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Ponatinib efficiently kills imatinib-resistant chronic eosinophilic leukemia cells harboring gatekeeper mutant T674I FIP1L1-PDGFRα: roles of Mcl-1 and β-catenin.

Jin Y, Ding K, Li H, Xue M, Shi X, Wang C, Pan J - Mol. Cancer (2014)

Bottom Line: Therefore, novel TKIs effective against T674I FIP1L1-PDGFRα are needed.The purpose of this study was to examine the effect of ponatinib on T674I FIP1L1-PDGFRα.It induced apoptosis in CEL cells with caspase-3-dependent cleavage of Mcl-1, and inhibited tyrosine phosphorylation of β-catenin to decrease its stability and pro-survival functions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathophysiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China. panjx2@mail.sysu.edu.cn.

ABSTRACT

Background: T674I FIP1L1-PDGFRα in a subset of chronic eosinophilic leukemia (CEL) is a gatekeeper mutation that is resistant to many tyrosine kinase inhibitors (TKIs) (e.g., imatinib, nilotinib and dasatinib), similar to T315I Bcr-Abl. Therefore, novel TKIs effective against T674I FIP1L1-PDGFRα are needed. Ponatinib (AP24534) is a novel orally bioavailable TKI against T315I Bcr-Abl, but it is not clear whether ponatinib is effective against T674I FIP1L1-PDGFRα. The purpose of this study was to examine the effect of ponatinib on T674I FIP1L1-PDGFRα.

Methods: Molecular docking analysis in silico was performed. The effects of ponatinib on PDGFRα signaling pathways, apoptosis and cell cycling were examined in EOL-1, BaF3 cells expressing either wild type (WT) or T674I FIP1L1-PDGFRα. The in vivo antitumor activity of ponatinib was evaluated with xenografted BaF3-T674I FIP1L1-PDGFRα cells in nude mice models.

Results: Molecular docking analysis revealed that ponatinib could bind to the DFG (Asp-Phe-Gly)-out state of T674I PDGFRα. Ponatinib potently inhibited the phosphorylation of WT and T674I FIP1L1-PDGFRα and their downstream signaling molecules (e.g., Stat3, Stat5). Ponatinib strikingly inhibited the growth of both WT and T674I FIP1L1-PDGFRα-carrying CEL cells (IC50: 0.004-2.5 nM). It induced apoptosis in CEL cells with caspase-3-dependent cleavage of Mcl-1, and inhibited tyrosine phosphorylation of β-catenin to decrease its stability and pro-survival functions. In vivo, ponatinib abrogated the growth of xenografted BaF3-T674I FIP1L1-PDGFRα cells in nude mice.

Conclusions: Ponatinib is a pan-FIP1L1-PDGFRα inhibitor, and clinical trials are warranted to investigate its efficacy in imatinib-resistant CEL.

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Related in: MedlinePlus

Inhibition of tyrosine kinase activity of PDGFRα by ponatinib attenuates signaling of β-catenin by lowering its stability. (A) Ponatinib concentration-dependently lowered β-catenin. EOL-1 cells were incubated with ponatinib for 24 h, and cytoplasmic and nuclear extracts were determined by immunoblotting. (B) Analysis of β-catenin localization. EOL-1 cells were pretreated with 1 nM ponatinib for 24 h, immunofluorescence analysis was performed with anti-β-catenin. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). (C) EOL-1 cells were pretreated with the indicated concentrations of ponatinib for 24 h or 1 nM ponatinib for various durations; and the nuclear extracts were then assayed for TCF/LEF activation by EMSA. (D) Ponatinib increased β-catenin turnover rate. After pretreatment with or without 1 nM ponatinib for 16 h, EOL-1 cells were exposed to 5 μg/ml of CHX, followed by immunoblotting for β-catenin. (E) Inhibition of PDGFRα decreased β-catenin. EOL-1 cells were treated with 1 nM ponatinib for various times, then total and tyrosine-phosphorylated β-catenin were evaluated (Left) by immunoblotting. EOL-1 cells were transfected with mock siRNA or PDGFRα siRNA, and β-catenin was monitored by immunoblotting. (F) Ponatinib abrogated TCF/LEF-dependent luciferase activity. EOL-1 cells were transfected with TOPflash and FOPflash plasmids and pEFRenilla-luc. After 24 h, the cells were treated with ponatinib for another 24 h, then underwent luciferase activity assay. (G) Ponatinib decreased the expression of target genes of β-catenin. Immunoblotting analysis in EOL-1 cells that were exposed to ponatinib for 24 h. (H) Ectopically changing the levels of β-catenin affected the ponatinib-mediated apoptosis. Twenty-four hours after transfection with control or Mcl-1 siRNA, or empty vector, pcDNA3-β-catenin, EOL-1 cells were treated with ponatinib, and the relevant protein levels were evaluated by immunoblotting (left); parallel samples were examined by the trypan blue dye exclusion assay (right, *** P<0.0001, t test; error bars represent 95% confidence intervals).
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Figure 5: Inhibition of tyrosine kinase activity of PDGFRα by ponatinib attenuates signaling of β-catenin by lowering its stability. (A) Ponatinib concentration-dependently lowered β-catenin. EOL-1 cells were incubated with ponatinib for 24 h, and cytoplasmic and nuclear extracts were determined by immunoblotting. (B) Analysis of β-catenin localization. EOL-1 cells were pretreated with 1 nM ponatinib for 24 h, immunofluorescence analysis was performed with anti-β-catenin. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). (C) EOL-1 cells were pretreated with the indicated concentrations of ponatinib for 24 h or 1 nM ponatinib for various durations; and the nuclear extracts were then assayed for TCF/LEF activation by EMSA. (D) Ponatinib increased β-catenin turnover rate. After pretreatment with or without 1 nM ponatinib for 16 h, EOL-1 cells were exposed to 5 μg/ml of CHX, followed by immunoblotting for β-catenin. (E) Inhibition of PDGFRα decreased β-catenin. EOL-1 cells were treated with 1 nM ponatinib for various times, then total and tyrosine-phosphorylated β-catenin were evaluated (Left) by immunoblotting. EOL-1 cells were transfected with mock siRNA or PDGFRα siRNA, and β-catenin was monitored by immunoblotting. (F) Ponatinib abrogated TCF/LEF-dependent luciferase activity. EOL-1 cells were transfected with TOPflash and FOPflash plasmids and pEFRenilla-luc. After 24 h, the cells were treated with ponatinib for another 24 h, then underwent luciferase activity assay. (G) Ponatinib decreased the expression of target genes of β-catenin. Immunoblotting analysis in EOL-1 cells that were exposed to ponatinib for 24 h. (H) Ectopically changing the levels of β-catenin affected the ponatinib-mediated apoptosis. Twenty-four hours after transfection with control or Mcl-1 siRNA, or empty vector, pcDNA3-β-catenin, EOL-1 cells were treated with ponatinib, and the relevant protein levels were evaluated by immunoblotting (left); parallel samples were examined by the trypan blue dye exclusion assay (right, *** P<0.0001, t test; error bars represent 95% confidence intervals).

Mentions: β-catenin, a critical effector in the canonical Wnt/β-catenin signaling cascade widely involved in cell proliferation, differentiation, escape of apoptosis and transformation [25], is a substrate of tyrosine kinases such as PDGFRα, Bcr-Abl, Flt3, and KIT [26-28]. Tyrosine phosphorylation of β-catenin leads to increased protein stability, keeping β-catenin active [26-29]. We therefore examined the potential change in tyrosine phosphorylation of β-catenin as a result of inhibition of PDGFRα by ponatinib. Because nuclear translocation of β-catenin is required for its functions (i.e., to activate TCF/LEF), we first examined whether ponatinib affected the subcellular distribution of β-catenin. With standard subcellular fractionation protocols, the levels of β-catenin in cytosolic and nuclear fractions were dose-dependently lowered by ponatinib (Figure 5A). Immunofluorescence analysis further confirmed that β-catenin was decreased by ponatinib in both cytosolic and nuclear compartments (Figure 5B). Electrophoretic mobility shift assay (EMSA) also revealed a concentration- and time-dependent decrease in nuclear β-catenin with ponatinib treatment (Figure 5C). Time chase experiments in the presence of CHX revealed that ponatinib led to an increased degradation rate of β-catenin (Figure 5D). Time-course study showed that the decrease in levels of β-catenin occurred concurrently with tyrosine dephosphorylation in β-catenin after inactivation of PDGFRα by ponatinib (Figure 5E). EOL-1 cells transfected with siRNA against PDGFRα displayed decreased levels in PDGFRα and β-catenin (Figure 5E, right), further supporting the specific effect of PDGFRα on β-catenin stability. These data support that tyrosine phosphorylation in β-catenin by PDGFRα directly promotes β-catenin stability.


Ponatinib efficiently kills imatinib-resistant chronic eosinophilic leukemia cells harboring gatekeeper mutant T674I FIP1L1-PDGFRα: roles of Mcl-1 and β-catenin.

Jin Y, Ding K, Li H, Xue M, Shi X, Wang C, Pan J - Mol. Cancer (2014)

Inhibition of tyrosine kinase activity of PDGFRα by ponatinib attenuates signaling of β-catenin by lowering its stability. (A) Ponatinib concentration-dependently lowered β-catenin. EOL-1 cells were incubated with ponatinib for 24 h, and cytoplasmic and nuclear extracts were determined by immunoblotting. (B) Analysis of β-catenin localization. EOL-1 cells were pretreated with 1 nM ponatinib for 24 h, immunofluorescence analysis was performed with anti-β-catenin. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). (C) EOL-1 cells were pretreated with the indicated concentrations of ponatinib for 24 h or 1 nM ponatinib for various durations; and the nuclear extracts were then assayed for TCF/LEF activation by EMSA. (D) Ponatinib increased β-catenin turnover rate. After pretreatment with or without 1 nM ponatinib for 16 h, EOL-1 cells were exposed to 5 μg/ml of CHX, followed by immunoblotting for β-catenin. (E) Inhibition of PDGFRα decreased β-catenin. EOL-1 cells were treated with 1 nM ponatinib for various times, then total and tyrosine-phosphorylated β-catenin were evaluated (Left) by immunoblotting. EOL-1 cells were transfected with mock siRNA or PDGFRα siRNA, and β-catenin was monitored by immunoblotting. (F) Ponatinib abrogated TCF/LEF-dependent luciferase activity. EOL-1 cells were transfected with TOPflash and FOPflash plasmids and pEFRenilla-luc. After 24 h, the cells were treated with ponatinib for another 24 h, then underwent luciferase activity assay. (G) Ponatinib decreased the expression of target genes of β-catenin. Immunoblotting analysis in EOL-1 cells that were exposed to ponatinib for 24 h. (H) Ectopically changing the levels of β-catenin affected the ponatinib-mediated apoptosis. Twenty-four hours after transfection with control or Mcl-1 siRNA, or empty vector, pcDNA3-β-catenin, EOL-1 cells were treated with ponatinib, and the relevant protein levels were evaluated by immunoblotting (left); parallel samples were examined by the trypan blue dye exclusion assay (right, *** P<0.0001, t test; error bars represent 95% confidence intervals).
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Figure 5: Inhibition of tyrosine kinase activity of PDGFRα by ponatinib attenuates signaling of β-catenin by lowering its stability. (A) Ponatinib concentration-dependently lowered β-catenin. EOL-1 cells were incubated with ponatinib for 24 h, and cytoplasmic and nuclear extracts were determined by immunoblotting. (B) Analysis of β-catenin localization. EOL-1 cells were pretreated with 1 nM ponatinib for 24 h, immunofluorescence analysis was performed with anti-β-catenin. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). (C) EOL-1 cells were pretreated with the indicated concentrations of ponatinib for 24 h or 1 nM ponatinib for various durations; and the nuclear extracts were then assayed for TCF/LEF activation by EMSA. (D) Ponatinib increased β-catenin turnover rate. After pretreatment with or without 1 nM ponatinib for 16 h, EOL-1 cells were exposed to 5 μg/ml of CHX, followed by immunoblotting for β-catenin. (E) Inhibition of PDGFRα decreased β-catenin. EOL-1 cells were treated with 1 nM ponatinib for various times, then total and tyrosine-phosphorylated β-catenin were evaluated (Left) by immunoblotting. EOL-1 cells were transfected with mock siRNA or PDGFRα siRNA, and β-catenin was monitored by immunoblotting. (F) Ponatinib abrogated TCF/LEF-dependent luciferase activity. EOL-1 cells were transfected with TOPflash and FOPflash plasmids and pEFRenilla-luc. After 24 h, the cells were treated with ponatinib for another 24 h, then underwent luciferase activity assay. (G) Ponatinib decreased the expression of target genes of β-catenin. Immunoblotting analysis in EOL-1 cells that were exposed to ponatinib for 24 h. (H) Ectopically changing the levels of β-catenin affected the ponatinib-mediated apoptosis. Twenty-four hours after transfection with control or Mcl-1 siRNA, or empty vector, pcDNA3-β-catenin, EOL-1 cells were treated with ponatinib, and the relevant protein levels were evaluated by immunoblotting (left); parallel samples were examined by the trypan blue dye exclusion assay (right, *** P<0.0001, t test; error bars represent 95% confidence intervals).
Mentions: β-catenin, a critical effector in the canonical Wnt/β-catenin signaling cascade widely involved in cell proliferation, differentiation, escape of apoptosis and transformation [25], is a substrate of tyrosine kinases such as PDGFRα, Bcr-Abl, Flt3, and KIT [26-28]. Tyrosine phosphorylation of β-catenin leads to increased protein stability, keeping β-catenin active [26-29]. We therefore examined the potential change in tyrosine phosphorylation of β-catenin as a result of inhibition of PDGFRα by ponatinib. Because nuclear translocation of β-catenin is required for its functions (i.e., to activate TCF/LEF), we first examined whether ponatinib affected the subcellular distribution of β-catenin. With standard subcellular fractionation protocols, the levels of β-catenin in cytosolic and nuclear fractions were dose-dependently lowered by ponatinib (Figure 5A). Immunofluorescence analysis further confirmed that β-catenin was decreased by ponatinib in both cytosolic and nuclear compartments (Figure 5B). Electrophoretic mobility shift assay (EMSA) also revealed a concentration- and time-dependent decrease in nuclear β-catenin with ponatinib treatment (Figure 5C). Time chase experiments in the presence of CHX revealed that ponatinib led to an increased degradation rate of β-catenin (Figure 5D). Time-course study showed that the decrease in levels of β-catenin occurred concurrently with tyrosine dephosphorylation in β-catenin after inactivation of PDGFRα by ponatinib (Figure 5E). EOL-1 cells transfected with siRNA against PDGFRα displayed decreased levels in PDGFRα and β-catenin (Figure 5E, right), further supporting the specific effect of PDGFRα on β-catenin stability. These data support that tyrosine phosphorylation in β-catenin by PDGFRα directly promotes β-catenin stability.

Bottom Line: Therefore, novel TKIs effective against T674I FIP1L1-PDGFRα are needed.The purpose of this study was to examine the effect of ponatinib on T674I FIP1L1-PDGFRα.It induced apoptosis in CEL cells with caspase-3-dependent cleavage of Mcl-1, and inhibited tyrosine phosphorylation of β-catenin to decrease its stability and pro-survival functions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathophysiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China. panjx2@mail.sysu.edu.cn.

ABSTRACT

Background: T674I FIP1L1-PDGFRα in a subset of chronic eosinophilic leukemia (CEL) is a gatekeeper mutation that is resistant to many tyrosine kinase inhibitors (TKIs) (e.g., imatinib, nilotinib and dasatinib), similar to T315I Bcr-Abl. Therefore, novel TKIs effective against T674I FIP1L1-PDGFRα are needed. Ponatinib (AP24534) is a novel orally bioavailable TKI against T315I Bcr-Abl, but it is not clear whether ponatinib is effective against T674I FIP1L1-PDGFRα. The purpose of this study was to examine the effect of ponatinib on T674I FIP1L1-PDGFRα.

Methods: Molecular docking analysis in silico was performed. The effects of ponatinib on PDGFRα signaling pathways, apoptosis and cell cycling were examined in EOL-1, BaF3 cells expressing either wild type (WT) or T674I FIP1L1-PDGFRα. The in vivo antitumor activity of ponatinib was evaluated with xenografted BaF3-T674I FIP1L1-PDGFRα cells in nude mice models.

Results: Molecular docking analysis revealed that ponatinib could bind to the DFG (Asp-Phe-Gly)-out state of T674I PDGFRα. Ponatinib potently inhibited the phosphorylation of WT and T674I FIP1L1-PDGFRα and their downstream signaling molecules (e.g., Stat3, Stat5). Ponatinib strikingly inhibited the growth of both WT and T674I FIP1L1-PDGFRα-carrying CEL cells (IC50: 0.004-2.5 nM). It induced apoptosis in CEL cells with caspase-3-dependent cleavage of Mcl-1, and inhibited tyrosine phosphorylation of β-catenin to decrease its stability and pro-survival functions. In vivo, ponatinib abrogated the growth of xenografted BaF3-T674I FIP1L1-PDGFRα cells in nude mice.

Conclusions: Ponatinib is a pan-FIP1L1-PDGFRα inhibitor, and clinical trials are warranted to investigate its efficacy in imatinib-resistant CEL.

Show MeSH
Related in: MedlinePlus