SUMOylation Inhibition Enhances Protein Transcription under CMV Promoter: A Lesson from a Study with the F508del-CFTR Mutant.

Cystic fibrosis (CF) is a genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a selective anion channel expressed in the epithelium of various organs. The most frequent mutation is F508del. This mutation leads to a misfolded CFTR protein quickly degraded via ubiquitination in the endoplasmic reticulum. Although preventing ubiquitination stabilizes the protein, functionality is not restored due to impaired plasma membrane transport. However, inhibiting the ubiquitination process can improve the effectiveness of correctors which act as chemical chaperones, facilitating F508del CFTR trafficking to the plasma membrane. Previous studies indicate a crosstalk between SUMOylation and ubiquitination in the regulation of CFTR. In this study, we investigated the potential of inhibiting SUMOylation to increase the effects of correctors and enhance the rescue of the F508del mutant across various cell models. In the widely used CFBE41o-cell line expressing F508del-CFTR, inhibiting SUMOylation substantially boosted F508del expression, thereby increasing the efficacy of correctors. Interestingly, this outcome did not result from enhanced stability of the mutant channel, but rather from augmented cytomegalovirus (CMV) promoter-mediated gene expression of F508del-CFTR. Notably, CFTR regulated by endogenous promoters in multiple cell lines or patient cells was not influenced by SUMOylation inhibitors.

Analysis of the phosphoproteome of CK2α(-/-)/Δα' C2C12 myoblasts compared to the wild-type cells.

CK2 is a Ser/Thr protein kinase composed of two catalytic (α/α') subunits and a non-catalytic ß-subunit dimer, whose activity is often abnormally high in cancer cells. The concept that CK2 may be dispensable for cell survival has been challenged by the finding that viable CK2α/α' knock-out myoblast clones still express small amounts of an N-terminally deleted α' subunit generated during the CRISPR/Cas9 procedure. Here we show that, although the overall CK2 activity of these CK2α(-/-)/Δα' (KO) cells is less than 10% compared to wild-type (WT) cells, the number of phosphosites with the CK2 consensus is comparable to that of WT cells. A more in-depth analysis, however, reveals that the two phosphoproteomes are not superimposable according to a number of criteria, notably a functional analysis of the phosphoproteome found in the two types of cells, and variable sensitivity of the phosphosites to two structurally unrelated CK2 inhibitors. These data support the idea that a minimal CK2 activity, as in KO cells, is sufficient to perform basic housekeeping functions essential for cell survival, but not to accomplish several specialized tasks required upon cell differentiation and transformation. From this standpoint, a controlled downregulation of CK2 would represent a safe and valuable anti-cancer strategy.

KDM2A and KDM3B as Potential Targets for the Rescue of F508del-CFTR.

Cystic fibrosis (CF) is caused by mutations in the gene encoding of the cystic fibrosis transmembrane conductance regulator (CFTR), an anion-selective plasma membrane channel that mainly regulates chloride transport in a variety of epithelia. More than 2000 mutations, most of which presumed to be disease-relevant, have been identified in the CFTR gene. The single CFTR mutation F508del (deletion of phenylalanine in position 508) is present in about 90% of global CF patients in at least one allele. F508del is responsible for the defective folding and processing of CFTR, failing to traffic to the plasma membrane and undergoing premature degradation via the ubiquitin-proteasome system. CFTR is subjected to different post-translational modifications (PTMs), and the possibility to modulate these PTMs has been suggested as a potential therapeutic strategy for the functional recovery of the disease-associated mutants. Recently, the PTM mapping of CFTR has identified some lysine residues that may undergo methylation or ubiquitination, suggesting a competition between these two PTMs. Our work hypothesis moves from the idea that favors methylation over ubiquitination, e.g., inhibiting demethylation could be a successful strategy for preventing the premature degradation of unstable CFTR mutants. Here, by using a siRNA library against all the human demethylases, we identified the enzymes whose downregulation increases F508del-CFTR stability and channel function. Our results show that KDM2A and KDM3B downregulation increases the stability of F508del-CFTR and boosts the functional rescue of the channel induced by CFTR correctors.

Mechanism of CK2 Inhibition by a Ruthenium-Based Polyoxometalate.

CK2 is a Ser/Thr protein kinase involved in many cellular processes such as gene expression, cell cycle progression, cell growth and differentiation, embryogenesis, and apoptosis. Aberrantly high CK2 activity is widely documented in cancer, but the enzyme is also involved in several other pathologies, such as diabetes, inflammation, neurodegeneration, and viral infections, including COVID-19. Over the last years, a large number of small-molecules able to inhibit the CK2 activity have been reported, mostly acting with an ATP-competitive mechanism. Polyoxometalates (POMs), are metal-oxide polyanionic clusters of various structures and dimensions, with unique chemical and physical properties. POMs were identified as nanomolar CK2 inhibitors, but their mechanism of inhibition and CK2 binding site remained elusive. Here, we present the biochemical and biophysical characterizing of the interaction of CK2α with a ruthenium-based polyoxometalate, [Ru4(µ-OH)2(µ-O)4(H2O)4 (γ-SiW10O36)2]10- (Ru4POM), a potent inhibitor of CK2. Using analytical Size-Exclusion Chromatography (SEC), Isothermal Titration Calorimetry (ITC), and SAXS we were able to unravel the mechanism of inhibition of Ru4POM. Ru4POM binds to the positively-charged substrate binding region of the enzyme through electrostatic interactions, triggering the dimerization of the enzyme which consequently is inactivated. Ru4POM is the first non-peptide molecule showing a substrate-competitive mechanism of inhibition for CK2. On the basis of SAXS data, a structural model of the inactivated (CK2α)2(Ru4POM)2 complex is presented.

Targeting the E1 ubiquitin-activating enzyme (UBA1) improves elexacaftor/tezacaftor/ivacaftor efficacy towards F508del and rare misfolded CFTR mutants.

The advent of Trikafta (Kaftrio in Europe) (a triple-combination therapy based on two correctors-elexacaftor/tezacaftor-and the potentiator ivacaftor) has represented a revolution for the treatment of patients with cystic fibrosis (CF) carrying the most common misfolding mutation, F508del-CFTR. This therapy has proved to be of great efficacy in people homozygous for F508del-CFTR and is also useful in individuals with a single F508del allele. Nevertheless, the efficacy of this therapy needs to be improved, especially in light of the extent of its use in patients with rare class II CFTR mutations. Using CFBE41o- cells expressing F508del-CFTR, we provide mechanistic evidence that targeting the E1 ubiquitin-activating enzyme (UBA1) by TAK-243, a small molecule in clinical trials for other diseases, boosts the rescue of F508del-CFTR induced by CFTR correctors. Moreover, TAK-243 significantly increases the F508del-CFTR short-circuit current induced by elexacaftor/tezacaftor/ivacaftor in differentiated human primary airway epithelial cells, a gold standard for the pre-clinical evaluation of patients' responsiveness to pharmacological treatments. This new combinatory approach also leads to an improvement in CFTR conductance on cells expressing other rare CF-causing mutations, including N1303K, for which Trikafta is not approved. These findings show that Trikafta therapy can be improved by the addition of a drug targeting the misfolding detection machinery at the beginning of the ubiquitination cascade and may pave the way for an extension of Trikafta to low/non-responding rare misfolded CFTR mutants.

Targeting CK2 in cancer: a valuable strategy or a waste of time?

CK2 is a protein kinase involved in several human diseases (ranging from neurological and cardiovascular diseases to autoimmune disorders, diabetes, and infections, including COVID-19), but its best-known implications are in cancer, where it is considered a pharmacological target. Several CK2 inhibitors are available and clinical trials are underway in different cancer types. Recently, the suitability of CK2 as a broad anticancer target has been questioned by the finding that a newly developed compound, named SGC-CK2-1, which is more selective than any other known CK2 inhibitor, is poorly effective in reducing cell growth in different cancer lines, prompting the conclusion that the anticancer efficacy of CX-4945, the commonly used clinical-grade CK2 inhibitor, is to be attributed to its off-target effects. Here we perform a detailed scrutiny of published studies on CK2 targeting and a more in-depth analysis of the available data on SGC-CK2-1 vs. CX-4945 efficacy, providing a different perspective about the actual reliance of cancer cells on CK2. Collectively taken, our arguments would indicate that the pretended dispensability of CK2 in cancer is far from having been proved and warn against premature conclusions, which could discourage ongoing investigations on a potentially valuable drug target.

Protein kinase CK2: a potential therapeutic target for diverse human diseases.

CK2 is a constitutively active Ser/Thr protein kinase, which phosphorylates hundreds of substrates, controls several signaling pathways, and is implicated in a plethora of human diseases. Its best documented role is in cancer, where it regulates practically all malignant hallmarks. Other well-known functions of CK2 are in human infections; in particular, several viruses exploit host cell CK2 for their life cycle. Very recently, also SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has been found to enhance CK2 activity and to induce the phosphorylation of several CK2 substrates (either viral and host proteins). CK2 is also considered an emerging target for neurological diseases, inflammation and autoimmune disorders, diverse ophthalmic pathologies, diabetes, and obesity. In addition, CK2 activity has been associated with cardiovascular diseases, as cardiac ischemia-reperfusion injury, atherosclerosis, and cardiac hypertrophy. The hypothesis of considering CK2 inhibition for cystic fibrosis therapies has been also entertained for many years. Moreover, psychiatric disorders and syndromes due to CK2 mutations have been recently identified. On these bases, CK2 is emerging as an increasingly attractive target in various fields of human medicine, with the advantage that several very specific and effective inhibitors are already available. Here, we review the literature on CK2 implication in different human pathologies and evaluate its potential as a pharmacological target in the light of the most recent findings.

How can a traffic light properly work if it is always green? The paradox of CK2 signaling.

CK2 is a constitutively active protein kinase that assuring a constant level of phosphorylation to its numerous substrates supports many of the most important biological functions. Nevertheless, its activity has to be controlled and adjusted in order to cope with the varying needs of a cell, and several examples of a fine-tune regulation of its activity have been described. More importantly, aberrant regulation of this enzyme may have pathological consequences, e.g. in cancer, chronic inflammation, neurodegeneration, and viral infection. Our review aims at summarizing our current knowledge about CK2 regulation. In the first part, we have considered the most important stimuli shown to affect protein kinase CK2 activity/expression. In the second part, we focus on the molecular mechanisms by which CK2 can be regulated, discussing controversial aspects and future perspectives.

A mutational approach to dissect the functional role of the putative CFTR "PTM-CODE".

Deletion of Phe at position 508 (F508del) in CFTR is the commonest cause of Cystic Fibrosis; this mutation affects the fate of the protein, since most of the F508del-CFTR is retained in the endoplasmic reticulum, ubiquitylated and degraded. CFTR is subjected to different post-translational modifications (PTMs) and the possibility to modulate these PTMs has been suggested as a potential therapeutic strategy for the functional recovery of F508del-CFTR. Recently, it has been suggested the presence of a PTM signature (phosphorylation, methylation and ubiquitylation) in the regulatory insertion element of the CFTR, named PTM-code, which is associated with CFTR maturation and F508del-CFTR recovery. However, the real contribution of these PTMs is still to be deciphered. Here, by using a mutational approach, we show that the PTM-code is dispensable for the functional recovery of F508del-CFTR and therefore its regulation would not be essential in the light of a therapeutical approach.

Protein kinase CK2 inhibition as a pharmacological strategy.

CK2 is a constitutively active Ser/Thr protein kinase which phosphorylates hundreds of substrates. Since they are primarily related to survival and proliferation pathways, the best-known pathological roles of CK2 are in cancer, where its targeting is currently being considered as a possible therapy. However, CK2 activity has been found instrumental in many other human pathologies, and its inhibition will expectably be extended to different purposes in the near future. Here, after a description of CK2 features and implications in diseases, we analyze the different inhibitors and strategies available to target CK2, and update the results so far obtained by their in vivo application.

Comparing the efficacy and selectivity of Ck2 inhibitors. A phosphoproteomics approach.

CK2 (an acronym derived from the misnomer "casein kinase 2") denotes a ubiquitous, highly pleiotropic protein kinase which has been implicated in global human pathologies, with special reference to cancer. A large spectrum of fairly selective, cell permeable CK2 inhibitors are available, one of which, CX4945 is already in clinical trials for the treatment of neoplasia. Another recently developed CK2 inhibitor, GO289, displays in vitro potency and selectivity comparable to CX4945. Here the cellular efficiency of these two inhibitors has been evaluated by treating C2C12 myoblasts for 5 h with each of them at 4 µM concentration and running a quantitative phosphoproteomics analysis of phosphosites affected by the two compounds. A small but significant proportion of the quantified phosphosites is decreased by treatment with CX4945 and, even more with GO289. This figure substantially increases if a subset of quantified phosphosites conforming to the CK2 consensus (pS/pT-x-x-D/E/pS/pT) is considered. Also in this case GO289 is more effective than CX4945. By adopting stringent criteria two shortlists of 70 and 35 sites whose phosphorylation is decreased >50% by GO289 and CX4945, respectively, have been generated. All these phosphosites conform to the consensus of CK2 with just sporadic exceptions. Their WebLogos are indistinguishable from that of bona fide CK2 phosphosites and their Two-Sample Logos rule out any significant contribution of Pro-directed and basophilic protein kinases to their generation. To sum up, we can conclude that by treating C2C12 cells for 5 h with either CX4945 or GO289 off-target effects are negligible since almost all the phosphosites undergoing a substantial reduction are attributable to CK2, with a higher inhibitory efficacy displayed by GO289. CX4945 and GO289 provide highly selective tools to control the CK2-dependent phosphoproteome compared with previously developed CK2 inhibitors.

Contribution of the CK2 Catalytic Isoforms α and α' to the Glycolytic Phenotype of Tumor Cells.

CK2 is a Ser/Thr protein kinase overexpressed in many cancers. It is usually present in cells as a tetrameric enzyme, composed of two catalytic (α or α') and two regulatory (ß) subunits, but it is active also in its monomeric form, and the specific role of the different isoforms is largely unknown. CK2 phosphorylates several substrates related to the uncontrolled proliferation, motility, and survival of cancer cells. As a consequence, tumor cells are addicted to CK2, relying on its activity more than healthy cells for their life, and exploiting it for developing multiple oncological hallmarks. However, little is known about CK2 contribution to the metabolic rewiring of cancer cells. With this study we aimed at shedding some light on it, especially focusing on the CK2 role in the glycolytic onco-phenotype. By analyzing neuroblastoma and osteosarcoma cell lines depleted of either one (α) or the other (α') CK2 catalytic subunit, we also aimed at disclosing possible pro-tumor functions which are specific of a CK2 isoform. Our results suggest that both CK2 α and α' contribute to cell proliferation, survival and tumorigenicity. The analyzed metabolic features disclosed a role of CK2 in tumor metabolism, and suggest prominent functions for CK2 α isoform. Results were also confirmed by CK2 pharmacological inhibition. Overall, our study provides new information on the mechanism of cancer cells addiction to CK2 and on its isoform-specific functions, with fundamental implications for improving future therapeutic strategies based on CK2 targeting.

White Adipose Tissue Expansion in Multiple Symmetric Lipomatosis Is Associated with Upregulation of CK2, AKT and ERK1/2.

Multiple symmetric lipomatosis (MSL) is a rare disorder characterized by overgrowing lipomatous tissue (LT) in the subcutaneous adipose tissue (SAT). What LT is and how it expands are not completely understood; previous data suggested that it could derive from brown AT precursors. In six MSL type I patients, we compared LT morphology by histological and immunohistochemistry (IHC) analysis, gene expression, by qPCR, kinase activity, by Western Blot and in vitro assay to paired-control SAT using AT from patients with pheochromocytoma as a human browning reference. In the stromal vascular fraction (SVF), we quantified adipose stem cells (ASCs) by flow cytometry, the proliferation rate, white and beige adipogenic potential and clonogenicity and adipogenicity by a limiting dilution assay. LT displayed white AT morphology and expression pattern and did not show increased levels of the brown-specific marker UCP1. In LT, we evidenced AKT, CK2 and ERK1/2 hyperactivation. LT-SVF contained increased ASCs, proliferated faster, sprouted clones and differentiated into adipocytes better than the control, displaying enhanced white adipogenic potential but not increased browning compared to SAT. In conclusion, LT is a white AT depot expanding by hyperplasia through increased stemness and enhanced white adipogenesis upregulating AKT, CK2 and ERK1/2, which could represent new targets to counteract MSL.

Role of CK2 inhibitor CX-4945 in anti-cancer combination therapy - potential clinical relevance.

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.

"Janus" efficacy of CX-5011: CK2 inhibition and methuosis induction by independent mechanisms.

Methuosis has been described as a distinctive form of cell death characterized by the displacement of large fluid-filled vacuoles derived from uncontrolled macropinocytosis. Its induction has been proposed as a new strategy against cancer cells. Small molecules, such as indole-based calchones, have been identified as methuosis inducers and, recently, the CK2 inhibitor CX-4945 has been shown to have a similar effect on different cell types. However, the contribution of protein kinase CK2 to methuosis signalling is still controversial. Here we show that methuosis is not related to CK2 activity since it is not affected by structurally unrelated CK2 inhibitors and genetic reduction/ablation of CK2 subunits. Interestingly, CX-5011, a CK2 inhibitor related to CX-4945, behaves as a CK2-independent methuosis inducer, four times more powerful than its parental compound and capable to promote the formation on enlarged cytosolic vacuoles at low micromolar concentrations. We show that pharmacological inhibition of the small GTPase Rac-1, its downregulation by siRNA treatment, or the over-expression of the dominant-negative mutated form of Rac-1 (Rac-1 T17N), impairs CX-5011 ability to induce methuosis. Furthermore, cell treatment with CX-5011 induces a durable activation of Rac-1 that persists for at least 24 h. Worthy of note, CX-5011 is able to promote macropinocytosis not only in mammalian cells, but also in an in-vivo zebrafish model. Based on these evidences, CX-5011 is, therefore, proposed as a potential promising compound for cancer therapies for its dual efficacy as an inhibitor of the pro-survival kinase CK2 and inducer of methuosis.

A N-terminally deleted form of the CK2α' catalytic subunit is sufficient to support cell viability.

Viable clones of C2C12 myoblasts where both catalytic subunits of protein kinase CK2 had been knocked out by the CRISPR/Cas9 methodology have recently been generated, thus challenging the concept that CK2 is essential for cell viability. Here we present evidence that these cells are still endowed with a residual "CK2-like" activity that is able to phosphorylate Ser-13 of endogenous CDC37. Searching for a molecular entity accounting for such an activity we have identified a band running slightly ahead of CK2α' on SDS-PAGE. This band is not detectable by in-gel casein kinase assay but it co-immuno-precipitates with the ß-subunit being downregulated by specific CK2α' targeting siRNA treatment. Its size and biochemical properties are consistent with those of CK2α' mutants deleted upstream of Glu-15 generated during the knockout process. This mutant sheds light on the role of the CK2 N-terminal segment as a regulator of activity and stability. Comparable cytotoxic efficacy of two selective and structurally unrelated CK2 inhibitors support the view that survival of CK2α/α'-/- cells relies on this deleted form of CK2α', whose discovery provides novel perspectives about the biological role of CK2.

Effects of CK2ß subunit down-regulation on Akt signalling in HK-2 renal cells.

The PI3K/Akt pathway is interconnected to protein kinase CK2, which directly phosphorylates Akt1 at S129. We have previously found that, in HK-2 renal cells, downregulation of the CK2 regulatory subunit ß (shCK2ß cells) reduces S129 Akt phosphorylation. Here, we investigated in more details how the different CK2 isoforms impact on Akt and other signaling pathways. We found that all CK2 isoforms phosphorylate S129 in vitro, independently of CK2ß. However, in HK-2 cells the dependence on CK2ß was confirmed by rescue experiments (CK2ß re-expression in shCK2ß HK-2 cells), suggesting the presence of additional components that drive Akt recognition by CK2 in cells. We also found that CK2ß downregulation altered the phosphorylation ratio between the two canonical Akt activation sites (pT308 strongly reduced, pS473 slightly increased) in HK-2 cells. Similar results were found in other cell lines where CK2ß was stably knocked out by CRISPR-Cas9 technology. The phosphorylation of rpS6 S235/S236, a downstream effector of Akt, was strongly reduced in shCK2ß HK-2 cells, while the phosphorylation of two Akt direct targets, PRAS40 T246 and GSK3ß S9, was increased. Differently to what observed in response to CK2ß down-regulation, the chemical inhibition of CK2 activity by cell treatment with the specific inhibitor CX-4945 reduced both the Akt canonical sites, pT308 and pS473. In CX-4945-treated cells, the changes in rpS6 pS235/S236 and GSK3ß pS9 mirrored those induced by CK2ß knock-down (reduction and slight increase, respectively); on the contrary, the effect on PRAS40 pT246 phosphorylation was sharply different, being strongly reduced by CK2 inhibition; this suggests that this Akt target might be dependent on Akt pS473 status in HK-2 cells. Since PI3K/Akt and ERK1/2/p90rsk pathways are known to be interconnected and both modulated by CK2, with GSK3ß pS9 representing a convergent point, we investigated if ERK1/2/p90rsk signaling was affected by CK2ß knock-down and CX-4945 treatment in HK-2 cells. We found that p90rsk was insensitive to any kind of CK2 targeting; therefore, the observation that, similarly, GSK3ß pS9 was not reduced by CK2 blockade suggests that GSK3ß phosphorylation is mainly under the control of p90rsk in these cells. However, we found that the PI3K inhibitor LY294002 reduced GSK3ß pS9, and concomitantly decreased Snail1 levels (a GSK3ß target and Epithelial-to-Mesenchymal transition marker). The effects of LY294002 were observed also in CK2ß-downregulated cells, suggesting that reducing GSK3ß pS9 could be a strategy to control Snail1 levels in any situation where CK2ß is defective, as possibly occurring in cancer cells.

Deciphering the role of protein kinase CK2 in the maturation/stability of F508del-CFTR.

F508del-CFTR, the most common mutation in cystic fibrosis (CF) patients, impairs CFTR trafficking to plasma membrane leading to its premature proteasomal degradation. Several post-translational modifications have been identified on CFTR with multiple roles in stability, localization and channel function, and the possibility to control the enzymes responsible of these modifications has been long considered a potential therapeutic strategy. Protein kinase CK2 has been previously suggested as an important player in regulating CFTR functions and it has been proposed as a pharmacological target in a combinatory therapy to treat CF patients. However, the real implication of CK2 in F508del-CFTR proteostasis, and in particular the hypothesis that its inhibition could be important in CF therapies, is still elusive. Here, by using immortalized cell lines, primary human cells, and knockout cell lines deprived of CK2 subunits, we do not disclose any direct correlation between F508del-CFTR proteostasis and CK2 expression/activity. Rather, our data indicate that the CK2α' catalytic subunit should be preserved rather than inhibited for F508del rescue by the correctors of class-1, such as VX-809, disclosing new important features in CF therapeutic approaches.

Protein Kinase CK2 Subunits Differentially Perturb the Adhesion and Migration of GN11 Cells: A Model of Immature Migrating Neurons.

Protein kinase CK2 (CK2) is a highly conserved and ubiquitous kinase is involved in crucial biological processes, including proliferation, migration, and differentiation. CK2 holoenzyme is a tetramer composed by two catalytically active (α/α') and two regulatory (ß) subunits and exerts its function on a broad range of targets. In the brain, it regulates different steps of neurodevelopment, such as neural differentiation, neuritogenesis, and synaptic plasticity. Interestingly, CK2 mutations have been recently linked to neurodevelopmental disorders; however, the functional requirements of the individual CK2 subunits in neurodevelopment have not been yet investigated. Here, we disclose the role of CK2 on the migration and adhesion properties of GN11 cells, an established model of mouse immortalized neurons, by different in vitro experimental approaches. Specifically, the cellular requirement of this kinase has been assessed pharmacologically and genetically by exploiting CK2 inhibitors and by generating subunit-specific CK2 knockout GN11 cells (with a CRISPR/Cas9-based approach). We show that CK2α' subunit has a primary role in increasing cell adhesion and reducing migration properties of GN11 cells by activating the Akt-GSK3ß axis, whereas CK2α subunit is dispensable. Further, the knockout of the CK2ß regulatory subunits counteracts cell migration, inducing dramatic alterations in the cytoskeleton not observed in CK2α' knockout cells. Collectively taken, our data support the view that the individual subunits of CK2 play different roles in cell migration and adhesion properties of GN11 cells, supporting independent roles of the different subunits in these processes.

The protein kinase CK2 contributes to the malignant phenotype of cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) is a particularly aggressive hepatobiliary malignancy, for which the molecular mechanisms underlying the malignant phenotype are still poorly understood, and novel and effective therapeutic strategies are limited. The pro-survival protein kinase CK2 is frequently overexpressed in cancer and is receiving increasing interest as an anti-tumor drug target. Its precise role in CCA biology is still largely unknown. Here we show that expression of the CK2α and α' catalytic subunits and of the ß regulatory subunit is increased in human CCA samples. Increased expression of CK2 subunits was shown in CCA cell lines compared to non-transformed cholangiocytes. We used chemical inhibition of CK2 and genetic modification by CRISPR/Cas9 to explore the contribution of CK2 to the malignant phenotype of CCA cells. Disruption of CK2 activity results in cell death through apoptosis, reduced invasion and migration potential, and G0/G1 cell cycle arrest. Importantly, CCA cells with a reduced CK2 activity are more sensitive to chemotherapy. Altogether, our results demonstrate that CK2 significantly contributes to increased proliferative potential and augmented growth of CCA cells and indicate the rationale for its targeting as a promising pharmacologic strategy for cholangiocarcinoma.