Limits...
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.

Show MeSH

Related in: MedlinePlus

CK2 inhibition hampers STAT3 activation upon daunorubicin treatment of AML cells. (A) Representative immunoblot analysis of phospho Ser727 STAT3 (pSTAT3 Ser727), total STAT3, MCL1, SOCS3 protein levels in ML-2 cells untreated, exposed to increasing concentrations of daunorubicin (0.05-0.1-0.15 μM) in the absence or presence of CX-4945 5 μM (top panels) or K27 4 μM (bottom panels). (B) Real time quantitative PCR analysis of MCL1 (lefmost panels) and SOCS3 (rightmost panels) mRNA in ML-2 cells untreated, exposed to increasing concentrations of daunorubicin (0.05 and 0.15 μM) in the absence or presence of CX-4945 5 μM (top panels) or K27 4 μM (bottom panels). (C) Immunoblot analysis of phospho Ser727 STAT3 (pSTAT3 Ser727), total STAT3 and MCL1 in freshly isolated AML blasts from a patient left untreated or exposed to increasing concentrations of daunorubicin (0.05-0.1 μM) in the absence or presence of CX-4945 5 μM. In all the experiments data represent mean ± SD, n = 3. * indicates p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3852751&req=5

Figure 7: CK2 inhibition hampers STAT3 activation upon daunorubicin treatment of AML cells. (A) Representative immunoblot analysis of phospho Ser727 STAT3 (pSTAT3 Ser727), total STAT3, MCL1, SOCS3 protein levels in ML-2 cells untreated, exposed to increasing concentrations of daunorubicin (0.05-0.1-0.15 μM) in the absence or presence of CX-4945 5 μM (top panels) or K27 4 μM (bottom panels). (B) Real time quantitative PCR analysis of MCL1 (lefmost panels) and SOCS3 (rightmost panels) mRNA in ML-2 cells untreated, exposed to increasing concentrations of daunorubicin (0.05 and 0.15 μM) in the absence or presence of CX-4945 5 μM (top panels) or K27 4 μM (bottom panels). (C) Immunoblot analysis of phospho Ser727 STAT3 (pSTAT3 Ser727), total STAT3 and MCL1 in freshly isolated AML blasts from a patient left untreated or exposed to increasing concentrations of daunorubicin (0.05-0.1 μM) in the absence or presence of CX-4945 5 μM. In all the experiments data represent mean ± SD, n = 3. * indicates p < 0.05.

Mentions: Previous work by others’ and our group has demonstrated that CK2 may favour STAT3 activation [31]. STAT3 transcription factors could lend to malignant cells the ability to escape apoptosis induced by a variety of external stimuli, including chemotherapeutic drugs, such as doxorubicin [32-34], and have been described to be important in the pathogenesis of myeloid malignancies [35,36]. Thus, we investigated whether CK2 could regulate STAT3 activation and transcriptional activity, which could account for resistance to daunorubicin in AML cells. As shown in the representative immunoblots in Figure 7A, daunorubicin slightly triggered STAT3 phosphorylation on Ser727. Remarkably, this phosphorylation was nearly completely abrogated by the inhibition of CK2 with either 5 μM CX-4945 (top panels) or 4 μM K27 (bottom panels). The STAT3 pathway may exert its anti-apoptotic function at least in part through the transcriptional up regulation of anti-apoptotic genes, like MCL1. Moreover, a reliable STAT3 target gene is SOCS3, a repressor of cytokine signaling which buffer down the JAK/STAT pathway in a negative feedback loop [37-39]. The immunoblot (Figure 7A) as well as QRT-PCR analysis (Figure 7B) of the expression of these two downstream STAT3 targets demonstrated that both CK2 inhibitors were able to strongly down regulate the transcription and protein expression of MCL1 and SOCS3 (Figure 7A and B) (p < 0.05, n = 3). Thus, these results clearly suggest that CK2 inhibition hampers the STAT3-dependent, daunorubicin-elicited anti-apoptotic response in AML cells.


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 inhibition hampers STAT3 activation upon daunorubicin treatment of AML cells. (A) Representative immunoblot analysis of phospho Ser727 STAT3 (pSTAT3 Ser727), total STAT3, MCL1, SOCS3 protein levels in ML-2 cells untreated, exposed to increasing concentrations of daunorubicin (0.05-0.1-0.15 μM) in the absence or presence of CX-4945 5 μM (top panels) or K27 4 μM (bottom panels). (B) Real time quantitative PCR analysis of MCL1 (lefmost panels) and SOCS3 (rightmost panels) mRNA in ML-2 cells untreated, exposed to increasing concentrations of daunorubicin (0.05 and 0.15 μM) in the absence or presence of CX-4945 5 μM (top panels) or K27 4 μM (bottom panels). (C) Immunoblot analysis of phospho Ser727 STAT3 (pSTAT3 Ser727), total STAT3 and MCL1 in freshly isolated AML blasts from a patient left untreated or exposed to increasing concentrations of daunorubicin (0.05-0.1 μM) in the absence or presence of CX-4945 5 μM. In all the experiments data represent mean ± SD, n = 3. * indicates p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852751&req=5

Figure 7: CK2 inhibition hampers STAT3 activation upon daunorubicin treatment of AML cells. (A) Representative immunoblot analysis of phospho Ser727 STAT3 (pSTAT3 Ser727), total STAT3, MCL1, SOCS3 protein levels in ML-2 cells untreated, exposed to increasing concentrations of daunorubicin (0.05-0.1-0.15 μM) in the absence or presence of CX-4945 5 μM (top panels) or K27 4 μM (bottom panels). (B) Real time quantitative PCR analysis of MCL1 (lefmost panels) and SOCS3 (rightmost panels) mRNA in ML-2 cells untreated, exposed to increasing concentrations of daunorubicin (0.05 and 0.15 μM) in the absence or presence of CX-4945 5 μM (top panels) or K27 4 μM (bottom panels). (C) Immunoblot analysis of phospho Ser727 STAT3 (pSTAT3 Ser727), total STAT3 and MCL1 in freshly isolated AML blasts from a patient left untreated or exposed to increasing concentrations of daunorubicin (0.05-0.1 μM) in the absence or presence of CX-4945 5 μM. In all the experiments data represent mean ± SD, n = 3. * indicates p < 0.05.
Mentions: Previous work by others’ and our group has demonstrated that CK2 may favour STAT3 activation [31]. STAT3 transcription factors could lend to malignant cells the ability to escape apoptosis induced by a variety of external stimuli, including chemotherapeutic drugs, such as doxorubicin [32-34], and have been described to be important in the pathogenesis of myeloid malignancies [35,36]. Thus, we investigated whether CK2 could regulate STAT3 activation and transcriptional activity, which could account for resistance to daunorubicin in AML cells. As shown in the representative immunoblots in Figure 7A, daunorubicin slightly triggered STAT3 phosphorylation on Ser727. Remarkably, this phosphorylation was nearly completely abrogated by the inhibition of CK2 with either 5 μM CX-4945 (top panels) or 4 μM K27 (bottom panels). The STAT3 pathway may exert its anti-apoptotic function at least in part through the transcriptional up regulation of anti-apoptotic genes, like MCL1. Moreover, a reliable STAT3 target gene is SOCS3, a repressor of cytokine signaling which buffer down the JAK/STAT pathway in a negative feedback loop [37-39]. The immunoblot (Figure 7A) as well as QRT-PCR analysis (Figure 7B) of the expression of these two downstream STAT3 targets demonstrated that both CK2 inhibitors were able to strongly down regulate the transcription and protein expression of MCL1 and SOCS3 (Figure 7A and B) (p < 0.05, n = 3). Thus, these results clearly suggest that CK2 inhibition hampers the STAT3-dependent, daunorubicin-elicited anti-apoptotic response in AML cells.

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