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Background: Lung cancer constitutes the leading cause of cancer mortality. High levels of endothelin-1 (ET-1), its cognate receptor ETAR and its activating enzyme, the endothelin-converting enzyme-1 (ECE-1), have been reported in several cancer types, including lung cancer. ECE-1 comprises four isoforms, which only differ in their cytoplasmic N-terminus. Protein kinase CK2 phosphorylates the N-terminus of isoform ECE-1c, increasing its stability and leading to enhanced invasiveness in glioblastoma and colorectal cancer cells, which is believed to be mediated by the amino acid residue Lys-6, a conserved putative ubiquitination site neighboring the CK2-phosphorylated residues Ser-18 and Ser-20. Whether Lys-6 is linked to the acquisition of a cancer stem cell (CSC)-like phenotype and aggressiveness in human non–small cell lung cancer (NSCLC) cells has not been studied. Methods: In order to establish the role of Lys-6 in the stability of ECE-1c and its involvement in lung cancer aggressiveness, we mutated this residue to a non-ubiquitinable arginine and constitutively expressed the wild-type (ECE-1cWT) and mutant (ECE-1cK6R) proteins in A549 and H1299 human NSCLC cells by lentiviral transduction. We determined the protein stability of these clones alone or in the presence of the CK2 inhibitor silmitasertib, compared to ECE-1cWT and mock-transduced cells. In addition, the concentration of secreted ET-1 in the growth media was determined by ELISA. Expression of stemness genes were determined by Western blot and RT-qPCR. Chemoresistance to cisplatin was studied by MTS viability assay. Migration and invasion were measured through transwell and Matrigel assays, respectively, and the side-population was determined using flow cytometry. Results: ECE-1cK6R displayed higher stability in NSCLC cells compared to ECE-1cWT-expressing cells, but ET-1 secreted levels showed no difference up to 48 h. Most importantly, ECE-1cK6R promoted expression of the stemness genes c-Myc, Sox-2, Oct-4, CD44 and CD133, which enhance cellular self-renewal capability. Also, the ECE-1cK6R-expressing cells showed higher cisplatin chemoresistance, correlating with an augmented side-population abundance due to the increased expression of the ABCG2 efflux pump. Finally, the ECE-1cK6R-expressing cells showed enhanced invasiveness, which correlated with the regulated expression of known EMT markers. Conclusions: Our findings suggest an important role of ECE-1c in lung cancer. ECE-1c is key in a non-canonical ET-1-independent mechanism which triggers a CSC-like phenotype, leading to enhanced lung cancer aggressiveness. Underlying this mechanism, ECE-1c is stabilized upon phosphorylation by CK2, which is upregulated in many cancers. Thus, phospho-ECE-1c may be considered as a novel prognostic biomarker of recurrence, as well as the CK2 inhibitor silmitasertib as a potential therapy for lung cancer patients. [ABSTRACT FROM AUTHOR]

Background: Protein kinase CK2 is a ubiquitous and highly conserved protein Ser/Thr kinase with diverse cell functions. CK2 is upregulated in various cancers and affects numerous aspects of their underlying pathobiology. The important role of microRNAs (miRNAs) referred to as oncomirs is also recognized in various cancers. Elevation of both CK2 and altered miRNA expression in cancers raised the question whether there was a connection between CK2 function and oncomirs in cancer. Methods: PCR array analysis was used to examine the effects of CK2 siRNA-mediated downregulation on miRNA levels in C4-2 prostate cancer cells. We employed prostate cancer, breast cancer, and head and neck squamous cell carcinoma (HNSCC) cells as well as a prostate cancer xenograft orthotopic tumor model to examine the effects of CK2 siRNA-mediated downregulation or chemical inhibition on oncomir cluster miR-17 ~ 92 and miR-106b ~ 25 constituent miRNAs by quantitative reverse-transcriptase stem-loop PCR. Pri-miRNAs were measured in cancer cell lines by quantitative reverse-transcriptase PCR. Protein levels were assessed by western blot. PC3-LN4 prostate cancer orthotopic xenograft tumors and blood were collected from nude mice following repeated treatments with tenfibgen ligand nanocapsules containing RNAi-CK2 or RNAi-Control cargoes. Results: PCR array analysis demonstrated effect on a subset of miRNAs following CK2 downregulation; we focused our investigation on CK2 regulation of miR-17 ~ 92 and 106b ~ 25 oncomir clusters. Chemical inhibition or molecular downregulation of CK2 greatly reduced expression of miR-17 ~ 92 and 106b ~ 25 in prostate, breast and head and neck cancer cells in vitro. CK2α and CK2α´ protein levels were significantly correlated with many of the miR-17 ~ 92 and some of the miR-106b ~ 25 constituent members in prostate cancer cells. Decreased pri-miRNA levels for the miR-17 ~ 92 gene cluster transcript were observed for 5 of 6 cancer cell lines tested following CK2 downregulation. Nanocapsule-mediated delivery of RNAi-CK2 reduced CK2 protein expression in orthotopic prostate xenograft tumors and decreased intra-tumoral and serum levels of the oncomirs. Conclusions: Targeting CK2 for the development of new cancer therapies is under active investigation in many laboratories and pharmaceutical companies. Our data suggest a new role for CK2 in cell signaling and survival in multiple cancer types through maintenance of miR-17 ~ 92 and 106b ~ 25 biogenesis. [ABSTRACT FROM AUTHOR]

CK2 is a protein kinase that plays an important role in numerous cellular pathways involved in cell growth, differentiation, proliferation, and death. Consequently, upregulation of CK2 is implicated in many disease types, in particular cancer. As such, CK2 has gained significant attention as a potential therapeutic target in cancer, and over 40 chemical probes targeting CK2 have been developed in the past decade. In this review, we highlighted several chemical probes that target sites outside the conventional ATP-binding site. These chemical probes belong to different classes of molecules, from small molecules to peptides, and possess different mechanisms of action. Many of the chemical probes discussed in this review could serve as promising new candidates for drugs selectively targeting CK2. [ABSTRACT FROM AUTHOR]

Fibrotic scars play important roles in tissue reconstruction and functional recovery in the late stage of nervous system injury. However, the mechanisms underlying fibrotic scar formation and regulation remain unclear. Casein kinase II (CK2) is a protein kinase that regulates a variety of cellular functions through the phosphorylation of proteins, including bromodomain-containing protein 4 (BRD4). CK2 and BRD4 participate in fibrosis formation in a variety of tissues. However, whether CK2 affects fibrotic scar formation remains unclear, as do the mechanisms of signal regulation after cerebral ischemic injury. In this study, we assessed whether CK2 could modulate fibrotic scar formation after cerebral ischemic injury through BRD4. Primary meningeal fibroblasts were isolated from neonatal rats and treated with transforming growth factor-β1 (TGF-β1), SB431542 (a TGF-β1 receptor kinase inhibitor) or TBB (a highly potent CK2 inhibitor). Adult SD rats were intraperitoneally injected with TBB to inhibit CK2 after MCAO/R. We found that CK2 expression was increased in vitro in the TGF-β1-induced fibrosis model and in vivo in the MCAO/R injury model. The TGF-β1 receptor kinase inhibitor SB431542 decreased CK2 expression in fibroblasts. The CK2 inhibitor TBB reduced the increases in proliferation, migration and activation of fibroblasts caused by TGF-β1 in vitro, and it inhibited fibrotic scar formation, ameliorated histopathological damage, protected Nissl bodies, decreased infarct volume and alleviated neurological deficits after MCAO/R injury in vivo. Furthermore, CK2 inhibition decreased BRD4 phosphorylation both in vitro and in vivo. The findings of the present study suggested that CK2 may control BRD4 phosphorylation to regulate fibrotic scar formation, to affecting outcomes after ischemic stroke. [ABSTRACT FROM AUTHOR]

Immune cells can protect against tumor progression by killing cancer cells, while aberrant expression of the immune checkpoint protein PD‐L1 (programmed death ligand 1) in cancer cells facilitates tumor immune escape and inhibits anti‐tumor immunotherapy. As a serine/threonine kinase, CK2 (casein kinase 2) regulates tumor progression by multiple pathways, while it is still unclear the effect of CK2 on tumor immune escape. Here it is found that ING4 induced PD‐L1 autophagic degradation and inhibites non‐small cell lung cancer (NSCLC) immune escape by increasing T cell activity. However, clinical analysis suggests that high expression of CK2 correlates with low ING4 protein level in NSCLC. Further analysis shows that CK2 induce ING4‐S150 phosphorylation leading to ING4 ubiquitination and degradation by JFK ubiquitin ligase. In contrast, CK2 gene knockout increases ING4 protein stability and T cell activity, subsequently, inhibites NSCLC immune escape. Furthermore, the combined CK2 inhibitor with PD‐1 antibody effectively enhances antitumor immunotherapy. These findings provide a novel strategy for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

At the feto-maternal interface, fetal membranes (FM) play a crucial role throughout pregnancy. FM rupture at term implicates different sterile inflammation mechanisms including pathways activated by the transmembrane glycoprotein receptor for advanced glycation end-products (RAGE) belonging to the immunoglobulin superfamily. As the protein kinase CK2 is also implicated in the inflammation process, we aimed to characterize the expressions of RAGE and the protein kinase CK2 as a candidate regulator of RAGE expression. The amnion and choriodecidua were collected from FM explants and/or primary amniotic epithelial cells throughout pregnancy and at term in spontaneous labor (TIL) or term without labor (TNL). The mRNA and protein expressions of RAGE and the CK2α, CK2α′, and CK2β subunits were investigated using reverse transcription quantitative polymerase chain reaction and Western blot assays. Their cellular localizations were determined with microscopic analyses, and the CK2 activity level was measured. RAGE and the CK2α, CK2α′, and CK2β subunits were expressed in both FM layers throughout pregnancy. At term, RAGE was overexpressed in the amnion from the TNL samples, whereas the CK2 subunits were expressed at the same level in the different groups (amnion/choriodecidua/amniocytes, TIL/TNL), without modification of the CK2 activity level and immunolocalization. This work paves the way for future experiments regarding the regulation of RAGE expression by CK2 phosphorylation. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is the most common and aggressive type of brain tumor due to its elevated recurrence following treatments. This is mainly mediated by a subpopulation of cells with stemness traits termed glioblastoma stem-like cells (GSCs), which are extremely resistant to anti-neoplastic drugs. Thus, an advancement in the understanding of the molecular processes underlying GSC occurrence should contribute significantly towards progress in reducing aggressiveness. High levels of endothelin-converting enzyme-1 (ECE1), key for endothelin-1 (ET-1) peptide activation, have been linked to the malignant progression of GBM. There are four known isoforms of ECE1 that activate ET-1, which only differ in their cytoplasmic N-terminal sequences. Isoform ECE1c is phosphorylated at Ser-18 and Ser-20 by protein kinase CK2, which increases its stability and hence promotes aggressiveness traits in colon cancer cells. In order to study whether ECE1c exerts a malignant effect in GBM, we designed an ECE1c mutant by switching a putative ubiquitination lysine proximal to the phospho-serines Lys-6-to-Arg (i.e., K6R). This ECE1cK6R mutant was stably expressed in U87MG, T98G, and U251 GBM cells, and their behavior was compared to either mock or wild-type ECE1c-expressing clone cells. ECE1cK6R behaved as a highly stable protein in all cell lines, and its expression promoted self-renewal and the enrichment of a stem-like population characterized by enhanced neurospheroid formation, as well as increased expression of stem-like surface markers. These ECE1cK6R-derived GSC-like cells also displayed enhanced resistance to the GBM-related chemotherapy drugs temozolomide and gemcitabine and increased expression of the ABCG2 efflux pump. In addition, ECE1cK6R cells displayed enhanced metastasis-associated traits, such as the modulation of adhesion and the enhancement of cell migration and invasion. In conclusion, the acquisition of a GSC-like phenotype, together with heightened chemoresistance and invasiveness traits, allows us to suggest phospho-ECE1c as a novel marker for poor prognosis as well as a potential therapeutic target for GBM. [ABSTRACT FROM AUTHOR]

Background and Purpose: Liver fibrosis is a serious cause of morbidity and mortality worldwide characterized by accumulation of extracellular matrix produced by hepatic stellate cells (HSCs). The protein kinase CK2 is a pro‐survival kinase overexpressed in human tumours. However, the biological role of CK2 in liver fibrosis is largely unknown. We aimed to investigate the mechanism by which CK2 promotes liver fibrosis. Experimental Approach: In vitro, LX‐2 cells were stimulated with transforming growth factor‐β (TGF‐β). HSCs were also isolated for research. In vivo, the adeno‐associated virus AAV‐sh‐csnk2a1 was used to knockdown CK2α specifically in HSCs, and CX‐4945 was used to pharmacologically inhibit the enzymatic activity of CK2 in murine models of fibrosis induced by carbon tetrachloride (CCl4) and a 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) diet. Histological and biochemical analyses were performed to study the involvement of CK2 in regulation of fibrogenic and fibrolytic factors as well as activation properties of HSCs. Key Results: HSC‐specific genetic invalidation of CK2α or pharmacological inhibition of CK2 protected mice treated with CCl4 or fed a DDC diet against liver fibrosis and HSC accumulation. Mechanistically, CK2α, which bound to Smoothened (SMO), was a positive regulator of the Hedgehog signal transduction pathway. CK2 prevented ubiquitination and proteasomal degradation of SMO, which was abolished by knockdown of CK2α or pharmacological inhibition of CK2. Conclusions and Implications: CK2 activation is critical to sustain the activated and fibrogenic phenotype of HSCs via SMO stabilization. Therefore, inactivation of CK2 by CX‐4945 may be of therapeutic interest for liver fibrotic diseases. [ABSTRACT FROM AUTHOR]

Protein kinase 2 (CK2) is an enzyme ubiquitously present and exhibits extensive kinase activity. It has been strongly linked to tumor progression through the abnormal phosphorylation of key proteins. Research has consistently demonstrated that CK2 is deregulated in various cancer types, with enhanced protein expression and nuclear distribution in tumor cells. CK2 plays a crucial role in a complex network that promotes cell infiltration, migration, proliferation, apoptosis, and cancer progression through multiple pathways, including PI3K/AKT, JAK2/STAT3, ATF4/CDKN1, and HSP90/Cdc37. In addition to its role in cancer growth, there is mounting evidence that CK2 may also affect the immunological dynamics of cancer by altering immune cell functions within the tumor microenvironment, thus facilitating tumor immune evasion. Recent research has increasingly focused on CK2, recognizing it as a therapeutic objective for oncological interventions. This review will critically examine the structure and signaling pathways of CK2, highlighting the significance of further research aimed at enhancing our understanding of the CK2 machinery. Finally, we conclude by refining therapeutic options, notably transitioning from non-pharmacological techniques to strategic CK2 inhibitor use. This development shortens the path to the desired outcome, establishing a pioneering standard in cancer therapy. [Display omitted] • Protein kinase CK2 is a pleiotropic kinase within the human kinase family. • A key player in tumor formation across numerous forms of cancer through aberrant phosphorylation of essential proteins. • Elevated CK2 activity triggers tumorigenic. signalling • CK2 plays a pivotal role in immune responses, impacting both innate and adaptive immunity. • CK2 inhibitors with non-pharmacological methods to modulate kinase activity represents a significant breakthrough. [ABSTRACT FROM AUTHOR]

DNA polymerase β plays a fundamental role in the life cycle of Trypanosoma cruzi since it participates in the kinetoplast DNA repair and replication. This enzyme can be found in two forms in cell extracts of T. cruzi epimastigotes form. The H form is a phosphorylated form of DNA polymerase β, while the L form is not phosphorylated. The protein kinases which are able to in vivo phosphorylate DNA polymerase β have not been identified yet. In this work, we purified the H form of this DNA polymerase and identified the phosphorylation sites. DNA polymerase β is in vivo phosphorylated at several amino acid residues including Tyr35, Thr123, Thr137 and Ser286. Thr123 is phosphorylated by casein kinase 2 and Thr137 and Ser286 are phosphorylated by protein kinase C-like enzymes. Protein kinase C encoding genes were identified in T. cruzi, and those genes were cloned, expressed in bacteria and the recombinant protein was purified. It was found that T. cruzi possesses three different protein kinase C-like enzymes named TcPKC1, TcPKC2, and TcPKC3. Both TcPKC1 and TcPKC2 were able to in vitro phosphorylate recombinant DNA polymerase β, and in addition, TcPKC1 gets auto phosphorylated. Those proteins contain several regulatory domains at the N-terminus, which are predicted to bind phosphoinositols, and TcPKC1 contains a lipocalin domain at the C-terminus that might be able to bind free fatty acids. Tyr35 is phosphorylated by an unidentified protein kinase and considering that the T. cruzi genome does not contain Tyr kinase encoding genes, it is probable that Tyr35 could be phosphorylated by a dual protein kinase. Wee1 is a eukaryotic dual protein kinase involved in cell cycle regulation. We identified a Wee1 homolog in T. cruzi and the recombinant kinase was assayed using DNA polymerase β as a substrate. T. cruzi Wee1 was able to in vitro phosphorylate recombinant DNA polymerase β, although we were not able to demonstrate specific phosphorylation on Tyr35. Those results indicate that there exists a cell signaling pathway involving PKC-like kinases in T. cruzi. [ABSTRACT FROM AUTHOR]

Cancer is a leading cause of death worldwide. Casein kinase 2 (CK2) is commonly dysregulated in cancer, impacting diverse molecular pathways. CK2 is a highly conserved serine/threonine kinase, constitutively active and ubiquitously expressed in eukaryotes. With over 500 known substrates and being estimated to be responsible for up to 10% of the human phosphoproteome, it is of significant importance. A broad spectrum of diverse types of cancer cells has been already shown to rely on disturbed CK2 levels for their survival. The hallmarks of cancer provide a rationale for understanding cancer's common traits. They constitute the maintenance of proliferative signaling, evasion of growth suppressors, resisting cell death, enabling of replicative immortality, induction of angiogenesis, the activation of invasion and metastasis, as well as avoidance of immune destruction and dysregulation of cellular energetics. In this work, we have compiled evidence from the literature suggesting that CK2 modulates all hallmarks of cancer, thereby promoting oncogenesis and operating as a cancer driver by creating a cellular environment favorable to neoplasia. [ABSTRACT FROM AUTHOR]

Huntington's Disease (HD) is a devastating neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the HTT gene, for which no disease modifying therapies are currently available. Much of the recent research has focused on developing therapies to directly lower HTT expression, and while promising, these therapies have presented several challenges regarding administration and efficacy. Another promising therapeutic approach is the modulation of HTT post-translational modifications (PTMs) that are dysregulated in disease and have shown to play a key role in HTT toxicity. Among all PTMs, modulation of HTT phosphorylation has been proposed as an attractive therapeutic option due to the possibility of orally administering specific kinase effectors. One of the kinases described to participate in HTT phosphorylation is Protein Kinase CK2. CK2 has recently emerged as a target for the treatment of several neurological and psychiatric disorders, although its role in HD remains controversial. While pharmacological studies in vitro inhibiting CK2 resulted in reduced HTT phosphorylation and increased toxicity, genetic approaches in mouse models of HD have provided beneficial effects. In this review we discuss potential therapeutic approaches related to the manipulation of HTT-PTMs with special emphasis on the role of CK2 as a therapeutic target in HD. [ABSTRACT FROM AUTHOR]

CIGB-300 is an antitumor peptide that inhibits the CK2-mediated phosphorylation by direct targeting of substrates. This paper aims to describe breakthroughs about the CIGB-300 antineoplastic mechanism related to the inhibition of CK2-mediated phosphorylation and the synergism with anticancer drugs. CK2 phosphorylation assays were performed with catalytic CK2α subunit or the CK2 holoenzyme in presence or not of CIGB-300. CIGB-300/CK2 interaction was verified by pull-down experiments, in situ colocalization and phosphoproteomic analysis of CIGB-300-treated lung cancer cells were also performed. Synergism of the peptide with anticancer drugs was evaluated in vitro and for Cisplatin; it was also tested in vivo. Besides, comparative proteomics of CIGB-300 combined with Cisplatin was conducted. CIGB-300 targeted the CK2α subunit and inhi-bited the enzymatic activity of the holoenzyme in different experimental settings. Likewise, phosphoproteomic and Western Blot analysis allowed for knowing the early CK2 inhibition profile elicited by CIGB-300 at 10 and 30 min of treatment. Moreover, CIGB-300 did synergize with chemotherapeutics and EGFR inhibitors, and molecular events that reportedly support such synergistic interactions were also known. CIGB-300 inhibits the CK2-mediated phosphorylation by using an alternative mechanism of direct interaction with the enzyme itself. Besides, CIGB-300 synergizes with chemotherapeutics and EGFR inhibitors by modulating different proteins related to drug resistance. [ABSTRACT FROM AUTHOR]

Casein kinase 2 (CK2) is a ubiquitously expressed serine/threonine kinase and is upregulated in human obesity. CX-4945 (Silmitasertib) is a CK2 inhibitor with anti-cancerous and anti-adipogenic activities. However, the anti-adipogenic and pro-lipolytic effects and the mode of action of CX-4945 in (pre)adipocytes remain elusive. Here, we explored the effects of CX-4945 on adipogenesis and lipolysis in differentiating and differentiated 3T3-L1 cells, a murine preadipocyte cell line. CX-4945 at 15 μM strongly reduced lipid droplet (LD) accumulation and triglyceride (TG) content in differentiating 3T3-L1 cells, indicating the drug's anti-adipogenic effect. Mechanistically, CX-4945 reduced the expression levels of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and perilipin A in differentiating 3T3-L1 cells. Strikingly, CX-4945 further increased the phosphorylation levels of cAMP-activated protein kinase (AMPK) and liver kinase B-1 (LKB-1) while decreasing the intracellular ATP content in differentiating 3T3-L1 cells. In differentiated 3T3-L1 cells, CX-4945 had abilities to stimulate glycerol release and elevate the phosphorylation levels of hormone-sensitive lipase (HSL), pointing to the drug's pro-lipolytic effect. In addition, CX-4945 induced the activation of extracellular signal-regulated kinase-1/2 (ERK-1/2), and PD98059, an inhibitor of ERK-1/2, attenuated the CX4945-induced glycerol release and HSL phosphorylation in differentiated 3T3-L1 cells, indicating the drug's ERK-1/2-dependent lipolysis. In summary, this investigation shows that CX-4945 has strong anti-adipogenic and pro-lipolytic effects on differentiating and differentiated 3T3-L1 cells, mediated by control of the expression and phosphorylation levels of CK2, C/EBP-α, PPAR-γ, FAS, ACC, perilipin A, AMPK, LKB-1, ERK-1/2, and HSL. [ABSTRACT FROM AUTHOR]

Aims: Sonic hedgehog subtype medulloblastoma is featured with overactivation of hedgehog pathway and can be targeted by SMO‐specific inhibitors. However, the resistance is frequently developed leading to treatment failure of SMO inhibitors. W535L mutation of SMO (SMOW535L) is thought to be an oncogenic driver for Sonic hedgehog subtype MB and confer resistance to SMO inhibitors. The regulation network of SMOW535L remains to be explored in comparison with wild‐type SMO (SMOWT). Methods: In this study, we profiled transcriptomes, methylomes, and interactomes of MB cells expression SMOWT or SMOW535L in the treatment of DMSO or SMO inhibitor, respectively. Results: Analysis of transcriptomic data indicated that SMO inhibitor disrupted processes of endocytosis and cilium organization in MB cells with SMOWT, which are necessary for SMO activation. In MB cells with SMOW535L, however, SMO inhibitor did not affect the two processes‐related genes, implying resistance of SMOW535L toward SMO inhibitor. Moreover, we noticed that SMO inhibitor significantly inhibited metabolism‐related pathways. Our metabolic analysis indicated that nicotinate and nicotinamide metabolism, glycerolipid metabolism, beta‐alanine metabolism, and synthesis and degradation of ketone bodies might be involved in SMOW535L function maintenance. Interactomic analysis revealed casein kinase II (CK2) as an important SMO‐associated protein. Finally, we linked CK2 and AKT together and found combination of inhibitors targeting CK2 and AKT showed synergetic effects to inhibit the growth of MB cells with SMO constitutive activation mutation. Conclusions: Taken together, our work described SMO‐related transcriptomes, metabolomes, and interactomes under different SMO status and treatment conditions, identifying CK2 and AKT as therapeutic targets for SHH‐subtype MB cells with SMO inhibitor resistance. [ABSTRACT FROM AUTHOR]

Overexpression of casein kinase 2 (CK2) has an oncogenic and pro-survival role in many cancers. CX-4945 (Silmitasertib) is a CK2 inhibitor with anti-cancerous and anti-angiogenic effects. Up to date, the anti-cancer effect and mechanism of CX-4945 on human cholangiocarcinoma (CCA) remain unclear. This study investigated whether CX-4945 inhibits growth and induces apoptosis of HuCCT-1 cells, a human CCA cell line. Of note, treatment with CX-4945 at 20 μM markedly reduced survival and induced apoptosis of HuCCT-1 cells, as evidenced by nuclear DNA fragmentation, PARP cleavage, activation of caspase-9/3, and up-regulation of DR-4. Although CX-4945 did not affect the phosphorylation and expression of CK2, it vastly inhibited the phosphorylation of CK2 substrates, supporting the drug's efficacy in inhibiting CK2 and its downstream pathway. Importantly, knockdown of CK2 that partially suppressed the phosphorylation of CK2 substrates resulted in a significant reduction of HuCCT-1 cell survival. In addition, CX-4945 reduced the phosphorylation and expression of STAT-3 and STAT-5 in HuCCT-1 cells, and pharmacological inhibition or respective knockdown of these proteins resulted in significant growth suppression of HuCCT-1 cells. CX-4945 also had abilities to decrease Mcl-1 expression while increasing eIF-2α phosphorylation in HuCCT-1 cells. Furthermore, there was a time-differential negative regulation of HIF-1α expression by CX-4945 in HuCCT-1 cells, and knockdown of HIF-1α caused a significant reduction of the cell survival. In summary, these results demonstrated that CX-4945 has anti-growth, anti-angiogenic, and pro-apoptotic effects on HuCCT-1 cells, which are mediated through control of CK2, caspase-9/3, DR-4, STAT-3/5, Mcl-1, eIF-2α, and HIF-1α. [ABSTRACT FROM AUTHOR]

Juvenile angiofibroma (JA) is a rare fibrovascular neoplasm predominately found within the posterior nasal cavity of adolescent males. JA expresses the proteoglycan nerve–glial antigen (NG)2, which crucially determines the migratory capacity of distinct cancer cells. Moreover, it is known that the protein kinase CK2 regulates NG2 gene expression. Therefore, in the present study, we analyzed whether the inhibition of CK2 suppresses NG2-dependent JA cell proliferation and migration. For this purpose, we assessed the expression of NG2 and CK2 in patient-derived JA tissue samples, as well as in patient-derived JA cell cultures by Western blot, immunohistochemistry, flow cytometry and quantitative real-time PCR. The mitochondrial activity, proliferation and migratory capacity of the JA cells were determined by water-soluble tetrazolium (WST)-1, 5-bromo-2′-deoxyuridine (BrdU) and collagen sprouting assays. We found that NG2 and CK2 were expressed in both the JA tissue samples and cell cultures. The treatment of the JA cells with the two CK2 inhibitors, CX-4945 and SGC-CK2-1, significantly reduced NG2 gene and protein expression when compared to the vehicle-treated cells. In addition, the loss of CK2 activity suppressed the JA cell proliferation and migration. These findings indicate that the inhibition of CK2 may represent a promising therapeutic approach for the treatment of NG2-expressing JA. [ABSTRACT FROM AUTHOR]

• Mn activates YY1 through posttranslational phosphorylation in astrocytes. • Mn phosphorylates YY1 at serine residues via kinases Aurora B kinase (AurkB) and Casein kinase II (CK2). • Mn reduces EAAT2 expression via YY1 phosphorylation and subsequent YY1 nuclear translocation. Impairment of the astrocytic glutamate transporter excitatory amino acid transporter 2 (EAAT2) is associated with neurological disorders such as Parkinson's disease (PD), Alzheimer's disease (AD), and manganism, a neurological disorder caused by overexposure to manganese (Mn) which shares the features of sporadic PD. Mechanisms of Mn-induced neurotoxicity include dysregulation of EAAT2 following activation of the transcription factor Yin Yang 1 (YY1) by transcriptional upregulation, but the posttranslational mechanisms by which YY1 is activated to repress EAAT2 remain to be elucidated. In the present study, we tested if Mn activates YY1 through posttranslational phosphorylation in cultured H4 human astrocytes, leading to EAAT2 repression. The results demonstrate that Mn exposure induced phosphorylation of YY1 at serine residues via kinases Aurora B kinase (AurkB) and Casein kinase II (CK2), leading to YY1 nuclear translocation, YY1/HDAC interactions, binding to the EAAT2 promoter, and consequent decreases in EAAT2 promoter activity and mRNA/protein levels. Although further studies are warranted to fully elucidate the mechanisms of Mn-induced YY1 phosphorylation and resultant EAAT2 impairment, our findings indicate that serine phosphorylation of YY1 via AurkB and CK2 is critical, at least in part, to its activation and transcriptional repression of EAAT2. [ABSTRACT FROM AUTHOR]

Highly pleiotropic and constitutively active protein kinase CK2 is a key target in cancer therapy, but only one small‐molecule inhibitor has reached clinical trials—CX‐4945. In this study, we present the indeno[1,2‐b]indole derivative 5‐isopropyl‐4‐methoxy‐7‐methyl‐5,6,7,8‐tetrahydroindeno[1,2‐b]indole‐9,10‐dione (5a‐2) that decreased the intracellular CK2 activity in A431, A549, and LNCaP tumor cell lines analogous to CX‐4945 (> 75% inhibition at 20 µm) and similarly blocked CK2‐specific Akt phosphorylation in LNCaP cells. Cellular uptake analysis demonstrated higher intracellular concentrations of 5a‐2 (408.3 nm) compared with CX‐4945 (119.3 nm). This finding clarifies the comparable effects of both compounds on the intracellular CK2 activity despite their different inhibitory potency in vitro [IC50 = 25 nm (5a‐2) and 3.7 nm (CX‐4945)]. Examination of the effects of both CK2 inhibitors on cancer cells using live‐cell imaging revealed notable differences. Whereas CX‐4945 showed a stronger pro‐apoptotic effect on tumor cells, 5a‐2 was more effective in inhibiting tumor cell migration. Our results showed that 49% of intracellular CX‐4945 was localized in the nuclear fraction, whereas 71% of 5a‐2 was detectable in the cytoplasm. The different subcellular distribution, and thus the site of CK2 inhibition, provides a possible explanation for the different cellular effects. Our study indicates that investigating CK2 inhibition‐mediated cellular effects in relation to the subcellular sites of CK2 inhibition may help to improve our understanding of the preferential roles of CK2 within different cancer cell compartments. [ABSTRACT FROM AUTHOR]

Background. Oropharyngeal squamous cell carcinoma (OPSCC) incidence is rising worldwide, especially human papillomavirus (HPV)-associated disease. Historically, high levels of protein kinase CK2 were linked with poor outcomes in head and neck squamous cell carcinoma (HNSCC), without consideration of HPV status. This retrospective study examined tumor CK2α protein expression levels and related clinical outcomes in a cohort of Veteran OPSCC patient tumors which were determined to be predominantly HPV(+). Methods. Patients at the Minneapolis VA Health Care System with newly diagnosed primary OPSCC from January 2005 to December 2015 were identified. A total of 119 OPSCC patient tumors were stained for CK2α, p16 and Ki-67 proteins and E6/E7 RNA. CK2α protein levels in tumors and correlations with HPV status and Ki-67 index were assessed. Overall survival (OS) analysis was performed stratified by CK2α protein score and separately by HPV status, followed by Cox regression controlling for smoking status. To strengthen the limited HPV(-) data, survival analysis for HPV(-) HNSCC patients in the publicly available The Cancer Genome Atlas (TCGA) PanCancer RNA-seq dataset was determined for CSNK2A1. Results. The patients in the study population were all male and had a predominant history of tobacco and alcohol use. This cohort comprised 84 HPV(+) and 35 HPV(-) tumors. CK2α levels were higher in HPV(+) tumors compared to HPV(-) tumors. Higher CK2α scores positively correlated with higher Ki-67 index. OS improved with increasing CK2α score and separately OS was significantly better for those with HPV(+) as opposed to HPV(-) OPSCC. Both remained significant after controlling for smoking status. High CSNK2A1 mRNA levels from TCGA data associated with worse patient survival in HPV(-) HNSCC. Conclusions. High CK2α protein levels are detected in HPV(+) OPSCC tumors and demonstrate an unexpected association with improved survival in a strongly HPV(+) OPSCC cohort. Worse survival outcomes for high CSNK2A1 mRNA levels in HPV(-) HNSCC are consistent with historical data. Given these surprising findings and the rising incidence of HPV(+) OPSCC, further study is needed to understand the biological roles of CK2 in HPV(+) and HPV(-) HNSCC and the potential utility for therapeutic targeting of CK2 in these two disease states. [ABSTRACT FROM AUTHOR]

Background: Che-1/AATF (Che-1) is an RNA polymerase II binding protein involved in several cellular processes, including proliferation, apoptosis and response to stress. We have recently demonstrated that Che-1 is able to promote cell proliferation by sustaining global histone acetylation in multiple myeloma (MM) cells where it interacts with histone proteins and competes with HDAC class I members for binding. Methods: Site-directed Mutagenesis was performed to generate a Che-1 mutant (Che-1 3S) lacking three serine residues (Ser316, Ser320 and Ser321) in 308–325 aa region. Western blot experiments were conducted to examine the effect of depletion or over-expression of Che-1 and Che-1 3S mutant on histone acetylation, in different human cancer cell lines. Proliferation assays were assessed to estimate the change in cells number when Che-1 was over-expressed or deleted. Immunoprecipitation assays were performed to evaluate Che-1/histone H3 interaction when Ser316, Ser320 and Ser321 were removed. The involvement of CK2 kinase in Che-1 phosphorylation at these residues was analysed by in vitro kinase, 2D gel electrophoresis assays and mass spectrometry analysis. Results: Here, we confirmed that Che-1 depletion reduces cell proliferation with a concomitant general histone deacetylation in several tumor cell lines. Furthermore, we provided evidence that CK2 protein kinase phosphorylates Che-1 at Ser316, Ser320 and Ser321 and that these modifications are required for Che-1/histone H3 binding. These results improve our understanding onto the mechanisms by which Che-1 regulates histone acetylation and cell proliferation. Conclusions: Che-1 phosphorylation at Ser316, Ser320 and Ser321 by CK2 promotes the interaction with histone H3 and represents an essential requirement for Che-1 pro-proliferative ability. [ABSTRACT FROM AUTHOR]

CSNK2B has recently been implicated as a disease gene for neurodevelopmental disability (NDD) and epilepsy. Information about developmental outcomes has been limited by the young age and short follow‐up for many of the previously reported cases, and further delineation of the spectrum of associated phenotypes is needed. We present 25 new patients with variants in CSNK2B and refine the associated NDD and epilepsy phenotypes. CSNK2B variants were identified by research or clinical exome sequencing, and investigators from different centers were connected via GeneMatcher. Most individuals had developmental delay and generalized epilepsy with onset in the first 2 years. However, we found a broad spectrum of phenotypic severity, ranging from early normal development with pharmacoresponsive seizures to profound intellectual disability with intractable epilepsy and recurrent refractory status epilepticus. These findings suggest that CSNK2B should be considered in the diagnostic evaluation of patients with a broad range of NDD with treatable or intractable seizures. [ABSTRACT FROM AUTHOR]

Cognitive behavioral therapy, rooted in exposure therapy, is currently the primary approach employed in the treatment of anxiety-related conditions, including post-traumatic stress disorder (PTSD). In laboratory settings, fear extinction in animals is a commonly employed technique to investigate exposure therapy; however, the precise mechanisms underlying fear extinction remain elusive. Casein kinase 2 (CK2), which regulates neuroplasticity via phosphorylation of its substrates, has a significant influence in various neurological disorders, such as Alzheimer's disease and Parkinson's disease, as well as in the process of learning and memory. In this study, we adopted a classical Pavlovian fear conditioning model to investigate the involvement of CK2 in remote fear memory extinction and its underlying mechanisms. The results indicated that the activity of CK2 in the medial prefrontal cortex (mPFC) of mice was significantly upregulated after extinction training of remote cued fear memory. Notably, administration of the CK2 inhibitor CX-4945 prior to extinction training facilitated the extinction of remote fear memory. In addition, CX-4945 significantly upregulated the expression of p-ERK1/2 and p-CREB in the mPFC. Our results suggest that CK2 negatively regulates remote fear memory extinction, at least in part, by inhibiting the ERK-CREB pathway. These findings contribute to our understanding of the underlying mechanisms of remote cued fear extinction, thereby offering a theoretical foundation and identifying potential targets for the intervention and treatment of PTSD. • CK2 activity increased in mPFC after extinction training of remote cued fear memory. • CK2 inhibition facilitated the extinction of remote cued fear memory. • Inhibition of CK2 activated the ERK-CREB pathway in the mPFC. [ABSTRACT FROM AUTHOR]

Transcription factors have proven difficult to target with small molecules because they lack pockets necessary for potent binding. Disruption of protein expression can suppress targets and enable therapeutic intervention. To this end, we developed a drug discovery workflow that incorporates cell-line-selective screening and high-throughput expression profiling followed by regulatory network analysis to identify compounds that suppress regulatory drivers of disease. Applying this approach to neuroblastoma (NBL), we screened bioactive molecules in cell lines representing its MYC-dependent (MYCNA) and mesenchymal (MES) subtypes to identify selective compounds, followed by PLATESeq profiling of treated cells. This revealed compounds that disrupt a sub-network of MYCNA-specific regulatory proteins, resulting in MYCN degradation in vivo. The top hit was isopomiferin, a prenylated isoflavonoid that inhibited casein kinase 2 (CK2) in cells. Isopomiferin and its structural analogs inhibited MYC and MYCN in NBL and lung cancer cells, highlighting the general MYC-inhibiting potential of this unique scaffold. [Display omitted] • OncoTreat prioritized subtype-selective compounds that disrupt a regulatory module • Top hits included prenylated isoflavonoid molecules that suppress MYCN protein • Pomiferin suppressed MYCN in vivo , slowing growth of SK-N-Be2 NBL xenografts • VIKING algorithm identified putative targets, including casein kinase2 (CK2) Stokes and Vasciaveo et al. evaluated selective agents targeting MYCN-amplified neuroblastoma cells using PLATE-Seq and regulatory network analysis. This revealed prenylated isoflavonoids that suppress a tumor checkpoint module, causing MYCN depletion and cell death. This paper highlights a multifaceted approach that enables screening for agents targeting networked drivers of disease. [ABSTRACT FROM AUTHOR]

Protein phosphorylation is the most frequent post-translational modification by which the properties of eukaryotic proteins can be reversibly modified. In humans, over 500 protein kinases generate a huge phosphoproteome including more than 200,000 individual phosphosites, a figure which is still continuously increasing. The in vivo selectivity of protein kinases is the outcome of a multifaceted and finely tuned process where numerous factors play an integrated role. To gain information about the actual contribution to this process of local features that reflect the interaction of the protein targets with the catalytic site of the kinases, the prevalence of the commonest motifs determining the consensus sequence of Ser/Thr-specific kinases has been examined in the whole human phosphoproteome and in the phosphoproteomes generated by a panel of the 47 most pleiotropic protein kinases. Our analysis shows that: (1) most phosphosites do conform to at least one of the motifs considered, with a substantial proportion conforming to two or more of them; (2) some motifs, with special reference to the one recognized by protein kinase CK2 (pS/pT-x-x-E/D) are very promiscuous, being abundantly represented also at the phosphosites of all the other protein kinases considered; (3) by contrast, other phosphorylated motifs, notably pS/pT-P, pS/pT-Q and pS-x-E, are more discriminatory and selective, being nearly absent in the phosphosites that are not attributable to certain categories of kinases. The information provided will prove helpful to make reliable inferences based on the manual inspection of individual phosphosites. [ABSTRACT FROM AUTHOR]

Kinase activity plays an essential role in the regulation of immune cell defenses against pathogens. The protein kinase CK2 (formerly casein kinase II) is an evolutionarily conserved kinase with hundreds of identified substrates. CK2 is ubiquitously expressed in somatic and immune cells, but the roles of CK2 in regulation of immune cell function remain largely elusive. This reflects the essential role of CK2 in organismal development and limited prior work with conditional CK2 mutant murine models. Here, we generated mice with a conditional (floxed) allele of Csnk2a , which encodes the catalytic CK2α subunit of CK2. When crossed to Lyz2 -cre mice, excision of Csnk2a sequence impaired CK2α expression in myeloid cells but failed to detectably alter myeloid cell development. By contrast, deficiency for CK2α increased inflammatory myeloid cell recruitment, activation, and resistance following systemic Listeria monocytogenes (Lm) infection. Results from mixed chimera experiments indicated that CK2α deficiency in only a subset of myeloid cells was not sufficient to reduce bacterial burdens. Nor did cell-intrinsic deficiency for CK2α suffice to alter accumulation or activation of monocytes and neutrophils in infected tissues. These data suggest that CK2α expression by Lyz2 -expressing cells promotes inflammatory and anti-bacterial responses through effects in trans. Our results highlight previously undescribed suppressive effects of CK2 activity on inflammatory myeloid cell responses and illustrate that cell-extrinsic effects of CK2 can shape inflammatory and protective innate immune responses. [ABSTRACT FROM AUTHOR]

Background: Protein kinase CK2 inhibition has long been considered as an attractive anti-cancer strategy based on the following considerations: CK2 is a pro-survival kinase, it is frequently over-expressed in human tumours and its over-expression correlates with a worse prognosis. Preclinical evidence strongly supports the feasibility of this target and, although dozens of CK2 inhibitors have been described in the literature so far, CX-4945 (silmitasertib) was the first that entered into clinical trials for the treatment of both human haematological and solid tumours. However, kinase inhibitor monotherapies turned out to be effective only in a limited number of malignancies, probably due to the multifaceted causes that underlie them, supporting the emerging view that multi-targeted approaches to treat human tumours could be more effective. Conclusions: In this review, we will address combined anti-cancer therapeutic strategies described so far which involve the use of CX-4945. Data from preclinical studies clearly show the ability of CX-4945 to synergistically cooperate with different classes of anti-neoplastic agents, thereby contributing to an orchestrated anti-tumour action against multiple targets. Overall, these promising outcomes support the translation of CX-4945 combined therapies into clinical anti-cancer applications. [ABSTRACT FROM AUTHOR]

Background: CK2 (casein kinase 2) is a serine/threonine-selective protein kinase that has been involved in a variety of cellular processes such as DNA repair, cell cycle control and circadian rhythm regulation. However, its functional roles in oocyte meiosis have not been fully determined. Results: We report that CK2 is essential for porcine oocyte meiotic maturation by regulating spindle assembly checkpoint (SAC). Immunostaining and immunoblotting analysis showed that CK2 was constantly expressed and located on the chromosomes during the entire oocyte meiotic maturation. Inhibition of CK2 activity by its selective inhibitor CX-4945 impaired the first polar body extrusion and arrested oocytes at M I stage, accompanied by the presence of BubR1 at kinetochores, indicative of activated SAC. In addition, we found that spindle/chromosome structure was disrupted in CK2-inhibited oocytes due to the weakened microtubule stability, which is a major cause resulting in the activation of SAC. Last, we found that the level DNA damage as assessed by γH2A.X staining was considerably elevated when CK2 was inhibited, suggesting that DNA damage might be another critical factor leading to the SAC activation and meiotic failure of oocytes. Conclusions: Our findings demonstrate that CK2 promotes the porcine oocyte maturation by ensuring normal spindle assembly and DNA damage repair. [ABSTRACT FROM AUTHOR]

Casein kinase 2 (CK2) is a ubiquitous pleiotropic enzyme that is highly conserved across eukaryotic kingdoms. CK2 is singular amongst kinases as it is highly rigid and constitutively active. Arabidopsis thaliana is widely used as a model system in molecular plant research; the biological functions of A. thaliana CK2 are well studied in vivo and many of its substrates have been identified. Here, crystal structures of the α subunit of A. thaliana CK2 in three crystal forms and of its complex with the nonhydrolyzable ATP analog AMppNHp are presented. While the C‐lobe of the enzyme is highly rigid, structural plasticity is observed for the N‐lobe. Small but significant displacements within the active cleft are necessary in order to avoid steric clashes with the AMppNHp molecule. Binding of AMppNHp is influenced by a rigid‐body motion of the N‐lobe that was not previously recognized in maize CK2. [ABSTRACT FROM AUTHOR]

Background: Casein kinase II (CK2) is involved in multiple tumor-relevant signaling pathways affecting proliferation and apoptosis. CK2 is frequently upregulated in acute B-lymphoblastic leukemia (B-ALL) and can be targeted by the ATP-competitive CK2 inhibitor CX-4945. While reduced proliferation of tumor entities including B-ALL after CX-4945 incubation has been shown in vitro and in vivo, the detailed way of action is unknown. Here, we investigated the influence on the PI3K/AKT and apoptosis cascades in vivo and in vitro for further clarification.Methods: A B-ALL xenograft model in NSG mice was used to perform in vivo longitudinal bioluminescence imaging during six day CX-4945 treatment. CX-4945 serum levels were determined at various time points. Flow cytometry of bone marrow and spleen cells was performed to analyze CX-4945-induced effects on tumor cell proliferation and distribution in B-ALL engrafted mice. ALL cells were enriched and characterized by targeted RNA sequencing. In vitro, B-ALL cell lines SEM, RS4;11 and NALM-6 were incubated with CX-4945 and gene expression of apoptosis regulators BCL6 and BACH2 was determined.Results: In B-ALL-engrafted mice, overall tumor cell proliferation and distribution was not significantly influenced by CK2 inhibition. CX-4945 was detectable in serum during therapy and serum levels declined rapidly after cessation of CX-4945. While overall proliferation was not affected, early bone marrow and spleen blast frequencies seemed reduced after CK2 inhibition. Gene expression analyses revealed reduced expression of anti-apoptotic oncogene BCL6 in bone marrow blasts of CX-4945-treated animals. Further, BCL6 protein expression decreased in B-ALL cell lines exposed to CX-4945 in vitro. Surprisingly, levels of BCL6 opponent and tumor suppressor BACH2 also declined after prolonged incubation. Simultaneously, increased phosphorylation of direct CK2 target and tumor initiator AKT was detected at respective time points, even in initially pAKT-negative cell line NALM-6.Conclusions: The CK2 inhibitor CX-4945 has limited clinical effects in an in vivo B-ALL xenograft model when applied as a single drug over a six day period. However, gene expression in B-ALL cells was altered and suggested effects on apoptosis via downregulation of BCL6. Unexpectedly, the BCL6 opponent BACH2 was also reduced. Interactions and regulation loops have to be further evaluated. [ABSTRACT FROM AUTHOR]

Phosphorus is one of the most important macronutrients required for plant growth and development. The importance of phosphorylation modification in regulating phosphate (Pi) homeostasis in plants is emerging. We performed phosphoproteomic profiling to characterize proteins whose degree of phosphorylation is altered in response to Pi starvation in rice root. A subset of 554 proteins, including 546 down-phosphorylated and eight up-phosphorylated proteins, exhibited differential phosphorylation in response to Pi starvation. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with the differentially phosphorylated proteins indicated that RNA processing, transport, splicing and translation and carbon metabolism played critical roles in response to Pi starvation in rice. Levels of phosphorylation of four mitogen-activated protein kinases (MAPKs), including OsMAPK6, five calcium-dependent protein kinases (CDPKs) and OsCK2β3 decreased in response to Pi starvation. The decreased phosphorylation level of OsMAPK6 was confirmed by Western blotting. Mutation of OsMAPK6 led to Pi accumulation under Pi-sufficient conditions. Motif analysis indicated that the putative MAPK, casein kinase 2 (CK2) and CDPK substrates represented about 54.4%, 21.5% and 4.7%, respectively, of the proteins exhibiting differential phosphorylation. Based on the motif analysis, 191, 151 and 46 candidate substrates for MAPK, CK2 and CDPK were identified. These results indicate that modification of phosphorylation profiles provides complementary information on Pi-starvation-induced processes, with CK2, MAPK and CDPK protein kinase families playing key roles in these processes in rice. [ABSTRACT FROM AUTHOR]

Lung cancer negatively impacts global health, and the incidence of non-small cell lung cancer (NSCLC) is highest among all forms of lung cancer. Chemotherapy failure mainly occurs due to drug resistance; however, the associated molecular mechanism remains unclear. Casein kinase II (CK2), which plays important roles in the occurrence, development and metastasis of many tumours, regulates Wnt signaling by modulating β-catenin expression. In the present study the effects of the CK2 inhibitor, CX4945 on cisplatin [or cis-diamminedichloroplatinum (II); (DDP)]-resistant A549 cells (A549/DDP) were investigated to elucidate the underlying molecular mechanism. A549/DDP cells were divided into four groups (blank control, CX4945, cisplatin and CX4945+cisplatin). Cisplatin resistance was 5.16-fold greater in A549/DDP cells compared with that in A549 cells, with an optimal cisplatin concentration of 5 µg/ml. Moreover, levels of CK2, dishevelled-2 (DVL-2) phosphorylated (p) at Ser143 (p-DVL-2Ser143), and major Wnt-signaling proteins were significantly higher in A549/DDP cells compared with that in A549 cells (P<0.05), with these levels further increased following cisplatin treatment (P<0.05), whereas these levels significantly decreased in A549 cells after cisplatin treatment (P<0.05). Additionally, multidrug-resistance-associated protein 1 and lung resistance protein expression was significantly higher in A549/DDP cells compared with that in A549 cells (P<0.05), with these levels increasing further in A549/DDP (P<0.05) but not A549 cells upon cisplatin treatment (P>0.05). In addition, reduced expression of resistance proteins in A549/DDP cells was accompanied by a decline in the 50% growth inhibition after CX4945 pre-treatment. Furthermore, levels of p-DVL-2Ser143 and major Wnt-signaling proteins decreased significantly after treatment of A549/DDP cells with CX4945+cisplatin, whereas DVL-2 and p-DVL-2Thr224 levels remained unchanged. Additionally, significant elevations in apoptosis rates in the CX4945+cisplatin group relative to the control and cisplatin-only groups, was observed (P<0.001). These results suggested that inhibiting Wnt/β-catenin signaling with CX4945, which attenuates levels of drug-resistance-associated proteins and induces apoptosis, might reverse cisplatin resistance in NSCLC. [ABSTRACT FROM AUTHOR]

Background/Aims: The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) family. Upon stimulation, these kinases translocate from the cytoplasm to the nucleus, where they induce physiological processes such as proliferation and differentiation. The mechanism of translocation of this kinase involves phosphorylation of two Ser residues within a nuclear translocation signal (NTS), which allows binding to importin7 and a subsequent penetration via nuclear pores. However, the regulation of this process and the protein kinases involved are not yet clear. Methods: To answer this point we developed specific anti phospho-SPS antibody, used this and other antibodies in Western blots and crystalized the phospho-mimetic mutated ERK. Results: Here we show that the phosphorylation of both Ser residues is mediated mainly by casein kinase 2 (CK2) and that active ERK may assist in the phosphorylation of the N-terminal Ser. We also demonstrate that the phosphorylation is dependent on the release of ERK from cytoplasmic anchoring proteins. Crystal structure of the phosphomimetic ERK revealed that the NTS phosphorylation creates an acidic patch in ERK. Our model is that in resting cells ERK is bound to cytoplasmic anchors, which prevent its NTS phosphorylation. Upon stimulation, phosphorylation of the ERK TEY domain releases ERK and allows phosphorylation of its NTS by CK2 and active ERK to generate a negatively charged patch in ERK, binding to importin 7 and nuclear translocation. Conclusion: These results provide an important role of CK2 in regulating nuclear ERK activities. [ABSTRACT FROM AUTHOR]

Dihydrofolate reductase (DHFR) is a prominent molecular target in antitumor, antibacterial, antiprotozoan, and immunosuppressive chemotherapies, and CK2 protein kinase is an ubiquitous enzyme involved in many processes, such as tRNA and rRNA synthesis, apoptosis, cell cycle or oncogenic transformation. We show for the first time that CK2α subunit strongly interacted with and phosphorylated DHFR in vitro. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we determined DHFR-CK2α binding kinetic parameters (K d below 0.5 μM, k on = 10.31 × 104 M−1s−1 and k off = 1.40 × 10−3s−1) and calculated Gibbs free energy (−36.4 kJ/mol). In order to identify phosphorylation site(s) we used site-directed mutagenesis to obtain several DHFR mutants with predicted CK2-phosphorylable serine or threonine residues substituted with alanines. All enzyme forms were subjected to CK2α subunit catalytic activity and the results pointed to serine 168 as a phosphorylation site. Mass spectrometry analyses confirmed the presence of phosphoserine 168 and revealed additionally the presence of phosphoserine 145, although the latter phosphorylation was on a very low level. • Dihydrofolate reductase and CK2 kinase are molecular targets in chemotherapies. • CK2 strongly interacts with and phosphorylates dihydrofolate reductase. • Serines 168 and 145 were identified as CK2 phosphorylation sites. [ABSTRACT FROM AUTHOR]

Highlights • WM components express abundant levels of the CK2α subunit. • CK2 signaling differentially regulates the CDK5 and AKT/GSK3β signaling pathways to mediate WM ischemic injury. • Inhibition of CK2 and the active conformation of AKT provides a common therapeutic target against WM ischemia, independent of age. • Neuronal responses to CK2 signaling during ischemia vary, depending upon the experimental model used. Abstract Strokes occur predominantly in the elderly and white matter (WM) is injured in most strokes, contributing to the disability associated with clinical deficits. Casein kinase 2 (CK2) is expressed in neuronal cells and was reported to be neuroprotective during cerebral ischemia. Recently, we reported that CK2 is abundantly expressed by glial cells and myelin. However, in contrast to its role in cerebral (gray matter) ischemia, CK2 activation during ischemia mediated WM injury via the CDK5 and AKT/GSK3β signaling pathways (Bastian et al., 2018). Subsequently, CK2 inhibition using the small molecule inhibitor CX-4945 correlated with preservation of oligodendrocytes as well as conservation of axon structure and axonal mitochondria, leading to improved functional recovery. Notably, CK2 inhibition promoted WM function when applied before or after ischemic injury by differentially regulating the CDK5 and AKT/GSK3β pathways. Specifically, blockade of the active conformation of AKT conferred post-ischemic protection to young, aging, and old WM, suggesting a common therapeutic target across age groups. CK2 inhibitors are currently being used in clinical trials for cancer patients; therefore, it is important to consider the potential benefits of CK2 inhibitors during an ischemic attack. [ABSTRACT FROM AUTHOR]

A new series of 2-amino-4-phenyl-6-hydroxy-7-alkyl-pyranobenzoquinones was synthesized as ATP-competitive CK2 inhibitors. They were readily synthesized through a three-component Knoevenagel condensation-Michael addition-heterocyclization reaction from aldehydes, malononitrile, and 3-alkyl-2,5-dihydroxybenzoquinones. Some of the synthesized compounds presented interesting inhibitory activity with IC 50 values in the submicromolar range. A structure-activity relationship study was carried out and the mode of binding was analysed by docking studies and supported by ATP competition assays. [ABSTRACT FROM AUTHOR]

We have previously shown that casein kinase 2 (CK2) negatively regulates dopamine D1 and adenosine A2A receptor signaling in the striatum. Ablation of CK2 in D1 receptor-positive striatal neurons caused enhanced locomotion and exploration at baseline, whereas CK2 ablation in D2 receptor-positive neurons caused increased locomotion after treatment with A2A antagonist, caffeine. Because both, D1 and A2A receptors, play major roles in the cellular responses to i-DOPA in the striatum, these findings prompted us to examine the impact of CK2 ablation on the effects of l-DOPA treatment in the unilateral 6-OHDA lesioned mouse model of Parkinson's disease. We report here that knock-out of CK2 in striatonigral neurons reduces the severity of L-DOPA-induced dyskinesia (LID), a finding that correlates with lowered pERK but unchanged pPKA substrate levels in D1 medium spiny neurons as well as in cholinergic interneurons. In contrast, lack of CK2 in striatopallidal neurons enhances LID and ERK phosphorylation. Coadministration of caffeine with a low dose of l-DOPA reduces dyskinesia in animals with striatopallidal knock-out to wild-type levels, suggesting a dependence on adenosine receptor activity. We also detect reduced Golf levels in the striatonigral but not in the striatopallidal knock-out in response to l-DOPA treatment. Our work shows, in a rodent model of PD, that treatment-induced dyskinesia and striatal ERK activation are bidirectionally modulated by ablating CK2 in DI - or D2-positive projection neurons, in male and female mice. The results reveal that CK2 regulates signaling events critical to LID in each of the two main populations of striatal neurons. [ABSTRACT FROM AUTHOR]

Neointimal hyperplasia is a product of VSMC replication and consequent accumulation within the blood vessel wall. In this study, we determined whether inhibition of protein kinase CK2 and the resultant stabilisation of proline-rich homeodomain (PRH) could suppress VSMC proliferation. Both silencing and pharmacological inhibition of CK2 with K66 antagonised replication of isolated VSMCs. SiRNA-induced knockdown as well as ectopic overexpression of proline-rich homeodomain indicated that PRH disrupts cell cycle progression. Mutation of CK2 phosphorylation sites Ser 163 and Ser 177 within the PRH homeodomain enabled prolonged cell cycle arrest by PRH. Concomitant knockdown of PRH and inhibition of CK2 with K66 indicated that the anti-proliferative action of K66 required the presence of PRH. Both K66 and adenovirus-mediated gene transfer of S163C:S177C PRH impaired neointima formation in human saphenous vein organ cultures. Importantly, neither intervention had notable effects on cell cycle progression, cell survival or migration in cultured endothelial cells. [ABSTRACT FROM AUTHOR]

Objective: To find out whether Casein Kinase 2 (CK2) and Survivin co-express and correlate in prostate as well as in breast cancer. Study Design: A cross sectional analytical study. Place and Duration of Study: Study was done at Army Medical College and in collaboration with Armed Forces Institute of Pathology (AFIP). The duration of this research was two years. Material and Methods: The research was authorized by the Ethical Committee of AFIP. CK2 expression was determined by immunohistochemistry in paraffin embedded tissues from established patients of prostate cancer (n=30) and breast adenocarcinoma (n=30). Correlation of CK2 with Survivin was evaluated. Data were analyzed through SPSS version 20. For studying the correlation between the two proteins, pearson correlation coefficient was calculated. Data was considered at p-value =0.05. Results: An expressively strong and affirmative correlation was found among expression of total CK2 and total Survivin in invasive along with non-invasive cases of both prostate and breast cancers. A significantly strong and positive correlation was found between nuclear CK2 and Survivin expression in non-invasive cases of prostate cancer and invasive as well as non-invasive cases of breast cancer. A significantly strong and positive correlation was found only between cytoplasmic CK2 and Survivin expression in non-invasive cases of prostate cancer. Conclusion: CK2 and Survivin expressions have strong and positive correlation in both prostate and breast cancer particularly in non-invasive stage of the cancer. [ABSTRACT FROM AUTHOR]

Casein kinase II (CK2) is an ubiquitous and pleiotropic serine/threonine protein kinase able to phosphorylate hundreds of substrates. Being implicated in several human diseases, from neurodegeneration to cancer, the biological roles of CK2 have been intensively studied. Upregulation of CK2 has been shown to be critical to tumor progression, making this kinase an attractive target for cancer therapy. Several CK2 inhibitors have been developed so far, the first being discovered by "trial and error testing". In the last decade, the development of in silico rational drug design has prompted the discovery, de novo design and optimization of several CK2 inhibitors, active in the low nanomolar range. The screening of big chemical libraries and the optimization of hit compounds by Structure Based Drug Design (SBDD) provide telling examples of a fruitful application of rational drug design to the development of CK2 inhibitors. Ligand Based Drug Design (LBDD) models have been also applied to CK2 drug discovery, however they were mainly focused on methodology improvements rather than being critical for de novo design and optimization. This manuscript provides detailed description of in silico methodologies whose applications to the design and development of CK2 inhibitors proved successful and promising. [ABSTRACT FROM AUTHOR]

Protein kinase CK2, initially designated as casein kinase 2, is an ubiquitously expressed serine/threonine kinase. This enzyme, implicated in many cellular processes, is highly expressed and active in many tumor cells. A large number of compounds has been developed as inhibitors comprising different backbones. Beside others, structures with an indeno[1,2-b]indole scaffold turned out to be potent new leads. With the aim of developing new inhibitors of human protein kinase CK2, we report here on the generation of common feature pharmacophore model to further explain the binding requirements for human CK2 inhibitors. Nine common chemical features of indeno[1,2-b]indole-type CK2 inhibitors were determined using MOE software (Chemical Computing Group, Montreal, Canada). This pharmacophore model was used for database mining with the aim to identify novel scaffolds for developing new potent and selective CK2 inhibitors. Using this strategy several structures were selected by searching inside the ZINC compound database. One of the selected compounds was bikaverin (6,11-dihydroxy-3,8-dimethoxy-1-methylbenzo[b]xanthene-7,10,12-trione), a natural compound which is produced by several kinds of fungi. This compound was tested on human recombinant CK2 and turned out to be an active inhibitor with an IC50 value of 1.24 μM. [ABSTRACT FROM AUTHOR]

CK2 genes are overexpressed in many human cancers, and most often overexpression is associated with worse prognosis. Site-specific expression in mice leads to cancer development (e.g., breast, lymphoma) indicating the oncogenic nature of CK2. CK2 is involved in many key aspects of cancer including inhibition of apoptosis, modulation of signaling pathways, DNA damage response, and cell cycle regulation. A number of CK2 inhibitors are now available and have been shown to have activity against various cancers in vitro and in pre-clinical models. Some of these inhibitors are now undergoing exploration in clinical trials as well. In this review, we will examine some of the major cancers in which CK2 inhibition has promise based on in vitro and pre-clinical studies, the proposed cellular and signaling mechanisms of anti-cancer activity by CK2 inhibitors, and the current or recent clinical trials using CK2 inhibitors. [ABSTRACT FROM AUTHOR]

Protein kinase CK2 has received a surge of attention in recent years due to the evidence of its overexpression in a variety of solid tumors and multiple myelomas as well as its participation in cell survival pathways. CK2 is also upregulated in the most prevalent and aggressive cancer of brain tissue, glioblastoma multiforme, and in preclinical models, pharmacological inhibition of the kinase has proven successful in reducing tumor size and animal mortality. CK2 is highly expressed in the mammalian brain and has many bona fide substrates that are crucial in neuronal or glial homeostasis and signaling processes across synapses. Full and conditional CK2 knockout mice have further elucidated the importance of CK2 in brain development, neuronal activity, and behavior. This review will discuss recent advances in the field that point to CK2 as a regulator of neuronal functions and as a potential novel target to treat neurological and psychiatric disorders. [ABSTRACT FROM AUTHOR]

Compelling evidence indicates that protein kinase CK2 plays an important role in many steps of cancer initiation and progression, therefore, the development of effective and cell-permeable inhibitors targeting this kinase has become an important objective for the treatment of a variety of cancer types including glioblastoma. We have recently identified 1,3-dichloro-6-[(E)-((4-methoxyphenyl)imino)methyl]dibenzo(b,d)furan-2,7-diol (D11) as a potent and selective inhibitor of protein kinase CK2. In this study, we have further characterized this compound and demonstrated that it suppresses CK2 kinase activity by mixed type inhibition (KI 7.7 nM, KI 0 42 nM). Incubation of glioblastoma cells with D11 induces cell death and upon hypoxia the compound leads to HIF-1α destabilization. The analysis of differential mRNA expression related to human hypoxia signaling pathway revealed that D11-mediated inhibition of CK2 caused strong down-regulation of genes associated with the hypoxia response including ANGPTL4, CA9, IGFBP3, MMP9, SLC2A1 and VEGFA. Taken together, the results reported here support the notion that including D11 in future treatment regimens might turn out to be a promising strategy to target tumor hypoxia to overcome resistance to radio- and chemotherapy. [ABSTRACT FROM AUTHOR]

CK2 is a Ser/Thr protein kinase that is highly conserved amongst all eukaryotes. It is a well-known oncogenic kinase that regulates vital cell autonomous functions and animal development. Genetic studies in the fruit fly Drosophila are providing unique insights into the roles of CK2 in cell signaling, embryogenesis, organogenesis, neurogenesis, and the circadian clock, and are revealing hitherto unknown complexities in CK2 functions and regulation. Here, we review Drosophila CK2 with respect to its structure, subunit diversity, potential mechanisms of regulation, developmental abnormalities linked to mutations in the gene encoding CK2 subunits, and emerging roles in multiple aspects of eye development. We examine the Drosophila CK2 "interaction map" and the eye-specific "transcriptome" databases, which raise the prospect that this protein kinase has many additional targets in the developing eye. We discuss the possibility that CK2 functions during early retinal neurogenesis in Drosophila and mammals bear greater similarity than has been recognized, and that this conservation may extend to other developmental programs. Together, these studies underscore the immense power of the Drosophila model organism to provide new insights and avenues to further investigate developmentally relevant targets of this protein kinase. [ABSTRACT FROM AUTHOR]

[Display omitted] Positron emission tomography (PET) is a powerful imaging tool that enables early in vivo detection of Alzheimer's disease (AD). For this purpose, various PET ligands have been developed to image β-amyloid and tau protein aggregates characteristically found in the brain of AD patients. In this study, we initiated to develop another type of PET ligand that targets protein kinase CK2 (formerly termed as casein kinase II), because its expression level is known to be altered in postmortem AD brains. CK2 is a serine/threonine protein kinase, an important component of cellular signaling pathways that control cellular degeneration. In AD, the CK2 level in the brain is thought to be elevated by its involvement in both phosphorylation of proteins such as tau and neuroinflammation. Decreased CK2 activity and expression levels lead to β-amyloid accumulation. In addition, since CK2 also contributes to the phosphorylation of tau protein, the expression level and activity of CK2 is expected to undergo significant changes during the progression of AD pathology. Furthermore, CK2 could act as a potential target for modulating the inflammatory response in AD. Therefore, PET imaging targeting CK2 expressed in the brain could be a useful another imaging biomarker for AD. We synthesized and radiolabeled a CK2 inhibitor, [11C]GO289, in high yields from its precursor and [11C]methyl iodide under basic conditions. On autoradiography, [11C]GO289 specifically bound to CK2 in both rat and human brain sections. On baseline PET imaging, this ligand entered and rapidly washed out of the rat brain with its peak activity rather being small (SUV < 1.0). However, on blocking, there was no detectable CK2 specific binding signal. Thus, [11C]GO289 may be useful in vitro but not so in vivo in its current formulation. The lack of detectable specific binding signal in the latter may be due to a relatively high component of nonspecific binding signal in the overall rather weak PET signal, or it may also be related to the known fact that ATP can competitively binds to subunits of CK2, reducing its availability for this ligand. In the future, it will be necessary for PET imaging of CK2 to try out different non-ATP competitive formulations of CK2 inhibitor that can also provide significantly higher in vivo brain penetration. [ABSTRACT FROM AUTHOR]

As anchoring Casein Kinase 2 (CK2) in several human tumors, DN701 as a novel CK2 inhibitor was applied to reverse chemo-resistance via its antitumor effect synergized with oxaliplatin. Recently, translesion DNA synthesis (TLS) has attracted our attention for its association with chemo-resistance, as demonstrated by previous clinical data. The in vitro cell-based properties supported that oxaliplatin combined with DN701 could reverse drug resistance via blockading CK2-mediated aryl hydrocarbon receptor (AhR) and translesion DNA synthesis (TLS)-induced DNA damage repair. Moreover, pharmacologic or genetic inhibition on REV3L (Protein reversion less 3-like) greatly impaired TLS-induced genomic instability. Mechanistically, combination of oxaliplatin with DN701 was found to inhibit CK2 expression and AhR-TLS-REV3L axis signaling, implying the potential decrease of genomic instability. In addition, the combination of oxaliplatin with DN701 could reduce CK2-AhR-TLS-related genomic instability, leading to potent antitumor effects in vivo. Our study presents an underlying mechanism that DN701 could attenuate tumoral chemo-resistance via decaying CK2-mediated AhR and TLS genomic instability, suggesting a potential cancer chemotherapeutic modality to prolong survival in chemo-resistant patients. Due to the resistance of colon cancer to oxaliplatin, CK2-induced translocation DNA synthesis (TLS) leads to DNA damage repair and the decrease of cell sensitivity. However, oxaliplatin combined with a novel CK2 inhibitor DN701 could suppress CK2-AhR-REV3L axis and TLS-mediated genomic instability, thus inhibiting DNA damage repair and circumventing acquired drug resistance. [Display omitted] • DN701 combined with oxaliplatin displayed superior anti-proliferative cytotoxicity. • The combined treatment reduced activation of CK2-AhR-REV3L axis with tumor suppression. • The combined treatment reduced TLS-mediated genomic instability with cancer inhibition. [ABSTRACT FROM AUTHOR]

Dopamine plays a central role in the regulation of psychomotor functions in the brain. Furthermore, the dopaminergic system is involved in the ictogenesis in human patients and animal models of epilepsy. Dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32) plays an important role in the regulation of interactions between dopamine and glutamate receptors in neurons. Indeed, SKF 83822 (a specific D1 receptor agonist) facilitates DARPP-32-mediated protein phosphatase 1 (PP1) inhibition leading to the increase in phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR), which potentiates channel activities and currents and thereby generates seizure activity. In the present study, we found that pyridoxal-5′-phosphate phosphatase/chronophin (PLPP/CIN), a selective phosphatase for serine (S) residues, attenuated seizure susceptibility in response to SKF 83822 by dephosphorylating DARPP-32 S97 site. Similarly, inhibition of DARPP-32 S97 phosphorylation by 2-[4,5,6,7-Tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazole-1-yl]acetic acid (TMCB; a selective casein kinase 2 inhibitor) attenuated SKF 83822-induced seizure activity. These inhibitory effects of PLPP/CIN and TMCB were relevant to the regulations of DARPP-32-PP1-AMPAR signaling pathway. Therefore, our findings suggest that PLPP/CIN may be a modulator in dopaminergic neurotransmission as well as glutamatergic systems, and that the PLPP/CIN-mediated DARPP-32 regulation may be one of the potential therapeutic targets for medication of seizure or epilepsy induced by D1 receptor hyperactivation. [Display omitted] • PLPP/CIN reduces seizure susceptibility in response to SKF83822. • PLPP/CIN relieves PP1-DARPP-32 binding by dephosphorylating DARPP-32 S97 site. • PLPP/CIN facilitates PP1-mediated GRIA1 S845 dephosphorylation. • CK2 inhibitor reduces seizure susceptibility and its intensity in response to SKF 83822. • CK2 inhibitor decreases SKF 83822-induced DARPP-32 T34 and S97 phosphorylations. [ABSTRACT FROM AUTHOR]

Cellular functions are regulated by signal transduction pathway networks consisting of protein-modifying enzymes that control the activity of many downstream proteins. Protein kinases and phosphatases regulate gene expression by reversible phosphorylation of transcriptional factors, which are their direct substrates. Casein kinase II (CK2) is a serine/threonine kinase that phosphorylates a large number of proteins that have critical roles in cellular proliferation, metabolism and survival. Altered function of CK2 has been associated with malignant transformation, immunological disorders and other types of diseases. Protein phosphatase 1 (PP1) is a serine/threonine phosphatase, which regulates the phosphorylation status of many proteins that are essential for cellular functions. IKAROS is a DNA-binding protein, which functions as a regulator of gene transcription in hematopoietic cells. CK2 directly phosphorylates IKAROS at multiple phosphosites which determines IKAROS activity as a regulator of gene expression. PP1 binds to IKAROS via the PP1-consensus recognition site and dephosphorylates serine/threonine residues that are phosphorylated by CK2. Thus, the interplay between CK2 and PP1 signaling pathways have opposing effects on the phosphorylation status of their mutual substrate – IKAROS. This review summarizes the effects of CK2 and PP1 on IKAROS role in regulation of gene expression and its function as a tumor suppressor in leukemia. [ABSTRACT FROM AUTHOR]