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Inhibition of protein kinase CK2 with the clinical-grade small ATP-competitive compound CX-4945 or by RNA interference unveils its role in acute myeloid leukemia cell survival, p53-dependent apoptosis and daunorubicin-induced cytotoxicity.

Quotti Tubi L, Gurrieri C, Brancalion A, Bonaldi L, Bertorelle R, Manni S, Pavan L, Lessi F, Zambello R, Trentin L, Adami F, Ruzzene M, Pinna LA, Semenzato G, Piazza F - J Hematol Oncol (2013)

Bottom Line: CK2a was found highly expressed in the majority of samples across the different acute myeloid leukemia prognostic subgroups as compared to normal CD34+ hematopoietic and bone marrow cells.CK2 inhibition was associated with a synergistic increase of the cytotoxic effects of daunorubicin.These results suggest that CK2 is over expressed across the different acute myeloid leukemia subsets and acts as an important regulator of acute myeloid leukemia cell survival.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: The involvement of protein kinase CK2 in sustaining cancer cell survival could have implications also in the resistance to conventional and unconventional therapies. Moreover, CK2 role in blood tumors is rapidly emerging and this kinase has been recognized as a potential therapeutic target. Phase I clinical trials with the oral small ATP-competitive CK2 inhibitor CX-4945 are currently ongoing in solid tumors and multiple myeloma.

Methods: We have analyzed the expression of CK2 in acute myeloid leukemia and its function in cell growth and in the response to the chemotherapeutic agent daunorubicin We employed acute myeloid leukemia cell lines and primary blasts from patients grouped according to the European LeukemiaNet risk classification. Cell survival, apoptosis and sensitivity to daunorubicin were assessed by different means. p53-dependent CK2-inhibition-induced apoptosis was investigated in p53 wild-type and mutant cells.

Results: CK2a was found highly expressed in the majority of samples across the different acute myeloid leukemia prognostic subgroups as compared to normal CD34+ hematopoietic and bone marrow cells. Inhibition of CK2 with CX-4945, K27 or siRNAs caused a p53-dependent acute myeloid leukemia cell apoptosis. CK2 inhibition was associated with a synergistic increase of the cytotoxic effects of daunorubicin. Baseline and daunorubicin-induced STAT3 activation was hampered upon CK2 blockade.

Conclusions: These results suggest that CK2 is over expressed across the different acute myeloid leukemia subsets and acts as an important regulator of acute myeloid leukemia cell survival. CK2 negative regulation of the protein levels of tumor suppressor p53 and activation of the STAT3 anti-apoptotic pathway might antagonize apoptosis and could be involved in acute myeloid leukemia cell resistance to daunorubicin.

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CK2 expression and activity in AML cells. (A) Real-time quantitative PCR analysis of CK2α mRNA expression in a panel of AML cell lines (K562, HL-60, NB4, ML2) and in normal CD34+ hematopoietic stem cells. (B) Top: representative western blot analysis of CK2α protein expression in a panel of AML cell lines (K562, HL-60, NB4, ML2) and in normal CD34+ hematopoietic stem cells; bottom: corresponding densitometric analysis. (C) In vitro kinase assay measuring CK2 kinase activity against a synthetic peptide using cell lysates from AML cell lines and normal CD34+ hematopoietic stem cells. (D) Western blot analysis of CK2α expression in normal - or patient derived AML - peripheral blood or bone marrow cells. Thirty-one AML cases were divided according to the European Leukemia Net classification of risk groups in favourable, intermediate-I, intermediate-II and unfavourable. Immunoblots are shown on the upper panel while the corresponding densitometric analysis is shown on the lower panel.
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Figure 1: CK2 expression and activity in AML cells. (A) Real-time quantitative PCR analysis of CK2α mRNA expression in a panel of AML cell lines (K562, HL-60, NB4, ML2) and in normal CD34+ hematopoietic stem cells. (B) Top: representative western blot analysis of CK2α protein expression in a panel of AML cell lines (K562, HL-60, NB4, ML2) and in normal CD34+ hematopoietic stem cells; bottom: corresponding densitometric analysis. (C) In vitro kinase assay measuring CK2 kinase activity against a synthetic peptide using cell lysates from AML cell lines and normal CD34+ hematopoietic stem cells. (D) Western blot analysis of CK2α expression in normal - or patient derived AML - peripheral blood or bone marrow cells. Thirty-one AML cases were divided according to the European Leukemia Net classification of risk groups in favourable, intermediate-I, intermediate-II and unfavourable. Immunoblots are shown on the upper panel while the corresponding densitometric analysis is shown on the lower panel.

Mentions: CK2 is over expressed in several solid tumor cells. Kim et al. reported high expression of CK2 also in a subset of AML [22]. In this report, AML cases were grouped according to normal and abnormal karyotype and no differential CK2 expression was observed among the subgroups with abnormal karyotype. Here, we analyzed CK2 expression in AML cell lines and AML cells from patients classified according to the European LeukemiaNet (ELN) classification, which distinguishes different prognostic groups according to cytogenetic alterations and mutations to specific genes [23]. Firstly, quantitative RT-PCR was performed in different cell lines, including K562, NB4, HL-60 and ML2, and normal CD34+ hematopoietic cells in order to assess CK2α mRNA levels. As shown in Figure 1A, CK2α mRNA was much higher in AML cell lines as compared to normal CD34+ hemopoietic cells. Among the different AML cell lines, K562 was the one displaying the highest CK2α mRNA levels (up to fourteen-fold more as compared to CD34+ stem cells); NB4, HL-60 and ML2 showed intermediate (up to seven-fold more as compared to CD34+ cells) CK2α levels. CK2α protein levels and CK2 kinase activity were also measured in AML cell lines and CD34+ cells (Figure 1B and C). Differently than for the mRNA levels, CK2α protein and activity were found high in K562, ML2 and NB4 but much lower in HL-60 cells. Similar results were obtained when CK2α mRNA and protein levels were compared in AML cells lines and in peripheral blood or bone marrow mononuclear cells (Additional file 1: Figure S1). Next, by Western blot (WB) analysis we analyzed CK2α protein expression across normal peripheral blood or bone marrow cells and primary AML blasts from AML patients. The clinical, biological and genetic features of the samples analyzed are summarized in Table 1. As shown in Figure 1D, CK2α expression was higher in blasts of most of the AML cases, but not all, as compared to normal cells. These results are in accordance with previous observations cited above [22]. Perhaps due to the relatively low number of patients analyzed, we could not detect statistically significant differences among the different ELN AML subgroups upon quantification and densitometric analysis. To note, due to the collection of the samples over different times, four different blots are shown, each created and analyzed by densitometry separately. Thus, the values shown are quite different among the four experiments.


Inhibition of protein kinase CK2 with the clinical-grade small ATP-competitive compound CX-4945 or by RNA interference unveils its role in acute myeloid leukemia cell survival, p53-dependent apoptosis and daunorubicin-induced cytotoxicity.

Quotti Tubi L, Gurrieri C, Brancalion A, Bonaldi L, Bertorelle R, Manni S, Pavan L, Lessi F, Zambello R, Trentin L, Adami F, Ruzzene M, Pinna LA, Semenzato G, Piazza F - J Hematol Oncol (2013)

CK2 expression and activity in AML cells. (A) Real-time quantitative PCR analysis of CK2α mRNA expression in a panel of AML cell lines (K562, HL-60, NB4, ML2) and in normal CD34+ hematopoietic stem cells. (B) Top: representative western blot analysis of CK2α protein expression in a panel of AML cell lines (K562, HL-60, NB4, ML2) and in normal CD34+ hematopoietic stem cells; bottom: corresponding densitometric analysis. (C) In vitro kinase assay measuring CK2 kinase activity against a synthetic peptide using cell lysates from AML cell lines and normal CD34+ hematopoietic stem cells. (D) Western blot analysis of CK2α expression in normal - or patient derived AML - peripheral blood or bone marrow cells. Thirty-one AML cases were divided according to the European Leukemia Net classification of risk groups in favourable, intermediate-I, intermediate-II and unfavourable. Immunoblots are shown on the upper panel while the corresponding densitometric analysis is shown on the lower panel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: CK2 expression and activity in AML cells. (A) Real-time quantitative PCR analysis of CK2α mRNA expression in a panel of AML cell lines (K562, HL-60, NB4, ML2) and in normal CD34+ hematopoietic stem cells. (B) Top: representative western blot analysis of CK2α protein expression in a panel of AML cell lines (K562, HL-60, NB4, ML2) and in normal CD34+ hematopoietic stem cells; bottom: corresponding densitometric analysis. (C) In vitro kinase assay measuring CK2 kinase activity against a synthetic peptide using cell lysates from AML cell lines and normal CD34+ hematopoietic stem cells. (D) Western blot analysis of CK2α expression in normal - or patient derived AML - peripheral blood or bone marrow cells. Thirty-one AML cases were divided according to the European Leukemia Net classification of risk groups in favourable, intermediate-I, intermediate-II and unfavourable. Immunoblots are shown on the upper panel while the corresponding densitometric analysis is shown on the lower panel.
Mentions: CK2 is over expressed in several solid tumor cells. Kim et al. reported high expression of CK2 also in a subset of AML [22]. In this report, AML cases were grouped according to normal and abnormal karyotype and no differential CK2 expression was observed among the subgroups with abnormal karyotype. Here, we analyzed CK2 expression in AML cell lines and AML cells from patients classified according to the European LeukemiaNet (ELN) classification, which distinguishes different prognostic groups according to cytogenetic alterations and mutations to specific genes [23]. Firstly, quantitative RT-PCR was performed in different cell lines, including K562, NB4, HL-60 and ML2, and normal CD34+ hematopoietic cells in order to assess CK2α mRNA levels. As shown in Figure 1A, CK2α mRNA was much higher in AML cell lines as compared to normal CD34+ hemopoietic cells. Among the different AML cell lines, K562 was the one displaying the highest CK2α mRNA levels (up to fourteen-fold more as compared to CD34+ stem cells); NB4, HL-60 and ML2 showed intermediate (up to seven-fold more as compared to CD34+ cells) CK2α levels. CK2α protein levels and CK2 kinase activity were also measured in AML cell lines and CD34+ cells (Figure 1B and C). Differently than for the mRNA levels, CK2α protein and activity were found high in K562, ML2 and NB4 but much lower in HL-60 cells. Similar results were obtained when CK2α mRNA and protein levels were compared in AML cells lines and in peripheral blood or bone marrow mononuclear cells (Additional file 1: Figure S1). Next, by Western blot (WB) analysis we analyzed CK2α protein expression across normal peripheral blood or bone marrow cells and primary AML blasts from AML patients. The clinical, biological and genetic features of the samples analyzed are summarized in Table 1. As shown in Figure 1D, CK2α expression was higher in blasts of most of the AML cases, but not all, as compared to normal cells. These results are in accordance with previous observations cited above [22]. Perhaps due to the relatively low number of patients analyzed, we could not detect statistically significant differences among the different ELN AML subgroups upon quantification and densitometric analysis. To note, due to the collection of the samples over different times, four different blots are shown, each created and analyzed by densitometry separately. Thus, the values shown are quite different among the four experiments.

Bottom Line: CK2a was found highly expressed in the majority of samples across the different acute myeloid leukemia prognostic subgroups as compared to normal CD34+ hematopoietic and bone marrow cells.CK2 inhibition was associated with a synergistic increase of the cytotoxic effects of daunorubicin.These results suggest that CK2 is over expressed across the different acute myeloid leukemia subsets and acts as an important regulator of acute myeloid leukemia cell survival.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: The involvement of protein kinase CK2 in sustaining cancer cell survival could have implications also in the resistance to conventional and unconventional therapies. Moreover, CK2 role in blood tumors is rapidly emerging and this kinase has been recognized as a potential therapeutic target. Phase I clinical trials with the oral small ATP-competitive CK2 inhibitor CX-4945 are currently ongoing in solid tumors and multiple myeloma.

Methods: We have analyzed the expression of CK2 in acute myeloid leukemia and its function in cell growth and in the response to the chemotherapeutic agent daunorubicin We employed acute myeloid leukemia cell lines and primary blasts from patients grouped according to the European LeukemiaNet risk classification. Cell survival, apoptosis and sensitivity to daunorubicin were assessed by different means. p53-dependent CK2-inhibition-induced apoptosis was investigated in p53 wild-type and mutant cells.

Results: CK2a was found highly expressed in the majority of samples across the different acute myeloid leukemia prognostic subgroups as compared to normal CD34+ hematopoietic and bone marrow cells. Inhibition of CK2 with CX-4945, K27 or siRNAs caused a p53-dependent acute myeloid leukemia cell apoptosis. CK2 inhibition was associated with a synergistic increase of the cytotoxic effects of daunorubicin. Baseline and daunorubicin-induced STAT3 activation was hampered upon CK2 blockade.

Conclusions: These results suggest that CK2 is over expressed across the different acute myeloid leukemia subsets and acts as an important regulator of acute myeloid leukemia cell survival. CK2 negative regulation of the protein levels of tumor suppressor p53 and activation of the STAT3 anti-apoptotic pathway might antagonize apoptosis and could be involved in acute myeloid leukemia cell resistance to daunorubicin.

Show MeSH
Related in: MedlinePlus