Prosurvival autophagy is regulated by protein kinase CK1 alpha in multiple myeloma

Multiple myeloma (MM) is a tumor of plasma cells (PCs). Due to the intense immunoglobulin secretion, PCs are prone to endoplasmic reticulum stress and activate several stress-managing pathways, including autophagy. Indeed, autophagy deregulation is maladaptive for MM cells, resulting in cell death. CK1α, a pro-survival kinase in MM, has recently been involved as a regulator of the autophagic flux and of the transcriptional competence of the autophagy-related transcription factor FOXO3a in several cancers. In this study, we investigated the role of CK1α in autophagy in MM. To study the autophagic flux we generated clones of MM cell lines expressing the mCherry-eGFP-LC3B fusion protein. We observed that CK1 inhibition with the chemical ATP-competitive CK1 α/δ inhibitor D4476 resulted in an impaired autophagic flux, likely due to an alteration of lysosomes acidification. However, D4476 caused the accumulation of the transcription factor FOXO3a in the nucleus, and this was paralleled by the upregulation of mRNA coding for autophagic genes. Surprisingly, silencing of CK1α by RNA interference triggered the autophagic flux. However, FOXO3a did not shuttle into the nucleus and the transcription of autophagy-related FOXO3a-dependent genes was not observed. Thus, while the chemical inhibition with the dual CK1α/δ inhibitor D4476 induced cell death as a consequence of an accumulation of ineffective autophagic vesicles, on the opposite, CK1α silencing, although it also determined apoptosis, triggered a full activation of the early autophagic flux, which was then not supported by the upregulation of autophagic genes. Taken together, our results indicate that the family of CK1 kinases may profoundly influence MM cells survival also through the modulation of the autophagic pathway

Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death

BACKGROUND: Glycogen Synthase Kinase-3 (GSK-3) \u3b1 and \u3b2 are two serine-threonine kinases controlling insulin, Wnt/\u3b2-catenin, NF-\u3baB signaling and other cancer-associated transduction pathways. Recent evidence suggests that GSK-3 could function as growth-promoting kinases, especially in malignant cells. In this study, we have investigated GSK-3\u3b1 and GSK-3\u3b2 function in multiple myeloma (MM). METHODS: GSK-3 \u3b1 and \u3b2 expression and cellular localization were investigated by Western blot (WB) and immunofluorescence analysis in a panel of MM cell lines and in freshly isolated plasma cells from patients. MM cell growth, viability and sensitivity to bortezomib was assessed upon treatment with GSK-3 specific inhibitors or transfection with siRNAs against GSK-3 \u3b1 and \u3b2 isoforms. Survival signaling pathways were studied with WB analysis. RESULTS: GSK-3\u3b1 and GSK-3\u3b2 were differently expressed and phosphorylated in MM cells. Inhibition of GSK-3 with the ATP-competitive, small chemical compounds SB216763 and SB415286 caused MM cell growth arrest and apoptosis through the activation of the intrinsic pathway. Importantly, the two inhibitors augmented the bortezomib-induced MM cell cytotoxicity. RNA interference experiments showed that the two GSK-3 isoforms have distinct roles: GSK-3\u3b2 knock down decreased MM cell viability, while GSK-3\u3b1 knock down was associated with a higher rate of bortezomib-induced cytotoxicity. GSK-3 inhibition caused accumulation of \u3b2-catenin and nuclear phospho-ERK1, 2. Moreover, GSK-3 inhibition and GSK-3\u3b1 knockdown enhanced bortezomib-induced AKT and MCL-1 protein degradation. Interestingly, bortezomib caused a reduction of GSK-3 serine phosphorylation and its nuclear accumulation with a mechanism that resulted partly dependent on GSK-3 itself. CONCLUSIONS: These data suggest that in MM cells GSK-3\u3b1 and \u3b2 i) play distinct roles in cell survival and ii) modulate the sensitivity to proteasome inhibitors

A new feature of CK2 function: it's role in granulocytic differentiation induced by retinoic acid

ABSTRACT The Ser/Thr kinase CK2 promotes cell survival and proliferation 1; it also modulates a number of molecules involved in cell development 2, 3, 4, 5, 6. In this study, we aimed to study a potential novel role for CK2 in myeloid cell differentiation. We took advantage of the well-established model of acute promyelocytic leukemia cells (APL) maturation induced by retinoic acid (RA). Both CK2 expression and activity were high in APL cell lines. RA caused a modulation of CK2 subunits expression. CK2 inhibition, with chemicals or RNA interference 1) prevented RA-induced cell cycle arrest in the G1 phase 2) blocked the phenotypical, morphological and functional differentiation of APL cells 3) impaired the transcription of RARα target genes p21 and CEBPε. Interestingly, CK2 blockade determined delocalization of RARα from the nucleus to the cytoplasm even in presence of RA. Upon CK2 inhibition, lower-molecular weight RARα specific bands appeared, which likely correspond to dephosphorylated forms of the receptor. Moreover, CK2 blockade resulted to slow down the decrease of RARα expression due to degradation triggered by RA. Experiments performed after protein synthesis inhibition showed that RARα turnover is fast and blockade of proteasome with MG132 partially restores RARα expression. Thus, other mechanisms of degradation could operate on RARα protein. Remarkably, the combined treatment with CK2 inhibitors and MG132 favoured the shift from higher forms to lower forms of RARα. CK2 blockade in the presence of RA and MG132, did not increase significantly the huge accumulation of the lighter forms of RARα already produced by retinoic acid treatment. Taken together our results indicate that CK2 is essential in granulocytic differentiation upon RA treatment and could operate at several levels: (1) modulation of RARα transcriptional activity; (2) retention of RARα in the nucleus; (3) phosphorylation of RARα and consequent RARα localization, stability, and control of its turnover during differentiation. Further investigation is needed to clarify the mechanism of CK2-RARα interactions; the kinase could perform its activity in a direct or most likely in an indirect way through the modulation of other kinases or phosphateses. Moreover, it would be interesting to elucidate the possibility that CK2 could regulate proteins involved in RARα shuttling from nucleus to cytoplasm compartments, influencing also in this way its transcriptional activity.La Ser/Thr kinase CK2 promuove la sopravvivenza cellulare e la proliferazione; modula anche una serie di molecole coinvolte nello sviluppo cellulare2,3,4,5,6. In questo studio, ci siamo prefissati di indagare un potenziale nuovo ruolo di CK2 nel differenziamento mieloide. Ci siamo avvalsi del modello di maturazione indotto da acido retinoico (AR) di cellule di leucemia promielocitica acuta (LPA). Sia l'espressione che l'attività di CK2 erano elevate in cellule di LPA. L' AR ha determinato una modulazione dell'espressione delle subunità di CK2. L'inibizione di CK2, con agenti chimici o l'RNA interference 1) ha impedito l'arresto del ciclo cellulare in fase G0/G1, promosso dall'AR; 2) ha bloccato il differenziamento dei blasti di LPA sia a livello fenotipico, morfologico e funzionale; 3) ha inibito la trascrizione di geni target di RARα quali p21 e CEBPε. E' stato interessante notare come il blocco di CK2 abbia determinato la delocalizzazione di RARα dal nucleo al citoplasma nonostante la presenza di AR.. In seguito all'inibizione di CK2, sono state rilevate in western blot la comparsa di bande a più basso peso molecolare di RARα, che verosimilmente corrispondono a forme defosforilate del recettore. Inoltre l'inibizione di CK2 è risultata rallentare la diminuzione dei livelli di RARα dovuta al processo di degradazione innescato dall'AR. Esperimenti condotti dopo inibizione della sintesi proteica hanno mostrato che il turnover di RARα è rapido e il blocco del proteasoma con MG132 ripristina parzialmente l'espressione di RARα. Quindi altri meccanismi di degradazione possono agira su RARα. E' da sottolineare che il trattamento combinato di inibitori di CK2 con MG132, ha favorito lo shift delle forme ad alto peso molecolare di RARα verso forme a più basso peso molecolare. Il blocco di Ck2 in presenza di AR e MG132 non ha causato un aumento significativo della quota già abbondante di forme defosforilate prodotte in seguito a trattamento con AR. Riassumendo questi risultati dimostrano che CK2 è essenziale nel differenziamento granulocitico indotto da AR e può agire su più livelli: (1) modulazione dell'attività trascrizionale di RARα ; (2) ritenzione di RARα nel nucleo; (3) fosforilazione di RARα che potrebbe influenzare la localizzazione, la stabilità,e il turnover del recettore durante il differenziamento. Ulteriori indagini saranno necessarie per chiarire i meccanismi dell'interazione tra CK2 e RARα; la chinasi potrebbe agire in modo diretto o più probabilmente in odo indiretto modulando altre chinasi o fosfatasi. Inoltre sarà interessante capire se CK2 possa regolare proteine coinvolte nello shuttling di RARα dal nucleo al citoplasma, influenzando anche in questo modo l'attività trascrizionale del recettore

Metabolic control of epigenetic rearrangements in B cell pathophysiology

: Both epigenetic and metabolic reprogramming guide lymphocyte differentiation and can be linked, in that metabolic inputs can be integrated into the epigenome to inform cell fate decisions. This framework has been thoroughly investigated in several pathophysiological contexts, including haematopoietic cell differentiation. In fact, metabolite availability dictates chromatin architecture and lymphocyte specification, a multi-step process where haematopoietic stem cells become terminally differentiated lymphocytes (effector or memory) to mount the adaptive immune response. B and T cell precursors reprogram their cellular metabolism across developmental stages, not only to meet ever-changing energetic demands but to impose chromatin accessibility and regulate the function of master transcription factors. Metabolic control of the epigenome has been extensively investigated in T lymphocytes, but how this impacts type-B life cycle remains poorly appreciated. This assay will review our current understanding of the connection between cell metabolism and epigenetics at crucial steps of B cell maturation and how its dysregulation contributes to malignant transformation

Targeting Protein Kinases in Blood Cancer: Focusing on CK1\u3b1 and CK2

Disturbance of protein kinase activity may result in dramatic consequences that often lead to cancer development and progression. In tumors of blood origin, both tyrosine kinases and ser-ine/threonine kinases are altered by different types of mutations, critically regulating cancer hallmarks. CK1\u3b1 and CK2 are highly conserved, ubiquitously expressed and constitutively active pleiotropic kinases, which participate in multiple biological processes. The involvement of these kinases in solid and blood cancers is well documented. CK1\u3b1 and CK2 are overactive in multiple myeloma, leukemias and lymphomas. Intriguingly, they are not required to the same degree for the viability of normal cells, corroborating the idea of \u201cdruggable\u201d kinases. Different to other kinases, mutations on the gene encoding CK1\u3b1 and CK2 are rare or not reported. Actually, these two kinases are outside the paradigm of oncogene addiction, since cancer cells\u2019 dependency on these proteins resembles the phenomenon of \u201cnon-oncogene\u201d addiction. In this review, we will summarize the general features of CK1\u3b1 and CK2 and the most relevant oncogenic and stress-related signaling nodes, regulated by kinase phosphorylation, that may lead to tumor progression. Finally, we will report the current data, which support the positioning of these two kinases in the therapeutic scene of hematological cancers

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

Background. Leukemia stem cells (LSC) constitute the reservoir of acute and chronic leukemias, from which the relapse of the disease takes origin. The Wnt/\u3b2catenin 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: CK1\u3b1, CK1\u3b3, CK2\u3b1 (catalytic) and CK2\u3b2 (regulatory subunit); Lef1, FoxO1, FoxO3, CyclinD1 (Wnt targets); Ptch, Gli1, Gli2, Gli3, Bmi1 (Hh targets); western blot analysis was performed to determine CK1\u3b1, CK2\u3b1, CK2\u3b2, p53, phospho-Ser529 NF-\u3baB 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. CK1\u3b1, CK2\u3b1 and CK2\u3b2 mRNA and proteins were found overexpressed in LSC and in AML blasts. CK1\u3b3 mRNA levels were barely detectable. Notably, some AML cases displayed low levels of CK2\u3b2 but high levels of CK2\u3b1, indicating a possible CK2\u3b2-independent function of CK2\u3b1. 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 CK1\u3b1 and CK2 and high-risk cytogenetic groups was observed. Also, in most of CK2\u3b1 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 (\u3b1) and CK2 (\u3b1 and \u3b2) mRNA and protein levels were higher in LSC (especially from high risk groups) than in HSC. CK1\u3b3 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

CK2 Kinase Inhibitors Display Anti-Myeloma Effects and Antagonize Osteoclast Activity in Models of Multiple Myeloma Bone Marrow Microenvironment

Background. 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-\u3baB 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-\u3baB 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-\u3baB activity evaluated in MM cells was found upon CK2 inhibition, with a parallel reduction of the production of NF-\u3baB-dependent cytokines. When assayed on osteoprogenitors, CX4945 displayed an inhibitory effect on osteoclast formation from CD14+ monocytes even at low concentrations (1 \u3bcM up to 7 \u3bcM, comparable with the effects of zolendronate 1 \u3bcM), whereas it inhibited the formation of osteoblasts from BM colonies at day 14 at fairly higher concentrations (>5 \u3bcM). Moreover, CX4945 inhibited osteoblast proliferation at even higher concentration (>7.5 \u3bcM). 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

CX-4945, a selective inhibitor of casein kinase 2, synergizes with B cell receptor signaling inhibitors in inducing diffuse large B cell lymphoma cell death

Approximately one third of Diffuse Large B cell Lymphomas (DLBCL) are refractory or relapse. Novel therapeutic approaches under scrutiny include inhibitors of B-cell receptor (BCR) signaling. Protein kinase CK2 propels survival, proliferation and stress response in solid and hematologic malignancies and promotes a "non-oncogene addiction" phenotype. Whether this kinase regulates BCR signaling thus being a suitable pharmacological target in DLBCL is unknown