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Microenvironmental hypoxia regulates FLT3 expression and biology in AML.

Sironi S, Wagner M, Kuett A, Drolle H, Polzer H, Spiekermann K, Rieger C, Fiegl M - Sci Rep (2015)

Bottom Line: Hence, effects of hypoxia on FLT3 expression and biology could provide novel insight into AML biology.Hypoxia-mediated down-regulation was specific for FLT3, reversible and proteasome-dependent; with FLT3 half-life being significantly shorter at hypoxia.In conclusion, FLT3 expression in AML is dependent on the oxygen partial pressure, but response to hypoxia differs.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine III, Klinikum der Universität München, Munich, Germany.

ABSTRACT
Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase constitutively expressed by acute myeloid leukaemia (AML) blasts. In addition, 25% of AML patients harbour a FLT3-ITD mutation, associated with inferior outcome due to increased relapse rate. Relapse might be propagated by interactions between AML blasts and the bone marrow microenvironment. Besides cellular elements of the microenvironment (e.g. mesenchymal stromal cells), bone marrow hypoxia has emerged as an additional crucial component. Hence, effects of hypoxia on FLT3 expression and biology could provide novel insight into AML biology. Here we show that 25% of AML patients down-regulate FLT3 expression on blasts in response to in vitro hypoxia (1% O2), which was independent of its mutational state. While virtually no AML cell lines regulate FLT3 in response to hypoxia, the down-regulation could be observed in Ba/F3 cells stably transfected with different FLT3 mutants. Hypoxia-mediated down-regulation was specific for FLT3, reversible and proteasome-dependent; with FLT3 half-life being significantly shorter at hypoxia. Also, PI-3K inhibition could partially abrogate down-regulation of FLT3. Hypoxia-mediated down-regulation of FLT3 conferred resistance against cytarabine in vitro. In conclusion, FLT3 expression in AML is dependent on the oxygen partial pressure, but response to hypoxia differs.

No MeSH data available.


Related in: MedlinePlus

FLT3 down-regulation is a specific phenomenon.(A) Western blot of whole lysates of Ba/F3 cell line stably transfected with different FLT3 ITD constructs (W51, W78, NPOS) show down-regulation of FLT3 at hypoxic conditions. (B) Ba/F3 cells stably transfected with the kinase inactive FLT3 K644R mutant also down-regulated FLT3 under 1% O2. (C) No regulation of EGF-R by hypoxia of 1% O2 within 72 hours in Ba/F3 cells. (D) Ba/F3 cells stably transfected with Epo-R show no regulation of Epo-R by hypoxia. (E) Fold change of FLT3 OD normalized to β-actin in Ba/F3 W51, Ba/F3 W78 and Ba/F3 NPOS cells shows a statistical significance of FLT3 down-regulation under hypoxic conditions, whereas fold change of EGF-r and EPO-r OD do not show a significant down-regulation of this receptors under hypoxia. (F) No regulation of EGF-R and KIT receptor in n = 4 AML patient samples previously tested for FLT3 down-regulation under hypoxia (n = 3 down-regulating patients, n = 1 not regulating patient). (G) Optical density (OD) of KIT receptor and EGF-r western blot bands normalized to β-actin shows no statistical significance regulation under hypoxia in n = 4 AML patient samples.
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f4: FLT3 down-regulation is a specific phenomenon.(A) Western blot of whole lysates of Ba/F3 cell line stably transfected with different FLT3 ITD constructs (W51, W78, NPOS) show down-regulation of FLT3 at hypoxic conditions. (B) Ba/F3 cells stably transfected with the kinase inactive FLT3 K644R mutant also down-regulated FLT3 under 1% O2. (C) No regulation of EGF-R by hypoxia of 1% O2 within 72 hours in Ba/F3 cells. (D) Ba/F3 cells stably transfected with Epo-R show no regulation of Epo-R by hypoxia. (E) Fold change of FLT3 OD normalized to β-actin in Ba/F3 W51, Ba/F3 W78 and Ba/F3 NPOS cells shows a statistical significance of FLT3 down-regulation under hypoxic conditions, whereas fold change of EGF-r and EPO-r OD do not show a significant down-regulation of this receptors under hypoxia. (F) No regulation of EGF-R and KIT receptor in n = 4 AML patient samples previously tested for FLT3 down-regulation under hypoxia (n = 3 down-regulating patients, n = 1 not regulating patient). (G) Optical density (OD) of KIT receptor and EGF-r western blot bands normalized to β-actin shows no statistical significance regulation under hypoxia in n = 4 AML patient samples.

Mentions: Results in primary AML blasts and in Ba/F3 WT and W51 cells suggested that FLT3 down-regulation is independent from the mutational state of the receptor. To further validate this hypothesis, we investigated the expression of FLT3 at 1% O2 in 2 additional Ba/F3 FLT3-ITD mutants (Ba/F3 W78 and Ba/F3 NPOS) and in the kinase-inactive Ba/F3 K644R mutant. The hypoxia mediated down-regulation was also observed in each of these Ba/F3 cells (Fig. 4A,B). Next, we investigated whether hypoxia-mediated down-regulation is a general phenomenon applying to other tyrosine kinase receptors (TKR) and cytokine receptors as well; especially receptors that have a key role in haematopoiesis and leukaemogenesis as EGF-R, c-KIT and Epo-R. Ba/F3 cells stably transfected with EGF-R and Epo-R, were analysed for a 3-day time kinetic at 21% O2 and 1% O2 and, as shown in Fig. 4C,D, we failed to observe any hypoxia-induced regulation of the respective receptors.


Microenvironmental hypoxia regulates FLT3 expression and biology in AML.

Sironi S, Wagner M, Kuett A, Drolle H, Polzer H, Spiekermann K, Rieger C, Fiegl M - Sci Rep (2015)

FLT3 down-regulation is a specific phenomenon.(A) Western blot of whole lysates of Ba/F3 cell line stably transfected with different FLT3 ITD constructs (W51, W78, NPOS) show down-regulation of FLT3 at hypoxic conditions. (B) Ba/F3 cells stably transfected with the kinase inactive FLT3 K644R mutant also down-regulated FLT3 under 1% O2. (C) No regulation of EGF-R by hypoxia of 1% O2 within 72 hours in Ba/F3 cells. (D) Ba/F3 cells stably transfected with Epo-R show no regulation of Epo-R by hypoxia. (E) Fold change of FLT3 OD normalized to β-actin in Ba/F3 W51, Ba/F3 W78 and Ba/F3 NPOS cells shows a statistical significance of FLT3 down-regulation under hypoxic conditions, whereas fold change of EGF-r and EPO-r OD do not show a significant down-regulation of this receptors under hypoxia. (F) No regulation of EGF-R and KIT receptor in n = 4 AML patient samples previously tested for FLT3 down-regulation under hypoxia (n = 3 down-regulating patients, n = 1 not regulating patient). (G) Optical density (OD) of KIT receptor and EGF-r western blot bands normalized to β-actin shows no statistical significance regulation under hypoxia in n = 4 AML patient samples.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4663471&req=5

f4: FLT3 down-regulation is a specific phenomenon.(A) Western blot of whole lysates of Ba/F3 cell line stably transfected with different FLT3 ITD constructs (W51, W78, NPOS) show down-regulation of FLT3 at hypoxic conditions. (B) Ba/F3 cells stably transfected with the kinase inactive FLT3 K644R mutant also down-regulated FLT3 under 1% O2. (C) No regulation of EGF-R by hypoxia of 1% O2 within 72 hours in Ba/F3 cells. (D) Ba/F3 cells stably transfected with Epo-R show no regulation of Epo-R by hypoxia. (E) Fold change of FLT3 OD normalized to β-actin in Ba/F3 W51, Ba/F3 W78 and Ba/F3 NPOS cells shows a statistical significance of FLT3 down-regulation under hypoxic conditions, whereas fold change of EGF-r and EPO-r OD do not show a significant down-regulation of this receptors under hypoxia. (F) No regulation of EGF-R and KIT receptor in n = 4 AML patient samples previously tested for FLT3 down-regulation under hypoxia (n = 3 down-regulating patients, n = 1 not regulating patient). (G) Optical density (OD) of KIT receptor and EGF-r western blot bands normalized to β-actin shows no statistical significance regulation under hypoxia in n = 4 AML patient samples.
Mentions: Results in primary AML blasts and in Ba/F3 WT and W51 cells suggested that FLT3 down-regulation is independent from the mutational state of the receptor. To further validate this hypothesis, we investigated the expression of FLT3 at 1% O2 in 2 additional Ba/F3 FLT3-ITD mutants (Ba/F3 W78 and Ba/F3 NPOS) and in the kinase-inactive Ba/F3 K644R mutant. The hypoxia mediated down-regulation was also observed in each of these Ba/F3 cells (Fig. 4A,B). Next, we investigated whether hypoxia-mediated down-regulation is a general phenomenon applying to other tyrosine kinase receptors (TKR) and cytokine receptors as well; especially receptors that have a key role in haematopoiesis and leukaemogenesis as EGF-R, c-KIT and Epo-R. Ba/F3 cells stably transfected with EGF-R and Epo-R, were analysed for a 3-day time kinetic at 21% O2 and 1% O2 and, as shown in Fig. 4C,D, we failed to observe any hypoxia-induced regulation of the respective receptors.

Bottom Line: Hence, effects of hypoxia on FLT3 expression and biology could provide novel insight into AML biology.Hypoxia-mediated down-regulation was specific for FLT3, reversible and proteasome-dependent; with FLT3 half-life being significantly shorter at hypoxia.In conclusion, FLT3 expression in AML is dependent on the oxygen partial pressure, but response to hypoxia differs.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine III, Klinikum der Universität München, Munich, Germany.

ABSTRACT
Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase constitutively expressed by acute myeloid leukaemia (AML) blasts. In addition, 25% of AML patients harbour a FLT3-ITD mutation, associated with inferior outcome due to increased relapse rate. Relapse might be propagated by interactions between AML blasts and the bone marrow microenvironment. Besides cellular elements of the microenvironment (e.g. mesenchymal stromal cells), bone marrow hypoxia has emerged as an additional crucial component. Hence, effects of hypoxia on FLT3 expression and biology could provide novel insight into AML biology. Here we show that 25% of AML patients down-regulate FLT3 expression on blasts in response to in vitro hypoxia (1% O2), which was independent of its mutational state. While virtually no AML cell lines regulate FLT3 in response to hypoxia, the down-regulation could be observed in Ba/F3 cells stably transfected with different FLT3 mutants. Hypoxia-mediated down-regulation was specific for FLT3, reversible and proteasome-dependent; with FLT3 half-life being significantly shorter at hypoxia. Also, PI-3K inhibition could partially abrogate down-regulation of FLT3. Hypoxia-mediated down-regulation of FLT3 conferred resistance against cytarabine in vitro. In conclusion, FLT3 expression in AML is dependent on the oxygen partial pressure, but response to hypoxia differs.

No MeSH data available.


Related in: MedlinePlus