Regulation of Ikaros Function by CK2 Kinase During Lymphocyte Differentiation and Leukemia

Abstract Abstract 4196 Ikaros encodes a DNA-binding zinc finger protein that functions as a master regulator of lymphocyte differentiation and acts as a tumor suppressor in leukemia. The loss of Ikaros activity has been associated with both B and T cell leukemia and deletion of Ikaros has been associated with poor outcome in childhood ALL. Ikaros function depends on its ability to localize to pericentromeric heterochromatin (PC-HC). Ikaros protein binds to the upstream regulatory elements of target genes, aids in their recruitment to PC-HC, and regulates their transcription. Ikaros protein interacts with histone deacetylation complex and represses transcription of its target genes via chromatin remodeling. We have previously reported that Ikaros' activity and protein stability is regulated by pro-oncogenic CK2 kinase (Popescu et al. J Biol Chem 2009 284:13869). Here we studied the effect of CK2-mediated phosphorylation on Ikaros function in primary thymocytes. Using Ikaros phosphomimetic and phosphoresistant mutants of CK2 phosphorylation sites we demonstrate that Ikaros proteins with phosphomimetic mutations at CK2 phosphorylation sites 1) have decreased DNA-binding affinity for the promoter of the terminal deoxynucleotidetransferase (TdT) gene, and other Ikaros target genes involved in lymphocyte differentiation; 2) lose the ability to associate with Sin3a, a component of the NuRD histone deacetylase complex, as indicated by co-immunoprecipitation assays and 3) fail to repress genes involved in thymocyte differentiation as indicated by luciferase reporter assay. The introduction of phosphoresistant mutations at five N-terminal CK2 phosphorylation sites on the Ikaros protein restored Ikaros' association with Sin3a, as well as wild-type levels of repressor activity. Treatment of primary thymocytes with specific inhibitors of CK2 kinase (TBB or DMAT) resulted in increased Ikaros' DNA-binding affinity to the promoter regions of its target genes as measured by quantitative chromatin immunoprecipitation, similar to the results with phosphoresistant mutants. We tested the effect of CK2 kinase inhibition on Ikaros' activity in human leukemia cells. Inhibition of CK2 kinase with TBB led to increased Ikaros' DNA-binding affinity and increased repression of Ikaros target genes. Increased Ikaros activity following inhibition of CK2 kinase was associated with increased sensitivity of human leukemia cells to Doxorubicin or radiation. In summary, these results demonstrate that CK2 kinase regulates thymocyte differentiation by controlling Ikaros' association with chromatin remodeling complexes and its ability to repress the transcription of developmentally regulated genes. Results suggest that CK2 kinase exerts its pro-oncogenic activity in human leukemia cells by inhibiting Ikaros' function as a tumor suppressor. Inhibition of CK2 kinase restores Ikaros function in thymocyte differentiation, along with its tumor suppressor activity and led to increased sensitivity of human leukemia cells to chemotherapy and/or radiation treatment. Thus, inhibition of the CK2 kinase pathway is a promising therapeutic target for human leukemia. Disclosures: No relevant conflicts of interest to declare.

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DNA binding ability and cytotoxicity, cell cycle arrest and apoptosis inducing properties of a benzochromene derivative against K562 human leukemia cells

Nucleosides Nucleotides & Nucleic Acids ◽

10.1080/15257770.2021.1937644 ◽

2021 ◽

pp. 1-22

Author(s):

Mina Hanifeh Ahagh ◽

Gholamreza Dehghan ◽

Majid Mahdavi ◽

Mohammad Ali Hosseinpour Feizi ◽

Reza Teimuri-Mofrad ◽

...

Keyword(s):

Cell Cycle ◽

Dna Binding ◽

Cell Cycle Arrest ◽

Leukemia Cells ◽

Human Leukemia ◽

Binding Ability ◽

Cycle Arrest ◽

Human Leukemia Cells

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Enforced expression of the tumor suppressor p53 renders human leukemia cells (U937) more sensitive to 1-[β-d-arabinofuranosyl]cytosine (ara-C)-induced apoptosis

Biochemical Pharmacology ◽

10.1016/s0006-2952(03)00149-7 ◽

2003 ◽

Vol 65 (12) ◽

pp. 1997-2008 ◽

Cited By ~ 4

Author(s):

Roy H. Decker ◽

Jay Levin ◽

Lora B. Kramer ◽

Yun Dai ◽

Steven Grant

Keyword(s):

Tumor Suppressor ◽

Leukemia Cells ◽

Human Leukemia ◽

Tumor Suppressor P53 ◽

Human Leukemia Cells ◽

Induced Apoptosis

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c-Myb-Interacting Proteins and Their Functions.

Blood ◽

10.1182/blood.v108.11.1423.1423 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1423-1423

Author(s):

Shenghao Jin ◽

Huiwu Zhao ◽

Alan M. Gewirtz

Keyword(s):

Dna Binding ◽

Molecular Mechanisms ◽

Histone Methyltransferase ◽

Leukemic Cells ◽

Leukemia Cells ◽

Luciferase Reporter ◽

Human Leukemia ◽

New Paradigm ◽

Avian Myeloblastosis Virus ◽

Human Leukemia Cells

Abstract Post-translational modifications of chromatin have fundamental roles in many biological processes including transcriptional regulation, heterochromatin organization, and x-chromosome inactivation. One of these modifications, histone methylation, is recognized as an important component of an epigenetic indexing system demarcating transcriptionally active and inactive domains. It is possible that methylation induces alterations in chromatin architecture, either condensing or relaxing its structure. These alterations not only regulate accessibility for DNA binding proteins, but also create binding sites for regulatory proteins that contain specialized binding domains. The c-Myb gene is the normal cellular counterpart of an oncogene transduced by the avian myeloblastosis virus. It is a nuclear protein that functions as a DNA binding transcription factor and a number of complementary gene silencing strategies have demonstrated that it plays a pivotal role during hematopoietic cell development and is an obligate requirement for adult hematopoiesis. c-Myb is highly expressed in immature hematopoietic cells and down-regulated upon differentiation. It has also been shown that c-Myb expression is important in promoting the proliferation of leukemic cells. However, the biologically relevant molecular mechanisms that regulate c-Myb activity during normal and leukemic hematopoiesis remain unclear. In order to investigate c-Myb function in leukemic vs non-leukemic cells, and ultimately in normal cells, more mechanistically, we expressed epitope-tagged c-Myb in transiently transfected 293T cells, and both transiently and stably transfected K562 human leukemia cells. We then carried out a series of immunoprecipitation experiments to examine proteins that c-Myb might interact with in both environments. In accord with prior reports, we found that c-Myb is associated with the tumor suppressor protein menin, a product of the MEN1 gene that is mutated in familial multiple endocrine neoplasia type 1. Menin has been reported to associate with a Trithorax family histone methyltransferase complex, and to be an essential oncogenic cofactor for MLL-associated leukemogenesis. Stable interaction between c-Myb and menin was confirmed by reciprocal immunoprecipitation in co-transfected 293T cells and retrovirus infected K562 human leukemia cells. We also examined whether c-Myb is associated with the histone methyltransferase complex in transfected 293T cells. Again, using an immunoprecipitation strategy we found that c-Myb is associated with both WDR5 and RbBp5, which are common components of multiple H3 K4 methyltransferase complexes. A Luciferase reporter assay revealed that transfection of 100 ng of WDR5 with 200 ng of c-Myb resulted in 2-fold activation of the reporter, as compared to signal obtained from cells transfected with 200 ng of c-Myb cDNA alone. Furthermore, c-Myb-associated methyltransferase complexes, isolated from c-Myb transfected 293T cells, K562 human leukemia cells expressing epitope-tagged c-Myb, as well as untransfected K562 cells, were able to methylate histone H3. In aggregate, these results suggest that in addition to regulating gene transcription by binding to DNA, c-Myb may also regulate transcription via a new paradigm, that of covalent histone modification.

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1,25-Dihydroxyvitamin D3 induces the expression of the tumor suppressor protein CYLD in human leukemia cells

Journal of Biotechnology ◽

10.1016/j.jbiotec.2017.06.649 ◽

2017 ◽

Vol 256 ◽

pp. S29

Author(s):

Maria Vaiou ◽

George Mosialos ◽

Anargyros N. Moulas

Keyword(s):

Tumor Suppressor ◽

Tumor Suppressor Protein ◽

Leukemia Cells ◽

Human Leukemia ◽

Dihydroxyvitamin D3 ◽

1,25 Dihydroxyvitamin D3 ◽

Human Leukemia Cells

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Protein Phosphatase 1 Regulates the Tumor Suppressor Function of Ikaros and Radiation Resistance in Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v118.21.2465.2465 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2465-2465

Author(s):

Sinsa Dovat ◽

Kimberly J Payne ◽

Chunhua Song

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Tumor Suppressor ◽

Dna Binding ◽

Binding Affinity ◽

Chromatin Remodeling ◽

Target Genes ◽

Radiation Treatment ◽

Lymphoblastic Leukemia ◽

Suppressor Activity ◽

Dna Binding Affinity

Abstract Abstract 2465 The Ikaros gene encodes a DNA-binding zinc finger protein that acts as a master regulator of hematopoiesis and a tumor suppressor in acute lymphoblastic leukemia (ALL). Ikaros exerts its tumor suppressor activity by binding to the upstream regulatory regions of its target genes resulting in chromatin remodeling and transcriptional repression of the target gene. We have previously reported that Ikaros is a substrate for Protein Phosphatase 1 (PP1), and that PP1 regulates the DNA-binding affinity of Ikaros and its subcellular localization to pericentromeric heterochromatin. PP1 has been postulated to possess tumor suppressor activity, although the mechanisms were unknown. We hypothesize that PP1 positively regulates the tumor suppressor function of Ikaros in ALL. In this report, we studied the role of PP1 in regulating Ikaros function as a transcriptional regulator of its target genes in acute lymphoblastic leukemia (ALL), and in controlling the sensitivity of leukemia cells to radiation. An Ikaros construct with a mutated PP1 interaction site (IK 465/7A) was tested for: 1) association with histone deacetylase (HDAC) chromatin remodeling complex by co-immunoprecipitation; 2) in vivo DNA-binding to promoter regions of known Ikaros target genes by quantitative chromatin immunoprecipitation (qChIP); and 3) direct transcriptional repression of Ikaros target genes, by transient luciferase reporter assay. Our results demonstrate that the loss of interaction with PP1 results in hyperphosphorylation of Ikaros protein resulting in an inability of Ikaros to interact with the HDAC chromatin remodeling complex. The loss of PP1 interaction impaired Ikaros' ability to function as transcriptional repressor due to poor DNA-binding affinity toward the promoters of Ikaros target genes. The introduction of phosphoresistant (alanine) mutations at CK2 kinase phosphorylation sites on the Ikaros protein (IK 465/7A+A11 mutant) restored Ikaros' ability to bind the histone deacetylase complex (including HDAC1 and HDAC2 proteins), as well as its transcriptional repressor function and DNA-binding affinity toward promoters of its target genes. These data strongly suggest that dephosphorylation of Ikaros by PP1 is essential for its function in chromatin remodeling and regulation of gene expression. To study the role of PP1 in ALL, we treated primary ALL cells with specific inhibitors of PP1 and studied the impact PP1 inhibition on ALL cells. Our data demonstrate that the inhibition of PP1 activity results in decreased sensitivity of ALL cells to radiation treatment, and that these changes correlate with a decrease in Ikaros' DNA-binding affinity (as evidenced by qChIP), and a loss of Ikaros function as a direct regulator of target gene transcription. These studies identified PP1 as an important signal transduction pathway that controls the proliferation of ALL cells. Our results suggest that PP1-mediated dephosphorylation and CK2 kinase-mediated phosphorylation are two opposing signaling pathways that regulate Ikaros function as a tumor suppressor in ALL, as well as the resistance of leukemia cells to radiation treatment. Disclosures: No relevant conflicts of interest to declare.

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