Stem cell factor receptor (c‐kit, CD117) is expressed on blast cells from most immature types of acute myeloid malignancies but is also a characteristic of a subset of acute promyelocytic leukaemia

Abstract Mutations in the extracellular portion of the KIT receptor tyrosine kinase (exon 8 mutations) are strongly associated with core binding factor (CBF) - acute myeloid leukemia (AML), but the functional role of these mutations has not been elucidated. In 93% of cases, codon Asp419 is deleted and exon 8 mutations were reported to confer an impaired prognosis to patients with CBF-AML. In this study, we are the first to report pro-proliferative and antiapoptotic potential of representative KIT exon 8 mutations in a cell culture model and to show a significant difference to KIT wildtype (KIT-WT). Three representative exon 8 mutants including a single deletion of codon 419 were created by in vitro site-directed mutagenesis. The integrity of all constructs was assessed by complete nucleotide sequencing. After stable expression in IL-3 dependent Ba/F3 cells (confirmed by FACS analysis and immunoblotting), exon 8 KIT mutants were characterized by a hypersensitivity to stem cell factor (SCF) stimulation in terms of proliferation and resistance to apoptotic cell death. The differences to KIT-WT occurred in the physiological range of SCF from 1 to 10ng/ml. The proliferative response caused by stimulation with SCF was reversed in KIT-WT and exon 8 mutants in the presence of Imatinib® (Novartis) in contrast to the activation loop mutant D816V which could not be inhibited. These biological effects were confirmed by demonstrating increased phosphorylation of the KIT downstream targets mitogen-activated protein kinase (MAPK) and AKT after SCF stimulation compared to the KIT-WT receptor. Furthermore, the MEK inhibitor PD98059 and the PI3 kinase inhibitor LY294002 resulted in a dose dependent inhibition of SCF induced proliferation in exon 8 mutants. Our data show that KIT exon 8 mutations represent gain-of-function mutations by inducing receptor hypersensitivity to its ligand SCF by activation of MAPK and PI3K and might represent a new molecular target for treatment of CBF leukemias.

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KIT exon 8 mutations associated with core-binding factor (CBF)–acute myeloid leukemia (AML) cause hyperactivation of the receptor in response to stem cell factor

Blood ◽

10.1182/blood-2004-06-2068 ◽

2005 ◽

Vol 105 (8) ◽

pp. 3319-3321 ◽

Cited By ~ 66

Author(s):

Tobias M. Kohl ◽

Susanne Schnittger ◽

Joachim W. Ellwart ◽

Wolfgang Hiddemann ◽

Karsten Spiekermann

Keyword(s):

Acute Myeloid Leukemia ◽

Stem Cell ◽

Stem Cell Factor ◽

Myeloid Leukemia ◽

Apoptotic Cell ◽

Mitogen Activated Protein Kinase ◽

Core Binding Factor ◽

Binding Factor ◽

Mitogen Activated Protein ◽

Acute Myeloid

AbstractKIT exon 8 mutations are located in the extracellular portion of the receptor and are strongly associated with core-binding factor (CBF)-acute myeloid leukemia (AML). To characterize the functional role of these mutants, we analyzed the proproliferative and antiapoptotic potential of 3 KIT exon 8 mutations in interleukin 3 (IL-3)-dependent Ba/F3 cells. All KIT exon 8 mutants induced receptor hyperactivation in response to stem cell factor (SCF) stimulation in terms of proliferation and resistance toward apoptotic cell death. A representative KIT exon 8 mutant showed spontaneous receptor dimerization, phosphorylation of mitogen-activated protein kinase (MAPK), and conferred IL-3-independent growth to Ba/F3 cells. MAPK and phosphatidylinositol 3-kinase (PI3-kinase) activation was essential for the phenotype of this mutant. Additionally, imatinib inhibited proliferation of KIT exon 8 mutant-expressing Ba/F3 cells. Our data show that KIT exon 8 mutations represent gain-of-function mutations and might represent a new molecular target for treatment of CBF leukemias. (Blood. 2005;105:3319-3321)

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Effects of Stem Cell Factor on Hypoxia-Inducible Factor 1 Alpha Accumulation in Human Acute Myeloid Leukaemia and LAD2 Mast Cells

PLoS ONE ◽

10.1371/journal.pone.0022502 ◽

2011 ◽

Vol 6 (7) ◽

pp. e22502 ◽

Cited By ~ 17

Author(s):

Bernhard F. Gibbs ◽

Inna M. Yasinska ◽

Abraham E. Oniku ◽

Vadim V. Sumbayev

Keyword(s):

Stem Cell ◽

Mast Cells ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Stem Cell Factor ◽

Hypoxia Inducible Factor ◽

Hypoxia Inducible Factor 1 ◽

Acute Myeloid

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The key role of stem cell factor/KIT signaling in the proliferation of blast cells from Down syndrome-related leukemia

Leukemia ◽

10.1038/leu.2008.267 ◽

2008 ◽

Vol 23 (1) ◽

pp. 95-103 ◽

Cited By ~ 14

Author(s):

T Toki ◽

R Kanezaki ◽

S Adachi ◽

H Fujino ◽

G Xu ◽

...

Keyword(s):

Down Syndrome ◽

Stem Cell ◽

Stem Cell Factor ◽

Blast Cells

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E2a/Pbx1 Induces the Rapid Proliferation of Stem Cell Factor-Dependent Murine Pro-T Cells That Cause Acute T-Lymphoid or Myeloid Leukemias in Mice

Molecular and Cellular Biology ◽

10.1128/mcb.24.3.1256-1269.2004 ◽

2004 ◽

Vol 24 (3) ◽

pp. 1256-1269 ◽

Cited By ~ 19

Author(s):

David B. Sykes ◽

Mark P. Kamps

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Stem Cell ◽

Cell Division ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Stem Cell Factor ◽

Myeloid Leukemia ◽

Lymphoblastic Leukemia ◽

Acute Myeloid

ABSTRACT Oncoprotein E2a/Pbx1 is produced by the t(1;19) chromosomal translocation of human pre-B acute lymphoblastic leukemia. E2a/Pbx1 blocks differentiation of primary myeloid progenitors but, paradoxically, induces apoptosis in established pre-B-cell lines, and no transforming function of E2a/Pbx1 has been reported in cultured lymphoid progenitors. Here, we demonstrate that E2a/Pbx1 induces immortal proliferation of stem cell factor (SCF)-dependent pro-T thymocytes by a mechanism dependent upon both its transactivation and DNA-binding functions. E2a-Pbx1 cooperated with cytokines or activated signaling oncoproteins to induce cell division, as inactivation of conditional E2a/Pbx1 in either factor-dependent pro-T cells or pro-T cells made factor independent by expression of Bcr/Abl resulted in pro-T-cell quiescence, while reactivation of E2a/Pbx1 restored cell division. Infusion of E2a/Pbx1 pro-T cells in mice caused T lymphoblastic leukemia and, unexpectedly, acute myeloid leukemia. The acute lymphoblastic leukemia did not evidence further maturation, suggesting that E2a/Pbx1 establishes an early block in pro-T-cell development that cannot be overcome by marrow or thymic microenvironments. In an E2a/Pbx1 pro-T thymocyte clone that induced only pro-T acute lymphoblastic leukemia, coexpression of Bcr/Abl expanded its leukemic phenotype to include acute myeloid leukemia, suggesting that unique functions of cooperating signaling oncoproteins can influence the lymphoid versus myeloid character of E2a/Pbx1 leukemia and may cooperate with E2a/Pbx1 to dictate the pre-B-cell phenotype of human leukemia containing t(1;19).

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Stem cell factor directly stimulates the development of enriched granulocyte-macrophage colony-forming cells and promotes the effects of other colony-stimulating factors

Blood ◽

10.1182/blood.v80.9.2230.bloodjournal8092230 ◽

1992 ◽

Vol 80 (9) ◽

pp. 2230-2236

Author(s):

CM Heyworth ◽

AD Whetton ◽

S Nicholls ◽

K Zsebo ◽

TM Dexter

Keyword(s):

Stem Cell ◽

Stem Cell Factor ◽

Secondary Growth ◽

Marked Change ◽

Soft Agar ◽

Kit Ligand ◽

Blast Cells ◽

Macrophage Colony Stimulating Factor ◽

Macrophage Colony ◽

Stimulating Factor

The effects of the c-kit ligand (stem cell factor [SCF]) on the development of a highly enriched population of granulocyte-macrophage colony-forming cells (GM-CFC) were assessed. In soft agar assays, both in serum-containing and in serum-deprived cultures, SCF promoted the formation of colonies that contained predominantly granulocytic cells with some blast cells also present. The size of these colonies was far smaller than observed in the presence of interleukin-3 (IL-3). In serum- deprived conditions, no colonies were formed in the presence of macrophage colony-stimulating factor (M-CSF), but when M-CSF was combined with SCF, a marked change was noted in that large colonies were produced containing predominantly macrophages. When GM-CFC were cultured in the presence of IL-3 and SCF, colonies were formed that contained blast cells, granulocytes, and macrophages. A synergistic interaction was also seen using a combination of G-CSF plus SCF in either serum-containing or serum-deprived cultures. The addition of SCF to colony-forming assays markedly reduced the concentration of IL-3 or G-CSF required for optimal levels of colony formation. Furthermore, SCF was capable of promoting the survival of GM-CFC for several days, after which large colonies containing mature cells were formed upon the addition of a secondary growth factor such as G-CSF or IL-3. Thus, SCF can directly act on highly enriched committed progenitor cells in serum- deprived conditions to promote survival, proliferation, and development.

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Enhancement of murine blast cell colony formation in culture by recombinant rat stem cell factor, ligand for c-kit

Blood ◽

10.1182/blood.v78.5.1223.bloodjournal7851223 ◽

1991 ◽

Vol 78 (5) ◽

pp. 1223-1229 ◽

Cited By ~ 1

Author(s):

K Tsuji ◽

KM Zsebo ◽

M Ogawa

Keyword(s):

Stem Cell ◽

Colony Formation ◽

Stem Cell Factor ◽

Blast Cell ◽

Spleen Cells ◽

Dormant Period ◽

Kit Ligand ◽

Rat Liver Cells ◽

Blast Cells ◽

Stimulating Factor

Mice with W mutations characterized by hypopigmentation, sterility, anemia, and mast cell deficiency have abnormalities in c-kit, a receptor with tyrosine kinase activity. Recently, the ligand for c-kit was cloned by investigators in several laboratories. Zsebo et al identified and cloned a gene for a cytokine termed stem cell factor (SCF) in the medium conditioned by buffalo rat liver cells, and this cytokine proved to be c-kit ligand. We have examined the effects of recombinant rat SCF (rrSCF) on colony formation from primitive hematopoietic progenitors in culture. rrSCF and erythropoietin (Ep) supported formation of granulocyte/macrophage (GM) colonies as well as a small number of multilineage and blast cell colonies from marrow cells of normal mice. We then examined the effects of rrSCF using marrow and spleen cells of mice that had been treated with 150 mg/kg 5- fluorouracil (5-FU). Unlike single factors, combinations of factors such as rrSCF plus interleukin-3 (IL-3), rrSCF plus IL-6, and rrSCF plus granulocyte colony-stimulating factor (G-CSF) markedly stimulated the growth of multilineage colonies. In contrast to these factor combinations and a combination of IL-3 and IL-6, a combination of rrSCF and IL-4 did not support multilineage colony formation. Mapping studies of the development of multipotential blast cell colonies further indicated that rrSCF, like IL-6, G-CSF, and IL-11, shortens the dormant period in which the stem cells reside. When we tested the effects of rrSCF using pooled blast cells, which are highly enriched for progenitors and are devoid of stromal cells, rrSCF plus Ep supported formation of only a few multilineage colonies, indicating that rrSCF itself is ineffective in support of the proliferation of multipotential progenitors. However, rrSCF supported formation of a significant number of neutrophil and neutrophil/macrophage colonies from pooled blast cells, indicating that rrSCF is able to support directly the proliferation of progenitors in neutrophil/monocyte lineages. c-kit ligand may play important roles in adult hematopoiesis.

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