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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, with a predominant implication of Pim-2, regulate FLT3i-naïve and FLT3i-resistant AML cell survival.(A) AML cell lines (MOLM-14, MV4-11, HL-60, OCI-AML3, and THP-1) were transduced via lentivirus with a vector promoting the expression of an anti–Pim-2 shRNA after induction with Dox (200 ng/ml). Apoptosis was quantified by annexin V staining after 4 days of shRNA induction (n = 3). The extent of Pim-2 knockdown in each cell line was determined by Western blot (bottom). (B) Western blot for Pim-2 expression (top) and annexin V staining in normal CD34+ hematopoietic progenitor cells lentivirally transduced with scrambled (−) or Pim-2 (+) shRNA (n = 3). (C) Pim-2 shRNA–transduced MOLM-14 cells were cotransfected with Pim2 or Pim2KD as indicated. Cell viability after Dox treatment was assessed by staining with the UptiBlue fluorescent reagent (n = 6). (D to G) Tumor growth and survival (Kaplan-Meier curve) of MOLM-14 cells transfected with scrambled or Pim-2 shRNA and xenografted into nude mice. Animals were treated with vehicle [phosphate-buffered saline (PBS), black line, n = 8] or Dox (200 μg/ml) (Pim-2 shRNA in red, scrambled shRNA in blue; n = 8 for each). (H) Tumor sections stained by HES and TUNEL or labeled with Pim-1, Pim-2, and phospho-4E-BP1 (S65) antibodies. Representative images from three separate experiments are shown. (I) MOLM-14 cells were harvested from AC220-treated xenografted mice as depicted in Fig. 2, E and F. Pim-2 knockdown was induced with Dox (200 ng/ml) for 24 hours, and 2 nM AC220 was then added to the culture for an additional 24 hours. Apoptosis was quantified by annexin V staining. 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 3: Pim kinases, with a predominant implication of Pim-2, regulate FLT3i-naïve and FLT3i-resistant AML cell survival.(A) AML cell lines (MOLM-14, MV4-11, HL-60, OCI-AML3, and THP-1) were transduced via lentivirus with a vector promoting the expression of an anti–Pim-2 shRNA after induction with Dox (200 ng/ml). Apoptosis was quantified by annexin V staining after 4 days of shRNA induction (n = 3). The extent of Pim-2 knockdown in each cell line was determined by Western blot (bottom). (B) Western blot for Pim-2 expression (top) and annexin V staining in normal CD34+ hematopoietic progenitor cells lentivirally transduced with scrambled (−) or Pim-2 (+) shRNA (n = 3). (C) Pim-2 shRNA–transduced MOLM-14 cells were cotransfected with Pim2 or Pim2KD as indicated. Cell viability after Dox treatment was assessed by staining with the UptiBlue fluorescent reagent (n = 6). (D to G) Tumor growth and survival (Kaplan-Meier curve) of MOLM-14 cells transfected with scrambled or Pim-2 shRNA and xenografted into nude mice. Animals were treated with vehicle [phosphate-buffered saline (PBS), black line, n = 8] or Dox (200 μg/ml) (Pim-2 shRNA in red, scrambled shRNA in blue; n = 8 for each). (H) Tumor sections stained by HES and TUNEL or labeled with Pim-1, Pim-2, and phospho-4E-BP1 (S65) antibodies. Representative images from three separate experiments are shown. (I) MOLM-14 cells were harvested from AC220-treated xenografted mice as depicted in Fig. 2, E and F. Pim-2 knockdown was induced with Dox (200 ng/ml) for 24 hours, and 2 nM AC220 was then added to the culture for an additional 24 hours. Apoptosis was quantified by annexin V staining. 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: Pim-2 knockdown promoted apoptosis in two FLT3-ITD+ AML cell lines (MOLM-14 and MV4-11; Fig. 3A). By contrast, it had no effect on annexin V binding in FLT3 wild-type AML cell lines (THP-1, OCI-AML3, and HL-60) or normal CD34+ hematopoietic progenitor cells (Fig. 3, A and B). In contrast to other models (10) and to our observations in normal CD34+ hematopoietic progenitor cells, no compensatory increase of other Pim kinase family members was seen after Pim-1 or Pim-2 knockdown in AML cells (fig. S3A). In MOLM-14 cells, Pim-1 knockdown did not induce annexin V binding (fig. S3, B and C) and reduced cell proliferation and viability, but to a lesser extent than did Pim-2 depletion (fig. S3D), suggesting the absence of functional compensation between these kinases in AML. Decreased cell viability induced by Pim-2 knockdown was rescued by ectopic murine Pim-2 expression but not by a mutant Pim2 allele devoid of kinase activity [kinase-dead Pim2 mutant (Pim2KD)] in MOLM-14 cells (Fig. 3C). These results suggest that Pim kinases, particularly Pim-2, are essential to the survival of FLT3-ITD+ AML cells.


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, with a predominant implication of Pim-2, regulate FLT3i-naïve and FLT3i-resistant AML cell survival.(A) AML cell lines (MOLM-14, MV4-11, HL-60, OCI-AML3, and THP-1) were transduced via lentivirus with a vector promoting the expression of an anti–Pim-2 shRNA after induction with Dox (200 ng/ml). Apoptosis was quantified by annexin V staining after 4 days of shRNA induction (n = 3). The extent of Pim-2 knockdown in each cell line was determined by Western blot (bottom). (B) Western blot for Pim-2 expression (top) and annexin V staining in normal CD34+ hematopoietic progenitor cells lentivirally transduced with scrambled (−) or Pim-2 (+) shRNA (n = 3). (C) Pim-2 shRNA–transduced MOLM-14 cells were cotransfected with Pim2 or Pim2KD as indicated. Cell viability after Dox treatment was assessed by staining with the UptiBlue fluorescent reagent (n = 6). (D to G) Tumor growth and survival (Kaplan-Meier curve) of MOLM-14 cells transfected with scrambled or Pim-2 shRNA and xenografted into nude mice. Animals were treated with vehicle [phosphate-buffered saline (PBS), black line, n = 8] or Dox (200 μg/ml) (Pim-2 shRNA in red, scrambled shRNA in blue; n = 8 for each). (H) Tumor sections stained by HES and TUNEL or labeled with Pim-1, Pim-2, and phospho-4E-BP1 (S65) antibodies. Representative images from three separate experiments are shown. (I) MOLM-14 cells were harvested from AC220-treated xenografted mice as depicted in Fig. 2, E and F. Pim-2 knockdown was induced with Dox (200 ng/ml) for 24 hours, and 2 nM AC220 was then added to the culture for an additional 24 hours. Apoptosis was quantified by annexin V staining. 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 3: Pim kinases, with a predominant implication of Pim-2, regulate FLT3i-naïve and FLT3i-resistant AML cell survival.(A) AML cell lines (MOLM-14, MV4-11, HL-60, OCI-AML3, and THP-1) were transduced via lentivirus with a vector promoting the expression of an anti–Pim-2 shRNA after induction with Dox (200 ng/ml). Apoptosis was quantified by annexin V staining after 4 days of shRNA induction (n = 3). The extent of Pim-2 knockdown in each cell line was determined by Western blot (bottom). (B) Western blot for Pim-2 expression (top) and annexin V staining in normal CD34+ hematopoietic progenitor cells lentivirally transduced with scrambled (−) or Pim-2 (+) shRNA (n = 3). (C) Pim-2 shRNA–transduced MOLM-14 cells were cotransfected with Pim2 or Pim2KD as indicated. Cell viability after Dox treatment was assessed by staining with the UptiBlue fluorescent reagent (n = 6). (D to G) Tumor growth and survival (Kaplan-Meier curve) of MOLM-14 cells transfected with scrambled or Pim-2 shRNA and xenografted into nude mice. Animals were treated with vehicle [phosphate-buffered saline (PBS), black line, n = 8] or Dox (200 μg/ml) (Pim-2 shRNA in red, scrambled shRNA in blue; n = 8 for each). (H) Tumor sections stained by HES and TUNEL or labeled with Pim-1, Pim-2, and phospho-4E-BP1 (S65) antibodies. Representative images from three separate experiments are shown. (I) MOLM-14 cells were harvested from AC220-treated xenografted mice as depicted in Fig. 2, E and F. Pim-2 knockdown was induced with Dox (200 ng/ml) for 24 hours, and 2 nM AC220 was then added to the culture for an additional 24 hours. Apoptosis was quantified by annexin V staining. 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: Pim-2 knockdown promoted apoptosis in two FLT3-ITD+ AML cell lines (MOLM-14 and MV4-11; Fig. 3A). By contrast, it had no effect on annexin V binding in FLT3 wild-type AML cell lines (THP-1, OCI-AML3, and HL-60) or normal CD34+ hematopoietic progenitor cells (Fig. 3, A and B). In contrast to other models (10) and to our observations in normal CD34+ hematopoietic progenitor cells, no compensatory increase of other Pim kinase family members was seen after Pim-1 or Pim-2 knockdown in AML cells (fig. S3A). In MOLM-14 cells, Pim-1 knockdown did not induce annexin V binding (fig. S3, B and C) and reduced cell proliferation and viability, but to a lesser extent than did Pim-2 depletion (fig. S3D), suggesting the absence of functional compensation between these kinases in AML. Decreased cell viability induced by Pim-2 knockdown was rescued by ectopic murine Pim-2 expression but not by a mutant Pim2 allele devoid of kinase activity [kinase-dead Pim2 mutant (Pim2KD)] in MOLM-14 cells (Fig. 3C). These results suggest that Pim kinases, particularly Pim-2, are essential to the survival of FLT3-ITD+ AML cells.

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