NKX3.1 Is a Direct TAL1 Target Gene That Mediates the TAL1 Dependent Proliferation of Human T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Abstract The TAL1/SCL gene encodes a bHLH (basic Helix-Loop-helix) protein that acts as a master gene in hematopoiesis. The TAL1/SCL gene is also the most frequently activated gene in human T-ALL but the oncogenic transcriptional programs, downstream of TAL1 in human T-ALL, are not well characterized. Using RNA interference to knockdown TAL1 expression, we show that TAL1 regulates both cell proliferation and death of human T-ALL cells. To determine the TAL1 target genes in human T-ALL, we combine TAL1 knockdown and gene expression profiling and show that TAL1 activates and repress a common subset of genes in cell lines. This subset includes known TAL1 target genes but also the NKX3.1 gene that is a homeobox gene, specifically expressed in the prostate epithelium during prostate development and in adulthood. NKX3.1 gene inactivation is one of the earliest events that occur in prostate cancer initiation, defining NKX3.1 as a major tumor suppressor gene of this cancer. TAL1 expression is associated with NKX3.1 activation in human T-ALL cell lines and NKX3.1 is expressed in TAL1 expressing human T-ALL blasts. TAL1 and GATA-3 are specifically bound in vivo to the [−870/−570] region of the human NKX3.1 gene promoter, and ex vivo, TAL1 can either directly binds an E-box [position −738] or be recruited by GATA-3 on a GATA binding site [position −697]. Finally, functional analyses of the NKX3.1 promoter indicate that these binding sites mediate the transcriptional activity of this promoter in T-cell lines. Sequences analysis of the human and mouse NKX3.1 promoters show that the regulatory sequences involved in the TAL1 activation of the human NKX3.1 gene are not conserved in the mouse gene, indicating why the NKX3.1 gene is not expressed in mouse models of TAL1 mediated leukemogenesis. NKX3.1 knockdown shows that NKX3.1 is necessary for the proliferation of TAL1 expressing T-ALL cell lines and NKX3.1 overexpression can complement the proliferation defects associated with TAL1 knockdown in T-ALL cell lines. Microarray analyses show that TAL1 and NKX3.1 regulate a common subset of genes in T-ALL that includes numerous genes encoding proteins known to be involved in T-cell proliferation and/or signaling. Finally, using a new culture system that enables proliferation of primary human leukemic cells, we show that the NKX3.1 gene is specifically activated in human TAL1 expressing T-ALL together with the defined potential TAL1 and/or NKX3.1 target genes. These results characterize NKX3.1 as the first gene directly activated by TAL1 and involved in the TAL1 dependent proliferation of human T-cell Acute Lymphoblastic Leukemia.

Download Full-text

Does activation of the TAL1 gene occur in a majority of patients with T- cell acute lymphoblastic leukemia? A pediatric oncology group study

Blood ◽

10.1182/blood.v86.2.666.bloodjournal862666 ◽

1995 ◽

Vol 86 (2) ◽

pp. 666-676 ◽

Cited By ~ 114

Author(s):

RO Bash ◽

S Hall ◽

CF Timmons ◽

WM Crist ◽

M Amylon ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Lymphoblastic Leukemia ◽

Ectopic Expression ◽

Leukemic Cells ◽

Monoallelic Expression ◽

Regulatory Sequences ◽

Cis Acting ◽

Polymorphic Dinucleotide ◽

Cell Acute Lymphoblastic Leukemia

Almost 25% of patients with T-cell acute lymphoblastic leukemia (T-ALL) have tumor-specific rearrangements of the TAL1 gene. Although TAL1 expression has not been observed in normal lymphocytes, TAL1 gene products are readily detected in leukemic cells that harbor a rearranged TAL1 allele. Hence, it has been proposed that ectopic expression of TAL1 promotes the development of T-ALL. In this report, we show that TAL1 is expressed in the leukemic cells of most patients with T-ALL, including many that do not display an apparent TAL1 gene alteration. A polymorphic dinucleotide repeat in the transcribed sequences of TAL1 was used to determine the allele specificity of TAL1 transcription in primary T-ALL cells. Monoallelic expression of TAL1 was observed in the leukemic cells of all patients (8 of 8) bearing a TAL1 gene rearrangement. In the leukemic cells of patients without detectable TAL1 rearrangements, TAL1 transcription occurred in either a monoallelic (3 of 7 patients) or a biallelic (4 of 7 patients) fashion. Thus, TAL1 activation in these patients may result from subtle alterations in cis-acting regulatory sequences (affecting expression of a single TAL1 allele) or changes in trans-acting factors that control TAL1 transcription (affecting expression of both TAL1 alleles).

Download Full-text

Gain-of-Function NOTCH1 Mutations Occur Frequently in Human T Cell Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v104.11.4.4 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4-4

Author(s):

Andrew P. Weng ◽

Adolfo A. Ferrando ◽

Woojoong Lee ◽

John P. Morris ◽

Lewis B. Silverman ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Cell Lines ◽

Reporter Gene ◽

Lymphoblastic Leukemia ◽

Reporter Gene Activity ◽

Human T Cell ◽

Cell Acute Lymphoblastic Leukemia ◽

In Cis ◽

Notch1 Mutations

Abstract NOTCH1 was discovered originally through its involvement in a rare (7;9) translocation found in human T cell acute lymphoblastic leukemia (T-ALL). Here, we report that >50% of human T-ALLs have activating NOTCH1 mutations, occurring as amino acid substitutions in an extracellular heterodimerization (HD) domain and/or as frameshift and stop codon mutations that result in the deletion of a C-terminal PEST destruction box. Normal pro-NOTCH1 is processed into a heterodimer consisting of an extracellular subunit and a transmembrane subunit, which associate non-covalently through the HD domain. NOTCH1 activation is triggered by binding of Serrate or Delta-like ligands to the extracellular subunit, which induces successive proteolytic cleavages in the transmembrane subunit that are dependent on i) metalloproteases and ii) gamma-secretase. The γ-secretase cleavage releases intracellular NOTCH1 (ICN1), which translocates to the nucleus and forms a transcriptional activation complex with the transcription factor CSL and co-activators of the Mastermind family. Normal turnover of ICN1 is regulated by the C-terminal PEST sequence. Data pointing to the existence of frequent abnormalities of NOTCH1 in T-ALL stemmed from a functional screen of 30 T-ALL cell lines. This identified five T-ALL cell lines that underwent growth arrest in response to i) treatment with an inhibitor γ-secretase, and ii) retroviral transduction of dominant negative Mastermind-like-1. Sequencing of of cDNAs from 4 of these 5 cell lines demonstrated both a missense mutation in the HD domain and a frameshift mutation in the PEST domain lying in cis in the same NOTCH1 allele. Subsequent sequencing of genomic DNA obtained from bone marrow lymphoblasts of 96 children and adolescents with T-ALL demonstrated identical or similar mutations in NOTCH1 in 53 samples (55.2%). Mutations in the HD domain alone were observed in 26 cases (27.1%), in the PEST domain alone in 11 cases (11.4%), and in both the HD and PEST domains in 16 cases (16.7%). Mutations were observed in tumors associated with expression of HOX11 (2/3), HOX11L2 (10/13; 77%), TAL1 (12/31; 39%), LYL1 (9/14; 64%), MLL-ENL (1/3) or CALM-AF10 (1/2), which span the major molecular T-ALL subtypes. In contrast, NOTCH1 mutations were not observed in genomic DNAs samples obtained from B-ALL lymphoblasts (N=89), or from T-ALL patients with NOTCH1-associated disease at the time of clinical remission (N=4). Reporter gene assays conducted with plasmids expressing normal and mutated forms of NOTCH1 showed that a PEST deletion or various HD mutations alone caused ~1.5-fold and 3–9-fold stimulations of reporter gene activity, respectively, whereas normal NOTCH1 lacked intrinsic signaling activity. More strikingly, the combination of various HD mutations and a PEST deletion in cis caused synergistic 20–40-fold stimulations of reporter gene activity that were completely abrogated by a γ-secretase inhibitor, indicating that signaling depends on proteolysis. These results suggest a model in which HD domain mutations promote ICN1 production, and PEST domain mutations enhance ICN1 stability. Our findings greatly expand the role of NOTCH1 in the pathogenesis of human T-ALL, and provide a rationale for targeted therapies that interfere with NOTCH signaling.

Download Full-text

Collaboration Between RUNX and NOTCH Pathways in T-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v120.21.1279.1279 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1279-1279 ◽

Cited By ~ 1

Author(s):

Christopher R Jenkins ◽

Hongfang Wang ◽

Olena O Shevchuk ◽

Sonya H Lam ◽

Vincenzo Giambra ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Notch Signaling ◽

Cell Lines ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Brdu Incorporation ◽

Knock Down ◽

Primary Mouse ◽

Cell Acute Lymphoblastic Leukemia

Abstract Abstract 1279 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy characterized by the clonal outgrowth of developmentally arrested T-lymphoid blasts. Notch signaling is activated by mutation of NOTCH1 and/or FBW7 in over half of cases, and ultimately results in increased expression of target genes via the NOTCH/CSL transcriptional complex. Enforced expression of activated NOTCH1 in mouse hematopoietic progenitors leads to the development of clonal T-cell leukemias, suggesting that collaborating mutations are required for establishment and/or propagation of malignant clones. To identify candidate collaborating loci, Beverly and Capobianco performed a retroviral insertional mutagenesis screen in mice expressing a relatively weak activated Notch1 transgene and found recurrent insertions into Ikaros (Ikzf1). These insertions resulted in expression of dominant negative isoforms of Ikaros and thus potentiated Notch signaling since Ikaros and Notch/CSL compete for occupancy at target gene regulatory elements. In an attempt to identify collaborating mutations outside of the Notch pathway, we performed a similar screen, but employed instead a very potent activated NOTCH1 allele (ΔE) in hopes of saturating the Notch signaling pathway. We thus cloned out the insertion sites from 88 primary mouse leukemias generated by transduction of bone marrow with ΔE retrovirus. While recurrent insertions into Ikzf1 were again identified, we also observed frequent insertions into other regions including the Runx3 locus. The Runx3 integrations were tightly clustered in a region 40–60kb upstream of the transcriptional start site, suggesting the retroviral LTR might be inducing an increase in Runx3 expression. A single integration upstream of Runx1 was also identified in a region frequently mutated in similar screens. Of note, analysis of publically available gene expression profile data revealed that RUNX1 and RUNX3 are ubiquitously expressed in patient T-ALL samples. In order to functionally characterize the roles of RUNX1 and RUNX3 in T-ALL, we utilized lentiviral shRNAs to knock down RUNX1 and/or RUNX3 across a broad panel of 26 human T-ALL cell lines. Despite recent studies suggesting RUNX1 may act as a tumor suppressor in T-ALL, we observed the overwhelming majority of cell lines to show substantial growth defects after knock-down of RUNX1/3 as measured by competitive growth assay. These results were confirmed in a subset of cell lines and also in xenograft-expanded primary T-ALL samples by BrdU incorporation/DNA content assays which showed reduced proliferation/G1 cell cycle arrest following RUNX1/3 knock-down. Conversely, overexpression of RUNX3 induced T-ALL cells to proliferate more rapidly and to resist ABT-263-induced apoptosis. To explore potential target genes responsible for these pro-growth/survival effects, we mined available ChIP-Seq data and found NOTCH1/CSL and RUNX1 binding sites to co-localize within IGF1R and IL7R loci at intronic enhancer-like regions with associated H3K4me1>H3K4me3 marks and reduced H3K27me3 marks. Importantly, we show that NOTCH1 and RUNX factors co-regulate surface protein expression of IGF1R and IL7R in a synergistic/additive manner. As we and others have previously demonstrated important roles for both IGF1R and IL7R in T-ALL cell growth and leukemia-initiating activity, these studies reveal a novel collaborative mechanism between NOTCH1 and RUNX proteins in supporting propagation of established T-ALL disease. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

RUNX1 promotes cell growth in human T-cell acute lymphoblastic leukemia by transcriptional regulation of key target genes

Experimental Hematology ◽

10.1016/j.exphem.2018.04.008 ◽

2018 ◽

Vol 64 ◽

pp. 84-96 ◽

Cited By ~ 2

Author(s):

Catherine E. Jenkins ◽

Samuel Gusscott ◽

Rachel J. Wong ◽

Olena O. Shevchuk ◽

Gurneet Rana ◽

...

Keyword(s):

Transcriptional Regulation ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Cell Growth ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Human T Cell ◽

Cell Acute Lymphoblastic Leukemia

Download Full-text

The TAL1 Complex Represses the FBXW7 Tumor Suppressor Through Mir-223 in Human T-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v120.21.1296.1296 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1296-1296

Author(s):

Marc R Mansour ◽

Takaomi Sanda ◽

Lee N Lawton ◽

Xiaoyu Li ◽

Taras Kreslavsky ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Tumor Suppressor ◽

Protein Expression ◽

Growth Inhibition ◽

Cell Lines ◽

Mirna Expression ◽

Lymphoblastic Leukemia ◽

Human T Cell ◽

Cell Acute Lymphoblastic Leukemia

Abstract Abstract 1296 The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T-cell acute lymphoblastic leukemia (T-ALL) and causes T-ALL in murine transgenic models, emphasizing its importance in the molecular pathogenesis of this disease. However, the mechanism by which TAL1 leads to transformation of thymocytes is unclear. Dysregulation of miRNAs play an important role in tumorigenesis in diverse cancer types. A recent study identified miR-223 as the most abundant miRNA in T-ALL patient samples and was oncogenic by virtue of its ability to accelerate Notch-induced T-ALL in a murine model (Mavrakis et al. Nature Genetics 2011). However, the underlying mechanisms leading to dysregulated miRNA expression in T-ALL remain poorly understood. In order to explore the hypothesis that aberrant expression of miRNAs is mediated by the TAL1 oncogene in T-ALL, we generated high-resolution maps of the genome-wide occupancy of the TAL1 complex, including E2A, HEB, GATA3, LMO2 and RUNX1 by chromatin immunoprecipitation coupled to massively parallel DNA sequencing (ChIP-seq). Analysis of binding sites in two TAL1-positive T-ALL cell lines (Jurkat and CCRF-CEM cells) and two primary T-ALL samples identified 54 miRNAs where binding of the TAL1 complex was within 10 kb of either the transcriptional start sites or the start sites of genes that contain miRNAs in their intronic regions. To determine which of these miRNAs were not only directly bound, but also regulated by the TAL1 complex, we analyzed global changes in miRNAs after knockdown of TAL1 in Jurkat cells using two independent shRNAs. By miRNA expression profiling, we identified significant changes in expression of 25 miRNAs, of which nine were down-regulated on TAL1 knockdown (and thus positively regulated by TAL1) and 16 were up-regulated on TAL1 knockdown (and thus negatively regulated by TAL1). Of these 25 miRNAs, four (miR-223, miR181a*, miR-26a and miR-29c) were shown to be direct targets of the TAL1 complex based on our ChIP-seq data. We chose to focus on miR-223 because it exhibited the most dynamic down-regulation after TAL1 knockdown. ChIP-qPCR validated binding of the TAL1 complex to a region within 4 kb of the miR-223 transcriptional start site. Analysis of RNA polymerase II and CBP binding showed significant enrichment, and high levels of H3K4M3 and H3K79M2 modification were detected indicative of transcriptional initiation and elongation of this locus. Furthermore, expression of miR-223 was significantly higher in the TAL1-positive cell lines (n=13) as compared to the TAL1-low cells (n=10) (P<0.0001). miR-223 levels also closely mirrored TAL1 levels in murine thymic subsets, with marked down-regulation after the DN2 stage, suggesting miR-223 is a physiological target of TAL1 during normal thymic development, and that its overexpression in TAL1-positive T-ALL cells, arrested at the double-positive (DP) stage, is aberrant compared to their normal DP counterpart. To test the hypothesis that the growth inhibition observed after TAL1 knockdown is mediated by decreases in miR-223 expression, we retrovirally infected Jurkat and RPMI-8402 T-ALL cell lines with a miR-223 construct, such that miR-223 expression was no longer under the control of TAL1 in these cells. Forced expression of miR-223 partially rescued the growth inhibitory effects induced by TAL1 knockdown, in both a lentiviral and doxycycline-inducible shRNA system. Additionally, inhibition of mature miR-223 by lentiviral infection of a miR-223 shRNA construct led to significant growth inhibition of TAL1-positive cell lines through the induction of apoptosis. Thus, maintenance of miR-223 expression is required for optimal growth of TAL1-positive T-ALL cells. The highest ranked predicted target of miR-223 by Targetscan is the FBXW7 tumor suppressor, a ubiquitin ligase that is mutated in a significant proportion of T-ALL patients and targets oncogenes such as c-MYC, NOTCH and mTOR for degradation. Accordingly, overexpression of miR-223 in TAL1-low miR-223-low T-ALL cells markedly down-regulated FBXW7 protein expression. Furthermore, the up-regulation of FBXW7 protein expression observed on knockdown of TAL1 in TAL1-positive cell lines could be prevented by retroviral miR-223 expression. Thus, miR-223 is an important target of TAL1 and links the TAL1 oncogene to repression of the FBXW7 tumor suppressor. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

miR106a-363 Cluster Has Oncogenic Potential in Childhood T-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2018-99-111103 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5142-5142

Author(s):

Monika Drobna ◽

Bronislawa Szarzynska-Zawadzka ◽

Maria Kosmalska ◽

Roman Jaksik ◽

Tomasz Szczepanski ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Cell Lines ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Luciferase Reporter ◽

Pathway Enrichment Analysis ◽

Thermo Fisher Scientific ◽

Cell Acute Lymphoblastic Leukemia ◽

Fisher Scientific

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy originating from T-cell precursors and is characterized by high genetic, immunophenotypic, and clinical heterogeneity. MicroRNAs (miRNAs) belong to the class of small noncoding RNAs and are implicated in the regulation of hematopoiesis and in the development of leukemia. miRNAs control expression of their target genes at the post-transcriptional level by blocking translation of messenger RNAs (mRNAs) or promoting their degradation. Some miRNAs are encoded within clusters, giving rise to policistronic transcripts. Such miRNAs are co-expressed and may co-regulate the expression of genes involved in certain biological processes and pathways. In our recent study we performed miRNA profiling in pediatric T-ALL using Next-Generation Sequencing (Dawidowska M et al. Blood 2017; 130:1443) and identified miRNAs differentially expressed in T-ALL. The set of overexpressed miRNAs included, among others, miR-20b-5p, miR-363-3p and miR-92a-2-5p, belonging to a cluster of six miRNAs: miR-106a-363 (ChrXq26.2). miR-106a-363 cluster is a paralog of miR-17-92 cluster (Chr13q31.3), a prototypic oncogenic cluster of eminent importance in human hematopoietic cancers, with reported role in T-ALL pathogenesis (Mavrakis KJ et al., Nature Cell Biology 2010, 12:4). Despite the similarity of seed sequences between miRNAs from miR-17-92 and miR-106a-363 clusters, the significance of miR-106a-363 cluster in T-ALL remains to be elucidated. In this study we investigated the expression of the miR-20b-5p, miR-363-3p and miR-92a-2-5p in children with T-ALL, healthy donor thymocytes, normal bone marrow samples and 6 T-ALL cell lines. RT-qPCR analysis (TaqMan Advanced miRNA Assays; Thermo Fisher Scientific) confirmed overexpression of 2 miRNAs from cluster miR-106a-363 (miR-20b-5p and miR-363-3p) in children with T-ALL and in T-ALL cell lines, suggesting their oncogenic function. To predict potential target genes of overexpressed miRNAs belonging to miR106a-363 cluster, we applied 8 target prediction algorithms and pathway enrichment analysis. This revealed the enrichment of miR-20b-5p and miR-363-3p target genes in GO term: positive regulation of apoptosis. We further validated predicted miRNA-mRNA interactions (Dual Luciferase Reporter Assays; Promega) confirming the majority of them (e.g. PTEN, FBXW7, BCL2L11). Finally, we assessed the effect of mimicry/inhibition (miRVana, Thermo Fisher Scientific) of overexpressed miRNAs from miR-106a-363 cluster on proliferation, cell cycle distribution and apoptosis in 3 T-ALL cell lines. Overexpression of miR-20b-5p and miR-363-3p in CCRF-CEM, DND-41 and P12-Ichikawa cells resulted in increased proliferation and inhibited apoptosis. To summarize, in this study we showed that miRNAs belonging to miR-106a-363 cluster directly interact with mRNAs implicated in the regulation of apoptosis and that miR-20b-5p and miR-363-3p have pro-proliferative and anti-apoptotic effects in T-ALL cells in vitro. These results indicate that miR-106a-363 cluster may have an oncogenic role in the pathogenesis of T-ALL via suppression of pro-apoptotic genes. Research funded by National Science Centre, Poland grants: 2014/15/B/NZ2/03394, 2017/25/N/NZ2/01132 and National Centre of Research and Development (NCRD) grant STARTEGMED3/304586/5/NCBR/2017. Disclosures No relevant conflicts of interest to declare.

Download Full-text

The Pias-Like Coactivator ZMIZ1 Drives C-MYC Induction in Collaboration with NOTCH1 in T-Cell Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v120.21.2380.2380 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2380-2380

Author(s):

Margaret Decker ◽

Choi Li ◽

Lesley A Rakowski ◽

Tomasz Cierpicki ◽

Mark Y. Chiang

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Cell Line ◽

Mouse Models ◽

Target Genes ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Ectopic Expression ◽

Human T Cell ◽

Cell Acute Lymphoblastic Leukemia

Abstract Abstract 2380 Activating NOTCH1 mutations are found in 50–60% of human T-cell acute lymphoblastic leukemia (T-ALL) samples. In mouse models, these mutations generally fail to induce leukemia. Cooperating oncogenes must be recruited by NOTCH1 to fully induce leukemia. Murine insertional mutagenesis screens previously implicated ZMIZ1 as a possible NOTCH1 collaborator in leukemia (Uren et al., Cell, 2008; Dupuy et al., Nature, 2005; Berquam-Vrieze et al., Blood, 2011). ZMIZ1 is a transcriptional co-activator of the Protein Inhibitor of Activated STAT (PIAS)-like family. It shares a zinc finger domain, the MIZ domain, with PIAS proteins. The MIZ domain mediates interactions with DNA-binding transcription factors and sumoylation. Previously, we showed that ZMIZ1 promotes T-ALL in collaboration with leukemia-associated NOTCH1 alleles in mouse models. ZMIZ1 and activated NOTCH1 were co-expressed in a subset of human patients. Genetic ZMIZ1 inhibition slowed leukemic cell growth and overcame resistance of some T-ALL cell lines to NOTCH inhibitors. ZMIZ1 may be a new clinically relevant oncogene. Here we sought to determine the downstream target genes of ZMIZ1 in leukemia. Validation of gene expression profiling data identified C-MYC and IL7RA as downstream targets of ZMIZ1. Targeting the C-MYC or IL-7 pathways using genetic and pharmacological inhibitors partly phenocopied the growth inhibitory effects we previously saw with ZMIZ1 inhibition. In order to determine whether these genes are direct or indirect targets of ZMIZ1, we generated an estrogen fusion protein, ZMIZ1-ER. ZMIZ1-ER induced C-MYC and IL7RA expression in the presence of tamoxifen, but failed to induce these genes with the addition of cycloheximide. These data suggest that C-MYC and IL-7RA are indirect targets. Like the PIAS proteins, ZMIZ1 appeared to have a broad effect on transcription to exert its functions. We next sought to elucidate the biochemical mechanism of ZMIZ1. Ectopic expression of ZMIZ1 or NOTCH1 had weak effects on endogenous c-Myc expression and failed to rescue a C-MYC-dependent T-ALL cell line after withdrawal of ectopic C-MYC. In contrast, ZMIZ1 in combination with NOTCH1 dramatically induced C-MYC expression by several fold and rescued the C-MYC dependent cell line. ZMIZ1 enhanced the ability of even weak NOTCH1 mutants to induce C-MYC, suggesting a mechanism by which ZMIZ1 may increase resistance to NOTCH inhibitors. ZMIZ1 did not influence C-MYC expression post-transcriptionally. It functioned primarily as a transcriptional activator. Although both C-MYC and IL7RA are both NOTCH1 target genes, ZMIZ1 did not directly interact with NOTCH1 or influence the expression of several other NOTCH1 target genes such Ptcra, Hes1, Dtx1, and Cd25. Thus, ZMIZ1 did not pan-activate NOTCH signaling. Based on bioinformatic analysis, we generated mutants that deleted individual domains of ZMIZ1. All mutants expressed at high levels by Western blot. Deletion of the transcriptional activation domain or the N-terminal domain (NTD) abolished the ability of ZMIZ1 to induce c-Myc and drive proliferation. Surprisingly, deletion of the PAT-like, Proline-rich, and MIZ domains or all three domains simultaneously had no effect on ZMIZ1 function. The 120-amino acid NTD has a predicted helical structure without significant sequence homology to any known domain. It is not found in ZMIZ2 or PIAS proteins. In summary, the mechanism of ZMIZ1 appears to be novel, indirect, transcriptional, and independent of canonical NOTCH and PIAS functions. Our study demonstrates the importance of characterizing genetic collaborations between parallel leukemic pathways that may be therapeutically targeted. They also raise new inquiries into potential NOTCH-ZMIZ1 collaboration in a variety of C-MYC-driven cancers. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

DNA Methylation in T-Cell Acute Lymphoblastic Leukemia: In Search for Clinical and Biological Meaning

International Journal of Molecular Sciences ◽

10.3390/ijms22031388 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1388

Author(s):

Natalia Maćkowska ◽

Monika Drobna-Śledzińska ◽

Michał Witt ◽

Małgorzata Dawidowska

Keyword(s):

Dna Methylation ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Epigenetic Modification ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Global Methylation ◽

Current State ◽

History Of ◽

Cell Acute Lymphoblastic Leukemia

Distinct DNA methylation signatures, related to different prognosis, have been observed across many cancers, including T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematological neoplasm. By global methylation analysis, two major phenotypes might be observed in T-ALL: hypermethylation related to better outcome and hypomethylation, which is a candidate marker of poor prognosis. Moreover, DNA methylation holds more than a clinical meaning. It reflects the replicative history of leukemic cells and most likely different mechanisms underlying leukemia development in these T-ALL subtypes. The elucidation of the mechanisms and aberrations specific to (epi-)genomic subtypes might pave the way towards predictive diagnostics and precision medicine in T-ALL. We present the current state of knowledge on the role of DNA methylation in T-ALL. We describe the involvement of DNA methylation in normal hematopoiesis and T-cell development, focusing on epigenetic aberrations contributing to this leukemia. We further review the research investigating distinct methylation phenotypes in T-ALL, related to different outcomes, pointing to the most recent research aimed to unravel the biological mechanisms behind differential methylation. We highlight how technological advancements facilitated broadening the perspective of the investigation into DNA methylation and how this has changed our understanding of the roles of this epigenetic modification in T-ALL.

Download Full-text

PTEN microdeletions in T-cell acute lymphoblastic leukemia are caused by illegitimate RAG-mediated recombination events

Blood ◽

10.1182/blood-2014-03-562751 ◽

2014 ◽

Vol 124 (4) ◽

pp. 567-578 ◽

Cited By ~ 51

Author(s):

Rui D. Mendes ◽

Leonor M. Sarmento ◽

Kirsten Canté-Barrett ◽

Linda Zuurbier ◽

Jessica G. C. A. M. Buijs-Gladdines ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Lymphoblastic Leukemia ◽

Human T Cell ◽

Key Points ◽

Inactivation Mechanism ◽

Cell Acute Lymphoblastic Leukemia

Key Points Microdeletions represent an additional inactivation mechanism for PTEN in human T-cell acute lymphoblastic leukemia. PTEN microdeletions are RAG-mediated aberrations.

Download Full-text

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

Blood ◽

10.1182/blood-2006-05-025221 ◽

2006 ◽

Vol 109 (8) ◽

pp. 3417-3423 ◽

Cited By ~ 55

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.

Download Full-text