Publicação
CK2 as a potencial novel therapeutic target in chronic lymphocytic leukemia
| Resumo: | Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and is characterized by the accumulation of mature, monoclonal B cells in the blood, bone marrow and secondary lymphoid organs. Clinically, CLL is a very heterogeneous disease with overall survival raging from a few years to decades. Prognostic indicators include immunoglobulin heavy chain variable region (IGVH) mutation status, cytogenetic abnormalities, clinical stage, lymphocyte doubling time, peripheral blood leukocyte counts and β 2 microglobulin levels, and ZAP-70 and CD38 expression. Important advances have been made in identifying inherited and acquired genetic mutations, exploring the role of B cell receptor (BCR) signaling, and understanding the relation between CLL cells and the tumor microenvironment. These advances reveal CLL to be a disease that is reliant on the interplay between inherited, environmental, and host factors. New therapeutic approaches have had a dramatic impact on the outcome of patients with CLL improving overall survival. However, not all patients can tolerate aggressive regimens, high risk groups remain having poor response to treatment, and patients continue to relapse. This defines CLL as an incurable malignancy warranting the development of new therapeutic strategies. In this context, specific inhibition of signaling elements essential for leukemia cell survival offers great promise for the design of more efficient and selective therapies that can bypass resistance mechanisms. The ubiquitous serine/threonine protein kinase CK2, a tetramer consisting of 2 catalytic (α and/or α’) and 2 regulatory β subunits, is highly pleiotropic and intervenes laterally on many signaling pathways. More than 300 CK2 substrates have been identified so far but it is predictable that they can be much more, and that CK2 alone can contribute to the generation of the eukaryotic phosphoproteome more than any other individual protein kinase. CK2 can drive tumorigenesis by different mechanisms, playing a global anti-apoptotic role, enhancing multi-drug resistance, activating the chaperone machinery that protects the onco-kinome, and sustaining neo-vascularization. Overexpression of CK2 has been consistently observed in human cancers, including lung, kidney, head and neck, prostate and breast. Importantly, inhibition of CK2 activity, using specific pharmacological inhibitors or silencing the catalytic subunit, has been shown to decrease the survival of multiple myeloma, acute myeloid leukemia and T-cell acute lymphoblastic leukemia cells. In CLL, the genes that code for CK2α (CSNK2A1) and CK2β (CSNK2B) were identified as a part of a poor prognosis cluster associated with shorter treatment-free survival. Primary CLL cells display constitutive activation of PI3K kinase activity, which appears to be critical for CLL cell survival. PTEN, the main negative regulator of PI3K signaling pathway, can be phosphorylated by CK2 at the C terminus, leading to PTEN functional inactivation and concomitant increased protein stability. We previously showed that CK2 overexpression/hyperactivation in primary T-cell acute lymphoblastic leukemia cells induces PTEN non-deletional posttranslational inactivation by phosphorylation and consequent PI3K pathway hyperactivation. However, the relative expression and functional impact of CK2 in CLL remains to be established. In the first part of this study (Chapter 2), we demonstrate that CK2 plays a critical role in the maintenance of CLL cell viability. Primary CLL cells displayed significantly higher CK2 expression and activity than normal B cells. Furthermore, inhibition of CK2 activity induced apoptosis of leukemia cells without significantly affecting normal B and T lymphocytes. Importantly, this effect is not reversed by co-culture with stromal cells, which are otherwise capable of rescuing CLL cells from in vitro spontaneous apoptosis. CLL cell death upon CK2 inhibition was mediated by inactivation of PKC, a PI3K downstream target, and correlated with increased PTEN activity, indicating that CK2 promotes CLL cell survival at least in part via PI3K-dependent signaling. Although CK2 antagonists induced significant apoptosis of CLL cells in all patient samples analyzed, sensitivity to CK2 blockade positively correlated with the percentage of CLL cells in the peripheral blood, β2 microglobulin serum levels and clinical stage. These data suggest that subsets of patients with aggressive and advanced stage disease may especially benefit from therapeutic strategies targeting CK2 function. Overall, our results identify CK2 as a critical regulator of CLL cell viability and originated two publications in peerreviewed international journals (Martins et al, Blood 2010; Martins et al, Mol Cell Biochem 2011). Given these promising results, we sought to validate CK2 as a therapeutic target for intervention in CLL by testing, in vitro and in mouse models, CK2 inhibitors that are currently in phase I clinical trials for other cancers (Chapter 3). Several inhibitors have been used to modulate CK2 activity in vitro but only two have entered clinical trials, CX4945 (Cylene Pharmaceuticals, USA) and CIGB300 (Center for Genetic Engineering and Biotechnology, Cuba). CX4945 is an orally available ATP-competitive inhibitor of CK2 with a potent and highly specific inhibition of CK2 enzymatic activity. It decreases the viability and proliferation of a broad spectrum of cancer cell lines, and shows antitumor efficacy in subcutaneous xenograft mouse models. In phase I clinical trials, CX4945 induced stable disease in 20% of patients with a variable spectrum of solid tumors. Importantly, adverse effects reported were generally of mild to moderate intensities. CIGB300 is a cyclic peptide which inhibits CK2 phosphorylation by binding to the acidic phosphoacceptor site on CK2 substrates. CIGB300 demonstrated an antiproliferative and pro-apoptotic effect in a variety of tumor cell lines, and elicited significant antitumor effect both in subcutaneous murine syngenic tumors and subcutaneous xenograft mouse models. Notably, phase I clinical trials in cervical cancer showed a significant decrease of the tumor, and the peptide was found to be safe and well tolerated in the dose range studied. In our study, both CX4945 and CIGB300 induced a significant decrease in the viability and proliferation of CLL cell lines with clinically suitable IC50 values. One of these cell lines, MO1043, was further injected subcutaneously in Nude mice and both inhibitors were able to significantly delay tumor development. Importantly, in vivo combination of CX4945 with fludarabine, a commonly used drug in CLL, significantly improved treatment outcome when compared with fludarabine alone, suggesting a potential therapeutic synergism between CX4945 and fludarabine. This could enable a decrease in the administration of fludarabine to patients, and consequently in its adverse effects. In addition, it could also increase the sensitivity of CLL cells to fludarabine – a finding of particular importance for drug-refractory patients. Moreover, both inhibitors showed to promote apoptosis in all primary CLL samples analyzed, in a dose and time-dependent manner, independently of the presence of genetic abnormalities such as 11q deletion. Consistent with our previous findings, clinically-relevant CK2 inhibitors had a greater effect on cells from patients with advance disease and/or high tumor burden. Finally, CK2 inhibition increased PTEN activation and down-modulated the activity of Akt and PKC, two major downstream targets of PI3K oncogenic pathway. Overall, our data indicates that CK2 may be a valuable therapeutic target in CLL and further provide rationale for the initiation of human clinical trials. |
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| Autores principais: | Martins, Leila Raquel Galveias Casquinha Pires |
| Assunto: | Leukemia, Lymphocytic, Chronic, B-Cell Molecular biology Cell biology Therapeutics Teses de doutoramento - 2012 |
| Ano: | 2012 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and is characterized by the accumulation of mature, monoclonal B cells in the blood, bone marrow and secondary lymphoid organs. Clinically, CLL is a very heterogeneous disease with overall survival raging from a few years to decades. Prognostic indicators include immunoglobulin heavy chain variable region (IGVH) mutation status, cytogenetic abnormalities, clinical stage, lymphocyte doubling time, peripheral blood leukocyte counts and β 2 microglobulin levels, and ZAP-70 and CD38 expression. Important advances have been made in identifying inherited and acquired genetic mutations, exploring the role of B cell receptor (BCR) signaling, and understanding the relation between CLL cells and the tumor microenvironment. These advances reveal CLL to be a disease that is reliant on the interplay between inherited, environmental, and host factors. New therapeutic approaches have had a dramatic impact on the outcome of patients with CLL improving overall survival. However, not all patients can tolerate aggressive regimens, high risk groups remain having poor response to treatment, and patients continue to relapse. This defines CLL as an incurable malignancy warranting the development of new therapeutic strategies. In this context, specific inhibition of signaling elements essential for leukemia cell survival offers great promise for the design of more efficient and selective therapies that can bypass resistance mechanisms. The ubiquitous serine/threonine protein kinase CK2, a tetramer consisting of 2 catalytic (α and/or α’) and 2 regulatory β subunits, is highly pleiotropic and intervenes laterally on many signaling pathways. More than 300 CK2 substrates have been identified so far but it is predictable that they can be much more, and that CK2 alone can contribute to the generation of the eukaryotic phosphoproteome more than any other individual protein kinase. CK2 can drive tumorigenesis by different mechanisms, playing a global anti-apoptotic role, enhancing multi-drug resistance, activating the chaperone machinery that protects the onco-kinome, and sustaining neo-vascularization. Overexpression of CK2 has been consistently observed in human cancers, including lung, kidney, head and neck, prostate and breast. Importantly, inhibition of CK2 activity, using specific pharmacological inhibitors or silencing the catalytic subunit, has been shown to decrease the survival of multiple myeloma, acute myeloid leukemia and T-cell acute lymphoblastic leukemia cells. In CLL, the genes that code for CK2α (CSNK2A1) and CK2β (CSNK2B) were identified as a part of a poor prognosis cluster associated with shorter treatment-free survival. Primary CLL cells display constitutive activation of PI3K kinase activity, which appears to be critical for CLL cell survival. PTEN, the main negative regulator of PI3K signaling pathway, can be phosphorylated by CK2 at the C terminus, leading to PTEN functional inactivation and concomitant increased protein stability. We previously showed that CK2 overexpression/hyperactivation in primary T-cell acute lymphoblastic leukemia cells induces PTEN non-deletional posttranslational inactivation by phosphorylation and consequent PI3K pathway hyperactivation. However, the relative expression and functional impact of CK2 in CLL remains to be established. In the first part of this study (Chapter 2), we demonstrate that CK2 plays a critical role in the maintenance of CLL cell viability. Primary CLL cells displayed significantly higher CK2 expression and activity than normal B cells. Furthermore, inhibition of CK2 activity induced apoptosis of leukemia cells without significantly affecting normal B and T lymphocytes. Importantly, this effect is not reversed by co-culture with stromal cells, which are otherwise capable of rescuing CLL cells from in vitro spontaneous apoptosis. CLL cell death upon CK2 inhibition was mediated by inactivation of PKC, a PI3K downstream target, and correlated with increased PTEN activity, indicating that CK2 promotes CLL cell survival at least in part via PI3K-dependent signaling. Although CK2 antagonists induced significant apoptosis of CLL cells in all patient samples analyzed, sensitivity to CK2 blockade positively correlated with the percentage of CLL cells in the peripheral blood, β2 microglobulin serum levels and clinical stage. These data suggest that subsets of patients with aggressive and advanced stage disease may especially benefit from therapeutic strategies targeting CK2 function. Overall, our results identify CK2 as a critical regulator of CLL cell viability and originated two publications in peerreviewed international journals (Martins et al, Blood 2010; Martins et al, Mol Cell Biochem 2011). Given these promising results, we sought to validate CK2 as a therapeutic target for intervention in CLL by testing, in vitro and in mouse models, CK2 inhibitors that are currently in phase I clinical trials for other cancers (Chapter 3). Several inhibitors have been used to modulate CK2 activity in vitro but only two have entered clinical trials, CX4945 (Cylene Pharmaceuticals, USA) and CIGB300 (Center for Genetic Engineering and Biotechnology, Cuba). CX4945 is an orally available ATP-competitive inhibitor of CK2 with a potent and highly specific inhibition of CK2 enzymatic activity. It decreases the viability and proliferation of a broad spectrum of cancer cell lines, and shows antitumor efficacy in subcutaneous xenograft mouse models. In phase I clinical trials, CX4945 induced stable disease in 20% of patients with a variable spectrum of solid tumors. Importantly, adverse effects reported were generally of mild to moderate intensities. CIGB300 is a cyclic peptide which inhibits CK2 phosphorylation by binding to the acidic phosphoacceptor site on CK2 substrates. CIGB300 demonstrated an antiproliferative and pro-apoptotic effect in a variety of tumor cell lines, and elicited significant antitumor effect both in subcutaneous murine syngenic tumors and subcutaneous xenograft mouse models. Notably, phase I clinical trials in cervical cancer showed a significant decrease of the tumor, and the peptide was found to be safe and well tolerated in the dose range studied. In our study, both CX4945 and CIGB300 induced a significant decrease in the viability and proliferation of CLL cell lines with clinically suitable IC50 values. One of these cell lines, MO1043, was further injected subcutaneously in Nude mice and both inhibitors were able to significantly delay tumor development. Importantly, in vivo combination of CX4945 with fludarabine, a commonly used drug in CLL, significantly improved treatment outcome when compared with fludarabine alone, suggesting a potential therapeutic synergism between CX4945 and fludarabine. This could enable a decrease in the administration of fludarabine to patients, and consequently in its adverse effects. In addition, it could also increase the sensitivity of CLL cells to fludarabine – a finding of particular importance for drug-refractory patients. Moreover, both inhibitors showed to promote apoptosis in all primary CLL samples analyzed, in a dose and time-dependent manner, independently of the presence of genetic abnormalities such as 11q deletion. Consistent with our previous findings, clinically-relevant CK2 inhibitors had a greater effect on cells from patients with advance disease and/or high tumor burden. Finally, CK2 inhibition increased PTEN activation and down-modulated the activity of Akt and PKC, two major downstream targets of PI3K oncogenic pathway. Overall, our data indicates that CK2 may be a valuable therapeutic target in CLL and further provide rationale for the initiation of human clinical trials. |
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