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Protein kinase CK2: analysis of its role in acute myeloid leukemia and conditional KO in mouse hematopoiesis
Get PDFHematopoiesis is a tightly controlled, hierarchically organized developmental process whereby hematopoietic stem cells (HSC) give rise to highly specialized, differentiated blood cells.
HSC are characterized by the capacity of both self-renewing and differentiating in downstream committed hematopoietic progenitor cells. In vertebrates, hematopoiesis in embryonic and fetal life occurs in primitive hematopoietic organs whereas in the adult organism the definitive site of blood cell production is the bone marrow (BM). HSC are quiescent cells that reside most of the time in G0-G1 phases of the cell cycle, dividing during their lifespan as much as it is needed to maintain their own pool and give rise to committed precursors.
Leukemia formation is characterized by the block of this process and accumulation of immature cells in the bone marrow and peripheral blood.
In particular, acute myeloid leukaemia (AML) is characterized by the accumulation of large numbers of abnormal cells that fail to differentiate into functional mature blood cells. Leukemic blasts have a limited, exhaustible proliferative potential, suggesting that, in order to maintain leukemia growth, a small subpopulation of leukemic stem cells (LSCs) must display an inexhaustible proliferative capacity and self-renewal potential. No definitive proof of LSCs was available, however, until Dick and colleagues showed that the engraftment of NOD/SCID mice with primary AML samples could only be accomplished using cells that were phenotypically similar to normal hematopoietic stem cells (HSCs) by expressing CD34 and lacking markers of lineage commitment such as CD38 .
HSCs and LSCs share common features: self-renewal, the capacity to differentiate, resistance to apoptosis, and limitless proliferative potential.
The pathway regulating these functional properties can be categorized into self-renewal, developmental, and miscellaneous pathways, each of which is governed by a distinct set of critical genes that have emerged from molecular profiling and can be associated with stemness.
In particular Wingless (Wnt)/β catenin and PI3K/AKT pathways are crucial for the control of both HSCs and LSCs biology, because they regulate proliferation, self renewal and differentiation of HSCs and they are involved in maintenance of LSCs.
Phosphorylation is one of the molecular mechanism responsible for the signal transduction modulation of these two pathways and some molecules belonging to these pathways are phosphorylated by serin-threonin kinase CK2. Thus, this protein can have an important role in the biology of both HSCs and LSCs.
CK2 is a pleiotropic and constitutive activated kinase, which has a tetrameric structure composed by two alpha catalytic subunits and two beta regulatory subunits, but these two components can work also separately. CK2 is essential for cell survival and proliferation, and is more and more evident its involvement in various aspects of tumor transformation. High levels of CK2 are found in different tumor type such as breast, lung, kidney cancer and also blood cancer. CK2 promotes the activity of proto-oncogenes (c-myc, C-Jun, A-Raf), drives cell cycle progression, stimulates beta-catenin activity, inhibits the onco-suppressors p53, PML and PTEN, and it can exert an anti-apoptotic effect through the inhibition of caspase activity.
For this reasons, the principal aim of our research project is obtaining experimental evidence of a role of CK2 in normal hematopoiesis and in LSCs through the analysis of AML cell lines and samples collected from patients, and of a conditional knockout mouse model for CK2β in the hematopoietic system.
As far as the role of CK2 in the hematopoiesis is concerned, our conditional KO mouse model for CK2β in the hematopoietic compartment, demonstrate that the regulatory subunit of CK2 has an important role in hematopoiesis. In particular, the KO of CK2β induces an impairment in hematopoiesis, especially in erythropoiesis. However, CK2β KO seems not to influence the HSCs pool but instead the hematopoietic progenitors.
As far as the analysis of LSCs from AML samples, our analysis performed both on AML samples derive from patients and on Kasumi-1 cell line demonstrated that CK2 does not affect the Wnt/ β catenin and HH pathways, whereas it is able to modulate the PI3K/AKT pathway. Moreover, we have demonstrated that CK2 is important in LSCs survival as its inhibition increases the apoptosis and potentiates the effects of Daunorubicin, a drug currently used for AML treatment in clinic.
Taken together, all our results indicate that CK2 possesses an important role both in hematopoiesis and in the biology of LSCs
Analysis of Wnt and Hedgehog Pathways Regulating Protein Kinases CK1 and CK2 in Acute Myeloid Leukemia Cells and Stem Cells: Correlation with the Expression of Wnt and Hedgehog Targets and Biological and Clinical Features
No full textBackground. Leukemia stem cells (LSC) constitute the reservoir of acute and chronic leukemias, from which the relapse of the disease takes origin. The Wnt/βcatenin and the Hedgehog (Hh) signaling pathways regulate the balance between self-renewal and commitment of both normal hematopoietic stem cells (HSC) and LSC. Serine-Threonine protein kinases CK1 and CK2 phosphorylate members of Wnt and Hh and are therefore potential regulators of HSC and LSC biology; however, their function in HSC and LSC is unknown.
Purpose. In the present work we have investigated the mRNA and protein levels of CK1 and CK2 in acute myeloid leukemia (AML) blasts and LSC isolated from patients. We have also integrated the expression data of CK1 and CK2 with the expression data of Wnt and Hh signaling pathways gene targets and with biological and clinical parameters of a group of AML patients and cell lines. We also tested the effects on the growth of AML blasts of CK1 and CK2 small chemical inhibitors. The aim of this study was to gain insights into the function of these pivotal protein kinases and on their expression levels relative to biological, clinical and prognostic variables that dictate the outcome of AML patients.
Methods. mRNA was extracted from HSC and FACS-sorted LSC (as defined as Lineage- CD34+ CD38- CD123+ CD90+); proteins were obtained from the bulk of blasts from fifteen AML patients. Quantitative RT-PCR was employed to assess mRNA levels of: CK1α, CK1γ, CK2α (catalytic) and CK2β (regulatory subunit); Lef1, FoxO1, FoxO3, CyclinD1 (Wnt targets); Ptch, Gli1, Gli2, Gli3, Bmi1 (Hh targets); western blot analysis was performed to determine CK1α, CK2α, CK2β, p53, phospho-Ser529 NF-κB and phospho-Ser13 Cdc37 (two direct CK2 target sites) protein levels. Normal CD34+ bone marrow cells were used as controls. CK2 chemical inhibitors (CX4945 and tTBB) were assayed on LSC and AML cells.
Results. CK1α, CK2α and CK2β mRNA and proteins were found overexpressed in LSC and in AML blasts. CK1γ mRNA levels were barely detectable. Notably, some AML cases displayed low levels of CK2β but high levels of CK2α, indicating a possible CK2β-independent function of CK2α. FoxO1, FoxO3 mRNAs were found upregulated while Lef1 and CyclinD1 ones were found slightly and Axin2 one strongly diminished, respectively. Ptch, Smo, Gli1, Gli2 and Gli3 mRNAs were unchanged or reduced, while we observed a strong upregulation of that of Bmi1. A correlation between the overexpression of CK1α and CK2 and high-risk cytogenetic groups was observed. Also, in most of CK2α high-expressing cases, TP53 was found downregulated, while phospho-Ser529 p65 upregulated. Importantly, inhibition of CK2 with selective compounds caused AML cell and LSC growth arrest, a restoration of TP53 and a downregulation of phospho-Ser529-NF-kB p65 and phospho-Ser13 Cdc37 protein levels.
Conclusions. The present study is the first to report on the expression of CK1 and CK2 kinases in normal HSC and LSC. CK1 (α) and CK2 (α and β) mRNA and protein levels were higher in LSC (especially from high risk groups) than in HSC. CK1γ mRNA levels were low. Wnt and Hh pathways genes were differentially upregulated pointing to a gene specific LSC-associated function. Moreover, our data with CK2 inhibitors suggest that CK2 could be a suitable therapeutic target to eradicate residual AML LSCs. Future research will assess more in depth the in vivo functional role of CK1 and CK2 in HSC and LSC
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
CK2 Kinase Inhibitors Display Anti-Myeloma Effects and Antagonize Osteoclast Activity in Models of Multiple Myeloma Bone Marrow Microenvironment
No full textBackground. Multiple myeloma (MM) plasma cell growth in the bone marrow (BM) microenvironment is fueled by survival signals delivered by the surrounding non-malignant cells (stromal and other types) and through contacts with the extracelllar matrix. Interactions of MM cells with osteoclasts and osteoblasts generate a milieu, in which bone resorption and bone loss occur more rapidly than bone deposition. Novel agents, such as bortezomib and lenalidomide, which target the MM BM microenvironment, have shown unprecedented anti-myeloma efficacy in part due to their ability to somewhat revert these microenvironmental alterations. However, often resistance occurs also to novel drugs and the disease progresses. We have described that targeting protein kinase CK2 with chemical inhibitors or RNA interference causes MM cell death, increases the sensitivity to chemotherapeutics and compromises the NF-κB and STAT3 activity (Piazza FA et al. 2006, Blood; 108: 1698). We also found that CK2 inhibitors synergize with Hsp90 inhibitors (Manni S et al. 2012, Clinical Cancer Res; 18: 1888) and bortezomib (Manni S et al., Blood (2011 ASH Annual Meeting Abstracts); 118; 1849) in inducing MM cell death. Moreover, a phase I clinical trial is ongoing in USA (ID: NCT01199718) testing the oral CK2 inhibitor CX4945 (Cylene Pharmaceuticals, CA, USA) in MM patients.
Purpose. We investigated whether and how CK2 inhibition with ATP-competitive CX4945 and tTBB inhibitors could affect the growth of MM cells and of osteoprogenitors in models of BM microenvironemnt. The aim of the study was to provide further insights into the mechanism of action of CK2 inhibitors also in the MM microenvironment, in particular on the stromal cell-mediated MM cell survival and on the unbalanced bone metabolism. We ultimately aimed at generating original data useful for the design of novel rational combination therapies incorporating CK2 inhibitors in the therapy of MM and of MM-bone disease.
Methods. MM plasma cells from patients and MM cell lines were cultured in the presence of BM stromal cells obtained from MM patients or BM stromal cell lines or in the presence of osteoclasts. ATP-competitive CK2 inhibitors were added to the co-cultures or to cultures of osteoblast cell lines or progenitors. Cell growth was evaluated with different means and signaling pathways were studied in MM plasma cells and in the stromal cells. NF-κB target gene expression and DNA binding was tested with microplate arrays. For osteoclast generation, CD14+ peripheral blood monocytes were stimulated in alpha-MEM medium with 10% FBS plus RANKL (60ng/ml) plus M-CSF (25ng/ml) for 28 days; early-osteoblasts colonies were obtained from BM cells stimulated under appropriate conditions.
Results. CK2 inhibition with CX4945 or tTBB caused apoptosis of MM cells (either freshly isolated from patients or cell lines) cultured on patient-derived mesenchymal stromal cells (MSC) or on the BM stromal cell line HS-5. The inhibitors did not significantly affect MSC viability. A reduction of NF-κB activity evaluated in MM cells was found upon CK2 inhibition, with a parallel reduction of the production of NF-κB-dependent cytokines. When assayed on osteoprogenitors, CX4945 displayed an inhibitory effect on osteoclast formation from CD14+ monocytes even at low concentrations (1 μM up to 7 μM, comparable with the effects of zolendronate 1 μM), whereas it inhibited the formation of osteoblasts from BM colonies at day 14 at fairly higher concentrations (>5 μM). Moreover, CX4945 inhibited osteoblast proliferation at even higher concentration (>7.5 μM). The anti-myeloma effect of CK2 inhibitors was present also when MM cells (INA-6 cell line) were cultured in the presence of osteoclasts generated from CD14+ monocytes.
Conclusions. Our study shows that inhibition of CK2 could profoundly affect the growth of MM cells in models of BM microenvironment while substantially sparing the normal cellular stromal counterparts and osteoblasts and suggests that CK2 inhibitors could be exploited to target the hyperactivity of osteoclast seen in MM bone disease
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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