GATA3 Silencing Defines a Distinct Subset of ETP-ALL.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2389-2389
Author(s):  
Lars Fransecky ◽  
Martin Neumann ◽  
Sandra Heesch ◽  
Marco Seehawer ◽  
Cornelia Schlee ◽  
...  
Keyword(s):  
T Cell ◽  
Mrna Expression ◽  
Lymphoblastic Leukemia ◽  
T Cell Development ◽  
Demethylating Agents ◽  
Expression Arrays ◽  
Gata3 Expression ◽  
Gata3 Mrna

Abstract Abstract 2389 Introduction The transcription factor GATA3 plays an important role in normal T cell development. Its role in mature T cells is well understood, but its function in earlier stages of T cell development remains unclear. Whereas GATA3 levels are precisely regulated for the T cell differentiation program, aberrant expression of GATA3 has been linked to tumorigenesis. Based on these observations, we investigated the role of GATA3 in Early Thymic Progenitor Acute Lymphoblastic Leukemia (ETP-ALL), a newly defined high-risk subgroup of T-ALL, characterized by a specific gene expression profile and distinct immunophenotype. Patients and Methods Eighty-six bone marrow samples from adult patients with newly diagnosed T-ALL, including ETP-ALL (n=17) enrolled into the German Multicenter Acute Lymphoblastic Leukemia (GMALL) trials, were studied for GATA3 expression by oligonucleotide expression arrays (HG-U133 plus 2.0) within the Microarray Innovations in LEukemia study. We identified additional 71 ETP-ALL adult patients and 94 T-ALL patients enrolled on the GMALL protocol, in which GATA3 mRNA expression was measured by quantitative polymerase chain reaction (RT-PCR). Combining ETP-ALL and T-ALL cases (n=165), we defined two GATA3 expression groups GATA3null and GATA3high based on a biological gap (GATA3 expression of 0.2). DNA methylation was analyzed in both T-ALL (n=11) and ETP-ALL (n=69) samples by pyrosequencing with primers designed to include seven CpG sites of Exon 2/Intron 3 of GATA3. Samples were grouped into GATA3 high vs. low methylation according to their mean methylation being below or above 40%. Results Based on gene expression arrays we observed a high proportion of ETP-ALL (11/17) that lacked GATA3 expression, whereas only a small fraction of the remaining T-ALL cases (3/69) had no GATA3 expression. These results were validated by RT-PCR in a larger cohort: 26% of ETP-ALL (19/71) were GATA3null, but only 2% of T-ALL (2/94) were in the GATA3null expression group. To explore the regulation of this specific expression pattern, epigenetic regulation of GATA3 was analyzed by pyrosequencing. While unselected T-ALL samples were hypomethylated (< 6% methylated CpG), ETP-ALL samples had a higher GATA3 methylation status (28% methylated CpG, p<0.001). ETP-ALL cases were further categorized into high methylated (18/69) and low methylated samples (51/69) and correlated to mRNA expression. GATA3null samples showed a higher degree of GATA3 methylation (41% methylated CpGs) compared to GATA3high samples (8% methylated CpGs, p < 0.001). In an in-vitro assay of T-cell leukemia cell lines demethylating agents increased GATA3 mRNA expression by up to 5-fold. In murine hematopoetic stem cells it was shown that loss of DNMT3A induced GATA3 expression via hypomethylation. In ETP-ALL, we identified 11 DNMT3A mutations in 69 samples (16%) and correlated the DNMT3A mutation status to GATA3 methylation. Ten of 11 (91%) DNMT3A mutated samples showed low level GATA3 methylation, whereas 17 (29%) of the 58 DNMT3A wildtype cases had high methylation. Conclusion ETP-ALL is a subgroup of adult T-ALL with a distinct molecular profile. Here we show that within ETP-ALL a separate molecular entity can be defined by GATA3 silencing due to DNA methylation. In-vitro studies showed that GATA3 expression can be restored by the use of demethylating agents. As loss of function mutations in DNMT3A correlate with low GATA3 methylation in ETP-ALL, a potential role of DNMT3A in the epigenetic silencing of GATA3 is suspected. So far, the number of targeted drugs available for T-ALL is limited. Therefore, incorporating demethylating agents may resolve the T-cell differentiation block in T-ALL by increasing GATA3 expression. Future work will explore downstream effects of GATA3 in acute leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals miR-22-3p Negatively Affects Tumor Progression in T-Cell Acute Lymphoblastic Leukemia

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1726
Author(s):  
Valentina Saccomani ◽  
Angela Grassi ◽  
Erich Piovan ◽  
Deborah Bongiovanni ◽  
Ludovica Di Martino ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Target Genes ◽  
Cell Transformation ◽  
Lymphoblastic Leukemia ◽  
T Cell Development ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Notch1 Signaling Pathway

T-cell acute lymphoblastic leukemia (T-ALL) is a rare, aggressive disease arising from T-cell precursors. NOTCH1 plays an important role both in T-cell development and leukemia progression, and more than 60% of human T-ALLs harbor mutations in components of the NOTCH1 signaling pathway, leading to deregulated cell growth and contributing to cell transformation. Besides multiple NOTCH1 target genes, microRNAs have also been shown to regulate T-ALL initiation and progression. Using an established mouse model of T-ALL induced by NOTCH1 activation, we identified several microRNAs downstream of NOTCH1 activation. In particular, we found that NOTCH1 inhibition can induce miR-22-3p in NOTCH1-dependent tumors and that this regulation is also conserved in human samples. Importantly, miR-22-3p overexpression in T-ALL cells can inhibit colony formation in vitro and leukemia progression in vivo. In addition, miR-22-3p was found to be downregulated in T-ALL specimens, both T-ALL cell lines and primary samples, relative to immature T-cells. Our results suggest that miR-22-3p is a functionally relevant microRNA in T-ALL whose modulation can be exploited for therapeutic purposes to inhibit T-ALL progression.

The in Vitro and In Vivo Oncogenic Activity of Homodimeric Mutant of Interleukin-7 Receptor a Chain (IL7Rα) Highlights the Significance of the IL7Rα/Jak1 Pathway in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 73-73
Author(s):  
Kazuaki Yokoyama ◽  
Nozomi Yokoyama ◽  
Kiyoko Izawa ◽  
Ai Kotani ◽  
Ratanakanit Harnprasopwat ◽  
...  
Keyword(s):  
T Cell ◽  
Lymphoblastic Leukemia ◽  
T Cell Development ◽  
Cell Development ◽  
Gamma Delta ◽  
Interleukin 7 ◽  
Signaling Axis ◽  
A Chain

Abstract Abstract 73 Interleukin-7 (IL7) is essential for T cell development and homeostasis. Dysregulation of signals that control normal T-cell development has been implicated in the onset of T-cell acute lymphoblastic leukemia (T-ALL). By analogy to activating mutations in the Notch pathways, we hypothesized that any mutations in the IL7 signaling axis might also contribute to T-ALL. Direct sequencing of human IL7 receptor a chain (hIL7RA) gene in a panel of 16 T-ALL cell lines identified two types of mutations in two different cell lines. One was an insertion mutation of 4 amino acids (LSRC) in the transmembrane region (INS, Fig.1A) from DND-41, a gamma-delta TCR+ T-ALL cell line, and the other was a truncated, loss-of-function, mutation in the cytoplasmic region from MOLT-4. We demonstrated that hIL7RA-INS mutant spontaneously formed a homodimer and constitutively activated downstream signals including Stat family members (1, 3 and 5), Akt and Erk via Jak1, but not Jak3. Next, we investigated oncogenic activity of hIL7RA-INS in primary hematopoietic progenitor cells. To this aim, lin− E.14 Balb/c fetal liver (FL) cells were retrovirally transduced with hIL7RA-INS in parallel with hIL7RA-wild type (WT), and then tested for their cytokine dependence in vitro. As expected, only hIL7RA-INS-transduced lin−FL cells showed abrogation of cytokine dependence. hIL7RA-transduced lin−FL cells were also transplanted into lethally irradiated syngeneic mice. Within 7–9 weeks after transplantation of lin−FL cells transduced with hIL7RA-INS, but not with hIL7RA-WT, recipient mice developed well-tolerated myelo- and lymphoproliferative disorders, characterized by marked leukocytosis, systemic lymphadenopathy and splenomegaly (Fig.1B). Notably, concomitant increase in hIL7RA+gamma-delta TCR+ T cells and decrease in B cells were observed in peripheral blood (Fig.1C). Histological examination of bone marrow, spleen and liver specimens from diseased mice revealed moderate to severe myeloid hyperplasia, disrupted splenic architecture by disseminated mature myeloid cells and infiltration of both myeloid and mononuclear cells into hepatic parenchyma, respectively. In addition, recipient mice for hIL7RA-INS-transduced lin−FL cells frequently manifested ruffled fur as well as mononuclear cell infiltration into salivary gland and pericardium, suggesting an autoimmune-like disorder. However, during median follow-up of 11 weeks, these recipient mice did not develop either overt leukemia or lymphoma, indicating that additional transforming events are required for evolution to aggressive hematological malignancies. These in vivo findings highlighted the possibility that aberrant signals via IL7RA in hematopoietic stem/progenitor cells might preferentially stimulate myelopoiesis over lymphopoiesis, and also confirmed the essential role of IL7RA in gamma-delta TCR+ T cell development, previously shown by IL7RA-knockout mice. Taken together, we speculated that dysregulated IL7RA signaling axis might be involved in the onset of T-ALL, especially with gamma-delta TCR+ phenotype. Finally, the present study, together with the recent report (JEM 208:901, 2011), emphasizes the significance of the sequential Notch-IL7RA pathways in the pathogenesis of T-ALL as well as the dominant role of the IL7RA/Jak1 axis in IL7 proliferative signal. Disclosures: No relevant conflicts of interest to declare.

L-arginine depletion by PEG-arginase I, a new potential therapy for acute lymphoblastic leukemia.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 6580-6580
Author(s):  
Ofelia Crombet Ramos ◽  
Claudia Hernandez ◽  
Kevin Morrow ◽  
John T. Cole ◽  
Paulo Rodriguez
Keyword(s):  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
De Novo ◽  
Remission Rate ◽  
Lymphoblastic Leukemia ◽  
Arginase I

6580 Background: Advances in therapies have resulted in an overall complete remission rate of approximately 85% for childhood acute lymphoblastic leukemia (ALL). In contrast, the overall remission rate of adults with leukemia continues to be poor, only about 40% in cases of T cell-ALL (T-ALL). Therefore, it is imperative to generate new therapies that alone or in combination with other treatments could potentially increase the percentages of complete responders or be used to treat the refractory ALL population. Our published results show that a pegylated form of human arginase I (peg-Arg I) prevented T-ALL cell proliferation in vitro and in vivo through the induction of tumor cell apoptosis. Interestingly, the anti-leukemic effects induced by peg-Arg I did not affect the anti-tumor activity of normal T cells, suggesting an anti-tumor specific effect. Our hypothesis states that peg-Arg I has an anti-tumoral effect on B-ALL and T-ALL cells in vitro and that the sensitivity of ALL cells to peg-Arg I depends on their expression of argininosuccinate synthase (ASS) and their ability to produce L-arginine de novo from citrulline. Methods: Malignant T cell proliferation was tested using nonradioactive cell proliferation yellow tretrazolium salt kit. Apoptosis studies were based on the expression of annexin V. Western blot assays were conducted to determine enzymatic expression in different cell lines. Results: The results of our in vitro experiments showed that peg-Arg I had a pro-apoptotic and anti-proliferattive effect on B-ALL cells similar to the one previously seen on T-ALL cells. These effects can be overcome in cell lines able that express ASS and therefore to produce L-arginine de novo. Conclusions: Our results suggest the role of ASS in the ALL-apoptosis induced by peg-Arg-I. Our next steps include: _Understand why ASS-expressing ALL cells do not undergo apoptosis when cultured with peg-Arg-I_Determine the role of ASS in the anti-leukemic effect induced by peg-Arg-I in vivo. Completion of this research is expected to lead to a better understanding of how peg-Arg-I kills ALL cells and could provide the foundation for a novel therapy for ALL patients.

scholarly journals Noncanonical Wnt signaling promotes apoptosis in thymocyte development

2007 ◽  
Vol 204 (13) ◽  
pp. 3077-3084 ◽  
Author(s):  
Huiling Liang ◽  
Andrew H. Coles ◽  
Zhiqing Zhu ◽  
Jennifer Zayas ◽  
Roland Jurecic ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Wnt Signaling ◽  
T Cell Development ◽  
Cell Development ◽  
Thymocyte Development ◽  
Growth And Survival ◽  
Canonical Wnt

The Wnt–β-catenin signaling pathway has been shown to govern T cell development by regulating the growth and survival of progenitor T cells and immature thymocytes. We explore the role of noncanonical, Wnt–Ca2+ signaling in fetal T cell development by analyzing mice deficient for Wnt5a. Our findings reveal that Wnt5a produced in the thymic stromal epithelium does not alter the development of progenitor thymocytes, but regulates the survival of αβ lineage thymocytes. Loss of Wnt5a down-regulates Bax expression, promotes Bcl-2 expression, and inhibits apoptosis of CD4+CD8+ thymocytes, whereas exogenous Wnt5a increases apoptosis of fetal thymocytes in culture. Furthermore, Wnt5a overexpression increases apoptosis in T cells in vitro and increases protein kinase C (PKC) and calmodulin-dependent kinase II (CamKII) activity while inhibiting β-catenin expression and activity. Conversely, Wnt5a deficiency results in the inhibition of PKC activation, decreased CamKII activity, and elevation of β-catenin amounts in thymocytes. These results indicate that Wnt5a induction of the noncanonical Wnt–Ca2+ pathway alters canonical Wnt signaling and is critical for normal T cell development.

scholarly journals T-Cell mRNA Expression in Response to Mycobacterium bovis BCG Vaccination and Mycobacterium bovis Infection of White-Tailed Deer

2009 ◽  
Vol 16 (8) ◽  
pp. 1139-1145 ◽  
Author(s):  
Tyler C. Thacker ◽  
Mitchell V. Palmer ◽  
W. Ray Waters
Keyword(s):  
T Cell ◽  
Mrna Expression ◽  
Mycobacterium Bovis ◽  
Lesion Group ◽  
Effective Vaccine ◽  
Blood Leukocytes ◽  
Visible Lesion ◽  
Gata3 Expression ◽  
Ifn Γ

ABSTRACT Understanding immune responses of white-tailed deer (WTD) to infection with Mycobacterium bovis provides insight into mechanisms of pathogen control and may provide clues to development of effective vaccine strategies. WTD were vaccinated with either M. bovis BCG strain Pasteur or BCG strain Danish. Both vaccinees and unvaccinated controls were subsequently inoculated with virulent M. bovis via the intratonsillar route. Real-time PCR was used to assess T-cell mRNA expression in peripheral blood leukocytes (PBL) from animals following vaccination and infection. Recall T-cell responses were measured by assessing the relative expression of gamma interferon (IFN-γ), T-cell-specific T-box transcription factor (Tbet), interleukin 12p40 (IL-12p40), IL-12p35, IL-23p19, FoxP3, IL-17, and GATA3 in PBL stimulated in vitro with purified protein derivative (PPD) of M. bovis or a recombinant fusion protein, ESAT6-CFP10. Animals vaccinated with BCG Danish expressed more IFN-γ and Tbet than either BCG Pasteur-vaccinated animals or unvaccinated controls. BCG Pasteur-vaccinated animals expressed more GATA3 than either group. After infection, unvaccinated controls expressed more Tbet and IL-12p40 than vaccinated animals. BCG Pasteur-vaccinated animals expressed more GATA3 than either the unvaccinated controls or the BCG Danish-vaccinated animals after infection. Animals were divided into pathology groups to correlate gene expression with severity of pathology. Animals in the visible lesion group expressed more Tbet and IFN-γ than animals that were culture negative, while Tbet and IFN-γ expression in the culture-positive, no-visible-lesion group was intermediate. GATA3 expression inversely correlated with pathology. Overall, expression of immune response genes correlated more closely with pathology than vaccination treatment.

scholarly journals Genome-Wide Analysis Identifies Rag1 and Rag2 as Novel Notch1 Transcriptional Targets in Thymocytes

2021 ◽  
Vol 9 ◽  
Author(s):  
Yang Dong ◽  
Hao Guo ◽  
Donghai Wang ◽  
Rongfu Tu ◽  
Guoliang Qing ◽  
...  
Keyword(s):  
T Cell ◽  
Lymphoblastic Leukemia ◽  
T Cell Development ◽  
Antigen Receptors ◽  
Transcriptional Targets ◽  
Recombination Activating Genes ◽  
Genome Wide ◽  
Element Bearing ◽  
Transcriptional Complexes

Recombination activating genes 1 (Rag1) and Rag2 are expressed in immature lymphocytes and essential for generating the vast repertoire of antigen receptors. Yet, the mechanisms governing the transcription of Rag1 and Rag2 remain to be fully determined, particularly in thymocytes. Combining cDNA microarray and ChIP-seq analysis, we identify Rag1 and Rag2 as novel Notch1 transcriptional targets in acute T-cell lymphoblastic leukemia (T-ALL) cells. We further demonstrate that Notch1 transcriptional complexes directly bind the Rag1 and Rag2 locus in not only T-ALL but also primary double negative (DN) T-cell progenitors. Specifically, dimeric Notch1 transcriptional complexes activate Rag1 and Rag2 through a novel cis-element bearing a sequence-paired site (SPS). In T-ALL and DN cells, dimerization-defective Notch1 causes compromised Rag1 and Rag2 expression; conversely, dimerization-competent Notch1 achieves optimal upregulation of both. Collectively, these results reveal Notch1 dimerization-mediated transcription as one of the mechanisms for activating Rag1 and Rag2 expression in both primary and transformed thymocytes. Our data suggest a new role of Notch1 dimerization in compelling efficient TCRβ rearrangements in DN progenitors during T-cell development.

scholarly journals Spontaneous Intestinal Tumorigenesis inApc/Min+Mice Requires Altered T Cell Development with IL-17A

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Wook-Jin Chae ◽  
Alfred L. M. Bothwell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Mutation ◽  
Adoptive Transfer ◽  
Tumor Regression ◽  
T Cell Development ◽  
Cell Development ◽  
Intestinal Tumorigenesis

The control of inflammatory diseases requires functional regulatory T cells (Tregs) with significant Gata-3 expression. Here we address the inhibitory role of Tregs on intestinal tumorigenesis in theApc/Min+mouse model that resembles human familial adenomatous polyposis (FAP).Apc/Min+mice had a markedly increased frequency of Foxp3+ Tregs and yet decreased Gata-3 expression in the lamina propria. To address the role of heterozygousApcgene mutation in Tregs, we generatedFoxp3-Cre,Apcflox/+mice. Tregs from these mice effectively inhibited tumorigenesis comparable to wild type Tregs after adoptive transfer intoApc/Min+mice, demonstrating that the heterozygousApcgene mutation in Tregs does not induce the loss of control over tumor microenvironment. Adoptive transfer of in vitro generatedApc/Min+iTregs (inducible Tregs) failed to inhibit intestinal tumorigenesis, suggesting that naïve CD4 T cells generated fromApc/Min+mice thymus were impaired. We also showed that adoptively transferred IL-17A-deficientApc/Min+Tregs inhibited tumor growth, suggesting that IL-17A was critical to impair the tumor regression function ofApc/Min+Tregs. Taken together, our results suggest that both T cell development in a functional thymus and IL-17A control the ability of Treg to inhibit intestinal tumorigenesis inApc/Min+mice.

12 Development of an in vitro assay to assess bispecific T cell engager using T cells from CD3e humanized mice

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A12-A12
Author(s):  
Jun Zhou ◽  
Shuang Zhu ◽  
Hongjuan Zhang ◽  
Lei Zheng ◽  
Mingfa Zang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Lymphoblastic Leukemia ◽  
Humanized Mice ◽  
Activation Markers ◽  
In Vivo Models ◽  
Vitro Assay ◽  
Bite Antibody

BackgroundBispecific T cell engagers (BiTE) is a fast-growing class of immunotherapies. They are bispecific antibody that bind to T cell-surface protein (for example, CD3e) and a specific tumor associate antigen (TAA) on tumor cells, by which to redirect T cells against tumor cells in a MHC-independent manner. A successful example in the clinical is Blinatumomab, a BiTE antibody against CD3/CD19 approved in 2014 to treat acute lymphoblastic leukemia. Currently, many CD3-based BiTE are in clinical trials, including BCMAxCD3, Her2xCD3, CEAxCD3, and PSMAxCD3. To evaluate the efficacy of BiTE in vitro, human peripheral blood monocyte cells (hPBMC) are commonly being used as a source of T cells to co-culture with tumor cells. The disadvantage of using hPBMC is donor-to-donor variability and the availability of the original donor if a study needs to be repeated.MethodsTo overcome this, we proposed to replace hPBMC with T cells from human CD3e (hCD3) genetically engineered mouse models mice (GEMM) for in in vitro coculture assay. T cells were isolated from hCD3 GEMM mice using negative selection mouse T cell isolation kit. Conventional tumor cell lines or luciferase-engineered patient-derived-xenograft (PDX)-derived organoids (PDXO) expressing specific antigens are co-cultured with hCD3 T cells in 96-well plates in the presence of BiTE antibody.ResultsWe measured the killing of tumor cells using either flow cytometry or luciferase activity as readouts. To analyze tumor-reactivity of T cells to cancer cell line or organoids, IFN-gamma in the culture medium was measured and activation markers on T cells was assessed.ConclusionsOur data showed the feasibility of using humanized mice T cells as a replacement for hPBMCs to assess BiTE antibody in vitro. We are further validating the application of murine hCD3 T cells for in vivo models to test bispecific T cell engagers.

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