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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 kinases are FLT3-ITD targets involved in resistance to FLT3 inhibition in AML.(A) AML cell lines (HL-60, OCI-AML3, MV4-11, and MOLM-14) were transduced via lentivirus with Dox-inducible anti-FLT3 shRNA vectors. shRNA induction was achieved with Dox (200 ng/ml). Western blots were performed using FLT3, Pim-1, and Pim-2 antibodies. WT, wild type. (B) MOLM-14 and OCI-AML3 cells were cultured with FLT3-L and/or 5 nM AC220. Tyrosine phosphorylation was evaluated in FLT3 immunoprecipitates. Pim-1, Pim-2, phospho-STAT5 (Y694), and STAT5 levels were detected in whole-cell lysates by immunoblotting. (C) MOLM-14 cells were treated for 24 hours with 5 nM AC220 (left), and MOLM-14 and MV4-11 cells were transduced with inducible shFLT3 and treated with Dox (200 ng/ml) for 48 hours (right). Pim-1 and Pim-2 mRNA levels were quantified by real-time polymerase chain reaction. Gene expression was normalized to the HPRT (hypoxanthine-guanine phosphoribosyltransferase) levels (n = 3). (D) STAT5A/B gain (right) or loss (left) of function in MOLM-14 cells transduced with lentivirus. STAT5A, STAT5B, Pim-1, Pim-2, and Bcl-xL protein levels were evaluated by immunoblotting. (E) MOLM-14 cells were separately transduced with a PIM1- or PIM2-expressing vector or an empty vector. Ectopic expression of Pim-1 and Pim-2 was verified by immunoblotting (right). Cells were treated with vehicle or 1 nM AC220 for 48 hours, and cell viability was assessed by an UptiBlue assay (left) (n = 3). (F) MOLM-14 cells expressing an FLT3 shRNA in a Dox-inducible manner were transduced with a Pim2 (murine Pim-2) allele or a control vector. After 48 hours of culture in the presence of Dox (200 ng/ml), FLT3 and murine Pim-2 expression was assessed by immunoblotting, and apoptosis was measured by annexin V staining. (G and H) MOLM-14 cells were transduced via lentivirus with a control vector (Dox-inducible anti–Pim-2 shRNA vector) or with Pim2 lentivirus and xenografted into nude mice. (E) Tumor growth was assessed in mice treated with AC220 (1 mg/kg) (initiated once the tumor size reached 100 mm3) with (black circle) or without (white circle) Pim2 transduction. Growth of untreated xenotransplanted MOLM-14 cells is also provided (white square box). The means of the individual tumor sizes are plotted (n = 8). (F) Kaplan-Meier survival curve analysis of MOLM-14 cells transduced (dashed line) or not (solid line) with Pim2, transplanted into nude mice, and treated with AC220 (n = 8). Results are expressed as means ± SEM. β-Actin was used as a loading control for all Western blots. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
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Figure 2: Pim kinases are FLT3-ITD targets involved in resistance to FLT3 inhibition in AML.(A) AML cell lines (HL-60, OCI-AML3, MV4-11, and MOLM-14) were transduced via lentivirus with Dox-inducible anti-FLT3 shRNA vectors. shRNA induction was achieved with Dox (200 ng/ml). Western blots were performed using FLT3, Pim-1, and Pim-2 antibodies. WT, wild type. (B) MOLM-14 and OCI-AML3 cells were cultured with FLT3-L and/or 5 nM AC220. Tyrosine phosphorylation was evaluated in FLT3 immunoprecipitates. Pim-1, Pim-2, phospho-STAT5 (Y694), and STAT5 levels were detected in whole-cell lysates by immunoblotting. (C) MOLM-14 cells were treated for 24 hours with 5 nM AC220 (left), and MOLM-14 and MV4-11 cells were transduced with inducible shFLT3 and treated with Dox (200 ng/ml) for 48 hours (right). Pim-1 and Pim-2 mRNA levels were quantified by real-time polymerase chain reaction. Gene expression was normalized to the HPRT (hypoxanthine-guanine phosphoribosyltransferase) levels (n = 3). (D) STAT5A/B gain (right) or loss (left) of function in MOLM-14 cells transduced with lentivirus. STAT5A, STAT5B, Pim-1, Pim-2, and Bcl-xL protein levels were evaluated by immunoblotting. (E) MOLM-14 cells were separately transduced with a PIM1- or PIM2-expressing vector or an empty vector. Ectopic expression of Pim-1 and Pim-2 was verified by immunoblotting (right). Cells were treated with vehicle or 1 nM AC220 for 48 hours, and cell viability was assessed by an UptiBlue assay (left) (n = 3). (F) MOLM-14 cells expressing an FLT3 shRNA in a Dox-inducible manner were transduced with a Pim2 (murine Pim-2) allele or a control vector. After 48 hours of culture in the presence of Dox (200 ng/ml), FLT3 and murine Pim-2 expression was assessed by immunoblotting, and apoptosis was measured by annexin V staining. (G and H) MOLM-14 cells were transduced via lentivirus with a control vector (Dox-inducible anti–Pim-2 shRNA vector) or with Pim2 lentivirus and xenografted into nude mice. (E) Tumor growth was assessed in mice treated with AC220 (1 mg/kg) (initiated once the tumor size reached 100 mm3) with (black circle) or without (white circle) Pim2 transduction. Growth of untreated xenotransplanted MOLM-14 cells is also provided (white square box). The means of the individual tumor sizes are plotted (n = 8). (F) Kaplan-Meier survival curve analysis of MOLM-14 cells transduced (dashed line) or not (solid line) with Pim2, transplanted into nude mice, and treated with AC220 (n = 8). Results are expressed as means ± SEM. β-Actin was used as a loading control for all Western blots. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Mentions: We used a doxycycline (Dox)–inducible short hairpin RNA (shRNA) to achieve targeted FLT3 knockdown in AML cell lines. FLT3 protein expression was efficiently suppressed in all cell lines tested (HL-60, OCI-AML3, MV4-11, and MOLM-14) but correlated with reduced Pim-1 and Pim-2 expression only in FLT3-ITD+ cell lines (MV4-11 and MOLM-14, Fig. 2A). In MOLM-14 and MV4-11 cells, FLT3 knockdown increased annexin V binding, in contrast to the results observed in two FLT3 wild-type AML cell lines, OCI-AML3 and HL-60 (fig. S2A), suggesting an addiction to FLT3-ITD signaling in these cell lines.


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 kinases are FLT3-ITD targets involved in resistance to FLT3 inhibition in AML.(A) AML cell lines (HL-60, OCI-AML3, MV4-11, and MOLM-14) were transduced via lentivirus with Dox-inducible anti-FLT3 shRNA vectors. shRNA induction was achieved with Dox (200 ng/ml). Western blots were performed using FLT3, Pim-1, and Pim-2 antibodies. WT, wild type. (B) MOLM-14 and OCI-AML3 cells were cultured with FLT3-L and/or 5 nM AC220. Tyrosine phosphorylation was evaluated in FLT3 immunoprecipitates. Pim-1, Pim-2, phospho-STAT5 (Y694), and STAT5 levels were detected in whole-cell lysates by immunoblotting. (C) MOLM-14 cells were treated for 24 hours with 5 nM AC220 (left), and MOLM-14 and MV4-11 cells were transduced with inducible shFLT3 and treated with Dox (200 ng/ml) for 48 hours (right). Pim-1 and Pim-2 mRNA levels were quantified by real-time polymerase chain reaction. Gene expression was normalized to the HPRT (hypoxanthine-guanine phosphoribosyltransferase) levels (n = 3). (D) STAT5A/B gain (right) or loss (left) of function in MOLM-14 cells transduced with lentivirus. STAT5A, STAT5B, Pim-1, Pim-2, and Bcl-xL protein levels were evaluated by immunoblotting. (E) MOLM-14 cells were separately transduced with a PIM1- or PIM2-expressing vector or an empty vector. Ectopic expression of Pim-1 and Pim-2 was verified by immunoblotting (right). Cells were treated with vehicle or 1 nM AC220 for 48 hours, and cell viability was assessed by an UptiBlue assay (left) (n = 3). (F) MOLM-14 cells expressing an FLT3 shRNA in a Dox-inducible manner were transduced with a Pim2 (murine Pim-2) allele or a control vector. After 48 hours of culture in the presence of Dox (200 ng/ml), FLT3 and murine Pim-2 expression was assessed by immunoblotting, and apoptosis was measured by annexin V staining. (G and H) MOLM-14 cells were transduced via lentivirus with a control vector (Dox-inducible anti–Pim-2 shRNA vector) or with Pim2 lentivirus and xenografted into nude mice. (E) Tumor growth was assessed in mice treated with AC220 (1 mg/kg) (initiated once the tumor size reached 100 mm3) with (black circle) or without (white circle) Pim2 transduction. Growth of untreated xenotransplanted MOLM-14 cells is also provided (white square box). The means of the individual tumor sizes are plotted (n = 8). (F) Kaplan-Meier survival curve analysis of MOLM-14 cells transduced (dashed line) or not (solid line) with Pim2, transplanted into nude mice, and treated with AC220 (n = 8). Results are expressed as means ± SEM. β-Actin was used as a loading control for all Western blots. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
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Figure 2: Pim kinases are FLT3-ITD targets involved in resistance to FLT3 inhibition in AML.(A) AML cell lines (HL-60, OCI-AML3, MV4-11, and MOLM-14) were transduced via lentivirus with Dox-inducible anti-FLT3 shRNA vectors. shRNA induction was achieved with Dox (200 ng/ml). Western blots were performed using FLT3, Pim-1, and Pim-2 antibodies. WT, wild type. (B) MOLM-14 and OCI-AML3 cells were cultured with FLT3-L and/or 5 nM AC220. Tyrosine phosphorylation was evaluated in FLT3 immunoprecipitates. Pim-1, Pim-2, phospho-STAT5 (Y694), and STAT5 levels were detected in whole-cell lysates by immunoblotting. (C) MOLM-14 cells were treated for 24 hours with 5 nM AC220 (left), and MOLM-14 and MV4-11 cells were transduced with inducible shFLT3 and treated with Dox (200 ng/ml) for 48 hours (right). Pim-1 and Pim-2 mRNA levels were quantified by real-time polymerase chain reaction. Gene expression was normalized to the HPRT (hypoxanthine-guanine phosphoribosyltransferase) levels (n = 3). (D) STAT5A/B gain (right) or loss (left) of function in MOLM-14 cells transduced with lentivirus. STAT5A, STAT5B, Pim-1, Pim-2, and Bcl-xL protein levels were evaluated by immunoblotting. (E) MOLM-14 cells were separately transduced with a PIM1- or PIM2-expressing vector or an empty vector. Ectopic expression of Pim-1 and Pim-2 was verified by immunoblotting (right). Cells were treated with vehicle or 1 nM AC220 for 48 hours, and cell viability was assessed by an UptiBlue assay (left) (n = 3). (F) MOLM-14 cells expressing an FLT3 shRNA in a Dox-inducible manner were transduced with a Pim2 (murine Pim-2) allele or a control vector. After 48 hours of culture in the presence of Dox (200 ng/ml), FLT3 and murine Pim-2 expression was assessed by immunoblotting, and apoptosis was measured by annexin V staining. (G and H) MOLM-14 cells were transduced via lentivirus with a control vector (Dox-inducible anti–Pim-2 shRNA vector) or with Pim2 lentivirus and xenografted into nude mice. (E) Tumor growth was assessed in mice treated with AC220 (1 mg/kg) (initiated once the tumor size reached 100 mm3) with (black circle) or without (white circle) Pim2 transduction. Growth of untreated xenotransplanted MOLM-14 cells is also provided (white square box). The means of the individual tumor sizes are plotted (n = 8). (F) Kaplan-Meier survival curve analysis of MOLM-14 cells transduced (dashed line) or not (solid line) with Pim2, transplanted into nude mice, and treated with AC220 (n = 8). Results are expressed as means ± SEM. β-Actin was used as a loading control for all Western blots. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Mentions: We used a doxycycline (Dox)–inducible short hairpin RNA (shRNA) to achieve targeted FLT3 knockdown in AML cell lines. FLT3 protein expression was efficiently suppressed in all cell lines tested (HL-60, OCI-AML3, MV4-11, and MOLM-14) but correlated with reduced Pim-1 and Pim-2 expression only in FLT3-ITD+ cell lines (MV4-11 and MOLM-14, Fig. 2A). In MOLM-14 and MV4-11 cells, FLT3 knockdown increased annexin V binding, in contrast to the results observed in two FLT3 wild-type AML cell lines, OCI-AML3 and HL-60 (fig. S2A), suggesting an addiction to FLT3-ITD signaling in these cell lines.

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