Casein kinase 2 controls the survival of normal thymic and leukemic γδ T cells via promotion of AKT signaling

Abstract

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.

Figure 1

Human γδ thymocytes have enhanced CK2 activity and are highly sensitive to CX-4945. (a) In vitro CK2α activity (kinase assay) in freshly isolated human thymic γδ and αβ T-cells (2 × 10⁶ cells per assay). CPM, counts per min. (b) Survival (% of live cells) of human thymic γδ, CD4⁺ and CD8⁺ T cells following 24 h of incubation with 5 μM of the CK2 inhibitor, CX-4945, analyzed by flow cytometry using Annexin-V/7-AAD staining. (c) Survival (% of live cells) of human thymic γδ, CD4⁺ and CD8⁺ T cells to different concentrations of CX-4945 (or vehicle), analyzed by Annexin-V/7-AAD staining following 7 days in culture with recombinant human IL-2 (rhIL-2) plus CD3+CD27 or CD3+CD28 stimulation of sorted thymic γδ or αβ T cells, respectively. Data in this figure are representative of at least three independent experiments; **P<0.01, ***P<0.001 (T-test).

Figure 2

CK2 activity in γδ thymocytes is modulated by TCR stimulation and activates AKT signaling. (a) In vitro CK2α activity in sorted γδ and αβ thymocytes (2 × 10⁶ cells per sample) after 6 h of stimulation with anti-CD3 antibodies (CD3), plus soluble CD27-ligand (CD3+CD27) or plus 5 μM CX-4945 (CD3+CD27+CX); values were normalized to unstimulated control (dashed line). (b) Western blot analysis of (phospho)proteins implicated in AKT signaling, in γδ and αβ thymocytes (1 × 10⁶ cells per sample) stimulated as in (a). (c) Proliferation (CFSE dilution assay) of γδ thymocytes after 7 days in culture with recombinant human IL-2 (rhIL-2) under the indicated conditions: medium only (Ctrl); anti-CD3 antibody stimulation (CD3); soluble CD27-ligand (CD27); their combination (CD3+CD27); and with 5 μM CX-4945 (CD3+CD27+CX). (d) Survival (% of live cells) of γδ thymocytes after 7 days of stimulation (or not, Ctrl for control) with anti-CD3 antibodies (CD3), plus soluble CD27-ligand (CD3+CD27), plus 5 μM of CX-4945 or 10 μM of MK-2206. Data in this figure are representative of at least three independent experiments; *P<0.05, ***P<0.001 (T-test).

Figure 3

γδ T-ALL cells display higher CK2 activity than αβ counterparts. In vitro CK2α activity (kinase assay; 6.6 × 10⁶ cells per assay) in freshly isolated γδ (n=4) and αβ (n=4) thymocyte samples; γδ (n=6) and αβ (n=14) T-cell samples obtained from T-ALL patients and expanded in NSG mice (as described in the Materials and methods); and the γδ T-ALL cell line, PEER (n=4). T-test, *P<0.05, ***P<0.001.

Figure 4

CK2 activity in γδ T-ALL cells is potentiated by CD27 costimulation and promotes AKT signaling. (a) CK2α activity in the γδ T-ALL cell line, PEER (2 × 10⁶ cells per condition), after 6 h of treatment with indicated concentrations of CX-4945. (b) CK2α activity in lysates from γδ T-ALL (PEER) cells (2 × 10⁶ cells per condition) after 6 h of stimulation under the indicated conditions (T-test, *P<0.05; **P<0.01). (c) Western blot analysis of (phospho)proteins implicated in AKT signaling, in γδ T-ALL (PEER) cells treated for 6 h with anti-CD3 antibodies (CD3), plus soluble CD27-ligand (CD3+CD27) or plus 5 μM CX-4945 (CD3+CD27+CX). Data are representative of five independent experiments. (d) Flow cytometry analysis of apoptosis (Annexin-V⁺; left panel), cell cycle/DNA staining (middle panel) and intracellular Bcl-2 protein staining (right panel; values indicate mean fluorescence intensity (MFI)) of γδ T-ALL (PEER) cells treated with CX-4945 (5 μM) during the indicated times. (e, f) Western blot analysis of phospho-AKT (and calnexin loading control) (e) and cell survival after 48 h (f) of PEER cells transduced with a bicistronic retroviral DNA construct: either empty vector (LZRS) expressing only IRES followed by eGFP (LZRS-IRES-eGFP) or vector co-expressing myrPKB/AKT and eGFP (LZRS-myrPKB/AKT-IRES-eGFP) (AKT^hi) and treated with 3 μM CX-4945 or vehicle (T-test, *P<0.05).

Figure 5

γδ T-ALL cells are more susceptible than αβ T-ALL to apoptosis induced by CX-4945. Flow cytometry analysis of the survival (Annexin-V/7-AAD staining) of (a) γδ (n=5) and αβ (n=5) T-cell blast samples (obtained from T-ALL patients and expanded in NSG mice) or (b) γδ (PEER) or αβ (MOLT-4) T-ALL cell lines, cultured for the indicated times with increasing concentrations of CX-4945 (T-test, *P<0.05; ***P<0.001).

Figure 6

CX-4945 treatment inhibits γδ T-ALL growth in vivo. (a) Tumor volume following injection of 2 × 10⁶ PEER γδ T-ALL cells subcutaneously into NRGS mice, treated with 75 mg/kg CX-4945 or vehicle (T-test, *P<0.05, **P<0.01). Day 0 refers to the start of treatment of mice bearing palpable tumors. (b–e) Tumor weight (b) or percentage of CD45⁺ CD7⁺ γδ T-ALL cells in the blood (c), bone marrow (d) or spleen (e) of mice killed after 18 days of treatment. Each dot represents an animal; T-test P-values are indicated.