Clinical-Grade Peptide-Based Inhibition of CK2 Blocks Viability and Proliferation of T-ALL Cells and Counteracts IL-7 Stimulation and Stromal Support

Abstract

Despite remarkable advances in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), relapsed cases are still a major challenge. Moreover, even successful cases often face long-term treatment-associated toxicities. Targeted therapeutics may overcome these limitations. We have previously demonstrated that casein kinase 2 (CK2)-mediated phosphatase and tensin homologue (PTEN) posttranslational inactivation, and consequent phosphatidylinositol 3-kinase (PI3K)/Akt signaling hyperactivation, leads to increased T-ALL cell survival and proliferation. We also revealed the existence of a crosstalk between CK2 activity and the signaling mediated by interleukin 7 (IL-7), a critical leukemia-supportive cytokine. Here, we evaluated the impact of CIGB-300, a the clinical-grade peptide-based CK2 inhibitor CIGB-300 on T-ALL biology. We demonstrate that CIGB-300 decreases the viability and proliferation of T-ALL cell lines and diagnostic patient samples. Moreover, CIGB-300 overcomes IL-7-mediated T-ALL cell growth and viability, while preventing the positive effects of OP9-delta-like 1 (DL1) stromal support on leukemia cells. Signaling and pull-down experiments indicate that the CK2 substrate nucleophosmin 1 (B23/NPM1) and CK2 itself are the molecular targets for CIGB-300 in T-ALL cells. However, B23/NPM1 silencing only partially recapitulates the anti-leukemia effects of the peptide, suggesting that CIGB-300-mediated direct binding to CK2, and consequent CK2 inactivation, is the mechanism by which CIGB-300 downregulates PTEN S380 phosphorylation and inhibits PI3K/Akt signaling pathway. In the context of IL-7 stimulation, CIGB-300 blocks janus kinase / signal transducer and activator of transcription (JAK/STAT) signaling pathway in T-ALL cells. Altogether, our results strengthen the case for anti-CK2 therapeutic intervention in T-ALL, demonstrating that CIGB-300 (given its ability to circumvent the effects of pro-leukemic microenvironmental cues) may be a valid tool for clinical intervention in this aggressive malignancy.

Keywords: CIGB-300; Casein kinase 2 (CK2); IL-7 receptor (IL-7R); IL-7-mediated signaling; Signaling therapies.; Stromal support; T-cell acute lymphoblastic leukemia (T-ALL).

Figure 1

CIGB-300 negatively impacts the viability and proliferation of T-ALL cells. The indicated T-ALL cell lines were cultured for 72 h with increasing concentrations of CIGB-300. (A) Viability/proliferation was evaluated by Alamar Blue. (B) Proliferation was measured by ³H-thymidine incorporation. (C) Viability of HPB-ALL and MOLT4 cells was evaluated by Annexin V-APC conjugate/ 7-aminoactinomycin D (7-AAD) staining after 16 h in the presence of CIGB-300 at 18 µM. (D) Detection of poly (ADP-ribose) polymerase (PARP) and caspase-3 cleavage was determined in HPB-ALL cells by western blot after treatment with CIGB-300 (18 µM), CX-4945 (12 µM) or control peptide F20-2 (18 µM) for the indicated time intervals. Relative densitometry analysis values of cleaved PARP and cleaved caspase 3 (17 kDa) bands, normalized to medium, are indicated. Results from (A,B) represent mean ± SD of 3 replicates, while those from (C,D) are representative of 2 independent experiments (2 replicates each).

Figure 2

Effect of CIGB-300 on CK2-mediated phosphorylation of Akt, PTEN, B23/NPM1 and their potential interactions in HPB-ALL cells. (A) Phosphorylated and total protein levels of CK2 substrates Akt, PTEN, and B23/NPM1, and PI3K/Akt pathway members, in HPB-ALL cells incubated with either 18 µM of CIGB-300 or the inhibitors CX-4945 (CX, 12 µM) and LY294002 (LY, 10 µM) for the indicated time. Actin was used as loading control. (B) Immunoblots from pull-down fractions using CIGB-300 conjugated to biotin as bait to capture interacting proteins. HPB-ALL cells were incubated for 1 h with biotin-tagged peptide (50 µM), subsequently lysed and submitted to SDS-PAGE and antibody detection as indicated. The experiments were performed in RPMI with or without 10% FBS (R10 or R0, respectively), as indicated. (C) Pull-down performed with cellular lysates of HPB-ALL cells as indicated above. CE, Cellular Extract. PD, Pull-Down fractions. PASS, flow-through fractions. NC, negative control (cellular lysate from HPB-ALL cells incubated with vehicle).

Figure 3

Silencing of NPM1 does not mimic the effects of CIGB-300 on HPB-ALL cells. HPB-ALL cells were transduced with mock vector or shRNA against NPM1 and analyzed by flow cytometry at indicated time intervals. (A) Percentage of transduced cells on live-cell populations, as identified by forward scatter (FSC) × side scatter (SSC) discrimination (R1 gate in dot plots on the left), was determined by analysis of GFP expression at day 9 post-infection (histograms on the right). Percentage of GFP-positive cells was calculated using untransduced cells as a negative control. (B) Immunoblot analysis of transduced cells showing B23/NPM1 protein knock down in total unsorted population (~60% decrease) and sorted GFP-positive cells (~90% decrease). Actin was used as a loading control. Relative densitometry analysis values of B23/NPM bands normalized to actin and then to either untransduced (unsorted cells) or LV-pLG (sorted cells) lanes are indicated. (C,D) Analysis of (C) GFP expression within the live cell population and (D) viability of HPB-ALL cells at the indicated time points after transduction. (E) Cytotoxic effect of CIGB-300 (18 µM) on LV-pLG or LV-shRNA NPM1 transduced HPB-ALL cells as assessed by propidium iodide (PI) staining and flow cytometry analysis.

Figure 4

CIGB-300 decreases the viability and proliferation of T-ALL cells irrespectively of IL-7-stimulation. (A) Evaluation of HPB-ALL cell viability by FSC × SSC discrimination and flow cytometry analysis after stimulation with IL-7 (50 ng/mL) for 48 h. (B) Evaluation of HPB-ALL cell viability by 7-AAD staining and flow cytometry analysis, after 48 h of incubation with 18 µM of CIGB-300 in the presence or absence of IL-7 (50 ng/mL). (C) Immunoblot analysis of PI3K/Akt and JAK/STAT signaling pathway activation. HPB-ALL cells cultured for 24 h in low serum (R1) were pre-incubated for 15 min with the CIGB-300 or F20-2 (18 µM) and then incubated with IL-7 (50 ng/mL) or medium for 45 min or 3h 45min (for a total of 1 or 4 h). Control cells were (pre)incubated with vehicle alone for a total of 1h. (D,E) TAIL7 cells were incubated with different concentrations of CIGB-300 with or without IL-7 (20 ng/mL) and cell viability and proliferation were measured by Alamar Blue (D) and ³H-thymidine incorporation (E) at 48 h. (F) Annexin V-APC/7-AAD dot plots of HPB-ALL cells treated as above and incubated with CIGB-300 at 18 µM for 16 h. (G) Effect of CIGB-300 on the phosphorylation of indicated proteins in TAIL7 cells stimulated or not with IL-7 (20 ng/mL). The cells were pre-incubated during 15 min with CIGB-300 (18 µM) or vehicle in medium (R1), and then IL-7 or medium were added to complete 1 h of incubation.

Figure 5

CIGB-300 decreases the viability of T-ALL cells co-cultured with OP9-hDL1 stromal cells. MOLT4 and HPB-ALL cells were cultured alone or with OP9-hDL1 cells for 48 h with or without the indicated concentrations of CIGB-300. Cell viability was determined by (A) Annexin V-APC/7-AAD staining and flow cytometry, or (B) Trypan blue staining and light microscopy. Results shown in (A) are mean ± SD of triplicates. At least two independent experiments were performed on each case. ns, not significant; **** p-value < 0.0001.

Figure 6

Primary T-ALL patient cells are sensitive to CIGB-300 even in the presence of IL-7. (A,B) T-ALL cells isolated from peripheral blood or bone marrow of pediatric patients at diagnosis were incubated with or without IL-7 and the indicated concentrations of CIGB-300 for 48 h and subsequently analyzed by flow cytometry to determine cell viability. (A) Percentage of viable cells for each patient sample in the indicated conditions. Impact of IL-7 stimulation on primary T-ALL cell viability is shown as fold change compared to unstimulated cells. (B) FSC × SSC dot plots of two representative primary T-ALL samples. Percentage of viable cells is indicated. (C) Immunoblots with indicated antibodies using lysates from primary T-ALL cells from Patient #179. Cells were pre-incubated for 15 min with or without CIGB-300 (30 µM) and then IL-7 (20 ng/mL) was added for 45 min.

Figure 7

Model of CIGB-300-mediated effects on T-ALL cells. (A) T-ALL cell viability and proliferation relies on cell-intrinsic signals and extracellular cues such as IL-7, which activate PI3K/Akt/mTOR and JAK/STAT pathways. CK2 phosphorylates and thereby inactivates PTEN leading to activation of PI3K/Akt/mTOR signaling [2]. This effect is reinforced by CK2-mediated direct phosphorylation of Akt [21]. In addition, CK2 binds to IL-7R (this interaction is not shown in the cartoon), which is essential for IL-7-mediated maximal signaling [25]. (B) In the present study, we showed that CIGB-300, a plasma membrane-crossing CK2 peptide antagonist [32], binds to and inhibits B23/NPM1 (which does not appear to be critical for CIGB-300 anti-T-ALL effects) and to CK2. The effects of CIGB-300 in T-ALL cells appear to be mediated mainly by its ability to inhibit CK2 activation, which results in reversion of cell-intrinsic and IL-7-mediated activation of PI3K/Akt/mTOR pathway as well as abrogation of IL-7-mediated JAK/STAT signaling. As a consequence of these effects, T-ALL cells enter apoptosis and no longer proliferate.