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

Blood ◽  
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.

scholarly journals Transcriptional Regulation of Genes by Ikaros Tumor Suppressor in Acute Lymphoblastic Leukemia

2020 ◽  
Vol 21 (4) ◽  
pp. 1377
Author(s):  
Pavan Kumar Dhanyamraju ◽  
Soumya Iyer ◽  
Gayle Smink ◽  
Yevgeniya Bamme ◽  
Preeti Bhadauria ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Tumor Suppressor ◽  
Chromatin Remodeling ◽  
Target Genes ◽  
Cellular Proliferation ◽  
Lymphoblastic Leukemia ◽  
Regulatory Elements ◽  
Binding Motif ◽  
Functional Inactivation

Regulation of oncogenic gene expression by transcription factors that function as tumor suppressors is one of the major mechanisms that regulate leukemogenesis. Understanding this complex process is essential for explaining the pathogenesis of leukemia as well as developing targeted therapies. Here, we provide an overview of the role of Ikaros tumor suppressor and its role in regulation of gene transcription in acute leukemia. Ikaros (IKZF1) is a DNA-binding protein that functions as a master regulator of hematopoiesis and the immune system, as well as a tumor suppressor in acute lymphoblastic leukemia (ALL). Genetic alteration or functional inactivation of Ikaros results in the development of high-risk leukemia. Ikaros binds to the specific consensus binding motif at upstream regulatory elements of its target genes, recruits chromatin-remodeling complexes and activates or represses transcription via chromatin remodeling. Over the last twenty years, a large number of Ikaros target genes have been identified, and the role of Ikaros in the regulation of their expression provided insight into the mechanisms of Ikaros tumor suppressor function in leukemia. Here we summarize the role of Ikaros in the regulation of the expression of the genes whose function is critical for cellular proliferation, development, and progression of acute lymphoblastic leukemia.

LEF1 Is a Tumor Suppressor in T Cell Acute Lymphoblastic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3802-3802
Author(s):  
Alejandro Gutierrez ◽  
Takaomi Sanda ◽  
Stuart Winter ◽  
Richard S. Larson ◽  
Lewis B. Silverman ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tumor Suppressor ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Suppressor Activity ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Expression Microarrays

Abstract In an effort to further unravel the molecular pathogenesis of T cell acute lymphoblastic leukemia (T-ALL), we performed array CGH on diagnostic specimens from 47 pediatric patients with T-ALL. The LEF1 transcription factor is best known as a positive mediator of oncogenic β-catenin signaling, and it is required for the expression of MYC, Cyclin D1 and Survivin in some contexts, including some normal hematopoietic progenitors. Additionally, Lef1 has been shown to be required for the survival of murine Notch-dependent T cell lymphoma cells. We were thus surprised to find highly focal deletions of LEF1 in 10.6 % (n = 5 of 47) of primary T-ALL patient samples, with 3 cases harboring homozygous deletions and 2 harboring heterozygous deletions of this locus. These deletions involved no other known genes. We then sequenced this gene in 45 of these cases and found heterozygous mutations in 3 additional T-ALL samples, including 2 cases with frameshift mutations predicted to truncate the protein prior to its context-dependent activation and HMG box domains, and one case with an Asp85Asn substitution. Seven of the eight samples harboring LEF1 abnormalities also had mutations in the heterodimerization or PEST domains of NOTCH1. In an effort to characterize the biologic consequences of LEF1 loss in T-ALL, we took advantage of expression microarrays that were previously performed on most of these samples. LEF1 loss defines a novel T-ALL subtype characterized by arrest at the CD1-positive early cortical stage, and whose gene expression profile resembles that of HOX11-positive cases, although expression of HOX11 was generally low in these samples. Loss of LEF1 was mutually exclusive to overexpression of TAL1 and of the HOXA/MEIS1 cluster. Interestingly, LEF1-negative T-ALL cases were associated with increased expression of MYC, and gene set enrichment analysis identified a significant association between LEF1 loss and upregulation of MYC target genes. In addition to its role as a transcriptional activator in the setting of active WNT/β-catenin signaling, LEF1 can also act as a transcriptional repressor in some cellular contexts. Experiments are currently underway to establish the mechanism mediating the tumor suppressor activity of LEF1 in T-ALL.

scholarly journals Nuclear Heparanase Regulates Chromatin Remodeling, Gene Expression and PTEN Tumor Suppressor Function

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2038
Author(s):  
Rada Amin ◽  
Kaushlendra Tripathi ◽  
Ralph D. Sanderson
Keyword(s):  
Tumor Suppressor ◽  
Chromatin Remodeling ◽  
Target Genes ◽  
Myeloma Cell ◽  
Chromatin Accessibility ◽  
Tumor Suppressor Function ◽  
Suppressor Activity ◽  
Downstream Target ◽  
Suppressor Function ◽  
Chromatin Opening

Heparanase (HPSE) is an endoglycosidase that cleaves heparan sulfate and has been shown in various cancers to promote metastasis, angiogenesis, osteolysis, and chemoresistance. Although heparanase is thought to act predominantly extracellularly or within the cytoplasm, it is also present in the nucleus, where it may function in regulating gene transcription. Using myeloma cell lines, we report here that heparanase enhances chromatin accessibility and confirm a previous report that it also upregulates the acetylation of histones. Employing the Multiple Myeloma Research Foundation CoMMpass database, we demonstrate that patients expressing high levels of heparanase display elevated expression of proteins involved in chromatin remodeling and several oncogenic factors compared to patients expressing low levels of heparanase. These signatures were consistent with the known function of heparanase in driving tumor progression. Chromatin opening and downstream target genes were abrogated by inhibition of heparanase. Enhanced levels of heparanase in myeloma cells led to a dramatic increase in phosphorylation of PTEN, an event known to stabilize PTEN, leading to its inactivity and loss of tumor suppressor function. Collectively, this study demonstrates that heparanase promotes chromatin opening and transcriptional activity, some of which likely is through its impact on diminishing PTEN tumor suppressor activity.

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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4196-4196
Author(s):  
Sinsa Dovat ◽  
Chunhua Song ◽  
Zafer Gurel ◽  
Kimberly J Payne ◽  
Marcela Popescu
Keyword(s):  
Tumor Suppressor ◽  
Dna Binding ◽  
Target Genes ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Phosphorylation Sites ◽  
Human Leukemia Cells ◽  
Dna Binding Affinity ◽  
Thymocyte Differentiation ◽  
Ck2 Kinase

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.

scholarly journals Regulation of Small GTPase Rab20 by Ikaros in B-Cell Acute Lymphoblastic Leukemia

2020 ◽  
Vol 21 (5) ◽  
pp. 1718
Author(s):  
Jonathon L Payne ◽  
Chunhua Song ◽  
Yali Ding ◽  
Pavan Kumar Dhanyamraju ◽  
Yevgeniya Bamme ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Dna Binding ◽  
B Cell ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Small Gtpase ◽  
Luciferase Reporter ◽  
Regulation Of Transcription ◽  
Full Cohort ◽  
Cell Acute Lymphoblastic Leukemia

Ikaros is a DNA-binding protein that regulates gene expression and functions as a tumor suppressor in B-cell acute lymphoblastic leukemia (B-ALL). The full cohort of Ikaros target genes have yet to be identified. Here, we demonstrate that Ikaros directly regulates expression of the small GTPase, Rab20. Using ChIP-seq and qChIP we assessed Ikaros binding and the epigenetic signature at the RAB20 promoter. Expression of Ikaros, CK2, and RAB20 was determined by qRT-PCR. Overexpression of Ikaros was achieved by retroviral transduction, whereas shRNA was used to knockdown Ikaros and CK2. Regulation of transcription from the RAB20 promoter was analyzed by luciferase reporter assay. The results showed that Ikaros binds the RAB20 promoter in B-ALL. Gain-of-function and loss-of-function experiments demonstrated that Ikaros represses RAB20 transcription via chromatin remodeling. Phosphorylation by CK2 kinase reduces Ikaros’ affinity toward the RAB20 promoter and abolishes its ability to repress RAB20 transcription. Dephosphorylation by PP1 phosphatase enhances both Ikaros’ DNA-binding affinity toward the RAB20 promoter and RAB20 repression. In conclusion, the results demonstrated opposing effects of CK2 and PP1 on expression of Rab20 via control of Ikaros’ activity as a transcriptional regulator. A novel regulatory signaling network in B-cell leukemia that involves CK2, PP1, Ikaros, and Rab20 is identified.

scholarly journals Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Krystyna Ślaska-Kiss ◽  
Nikolett Zsibrita ◽  
Mihály Koncz ◽  
Pál Albert ◽  
Ákos Csábrádi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Binding ◽  
Binding Affinity ◽  
Dna Methyltransferase ◽  
Restriction Enzymes ◽  
Dna Binding Protein ◽  
E Coli ◽  
Dna Binding Affinity ◽  
Higher Eukaryotes

AbstractTargeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

A novel PAX5-ELN fusion protein identified in B-cell acute lymphoblastic leukemia acts as a dominant negative on wild-type PAX5

Blood ◽  
2006 ◽  
Vol 109 (8) ◽  
pp. 3417-3423 ◽  
Author(s):  
Marina Bousquet ◽  
Cyril Broccardo ◽  
Cathy Quelen ◽  
Fabienne Meggetto ◽  
Emilienne Kuhlein ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Quantitative Polymerase Chain Reaction ◽  
Dominant Negative ◽  
Leukemic Cells ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Cell Acute Lymphoblastic Leukemia

Abstract We report a novel t(7;9)(q11;p13) translocation in 2 patients with B-cell acute lymphoblastic leukemia (B-ALL). By fluorescent in situ hybridization and 3′ rapid amplification of cDNA ends, we showed that the paired box domain of PAX5 was fused with the elastin (ELN) gene. After cloning the full-length cDNA of the chimeric gene, confocal microscopy of transfected NIH3T3 cells and Burkitt lymphoma cells (DG75) demonstrated that PAX5-ELN was localized in the nucleus. Chromatin immunoprecipitation clearly indicated that PAX5-ELN retained the capability to bind CD19 and BLK promoter sequences. To analyze the functions of the chimeric protein, HeLa cells were cotransfected with a luc-CD19 construct, pcDNA3-PAX5, and with increasing amounts of pcDNA3-PAX5-ELN. Thus, in vitro, PAX5-ELN was able to block CD19 transcription. Furthermore, real-time quantitative polymerase chain reaction (RQ-PCR) experiments showed that PAX5-ELN was able to affect the transcription of endogenous PAX5 target genes. Since PAX5 is essential for B-cell differentiation, this translocation may account for the blockage of leukemic cells at the pre–B-cell stage. The mechanism involved in this process appears to be, at least in part, through a dominant-negative effect of PAX5-ELN on the wild-type PAX5 in a setting ofPAX5 haploinsufficiency.

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