Protein Kinase CK2 Activity Research Articles

Influence of CK2 protein kinase activity on the interaction between Trypanosoma cruzi and its vertebrate and invertebrate hosts.

Chagas disease, endemic from Latin America, is caused by Trypanosoma cruzi and is transmitted by triatomine feces. This parasite undergoes complex morphological changes through its life cycle, promoted by significant changes in signal transduction pathways. The activity of protein kinase CK2 has been described in trypanosomatids. Using a specific peptide and radioactive ATP, we identified CK2 activity on the cellular surface and the cytoplasmic content in Trypanosoma cruzi, apart from the secreted form. Dephosphorylated casein promoted an increase of 48% in the secreted CK2 activity. Total extract of peritoneal macrophages from BALB/c and inactivated human serum promoted an increase of 67% and 36%, respectively, in this activity. The protein secreted by parasites was purified by HPLC and had shown compatibility with the catalytic subunit of mammalian CK2. Incubation of the parasites with CK2 inhibitors, added to the culture medium, prevented their growth. The opposite was observed when CK2 activators were used. Results of interaction between Trypanosoma cruzi and the gut of the vector have revealed that, in the presence of CK2 inhibitors, there is a reduction in the association rate. A similar inhibition profile was seen in the Trypanosoma cruzi-macrophages interaction, confirming the importance of this enzyme in the life cycle of this protozoan.

Abstract 4004: Targeting drug-resistant acute myeloid leukemia (AML) cells using novel casein kinase II (CK2) inhibitor

Abstract Introduction: Protein Kinase CK2 level and activity are high in Acute Myeloid Leukemia (AML) stem cells. Genetic inhibition of CK2 promotes apoptosis and shows synergistic cytotoxic activity with cytotoxic therapy. Here we report the anti-leukemia efficacy of a novel and potent small molecule inhibitor of CK2, BMS-135, in AML mouse models. Methods: AML cell lines (n=8) and primary AML cells (n=3) representing various AML genetic subtypes (MLL rearranged, FLT3-ITD, TP53 mutation) were tested in vitro. Cells were treated with serial dilution of BMS-135 for 24 to 48hrs, and cell viability was measured using calorimetric cell viability (WST) assay. Similarly, treated cells were analyzed for cell cycle distribution, apoptosis, and colony formation. We measured the mRNA and protein levels of known CK2 targets. RNA sequencing and gene expression analysis was done to evaluate gene expression changes following treatment with BMS-135. Cell line-derived xenograft and patient-derived xenograft of AML were treated with BMS-135 at a dose of 7.5mg/kg oral gavage twice daily for 21 days or vehicle. Results: Combined in vitro and in vivo experiments establish the efficacy of BMS-135. Cytotoxicity at inhibitory concentrations of 30-800nM, G0-G1 cell cycle arrest, increased apoptosis, and poor colony formation following treatment were noted consistently in all cells. Single-drug treatment with BMS-135 achieved 50-80% leukemia inhibition and significantly prolonged survival in treated mice after 3 weeks of therapy. Following in vivo treatment with BMS-135, we confirmed the inhibition of known CK2 targets (AKT, PI3K, Bcl-xL) in the bone marrow and spleen AML cells. No significant myelosuppression or organ toxicity was noted in tumor-bearing mice following 3 weeks of treatment when compared to vehicle-treated mice. BMS-135 shows synergistic cytotoxic activity with daunorubicin and cytarabine. Conclusions: Selective inhibitor of CK2 is well tolerated and shows superior in vivo efficacy and target inhibition in a series of AML cells and xenografts. BMS-135 works synergistically with cytotoxic agents like daunorubicin and cytarabine. These results support further pre-clinical characterization and clinical development of BMS-135 for treating AML in combination with cytotoxic therapy. Citation Format: Koby Duke, Katherine Mercer, Rajesh Rajaiah, Muhammad Daniyal, Yi Qiu, Ashok Purandare, Yasin Uzun, Lijun Zhang, Morgann Klink, Chandrika Gowda. Targeting drug-resistant acute myeloid leukemia (AML) cells using novel casein kinase II (CK2) inhibitor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4004.

CX-4945 and siRNA-Mediated Knockdown of CK2 Improves Cisplatin Response in HPV(+) and HPV(-) HNSCC Cell Lines.

Head and neck squamous cell carcinoma (HNSCC) can be categorized into human papillomavirus (HPV) positive or negative disease. Elevated protein kinase CK2 level and activity have been historically observed in HNSCC cells. Previous studies on CK2 in HNSCC did not generally include consideration of HPV(+) and HPV(−) status. Here, we investigated the response of HPV(+) and HPV(−) HNSCC cells to CK2 targeting using CX-4945 or siRNA downregulation combined with cisplatin treatment. HNSCC cell lines were examined for CK2 expression levels and activity and response to CX-4945, with and without cisplatin. CK2 levels and NFκB p65-related activity were high in HPV(+) HNSCC cells relative to HPV(−) HNSCC cells. Treatment with CX-4945 decreased viability and cisplatin IC50 in all cell lines. Targeting of CK2 increased tumor suppressor protein levels for p21 and PDCD4 in most instances. Further study is needed to understand the role of CK2 in HPV(+) and HPV(−) HNSCC and to determine how incorporation of the CK2-targeted inhibitor CX-4945 could improve cisplatin response in HNSCC.

Lysophosphatidylcholine triggers cell differentiation in the protozoan parasite Herpetomonas samuelpessoai through the CK2 pathway.

Protozoa are distantly related to vertebrates but present some features of higher eukaryotes, making them good model systems for studying the evolution of basic processes such as the cell cycle. Herpetomonas samuelpessoai is a trypanosomatid parasite isolated from the hemipteran insect Zelus leucogrammus. Lysophosphatidylcholine (LPC) is implicated in the transmission and establishment of Chagas disease, whose etiological agent is Trypanosoma cruzi. LPC is synthesized by T. cruzi and its vectors, the hemipteran Rhodnius prolixus and Triatoma infestans. Platelet-activating factor (PAF), a phospholipid with potent and diverse physiological and pathophysiological actions, is a powerful inducer of cell differentiation in Herpetomonas muscarum muscarum and T. cruzi. The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the 2-ester bond of 3-sn-phosphoglyceride, transforming phosphatidylcholine (PC) into LPC. In this study, we evaluated cellular differentiation, PLA2 activity and protein kinase CK2 activity of H. samuelpessoai in the absence and in the presence of LPC and PAF. We demonstrate that both PC and LPC promoted a twofold increase in the cellular differentiation of H. samuelpessoai, through CK2, with a concomitant inhibition of its cell growth. Intrinsic PLA2 most likely directs this process by converting PC into LPC. Our results suggest that the actions of LPC on H. samuelpessoai occur upon binding to a putative PAF receptor and that the protein kinase CK2 plays a major role in this process. Cartoon depicting a model for the synthesis and functions of LPC in Herpetomonas samuelpessoai, based upon our results regarding the role of LPC on the cell biology of Trypanosoma cruzi [28-32]. N nucleus, k kinetoplast, PC phosphatidylcholine, LPC lysophosphatidylcholine, PLA2 phospholipase A2, PAFR putative PAF receptor in trypanosomatids [65], CK2 protein kinase CK2 [16].

The effect of ionizing radiation on the subcellular localization and kinase activity of protein kinase CK2 in human non-small cell lung cancer cells

Protein kinase CK2 is a ubiquitously expressed kinase in eukaryotes, which is known to phosphorylate many protein substrates. Because CK2 is involved in the regulation of various signaling pathways, we wondered whether CK2 participated in the regulation of ionizing radiation (IR) induced biological process. In this study, we investigated the effect of IR on the subcellular localization and kinase activity in human non-small cell lung cancer (NSCLC) cells. Immunofluorescent results showed that CK2 subunits shuttle into the nucleus mostly beginning 1 h after IR and lasting more than 6 h. We also conducted in vitro kinase assay and observed an increase in CK2 kinase activity at 6 h after IR. Furthermore, an increase in S phase was observed at 6 h after IR. Colony formation assay results demonstrated that CK2 inhibitor CX-4945 significantly enhanced the effect of irradiation in NSCLC cells. These results indicated that CK2 may be implicated in the regulation of IR-induced biological process.

Non-mitogenic FGF2 protects cardiomyocytes from acute doxorubicin-induced toxicity independently of\xa0the protein kinase CK2/heme oxygenase-1 pathway

Doxorubicin (Dox)-induced cardiotoxicity, a limiting factor in the use of Dox to treat cancer, can be mitigated by the mitogenic factor FGF2 in vitro, via a heme oxygenase 1 (HO-1)-dependent pathway. HO-1 upregulation was reported to require protein kinase CK2 activity. We show that a mutant non-mitogenic FGF2 (S117A-FGF2), which does not activate CK2, is cardioprotective against acute cardiac ischemic injury. We now investigate the potential of S117A-FGF2 to protect cardiomyocytes against acute Dox injury and decrease Dox-induced upregulation of oxidized phospholipids. The roles of CK2 and HO-1 in cardiomyocyte protection are also addressed.Rat neonatal cardiomyocyte cultures were used as an established in vitro model of acute Dox toxicity. Pretreatment with S117A-FGF2 protected against Dox-induced: oxidative stress; upregulation of fragmented and non-fragmented oxidized phosphatidylcholine species, measured by LC/MS/MS; and cardiomyocyte injury and cell death measured by LDH release and a live-dead assay. CK2 inhibitors (TBB and Ellagic acid), did not affect protection by S117A-FGF2 but prevented protection by mitogenic FGF2. Furthermore, protection by S117A-FGF2, unlike that of FGF2, was not prevented by HO-1 inhibitors and S117A-FGF2 did not upregulate HO-1. Protection by S117A-FGF2 required the activity of FGF receptor 1 and ERK.We conclude that mitogenic and non-mitogenic FGF2 protect from acute Dox toxicity by common (FGFR1) and distinct, CK2/HO-1- dependent or CK2/HO-1-independent (respectively), pathways. Non-mitogenic FGF2 merits further consideration as a preventative treatment against Dox cardiotoxicity.

CK2 and PI3K are direct molecular targets of quercetin in chronic lymphocytic leukaemia.

Despite the encouraging results of the innovative therapeutic treatments, complete remission is uncommon in patients affected by chronic lymphocytic leukaemia, which remains an essentially incurable disease. Recently, clinical trials based on BH3-mimetic drugs showed positive outcomes in subjects with poor prognostic features. However, resistance to treatments occurs in a significant number of patients. We previously reported that the multi-kinase inhibitor quercetin, a natural flavonol, restores sensitivity to ABT-737, a BH3-mimetic compound, in both leukemic cell lines and B-cells isolated from patients. To identify the molecular target of quercetin, we employed a new cell line, HG3, obtained by immortalization of B-cells from a chronic lymphocytic leukaemia patient at the later stage of disease. We confirmed that quercetin in association with ABT-737 synergistically enhances apoptosis in HG3 (combination index < 1 for all fractions affected). We also reported that the cellular uptake of quercetin is extremely rapid, with an intracellular concentration of about 38.5 ng/106 cells, after treatment with 25 μM for 5 min. We demonstrated that the activity of protein kinase CK2, which positively triggers PI3K/Akt pathway by inactivating PTEN phosphatase, is inhibited by quercetin immediately after its addition to HG3 cells (0–2 min). PI3K activity was also inhibited by quercetin within 60 min from the treatment. The combined inhibition of CK2 and PI3K kinase activities by quercetin restored ABT-737 sensitivity and increased lethality in human leukemia cells.

Downregulation of protein kinase CK2 activity induces age-related biomarkers in C. elegans.

Studies show that a decrease in protein kinase CK2 (CK2) activity is associated with cellular senescence. However, the role of CK2 in organism aging is still poorly understood. Here, we investigated whether protein kinase CK2 (CK2) modulated longevity in Caenorhabditis elegans. CK2 activity decreased with advancing age in the worms. Knockdown of kin-10 (the ortholog of CK2β) led to a short lifespan phenotype and induced age-related biomarkers, including retardation of locomotion, decreased pharyngeal pumping rate, increased lipofuscin accumulation, and reduced resistance to heat and oxidative stress. The long lifespan of age-1 and akt-1 mutants was significantly suppressed by kin-10 RNAi, suggesting that CK2 acts downstream of AGE-1 and AKT-1. Kin-10 knockdown did not further shorten the short lifespan of daf-16 mutant worms but either decreased or increased the transcriptional activity of DAF-16 depending on the promoters of the target genes, indicating that CK2 is an upstream regulator of DAF-16 in C. elegans. Kin-10 knockdown increased production of reactive oxygen species (ROS) in the worms. Finally, the ROS scavenger N-acetyl-L-cysteine significantly counteracts the lifespan shortening and lipofuscin accumulation induced by kin-10 knockdown. Therefore, the present results suggest that age-dependent CK2 downregulation reduces longevity by associating with both ROS generation and the AGE-1-AKT-1-DAF-16 pathway in C. elegans.

Clinical Significance of Ck2 (CSNK2) and C-Myc Expression in Childhood Acute Lymphoblastic Leukemia

Background. Acute lymphoblastic leukemia (ALL) is the most common malignancy of childhood and a major cause of childhood cancer-related mortality. Although the cure rate now approaches 90%, certain pediatric ALL subgroups present subsequent relapse. For this reason, analyses of cell signaling pathways will help to identify new markers and/or targets for tailored therapy. PI3K/AKT/mTOR activation is frequently found in both B-ALL and T-ALL. Protein kinase Ck2 (CSNK2) activity in pediatric ALL was increased and its inhibition restored PTEN phosphatase activity with subsequent inactivation of AKT. Moreover, Ck2 may serve the activity of oncogenes such as BCR-ABL and c-MYC, control the activation of other critical signaling cascades (JAK-STAT), and sustain multiple cellular stress-elicited pathway such as the proteotoxic stress, unfolded protein and DNA-damage responses. Ck2 has also been shown to have an essential role in tuning signals derived from the stromal tumor microenvironment (Piazza F et al, Oncogene2016).Material and Methods. We analyzed cDNA collected from 46 patients with B-ALL [19 High Risk (HR) for Minimal Residual Disease (MRD) and 27 NON-HR] and 25 with T-ALL (8 HR and 17 NON-HR), respectively, diagnosed in our Center from 2000 to 2012. The latter subgroup was screened fro PTEN-Exon7 mutations and TXL3 rearrangements. We evaluated the gene expression of Ck2 and c-Myc genes using RQ-PCR with Sybr-Green and a relative quantification method (ΔΔCt method), comparing gene’s expression from patients with samples from 6 healty donors (HDs). In order to demonstrate the correlation between genetic alteration and signaling transduction, specifically in HR patients , we analyzed some phosphoproteins by Phospho-flow approach. We profiled 5 proteins (STAT3, STAT5, CREB, PTEN and pS6) in 4 T-ALL cases (3 with PTEN-Exon 7 mutation).Results. We observed a significant difference of Ck2 expression in T-ALL NON-HR patients vs HDs (Mean Ck2 Fold-Changes 3.494 vs 1.17, p=0.0315) and in T-ALL HR patients (6.384 vs 1.17, p=0.0219) vs HDs (Fig 1A and B). Comparing NON-HR vs HR cases, we found a statistically significant difference (p<0.0001) (Fig 1C). c-Myc mean expression was very similar between the two T-ALL subgroups. Moreover, among T-ALL cases, we identified 5 patients with PTEN-Exon7 mutations and 6 with TLX3 rearrangements. We observed that cases with PTEN-Exon7 mutation showed lower c-Myc expression than cases with PTEN-Exon7 wild-type (mean c-Myc 8.550 vs 1.920) whereas patients with TLX3 rearrangements showed higher c-Myc expression than TLX3 negative (mean c-Myc 18.260 vs 5.502) (p<0.005) (Fig 2A and B). We did not observe any correlation between these rearrangements and Ck2 expression. We also performed Ck2 and c-Myc expression analysis in B-ALL (NON-HR and HR) subgroups. We surprisingly observed a Ck2 overexpression in both NON-HR and HR B-ALLs compared to HDs. On the other side, we did not observed significant difference about c-Myc expression in cases with B-ALL vs HDs; whilst we observed an overexpression of c-Myc in HR vs NON-HR patients with B-ALL (mean 7.075 vs 2.095, respectively)(p<0.0004). Phospho-Flow analysis, in 3 cases with PTEN-Exon7 mutation (1 Ck2+/normal Myc, 1 normal Ck2/normal Myc, 1 Ck2-/normal Myc) showed PTEN null, very lower pS6 basal level and higher CREB basal level than in case with PTEN-Exon7 wild-type. Moreover, we observed that the latter patient, presented with a TLX3 rearrangements (Ck2+/Myc+) with higher c-Myc expression, showed higher STAT3 basal state level confirming that STAT3 induces the expression of c-Myc.Conclusions. Based on our preliminary findings, Ck2 could be considered as a marker and /or a potential candidate for targeted therapy, specifically in HR-ALL, as confirmed by the use of CK2 inhibitor (CX-4945) in ongoing clinical trials. c-Myc overexpression confirmed its association with HR features. The potential role as markers of both genes needs to be demonstrated in a larger population study. Combined application of genomic and phosphoproteomic strategies will lead us to better profile diagnostic samples of HR-ALL, addressing future tailored treatments. DisclosuresNo relevant conflicts of interest to declare.

Effect of a protein kinase CK2 inhibitor on intracellular levels of reactive oxygen species and DNA double-stand break in H460 cells

Objective To evaluate the effect of a protein kinase CK2 inhibitor on intracellular levels of reactive oxygen species and DNA double-stand break in human non-small cell lung cancer H460 cells. Methods H460 cells were exposed to 0, 12.5, 25.0, and 50.0 μmol/L quinalizarin, a specific inhibitor of protein kinase CK2, for 24 hours. The changes in protein and mRNA levels of CK2 subunits were measured. Flow cytometry was used to measure changes in the intracellular levels of reactive oxygen species in H460 cells after 4 or 24 hours of quinalizarin treatment. Immunofluorescence assays were performed to determine the effect of the CK2 inhibitor on γ-H2AX expression and the average fluorescent number of γ-H2AX foci in H460 cells. Comparison was made by analysis of variance and t test. Results There were no significant differences in protein or mRNA levels of CK2 subunits in H460 cells after quinalizarin treatment (CK2α, 0 μmol vs. 12.5 μmol/L, P=0.966; 0 μmol/L vs. 25 μmol/L, P=0.355; 0 μmol/L vs. 50 μmol/L, P=0.864, CK2α’, 0 μmol/L vs. 12.5 μmol/L, P=0.409; 0 μmol/L vs. 25 μmol/L, P=0.833; 0 μmol/L vs. 50 μmol/L, P=0.0.746, CK2β, 0 μmol/L vs. 12.5 μmol/L, P=0.532; 0 μmol/L vs. 25 μmol/L, P=0.830; 0 μmol/L vs. 50 μmol/L, P=0.061). The intracellular levels of reactive oxygen species were substantially elevated in H460 cells with the increase in quinalizarin concentration and treatment time. Different concentrations of quinalizarin resulted in dose-and time-dependent increases in the numbers of γ-H2AX foci after 4 and 24 hours of treatment (treated by Quianlizarin for 4 or 24 h, 0 μmol/L vs. 12.5 μmol/L, 12.5 μmol/L vs. 25 μmol/L, 25 μmol/L vs. 50 μmol/L, all P=0.000, concentration is 12.5 μmol/L, 25 μmol/L or 50 μmol/L, 4 h vs. 24 h, all all P=0.000). Conclusions Quinalizarin can increase the intracellular levels of reactive oxygen species and DNA double-stand break in H460 cells by inhibition of protein kinase CK2 activity. This study provides a theoretical basis for using quinalizarin as a potential radiosensitizer for lung cancer. Key words: Protein kinase CK2; Quinalizarin; Reactive oxygen species; DNA double-stand break

Surfactant Protein A Enhances Constitutive Immune Functions of Clathrin Heavy Chain and Clathrin Adaptor Protein 2.

NF-κB transcription factors are key regulators of pulmonary inflammatory disorders and repair. Constitutive lung cell type- and microenvironment-specific NF-κB/inhibitor κBα (IκB-α) regulation, however, is poorly understood. Surfactant protein (SP)-A provides both a critical homeostatic and lung defense control, in part by immune instruction of alveolar macrophages (AMs) via clathrin-mediated endocytosis. The central endocytic proteins, clathrin heavy chain (CHC) and the clathrin adaptor protein (AP) complex AP2, have pivotal alternative roles in cellular homeostasis that are endocytosis independent. Here, we dissect endocytic from alternative functions of CHC, the α-subunit of AP2, and dynamin in basal and SP-A-modified LPS signaling of macrophages. As revealed by pharmacological inhibition and RNA interference in primary AMs and RAW264.7 macrophages, respectively, CHC and α-adaptin, but not dynamin, prevent IκB-α degradation and TNF-α release, independent of their canonical role in membrane trafficking. Kinetics studies employing confocal microscopy, Western analysis, and immunomagnetic sorting revealed that SP-A transiently enhances the basal protein expression of CHC and α-adaptin, depending on early activation of protein kinase CK2 (former casein kinase II) and Akt1 in primary AMs from rats, SP-A(+/+), and SP-A(-/-) mice, as well as in vivo when intratracheally administered to SP-A(+/+) mice. Constitutive immunomodulation by SP-A, but not SP-A-mediated inhibition of LPS-induced NF-κB activity and TNF-α release, requires CHC, α-adaptin, and dynamin. Our data demonstrate that endocytic proteins constitutively restrict NF-κB activity in macrophages and provide evidence that SP-A enhances the immune regulatory capacity of these proteins, revealing a previously unknown pathway of microenvironment-specific NF-κB regulation in the lung.

Abstract 3791: The inhibition of protein kinase CK2 activity by the CIGB-300 synthetic peptide impairs the three-dimensional cell growth, Wnt and nuclear factor қB signaling pathways in lung cancer cells

Abstract CK2 is a serine/threonine kinase involved in cell growth, survival and apoptosis. The CIGB-300 is a synthetic peptide capable of binding to CK2 substrates thus preventing the enzyme activity. Previously we have determined that CIGB-300 presented an inhibitory concentration 50 dose (IC50) of 119±2.4 µM in NCI-H125 human lung cancer cells. Throughout an Annexin V-FITC assay we could determine that CIGB-300 is a potent apoptosis inductor since the treatment with this drug for 60 minutes induced apoptosis at a level comparable with that observed with etoposide (10 µM). Concomitantly with cell death induction, we could observe caspase-3 activation and the decreased expression of c-myc and cyclin D1 and D2. In this work we analyzed whether CIGB-300 is able to alter the ability of cells to grow in 3D and to modulate signaling pathways involved in tumor progression. First, we developed stable spheroids of NCI-H125 cells, which were able to grow in culture for at least 14 days. After 5 days of treatment, CIGB-300 induced a significant growth inhibition (median volume: 4.07x107±0.70x107 µm3 in control spheroids vs 2.13x107±0.35x107 µm3 in spheroids treated with the IC50 dose p&amp;lt;0.05). From the sixth day of treatment the size of the spheroids was dramatically reduced, returning to the initial volume on day eight. To analyze the effect of CIGB-300 on canonical Wnt signaling, this pathway was initially activated by incubating NCI-H125 cells with a conditioned media containing Wnt3a factor. This incubation led to a significant increase in the levels of cytoplasmic β-catenin. By Western blot we could observe that treatment with CIGB-300 blocked this increase, suggesting its role in the modulation of the Wnt pathway. In order to analyze the nuclear factor κB (NF-κB) dependent pathway, this transcription factor was activated by a phorbol ester treatment (PMA, 15 nM). Short treatment (15 min) with CIGB-300 induced an important reduction in NF-κB nuclear levels. However, this inhibition was reverted 24 h later as determined by Western blot and reporter gene expression assays. Our results indicate that the treatment with CIGB-300 induces a significant anti-proliferative response in a three-dimensional model, which resembles in vivo tumor growth conditions, also affecting key signaling pathways involved in tumor progression. Altogether, our data suggest that this peptide may become a new strategy for the treatment of lung cancer malignancies. Citation Format: Stéfano M. Cirigliano, María Inés Díaz Bessone, Carolina Flumian, Damián Emilio Berardi, Silvio E. Perea, Elisa Bal De Kier Joffé, Hernán Farina, Laura Todaro, Alejandro Urtreger. The inhibition of protein kinase CK2 activity by the CIGB-300 synthetic peptide impairs the three-dimensional cell growth, Wnt and nuclear factor қB signaling pathways in lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3791. doi:10.1158/1538-7445.AM2014-3791

Development of a high-throughput screening-compatible assay to identify inhibitors of the CK2α/CK2β interaction

Increased activity of protein kinase CK2 is associated with various types of cancer, neurodegenerative diseases, and chronic inflammation. In the search for CK2 inhibitors, attention has expanded toward compounds disturbing the interaction between CK2α and CK2β in addition to established active site-directed approaches. The current article describes the development of a fluorescence anisotropy-based assay that mimics the principle of CK2 subunit interaction by using CK2α1–335 and the fluorescent probe CF-Ahx-Pc as a CK2β analog. In addition, we identified new inhibitors able to displace the fluorescent probe from the subunit interface on CK2α1–335. Both CF-Ahx-Pc and the inhibitors I-Pc and Cl-Pc were derived from the cyclic peptide Pc, a mimetic of the C-terminal CK2α-binding motif of CK2β. The design of the two inhibitors was based on docking studies using the known crystal structure of the Pc/CK2α1–335 complex. The dissociation constants obtained in the fluorescence anisotropy assay for binding of all compounds to human CK2α1–335 were validated by isothermal titration calorimetry. I-Pc was identified as the tightest binding ligand with a KD value of 240nM and was shown to inhibit the CK2 holoenzyme-dependent phosphorylation of PDX-1, a substrate requiring the presence of CK2β, with an IC50 value of 92μM.

Prolonged Insulin Stimulation Down-regulates GLUT4 through Oxidative Stress-mediated Retromer Inhibition by a Protein Kinase CK2-dependent Mechanism in 3T3-L1 Adipocytes

Although insulin acutely stimulates glucose uptake by promotion of GLUT4 translocation from intracellular compartments to the plasma membrane in adipocytes and muscles, long term insulin stimulation causes GLUT4 depletion that is particularly prominent in the insulin-responsive GLUT4 storage compartment. This effect is caused mainly by accelerated lysosomal degradation of GLUT4, although the mechanism is not fully defined. Here we show that insulin acutely induced dissociation of retromer components from the low density microsomal membranes of 3T3-L1 adipocytes that was accompanied by disruption of the interaction of Vps35 with sortilin. This insulin effect was dependent on the activity of protein kinase CK2 but not phosphatidylinositol 3-kinase or extracellular signal-regulated kinase 1/2. Knockdown of Vps26 decreased GLUT4 to a level comparable with that with insulin stimulation for 4 h. Vps35 with a mutation in the CK2 phosphorylation motif (Vps35-S7A) was resistant to insulin-induced dissociation from the low density microsomal membrane, and its overexpression attenuated GLUT4 down-regulation with insulin. Furthermore, insulin-generated hydrogen peroxide was an upstream mediator of the insulin action on retromer and GLUT4. These results suggested that insulin-generated oxidative stress switches the GLUT4 sorting direction to lysosomes through inhibition of the retromer function in a CK2-dependent manner.

Activation of protein kinase CK2 attenuates FOXO3a functioning in a PML-dependent manner: implications in human prostate cancer

Protein kinase CK2 (also known as Caseine Kinase II) is an ubiquitous Ser/Thr protein kinase present in both the nucleus and cytoplasm of cells, targeting several key enzymes, growth factor receptors, transcription factors and cytoskeletal proteins. It is not only a key player in regulating cellular growth and proliferation, but also behaves as a potent suppressor of apoptosis. CK2 has been frequently found to be deregulated (mostly hyperactivated) in all cancers, prostate cancer being prominent of them. In the recent past, tumor suppressor PML (promyelocytic leukemia) has been shown to be a target of phosphorylation by CK2. This phosphorylation promotes the ubiquitin-mediated proteasomal degradation of PML thereby effectively curbing its role as a tumor suppressor. Among many others, PML has also been established to mediate its tumor suppressive role by mitigating the inactivation of active AKT (pAKT) inside the nucleus by assembling a dephosphorylating platform for nuclear pAKT. One of the immediate consequences, of this inactivation is the stabilization of FOXO3a, another well-established tumor suppressor, inside the nucleus and its downstream activities. Here, we propose a novel signaling axis apexed by deregulated CK2, dismantling the association of PML and PHLPP2 (we also report PHLPP2 to be a novel interacting partner of PML inside the nucleus), ultimately leading to the inactivation and nuclear exclusion of FOXO3a, thereby downregulating p21/p27/Bim in which degradation of PML and the concomitant stabilization of pAKT plays a cardinal part.

Anti-androgen receptor activity of apoptotic CK2 inhibitor CX4945 in human prostate cancer LNCap cells

Androgen receptor (AR) is crucial for transcriptional signaling in prostate cancers. The anti-cancer activity of protein kinase CK2 (formerly called casein kinase 2)-specific small molecule inhibitors have been reported in several cancers including prostate cancers. The orally available CX4945, a potent and selective small molecule inhibitor of CK2, has advanced into human clinical trials and has exhibited strong anti-tumor activity. The inhibition of CK2 leads to a down-regulation of the AR-dependent transcription, but the functional relevance of CX4945 to AR-dependent transcription in AR-positive LNCap cells has not been studied yet. Our observation of inhibitory effects of CX4945 on the expression or phosphorylation levels of CK2α, Akt and anti-apoptotic molecules including IAP family members agreed with a previous study showing the effect of CK2 inhibition in cancer cells. This study also provides novel information on the impact of CX4945 in the inhibition of AR-dependent transcriptional activation in LNCap cells via its down-regulation. Pharmacologic inhibition experiment revealed that CX4945 could exhibit its anti-cancer activity in LNCap cells via the independent inhibitions of AR and Akt-survivin signalings.

TF — A novel cell-permeable and selective inhibitor of human protein kinase CK2 induces apoptosis in the prostate cancer cell line LNCaP

BackgroundAbnormally high activity of protein kinase CK2 is linked to various diseases including cancer. Therefore, the inhibition of CK2 is a promising therapeutic strategy to fight this disease. MethodsWe screened a library of synthetic molecules concerning their capacity to inhibit CK2. The activity of CK2 and their IC50 and Ki values were determined by a capillary electrophoresis assay. The effects of the inhibitor in a cell culture model were analyzed by cell counting, a viability assay, cytofluorimetry and Western blot. ResultsThe best CK2 inhibitor found in this screen was 6,7-dichloro-1,4-dihydro-8-hydroxy-4-[(4-methylphenylamino)methylen]dibenzo [b,d]furan-3(2H)-one, which we refer to as “TF”. TF showed tight binding to CK2 with low IC50 (29nM) and Ki (15nM) values. TF inhibited only seven out of 61 human kinases tested (>70% inhibition). Incubation of LNCaP cells with 50μM TF for 48h decreased the intracellular CK2 activity by 50%, confirming that the inhibitor is membrane permeable. The decrease in activity was correlated with a severe reduction in cell viability. The reduction in cell viability is at least partly due to the induction of apoptosis. General significanceIn many cancers the protein kinase CK2 is significantly up-regulated and supports the neoplastic phenotype. New therapeutic strategies should be based on diverse reliable inhibitors to reverse the abnormal high levels to normal settings.

Design and synthesis of CK2 inhibitors

A series of new polybrominated benzimidazoles and benzotriazoles has been synthesized and their influence on the activity of protein kinase CK2 was evaluated. It was revealed that the most active inhibitors are those with methyl or ethyl substituent at benzene ring, namely 5,6,7-tribromo-4-methyl-1H-benzotriazole (38, IC(50) 0.51 μM) and 5,6,7-tribromo-4-ethyl-1H-benzotriazole (40, IC(50) 0.16 μM). The derivatives with large aromatic or heterocyclic substituents connected to benzimidazole or benzotriazole scaffold appeared to be less potent inhibitors.

Role of polyamines in determining the cellular response to chemotherapeutic agents: modulation of protein kinase CK2 expression and activity

Numerous studies have shown that platinum compounds stimulate the expression of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase resulting in anti-proliferative activity and apoptosis. As many cancer cell types including pancreatic cancer cells express high levels of polyamines, the possibility to develop anti-tumor strategies to deplete polyamine pools has drawn considerable attention in recent years. This has been effectively accomplished by treating cells with platinum drugs in combination with polyamine analogs such as N(1),N(11)-diethylnorspermine (DENSPM). The present study, examined the cytotoxic effects of oxaliplatin in combination with stimulators of polyamine catabolism in human pancreatic cancer cells, that are notoriously resistant to chemotherapeutic treatment, and colorectal cancer cells. Additionally, as protein kinase CK2 has been shown to be an anti-apoptotic and pro-survival enzyme regulated by the intracellular polyamine pools, we aimed to investigate the effect of combined DENSPM and oxaliplatin treatment on CK2-mRNA and -protein levels. Results reported here show that treatment with oxaliplatin and DENSPM in combination impairs cell viability particularly in the case of colorectal cancer cells. The analysis of CK2 expression and activity indicates that the response to a specific treatment may depend on the impact that individual compounds exert on pro-survival and pro-death proteins at the transcription and translation levels that should be carefully evaluated in view of subsequent clinical studies.

Repressing the Activity of Protein Kinase CK2 Releases Mitochondria-Mediated Apoptosis in Cancer Cells

Execution of the mitochondrial death signaling is paramount to an effective response of cancer cells to chemotherapeutic intervention. Therefore, factors that inhibit the engagement of the mitochondrial amplification pathway, such as the expression of the anti-apoptotic proteins of the Bcl2 family or inactivation of inducers of mitochondrial permeability, play a critical role in the acquisition of the resistant phenotype. Protein kinase CK2 (CK2) is a ubiquitous serine/threonine kinase that is highly conserved in eukaryotic cells. This multifunctional protein kinase has been shown to impact cell growth and proliferation, as numerous growth-related proteins are substrates of CK2. More importantly, experimental evidence linking increased expression and activity of the kinase to human cancers, underscores the relevance of CK2 biology to cellular transformation and carcinogenesis. Of note, among the many cellular substrates of CK2 are proteins involved in the efficient execution of the mitochondria-dependent cell death signaling, such as Bid, caspase-2, ARC and others. Supporting this, recent reports have demonstrated that genetic manipulation of CK2 expression as well as pharmacological inhibition of its enzymatic activity sensitizes cancer cells to apoptotic stimuli. Due to the critical regulatory role that this kinase plays in cell fate determination in cancer cells, there is a tremendous increase in activity geared at the development of CK2-specific therapies. Here we provide a brief review of CK2-mediated inhibition of mitochondrial death signaling in cancer cells and its implications for the design of novel target specific therapeutic strategies.