scholarly journals NKX3.1 is a direct TAL1 target gene that mediates proliferation of TAL1-expressing human T cell acute lymphoblastic leukemia

2010 ◽  
Vol 207 (10) ◽  
pp. 2141-2156 ◽  
Author(s):  
Sophie Kusy ◽  
Bastien Gerby ◽  
Nicolas Goardon ◽  
Nathalie Gault ◽  
Federica Ferri ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Notch Pathway ◽  
Gene Promoter ◽  
Leukemic Cells ◽  
Regulatory Sequence ◽  
Human T Cell ◽  
T Cell Leukemogenesis

TAL1 (also known as SCL) is expressed in >40% of human T cell acute lymphoblastic leukemias (T-ALLs). TAL1 encodes a basic helix-loop-helix transcription factor that can interfere with the transcriptional activity of E2A and HEB during T cell leukemogenesis; however, the oncogenic pathways directly activated by TAL1 are not characterized. In this study, we show that, in human TAL1–expressing T-ALL cell lines, TAL1 directly activates NKX3.1, a tumor suppressor gene required for prostate stem cell maintenance. In human T-ALL cell lines, NKX3.1 gene activation is mediated by a TAL1–LMO–Ldb1 complex that is recruited by GATA-3 bound to an NKX3.1 gene promoter regulatory sequence. TAL1-induced NKX3.1 activation is associated with suppression of HP1-α (heterochromatin protein 1 α) binding and opening of chromatin on the NKX3.1 gene promoter. NKX3.1 is necessary for T-ALL proliferation, can partially restore proliferation in TAL1 knockdown cells, and directly regulates miR-17-92. In primary human TAL1-expressing leukemic cells, the NKX3.1 gene is expressed independently of the Notch pathway, and its inactivation impairs proliferation. Finally, TAL1 or NKX3.1 knockdown abrogates the ability of human T-ALL cells to efficiently induce leukemia development in mice. These results suggest that tumor suppressor or oncogenic activity of NKX3.1 depends on tissue expression.

scholarly journals KDM2B in polycomb repressive complex 1.1 functions as a tumor suppressor in the initiation of T-cell leukemogenesis

2019 ◽  
Vol 3 (17) ◽  
pp. 2537-2549 ◽  
Author(s):  
Yusuke Isshiki ◽  
Yaeko Nakajima-Takagi ◽  
Motohiko Oshima ◽  
Kazumasa Aoyama ◽  
Mohamed Rizk ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Suppressor ◽  
Zinc Finger ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Dependent Manner ◽  
Polycomb Repressive Complex ◽  
Human T Cell ◽  
T Cell Leukemogenesis ◽  
Deficient Mice

Abstract KDM2B together with RING1B, PCGF1, and BCOR or BCORL1 comprise polycomb repressive complex 1.1 (PRC1.1), a noncanonical PRC1 that catalyzes H2AK119ub1. It binds to nonmethylated CpG islands through its zinc finger-CxxC DNA binding domain and recruits the complex to target gene loci. Recent studies identified the loss of function mutations in the PRC1.1 gene, BCOR and BCORL1 in human T-cell acute lymphoblastic leukemia (T-ALL). We previously reported that Bcor insufficiency induces T-ALL in mice, supporting a tumor suppressor role for BCOR. However, the function of BCOR responsible for tumor suppression, either its corepressor function for BCL6 or that as a component of PRC1.1, remains unclear. We herein examined mice specifically lacking the zinc finger-CxxC domain of KDM2B in hematopoietic cells. Similar to Bcor-deficient mice, Kdm2b-deficient mice developed lethal T-ALL mostly in a NOTCH1-dependent manner. A chromatin immunoprecipitation sequence analysis of thymocytes revealed the binding of KDM2B at promoter regions, at which BCOR and EZH2 colocalized. KDM2B target genes markedly overlapped with those of NOTCH1 in human T-ALL cells, suggesting that noncanonical PRC1.1 antagonizes NOTCH1-mediated gene activation. KDM2B target genes were expressed at higher levels than the others and were marked with high levels of H2AK119ub1 and H3K4me3, but low levels of H3K27me3, suggesting that KDM2B target genes are transcriptionally active or primed for activation. These results indicate that PRC1.1 plays a key role in restricting excessive transcriptional activation by active NOTCH1, thereby acting as a tumor suppressor in the initiation of T-cell leukemogenesis.

c-MYC Is a Major Downstream Target of NOTCH in T-Cell Acute Lymphoblastic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3005-3005
Author(s):  
Arthur Lau ◽  
Jon C. Aster ◽  
Andrew P. Weng
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Growth Arrest ◽  
Notch Pathway ◽  
Dominant Negative ◽  
Cellular Growth ◽  
Downstream Target ◽  
Human T Cell ◽  
Notch Inhibition

Abstract We recently reported that activating mutations in the NOTCH1 receptor occur in a high percentage of primary human T-cell acute lymphoblastic leukemias (T-ALL). Withdrawal of NOTCH signals by treatment with γ-secretase inhibitors (GSI) or by transduction with a dominant-negative Mastermind-like-1 polypeptide (a specific NOTCH pathway inhibitor) induces growth arrest of many T-ALL cell lines, suggesting that NOTCH supplies signals that are needed for maintenance of growth of T-ALL cells. In order to identify downstream targets of NOTCH that mediate these effects, we performed gene expression profiling on NOTCH signaling-dependent T-ALL cell lines before and after NOTCH inhibition. Among a number of identified candidate genes was c-MYC, which was of particular interest given its importance in promoting cellular growth and its known dysregulation in a number of hematolymphoid neoplasms. c-MYC was down-regulated following NOTCH inhibition, and rapidly up-regulated following release of NOTCH inhibition, even in the presence of protein synthesis inhibitors, suggesting that it is a direct NOTCH transcriptional target. Further, a subset of murine and human T-ALL cell lines were rescued from GSI-mediated growth arrest by c-MYC-expressing retroviruses. The failure of c-MYC to rescue some NOTCH-dependent cell lines likely stems from differences in cellular context, such as collaborating oncogenic lesions and/or the stage of T cell development the cell lines recapitulate. Nevertheless, these data implicate c-MYC as a major downstream target of NOTCH in T-ALL.

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

Blood ◽  
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.

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

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 748-748
Author(s):  
Sophie Kusy ◽  
Nicolas Goardon ◽  
Florence Armstrong ◽  
Francoise Pflumio ◽  
paul-Henri Romeo
Keyword(s):  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Regulatory Sequences ◽  
Human T Cell ◽  
Cell Acute Lymphoblastic Leukemia

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.

Adoptive T-Cell Therapy for B-Cell Acute Lymphoblastic Leukemia: Preclinical Studies

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3531-3540 ◽  
Author(s):  
Angelo A. Cardoso ◽  
J. Pedro Veiga ◽  
Paolo Ghia ◽  
Hernani M. Afonso ◽  
W. Nicholas Haining ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Adoptive T Cell Therapy ◽  
Leukemic Cells ◽  
Acute Leukemias ◽  
T Cell Lines

We have previously shown that leukemia-specific cytotoxic T cells (CTL) can be generated from the bone marrow of most patients with B-cell precursor acute leukemias. If these antileukemia CTL are to be used for adoptive immunotherapy, they must have the capability to circulate, migrate through endothelium, home to the bone marrow, and, most importantly, lyse the leukemic cells in a leukemia-permissive bone marrow microenvironment. We demonstrate here that such antileukemia T-cell lines are overwhelmingly CD8+ and exhibit an activated phenotype. Using a transendothelial chemotaxis assay with human endothelial cells, we observed that these T cells can be recruited and transmigrate through vascular and bone marrow endothelium and that these transmigrated cells preserve their capacity to lyse leukemic cells. Additionally, these antileukemia T-cell lines are capable of adhering to autologous stromal cell layers. Finally, autologous antileukemia CTL specifically lyse leukemic cells even in the presence of autologous marrow stroma. Importantly, these antileukemia T-cell lines do not lyse autologous stromal cells. Thus, the capacity to generate anti–leukemia-specific T-cell lines coupled with the present findings that such cells can migrate, adhere, and function in the presence of the marrow microenvironment enable the development of clinical studies of adoptive transfer of antileukemia CTL for the treatment of ALL.

Notch Signaling Represses Mir-223 in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4630-4630
Author(s):  
Samuel D Gusscott ◽  
Florian Kuchenbauer ◽  
Andrew P Weng
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Growth ◽  
Notch Signaling ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Gamma Secretase ◽  
Protein Levels ◽  
Cell Acute Lymphoblastic Leukemia ◽  
T Cell Leukemogenesis

Abstract Abstract 4630 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer of immature T cells that often shows aberrant activation of the Notch1 signaling pathway. Several studies have utilized mRNA expression profiling to identify downstream mediators of oncogenic Notch signaling in this context. Since microRNAs (miRNAs) have in recent years been shown to play important roles in hematological maliganancy, we performed a microarray-based screen for Notch-dependent miRNA expression in T-ALL. Jurkat and P12-Ichikawa cell lines were treated with gamma-secretase inhibitor to block Notch signaling vs. DMSO control for 4 days and profiled using Exigon miRCURY LNA miRNA microarrays. Surprisingly few miRNAs were found to be regulated by this approach; however, one of the hits, miR-223, showed consistent upregulation after gamma-secretase treatment in Jurkat cells and 5 additional human T-ALL cell lines assessed by miRNA qPCR. This observation was unique to human T-ALL as murine models of T-ALL showed no evidence for Notch-dependent miR-223 expression. Given that canonical Notch signaling results in transcriptional activation, our observation that Notch signaling is associated with reduced miR-223 expression suggests an intermediary repressor may be involved. miR-223 has been reported to play an important role in normal granulopoiesis, to be expressed relatively highly in T-ALL with myeloid-like gene features, and most recently to accelerate Notch-mediated T-cell leukemogenesis. To explore potential functional consequences for Notch-dependent miR-223 repression in T-ALL, candidate miR-223 targets identified by TargetScan software were analyzed with Ingenuity Pathway Analysis software, which indicated IGF-1, insulin receptor, PTEN, and ERK5 signaling pathways as the top hits. We recently reported IGF1R signaling to be important for growth and viability of bulk T-ALL cells as well as for leukemia-initiating cell activity. Additionally, we reported that Notch signaling directly upregulates IGF1R transcription by binding to an intronic enhancer which is present between exons 21/22 in the human, but not mouse IGF1R locus. As miR-223 has previously been reported to target IGF1R mRNA and reduce its translation, we hypothesized that Notch signaling may also upregulate net IGF1R protein expression by repressing miR-223. To test this hypothesis, we transduced several human T-ALL cell lines with miR-223 retrovirus and observed a modest decrease in total IGF1R protein levels by western blot; however, no significant change was observed in surface IGF1R levels as assessed by flow cytometry. Addtionally, knockdown of miR-223 by lentiviral expression miR-223 target sequences (miR-223 “sponge”) resulted in modestly increased total IGF1R protein levels, but again showed no demonstrable effect on surface IGF1R levels. Of note, we also observed no apparent effect of either overexpression or knockdown of miR-223 on bulk cell growth or viability. We interpret these findings to suggest that Notch signaling does not have major effects on the miR transcriptome, and that up- or down-modulation of miR-223 in established T-ALL cells does not have significant effects on overall cell growth/viability. Further studies will be required to determine if miR-223 may act in concert with other Notch target genes to modulate cell physiology. Disclosures: No relevant conflicts of interest to declare.

NKL Homeobox Gene MSX1 Reactivates An Oncogenic Network In Lymphoid and Myeloid Malignancies

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3765-3765
Author(s):  
Stefan Nagel ◽  
Stefan Ehrentraut ◽  
Corinna Meyer ◽  
Maren Kaufmann ◽  
Hans G. Drexler ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Notch Pathway ◽  
Molecular Cytogenetics ◽  
Homeobox Gene ◽  
In Silico Analysis ◽  
Reciprocal Regulation ◽  
Myeloid Malignancies

Abstract Homeobox genes encode conserved transcription factors (TFs) which regulate fundamental cellular processes during development. Many members of the NKL homeobox gene subfamily are aberrantly expressed in T-cell leukemia and compromise cell differentiation. NKL homeobox gene MSX1 is expressed during embryonic hematopoiesis and its deregulation in Hodgkin lymphoma suggests an oncogenic role of this gene in hematopoietic malignancies. After screening 114 leukemia/lymphoma cell lines by microarray profiling, we detected MSX1 overexpression in three examples each from T-cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL), and one from acute myeloid leukemia (AML). In silico analysis by R-based statistical tools identified conspicuous expression of MSX1 in 11% of pediatric T-ALL patients, and in 3% each of MCL and AML patients. Thus, we found aberrant MSX1 expression in subsets of both lymphoid and myeloid malignancies. Focusing on MCL and AML we excluded chromosomal rearrangements by classical and molecular cytogenetics at the MSX1 locus underlying overexpression in affected cell lines. However, comparative expression profiling data indicted aberrant histone acetylation involving PHF16 and RTN1, together with TFs FOXC1, HLXB9 and TAL1, as activators of MSX1 transcription. Their involvement was confirmed by siRNA-mediated knockdown and overexpression studies. Reciprocal regulation of MSX1 involved CCND1 and NOTCH signalling. Reporter gene analyses demonstrated that CCND1 and CDKN2D are direct transcriptional targets of MSX1 and its repressive cofactor histone H1C. Fluorescence in situ hybridization showed that t(11;14)(q13;q32) in MCL results in detachment of CCND1 from its corresponding repressive MSX1 binding site. In conclusion, we uncovered a regulatory network around MSX1 in leukemia/lymphoma cells, involving factors and pathways implicated in embryonic hematopoiesis. The reciprocal regulation of MSX1 and the NOTCH pathway in B-cells parallels that of MSX2 in T-cells. These data support the view of a recurrent genetic network involved in hematopoietic development which is reactivated in malignant transformation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression of common acute lymphoblastic leukemia antigen (CALLA) by lymphomas of B-cell and T-cell lineage

Blood ◽  
1981 ◽  
Vol 58 (3) ◽  
pp. 648-652 ◽  
Author(s):  
J Ritz ◽  
LM Nadler ◽  
AK Bhan ◽  
J Notis-McConarty ◽  
JM Pesando ◽  
...  
Keyword(s):  
Lymph Node ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Lymphoid Cells ◽  
Leukemic Cells ◽  
Hodgkin's Lymphomas ◽  
Leukemia Antigen

Previous studies have demonstrated that the common acute lymphoblastic leukemia antigen (CALLA) is expressed by leukemic cells from approximately 80% of patients with non-T-cell ALL and 30%-50% of patients with chronic myelocytic leukemia in blast crisis. A small number of normal bone marrow and fetal liver cells also express CALLA, but the functional role of this molecule is unknown. In the present study, we have used a monoclonal antibody (J5) specific for CALLA to study the expression of this antigen in non-Hodgkin's lymphomas. Within the B-cell lymphomas, it was found the CALLA was expressed by almost all Burkitt's and nodular poorly differentiated lymphocytic lymphomas. Within the T-cell lymphomas, CALLA was expressed in 40% of patients with lymphoblastic lymphoma. Three of 3 Burkitt's lymphoma cell lines and three of eight T-lymphoblast cell lines were also found to express CALLA. Normal spleen, lymph node, and thymus cells were not reactive with J5 antibody. These findings indicate that expression of CALLA is not limited to relatively undifferentiated leukemic lymphoblasts but also occurs in more differentiated lymphoid malignancies. However, normal differentiated lymphoid cells in lymph node, spleen, and thymus, which have a phenotype similar to that of lymphoma cells, do not appear to express CALLA.

scholarly journals Krüppel-like Factor 4 (KLF4) Suppresses T-Cell Acute Lymphoblastic Leukemia By Inhibiting Expression of MAP2K7 and Expansion of Leukemia Initiating Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3569-3569
Author(s):  
Ye Shen ◽  
Chun Shik Park ◽  
Koramit Suppipat ◽  
Takeshi Yamada ◽  
Toni-Ann Mistretta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Brdu Incorporation ◽  
Jnk Pathway

Abstract Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy in children. Although risk-adaptive therapy, CNS-directed chemotherapy and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs or novel multi-drug combinations are needed as frontline treatments for high-risk patients and as salvage agents for relapsed disease. T-cell ALL (T-ALL) is a subset of ALL that exhibits activating mutations of NOTCH1 in more than 50% of the patients. However, the use of gamma-secretase inhibitors to reduce NOTCH1 activity has not been successful in patients due to limited response and toxicity. Therefore, identification of genetic factors that cooperate with T-ALL leukemogenesis is needed for the development of alternative therapies. KLF4 is a transcription factor that functions as a tumor suppressor or an oncogene depending on cellular context. Our data showed significant reduction of KLF4 transcripts in lymphoblasts from T-ALL patients compared to blood and bone marrow cells from healthy individuals. In consistent with reduced KLF4 levels, these patients exhibit hyper-methylation of CpG islands located between nt -811 and +1190 relative to KLF4 transcription start site. From these findings we hypothesized that KLF4 has tumor suppressor function in T-ALL leukemogenesis. To test our hypothesis, we transduced 5-FU treated bone marrow (BM) cells from control (Klf4fl/fl), Klf4 null (Klf4fl/fl; Vav-iCre) and Klf4 heterozygous (Klf4fl/+; Vav-iCre) mice with retrovirus carrying a NOTCH1 activating mutant (L1601P-ΔP) and then transplanted these BM cells into irradiated recipient mice. In contrast to controls, mice transplanted with transduced Klf4-null BM cells developed T-ALL with significantly higher penetrance (Klf4 null 76.5% v.s. control 21.3%) and shorter latency (Klf4 null 93 days v.s. control 130 days). Interestingly, Klf4 heterozygous group shows similar survival kinetics as Klf4 null group, suggesting that Klf4 haploinsufficiency is enough to accelerate onset of leukemia. To investigate the effect of Klf4 deletion in established leukemia cells, we transplanted NOTCH1 L1601P-ΔP transduced BM cells from Klf4fl/fl; CreER+ mice to induce leukemia. Post-transplantation deletion of the Klf4 gene by tamoxifen administration was able to accelerate T-ALL development compared to mice injected with vehicle. On the cellular level, loss of KLF4 led to increased proliferation of leukemia cells as assessed by in vivo BrdU incorporation, which correlated with decreased levels of p21 protein. Limited dilution transplantation of primary leukemia cells into secondary recipients showed a 9-fold increase of leukemia initiating cells (LIC) frequency in Klf4null leukemia cells compared to controls, suggesting that KLF4 controls expansion of LIC in T-ALL. To elucidate molecular mechanism underlying KLF4 regulation in T-ALL cells, we performed microarray and ChIP-Seq in control and Klf4 null CD4+CD8+ leukemia cells. Combined analyses revealed 202 genes as KLF4 direct targets, of which 11 genes are also deregulated in human T-ALL cells by comparing with published microarray datasets. One of the top upregulated genes is Map2k7, which encodes a kinase upstream of the JNK pathway. Immunoblots in leukemia cells confirmed increased expression of MAP2K7 protein and enhanced phosphorylation of its downstream targets JNK and ATF2. To further investigate the role of JNK pathway in T-ALL, we tested JNK inhibitor SP600125 in human T-ALL cell lines (KOPTK1, DND41, CCRF-CEM, MOLT3). Interestingly, SP600125 showed dose-dependent cytotoxicity in all human T-ALL cell lines tested regardless of their NOTCH1 status. Overall our results showed for the first time that KLF4 functions as a tumor suppressor in T-ALL by regulating proliferation of leukemia cells and frequency of LIC. Additional study elucidated that KLF4 suppresses the JNK pathway via direct transcriptional regulation of MAP2K7. Moreover, the vulnerability of human T-ALL cell lines to JNK inhibition provides a novel target for future therapy in T-ALL patients. Disclosures No relevant conflicts of interest to declare.

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