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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 regulating mechanisms by hypoxia.(A) Representative experiment of FLT3 degradation in Ba/F3 W51 at 21% and 1% O2 after protein synthesis inhibition with CHX (50 μg/mL) showing faster and increased degradation during hypoxia (immature intracellular FLT3 receptor: 130 kDa, mature glycosylated FLT3 receptor: 155 kDa). (B) Fold change of FLT3 OD normalized to β-actin. t½ is shortened during hypoxia by 1 hour from 2.5 hours to 1.5 hours. (C) Degradation of FLT3 in Ba/F3 W51 cells remains proteasome dependent also at 1% O2, as treatment with proteasome inhibitor MG132 reverses degradation after protein synthesis blockade with CHX. Fold change of FLT3 OD for each time point is calculated compared to the 0 time point value and normalized to β-actin (D) mRNA levels for FLT3 were unaltered after exposure to 72 hours of hypoxia in Ba/F3 cells. (E) Effects of HSP90 inhibition, Hif1alpha activation and PI3-Kinase inhibition on Ba/F3 W51 cells (cells were adjusted at hypoxia for 24 hours and treated for 48 hours) FLT3 expression and FLT3 downstream pathway activation was analysed by western blot. Only PI3-K inhibition partially abrogated hypoxia-mediated down-regulation of FLT3.
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f3: FLT3 regulating mechanisms by hypoxia.(A) Representative experiment of FLT3 degradation in Ba/F3 W51 at 21% and 1% O2 after protein synthesis inhibition with CHX (50 μg/mL) showing faster and increased degradation during hypoxia (immature intracellular FLT3 receptor: 130 kDa, mature glycosylated FLT3 receptor: 155 kDa). (B) Fold change of FLT3 OD normalized to β-actin. t½ is shortened during hypoxia by 1 hour from 2.5 hours to 1.5 hours. (C) Degradation of FLT3 in Ba/F3 W51 cells remains proteasome dependent also at 1% O2, as treatment with proteasome inhibitor MG132 reverses degradation after protein synthesis blockade with CHX. Fold change of FLT3 OD for each time point is calculated compared to the 0 time point value and normalized to β-actin (D) mRNA levels for FLT3 were unaltered after exposure to 72 hours of hypoxia in Ba/F3 cells. (E) Effects of HSP90 inhibition, Hif1alpha activation and PI3-Kinase inhibition on Ba/F3 W51 cells (cells were adjusted at hypoxia for 24 hours and treated for 48 hours) FLT3 expression and FLT3 downstream pathway activation was analysed by western blot. Only PI3-K inhibition partially abrogated hypoxia-mediated down-regulation of FLT3.

Mentions: Half-life (t½) of FLT3 was reported as 2–3 hours20, however we found t½ of FLT3 at 1% O2 to be significantly shorter: as shown in Fig. 3A,B, degradation of FLT3 protein was fastened by 1 h at 1% O2, suggesting that hypoxia increases the turn-over of the protein. Treatment with the proteasome inhibitor MG132 (5 μM) reversed FLT3 degradation both at 1% O2 and at standard laboratory conditions (Fig. 3C), indicating that proteasome dependent degradation of FLT3 is also active during hypoxic conditions. Total levels of CBL were unaltered by hypoxia (data not shown). To investigate whether transcriptional mechanisms might also be involved in the different expression of FLT3 under hypoxic conditions, real-time PCR was performed for FLT3 mRNA levels at 1% O2 and 21% O2, but no significant differences were observed in FLT3 transcript level at the different oxygen conditions (Fig. 3D). ELISA-based analysis of the supernatant of FLT3 down-regulating cells did not show detectable levels of FLT3, making extracellular shedding of the protein, as a potential mechanism of loss of FLT3 protein by hypoxia, highly unlikely (data not shown). To further identify possible other molecular mechanisms, we investigated the effects of phosphatidylinositide 3-kinase (PI3-K), Heat shock Protein 90 (Hsp90) inhibition and HIF-hydroxylase inhibitor (i.e. HIF1alpha stabilization) on hypoxia-mediated FLT3 down-regulation. As shown in Fig. 3E, FLT3 down-regulation could partially be abrogated by PI3-K inhibition but was neither affected by Hsp90 inhibition nor by HIF-1 alpha activation, suggesting that hypoxia-mediated FLT3-down-regulation might be partially regulated by the PI3-K pathway, but independent from HIF1alpha.


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 regulating mechanisms by hypoxia.(A) Representative experiment of FLT3 degradation in Ba/F3 W51 at 21% and 1% O2 after protein synthesis inhibition with CHX (50 μg/mL) showing faster and increased degradation during hypoxia (immature intracellular FLT3 receptor: 130 kDa, mature glycosylated FLT3 receptor: 155 kDa). (B) Fold change of FLT3 OD normalized to β-actin. t½ is shortened during hypoxia by 1 hour from 2.5 hours to 1.5 hours. (C) Degradation of FLT3 in Ba/F3 W51 cells remains proteasome dependent also at 1% O2, as treatment with proteasome inhibitor MG132 reverses degradation after protein synthesis blockade with CHX. Fold change of FLT3 OD for each time point is calculated compared to the 0 time point value and normalized to β-actin (D) mRNA levels for FLT3 were unaltered after exposure to 72 hours of hypoxia in Ba/F3 cells. (E) Effects of HSP90 inhibition, Hif1alpha activation and PI3-Kinase inhibition on Ba/F3 W51 cells (cells were adjusted at hypoxia for 24 hours and treated for 48 hours) FLT3 expression and FLT3 downstream pathway activation was analysed by western blot. Only PI3-K inhibition partially abrogated hypoxia-mediated down-regulation of FLT3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663471&req=5

f3: FLT3 regulating mechanisms by hypoxia.(A) Representative experiment of FLT3 degradation in Ba/F3 W51 at 21% and 1% O2 after protein synthesis inhibition with CHX (50 μg/mL) showing faster and increased degradation during hypoxia (immature intracellular FLT3 receptor: 130 kDa, mature glycosylated FLT3 receptor: 155 kDa). (B) Fold change of FLT3 OD normalized to β-actin. t½ is shortened during hypoxia by 1 hour from 2.5 hours to 1.5 hours. (C) Degradation of FLT3 in Ba/F3 W51 cells remains proteasome dependent also at 1% O2, as treatment with proteasome inhibitor MG132 reverses degradation after protein synthesis blockade with CHX. Fold change of FLT3 OD for each time point is calculated compared to the 0 time point value and normalized to β-actin (D) mRNA levels for FLT3 were unaltered after exposure to 72 hours of hypoxia in Ba/F3 cells. (E) Effects of HSP90 inhibition, Hif1alpha activation and PI3-Kinase inhibition on Ba/F3 W51 cells (cells were adjusted at hypoxia for 24 hours and treated for 48 hours) FLT3 expression and FLT3 downstream pathway activation was analysed by western blot. Only PI3-K inhibition partially abrogated hypoxia-mediated down-regulation of FLT3.
Mentions: Half-life (t½) of FLT3 was reported as 2–3 hours20, however we found t½ of FLT3 at 1% O2 to be significantly shorter: as shown in Fig. 3A,B, degradation of FLT3 protein was fastened by 1 h at 1% O2, suggesting that hypoxia increases the turn-over of the protein. Treatment with the proteasome inhibitor MG132 (5 μM) reversed FLT3 degradation both at 1% O2 and at standard laboratory conditions (Fig. 3C), indicating that proteasome dependent degradation of FLT3 is also active during hypoxic conditions. Total levels of CBL were unaltered by hypoxia (data not shown). To investigate whether transcriptional mechanisms might also be involved in the different expression of FLT3 under hypoxic conditions, real-time PCR was performed for FLT3 mRNA levels at 1% O2 and 21% O2, but no significant differences were observed in FLT3 transcript level at the different oxygen conditions (Fig. 3D). ELISA-based analysis of the supernatant of FLT3 down-regulating cells did not show detectable levels of FLT3, making extracellular shedding of the protein, as a potential mechanism of loss of FLT3 protein by hypoxia, highly unlikely (data not shown). To further identify possible other molecular mechanisms, we investigated the effects of phosphatidylinositide 3-kinase (PI3-K), Heat shock Protein 90 (Hsp90) inhibition and HIF-hydroxylase inhibitor (i.e. HIF1alpha stabilization) on hypoxia-mediated FLT3 down-regulation. As shown in Fig. 3E, FLT3 down-regulation could partially be abrogated by PI3-K inhibition but was neither affected by Hsp90 inhibition nor by HIF-1 alpha activation, suggesting that hypoxia-mediated FLT3-down-regulation might be partially regulated by the PI3-K pathway, but independent from HIF1alpha.

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