Casein Kinase 2 Controls the Survival of Normal Thymic and Leukemic Γδ T Cells Via Promotion of AKT Signaling

OPEN Leukemia (2017) 31, 1603–1610 www.nature.com/leu ORIGINAL ARTICLE Casein kinase 2 controls the survival of normal thymic and leukemic γδ T cells via promotion of AKT signaling ST Ribeiro1, M Tesio2,3, JC Ribot1,3, E Macintyre2, JT Barata1 and B Silva-Santos1 The thymus is the major site for normal and leukemic T-cell development. The dissection of the molecular determinants of T-cell survival and differentiation is paramount for the manipulation of healthy or transformed T cells in cancer (immuno)therapy. Casein kinase 2 (CK2) is a serine/threonine protein kinase whose anti-apoptotic functions have been described in various hematological and solid tumors. Here we disclose an unanticipated role of CK2 in healthy human thymocytes that is selective to the γδ T-cell lineage. γδ thymocytes display higher (and T-cell receptor inducible) CK2 activity than their αβ counterparts, and are strikingly sensitive to death upon CK2 inhibition. Mechanistically, we show that CK2 regulates the pro-survival AKT signaling pathway in γδ thymocytes and, importantly, also in γδ T-cell acute lymphoblastic leukemia (T-ALL) cells. When compared with healthy thymocytes or leukemic αβ T cells, γδ T-ALL cells show upregulated CK2 activity, potentiated by CD27 costimulation, and enhanced apoptosis upon CK2 blockade using the chemical inhibitor CX-4945. Critically, this results in inhibition of tumor growth in a xenograft model of human γδ T-ALL. These data identify CK2 as a novel survival determinant of both healthy and leukemic γδ T cells, and may thus greatly impact their therapeutic manipulation. Leukemia (2017) 31, 1603–1610; doi:10.1038/leu.2016.363 INTRODUCTION very poorly studied and lack defined molecular targets, we have T cells develop in the thymus. The dissection of the cell-intrinsic here addressed the potential role of casein kinase 2 (CK2), a and -extrinsic signals that regulate thymocyte survival, prolifera- signaling effector molecule previously implicated in chronic tion and differentiation is critical to understand their potential for lymphocytic leukemia,13,14 multiple myeloma,15 B-ALL,16,17 transformation and to devise new therapies for T-cell acute T-ALL18,19 and other hematological disorders (reviewed in Piazza lymphoblastic leukemia (T-ALL). et al.20). T-cell commitment is coupled to somatic T-cell receptor (TCR) CK2 is a ubiquitous and constitutively activated serine/ rearrangements, generating thymocytes bearing either an αβ or a threonine protein kinase that regulates multiple pathways γδ TCR.1 The expression of a pre-TCR composed of TCRβ and including phosphatidylinositol 3-kinase/AKT and WNT signaling, the invariant pTα chain in αβ thymocyte progenitors results nuclear factor-κB transcription and the DNA damage response.20 in a massive proliferative burst (‘β-selection’) that dictates that CK2 displays pro-survival and anti-apoptotic functions that were αβ T cells largely outnumber their γδ counterparts. Likely a described in several cancer cell types. CK2 is frequently over- consequence, although significant progress has been made expressed or hyperactivated in both solid tumors and hematolo- αβ in our understanding of human T-cell development, gical malignancies, thus making it a promising target for cancer γδ the molecular determinants of thymocytes remain poorly treatment.20 In contrast, the physiological function of CK2 in characterized.1 γδ nontransformed cells is less established. Recent studies in mice Most of what we know about thymic T-cell differentiation have demonstrated that CK2 activity is necessary for peripheral comes from studies performed in mice, showing how various T-cell activation and function: interference with CK2 signaling receptors (namely, TCRγδ, CD27 and LTβR) and downstream impaired CD4+ T-cell activation and differentiation into T helper transcription factors (such as Id3, Sox13, TCF1 and Lef1) control 21 αβ type 2 (Th2) or Th17 cells, , whereas the genetic deletion of CK2 various maturation steps, from divergence from the lineage to + + fl in CD4 Foxp3 regulatory T cells abolished their suppressive the acquisition of effector functions such as pro-in ammatory 22 cytokine production.2–7 In contrast, much less is known about activity against allergy-promoting Th2 cells. However, no human thymic γδ T-cell differentiation. Notwithstanding, we functional role has yet been attributed to CK2 in the human recently showed that interleukin (IL-2) or IL-15 differentiate thymus. fi human γδ thymocytes into cytotoxic type 1 effector T cells, In this study we identi ed a novel role for CK2 in controlling the γδ γδ rendering them highly efficacious against leukemic cells in vitro survival of normal thymocytes and T-ALL cells. We analyzed γδ αβ and in vivo.8,9 CK2 activity in versus thymocytes and T-ALL cells, its γδ thymocytes can themselves transform into leukemic cells, regulation by cell-extrinsic signals, the downstream signaling causing a rare (o10% of all cases) form of T-ALL with distinctive mechanisms and the effect of its inhibition in vitro and in vivo in a clinical features.10–12 Given that malignant γδ T cells have been xenograft model of γδ T-ALL. 1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal and 2Institut Necker Enfants Malades, Hôpital Necker-Enfants Malades, APHP et Université Paris, Paris, France. Correspondence: Professor B Silva-Santos, Instituto de Medicina Molecular, Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal. E-mail: [email protected] 3These two authors contributed equally to this work. Received 17 September 2015; revised 12 October 2016; accepted 18 November 2016; accepted article preview online 30 November 2016; advance online publication, 16 December 2016 CK2 controls γδ thymocyte and T-ALL cell survival ST Ribeiro et al 1604 MATERIALS AND METHODS blasts was measured on samples that showed comparable percentages of Statement of Ethics human engraftment (defined by fluorescence-activated cell sorting analysis based on the expression of CD45 and CD7 antigens). Thymic specimens (from newborn to 15-year-old children) were obtained during pediatric corrective cardiac surgery after parents’ written informed consent. The study was approved by the Ethics Board of Faculdade de Western blot analysis Medicina da Universidade de Lisboa. Primary T-ALL blasts derived from Cell lysates were used for immunobloting as previously described.14 Briefly, diagnostic samples (peripheral blood or bone marrow), obtained the cells were lysed, at 4 °C, in cold lysis buffer (50 mM Tris (pH 7.6), 150 mM after informed content and amplified upon xenografting into NSG EDTA, 1% Nonidet P-40 in phosphate-buffered saline) enriched with (NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ) mice. protease and phosphatase inhibitor cocktails (Roche, Burgess Hill, UK). The total proteins were quantified using a Bradford assay (Bio-Rad, Hercules, ’ Isolation, cell culture and viral transduction CA, USA), following the manufacturer s instructions. Equal amounts of total protein was denatured in Laemmli buffer (Bio-Rad), boiled for 5 min at Thymic T cells were collected after thymus tissue dispersion and separation 95 °C and loaded in a 10% SDS–polyacrylamide gel electrophoresis. After by Histopaque-1077 (Sigma-Aldrich, St Louis, MO, USA) density gradient γδ 4 electrophoretic separation, the proteins were transferred to nitrocellulose separation. TCR -positive T cells were isolated (to 97% purity) by blotting paper (Amersham Biosciences, Little Chalfont, UK). The mem- magnetic positive selection; TCRαβ-positive T cells were isolated (to 496% γδ branes were blocked with 5% bovine serum albumin and 0.5% Tween-20 purity) by magnetic positive selection from the TCR -negative fraction (Sigma-Aldrich) in phosphate-buffered saline and probed with the (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were used as fresh or, indicated primary antibodies overnight. After rinse, the membranes were when indicted, cells were cultured at 37 °C with 5% CO2 in complete 23 probed using appropriate horseradish peroxidase-conjugated secondary RPMI-1640 as previously described on indicated conditions. For long- antibodies and developed by chemiluminescence using the ChemiDoc XRS term in vitro cell culture of thymocytes (7 days), recombinant human IL-2 + imaging system (Bio-Rad). was added to the medium. The PEER T-ALL (DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and MOLT-4T- ALL (ATCC CRL-1582) were cultured in 90% RPMI-1640+10% fetal bovine In vivo mouse experiments serum following the manufacturer’s instructions. When indicated, PEER cell All experimental procedures were performed in strict accordance with the line was transduced using a bicistronic retroviral DNA construct, either recommendations of the European Commission (Directive 2010/63/UE), empty vector (LZRS) expressing only IRES followed by eGFP (LZRS-IRES- French National Committee (87/848) and Portuguese authorities (Decreto- eGFP) or vector co-expressing myrPKB/AKT (constitutively activated AKT) Lei 113/2013) for the care and use of laboratory animals. TCRαβ- or TCRγδ- and eGFP (LZRS-myrPKB/AKT-IRES-eGFP) as previously described.24 To positive T-ALL cells obtained from patient diagnostic were injected into increase the percentage of transduced cells for the following experiments, 8–12-week-old NSG mice (1 × 106 cells/mouse, tail vein injections). Mice GFP+ cells were sorted (≈100% purity) using a FACSAria high-speed cell were monitored weekly by flow cytometry for human leukemic

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