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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

Functional consequences of FLT3 down-regulation.(A) Susceptibility of Ba/F3 W51 cells towards sorafenib (after 24 hours) was unaltered by the pO2. (B) No differences in STAT5 and AKT phosphorylation in Ba/F3 W51 cells cultured for 72 hours at 21% and 1% O2. (C) Ba/F3 cells transfected with an empty-vector and with Ba/F3 W51 cells were treated for 24 hours with increasing dose of cytarabine. FLT3-ITD affects susceptibility of Ba/F3 cells towards cytarabine at hypoxic conditions.
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f5: Functional consequences of FLT3 down-regulation.(A) Susceptibility of Ba/F3 W51 cells towards sorafenib (after 24 hours) was unaltered by the pO2. (B) No differences in STAT5 and AKT phosphorylation in Ba/F3 W51 cells cultured for 72 hours at 21% and 1% O2. (C) Ba/F3 cells transfected with an empty-vector and with Ba/F3 W51 cells were treated for 24 hours with increasing dose of cytarabine. FLT3-ITD affects susceptibility of Ba/F3 cells towards cytarabine at hypoxic conditions.

Mentions: The further characterize functional consequences of FLT3 hypoxia-mediated down-regulation, we tested the susceptibility of Ba/F3 cells towards the tyrosine kinase inhibitor sorafenib, according to the expression level of FLT3 (i.e. 21% O2 versus 1% O2). Interestingly, no differences in the anti-leukemic efficacy of the drug were observed between hypoxia and normoxia (IC50 5.06 μM at 21%O2 versus 3.93 μM at 1% O2, not significant, Fig. 5A). In line with this finding was the observation that both canonical down-stream pathways of FLT3 (STAT5 and AKT) were unaltered by hypoxia as shown in Fig. 5B. However, FLT3 seems to be involved in the pharmacodynamics of cytarabine: while Ba/F3 cells transfected with an empty vector (negative control) where equally susceptible to cytarabine at normoxic and hypoxic conditions (IC50 287.17 nM versus 452.86 nM, not significant, Fig. 5C), the presence of FLT3 ITD in Ba/F3 W51 substantially reduced the susceptibility of these cells towards cytarabine at hypoxia. Results are shown in Fig. 5D: in contrast to normoxic conditions (n = 4; IC50 50.45 nM) the IC50 was not reached in all the experiments under hypoxic conditions (n = 3; IC50 413.88 nM).


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)

Functional consequences of FLT3 down-regulation.(A) Susceptibility of Ba/F3 W51 cells towards sorafenib (after 24 hours) was unaltered by the pO2. (B) No differences in STAT5 and AKT phosphorylation in Ba/F3 W51 cells cultured for 72 hours at 21% and 1% O2. (C) Ba/F3 cells transfected with an empty-vector and with Ba/F3 W51 cells were treated for 24 hours with increasing dose of cytarabine. FLT3-ITD affects susceptibility of Ba/F3 cells towards cytarabine at hypoxic conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Functional consequences of FLT3 down-regulation.(A) Susceptibility of Ba/F3 W51 cells towards sorafenib (after 24 hours) was unaltered by the pO2. (B) No differences in STAT5 and AKT phosphorylation in Ba/F3 W51 cells cultured for 72 hours at 21% and 1% O2. (C) Ba/F3 cells transfected with an empty-vector and with Ba/F3 W51 cells were treated for 24 hours with increasing dose of cytarabine. FLT3-ITD affects susceptibility of Ba/F3 cells towards cytarabine at hypoxic conditions.
Mentions: The further characterize functional consequences of FLT3 hypoxia-mediated down-regulation, we tested the susceptibility of Ba/F3 cells towards the tyrosine kinase inhibitor sorafenib, according to the expression level of FLT3 (i.e. 21% O2 versus 1% O2). Interestingly, no differences in the anti-leukemic efficacy of the drug were observed between hypoxia and normoxia (IC50 5.06 μM at 21%O2 versus 3.93 μM at 1% O2, not significant, Fig. 5A). In line with this finding was the observation that both canonical down-stream pathways of FLT3 (STAT5 and AKT) were unaltered by hypoxia as shown in Fig. 5B. However, FLT3 seems to be involved in the pharmacodynamics of cytarabine: while Ba/F3 cells transfected with an empty vector (negative control) where equally susceptible to cytarabine at normoxic and hypoxic conditions (IC50 287.17 nM versus 452.86 nM, not significant, Fig. 5C), the presence of FLT3 ITD in Ba/F3 W51 substantially reduced the susceptibility of these cells towards cytarabine at hypoxia. Results are shown in Fig. 5D: in contrast to normoxic conditions (n = 4; IC50 50.45 nM) the IC50 was not reached in all the experiments under hypoxic conditions (n = 3; IC50 413.88 nM).

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