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Simple Summary: Estrogen Receptor α (ERα) is a key target for hormonal treatment of breast cancer. However, treatment with antihormonal therapies such as tamoxifen will eventually fail, presumably through hormone-independent signaling through estrogen receptors. It has been shown that a component of this resistance to hormonal therapy may be through expression of an alternative form of estrogen receptor, ERα36. Here, we show that targeting the enzyme CK2, either with the clinical stage CK2 inhibitor CX-4945, or by disrupting expression of its regulatory β subunit, can result in decreased expression of both the canonical form of ERα protein as well as the ERα36 isoform in tamoxifen-sensitive and tamoxifen-resistant breast cancer cells. Our studies provide a rationale for the clinical investigation of CX-4945 in the treatment of ERα-expressing breast cancers. Aberrant estrogen receptor (ERα) signaling mediates detrimental effects of tamoxifen including drug resistance and endometrial hyperplasia. ERα36, an alternative isoform of ERα, contributes to these effects. We have demonstrated that CK2 modulates ERα expression and function in breast cancer (BCa). Here, we assess if CX-4945 (CX), a clinical stage CK2 inhibitor, can disrupt ERα66 and ERα36 signaling in BCa. Using live cell imaging, we assessed the antiproliferative effects of CX in tamoxifen-sensitive and tamoxifen-resistant BCa cells in monolayer and/or spheroid cultures. CX-induced alterations in ERα66 and ERα36 mRNA and protein expression were assessed by RT-PCR and immunoblot. Co-immunoprecipitation was performed to determine the differential interaction of ERα isoforms with HSP90 and CK2 upon CX exposure. CX caused concentration-dependent decreases in proliferation in tamoxifen-sensitive MCF-7 and tamoxifen-resistant MCF-7 Tam1 cells and significantly repressed spheroid growth in 3D models. Additionally, CX caused dramatic decreases in endogenous or exogenously expressed ERα66 and ERα36 protein. Silencing of CK2β, the regulatory subunit of CK2, resulted in destabilization and decreased proliferation, similar to CX. Co-immunoprecipitation demonstrated that ERα66/36 show CK2 dependance for interaction with molecular chaperone HSP90. Our findings show that CK2 functions regulate the protein stability of ERα66 and ERα36 through a mechanism that is dependent on CK2β subunit and HSP90 chaperone function. CX may be a component of a novel therapeutic strategy that targets both tamoxifen-sensitive and tamoxifen-resistant BCa, providing an additional tool to treat ERα-positive BCa. [ABSTRACT FROM AUTHOR]

Transcription is extremely important for cellular processes but can be hindered by RNA polymerase II (RNAPII) pausing and stalling. Cockayne syndrome protein B (CSB) promotes the progression of paused RNAPII or initiates transcription-coupled nucleotide excision repair (TC-NER) to remove stalled RNAPII. However, the specific mechanism by which CSB initiates TC-NER upon damage remains unclear. In this study, we identified the indispensable role of the ARK2N-CK2 complex in the CSB-mediated initiation of TC-NER. The ARK2N-CK2 complex is recruited to damage sites through CSB and then phosphorylates CSB. Phosphorylation of CSB enhances its binding to stalled RNAPII, prolonging the association of CSB with chromatin and promoting CSA-mediated ubiquitination of stalled RNAPII. Consistent with this finding, Ark2n-/-mice exhibit a phenotype resembling Cockayne syndrome. These findings shed light on the pivotal role of the ARK2N-CK2 complex in governing the fate of RNAPII through CSB, bridging a critical gap necessary for initiating TC-NER. [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]

Casein kinase II (CK2) is an enzyme with pleiotropic kinase activity that catalyzes the phosphorylation of lots of substrates, including STAT3, p53, JAK2, PTEN, RELA, and AKT, leading to the regulation of diabetes, cardiovascular diseases, angiogenesis, and tumor progression. CK2 is observed to have high expression in multiple types of cancer, which is associated with poor prognosis. CK2 holds significant importance in the intricate network of pathways involved in promoting cell proliferation, invasion, migration, apoptosis, and tumor growth by multiple pathways such as JAK2/STAT3, PI3K/AKT, ATF4/p21, and HSP90/Cdc37. In addition to the regulation of cancer progression, increasing evidence suggests that CK2 could regulate tumor immune responses by affecting immune cell activity in the tumor microenvironment resulting in the promotion of tumor immune escape. Therefore, inhibition of CK2 is initially proposed as a pivotal candidate for cancer treatment. In this review, we discussed the role of CK2 in cancer progression and tumor therapy. [ABSTRACT FROM AUTHOR]

Simple Summary: Glioblastoma is the most common malignant brain cancer and is associated with poor prognosis, with an average survival after diagnosis of less than 18 months. Hundreds of clinical trials have been conducted, with more underway to make progress in treating glioblastoma. This review details conventional and emergent targeted therapies for glioblastoma. Glioblastoma is most commonly a primary brain tumor and the utmost malignant one, with a survival rate of approximately 12–18 months. Glioblastoma is highly heterogeneous, demonstrating that different types of cells from the same tumor can manifest distinct gene expression patterns and biological behaviors. Conventional therapies such as temozolomide, radiation, and surgery have limitations. As of now, there is no cure for glioblastoma. Alternative treatment methods to eradicate glioblastoma are discussed in this review, including targeted therapies to PI3K, NFKβ, JAK-STAT, CK2, WNT, NOTCH, Hedgehog, and TGFβ pathways. The highly novel application of oncolytic viruses and nanomaterials in combating glioblastoma are also discussed. Despite scores of clinical trials for glioblastoma, the prognosis remains poor. Progress in breaching the blood–brain barrier with nanomaterials and novel avenues for targeted and combination treatments hold promise for the future development of efficacious glioblastoma therapies. [ABSTRACT FROM AUTHOR]

Regulation of SIRT1 activity is vital to energy homeostasis and plays important roles in many diseases. We previously showed that insulin triggers the epigenetic regulator DBC1 to prime SIRT1 for repression by the multifunctional trafficking protein PACS‐2. Here, we show that liver DBC1/PACS‐2 regulates the diurnal inhibition of SIRT1, which is critically important for insulin‐dependent switch in fuel metabolism from fat to glucose oxidation. We present the x‐ray structure of the DBC1 S1‐like domain that binds SIRT1 and an NMR characterization of how the SIRT1 N‐terminal region engages DBC1. This interaction is inhibited by acetylation of K112 of DBC1 and stimulated by the insulin‐dependent phosphorylation of human SIRT1 at S162 and S172, catalyzed sequentially by CK2 and GSK3, resulting in the PACS‐2‐dependent inhibition of nuclear SIRT1 enzymatic activity and translocation of the deacetylase in the cytoplasm. Finally, we discuss how defects in the DBC1/PACS‐2‐controlled SIRT1 inhibitory pathway are associated with disease, including obesity and non‐alcoholic fatty liver disease. [ABSTRACT FROM AUTHOR]

The endothelin-1 (ET1) peptide has a pathological role in the activation of proliferation, survival and invasiveness pathways in different cancers. ET1's effects rely on its activation by the endothelin-converting enzyme-1 (ECE1), which is expressed as four isoforms, differing only in their cytoplasmic N-terminuses. We already demonstrated in colorectal cancer, glioblastoma, and preliminarily lung cancer, that the isoform ECE1c heightens aggressiveness by promoting cancer stem cell traits. This is achieved through a non-canonical ET1-independent mechanism of enhancement of ECE1c's stability upon CK2-dependent phosphorylation at S18 and S20. Here, a K6 residue is presumably responsible for ECE1c ubiquitination as its mutation to R impairs proteasomal degradation. However, how phosphorylation enhances ECE1c's stability and how this translates into aggressiveness are still open questions. In this brief report, by swapping residues to either phospho-mimetic or phospho-resistant amino acids, we propose that the N-terminus may also be phosphorylated at Y5 and/or T9 by an unknown kinase(s). In addition, N-terminus phosphorylation may lead to a blockage of K6 ubiquitination, increasing ECE1c's stability and presumably activating the Wnt/β-catenin signaling pathway. Thus, a novel CK2/ECE1c partnership may be emerging to promote aggressiveness and thus become a biomarker of poor prognosis and a potential therapeutic target for several cancers. [ABSTRACT FROM AUTHOR]

IntroductionIn classical Hodgkin lymphoma (cHL), the survival of neoplastic cells is mediated by the activation of NF-κB, JAK/STAT and PI3K/Akt signaling pathways. CK2 is a highly conserved serine/threonine kinase, consisting of two catalytic (α) and two regulatory (β) subunits, which is involved in several cellular processes and both subunits were found overexpressed in solid tumors and hematologic malignancies.Methods and resultsBiochemical analyses and in vitro assays showed an impaired expression of CK2 subunits in cHL, with CK2α being overexpressed and a decreased expression of CK2β compared to normal B lymphocytes. Mechanistically, CK2β was found to be ubiquitinated in all HL cell lines and consequently degraded by the proteasome pathway. Furthermore, at basal condition STAT3, NF-kB and AKT are phosphorylated in CK2-related targets, resulting in constitutive pathways activation. The inhibition of CK2 with CX-4945/silmitasertib triggered the de-phosphorylation of NF-κB-S529, STAT3-S727, AKT-S129 and -S473, leading to cHL cell lines apoptosis. Moreover, CX-4945/silmitasertib was able to decrease the expression of the immuno-checkpoint CD274/PD-L1 but not of CD30, and to synergize with monomethyl auristatin E (MMAE), the microtubule inhibitor of brentuximab vedotin.ConclusionsOur data point out a pivotal role of CK2 in the survival and the activation of key signaling pathways in cHL. The skewed expression between CK2α and CK2β has never been reported in other lymphomas and might be specific for cHL. The effects of CK2 inhibition on PD-L1 expression and the synergistic combination of CX-4945/silmitasertib with MMAE pinpoints CK2 as a high-impact target for the development of new therapies for cHL

Aberrant estrogen receptor (ERα) signaling mediates detrimental effects of tamoxifen including drug resistance and endometrial hyperplasia. ERα36, an alternative isoform of ERα, contributes to these effects. We have demonstrated that CK2 modulates ERα expression and function in breast cancer (BCa). Here, we assess if CX-4945 (CX), a clinical stage CK2 inhibitor, can disrupt ERα66 and ERα36 signaling in BCa. Using live cell imaging, we assessed the antiproliferative effects of CX in tamoxifen-sensitive and tamoxifen-resistant BCa cells in monolayer and/or spheroid cultures. CX-induced alterations in ERα66 and ERα36 mRNA and protein expression were assessed by RT-PCR and immunoblot. Co-immunoprecipitation was performed to determine the differential interaction of ERα isoforms with HSP90 and CK2 upon CX exposure. CX caused concentration-dependent decreases in proliferation in tamoxifen-sensitive MCF-7 and tamoxifen-resistant MCF-7 Tam1 cells and significantly repressed spheroid growth in 3D models. Additionally, CX caused dramatic decreases in endogenous or exogenously expressed ERα66 and ERα36 protein. Silencing of CK2β, the regulatory subunit of CK2, resulted in destabilization and decreased proliferation, similar to CX. Co-immunoprecipitation demonstrated that ERα66/36 show CK2 dependance for interaction with molecular chaperone HSP90. Our findings show that CK2 functions regulate the protein stability of ERα66 and ERα36 through a mechanism that is dependent on CK2β subunit and HSP90 chaperone function. CX may be a component of a novel therapeutic strategy that targets both tamoxifen-sensitive and tamoxifen-resistant BCa, providing an additional tool to treat ERα-positive BCa.

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]

MicroRNAs (miRNAs) together with Argonaute (AGO) proteins form the core of the RNA‐induced silencing complex (RISC) to regulate gene expression of their target RNAs post‐transcriptionally. Argonaute proteins are subjected to intensive regulation via various post‐translational modifications that can affect their stability, silencing efficacy and specificity for targeted gene regulation. We report here that in Caenorhabditis elegans, two conserved serine/threonine kinases – casein kinase 1 alpha 1 (CK1A1) and casein kinase 2 (CK2) – regulate a highly conserved phosphorylation cluster of 4 Serine residues (S988:S998) on the miRNA‐specific AGO protein ALG‐1. We show that CK1A1 phosphorylates ALG‐1 at sites S992 and S995, while CK2 phosphorylates ALG‐1 at sites S988 and S998. Furthermore, we demonstrate that phospho‐mimicking mutants of the entire S988:S998 cluster rescue the various developmental defects observed upon depleting CK1A1 and CK2. In humans, we show that CK1A1 also acts as a priming kinase of this cluster on AGO2. Altogether, our data suggest that phosphorylation of AGO within the cluster by CK1A1 and CK2 is required for efficient miRISC‐target RNA binding and silencing. Synopsis: Two conserved casein kinases, casein kinase 1 alpha 1 (CK1A1) and casein kinase 2 (CK2), phosphorylate a highly conserved serine cluster on microRNA‐specific Argonaute proteins. Argonaute phosphorylation by these kinases is essential to regulate microRNA‐mediated gene silencing in Caenorhabditis elegans. CK1A1 and CK2 are involved in the phosphorylation of the highly conserved serine cluster on the miRNA‐specific C. elegans AGO ALG‐1.Phosphorylation of the cluster on ALG‐1 by CK1A1 and CK2 is essential for microRNA function in C. elegans.CK1A1 acts as a priming kinase for this cluster on human AGO2.Phosphorylation of the cluster by CK1A1 and CK2 is required for efficient AGO‐miRISC binding and silencing of mRNA targets. [ABSTRACT FROM AUTHOR]

ABSTRACT The casein kinase 2 (CK2) complex has garnered extensive attention over the past decades as a potential therapeutic target for diverse human diseases, including cancer, diabetes, and obesity, due to its pivotal roles in eukaryotic growth, differentiation, and metabolic homeostasis. While CK2 is also considered a promising antifungal target, its role in fungal pathogens remains unexplored. In this study, we investigated the functions and regulatory mechanisms of the CK2 complex in Cryptococcus neoformans, a major cause of fungal meningitis. The cryptococcal CK2 complex consists of a single catalytic subunit, Cka1, and two regulatory subunits, Ckb1 and Ckb2. Our findings show that Cka1 plays a primary role as a protein kinase, while Ckb1 and Ckb2 have major and minor regulatory functions, respectively, in growth, cell cycle control, morphogenesis, stress response, antifungal drug resistance, and virulence factor production. Interestingly, triple mutants lacking all three subunits (cka1Δ ckb1Δ ckb2Δ) exhibited more severe phenotypic defects than the cka1Δ mutant alone, suggesting that Ckb1/2 may have Cka1-independent functions. In a murine model of systemic cryptococcosis, cka1Δ and cka1Δ ckb1Δ ckb2Δ mutants showed severely reduced virulence. Transcriptomic, proteomic, and phosphoproteomic analyses further revealed that the CK2 complex controls a wide array of effector proteins involved in transcriptional regulation, cell cycle control, nutrient metabolisms, and stress responses. Most notably, CK2 disruption led to dysregulation of key signaling cascades central to C. neoformans pathogenicity, including the Hog1, Mpk1 MAPKs, cAMP/PKA, and calcium/calcineurin signaling pathways. In summary, our study provides novel insights into the multifaceted roles of the fungal CK2 complex and presents a compelling case for targeting it in the development of new antifungal drugs.IMPORTANCEThe casein kinase 2 (CK2) complex, crucial for eukaryotic growth, differentiation, and metabolic regulation, presents a promising therapeutic target for various human diseases, including cancer, diabetes, and obesity. Its potential as an antifungal target is further highlighted in this study, which explores CK2’s functions in C. neoformans, a key fungal meningitis pathogen. The CK2 complex in C. neoformans, comprising the Cka1 catalytic subunit and Ckb1/2 regulatory subunits, is integral to processes like growth, cell cycle, morphogenesis, stress response, drug resistance, and virulence. Our findings of CK2’s role in regulating critical signaling pathways, including Hog1, Mpk1 MAPKs, cAMP/PKA, and calcium/calcineurin, underscore its importance in C. neoformans pathogenicity. This study provides valuable insights into the fungal CK2 complex, reinforcing its potential as a target for novel antifungal drug development and pointing out a promising direction for creating new antifungal agents.

Glioblastoma is most commonly a primary brain tumor and the utmost malignant one, with a survival rate of approximately 12–18 months. Glioblastoma is highly heterogeneous, demonstrating that different types of cells from the same tumor can manifest distinct gene expression patterns and biological behaviors. Conventional therapies such as temozolomide, radiation, and surgery have limitations. As of now, there is no cure for glioblastoma. Alternative treatment methods to eradicate glioblastoma are discussed in this review, including targeted therapies to PI3K, NFKβ, JAK-STAT, CK2, WNT, NOTCH, Hedgehog, and TGFβ pathways. The highly novel application of oncolytic viruses and nanomaterials in combating glioblastoma are also discussed. Despite scores of clinical trials for glioblastoma, the prognosis remains poor. Progress in breaching the blood–brain barrier with nanomaterials and novel avenues for targeted and combination treatments hold promise for the future development of efficacious glioblastoma therapies.

Abstract 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.

Human protein kinase CK2 is an important target for the development of anticancer drugs. Many inhibitors were developed in the last few decades, among them emodin, a natural inhibitor with anthracene-9,10-dione backbone. In this work the inhibitory activity of another natural compound endocrocin having similar chemical structure to emodin was evaluated and turned out to be a moderate inhibitor with IC50 value of 6 µM. Additionally, a series of anthracene-9,10-dione derivatives was synthesized and their inhibitory activity toward the target enzyme was evaluated and compared to the activity of emodin. Only one compound namely 1-amino-6-methylanthracene-9,10-dione (4i) resulted be a potent inhibitor with an IC50 value of 1.45 µM. The rest of derivatives showed moderate or no inhibitory activities when were tested at 10 µM concentration. Additionally, the effect of emodin, endocrocin and compound 4i was evaluated on breast cancer cells (MCF7). Using IncuCyte TM life cell monitoring, cell growth of MCF7 cells was blocked almost completely after addition of 30 µM of emodin, whereas endocrocin was able to reduce cell growth by around 45%. Endocrocin with a concentration of 30 µM was able to reduce the viability of MCF7 cells to around 70% after 48 h of incubation. While only 20% of MCF7 cells were still able to synthesize DNA upon incubation with 100 μM endocrocin for 24 h. On the other hand, compound 4i showed weak effect on MCF7 cell line.

Simple Summary: Acute myeloid leukemia (AML) is an aggressive blood cancer disease that can only be cured by intensive anticancer treatment, and for several patients, allogeneic stem cell transplantation is needed. The protein kinase casein kinase 2 (CK2) is an intracellular signaling molecule that supports cellular growth and survival. This is true both for normal and cancer cells, and the growth- and survival-supporting effects seem to be more important for many cancer cells (including AML cells) than for normal cells. Several pharmacological inhibitors of CK2 have been developed, and this therapeutic strategy is now tried in patients with various malignant diseases. In this article, we review available studies suggesting CK2 inhibition as an effective strategy also for the treatment of AML, either as monotherapy or as a part of drug combinations. The protein kinase CK2 (also known as casein kinase 2) is one of the main contributors to the human phosphoproteome. It is regarded as a possible therapeutic strategy in several malignant diseases, including acute myeloid leukemia (AML), which is an aggressive bone marrow malignancy. CK2 is an important regulator of intracellular signaling in AML cells, especially PI3K–Akt, Jak–Stat, NFκB, Wnt, and DNA repair signaling. High CK2 levels in AML cells at the first time of diagnosis are associated with decreased survival (i.e., increased risk of chemoresistant leukemia relapse) for patients receiving intensive and potentially curative antileukemic therapy. However, it is not known whether these high CK2 levels can be used as an independent prognostic biomarker because this has not been investigated in multivariate analyses. Several CK2 inhibitors have been developed, but CX-4945/silmitasertib is best characterized. This drug has antiproliferative and proapoptotic effects in primary human AML cells. The preliminary results from studies of silmitasertib in the treatment of other malignancies suggest that gastrointestinal and bone marrow toxicities are relatively common. However, clinical AML studies are not available. Taken together, the available experimental and clinical evidence suggests that the possible use of CK2 inhibition in the treatment of AML should be further investigated. [ABSTRACT FROM AUTHOR]

Circadian clocks orchestrate daily rhythms in organismal physiology and behavior to promote optimal performance and fitness. In Drosophila, key pacemaker proteins PERIOD (PER) and TIMELESS (TIM) are progressively phosphorylated to perform phase-specific functions. Whereas PER phosphorylation has been extensively studied, systematic analysis of site-specific TIM phosphorylation is lacking. Here, we identified phosphorylation sites of PER-bound TIM by mass spectrometry, given the importance of TIM as a modulator of PER function in the pacemaker. Among the 12 TIM phosphorylation sites we identified, at least two of them are critical for circadian timekeeping as mutants expressing non-phosphorylatable mutations exhibit altered behavioral rhythms. In particular, we observed that CK2-dependent phosphorylation of TIM(S1404) promotes nuclear accumulation of PER-TIM heterodimers by inhibiting the interaction of TIM and nuclear export component, Exportin 1 (XPO1). We propose that proper level of nuclear PER-TIM accumulation is necessary to facilitate kinase recruitment for the regulation of daily phosphorylation rhythm and phase-specific transcriptional activity of CLOCK (CLK). Our results highlight the contribution of phosphorylation-dependent nuclear export of PER-TIM heterodimers to the maintenance of circadian periodicity and identify a new mechanism by which the negative elements of the circadian clock (PER-TIM) regulate the positive elements (CLK-CYC). Finally, because the molecular phenotype of tim(S1404A) non-phosphorylatable mutant exhibits remarkable similarity to that of a mutation in human timeless that underlies familial advanced sleep phase syndrome (FASPS), our results revealed an unexpected parallel between the functions of Drosophila and human TIM and may provide new insights into the molecular mechanisms underlying human FASPS.

Exposure to ionizing radiation leads to oxidative damages in living cells. NADPH provides the indispensable reducing power to regenerate the reduced glutathione to maintain cellular redox equilibria. In mammalian cells, pentose phosphate pathway (PPP) is the major route to produce NADPH by using glycolytic intermediates, and the rate-limiting step of PPP is controlled by glucose-6-phosphate dehydrogenase (G6PD). Nevertheless, whether G6PD is timely co-opted under ionizing radiation to cope with oxidative stress remains elusive. Here we show that cellular G6PD activity is induced 30 min after ionizing radiation, while its protein expression is mostly unchanged. Mechanistically, casein kinase 2 (CK2) phosphorylates G6PD T145 under ionizing radiation, which consolidates the enzymatic activity of G6PD by facilitating G6PD binding with its substrate NADP+. Further, CK2-dependent G6PD T145 phosphorylation promotes NADPH production, decreases ROS level and supports cell proliferation under ionizing radiation. Our findings report a new anti-oxidative signaling route under ionizing radiation, by which CK2-mediated rapid activation of G6PD orchestrates NADPH synthesis to maintain redox homeostasis, thereby highlighting its potential value in the early treatment of ionizing radiation-induced injuries.

CK2 regulates receptor-mediated mitophagy that removes damaged mitochondria. The PINK1/Parkin pathways also involve mitochondrial clearance through mitophagy. However, it is not clear whether CK2 regulates PINK1/Parkin-dependent mitophagy in response to stress. Rotenone treatment showed a decrease of FUNDC1 expression in the mitochondrial fraction of SH-SY5Y and HeLa cells, but an increase of PINK1/Parkin expression only in SH-SY5Y cells. Interestingly, CK2 inhibition increased mitochondrial LC3II expression in rotenone-treated HeLa cells, whereas it decreased in SH-SY5Y cells, indicating that CK2 mediates rotenone-induced mitophagy in dopaminergic neurons. Furthermore, FUNDC1 expression increased in rotenone-treated SH-SY5Y cells by CK2 inhibition, whereas it decreased in HeLa cells. CK2 inhibition also blocked the increase of Drp1, PINK1 and Parkin translocation into mitochondria and decrease of PGAM5 expression in rotenone-treated SH-SY5Y cells. As expected, rotenone treatment in PGAM5-knockdown cells reduced the expression of PINK1 and Parkin and decrease of LC3II expression. Interestingly, we observed that knockdown of CK2α or PGAM5 induced a further increase in caspase-3 expression. These results suggest that PINK1/Parkin-dependent mitophagy was dominant over FUNDC1 receptor-mediated mitophagy. Collectively, our findings suggest that CK2 can positively induce PINK1/Parkin-dependent mitophagy, and that mitophagy regulates cytoprotective effects by CK2 signaling in dopaminergic neurons. All data generated or analyzed during this study are available upon request. • Rotenone decreases mitochondrial complex I activity, resulting in mitochondrial dysfunction. • Rotenone induces the FUNDC1 receptor-mediated signaling as well as PINK1/Parkin-dependent mitophagy in SH-SY5Y cells. • CK2 mediates PINK1/Parkin-dependent mitophagy though PGAM5 and Drp1. • Mitophagy can regulate rotenone-induced toxicity through CK2 signaling pathway in dopaminergic neurons. [ABSTRACT FROM AUTHOR]

Chromosome 1 open reading frame 109 (C1orf09) is a protein whose expression pattern and biological function in humans, particularly in malignant tumors, have not been explored. In this study, both bioinformatics and immunohistochemical staining revealed that C1orf109 was overexpressed in the cytoplasm of liver cancer cells, and the positive ratio of C1orf109 in liver cancer samples (42.5%, 37/87) was significantly higher than that in normal liver tissues (10%, 3/30, P = 0.0012). C1orf109 expression was correlated with an advanced TNM stage (P = 0.017) and vascular invasion (P = 0.023) and predicted the poor overall survival of patients with liver cancer (P = 0.001). C1orf109 facilitated tumor growth, colony formation, migration, and invasion by activating Wnt signaling by upregulating non-phosphorylated β-catenin and its downstream target genes such as CyclinD1, c-myc, and MMP7. Our results also suggest that C1orf109 interacts and co-localizes with casein kinase II (CK2) to activate Wnt signaling. Treatment with a CK2-specific inhibitor markedly counteracted the increased expression of CyclinD1, c-Myc, and MMP7, as well as the upregulation of tumor proliferation and invasion caused by C1orf109 overexpression. Taken together, our results indicate that C1orf109 accelerates liver cancer cell proliferation and invasion by strengthening the Wnt signaling pathway in a CK2-dependent manner. [ABSTRACT FROM AUTHOR]

Across metazoan animals, the effects of Notch signaling are mediated via the Enhancer of Split (E(spl)/HES) basic Helix–Loop–Helix–Orange (bHLH-O) repressors. Although these repressors are generally conserved, their sequence diversity is, in large part, restricted to the C-terminal domain (CtD), which separates the Orange (O) domain from the penultimate WRPW tetrapeptide motif that binds the obligate co-repressor Groucho. While the kinases CK2 and MAPK target the CtD and regulate Drosophila E(spl)-M8 and mammalian HES6, the generality of this regulation to other E(spl)/HES repressors has remained unknown. To determine the broader impact of phosphorylation on this large family of repressors, we conducted bioinformatics, evolutionary, and biochemical analyses. Our studies identify E(spl)-Mγ as a new target of native CK2 purified from Drosophila embryos, reveal that phosphorylation is specific to CK2 and independent of the regulatory CK2-β subunit, and identify that the site of phosphorylation is juxtaposed to the WRPW motif, a feature unique to and conserved in the Mγ homologues over 50 × 106 years of Drosophila evolution. Thus, a preponderance of E(spl) homologues (four out of seven total) in Drosophila are targets for CK2, and the distinct positioning of the CK2 and MAPK sites raises the prospect that phosphorylation underlies functional diversity of bHLH-O proteins. [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]

Iron scarcity provokes a cellular response consisting of the strong expression of high-affinity systems to optimize iron uptake and mobilization. Aft1 is a primary transcription factor involved in iron homeostasis and controls the expression of high-affinity iron uptake genes in Saccharomyces cerevisiae. Aft1 responds to iron deprivation by translocating from the cytoplasm to the nucleus. Here, we demonstrate that the AGC kinase Ypk1, as well as its upstream regulator TOR Complex 2 (TORC2), are required for proper Aft1 nuclear localization following iron deprivation. We exclude a role for TOR Complex 1 (TORC1) and its downstream effector Sch9, suggesting this response is specific for the TORC2 arm of the TOR pathway. Remarkably, we demonstrate that Aft1 nuclear localization and a robust transcriptional response to iron starvation also require biosynthesis of sphingolipids, including complex sphingolipids such as inositol phosphorylceramide (IPC) and upstream precursors, e.g., long-chain bases (LCBs) and ceramides. Furthermore, we observe the deficiency of Aft1 nuclear localization and impaired transcriptional response in the absence of iron when TORC2-Ypk1 is impaired is partially suppressed by exogenous addition of the LCB dihydrosphingosine (DHS). This latter result is consistent with prior studies linking sphingolipid biosynthesis to TORC2-Ypk1 signaling. Taken together, these results reveal a novel role for sphingolipids, controlled by TORC2-Ypk1, for proper localization and activity of Aft1 in response to iron scarcity. [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]

Large cell lung carcinoma (LCLC) is one form of NSCLC that spreads more aggressively than some other forms, and it represents an unmet medical need. Here, we investigated for the first time the effect of the anti-CK2 CIGB-300 peptide in NCI-H460 cells as an LCLC model. NCI-H460 cells were highly sensitive toward CIGB-300 cytotoxicity, reaching a peak of apoptosis at 6 h. Moreover, CIGB-300 slightly impaired the cell cycle of NCI-H460 cells. The CIGB-300 interactomics profile revealed in more than 300 proteins that many of them participated in biological processes relevant in cancer. Interrogation of the CK2 subunits targeting by CIGB-300 indicated the higher binding of the peptide to the CK2α′ catalytic subunit by in vivo pull-down assays plus immunoblotting analysis and confocal microscopy. The down-regulation of both phosphorylation and protein levels of the ribonuclear protein S6 (RPS6) was observed 48 h post treatment. Altogether, we have found that NCI-H460 cells are the most CIGB-300-sensitive solid tumor cell line described so far, and also, the findings we provide here uncover novel features linked to CK2 targeting by the CIGB-300 anticancer peptide. [ABSTRACT FROM AUTHOR]

As multicellular organisms grow, spatial and temporal patterns of gene expression are strictly regulated to ensure that developmental programs are invoked at appropriate stages. In this work, we describe a putative transcriptional regulator in Arabidopsis, TACO LEAF (TCO), whose overexpression results in the ectopic activation of reproductive genes during vegetative growth. Isolated as an activation-tagged allele, tco-1D displays gene misexpression and phenotypic abnormalities, such as curled leaves and early flowering, characteristic of chromatin regulatory mutants. A role for TCO in this mode of transcriptional regulation is further supported by the subnuclear accumulation patterns of TCO protein and genetic interactions between tco-1D and chromatin modifier mutants. The endogenous expression pattern of TCO and gene misregulation in tco loss-of-function mutants indicate that this factor is involved in seed development. We also demonstrate that specific serine residues of TCO protein are targeted by the ubiquitous kinase CK2. Collectively, these results identify TCO as a novel regulator of gene expression whose activity is likely influenced by phosphorylation, as is the case with many chromatin regulators.

As multicellular organisms grow, spatial and temporal patterns of gene expression are strictly regulated to ensure that developmental programs are invoked at appropriate stages. In this work, we describe a putative transcriptional regulator in Arabidopsis, TACO LEAF (TCO), whose overexpression results in the ectopic activation of reproductive genes during vegetative growth. Isolated as an activation-tagged allele, tco-1D displays gene misexpression and phenotypic abnormalities, such as curled leaves and early flowering, characteristic of chromatin regulatory mutants. A role for TCO in this mode of transcriptional regulation is further supported by the subnuclear accumulation patterns of TCO protein and genetic interactions between tco-1D and chromatin modifier mutants. The endogenous expression pattern of TCO and gene misregulation in tco loss-of-function mutants indicate that this factor is involved in seed development. We also demonstrate that specific serine residues of TCO protein are targeted by the ubiquitous kinase CK2. Collectively, these results identify TCO as a novel regulator of gene expression whose activity is likely influenced by phosphorylation, as is the case with many chromatin regulators.

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]

Osteoporosis (OP) is a bone disorder characterized by decreased bone mineral density (BMD). Bone Morphogenetic Protein-2 (BMP-2) injections are used to promote bone formation in OP patients. However, patients are unresponsive to BMP-2 while displaying an upregulation of BMP Receptor Type 1a (BMPRIa) and protein kinase CK2α (CK2α). A synthetically produced peptide named casein kinase 2.3 (CK2.3) utilizes the BMP-signaling pathway as it enhances osteogenesis of primary osteoblasts isolated from OP patients, whereas BMP-2 does not. Although shown in OP patients, there is currently no reliable mouse model to study BMP-2 and CK2.3 signaling. In this publication, we show that BMPRIa was required for CK2.3-mediated osteogenesis in C2C12 cells with a CRISPR-Cas9-mediated gene knockout for BMPRIa. We utilized the C57BL/6 (B6) mouse strain as an aging-model to study aberrant BMP-2 signaling, demonstrating that, like OP patients, in 15 and 20-month mice, BMP-2 did not increase bone growth and displayed upregulated BMPRIa and CK2α protein expression. Furthermore, CK2.3 enhanced osteogenesis and decreased osteoclastogenesis in all age groups, whereas BMP-2 only increased mineralization in 6-month mice while increasing osteoclast formation in all age groups. These data demonstrated that aging B6 mice were a reliable model and mimicked data obtained from OP patients. [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.

Background & Aims: NADPH oxidase 1 (NOX1) has emerged as a prime regulator of intestinal mucosa immunity and homeostasis. Dysregulation of NOX1 may cause inflammatory bowel disease (IBD). It is not clear how NOX1 is regulated in vivo under inflammatory conditions. We studied the role of CK2 in this process. Methods: The NOX1 organizer subunit, NADPH oxidase organizer 1 (NOXO1), was immunoprecipitated from cytokine-treated colon epithelial cells, and bound proteins were identified by mass spectrometry analysis. Sites on NOXO1 phosphorylated by CK2 were identified by nanoscale liquid chromatography coupled to tandem mass spectrometry. NOX1 activity was determined in colon epithelial cells and colonoids in the presence or absence of CX-4945, a CK2 specific inhibitor. Acute colitis was induced by administration of trinitrobenzenesulfonic acid in mice treated or not with CX-4945. Colon tissues were analyzed by histologic examination, quantitative polymerase chain reaction, and Western blots. CK2 activity, markers of inflammation, and oxidative stress were assessed. Results: We identified CK2 as a major partner of NOXO1 in colon epithelial cells under inflammatory conditions. CK2 directly binds NOXO1 at the C-terminus containing the Phox homology domain and phosphorylates NOXO1 on several sites. CX-4945 increased ROS generation by NOX1 in human colon epithelial cells and organoids. Strikingly, CK2 activity was reduced in trinitrobenzenesulfonic acid–induced acute colitis, and CX-4945 exacerbated colitis inflammation as shown by increased levels of CXCL1, ROS generation, lipid peroxidation, and colon damage. Conclusions: The ubiquitous protein kinase CK2 limits NOX1 activity via NOXO1 binding and phosphorylation in colonic epithelial cells and lessens experimental colitis. Loss of CK2 activity during acute colitis results in excessive ROS production, contributing to the pathogenesis. Strategies to activate CK2 could be an effective novel therapeutic approach in IBD.

The protein kinase CK2 (also known as casein kinase 2) is one of the main contributors to the human phosphoproteome. It is regarded as a possible therapeutic strategy in several malignant diseases, including acute myeloid leukemia (AML), which is an aggressive bone marrow malignancy. CK2 is an important regulator of intracellular signaling in AML cells, especially PI3K–Akt, Jak–Stat, NFκB, Wnt, and DNA repair signaling. High CK2 levels in AML cells at the first time of diagnosis are associated with decreased survival (i.e., increased risk of chemoresistant leukemia relapse) for patients receiving intensive and potentially curative antileukemic therapy. However, it is not known whether these high CK2 levels can be used as an independent prognostic biomarker because this has not been investigated in multivariate analyses. Several CK2 inhibitors have been developed, but CX-4945/silmitasertib is best characterized. This drug has antiproliferative and proapoptotic effects in primary human AML cells. The preliminary results from studies of silmitasertib in the treatment of other malignancies suggest that gastrointestinal and bone marrow toxicities are relatively common. However, clinical AML studies are not available. Taken together, the available experimental and clinical evidence suggests that the possible use of CK2 inhibition in the treatment of AML should be further investigated.

Metazoan development depends on tightly regulated gene expression programs that instruct progenitor cells to adopt specialized fates. Recent work found that posttranslational modifications, such as monoubiquitylation, can determine cell fate also independently of effects on transcription, yet how monoubiquitylation is implemented during development is poorly understood. Here, we have identified a regulatory circuit that controls monoubiquitylation-dependent neural crest specification by the E3 ligase CUL3 and its substrate adaptor KBTBD8. We found that CUL3KBTBD8 monoubiquitylates its essential targets only after these have been phosphorylated in multiple motifs by CK2, a kinase whose levels gradually increase during embryogenesis. Its dependency on multisite phosphorylation allows CUL3KBTBD8 to convert the slow rise in embryonic CK2 into decisive recognition of ubiquitylation substrates, which in turn is essential for neural crest specification. We conclude that multisite dependency of an E3 ligase provides a powerful mechanism for switch-like cell fate transitions controlled by monoubiquitylation.

Casein kinases are involved in a variety of signaling pathways, and also in inflammation, cancer, and neurological diseases. Therefore, they are regarded as potential therapeutic targets for drug design. Recent studies have highlighted the importance of the casein kinase 1 superfamily as well as protein kinase CK2 in the development of several neurodegenerative pathologies, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. CK1 kinases and their closely related tau tubulin kinases as well as CK2 are found to be overexpressed in the mammalian brain. Numerous substrates have been detected which play crucial roles in neuronal and synaptic network functions and activities. The development of new substances for the treatment of these pathologies is in high demand. The impact of these kinases in the progress of neurodegenerative disorders, their bona fide substrates, and numerous natural and synthetic compounds which are able to inhibit CK1, TTBK, and CK2 are discussed in this review.

Positive cofactor 4 (PC4) is a transcriptional coactivator that plays important roles in transcription and DNA replication. In mammals, PC4 is phosphorylated by CK2, and this event downregulates its RNA polymerase II (RNAPII) coactivator function. This work describes the effect of fission yeast PC4 phosphorylation on RNAPII transcription in a cell extract, which closely resembles the cellular context. We found that fission yeast PC4 is strongly phosphorylated by the catalytic subunit of CK2 (Cka1), while the regulatory subunit (Ckb1) downregulates the PC4 phosphorylation. The addition of Cka1 to an in vitro transcription assay can diminish the basal transcription from the Ad-MLP promoter; however, the addition of recombinant fission yeast PC4 or Ckb1 can stimulate the basal transcription in a cell extract. Fission yeast PC4 is phosphorylated in a domain which has consensus phosphorylation sites for CK2, and two serine residues were identified as critical for CK2 phosphorylation. Mutation of one of the serine residues in PC4 does not completely abolish the phosphorylation; however, when the two serine residues are mutated, CK2 is no longer able to phosphorylate PC4. The mutant which is not phosphorylated is able to stimulate transcription even though it is previously phosphorylated by Cka1, while the wild type and the point mutant are inactivated by Cka1 phosphorylation, and they cannot stimulate transcription by RNAPII in cell extracts. Those results demonstrate that CK2 can regulate the coactivator function of fission yeast PC4 and suggests that this event could be important in vivo as well. [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]

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]

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]

Okur-Chung neurodevelopmental syndrome (OCNDS) is an ultra-rare disorder caused by variants in the CSNK2A1 gene. CSNK2A1 encodes for the alpha subunit of casein kinase 2 (CK2), a serine/threonine kinase critical in neural development. CK2 is implicated in many human pathologies, including viral infections, cancer, inflammation, cardiovascular, neurodegenerative, and psychiatric diseases. However, the mechanism of action for the CSNK2A1 variants observed in OCNDS is not fully understood, although studies suggest a loss of function or altered substrate specificity. There are no approved treatments for OCNDS, and current treatments focus on symptom management. The CSNK2A1 Foundation was established in 2018 and aims to find a cure for OCNDS and provide support to affected individuals. OCNDS presents with symptoms at varying severity, including developmental delay/intellectual disabilities, autism, disrupted sleep, speech delays/inability to speak, short stature, and, in ~25% of cases, epilepsy. The foundation has developed a research toolbox that is readily available to researchers worldwide and has awarded ~$1 million in grant funding. These efforts have provided valuable insights into CK2 biology and the natural history of OCNDS. However, additional efforts are needed to fully characterize the disease mechanism and investigate potential treatment interventions. Continued investigation into CK2 and its role in neural development holds promise for a better understanding of OCNDS and related disorders in the future. To accelerate research, we have developed a research roadmap highlighting key focus areas of landscape analysis/toolbox expansion, biomarker development, and therapeutic testing through a series of steps that are nonlinear; we expect these efforts to guide decision-making for therapeutic exploration whether that be drug repurposing, gene therapy, novel drug discovery, or a combination. In this perspective article, we describe OCNDS and the CSNK2A1 gene, highlight gaps in OCNDS research, discuss the research roadmap, and offer the founder's perspective on our growth and future opportunities., Competing Interests: The authors declare that there is no conflict of interest., (© The Author(s), 2024.)

Abstract 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.

Mantle cell lymphoma (MCL) is an incurable B cell non-Hodgkin lymphoma, characterized by frequent relapses. In the last decade, the pro-survival pathways related to BCR signaling and Bcl-2 have been considered rational therapeutic targets in B cell derived lymphomas. The BTK inhibitor Ibrutinib and the Bcl-2 inhibitor Venetoclax are emerging as effective drugs for MCL. However, primary and acquired resistance also to these agents may occur. Protein Kinase CK2 is a S/T kinase overexpressed in many solid and blood-derived tumours. CK2 promotes cancer cell growth and clonal expansion, sustaining pivotal survival signaling cascades, such as the ones dependent on AKT, NF-κB, STAT3 and others, counteracting apoptosis through a “non-oncogene” addiction mechanism. We previously showed that CK2 is overexpressed in MCL and regulates the levels of activating phosphorylation on S529 of the NF-κB family member p65/RelA. In the present study, we investigated the effects of CK2 inactivation on MCL cell proliferation, survival and apoptosis and this kinase’s involvement in the BCR and Bcl-2 related signaling. By employing CK2 loss of function MCL cell models, we demonstrated that CK2 sustains BCR signaling (such as BTK, NF-κB and AKT) and the Bcl-2-related Mcl-1 expression. CK2 inactivation enhanced Ibrutinib and Venetoclax-induced cytotoxicity. The demonstration of a CK2-dependent upregulation of pathways that may antagonize the effect of these drugs may offer a novel strategy to overcome primary and secondary resistance.