of pathogenic variants in genes that alter these cellular functions. We conclude that VRK1-related distal hereditary neuropathies and motor neuron diseases represent a novel subgroup of Cajal body related neurological syndromes.

VRK1 Kinase Activity Modulating Histone H4K16 Acetylation Inhibited by SIRT2 and VRK-IN-1.

The accessibility of DNA to different cellular functions requires a dynamic regulation of chromatin organization that is mediated by different epigenetic modifications, which regulate chromatin accessibility and degree of compaction. These epigenetic modifications, particularly the acetylation of histone H4 in lysine 14 (H4K16ac), determine the degree of chromatin accessibility to different nuclear functions, as well as to DNA damage drugs. H4K16ac is regulated by the balance between two alternative histone modifications, acetylation and deacetylation, which are mediated by acetylases and deacetylases. Tip60/KAT5 acetylates, and SIRT2 deacetylates histone H4K16. However, the balance between these two epigenetic enzymes is unknown. VRK1 regulates the level of H4K16 acetylation by activating Tip60. We have shown that the VRK1 and SIRT2 are able to form a stable protein complex. For this work, we used in vitro interaction, pull-down and in vitro kinase assays. In cells, their interaction and colocalization were detected by immunoprecipitation and immunofluorescence. The kinase activity of VRK1 is inhibited by a direct interaction of its N-terminal kinase domain with SIRT2 in vitro. This interaction causes a loss of H4K16ac similarly to the effect of a novel VRK1 inhibitor (VRK-IN-1) or VRK1 depletion. The use of specific SIRT2 inhibitors in lung adenocarcinoma cells induces H4K16ac, contrary to the novel VRK-IN-1 inhibitor, which prevents H4K16ac and a correct DNA damage response. Therefore, the inhibition of SIRT2 can cooperate with VRK1 in the accessibility of drugs to chromatin in response to DNA damage caused by doxorubicin.

Circ VRK1/microRNA-17/PTEN axis modulates the angiogenesis of human brain microvascular endothelial cells to affect injury induced by oxygen-glucose deprivation/reperfusion.

BACKGROUND: Circular RNAs (circRNAs) can act as microRNA (miRNA) sponges, thus regulating gene expression. The role of circRNAs in the process of oxygen-glucose deprivation/reoxygenation (OGD/R) is unclear. Here, we explored the mechanism underlying Circ VRK1 in human brain microvascular endothelial cells (HBMVECs) injury induced by OGD/R. METHODS: The OGD/R cell model was established in HBMVECs. The microarray was applied to detect differentially expressed circRNAs, followed by subcellular fractionation assay. Colony formation assay, flow cytometry, ELISA, tube formation, Transwell and western blot assays were performed for loss-of-function assay. HE staining, TTC staining, immunohistochemistry and western blot were performed in an established mouse model. The relationships between Circ VRK1 and miR-17, and between miR-17 and PTEN were detected by bioinformatics and dual-luciferase assays. Rescue experiments were conducted in vitro and in vivo, and PI3K/AKT activity was detected by Western Blot. RESULTS: Circ VRK1, predominantly present in the cytoplasm of cells, was upregulated in the HBMVECs exposed to OGD/R. Circ VRK1 downregulation decreased proliferation, migration, tube formation, inflammatory factors and oxidative stress, while increased apoptosis in HBMVECs. Moreover, Circ VRK1 silencing reduced neurological damage, cerebral infarct size, CD34-positive cell counts and VEGF expression in mice. Circ VRK1 mediated PTEN expression and the PI3K/AKT pathway by targeting miR-17. Deletion of miR-17 inhibited the effects of Circ VRK1 siRNA, and silencing of PTEN suppressed the effects of miR-17 inhibitor. CONCLUSION: Circ VRK1 was upregulated during OGD/R. Circ VRK1 downregulation regulates PTEN expression by targeting miR-17, thereby promoting PI3K/AKT pathway activity to alleviate OGD/R injury.

The VRK1 chromatin kinase regulates the acetyltransferase activity of Tip60/KAT5 by sequential phosphorylations in response to DNA damage.

The regulation of histone epigenetic modifications mediates the adaptation of chromatin to different biological processes. DNA damage causes a local relaxation of chromatin associated to histone H4 acetylation in K16, mediated by Tip60/KAT5. In this work, we have studied the role that the VRK1 chromatin kinase plays on the activation of Tip60 during this process. In the DNA damage response induced by doxorubicin, VRK1 directly phosphorylates Tip60. However, the phosphorylated Tip60 residues and their functional roles are unknown. In DDR, we have identified these two Tip60 phosphorylated residues and the cooperation of the participating kinases. The T158 phosphorylation, mediated by VRK1, is early and transient, preceding that of S199, which is more sustained in time, and mediated by DNA-PK. The role of each phosphorylated residues was determined by using phosphomimetic and phosphonull mutants and their combination. T158 phosphorylation protects Tip60 from ubiquitin-mediated degradation, promotes its recruitment to chromatin from the nucleoplasm, and is necessary for its full trans-acetylase activity. The phosphorylation in S199 by DNA-PK directly facilitates Tip60 autoacetylation, but it is not enough for trans-acetylation of two of its targets, histone H4 and ATM, which requires a double phosphorylation of Tip60 in T158 and S199. DNA-PK inhibitors block the phosphorylation of S199. We propose a model in which the cooperation between VRK1 and DNA-PK mediates the sequential phosphorylation of Tip60/KAT5, and contributes to the recruitment of this protein to initiate the sequential remodeling of chromatin in DDR. Both proteins are candidates for novel synthetic lethality strategies in cancer treatment.

Combined HASPIN and mTOR inhibition is synergistic against KRAS-driven carcinomas.

BACKGROUND: Oncogenic mutations in the KRAS gene are very common in human cancers, resulting in cells with well-characterized selective advantages. For more than three decades, the development of effective therapeutics to inhibit KRAS-driven tumorigenesis has proved a formidable challenge and KRAS was considered 'undruggable'. Therefore, multi-targeted therapy may provide a reasonable strategy for the effective treatment of KRAS-driven cancers. Here, we assess the efficacy and mechanistic rationale for combining HASPIN and mTOR inhibition as a potential therapy for cancers carrying KRAS mutations. METHODS: We investigated the synergistic effect of a combination of mTOR and HASPIN inhibitors on cell viability, cell cycle, cell apoptosis, DNA damage, and mitotic catastrophe using a panel of human KRAS-mutant and wild-type tumor cell lines. Subsequently, the human transplant models were used to test the therapeutic efficacy and pharmacodynamic effects of the dual therapy. RESULTS: We demonstrated that the combination of mTOR and HASPIN inhibitors induced potent synergistic cytotoxic effects in KRAS-mutant cell lines and delayed the growth of human tumor xenograft. Mechanistically, we showed that inhibiting of mTOR potentiates HASPIN inhibition by preventing the phosphorylation of H3 histones, exacerbating mitotic catastrophe and DNA damage in tumor cell lines with KRAS mutations, and this effect is due in part to a reduction in VRK1. CONCLUSIONS: These findings indicate that increased DNA damage and mitotic catastrophe are the basis for the effective synergistic effect observed with mTOR and HASPIN inhibition, and support the clinical evaluation of this dual therapy in patients with KRAS-mutant tumors.

Ankle2 deficiency-associated microcephaly and spermatogenesis defects in zebrafish are alleviated by heterozygous deletion of vrk1.

Autosomal recessive primary microcephaly (MCPH) is a rare congenital disorder characterized by a below average brain volume at birth and is associated with neurodevelopmental disorders such as growth retardation and intellectual disability. Mutations in ANKLE2 have been identified as one of the causes of MCPH (MCPH16). ANKLE2 is a target molecule of the Zika virus NS4a protein that interferes with ANKLE2 function, resulting in severe microcephaly. ANKLE2 is essential for organizing the nuclear envelope and chromatin structures during the mitotic-end process via barrier to autointegration factor (BAF) dephosphorylation. However, the precise mechanism by which the loss of ANKLE2 function causes the pathogenesis of microcephaly remains unclear. In this study, we generated Ankle2-deficient zebrafish (ankle2-/-) with a significant reduction in brain size compared with that of their control siblings. The ankle2-/- brain showed a significant decrease in the number of radial glial progenitor cells, suggesting that Ankle2 deficiency in zebrafish causes neurogenesis defects. Furthermore, ankle2-/- male zebrafish showed infertility owing to defects in spermatogenesis. Notably, microcephaly was overcome by vrk1 morpholino knockdown or vrk1 heterozygous deletion. In addition, spermatogenesis in ankle2-/- zebrafish males was partially restored by the vrk1 heterozygous deletion, although infertility was not resolved. These results indicate that ANKLE2 and VRK1 coordinate with each other for BAF phosphorylation to maintain normal mitosis during neurogenesis and spermatogenesis.

Distal spinal muscular atrophy featured by predominant calf muscle involvement in VRK1 associated disease - Case series and review.

We describe the shared clinical, biochemical, radiological and myopathological characteristics of four patients with distal spinal muscular atrophy (dSMA) caused by vaccinia-related kinase 1 (VRK1) variants and provide a review of the literature on phenotype-genotype correlations in VRK1-related disease. The clinical phenotype was characterized by adult-onset dSMA with predominant calf muscle involvement and mildly elevated serum creatinine kinase (CK) levels. Muscle imaging showed predominant atrophy and fatty replacement of calf muscles. We identified the novel compound heterozygous variants c.607C>T (p.Arg203Trp) and c.858G>T (p.Met286Ile) in two siblings with adult-onset dSMA. Additionally, two unrelated patients both carried the known c.583T>G (p.Leu195Val) VRK1 variant, with either c.197C>G (p.Ala66Gly) or c.701A>G (p.Asn234Ser) as a second variant. We conclude that compound heterozygous VRK1 variants cause distal spinal muscular atrophy with predominant posterior leg muscle involvement.

VRK1 Predicts Poor Prognosis and Promotes Bladder Cancer Growth and Metastasis In Vitro and In Vivo.

Bladder cancer (BC) is one of the most common malignant tumors in the urinary system with growing morbidity and diagnostic rate in recent years. Therefore, identifying new molecular biomarkers that inhibit the progression of bladder cancer is needed for developing further therapeutics. This study found a new potential treatment target: vaccinia-related kinase 1 (VRK1) and explored the function and mechanism of VRK1 in the development of bladder cancer. First, TCGA database and tissue microarray analysis showed that VRK1 was significantly upregulated in bladder cancer. Kaplan-Meier survival analysis indicates that the OS and PFS of the VRK1 high expression group were significantly lower than the VRK1 low expression group (p = 0.002, p = 0.005). Cox multi-factor analysis results show that VRK1 expression is an independent risk factor affecting tumor progress. The maximum tumor diameter, staging, and adjuvant chemotherapy also have a certain impact on tumor progression (p < 0.05). In internal validation, the column C index is 0.841 (95% CI, 0.803-0.880). In addition, cell functional studies have shown that VRK1 can significantly inhibit the proliferation, migration, and invasiveness of bladder cancer cells. In vivo, nude mice transplanted tumors further prove that low VRK1 can significantly inhibit the proliferation capacity of bladder cancer cells. In summary, VRK1 expression is significantly related to the staging, grade, and poor prognosis of patients with bladder cancer. At the same time, in vivo and in vitro experiments have shown that downregulation of VRK1 can significantly inhibit the proliferation of bladder cancer cells. These findings provide a basis for using VRK1 as a potential therapeutic target for patients with bladder cancer.

Altered action potential waveform and shorter axonal initial segment in hiPSC-derived motor neurons with mutations in VRK1.

We recently described new pathogenic variants in VRK1, in patients affected with distal Hereditary Motor Neuropathy associated with upper motor neurons signs. Specifically, we provided evidences that hiPSC-derived Motor Neurons (hiPSC-MN) from these patients display Cajal Bodies (CBs) disassembly and defects in neurite outgrowth and branching. We here focused on the Axonal Initial Segment (AIS) and the related firing properties of hiPSC-MNs from these patients. We found that the patient's Action Potential (AP) was smaller in amplitude, larger in duration, and displayed a more depolarized threshold while the firing patterns were not altered. These alterations were accompanied by a decrease in the AIS length measured in patients' hiPSC-MNs. These data indicate that mutations in VRK1 impact the AP waveform and the AIS organization in MNs and may ultimately lead to the related motor neuron disease.

Interaction of obtusilactone B and related butanolide lactones with the barrier-to-autointegration factor 1 (BAF1). A computational study.

The barrier-to-autointegration factor 1 (BAF1) protein is a DNA-binding protein implicated in nuclear envelop repair and reformation after mitosis. This nuclear protein is frequently overexpressed in cancer cells and plays a role in the occurrence and development of different tumors. It is a potential therapeutic target for gastric cancer, breast cancer and other malignancies. For this reason, BAF1 inhibitors are searched. The butanolide lactone obtusilactone B (Ob-B) has been found to inhibit VRK1-dependent phosphorylation of BAF1, upon direct binding to the nuclear protein. Taking advantage of the known crystallographic structure of BAF1, we have elaborated molecular models of Ob-B bound to BAF1 to delimit the binding site and binding configuration. The long endoolefinic alkyl side chain of Ob-B extends into a small groove on the protein surface, and the adjacent exomethylene-γ-lactone moiety occupies a pocket comprising to the Ser-4 phosphorylation site of BAF1. Twenty butanolide lactones structurally close to ObB were screened for BAF1 binding. Several natural products with BAF1-binding capacity potentially superior to Ob-B were identified, including mahubanolide, kotomolide B, epilitsenolide D2, and a few other known anticancer plant natural products. Our study provides new ideas to guide the discovery and design of BAF1 inhibitors.