scholarly journals EZH2 knockdown upregulates expression of the genes involved in T-ALL cell differentiation

2021 ◽  
Author(s):  
Sahar Safaei ◽  
Behzad Baradaran ◽  
Behzad Mansoori ◽  
Masoumeh Fardi ◽  
Elham Baghbani ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Aberrant Expression ◽  
Expression Levels ◽  
Ezh2 Expression

Background: EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit), as one of the polycyclic group proteins (PcGs), is an epigenetic regulator that plays a crucial role in the pathophysiology of hematologic malignancies through regulating cell differentiation. Also, it is well known that aberrant expression of specific transcription factors can be involved in the pathogenesis of various cancers. Objective: Herein, we aimed to suppress EZH2 expression in MOLT-4 cells, T-ALL (T cell acute lymphoblastic leukemia) cell line, and evaluate the role of EZH2 on the expression of transcription factors that regulate T cell maturation, differentiation, and apoptosis. Methods: EZH2-siRNA was transfected into MOLT-4 cells, and the expression levels of EZH2, NOTCH1, TCF1, IKZF1, and NFATC1 were measured using real-time PCR. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was performed to study the effect of EZH2 knockdown on MOLT-4 cell viability. The apoptosis rate of EZH2-siRNA transfected cells was assessed by flow cytometry. The interaction of mentioned genes was investigated using STRING and GO (gene ontology). Results: Our results have shown that EZH2-siRNA transfection can substantially decrease EZH2 expression in MOLT-4 cells. Besides, EZH2 suppression can upregulate NOTCH1, TCF1, IKZF1, and NFATC1 expression levels. EZH2 knockdown does not affect the viability and apoptosis of MOLT-4 cells. The most remarkable protein-protein interaction of EZH2 has been with NOTCH1. Besides, GO analysis has demonstrated that EZH2, NOTCH1, TCF1, IKZF1, and NFATC1 were located within nucleoplasm and can regulate RNA polymerase II-mediated transcription. Conclusion: Our results have shown that MOLT-4 cells harbor increased expression of EZH2 in comparison with normal human T cells. EZH2 knockdown can upregulate the expression of the transcription factors involved in T cell differentiation. Thus, EZH2 can halt the differentiation of immature lymphoblastic T cells.

scholarly journals The oxidation of (−)-epigallocatechin-3-gallate inhibits T-cell acute lymphoblastic leukemia cell line HPB-ALL via the regulation of Notch1 expression

RSC Advances ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 1679-1684 ◽  
Author(s):  
Yu-Na Wang ◽  
Jing Wang ◽  
Hao-Nan Yang ◽  
Bang-Lei Zhang ◽  
Pan Zhang ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Line ◽  
Hematological Malignancy ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Activating Mutations ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia Cell Line

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy, and commonly associated with activating mutations in the Notch1 pathway.

Optimization of a BCR-ABL-Specific DNA Vaccine against Ph+ ALL Using a Nonviral Vector System and Nanoparticle Delivery

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4625-4625
Author(s):  
Yvonne Rott ◽  
Stefanie Arndt ◽  
Jordan Green ◽  
Daniel Anderson ◽  
Renata Stripecke ◽  
...  
Keyword(s):  
Overall Survival ◽  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Line ◽  
Dna Vaccine ◽  
Cd8 T Cells ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Gm Csf

Abstract Although 40–50% of adults and 70–80 % of children with acute lymphoblastic leukemia (ALL) can be cured by poly chemo therapy, the prognosis of patients with Philadelphia chromosome positive (Ph+) ALL remains poor. Therefore, new relapse prevention strategies are needed for patients with Ph+ ALL during remission. We have shown previously, that vaccination of mice with leukemia cell lines modified to express costimulatory molecules and cytokines induce a systemic immunity against the syngeneic BCR-ABLp185 expressing leukemia cell line BM185. However, the difficulties to culture and transfect human leukemia cells limit the clinical application of leukemia cell based vaccines. Thus, we evaluated the immunization of mice with DNA-based vaccines subsequently challenged by the cell line BM185. Combinations of minimalistic immunogenically defined gene expression (MIDGE) vectors encoding a BCR-ABLp185 fusion specific peptide, GM-CSF, IL12, IL27 or CD40L were used for in vivo transfection of murine skin. In addition, we used natural DNA-based double stem-loop immunomodulators (dSLIM), containing three CpG-motifs as non-specific immune adjuvant. In order to increase transfection efficacy, MIDGE-vectors were microencapsulated into poly(β-aminoester) nanoparticles with diameters of 200 nm. Mice immunized with the BCR-ABL/GM-CSF/dSLIM vaccine showed a significant longer mean tumor-free (p=0.019) and overall survival (p=0.008) compared to nonvaccinated mice. BCR-ABL specific sequences were required to prevent Ph+ acute lymphoblastic leukemia. Furthermore, CTL assays showed that specific lysis was significantly higher after vaccination with BCR-ABL/GM-CSF/dSLIM compared to GMCSF/dSLIM (p<0.05) and to naïve mice (p<0.005). The vaccine efficacy was clearly dosedependent. Microencapsulation of MIDGE vectors increased the efficacy of the vaccine compared to the naked DNA-vaccine. Mice immunized with the microencapsulated vaccine BCR-ABL/GM-CSF/dSLIM showed a significant longer mean tumor-free (p<0.0001) and overall survival (p<0.0001) compared to non-vaccinated mice and 70% survived and never developed leukemia. Cotransfection with IL27 or IL12 lead to significant longer tumor free (IL27: p=0.02; IL12: p<000.1) and overall survival (IL-27: p=0.03; IL12: p<000.1) compared to the vaccine BCR-ABL/GM-CSF/dSLIM. The best protection with a survival rate of 91% was observed in mice which received the vaccine BCR-ABL/GMCSF/IL12/dSLIM. We have shown previously in T-cell depletion studies that CD8+ T cells were the effector cells in the BM185 cell-based vaccine model and currently we evaluate whether CD8+ T cells also play a major role in the BM185 DNA-based vaccine model. In conclusion, we provide survival and functional data that show immunization and protection of mice with optimized leukemia specific DNA-vaccines.

scholarly journals T-cell translocation gene 1 (Ttg-1) encodes a nuclear protein normally expressed in neural lineage cells

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 599-606
Author(s):  
EA McGuire ◽  
AR Davis ◽  
SJ Korsmeyer
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Zinc Finger ◽  
Nuclear Protein ◽  
Zinc Finger Protein ◽  
Lymphoblastic Leukemia ◽  
Transcriptional Start Site ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Neural Lineage

We previously identified and cloned T-cell translocation gene 1 (Ttg- 1), a putative zinc finger protein, as a result of its deregulated expression in a T-cell acute lymphoblastic leukemia cell line (RPMI 8402) with a t(11;14)(p15;q11). We have now characterized its genomic organization and identified the major transcriptional start site to lie within an initiator-like motif. Ttg-1 is normally expressed in mouse brain and not in thymus. The mouse neuroblastoma cell line, N2a, also expresses Ttg-1. Antibodies raised against a TrpE-Ttg-1 fusion protein precipitate an 18-Kd nuclear protein from metabolically labeled 8402 cells. Immunofluorescence of N2a cells shows a nuclear pattern. The two potential zinc finger domains in Ttg-1 are highly homologous to similar regions in lin-11, mec-3, and lsl-1. This data suggests that Ttg-1 may be involved in gene regulation.

scholarly journals Endogenous TCR promotes in vivo persistence of CD19-CAR-T cells compared to a CRISPR/Cas9-mediated TCR knockout CAR

Blood ◽  
2020 ◽  
Vol 136 (12) ◽  
pp. 1407-1418 ◽  
Author(s):  
Dana Stenger ◽  
Tanja A. Stief ◽  
Theresa Kaeuferle ◽  
Semjon Willier ◽  
Felicitas Rataj ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Third Party ◽  
Leukemia Cell Line ◽  
Effector Memory ◽  
Childhood All ◽  
Car T Cells ◽  
Car T

Abstract Anti-CD19 chimeric antigen receptor (CAR) T cells showed significant antileukemic activity in B-precursor acute lymphoblastic leukemia (ALL). Allogeneic, HLA-mismatched off-the-shelf third-party donors may offer ideal fitness of the effector cells, but carry the risk of graft-versus-host disease. Knockout (KO) of the endogenous T-cell receptor (TCR) in CD19-CAR-T cells may be a promising solution. Here, we induced a CRISPR/Cas9-mediated KO of the TCRβ chain in combination with a second-generation retroviral CAR transduction including a 4-1BB costimulatory domain in primary T cells. This tandem engineering led to a highly functional population of TCR-KO-CAR-T cells with strong activation (CD25, interferon γ), proliferation, and specific killing upon CD19 target recognition. TCR-KO-CAR-T cells had a balanced phenotype of central memory and effector memory T cells. KO of the endogenous TCR in T cells strongly ablated alloreactivity in comparison with TCR-expressing T cells. In a patient-derived xenograft model of childhood ALL, TCR-KO-CAR-T cells clearly controlled CD19+ leukemia burden and improved survival in vivo. However, coexpression of endogenous TCR plus CAR led to superior persistence of T cells and significantly prolonged leukemia control in vivo, confirmed by a second in vivo model using the leukemia cell line NALM6. These results point toward an essential role of the endogenous TCR for longevity of the response at the price of alloreactivity. In conclusion, anti-CD19 CAR T cells with a CRISPR/Cas9-mediated TCR-KO are promising candidates for nonmatched third-party adoptive T-cell transfer with high antileukemic functionality in the absence of alloreactivity, but long-term persistence in vivo is better in the presence of the endogenous TCR.

Establishment of a NOD/SCID/γc−/−-Dependent Lymphoid Leukemia Cell Line, D593, with Novel RUNX1-LAF4 Fusion Gene.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4276-4276
Author(s):  
Akihiro Abe ◽  
Manabu Ninomiya ◽  
Shizuka Imagama ◽  
Momoko Suzuki ◽  
Fumihiko Hayakawa ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Cell Receptor ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Pcr Analysis ◽  
Lymphoid Leukemia

Abstract We established a NOD/SCID/γc−/−(NOG mouse)-dependent human lymphoid leukemia cell line, D593, by repeated xenotransplantation of pediatric T-cell acute lymphoblastic leukemia cells with the translocation t(2;21). The cell line, D-593, could be serially transplanted from mouse to mouse over a 2-year period. D593 had the same immuno-phenotype as the original leukemia cells: positive for CD2, 5, 7, 14, and 34, and negative for CD3, 4, 8, 19, and 41a. Cytoplasmic CD3 was positive and the rearrangement of T-cell receptor was detected by Southern blot analysis. A previously unreported translocation of t(2;21)(q11;q22) was observed in both the original patient sample and D593. The split signal of RUNX1 was detected by fluorescence in site hybridization in D593 indicating the involvement of RUNX1. Using 3′-RACE and RT-PCR analysis, we identified novel chimeric transcripts of RUNX1-LAF4 joining exon 7 of RUNX1 to exon 4 of LAF4. In the transplanted NOG mice, D593 homed into the trabecular endosteal region of bone marrow (BM), and proliferated from the endosteum to medulla. At the late stage of engraftment, the BM was filled with human lymphoblasts and metastases into the trabecular of the spleen and Glisson’s sheath of the liver were also observed. These findings suggest that D593 is a useful cell line to study not only the leukemia-related biology of RUNX1-LAF4 but also the novel therapeutic model against core-binding factor (CBF) leukemia.

NK-Like Homeodomain Proteins Regulate NOTCH3-Signaling in T-Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1194-1194
Author(s):  
Stefan Nagel ◽  
Letizia Venturini ◽  
Grzegorz K. Przybylski ◽  
Piotr Grabarczyk ◽  
Corinna Meyer ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Expression Patterns ◽  
Hematopoietic Cells ◽  
Notch Pathway ◽  
T Cell Differentiation ◽  
Homeodomain Proteins

Abstract Three NK-like (NKL) homeobox genes, TLX1/HOX11, TLX3/HOX11L2 and NKX2- 5/CSX, have been implicated in T-cell acute lymphoblastic leukemia (T-ALL). Here we screened further NKL genes in 24 T-ALL cell lines by RT-PCR and identified common expression of MSX2, highlighting this homeobox gene as a potential physiological family member in T-cells. Subsequent quantification of MSX2 confirmed expression in primary hematopoietic cells demonstrating higher levels in CD34+ stem cells when compared to peripheral blood cells or mature CD3+ T-cells. Analysis of core thymic factors in T-ALL cell lines, including IL7, BMP4, TGFbeta, NOTCH and T-cell receptor signaling, suggests their involvement in MSX2 regulation during T-cell differentiation. Chromosomal and genomic analysis of the MSX2 locus (at 5q35) uncovered deletion in t(5;14)(q35;q32) positive T-ALL cell lines associated with low expression levels of MSX2 and ectopic activation of TLX3 or NKX2-5, respectively. For functional analysis we lentivirally transduced T-ALL cells for overexpression of either MSX2 or oncogenic TLX1 and NKX2-5. These cells displayed transcriptional activation of NOTCH3-signaling, as indicated by expression array profiling and real-time PCR analysis of NOTCH3, HES1 and HEY1. The sensitivities to gamma-secretase inhibitor analyzed by MTT-assay of cells overexpressing MSX2, TLX1 or NKX2-5, respectively, were consistently decreased. Furthermore, in addition to MSX2, both TLX1 and NKX2-5 proteins interacted with repressor proteins of the NOTCH-pathway, SPEN/MINT and TLE1/GRG1, as shown by co-immunoprecipitation, probably representing one mechanism of (de)regulation. Elevated expression of NOTCH3 and HEY1 mRNA was detected in TLX1/3 positive T-ALL patients, confirming data obtained from cell lines. In conclusion, we have defined expression patterns, regulation and targets of MSX2 in hematopoietic cells, to reveal a novel modulatory activity in T-cell differentiation operating via NOTCH-signaling, and in leukemogenesis when replaced or supplemented by oncogenic NKL homeodomain proteins.

The Transcription Factor BATF Controls CD8+ T Cell Effector Differentiation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 189-189
Author(s):  
R. Anthony Barnitz ◽  
Makoto Kurachi ◽  
Madeleine E. Lemieux ◽  
Nir Yosef ◽  
Michael A. DiIorio ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Differentiation ◽  
Effector Cells ◽  
Cell Effector

Abstract Following activation by antigen, costimulation, and inflammation, naïve CD8+ T cells initiate a program of clonal expansion and differentiation resulting in wide-spread changes in expression of genes involved in cell-cycle, metabolism, effector function, apoptosis, and homing. Although, several key transcription factors (TFs) have been shown to be important in effector CD8+ T cell differentiation, the precise transcriptional regulation of this differentiation program remains poorly understood. The AP-1 family member BATF plays an important role in regulating differentiation and function in CD4+ Th17 cells, CD4+ follicular helper T cells, and in Ig class switching in B cells. We now show that BATF is also required for effector CD8+ T cell differentiation and regulates a core program of genes involved in effector differentiation. We found that BATF expression is rapidly up-regulated during effector CD8+ T cell differentiation in the mouse model of lymphocytic choriomeningitis virus (LCMV) infection. To examine the role of BATF in effector differentiation, we studied congenically distinct wild type (WT) and BATF knockout (KO) naïve P14 TCR transgenic CD8+ T cells co- transferred into a WT host. Upon infection, the BATF KO cells exhibited a profound, cell-intrinsic defect in effector CD8+ T cell differentiation, with a ∼400-fold decrease in peak number of effector cells. BATF KO effectors showed sustained activation and increased cell death by the mid-expansion phase of the immune response. To address the question of how loss of BATF causes such a severely diminished antigen-specific response, we profiled the binding sites of BATF throughout the genome by chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) in primary CD8+ effector cells. We found that BATF bound to regulatory regions in many genes critical for effector differentiation, including transcription factors (e.g. Tbx21, Eomes, Prdm1), genes involved in cytokine signaling (e.g. Il12rb2, Il2ra), homing (e.g. Sell, Selp, Ccr9), effector function (e.g. Gzmb, Ifng, Il2), apoptosis (e.g. Bcl2, Bcl2l1, Mcl1), and T cell activation (e.g. Ctla4, Cd247, Tnfrsf4), suggesting a major role for BATF in effector CD8+ T cell differentiation. Indeed, we found that genes bound by BATF were highly significantly overrepresented among genes that changed as a result of naïve CD8+ T cells differentiating into effectors in vivo (P = 10-27). Comparison of gene expression in in vitro WT and BATF KO effectors confirmed that BATF bound genes were perturbed by BATF loss of function. Analysis of the kinetics of gene expression during the first 72 hours of effector differentiation showed that loss of BATF perturbed the temporal sequence of expression of critical transcription factors, such as T-bet and Eomes, and resulted in inappropriately early cytokine expression. This suggests that BATF may be required to coordinate the earliest events in CD8+ T cell effector differentiation. To test this hypothesis, we used in vivo CFSE tracking to follow the early CD8+ T cell response during LCMV infection. We found that while BATF KO CD8+ T cells initiate cell division, there was a dramatic collapse in the ability to sustain proliferation and differentiation as early as day 3 post-infection. These results indicate that BATF ensures the orderly progression of a program of genes required by effector cells, restraining the expression of some and promoting the expression of others. More broadly, our results suggest that BATF may provide a common regulatory infrastructure for the development of effector cells in all T cell lineages. Disclosures: Wherry: Genentech: Patents & Royalties.

scholarly journals Ikaros Induces Quiescence and T-Cell Differentiation in a Leukemia Cell Line

2005 ◽  
Vol 25 (5) ◽  
pp. 1645-1654 ◽  
Author(s):  
Katie L. Kathrein ◽  
Rachelle Lorenz ◽  
Angela Minniti Innes ◽  
Erin Griffiths ◽  
Susan Winandy
Keyword(s):  
Cell Cycle ◽  
Cell Differentiation ◽  
T Cell ◽  
Tumor Suppressor ◽  
Cell Line ◽  
Tumor Suppressor Gene ◽  
Leukemia Cell ◽  
Suppressor Gene ◽  
T Cell Differentiation ◽  
Leukemia Cell Line

ABSTRACT Ikaros is a hematopoietic cell-specific zinc finger DNA binding protein that plays an important role in lymphocyte development. Genetic disruption of Ikaros results in T-cell transformation. Ikaros null mice develop leukemia with 100% penetrance. It has been hypothesized that Ikaros controls gene expression through its association with chromatin remodeling complexes. The development of leukemia in Ikaros null mice suggests that Ikaros has the characteristics of a tumor suppressor gene. In this report, we show that the introduction of Ikaros into an established mouse Ikaros null T leukemia cell line leads to growth arrest at the G0/G1 stage of the cell cycle. This arrest is associated with up-regulation of the cell cycle-dependent kinase inhibitor p27kip1, the induction of expression of T-cell differentiation markers, and a global and specific increase in histone H3 acetylation status. These studies provide strong evidence that Ikaros possesses the properties of a bona fide tumor suppressor gene for the T-cell lineage and offer insight into the mechanism of Ikaros's tumor suppressive activity.

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