Mol Cell Biochem (2011) 356:51–55 DOI 10.1007/s11010-011-0947-6
On CK2 regulation of chronic lymphocytic leukemia cell viability Leila R. Martins • Paulo Lu´cio • Milene C. Silva • Paula Gameiro • Maria G. Silva • Joa˜o T. Barata
Received: 13 June 2011 / Accepted: 24 June 2011 / Published online: 13 July 2011 Ó Springer Science+Business Media, LLC. 2011
Abstract Specific inhibition of signaling elements essential for the viability of B-cell chronic lymphocytic leukemia (CLL) cells offers great promise for the design of more efficient therapies. The protein serine/threonine kinase CK2 is frequently upregulated in cancer, and it is overexpressed and hyperactivated in primary CLL cells from untreated patients. We have shown that inhibition of CK2 induces apoptosis of CLL cells, whereas it does not significantly impact normal lymphocytes, demonstrating the selectivity of the CK2 inhibitors toward leukemia cells. Notably, although co-culture with OP9 stromal cells and BCR stimulation both promote leukemia cell survival in vitro, they do not prevent apoptosis of CLL cells treated with CK2 inhibitors. PI3K signaling pathway was previously shown to be essential for CLL cell viability, an observation we confirmed in all patient samples analyzed. Further, we observed that CK2 blockade decreases PTEN phosphorylation, leading to PTEN activation, and that apoptosis of CLL cells upon CK2 inhibition is mediated by PKC inactivation. This suggests that activation of PI3K/ PKC signaling pathway is involved in the pro-survival effects of CK2 in CLL cells. Sensitivity to CK2 inhibition does not correlate with expression of ZAP-70 or CD38, or with IGVH mutation status. However, it positively
L. R. Martins M. C. Silva J. T. Barata (&) Cancer Biology Unit, Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Av. Prof. Egas Moniz, Lisbon 1649-028, Portugal e-mail:
[email protected] P. Lu´cio P. Gameiro M. G. Silva Faculdade de Cieˆncias Me´dicas (FCM), Centro de Estudos de Doenc¸as Cro´nicas (CEDOC), Universidade Nova de Lisboa and Instituto Portugueˆs de Oncologia, Lisbon, Portugal
correlates with the percentage of CLL cells in the peripheral blood, b2 microglobulin levels, and Binet clinical stage. CK2 appears to play an important role in the biology of CLL and constitutes a promising target for the development of leukemia-specific therapies. Keywords CK2 Chronic lymphocytic leukemia Signaling therapy PI3K-PTEN IgM OP9 co-culture
Introduction Chronic lymphocytic leukemia (CLL) is the most prevalent form of leukemia in western countries. Despite major advances in the treatment of CLL in the past decade, it is still considered an incurable malignancy [1]. Chemoimmunotherapy combining fludarabine, cyclophosphamide, and rituximab appears to be the best treatment option, with significant improvement in response rates, duration of response, overall survival, and eradication of minimal residual disease [2]. However, none of the currently implemented treatments is strictly specific for CLL cells, resulting in increased toxicity and reduced bioavailability/ activity of the drug/antibody toward malignant cells. Specific inhibition of signaling effectors required for the viability of CLL cells should contribute to the development of more efficient and less detrimental therapies. The ubiquitous serine/threonine kinase CK2 is frequently overexpressed in cancer [3], including acute leukemia [4–6]. CK2 leads to constitutive activation of PI3K/Akt signaling pathway in T-cell acute lymphoblastic leukemia cells, due to phosphorylation and consequent functional inactivation of the tumor suppressor PTEN [5]. Activation of PI3K-mediated signaling has also been implicated in CLL [7]. We reasoned that a CK2–PI3K pathway link
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could exist in CLL that could be of biological and therapeutic relevance.
Materials and methods Patient samples Peripheral blood samples from CLL patients were collected after informed consent and ethical committee approval, and enriched in mononuclear cells (PBMCs) by Ficoll density grade centrifugation (Amersham Biosciences). Where indicated, CLL cells were purified ([90%) using RosetteSep human B-cell enrichment cocktail (StemCell Technologies) as indicated by the manufacturer. Patient phenotypic, clinical, and genetic features were determined as previously described [8]. Cell culture PBMCs and purified leukemia cells from CLL patients were cultured as 2 9 106 cells/ml in RPMI 1640 culture medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 1% heat-inactivated FBS (Invitrogen). Co-culture with OP9 stromal cells
Fig. 1 PBMCs from CLL patients were cultured with CK2 inhibitors and viability was assessed after 48 h by AnnexinV/7-AAD staining. a Viability of T cells and CLL cells upon treatment with 25 lM TBB. Left panel shows CD3/CD19 staining used to identify the different populations. Results are representative from 44 CLL patients analyzed. b Viability of CLL cells cultured with TBB (12.5 and 25 lM) or DRB (12.5 and 25 lM) (n = 38–44). Results are shown as mean ± SEM
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was done as previously described [8]. BCR stimulation was done in 96-well plate coated with goat anti-human IgM (10 lg/ml; SouthernBiotech) [9]. Where specified, cells were treated with the indicated concentrations of TBB (Merck) and DRB (Sigma-Aldrich). Cell viability analysis Cell viability was determined by double-staining with APC-conjugated AnnexinV (eBioscience) and 7-AAD (Becton–Dickinson) followed by analysis on a FACSCanto flow cytometer (Becton–Dickinson). Immunoblot Cells were lysed and western blot was performed as described [5], using the following antibodies: actin (Santa Cruz Biotechnology), P-PTEN (S380), PTEN, and P-PKCb (S660) (Cell signaling technology). Statistical analysis GraphPad Prism version 5.00 for Windows (GraphPad Software) was used for statistical analyses. Differences
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Fig. 2 Purified CLL cells were treated with 25 lM TBB and a co-cultured with OP9 stromal cells (n = 4) or b stimulated with an anti-IgM antibody (n = 13) for 48 h. Viability of CLL cells was assessed by AnnexinV/7-AAD staining. Results are shown as mean ± SEM
between mean values were evaluated using two-tailed Students t test, and considered significant for P \ 0.05.
Results and discussion Recently, we and others showed that CK2 is overexpressed and hyperactivated in CLL cells when compared to normal B cells from healthy age-matched donors [8, 10]. Culture of PBMCs from CLL patients with CK2 inhibitors leads to a notable increase in apoptosis of CLL cells, as assessed by AnnexinV/7-AAD staining (Fig. 1a, b), whereas the viability of normal T cells is not significantly affected (Fig. 1a). This consequently leads to a major increase in the percentage of T cells [8]. Our results are in line with earlier studies showing that normal or noncancer cells are significantly less sensitive to CK2 downregulation than prostate cancer cells [11]. This increased selectivity toward malignant cells highlights the potential of CK2 targeting strategies in cancer therapy. Microenvironmental cues appear to be essential in the triggering and maintenance of CLL [1, 12, 13]. In vitro studies using different stromal cell lines coincide in demonstrating their ability to promote survival of CLL cells [14, 15]. We used for the first time, as far as we are aware, OP9 mouse bone marrow stromal cells to evaluate the capacity of CK2 inhibitors to promote CLL cell death in an in vitro experimental context that mimics the pro-survival stimuli provided by the microenvironment. OP9 stroma coculture increases the viability of all CLL patient cells tested (Fig. 2a). However, CK2 inhibition significantly downregulates the viability of CLL cells even in the presence of OP9 cells (Fig. 2a). BCR stimuli can also increase the viability of CLL cells [14, 16]. Similar to co-culture with OP9 cells, IgM crosslinking upregulates the viability of CLL cells in vitro. However, this effect is completely abrogated upon CK2 pharmacological inhibition (Fig. 2b).
Fig. 3 PBMCs from CLL patients were cultured with 10 lM LY294002 (Ly) and viability of CLL cells was assessed after 48 h by AnnexinV/7-AAD staining (n = 32)
Fig. 4 Purified CLL cells were a incubated with 25 lM TBB in PBS and lysed after 2 h or b cultured in medium with 12.5 lM DRB and lysed after 24 and 48 h. Cell lysates were immunoblotted with antibodies against the indicated proteins and their phosphorylated forms. Short-term treatment results, illustrated by primary sample CLL#77, are representative of four patients analyzed; and culture results, illustrated by CLL#11, are representative of three patients analyzed
We next sought to characterize the molecular mechanisms by which CK2 regulates cell survival in CLL. PI3K signaling pathway was previously shown to be essential for CLL cell viability [17, 18]. In agreement, we found that treatment of primary CLL cells with the PI3K inhibitor LY294002 consistently results in leukemia cell apoptosis (Fig. 3). Moreover, CK2 inhibition decreases PTEN phosphorylation, indicative of PTEN activation, and in turn
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Fig. 5 PBMCs were cultured with 25 lM TBB and CLL cell viability was assessed after 48 h by AnnexinV/7-AAD staining. a ZAP70 negative (ZAP70 neg; n = 22) versus ZAP70 positive (ZAP70 pos; n = 17) patients. b IGVH mutated (mutated; n = 20)
versus IGVH unmutated (unmutated; n = 9) patients. c CD38 positive (CD38 pos; n = 14) versus CD38 negative (CD38 neg: n = 12). Results are shown as mean ± SEM. Similar results were obtained with purified CLL cells (data not shown)
this associates with decreased activation of the PI3K downstream target PKC (Fig. 4). Importantly, apoptosis of CLL cells upon CK2 inhibition is dependent on PKC inactivation, since treatment with the PKC activator PMA prevents leukemia cell death after treatment with CK2 antagonists [8]. These results suggest that the pro-survival effect of CK2 may rely, at least in part, on the activation of PI3K/PKC signaling pathway. CLL is characterized by biological, clinical, and cytogenetic heterogeneity. Several clinical and biologic prognostic factors have been used to estimate time to disease progression, need for therapy and overall survival of CLL patients. Among these factors are clinical staging, immunoglobulin heavy chain variable region (IGVH) mutation status, ZAP-70 and CD38 expression, b2 microglobulin levels, cytogenetic abnormalities, lymphocyte doubling time, and leukocyte counts [19]. We did not find any correlation between the effect of CK2 inhibition and ZAP-70 or CD38 expression, or IGVH mutation status (Fig. 5). In contrast, sensitivity to CK2 inhibition positively correlated with the percentage of CLL cells in the peripheral blood, b2 microglobulin serum levels, and Binet clinical stage [8]. Overall, our data indicate that CK2 regulates PI3Kdependent signaling, as determined by PTEN and PKC activation, in CLL cells and is a major modulator of CLL cell viability. These findings establish CK2 as promising target for specific therapeutic intervention in this incurable malignancy.
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Acknowledgments We are grateful to all the patients that made this study possible. This study was supported by the grant PIC/IC/83193/ 2007 from Fundac¸a˜o para a Cieˆncia e a Tecnologia. LRM has an FCT-SFRH PhD fellowship.
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