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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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Calculation of combination indexes indicates a synergistic cell growth arrest upon treatment of the AML cell line ML-2 with the association of daunorubicin and CK2 inhibitors. (A) Synergistic effect of CX-4945 and daunorubicin on ML-2 cell proliferation. Left graph: dose–response of ML-2 cells incubated for 48 hours with increasing concentrations of daunorubicin (black square) or daunorubicin plus a fixed dose (5 μM) of CX-4945 (white circle). Right graph: dose–response of ML-2 cells incubated for 48 hours with increasing concentrations of CX-4945 (black square) or CX-4945 plus a fixed dose (0.05 μM) of daunorubicin (white square). (B) Synergistic effect of K27 and daunorubicin on ML-2 cell proliferation. Left graph: dose–response of ML-2 cells incubated for 48 hours with increasing concentrations of daunorubicin (black square) or daunorubicin plus a fixed dose (5 μM) of K27 (white circle). Right graph: dose–response of ML-2 cells incubated for 48 hours with increasing concentrations of K27 (black square) or K27 plus a fixed dose (0.05 μM) of daunorubicin (white triangle). The combination index (CI), obtained according to the formula described in the Material and Methods section, was calculated as to be: 0.86 for the daunorubicin plus CX-4945 combination and 0.7 for the daunorubicin plus K27 combination, indicating a synergic effect. In all the experiments data represent mean ± SD, n = 3. * indicates p < 0.01.
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Figure 6: Calculation of combination indexes indicates a synergistic cell growth arrest upon treatment of the AML cell line ML-2 with the association of daunorubicin and CK2 inhibitors. (A) Synergistic effect of CX-4945 and daunorubicin on ML-2 cell proliferation. Left graph: dose–response of ML-2 cells incubated for 48 hours with increasing concentrations of daunorubicin (black square) or daunorubicin plus a fixed dose (5 μM) of CX-4945 (white circle). Right graph: dose–response of ML-2 cells incubated for 48 hours with increasing concentrations of CX-4945 (black square) or CX-4945 plus a fixed dose (0.05 μM) of daunorubicin (white square). (B) Synergistic effect of K27 and daunorubicin on ML-2 cell proliferation. Left graph: dose–response of ML-2 cells incubated for 48 hours with increasing concentrations of daunorubicin (black square) or daunorubicin plus a fixed dose (5 μM) of K27 (white circle). Right graph: dose–response of ML-2 cells incubated for 48 hours with increasing concentrations of K27 (black square) or K27 plus a fixed dose (0.05 μM) of daunorubicin (white triangle). The combination index (CI), obtained according to the formula described in the Material and Methods section, was calculated as to be: 0.86 for the daunorubicin plus CX-4945 combination and 0.7 for the daunorubicin plus K27 combination, indicating a synergic effect. In all the experiments data represent mean ± SD, n = 3. * indicates p < 0.01.

Mentions: To address whether the cooperation in inducing AML cell death between CK2 inhibition and daunorubicin was synergistic, we performed 3H-thymidine incorporation assays evaluating the rate of cell proliferation at increasing concentration of daunorubicin (range: 0.01-0.16 μM), CX-4945 (range: 1–40 μM) and K27 (range: 1–20 μM) and the combination of daunorubicin either with CX-4945 or K27. The results were analyzed to obtain the IC50 for the three agents and the constant ratio drug combination assay was performed, giving the combination indexes (CI) according to the method described in [30]. The results showed that treatment of ML2 AML cells with daunorubicin and CK2 inhibitors was synergic, as judged by the CI well below 1 (0.86 for the combination with CX-4945 and 0.7 for the combination with K27) (Figure 6A and B, respectively).


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)

Calculation of combination indexes indicates a synergistic cell growth arrest upon treatment of the AML cell line ML-2 with the association of daunorubicin and CK2 inhibitors. (A) Synergistic effect of CX-4945 and daunorubicin on ML-2 cell proliferation. Left graph: dose–response of ML-2 cells incubated for 48 hours with increasing concentrations of daunorubicin (black square) or daunorubicin plus a fixed dose (5 μM) of CX-4945 (white circle). Right graph: dose–response of ML-2 cells incubated for 48 hours with increasing concentrations of CX-4945 (black square) or CX-4945 plus a fixed dose (0.05 μM) of daunorubicin (white square). (B) Synergistic effect of K27 and daunorubicin on ML-2 cell proliferation. Left graph: dose–response of ML-2 cells incubated for 48 hours with increasing concentrations of daunorubicin (black square) or daunorubicin plus a fixed dose (5 μM) of K27 (white circle). Right graph: dose–response of ML-2 cells incubated for 48 hours with increasing concentrations of K27 (black square) or K27 plus a fixed dose (0.05 μM) of daunorubicin (white triangle). The combination index (CI), obtained according to the formula described in the Material and Methods section, was calculated as to be: 0.86 for the daunorubicin plus CX-4945 combination and 0.7 for the daunorubicin plus K27 combination, indicating a synergic effect. In all the experiments data represent mean ± SD, n = 3. * indicates p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Calculation of combination indexes indicates a synergistic cell growth arrest upon treatment of the AML cell line ML-2 with the association of daunorubicin and CK2 inhibitors. (A) Synergistic effect of CX-4945 and daunorubicin on ML-2 cell proliferation. Left graph: dose–response of ML-2 cells incubated for 48 hours with increasing concentrations of daunorubicin (black square) or daunorubicin plus a fixed dose (5 μM) of CX-4945 (white circle). Right graph: dose–response of ML-2 cells incubated for 48 hours with increasing concentrations of CX-4945 (black square) or CX-4945 plus a fixed dose (0.05 μM) of daunorubicin (white square). (B) Synergistic effect of K27 and daunorubicin on ML-2 cell proliferation. Left graph: dose–response of ML-2 cells incubated for 48 hours with increasing concentrations of daunorubicin (black square) or daunorubicin plus a fixed dose (5 μM) of K27 (white circle). Right graph: dose–response of ML-2 cells incubated for 48 hours with increasing concentrations of K27 (black square) or K27 plus a fixed dose (0.05 μM) of daunorubicin (white triangle). The combination index (CI), obtained according to the formula described in the Material and Methods section, was calculated as to be: 0.86 for the daunorubicin plus CX-4945 combination and 0.7 for the daunorubicin plus K27 combination, indicating a synergic effect. In all the experiments data represent mean ± SD, n = 3. * indicates p < 0.01.
Mentions: To address whether the cooperation in inducing AML cell death between CK2 inhibition and daunorubicin was synergistic, we performed 3H-thymidine incorporation assays evaluating the rate of cell proliferation at increasing concentration of daunorubicin (range: 0.01-0.16 μM), CX-4945 (range: 1–40 μM) and K27 (range: 1–20 μM) and the combination of daunorubicin either with CX-4945 or K27. The results were analyzed to obtain the IC50 for the three agents and the constant ratio drug combination assay was performed, giving the combination indexes (CI) according to the method described in [30]. The results showed that treatment of ML2 AML cells with daunorubicin and CK2 inhibitors was synergic, as judged by the CI well below 1 (0.86 for the combination with CX-4945 and 0.7 for the combination with K27) (Figure 6A and B, respectively).

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