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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

Ponatinib mediates caspase-3-dependent cleavage of Mcl-1. (A) Ponatinib precipitated in Mcl-1 turnover. After pretreatment with or without 1 nM ponatinib, EOL-1 cells were exposed to 5 μg/ml of cycloheximide (CHX), followed by Mcl-1 detection with immunoblotting. (B) MG-132 did not abrogate ponatinib-induced cleavage of Mcl-1. EOL-1 cells were treated with 1 nM ponatinib in the presence or absence of 0.5 μM MG-132 for 24 h. Mcl-1 level was then monitored with immunoblotting. (C) Mcl-1 cleavage occurred with onset of apoptosis after treatment with ponatinib. EOL-1 cells were treated with 1 nM ponatinib for different times, and the indicated proteins were measured with immunoblotting. (D) Mcl-1 cleaved in a caspase-3-dependent manner. EOL-1 cells were treated with 1 nM ponatinib for 24 h with or without 10 μM z-DEVD-fmk, then underwent immunoblotting. (E) Silencing Mcl-1 potentiated ponatinib-induced apoptosis in EOL-1 cells. Twenty-four hours after transfection with Mcl-1 siRNA or control (mock) siRNA, EOL-1 cells were treated with various concentrations of ponatinib, and levels of Mcl-1, PARP, and actin were evaluated by immunoblotting (top); parallel samples were examined for apoptosis by trypan blue staining (bottom, *** P < 0.0001, t test, error bars represent 95% confidence intervals; representative data from 3 independent experiments are shown). (F) Enforced overexpression of Mcl-1 abrogated the ponatinib-induced apoptosis. Twenty-four hours after transfection with pCMV5-flag empty vector or the plasmid expressing Mcl-1, EOL-1 cells were incubated with or without concentrations of ponatinib for another 24 h. Cell viability was evaluated by trypan blue dye exclusion (lower, *** P < 0.0001, t test, error bars represent 95% confidence intervals); Mcl-1 and PARP levels were detected by immunoblotting.
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Figure 4: Ponatinib mediates caspase-3-dependent cleavage of Mcl-1. (A) Ponatinib precipitated in Mcl-1 turnover. After pretreatment with or without 1 nM ponatinib, EOL-1 cells were exposed to 5 μg/ml of cycloheximide (CHX), followed by Mcl-1 detection with immunoblotting. (B) MG-132 did not abrogate ponatinib-induced cleavage of Mcl-1. EOL-1 cells were treated with 1 nM ponatinib in the presence or absence of 0.5 μM MG-132 for 24 h. Mcl-1 level was then monitored with immunoblotting. (C) Mcl-1 cleavage occurred with onset of apoptosis after treatment with ponatinib. EOL-1 cells were treated with 1 nM ponatinib for different times, and the indicated proteins were measured with immunoblotting. (D) Mcl-1 cleaved in a caspase-3-dependent manner. EOL-1 cells were treated with 1 nM ponatinib for 24 h with or without 10 μM z-DEVD-fmk, then underwent immunoblotting. (E) Silencing Mcl-1 potentiated ponatinib-induced apoptosis in EOL-1 cells. Twenty-four hours after transfection with Mcl-1 siRNA or control (mock) siRNA, EOL-1 cells were treated with various concentrations of ponatinib, and levels of Mcl-1, PARP, and actin were evaluated by immunoblotting (top); parallel samples were examined for apoptosis by trypan blue staining (bottom, *** P < 0.0001, t test, error bars represent 95% confidence intervals; representative data from 3 independent experiments are shown). (F) Enforced overexpression of Mcl-1 abrogated the ponatinib-induced apoptosis. Twenty-four hours after transfection with pCMV5-flag empty vector or the plasmid expressing Mcl-1, EOL-1 cells were incubated with or without concentrations of ponatinib for another 24 h. Cell viability was evaluated by trypan blue dye exclusion (lower, *** P < 0.0001, t test, error bars represent 95% confidence intervals); Mcl-1 and PARP levels were detected by immunoblotting.

Mentions: Because of the critical pro-survival role of Mcl-1 in leukemia [22,23], we explored its role in ponatinib-induced apoptosis of CEL cells. Mcl-1 mRNA levels did not significantly differ between ponatinib-treated CEL cells and controls (data not shown). Time chase experiments with inhibition of de novo protein synthesis by cycloheximide (CHX) revealed increased degradation of Mcl-1 level in ponatinib-treated CEL cells as compared with controls (Figure 4A). However, pretreatment with MG-132 (a specific proteasome inhibitor) did not prevent the ponatinib-induced degradation of Mcl-1 level (Figure 4B), which suggests that ponatinib decreased Mcl-1 level without involving proteasomes. Time-course study revealed that loss of Mcl-1 (p42) was accompanied by the appearance of a cleaved form Mcl-1 (p28) as apoptosis (specific cleavage of PARP) progressed (Figure 4C). These data are in agreement with a report of Mcl-1 being cleaved by caspase-3 at Asp127 to produce a 28-kDa fragment (Mcl-1128-350) [24]. To further confirm that ponatinib-induced Mcl-1 cleavage was caused by caspase-3 activation, CEL cells were treated with ponatinib in the absence or presence of the caspase-3 inhibitor z-DEVD-fmk. Immunoblotting revealed complete abrogation of decreased Mcl-1 level (p42) and appearance of the Mcl-1128-350 (p28) fragment (Figure 4D). Therefore, ponatinib-induced activation of caspase-3 may cleave and decrease the amount of Mcl-1.


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)

Ponatinib mediates caspase-3-dependent cleavage of Mcl-1. (A) Ponatinib precipitated in Mcl-1 turnover. After pretreatment with or without 1 nM ponatinib, EOL-1 cells were exposed to 5 μg/ml of cycloheximide (CHX), followed by Mcl-1 detection with immunoblotting. (B) MG-132 did not abrogate ponatinib-induced cleavage of Mcl-1. EOL-1 cells were treated with 1 nM ponatinib in the presence or absence of 0.5 μM MG-132 for 24 h. Mcl-1 level was then monitored with immunoblotting. (C) Mcl-1 cleavage occurred with onset of apoptosis after treatment with ponatinib. EOL-1 cells were treated with 1 nM ponatinib for different times, and the indicated proteins were measured with immunoblotting. (D) Mcl-1 cleaved in a caspase-3-dependent manner. EOL-1 cells were treated with 1 nM ponatinib for 24 h with or without 10 μM z-DEVD-fmk, then underwent immunoblotting. (E) Silencing Mcl-1 potentiated ponatinib-induced apoptosis in EOL-1 cells. Twenty-four hours after transfection with Mcl-1 siRNA or control (mock) siRNA, EOL-1 cells were treated with various concentrations of ponatinib, and levels of Mcl-1, PARP, and actin were evaluated by immunoblotting (top); parallel samples were examined for apoptosis by trypan blue staining (bottom, *** P < 0.0001, t test, error bars represent 95% confidence intervals; representative data from 3 independent experiments are shown). (F) Enforced overexpression of Mcl-1 abrogated the ponatinib-induced apoptosis. Twenty-four hours after transfection with pCMV5-flag empty vector or the plasmid expressing Mcl-1, EOL-1 cells were incubated with or without concentrations of ponatinib for another 24 h. Cell viability was evaluated by trypan blue dye exclusion (lower, *** P < 0.0001, t test, error bars represent 95% confidence intervals); Mcl-1 and PARP levels were detected by immunoblotting.
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Figure 4: Ponatinib mediates caspase-3-dependent cleavage of Mcl-1. (A) Ponatinib precipitated in Mcl-1 turnover. After pretreatment with or without 1 nM ponatinib, EOL-1 cells were exposed to 5 μg/ml of cycloheximide (CHX), followed by Mcl-1 detection with immunoblotting. (B) MG-132 did not abrogate ponatinib-induced cleavage of Mcl-1. EOL-1 cells were treated with 1 nM ponatinib in the presence or absence of 0.5 μM MG-132 for 24 h. Mcl-1 level was then monitored with immunoblotting. (C) Mcl-1 cleavage occurred with onset of apoptosis after treatment with ponatinib. EOL-1 cells were treated with 1 nM ponatinib for different times, and the indicated proteins were measured with immunoblotting. (D) Mcl-1 cleaved in a caspase-3-dependent manner. EOL-1 cells were treated with 1 nM ponatinib for 24 h with or without 10 μM z-DEVD-fmk, then underwent immunoblotting. (E) Silencing Mcl-1 potentiated ponatinib-induced apoptosis in EOL-1 cells. Twenty-four hours after transfection with Mcl-1 siRNA or control (mock) siRNA, EOL-1 cells were treated with various concentrations of ponatinib, and levels of Mcl-1, PARP, and actin were evaluated by immunoblotting (top); parallel samples were examined for apoptosis by trypan blue staining (bottom, *** P < 0.0001, t test, error bars represent 95% confidence intervals; representative data from 3 independent experiments are shown). (F) Enforced overexpression of Mcl-1 abrogated the ponatinib-induced apoptosis. Twenty-four hours after transfection with pCMV5-flag empty vector or the plasmid expressing Mcl-1, EOL-1 cells were incubated with or without concentrations of ponatinib for another 24 h. Cell viability was evaluated by trypan blue dye exclusion (lower, *** P < 0.0001, t test, error bars represent 95% confidence intervals); Mcl-1 and PARP levels were detected by immunoblotting.
Mentions: Because of the critical pro-survival role of Mcl-1 in leukemia [22,23], we explored its role in ponatinib-induced apoptosis of CEL cells. Mcl-1 mRNA levels did not significantly differ between ponatinib-treated CEL cells and controls (data not shown). Time chase experiments with inhibition of de novo protein synthesis by cycloheximide (CHX) revealed increased degradation of Mcl-1 level in ponatinib-treated CEL cells as compared with controls (Figure 4A). However, pretreatment with MG-132 (a specific proteasome inhibitor) did not prevent the ponatinib-induced degradation of Mcl-1 level (Figure 4B), which suggests that ponatinib decreased Mcl-1 level without involving proteasomes. Time-course study revealed that loss of Mcl-1 (p42) was accompanied by the appearance of a cleaved form Mcl-1 (p28) as apoptosis (specific cleavage of PARP) progressed (Figure 4C). These data are in agreement with a report of Mcl-1 being cleaved by caspase-3 at Asp127 to produce a 28-kDa fragment (Mcl-1128-350) [24]. To further confirm that ponatinib-induced Mcl-1 cleavage was caused by caspase-3 activation, CEL cells were treated with ponatinib in the absence or presence of the caspase-3 inhibitor z-DEVD-fmk. Immunoblotting revealed complete abrogation of decreased Mcl-1 level (p42) and appearance of the Mcl-1128-350 (p28) fragment (Figure 4D). Therefore, ponatinib-induced activation of caspase-3 may cleave and decrease the amount of Mcl-1.

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