Tcf1 is essential for initiation of oncogenic Notch1-driven chromatin topology in T-ALL

NOTCH1 is a well-established lineage specifier for T cells and amongst the most frequently mutated genes throughout all subclasses of T cell acute lymphoblastic leukemia (T-ALL). How oncogenic NOTCH1 signaling launches a leukemia-prone chromatin landscape during T-ALL initiation is unknown. Here we demonstrate an essential role for the high-mobility-group transcription factor Tcf1 in orchestrating chromatin accessibility and topology allowing aberrant Notch1 signaling to convey its oncogenic function. Although essential, Tcf1 is not sufficient to initiate leukemia. The formation of a leukemia-prone epigenetic landscape at the distal Notch1-regulated Myc enhancer, which is fundamental to this disease, is Tcf1-dependent and occurs within the earliest progenitor stage even before cells adopt a T lymphocyte or leukemic fate. Moreover, we discovered a unique evolutionarily conserved Tcf1-regulated enhancer element in the distal Myc-enhancer, which is important for the transition of pre-leukemic cells to full-blown disease.

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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.

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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.

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Inhibition of inflammatory signaling in Pax5 mutant cells mitigates B-cell leukemogenesis

Scientific Reports ◽

10.1038/s41598-020-76206-y ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Marta Isidro-Hernández ◽

Andrea Mayado ◽

Ana Casado-García ◽

Jorge Martínez-Cano ◽

Chiara Palmi ◽

...

Keyword(s):

B Cell ◽

Lymphoblastic Leukemia ◽

Neutralizing Antibody ◽

Leukemic Cells ◽

Enhanced Production ◽

Cell Acute Lymphoblastic Leukemia ◽

Neonatal Blood ◽

Mutant Cells ◽

Pharmacologic Inhibition

Abstract PAX5 is one of the most frequently mutated genes in B-cell acute lymphoblastic leukemia (B-ALL), and children with inherited preleukemic PAX5 mutations are at a higher risk of developing the disease. Abnormal profiles of inflammatory markers have been detected in neonatal blood spot samples of children who later developed B-ALL. However, how inflammatory signals contribute to B-ALL development is unclear. Here, we demonstrate that Pax5 heterozygosis, in the presence of infections, results in the enhanced production of the inflammatory cytokine interleukin-6 (IL-6), which appears to act in an autocrine fashion to promote leukemia growth. Furthermore, in vivo genetic downregulation of IL-6 in these Pax5 heterozygous mice retards B-cell leukemogenesis, and in vivo pharmacologic inhibition of IL-6 with a neutralizing antibody in Pax5 mutant mice with B-ALL clears leukemic cells. Additionally, this novel IL–6 signaling paradigm identified in mice was also substantiated in humans. Altogether, our studies establish aberrant IL6 expression caused by Pax5 loss as a hallmark of Pax5-dependent B-ALL and the IL6 as a therapeutic vulnerability for B-ALL characterized by PAX5 loss.

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DDX5 is a positive regulator of oncogenic NOTCH1 signaling in T cell acute lymphoblastic leukemia

Oncogene ◽

10.1038/onc.2012.482 ◽

2012 ◽

Vol 32 (40) ◽

pp. 4845-4853 ◽

Cited By ~ 51

Author(s):

S Lin ◽

L Tian ◽

H Shen ◽

Y Gu ◽

J-L Li ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Lymphoblastic Leukemia ◽

Positive Regulator ◽

Notch1 Signaling ◽

Cell Acute Lymphoblastic Leukemia

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Detection of minimal residual disease in T-cell acute lymphoblastic leukemia using polymerase chain reaction predicts impending relapse

Blood ◽

10.1182/blood.v78.3.739.739 ◽

1991 ◽

Vol 78 (3) ◽

pp. 739-747 ◽

Cited By ~ 69

Author(s):

GA Neale ◽

J Menarguez ◽

GR Kitchingman ◽

TJ Fitzgerald ◽

M Koehler ◽

...

Keyword(s):

T Cell ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Pcr Amplification ◽

Leukemic Cells ◽

Chain Reaction ◽

Leukemic Clone ◽

Cell Acute Lymphoblastic Leukemia ◽

Polymerase Chain

Abstract After achieving remission, approximately one-third of patients with T- cell acute lymphoblastic leukemia (T-ALL) relapse due to the resurgence of residual leukemic cells that cannot be detected in remission by morphologic methods. Thus, the early detection of residual disease is highly desirable to monitor the efficacy of therapy, or to institute an alternative mode of therapy. Toward this aim, we have examined the applicability of polymerase chain reaction (PCR) amplification in the detection of minimal residual disease (MRD) in bone marrow samples from patients with T-ALL in morphologic remission. Two different approaches were taken to identify leukemic clone-specific sequences that could be used as targets for PCR amplification. The first technique used T-cell receptor-delta (TCR-delta) gene rearrangements that were sequenced directly after PCR amplification of leukemic DNA. This method was successful in generating clone-specific probes for 76% of T-ALL patients screened. An alternative method was used to clone and sequence a TCR-beta chain gene from leukemic cells to generate a specific probe. The PCR assays that we used were specific for each patient's leukemic clone, and were capable of routinely detecting one leukemic cell in 10(4) normal cells. Using these sensitive PCR-based assays, we found no evidence for persistence of the leukemic clone in any of the bone marrow samples from four T-ALL patients who are in long-term (3.9 + to 8.1 + years) remission. In contrast, we detected residual disease in clinical remission samples from two patients who subsequently relapsed. In one patient, where we had appropriate samples, we observed a dramatic expansion of the leukemic clone 3 months before clinical relapse. These results suggest that PCR-based assays for detection of MRD in T-ALL patients have great potential in predicting impending relapse, and in determining the efficacy of the anti-leukemic therapy. These methods may also allow the identification of long-term survivors.

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Expression of Constitutively Activated Human c-Kit in Myb Transformed Early Myeloid Cells Leads to Factor Independence, Histiocytic Differentiation, and Tumorigenicity

Blood ◽

10.1182/blood.v90.11.4539 ◽

1997 ◽

Vol 90 (11) ◽

pp. 4539-4552 ◽

Cited By ~ 26

Author(s):

Petranel Ferrao ◽

Thomas J. Gonda ◽

Leonie K. Ashman

Keyword(s):

Myeloid Leukemia ◽

Lymphoblastic Leukemia ◽

Myeloid Cells ◽

Leukemic Cells ◽

Monocytic Leukemia ◽

Gm Csf ◽

Clonogenic Potential ◽

Cell Acute Lymphoblastic Leukemia ◽

Different Populations ◽

Macrophage Colony

Abstract The cDNAs encoding wild type (WT) human receptor tyrosine kinase c-Kit and a constitutively activated mutant, V816Kit, were introduced into granulocyte-macrophage colony-stimulating factor (GM-CSF )-dependent early murine hemopoietic cells, which had been transformed with activated Myb. WTKit cells were able to grow in the presence of the human ligand for Kit, stem cell factor (SCF ), but displayed reduced growth and clonogenic potential in either SCF or GM-CSF compared with the parental cells in GM-CSF. In contrast, V816Kit cells grew without factor at a higher rate than the parental cells in GM-CSF and displayed increased clonogenicity. Dissection of the growth characteristics in liquid culture showed that in the presence of appropriate factors, the different populations had similar proliferation rates, but that V816Kit profoundly increased cell survival compared with WTKit or parental cells. This suggests that the signals transduced by WTKit activated with SCF, and by V816Kit, were not identical. Also, WTKit and V816Kit-expressing cells both varied from the early myeloid progenitor phenotype of the parental cells and gave rise to a small number of large to giant adherent cells that expressed macrophage (α-naphthyl acetate) esterase and neutrophil (naphtol-AS-D-chloroacetate) esterase, were highly phagocytic and phenotypically resembled histiocytes. Thus, WTKit activated by SCF and V816Kit were able to induce differentiation in a proportion of Myb-transformed myeloid cells. The factor independent V816Kit cells, unlike the parental and WTKit expressing cells, were shown to produce tumors of highly mitotic, invasive cells at various stages of differentiation in syngeneic mice. These results imply that constitutively activated Kit can promote the development of differentiated myeloid tumors and that its oncogenic effects are not restricted to lineages (mast cell and B-cell acute lymphoblastic leukemia), which have been reported previously. Furthermore, the mixed populations of cells in culture and in the tumors phenotypically resembled the leukemic cells from patients with monocytic leukemia with histiocytic differentiation (acute myeloid leukemia-M5c), a newly proposed subtype of myeloid leukemia.

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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).

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A Critical Role of Blockade of Cell Differentiation in BCR-ABL-Induced B-Cell Acute Lymphoblastic Leukemia (B-ALL): Basis for Superb Therapeutic Effect on B-ALL in Mice by Promotion of Pro-B Leukemic Cell Differentiation and Treatment with Imatinib.

Blood ◽

10.1182/blood.v106.11.844.844 ◽

2005 ◽

Vol 106 (11) ◽

pp. 844-844

Author(s):

Yiguo Hu ◽

Linghong Kong ◽

Kevin Staples ◽

Kevin Mills ◽

John G. Monroe ◽

...

Keyword(s):

Cell Differentiation ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Myeloid Leukemia ◽

Lymphoblastic Leukemia ◽

Leukemic Cell ◽

Leukemic Cells ◽

B Cell Differentiation ◽

Cell Acute Lymphoblastic Leukemia

Abstract The BCR-ABL oncogene induces human Philadelphia-positive (Ph+) B-cell acute lymphoblastic leukemia (B-ALL) and chronic myeloid leukemia (CML) that advances to acute phase of CML called blast crisis. In this acute phase, CML patients can develop either B-ALL or acute myeloid leukemia. In B-ALL, differentiation of leukemic cells are blocked at pro-/pre-B stage, and the underlying mechanism is unknown. We hypothesize that this blockade of B-cell differentiation may be important for the development of B-ALL induced by BCR-ABL, and if so, promotion of B-leukemic cell differentiation would create a novel therapeutic strategy for B-ALL. To test this hypothesis, we first compared the percentages of IgM+ B-leukemic cells in BALB/c and C57BL/6 (B6) mice with BCR-ABL-induced B-ALL, because we have previously found that B-ALL develops more quickly in BALB/c mice than in B6 mice (Li et al, J. Exp. Med.189:1399–1412, 1999). We expressed BCR-ABL in bone marrow (BM) using retroviral transduction and transplantation in these two different strains of inbred mice to induce B-ALL. There were significantly more peripheral blood B220+ B cells in BALB/c B-ALL mice than those in B6 mice, correlating to faster B-ALL in BALB/c mice than in B6 mice. Among these B220+ cells, IgM+ cells were much less in BALB/c mice than in B6 mice. We also compared rearrangement of the B cell antigen receptor (BCR) heavy chains (m chains) between BALB/c and B6 backgrounds using BCR-ABL-expressing pro-B cell lines isolated from the B-ALL mice. Normal m chains rearrangement was found in B6 leukemic cells, but not in BALB/c leukemic cells. These results indicate that more differentiated B-leukemic cells are associated with less aggressive disease. To further demonstrate the role of blockade of B-cell differentiation in B-ALL development, we induced B-leukemic cell differentiation by co-expression of BCR-ABL and intact immunoregulatory tyrosine activation motifs (ITAM) contained in immunoglobulin (Ig)_/Igß complexes in BM cells of B-ALL mice, comparing to expression of BCR-ABL alone. We treated these mice with imatinib (orally, 100 mg/kg, twice a day). The treated mice with B-ALL induced by co-expression of BCR-ABL and ITAM lived three-week longer than those with B-ALL induced by BCR-ABL only, with some mice in long-term remission. Prolonged survival was associated with 50% increased B220+/IgM+ B-leukemic cells in peripheral blood of the mice. Taken together, our results demonstrate that blockade of B-cell differentiation is critical for the development of B-ALL induced by BCR-ABL, and provide a rationale for combination therapy of B-ALL with imatinib and induction of leukemic cell differentiation.

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Crac Channel Deletion in Leukemic Cells Delays Progression of Leukemia and Prolongs Survival of Mice with Notch-1-Induced T-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.1433.1433 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1433-1433

Author(s):

Shella Saint Fleur-Lominy ◽

Mate Maus ◽

Stefan Feske

Keyword(s):

Bone Marrow ◽

T Cell ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Wild Type ◽

Notch 1 ◽

Channel Function ◽

Crac Channels ◽

Crac Channel ◽

Cell Acute Lymphoblastic Leukemia

Abstract Introduction: Ca2+ release-activated Ca2+ (CRAC) channels and their activators stromal interaction molecule (STIM) 1 and 2 are the main regulators of calcium entry in T Lymphocytes through a process known as store-operated Ca2+ entry (SOCE). SOCE results in the activation of calcineurin and other downstream signals with important effects on lymphocyte function. Notch-1 is a protein that is essential for T lymphocyte development. Activating mutations of Notch-1 occurs in about 60% of T-cell acute lymphoblastic leukemia (T-ALL). Introduction of constitutively active forms of Notch-1 in hematopoietic stem cells (HSC) induces T-ALL in mice, providing a useful animal model for the study of leukemia. Methods: To study the role of CRAC channels in T-ALL, we used a mouse model in which c-kit+ HSC from wild-type (WT) and STIM1/STIM2-deficient mice (DKO) were retrovirally transduced with the intracellular Notch-1 domain (ICN1). Transduced HSC were injected into lethally irradiated C57BL/6 mice. Following leukemia development, mice were analyzed for survival and cellular and molecular activity of leukemic cells using various techniques including histology, flow cytometry, RT-PCR and gene array expression analysis. In addition, we used the human T-ALL cell line CEM, in which we introduced a dominant negative form of the CRAC channel subunit ORAI1 (ORAI1-DN) that abolishes CRAC channel function and SOCE, for coculture with the human bone marrow stromal cell line HS5. Results: Mice injected with wild-type HSC transduced with ICN1 succumbed from T-ALL characterized by the presence of CD4+ CD8+ leukemic T cell blasts in the blood, bone marrow and infiltrating organs within 3 to 4 weeks after transfer of HSC. By contrast, mice that had received ICN1 transduced STIM1/2 deficient HSC lived approximately twice as long. The survival benefit was not due to differences in leukemic cell numbers or in proliferation and apoptosis of leukemic cells. Histologies of the bone marrow and spleen of WT leukemic mice showed necrotic lesions, pronounced neutrophil infiltration, the presence of histiocytes engulfing red blood cells (RBC) indicative of severe inflammation. No signs of necrosis and inflammation were present in DKO leukemic mice. Paralleling the inflammation and destruction of the bone marrow environment, WT leukemic mice showed greatly diminished presence of erythroid precursors (EP) in the bone marrow whereas EP frequencies in DKO leukemic mice were similar to those in non-leukemic mice. In line with findings in mice, we observed that human leukemic CEM T cells reduced the viability of HS5 stromal cells in a contact-dependent manner. This cytotoxic effect of CEM cells depended on CRAC channel function as CEM cells transduced with ORAI1-DN had little effect on HS5 viability. Conclusion: These results suggest that CRAC channels are important for the function of T-ALL cells and their effects on the organs they infiltrate, most notably the bone marrow. Inhibition of CRAC channel function prolongs survival of mice with T-ALL potentially by attenuating the cytotoxic effects of leukemic T cells on their environment and on hematopoiesis. Further studies are underway to understand the mechanisms by which CRAC channels regulate leukemic T cell function. Disclosures Feske: Calcimedica: Consultancy, Equity Ownership, Honoraria, Patents & Royalties: CRAC Channel Inibitors.

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Leukemia-Associated Mutations within the NOTCH1 Heterodimerization Domain Fall into at Least Two Distinct Mechanistic Classes

Molecular and Cellular Biology ◽

10.1128/mcb.01655-05 ◽

2006 ◽

Vol 26 (12) ◽

pp. 4642-4651 ◽

Cited By ~ 168

Author(s):

Michael J. Malecki ◽

Cheryll Sanchez-Irizarry ◽

Jennifer L. Mitchell ◽

Gavin Histen ◽

Mina L. Xu ◽

...

Keyword(s):

Lymphoblastic Leukemia ◽

Proteolytic Activation ◽

Activating Mutations ◽

Notch1 Signaling ◽

Human T Cell ◽

Secretase Activity ◽

Cell Acute Lymphoblastic Leukemia ◽

Domain Stability ◽

Increased Susceptibility ◽

Notch1 Receptor

ABSTRACT The NOTCH1 receptor is cleaved within its extracellular domain by furin during its maturation, yielding two subunits that are held together noncovalently by a juxtamembrane heterodimerization (HD) domain. Normal NOTCH1 signaling is initiated by the binding of ligand to the extracellular subunit, which renders the transmembrane subunit susceptible to two successive cleavages within and C terminal to the heterodimerization domain, catalyzed by metalloproteases and γ-secretase, respectively. Because mutations in the heterodimerization domain of NOTCH1 occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL), we assessed the effect of 16 putative tumor-associated mutations on Notch1 signaling and HD domain stability. We show here that 15 of the 16 mutations activate canonical NOTCH1 signaling. Increases in signaling occur in a ligand-independent fashion, require γ-secretase activity, and correlate with an increased susceptibility to cleavage by metalloproteases. The activating mutations cause soluble NOTCH1 heterodimers to dissociate more readily, either under native conditions (n = 3) or in the presence of urea (n = 11). One mutation, an insertion of 14 residues immediately N terminal to the metalloprotease cleavage site, increases metalloprotease sensitivity more than all others, despite a negligible effect on heterodimer stability by comparison, suggesting that the insertion may expose the S2 site by repositioning it relative to protective NOTCH1 ectodomain residues. Together, these studies show that leukemia-associated HD domain mutations render NOTCH1 sensitive to ligand-independent proteolytic activation through two distinct mechanisms.

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