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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 expression in AML is regulated by the oxygen partial pressure.(A) Heterogeneous FLT3 basal expression in (n = 8; 4 WT, 4 ITD) AML patient samples was analyzed by western blot using whole cell lysates. (B) Expression of FLT3 was independent from its mutational state (optical density (OD) of FLT3 western blot bands normalized to GAPDH shown in Fig. 1; 4 WT, 4 ITD). (C) Western blot of representative examples (n = 4) of FLT3 down-regulating (sample 9 and 10) and non-regulating AML blasts (sample 11 and 12) after 48 h exposure to 1% O2. (D) Fold change of FLT3 OD normalized to β-actin. In FLT3 down-regulating AML cells (n = 8), down-regulation was 80% (p = 0.01), but in non-regulating cells (n = 13), FLT3 expression was virtually unchanged. (E) Clinical data available for n = 24 patients. Overall survival of patients without (n = 9) and with (n = 7) in vitro hypoxia-mediated FLT3 down-regulation (no detectable FLT3 in n = 8 patients). (F) Western blot analysis of whole cell lysates show down-regulation of FLT3 in healthy CD34+ cells in response to 1% O2.
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f1: FLT3 expression in AML is regulated by the oxygen partial pressure.(A) Heterogeneous FLT3 basal expression in (n = 8; 4 WT, 4 ITD) AML patient samples was analyzed by western blot using whole cell lysates. (B) Expression of FLT3 was independent from its mutational state (optical density (OD) of FLT3 western blot bands normalized to GAPDH shown in Fig. 1; 4 WT, 4 ITD). (C) Western blot of representative examples (n = 4) of FLT3 down-regulating (sample 9 and 10) and non-regulating AML blasts (sample 11 and 12) after 48 h exposure to 1% O2. (D) Fold change of FLT3 OD normalized to β-actin. In FLT3 down-regulating AML cells (n = 8), down-regulation was 80% (p = 0.01), but in non-regulating cells (n = 13), FLT3 expression was virtually unchanged. (E) Clinical data available for n = 24 patients. Overall survival of patients without (n = 9) and with (n = 7) in vitro hypoxia-mediated FLT3 down-regulation (no detectable FLT3 in n = 8 patients). (F) Western blot analysis of whole cell lysates show down-regulation of FLT3 in healthy CD34+ cells in response to 1% O2.

Mentions: In a first step, primary AML cells (n = 8) were analysed by western blot for their basal total FLT3 protein expression from uncultured cells. FLT3 expression levels were heterogeneous but independent from the mutational state of FLT3 (Fig. 1A,B). Next, we investigated by western blot analysis the effects of in vitro hypoxia of 1% O2 on FLT3 expression in AML patient samples (n = 33, 11 ITD, 22 WT). As expected, expression of FLT3 was highly variable: in 10 patients no FLT3 expression was observed (30%), while in 23 patients (70%), FLT3 levels could be detected. Surprisingly, in the latter group, we found two distinct behaviours concerning the effect of hypoxia on FLT3 expression (Fig. 1C): while 65% showed no difference in FLT3 expression after 48 hours of exposure to 1% O2 compared to 21%, in 35% of samples a significant down-regulation of FLT3 expression was observed (80% reduction at 1% O2, p = 0,01; Fig. 1D). The down-regulation of FLT3 was independent from the mutational state of FLT3 (down-regulators: ITD 57%, WT 42%, non-regulators: ITD 33%, WT 67%, not significant). Clinical data was available for 24 of these patients. Median age was 61.9 years [23.2–82.0] with 62.5% female patients. Seven samples showed in-vitro down-regulation of FLT3, 9 were non-regulators and in 8 patients no FLT3 was detected by western blotting. Adequate blast clearance (defined as <10% myeloblasts 7–10 days after completion of induction chemotherapy) was achieved in 100% of patients whose blasts showed hypoxia-mediated down-regulation of FLT3 in vitro and by 66% of not-regulating patients (not significant). Kaplan-Meier analysis revealed that patients with a regulating phenotype tended to live longer than patients without in vitro regulation of FLT3, however the difference was not significant (p = 0.09, Fig. 1E). The reason for the possible difference in survival was due to 3 early deaths, and interestingly, FLT3 non-regulators tended to have higher leukocyte count in the peripheral blood at the time point of initial diagnosis (107 G/l versus 35 G/l; p = 0.08). Additionally, FLT3 expression was analysed in normal CD34+ cells after 48 hours of exposure to 1% O2 and showed down-regulation of FLT3 as well (Fig. 1F).


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 expression in AML is regulated by the oxygen partial pressure.(A) Heterogeneous FLT3 basal expression in (n = 8; 4 WT, 4 ITD) AML patient samples was analyzed by western blot using whole cell lysates. (B) Expression of FLT3 was independent from its mutational state (optical density (OD) of FLT3 western blot bands normalized to GAPDH shown in Fig. 1; 4 WT, 4 ITD). (C) Western blot of representative examples (n = 4) of FLT3 down-regulating (sample 9 and 10) and non-regulating AML blasts (sample 11 and 12) after 48 h exposure to 1% O2. (D) Fold change of FLT3 OD normalized to β-actin. In FLT3 down-regulating AML cells (n = 8), down-regulation was 80% (p = 0.01), but in non-regulating cells (n = 13), FLT3 expression was virtually unchanged. (E) Clinical data available for n = 24 patients. Overall survival of patients without (n = 9) and with (n = 7) in vitro hypoxia-mediated FLT3 down-regulation (no detectable FLT3 in n = 8 patients). (F) Western blot analysis of whole cell lysates show down-regulation of FLT3 in healthy CD34+ cells in response to 1% O2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: FLT3 expression in AML is regulated by the oxygen partial pressure.(A) Heterogeneous FLT3 basal expression in (n = 8; 4 WT, 4 ITD) AML patient samples was analyzed by western blot using whole cell lysates. (B) Expression of FLT3 was independent from its mutational state (optical density (OD) of FLT3 western blot bands normalized to GAPDH shown in Fig. 1; 4 WT, 4 ITD). (C) Western blot of representative examples (n = 4) of FLT3 down-regulating (sample 9 and 10) and non-regulating AML blasts (sample 11 and 12) after 48 h exposure to 1% O2. (D) Fold change of FLT3 OD normalized to β-actin. In FLT3 down-regulating AML cells (n = 8), down-regulation was 80% (p = 0.01), but in non-regulating cells (n = 13), FLT3 expression was virtually unchanged. (E) Clinical data available for n = 24 patients. Overall survival of patients without (n = 9) and with (n = 7) in vitro hypoxia-mediated FLT3 down-regulation (no detectable FLT3 in n = 8 patients). (F) Western blot analysis of whole cell lysates show down-regulation of FLT3 in healthy CD34+ cells in response to 1% O2.
Mentions: In a first step, primary AML cells (n = 8) were analysed by western blot for their basal total FLT3 protein expression from uncultured cells. FLT3 expression levels were heterogeneous but independent from the mutational state of FLT3 (Fig. 1A,B). Next, we investigated by western blot analysis the effects of in vitro hypoxia of 1% O2 on FLT3 expression in AML patient samples (n = 33, 11 ITD, 22 WT). As expected, expression of FLT3 was highly variable: in 10 patients no FLT3 expression was observed (30%), while in 23 patients (70%), FLT3 levels could be detected. Surprisingly, in the latter group, we found two distinct behaviours concerning the effect of hypoxia on FLT3 expression (Fig. 1C): while 65% showed no difference in FLT3 expression after 48 hours of exposure to 1% O2 compared to 21%, in 35% of samples a significant down-regulation of FLT3 expression was observed (80% reduction at 1% O2, p = 0,01; Fig. 1D). The down-regulation of FLT3 was independent from the mutational state of FLT3 (down-regulators: ITD 57%, WT 42%, non-regulators: ITD 33%, WT 67%, not significant). Clinical data was available for 24 of these patients. Median age was 61.9 years [23.2–82.0] with 62.5% female patients. Seven samples showed in-vitro down-regulation of FLT3, 9 were non-regulators and in 8 patients no FLT3 was detected by western blotting. Adequate blast clearance (defined as <10% myeloblasts 7–10 days after completion of induction chemotherapy) was achieved in 100% of patients whose blasts showed hypoxia-mediated down-regulation of FLT3 in vitro and by 66% of not-regulating patients (not significant). Kaplan-Meier analysis revealed that patients with a regulating phenotype tended to live longer than patients without in vitro regulation of FLT3, however the difference was not significant (p = 0.09, Fig. 1E). The reason for the possible difference in survival was due to 3 early deaths, and interestingly, FLT3 non-regulators tended to have higher leukocyte count in the peripheral blood at the time point of initial diagnosis (107 G/l versus 35 G/l; p = 0.08). Additionally, FLT3 expression was analysed in normal CD34+ cells after 48 hours of exposure to 1% O2 and showed down-regulation of FLT3 as well (Fig. 1F).

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