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Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia.

Green AS, Maciel TT, Hospital MA, Yin C, Mazed F, Townsend EC, Pilorge S, Lambert M, Paubelle E, Jacquel A, Zylbersztejn F, Decroocq J, Poulain L, Sujobert P, Jacque N, Adam K, So JC, Kosmider O, Auberger P, Hermine O, Weinstock DM, Lacombe C, Mayeux P, Vanasse GJ, Leung AY, Moura IC, Bouscary D, Tamburini J - Sci Adv (2015)

Bottom Line: Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis.Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors.Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy.

View Article: PubMed Central - PubMed

Affiliation: Institut Cochin, Département Développement, Reproduction, Cancer, CNRS, UMR 8104, INSERM U1016, Paris 75014, France. ; Faculté de Médecine, Université Paris Descartes, Sorbonne Paris Cité, Paris 75005, France. ; Equipe Labellisée, Ligue Nationale Contre le Cancer (LNCC), Paris 75013, France. ; Department of Hematology, Charles Nicolle University Hospital, Rouen 76000, France.

ABSTRACT
Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors provide short-term disease control, but relapse invariably occurs within months. Pim protein kinases are oncogenic FLT3-ITD targets expressed in AML cells. We show that increased Pim kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. Ectopic Pim-2 expression induces resistance to FLT3 inhibition in both FLT3-ITD-induced myeloproliferative neoplasm and AML models in mice. Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors. Finally, dual inhibition of FLT3 and Pim kinases eradicates FLT3-ITD(+) cells including primary AML cells. Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy.

No MeSH data available.


Related in: MedlinePlus

Pim kinase inhibition directly facilitates FLT3-ITD receptor blockade by AC220.(A) Ba/F3 cells expressing FLT3-ITD, Pim2, and Pim2KD alleles as single or combined transfectants as indicated were stained with Fluo-4 AM and treated or not with FLT3-L (30 ng/ml). Variations in intracellular calcium concentrations ([Ca2+]i) were evaluated. Fluorescence (485-nm excitation/516-nm emission) was acquired over time to evaluate the kinetics of response. Variations are expressed as differences between the baseline and experimental [Ca2+]i elevations (ΔF/F0) (left), and [Ca2+]i elevations (ΔF/F0) are expressed as the mean area under the curves [change in relative fluorescence units (ΔRFU)] (right). Results are expressed as the mean of at least four independent samples. Thapsigargin (10 μM) was used as a control for calcium mobilization. (B) Protein extracts from MOLM-14 cells expressing a control vector, Pim2, or Pim2KD were assessed for FLT3 tyrosine phosphorylation (after FLT3 immunoprecipitation) as well as STAT5 (Y694) phosphorylation and Pim-2 and STAT5 expression by immunoblotting. (C) Parental Ba/F3 cells and Ba/F3 cells expressing FLT3-ITD or FLT3-ITD-D835Y alleles were treated with vehicle or 1 μM LGB321 for 1 hour, and calcium flux was measured as detailed in (A). (D) STAT5, Pim-2, and FLT3 recombinant proteins were mixed together in a kinase buffer without or with 1, 2, 5, 10, or 50 nM AC220 and without or with 1 μM LGB321 for 1 hour. Then, 200 μM ATP was added for 30 min, and proteins were solubilized in Laemmli buffer and analyzed by immunoblotting with a phospho-STAT5 Y694 antibody. A representative Western blot is provided (top). Signal intensity was quantified using MultiGauge software (Fujifilm), and results are presented with AC220 concentrations given in log scale and using the log(inhibitor) versus response variable slope (four parameters) function of GraphPad v6 software. Results of IC50 for STAT5 phosphorylation without (−) or with (+) LGB321 are provided (bottom) (n = 3). (E) Schematic representation of FLT3-ITD receptors and of Pim kinase consensus S935 site with either nonphosphomimetic or phosphomimetic amino acid substitutions. (F and G) Ba/F3 cells were transduced with FLT3-ITD receptors either unmodified (ITD) or harboring nonphosphomimetic (ITD-S935A) or phosphomimetic (ITD-S935D) amino acid substitutions. (F) Western blotting with phospho-FLT3 (Y591), phospho-ERK (T202/Y204), phospho-STAT5 (Y694), FLT3, STAT5, and ERK antibodies. (G) Ba/F3 cells were cultured for 48 hours with vehicle or 5 nM AC220. Apoptosis was measured by annexin V binding. Results are expressed as means ± SEM. β-Actin was used as a loading control for all Western blotting experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
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Figure 4: Pim kinase inhibition directly facilitates FLT3-ITD receptor blockade by AC220.(A) Ba/F3 cells expressing FLT3-ITD, Pim2, and Pim2KD alleles as single or combined transfectants as indicated were stained with Fluo-4 AM and treated or not with FLT3-L (30 ng/ml). Variations in intracellular calcium concentrations ([Ca2+]i) were evaluated. Fluorescence (485-nm excitation/516-nm emission) was acquired over time to evaluate the kinetics of response. Variations are expressed as differences between the baseline and experimental [Ca2+]i elevations (ΔF/F0) (left), and [Ca2+]i elevations (ΔF/F0) are expressed as the mean area under the curves [change in relative fluorescence units (ΔRFU)] (right). Results are expressed as the mean of at least four independent samples. Thapsigargin (10 μM) was used as a control for calcium mobilization. (B) Protein extracts from MOLM-14 cells expressing a control vector, Pim2, or Pim2KD were assessed for FLT3 tyrosine phosphorylation (after FLT3 immunoprecipitation) as well as STAT5 (Y694) phosphorylation and Pim-2 and STAT5 expression by immunoblotting. (C) Parental Ba/F3 cells and Ba/F3 cells expressing FLT3-ITD or FLT3-ITD-D835Y alleles were treated with vehicle or 1 μM LGB321 for 1 hour, and calcium flux was measured as detailed in (A). (D) STAT5, Pim-2, and FLT3 recombinant proteins were mixed together in a kinase buffer without or with 1, 2, 5, 10, or 50 nM AC220 and without or with 1 μM LGB321 for 1 hour. Then, 200 μM ATP was added for 30 min, and proteins were solubilized in Laemmli buffer and analyzed by immunoblotting with a phospho-STAT5 Y694 antibody. A representative Western blot is provided (top). Signal intensity was quantified using MultiGauge software (Fujifilm), and results are presented with AC220 concentrations given in log scale and using the log(inhibitor) versus response variable slope (four parameters) function of GraphPad v6 software. Results of IC50 for STAT5 phosphorylation without (−) or with (+) LGB321 are provided (bottom) (n = 3). (E) Schematic representation of FLT3-ITD receptors and of Pim kinase consensus S935 site with either nonphosphomimetic or phosphomimetic amino acid substitutions. (F and G) Ba/F3 cells were transduced with FLT3-ITD receptors either unmodified (ITD) or harboring nonphosphomimetic (ITD-S935A) or phosphomimetic (ITD-S935D) amino acid substitutions. (F) Western blotting with phospho-FLT3 (Y591), phospho-ERK (T202/Y204), phospho-STAT5 (Y694), FLT3, STAT5, and ERK antibodies. (G) Ba/F3 cells were cultured for 48 hours with vehicle or 5 nM AC220. Apoptosis was measured by annexin V binding. Results are expressed as means ± SEM. β-Actin was used as a loading control for all Western blotting experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Mentions: We measured intracellular calcium flow as a dynamic marker of FLT3-dependent signaling pathway activation. Calcium mobilization was promptly induced in Ba/F3-ITD cells—used as a minimal model of oncogene addiction to FLT3-ITD signaling (23)—after FLT3-L stimulation, consistent with the sustained sensitivity of FLT3-ITD receptors to FLT3-L in AML patients (Fig. 4A) (24). Ectopic expression of Pim2, but not Pim2KD, allele abrogated FLT3-L–induced signaling, whereas induction of calcium flux by thapsigargin was similar regardless of transfectant (Fig. 4A). Co-incubation of Pim-2 and FLT3 recombinant proteins decreased FLT3 tyrosine phosphorylation in vitro compared to the level of FLT3 autophosphorylation (fig. S4A), and MOLM-14 cells transfected with Pim2 exhibited reduced FLT3 and STAT5 tyrosine phosphorylation, in contrast to Pim2KD transfectants (Fig. 4B). These results suggest that Pim-2 activity inhibits FLT3-ITD signaling.


Pim kinases modulate resistance to FLT3 tyrosine kinase inhibitors in FLT3-ITD acute myeloid leukemia.

Green AS, Maciel TT, Hospital MA, Yin C, Mazed F, Townsend EC, Pilorge S, Lambert M, Paubelle E, Jacquel A, Zylbersztejn F, Decroocq J, Poulain L, Sujobert P, Jacque N, Adam K, So JC, Kosmider O, Auberger P, Hermine O, Weinstock DM, Lacombe C, Mayeux P, Vanasse GJ, Leung AY, Moura IC, Bouscary D, Tamburini J - Sci Adv (2015)

Pim kinase inhibition directly facilitates FLT3-ITD receptor blockade by AC220.(A) Ba/F3 cells expressing FLT3-ITD, Pim2, and Pim2KD alleles as single or combined transfectants as indicated were stained with Fluo-4 AM and treated or not with FLT3-L (30 ng/ml). Variations in intracellular calcium concentrations ([Ca2+]i) were evaluated. Fluorescence (485-nm excitation/516-nm emission) was acquired over time to evaluate the kinetics of response. Variations are expressed as differences between the baseline and experimental [Ca2+]i elevations (ΔF/F0) (left), and [Ca2+]i elevations (ΔF/F0) are expressed as the mean area under the curves [change in relative fluorescence units (ΔRFU)] (right). Results are expressed as the mean of at least four independent samples. Thapsigargin (10 μM) was used as a control for calcium mobilization. (B) Protein extracts from MOLM-14 cells expressing a control vector, Pim2, or Pim2KD were assessed for FLT3 tyrosine phosphorylation (after FLT3 immunoprecipitation) as well as STAT5 (Y694) phosphorylation and Pim-2 and STAT5 expression by immunoblotting. (C) Parental Ba/F3 cells and Ba/F3 cells expressing FLT3-ITD or FLT3-ITD-D835Y alleles were treated with vehicle or 1 μM LGB321 for 1 hour, and calcium flux was measured as detailed in (A). (D) STAT5, Pim-2, and FLT3 recombinant proteins were mixed together in a kinase buffer without or with 1, 2, 5, 10, or 50 nM AC220 and without or with 1 μM LGB321 for 1 hour. Then, 200 μM ATP was added for 30 min, and proteins were solubilized in Laemmli buffer and analyzed by immunoblotting with a phospho-STAT5 Y694 antibody. A representative Western blot is provided (top). Signal intensity was quantified using MultiGauge software (Fujifilm), and results are presented with AC220 concentrations given in log scale and using the log(inhibitor) versus response variable slope (four parameters) function of GraphPad v6 software. Results of IC50 for STAT5 phosphorylation without (−) or with (+) LGB321 are provided (bottom) (n = 3). (E) Schematic representation of FLT3-ITD receptors and of Pim kinase consensus S935 site with either nonphosphomimetic or phosphomimetic amino acid substitutions. (F and G) Ba/F3 cells were transduced with FLT3-ITD receptors either unmodified (ITD) or harboring nonphosphomimetic (ITD-S935A) or phosphomimetic (ITD-S935D) amino acid substitutions. (F) Western blotting with phospho-FLT3 (Y591), phospho-ERK (T202/Y204), phospho-STAT5 (Y694), FLT3, STAT5, and ERK antibodies. (G) Ba/F3 cells were cultured for 48 hours with vehicle or 5 nM AC220. Apoptosis was measured by annexin V binding. Results are expressed as means ± SEM. β-Actin was used as a loading control for all Western blotting experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
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Figure 4: Pim kinase inhibition directly facilitates FLT3-ITD receptor blockade by AC220.(A) Ba/F3 cells expressing FLT3-ITD, Pim2, and Pim2KD alleles as single or combined transfectants as indicated were stained with Fluo-4 AM and treated or not with FLT3-L (30 ng/ml). Variations in intracellular calcium concentrations ([Ca2+]i) were evaluated. Fluorescence (485-nm excitation/516-nm emission) was acquired over time to evaluate the kinetics of response. Variations are expressed as differences between the baseline and experimental [Ca2+]i elevations (ΔF/F0) (left), and [Ca2+]i elevations (ΔF/F0) are expressed as the mean area under the curves [change in relative fluorescence units (ΔRFU)] (right). Results are expressed as the mean of at least four independent samples. Thapsigargin (10 μM) was used as a control for calcium mobilization. (B) Protein extracts from MOLM-14 cells expressing a control vector, Pim2, or Pim2KD were assessed for FLT3 tyrosine phosphorylation (after FLT3 immunoprecipitation) as well as STAT5 (Y694) phosphorylation and Pim-2 and STAT5 expression by immunoblotting. (C) Parental Ba/F3 cells and Ba/F3 cells expressing FLT3-ITD or FLT3-ITD-D835Y alleles were treated with vehicle or 1 μM LGB321 for 1 hour, and calcium flux was measured as detailed in (A). (D) STAT5, Pim-2, and FLT3 recombinant proteins were mixed together in a kinase buffer without or with 1, 2, 5, 10, or 50 nM AC220 and without or with 1 μM LGB321 for 1 hour. Then, 200 μM ATP was added for 30 min, and proteins were solubilized in Laemmli buffer and analyzed by immunoblotting with a phospho-STAT5 Y694 antibody. A representative Western blot is provided (top). Signal intensity was quantified using MultiGauge software (Fujifilm), and results are presented with AC220 concentrations given in log scale and using the log(inhibitor) versus response variable slope (four parameters) function of GraphPad v6 software. Results of IC50 for STAT5 phosphorylation without (−) or with (+) LGB321 are provided (bottom) (n = 3). (E) Schematic representation of FLT3-ITD receptors and of Pim kinase consensus S935 site with either nonphosphomimetic or phosphomimetic amino acid substitutions. (F and G) Ba/F3 cells were transduced with FLT3-ITD receptors either unmodified (ITD) or harboring nonphosphomimetic (ITD-S935A) or phosphomimetic (ITD-S935D) amino acid substitutions. (F) Western blotting with phospho-FLT3 (Y591), phospho-ERK (T202/Y204), phospho-STAT5 (Y694), FLT3, STAT5, and ERK antibodies. (G) Ba/F3 cells were cultured for 48 hours with vehicle or 5 nM AC220. Apoptosis was measured by annexin V binding. Results are expressed as means ± SEM. β-Actin was used as a loading control for all Western blotting experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Mentions: We measured intracellular calcium flow as a dynamic marker of FLT3-dependent signaling pathway activation. Calcium mobilization was promptly induced in Ba/F3-ITD cells—used as a minimal model of oncogene addiction to FLT3-ITD signaling (23)—after FLT3-L stimulation, consistent with the sustained sensitivity of FLT3-ITD receptors to FLT3-L in AML patients (Fig. 4A) (24). Ectopic expression of Pim2, but not Pim2KD, allele abrogated FLT3-L–induced signaling, whereas induction of calcium flux by thapsigargin was similar regardless of transfectant (Fig. 4A). Co-incubation of Pim-2 and FLT3 recombinant proteins decreased FLT3 tyrosine phosphorylation in vitro compared to the level of FLT3 autophosphorylation (fig. S4A), and MOLM-14 cells transfected with Pim2 exhibited reduced FLT3 and STAT5 tyrosine phosphorylation, in contrast to Pim2KD transfectants (Fig. 4B). These results suggest that Pim-2 activity inhibits FLT3-ITD signaling.

Bottom Line: Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis.Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors.Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy.

View Article: PubMed Central - PubMed

Affiliation: Institut Cochin, Département Développement, Reproduction, Cancer, CNRS, UMR 8104, INSERM U1016, Paris 75014, France. ; Faculté de Médecine, Université Paris Descartes, Sorbonne Paris Cité, Paris 75005, France. ; Equipe Labellisée, Ligue Nationale Contre le Cancer (LNCC), Paris 75013, France. ; Department of Hematology, Charles Nicolle University Hospital, Rouen 76000, France.

ABSTRACT
Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors provide short-term disease control, but relapse invariably occurs within months. Pim protein kinases are oncogenic FLT3-ITD targets expressed in AML cells. We show that increased Pim kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. Ectopic Pim-2 expression induces resistance to FLT3 inhibition in both FLT3-ITD-induced myeloproliferative neoplasm and AML models in mice. Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors. Finally, dual inhibition of FLT3 and Pim kinases eradicates FLT3-ITD(+) cells including primary AML cells. Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy.

No MeSH data available.


Related in: MedlinePlus