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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 is down-regulated by hypoxia in Ba/F3 cells.(A) OCI-AML3 and OCI-AML5 do not regulate FLT3 under hypoxia. Fold chance Optical Density (OD) was calculated compared to the respective value under 21% O2 and normalized to β-actin. (B) Ba/F3 cell lines stably transfected with FLT3 WT and FLT3 W51 (ITD) reliably down-regulate FLT3 at hypoxic conditions. (C) Down-regulation of FLT3 occurs in Ba/F3 W51 cells only below oxygen levels of 6% O2. (D) Ba/F3 W51 cells down-regulate FLT3 after 48 hours of culture under hypoxia. Fold change Optical Density (OD) was calculated for each time point compared to the respective value under 21% O2 and normalized to β-actin. (E) FLT3 down-regulation in Ba/F3 W51 cells is not a short-term stress hypoxic effect but is maintained for >9 days of culture under hypoxia. (F) Restoration of FLT3 expression after re-oxygenation of hypoxic Ba/F3 W51 cells with 21% O2. (G) Optical Density (OD) of FLT3 was calculated at different conditions compared to the respective value under 21% O2 at 72 hours and normalised to β-actin. (H) Significant decrease in proliferation in Ba/F3 W51 cells after 48 h and 72 h of culture at 1% O2. (I) No increased induction of cell death by hypoxia of 1% O2 in Ba/F3 W51 cells after 72 hours as compared to cells at 21% O2.
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f2: FLT3 is down-regulated by hypoxia in Ba/F3 cells.(A) OCI-AML3 and OCI-AML5 do not regulate FLT3 under hypoxia. Fold chance Optical Density (OD) was calculated compared to the respective value under 21% O2 and normalized to β-actin. (B) Ba/F3 cell lines stably transfected with FLT3 WT and FLT3 W51 (ITD) reliably down-regulate FLT3 at hypoxic conditions. (C) Down-regulation of FLT3 occurs in Ba/F3 W51 cells only below oxygen levels of 6% O2. (D) Ba/F3 W51 cells down-regulate FLT3 after 48 hours of culture under hypoxia. Fold change Optical Density (OD) was calculated for each time point compared to the respective value under 21% O2 and normalized to β-actin. (E) FLT3 down-regulation in Ba/F3 W51 cells is not a short-term stress hypoxic effect but is maintained for >9 days of culture under hypoxia. (F) Restoration of FLT3 expression after re-oxygenation of hypoxic Ba/F3 W51 cells with 21% O2. (G) Optical Density (OD) of FLT3 was calculated at different conditions compared to the respective value under 21% O2 at 72 hours and normalised to β-actin. (H) Significant decrease in proliferation in Ba/F3 W51 cells after 48 h and 72 h of culture at 1% O2. (I) No increased induction of cell death by hypoxia of 1% O2 in Ba/F3 W51 cells after 72 hours as compared to cells at 21% O2.

Mentions: Next, we screened a panel of human leukemic AML cell lines (OCI-AML3, OCI-AML5, Mono-Mac-6, KG1, Kasumi-1, NB-4, MV-4/11, Molm-13, HEL, CMK, Mo7E, HL60) for FLT3 protein basal expression, which was not detectable in most of the cell lines. The cell lines with good detectable base line expression (Mono-Mac-6, OCI-AML3, OCI-AML5) were further investigated for FLT3 expression at 1% O2, however no FLT3 down-regulation was observed (two representative examples are shown in Fig. 2A). We than switched to Ba/F3 cells and analysed FLT3 expression in cells stably transfected with either FLT3-WT or FLT3-ITD construct W51. In this model, hypoxia induced down-regulation of FLT3 was reliably observed in all Ba/F3 cells independently from the mutational state of the receptor (Fig. 2B). As we were unable to detect significant differences between FLT3-WT and the FLT3-ITD mutant with respect to FLT3 down-regulation, Ba/F3 W51 (FLT3-ITD) cells were chosen for subsequent experiments due to their long-term IL-3 independent in vitro growth. With this in vitro model of hypoxia-mediated down-regulation of FLT3, we established the oxygen threshold required for down-regulation between 1% and 6% O2 (Fig. 2C) and the time window for down-regulation between 48 and 72 hours of hypoxia (Fig. 2D). FLT3 down-regulating phenotype was maintained after 9 and also after 12 days of hypoxia, suggesting that hypoxia-mediated FLT3 down-regulation is not a short-term stress reaction of AML cells towards 1% O2 (Fig. 2E). However, FLT3 down-regulation is reversible. Ba/F3 cells cultured for 3 days in hypoxic conditions showed a restoration of total protein within the first 24 hours after re-oxygenation with 21% O2 (Fig. 2F,G) and also exposure to fresh medium (data not shown). As expected, exposure to 1% O2 decreases proliferation of Ba/F3 cells (cell counts after 72 hours: 2.1 × 105/ml for 21% O2, 1.3 × 105/ml for 1% O2; p = 0.007, Fig. 2H), but viability was unaltered (88.7% viable cells at 21% O2 versus 85.4% at 1% O2 ; not significant, Figure I).


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 is down-regulated by hypoxia in Ba/F3 cells.(A) OCI-AML3 and OCI-AML5 do not regulate FLT3 under hypoxia. Fold chance Optical Density (OD) was calculated compared to the respective value under 21% O2 and normalized to β-actin. (B) Ba/F3 cell lines stably transfected with FLT3 WT and FLT3 W51 (ITD) reliably down-regulate FLT3 at hypoxic conditions. (C) Down-regulation of FLT3 occurs in Ba/F3 W51 cells only below oxygen levels of 6% O2. (D) Ba/F3 W51 cells down-regulate FLT3 after 48 hours of culture under hypoxia. Fold change Optical Density (OD) was calculated for each time point compared to the respective value under 21% O2 and normalized to β-actin. (E) FLT3 down-regulation in Ba/F3 W51 cells is not a short-term stress hypoxic effect but is maintained for >9 days of culture under hypoxia. (F) Restoration of FLT3 expression after re-oxygenation of hypoxic Ba/F3 W51 cells with 21% O2. (G) Optical Density (OD) of FLT3 was calculated at different conditions compared to the respective value under 21% O2 at 72 hours and normalised to β-actin. (H) Significant decrease in proliferation in Ba/F3 W51 cells after 48 h and 72 h of culture at 1% O2. (I) No increased induction of cell death by hypoxia of 1% O2 in Ba/F3 W51 cells after 72 hours as compared to cells at 21% O2.
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f2: FLT3 is down-regulated by hypoxia in Ba/F3 cells.(A) OCI-AML3 and OCI-AML5 do not regulate FLT3 under hypoxia. Fold chance Optical Density (OD) was calculated compared to the respective value under 21% O2 and normalized to β-actin. (B) Ba/F3 cell lines stably transfected with FLT3 WT and FLT3 W51 (ITD) reliably down-regulate FLT3 at hypoxic conditions. (C) Down-regulation of FLT3 occurs in Ba/F3 W51 cells only below oxygen levels of 6% O2. (D) Ba/F3 W51 cells down-regulate FLT3 after 48 hours of culture under hypoxia. Fold change Optical Density (OD) was calculated for each time point compared to the respective value under 21% O2 and normalized to β-actin. (E) FLT3 down-regulation in Ba/F3 W51 cells is not a short-term stress hypoxic effect but is maintained for >9 days of culture under hypoxia. (F) Restoration of FLT3 expression after re-oxygenation of hypoxic Ba/F3 W51 cells with 21% O2. (G) Optical Density (OD) of FLT3 was calculated at different conditions compared to the respective value under 21% O2 at 72 hours and normalised to β-actin. (H) Significant decrease in proliferation in Ba/F3 W51 cells after 48 h and 72 h of culture at 1% O2. (I) No increased induction of cell death by hypoxia of 1% O2 in Ba/F3 W51 cells after 72 hours as compared to cells at 21% O2.
Mentions: Next, we screened a panel of human leukemic AML cell lines (OCI-AML3, OCI-AML5, Mono-Mac-6, KG1, Kasumi-1, NB-4, MV-4/11, Molm-13, HEL, CMK, Mo7E, HL60) for FLT3 protein basal expression, which was not detectable in most of the cell lines. The cell lines with good detectable base line expression (Mono-Mac-6, OCI-AML3, OCI-AML5) were further investigated for FLT3 expression at 1% O2, however no FLT3 down-regulation was observed (two representative examples are shown in Fig. 2A). We than switched to Ba/F3 cells and analysed FLT3 expression in cells stably transfected with either FLT3-WT or FLT3-ITD construct W51. In this model, hypoxia induced down-regulation of FLT3 was reliably observed in all Ba/F3 cells independently from the mutational state of the receptor (Fig. 2B). As we were unable to detect significant differences between FLT3-WT and the FLT3-ITD mutant with respect to FLT3 down-regulation, Ba/F3 W51 (FLT3-ITD) cells were chosen for subsequent experiments due to their long-term IL-3 independent in vitro growth. With this in vitro model of hypoxia-mediated down-regulation of FLT3, we established the oxygen threshold required for down-regulation between 1% and 6% O2 (Fig. 2C) and the time window for down-regulation between 48 and 72 hours of hypoxia (Fig. 2D). FLT3 down-regulating phenotype was maintained after 9 and also after 12 days of hypoxia, suggesting that hypoxia-mediated FLT3 down-regulation is not a short-term stress reaction of AML cells towards 1% O2 (Fig. 2E). However, FLT3 down-regulation is reversible. Ba/F3 cells cultured for 3 days in hypoxic conditions showed a restoration of total protein within the first 24 hours after re-oxygenation with 21% O2 (Fig. 2F,G) and also exposure to fresh medium (data not shown). As expected, exposure to 1% O2 decreases proliferation of Ba/F3 cells (cell counts after 72 hours: 2.1 × 105/ml for 21% O2, 1.3 × 105/ml for 1% O2; p = 0.007, Fig. 2H), but viability was unaltered (88.7% viable cells at 21% O2 versus 85.4% at 1% O2 ; not significant, Figure I).

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