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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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Ponatinib induces apoptosis in FIP1LI-PDGFRα-expressing cells. (A) EOL-1 and BaF3-WT or -T674I FIP1L1-PDGFRα cells were exposed to increasing concentrations of ponatinib for 24 h, apoptotic cells were assayed with flow cytometry by PI/Annexin V-FITC (EOL-1) or 7-AAD/Annexin V-PE (BaF3-WT or -T674I FIP1L1-PDGFRα cells) staining. Left, representative histograms; Right, statistical data of 3 independent experiments, the vertical axis stands for the sum of all dead cells. Error bars represent 95% confidence intervals. **, P < 0.01; ***, P < 0.0001, one-way ANOVA, post hoc comparisons, Tukey’s test. (B) The indicated cells were treated with or without ponatinib (1 nM for EOL-1, 300 nM for BaF3-WT and -T674I FIP1L1-PDGFRα cells, respectively) for 24 h, washed with PBS and fixed with 2% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3). Representative photographs (9700×) were acquired under transmission electron microscopy. (C) The concentration- (for 24 h) and time-dependent (1 nM for EOL-1, 300 nM for BaF3-WT and -T674I FIP1L1-PDGFRα cells) cleavage of PARP and caspase-3 triggered by ponatinib was analyzed by immunoblotting. (D) Ponatinib elicited release of AIF and cytochrome c into the cytosol. Cells were treated with 1 nM ponatinib for the indicated durations and the cytosolic fraction was extracted with digitonin buffer. Levels of AIF and Cytochrome c (Cyto c) were detected by immunoblotting. (E) Immunoblotting of apoptosis-related proteins in CEL cells after treatment for 24 h.
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Figure 3: Ponatinib induces apoptosis in FIP1LI-PDGFRα-expressing cells. (A) EOL-1 and BaF3-WT or -T674I FIP1L1-PDGFRα cells were exposed to increasing concentrations of ponatinib for 24 h, apoptotic cells were assayed with flow cytometry by PI/Annexin V-FITC (EOL-1) or 7-AAD/Annexin V-PE (BaF3-WT or -T674I FIP1L1-PDGFRα cells) staining. Left, representative histograms; Right, statistical data of 3 independent experiments, the vertical axis stands for the sum of all dead cells. Error bars represent 95% confidence intervals. **, P < 0.01; ***, P < 0.0001, one-way ANOVA, post hoc comparisons, Tukey’s test. (B) The indicated cells were treated with or without ponatinib (1 nM for EOL-1, 300 nM for BaF3-WT and -T674I FIP1L1-PDGFRα cells, respectively) for 24 h, washed with PBS and fixed with 2% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3). Representative photographs (9700×) were acquired under transmission electron microscopy. (C) The concentration- (for 24 h) and time-dependent (1 nM for EOL-1, 300 nM for BaF3-WT and -T674I FIP1L1-PDGFRα cells) cleavage of PARP and caspase-3 triggered by ponatinib was analyzed by immunoblotting. (D) Ponatinib elicited release of AIF and cytochrome c into the cytosol. Cells were treated with 1 nM ponatinib for the indicated durations and the cytosolic fraction was extracted with digitonin buffer. Levels of AIF and Cytochrome c (Cyto c) were detected by immunoblotting. (E) Immunoblotting of apoptosis-related proteins in CEL cells after treatment for 24 h.

Mentions: We next assessed whether ponatinib induced apoptosis. The FIP1L1-PDGFRα-expressing cells were exposed to increasing concentrations of ponatinib for 24 h, and apoptosis was measured by Annexin V binding. Ponatinib led to remarkable apoptotic cell death in all 3 cell lines (Figure 3A). Further, condensation of chromatin in the periphery of the nuclei was observed by transmission electron microscopy, and this was consistent with the induction of apoptosis by ponatinib (Figure 3B). Moreover, ponatinib induced concentration- and time-dependent specific cleavage of PARP and caspase-3 activation in all three FIP1L1-PDGFRα-expressing cell lines (Figure 3C). Therefore, ponatinib could efficiently induce apoptosis in CEL cells harboring WT or T674I FIP1L1-PDGFRα.


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 induces apoptosis in FIP1LI-PDGFRα-expressing cells. (A) EOL-1 and BaF3-WT or -T674I FIP1L1-PDGFRα cells were exposed to increasing concentrations of ponatinib for 24 h, apoptotic cells were assayed with flow cytometry by PI/Annexin V-FITC (EOL-1) or 7-AAD/Annexin V-PE (BaF3-WT or -T674I FIP1L1-PDGFRα cells) staining. Left, representative histograms; Right, statistical data of 3 independent experiments, the vertical axis stands for the sum of all dead cells. Error bars represent 95% confidence intervals. **, P < 0.01; ***, P < 0.0001, one-way ANOVA, post hoc comparisons, Tukey’s test. (B) The indicated cells were treated with or without ponatinib (1 nM for EOL-1, 300 nM for BaF3-WT and -T674I FIP1L1-PDGFRα cells, respectively) for 24 h, washed with PBS and fixed with 2% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3). Representative photographs (9700×) were acquired under transmission electron microscopy. (C) The concentration- (for 24 h) and time-dependent (1 nM for EOL-1, 300 nM for BaF3-WT and -T674I FIP1L1-PDGFRα cells) cleavage of PARP and caspase-3 triggered by ponatinib was analyzed by immunoblotting. (D) Ponatinib elicited release of AIF and cytochrome c into the cytosol. Cells were treated with 1 nM ponatinib for the indicated durations and the cytosolic fraction was extracted with digitonin buffer. Levels of AIF and Cytochrome c (Cyto c) were detected by immunoblotting. (E) Immunoblotting of apoptosis-related proteins in CEL cells after treatment for 24 h.
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Figure 3: Ponatinib induces apoptosis in FIP1LI-PDGFRα-expressing cells. (A) EOL-1 and BaF3-WT or -T674I FIP1L1-PDGFRα cells were exposed to increasing concentrations of ponatinib for 24 h, apoptotic cells were assayed with flow cytometry by PI/Annexin V-FITC (EOL-1) or 7-AAD/Annexin V-PE (BaF3-WT or -T674I FIP1L1-PDGFRα cells) staining. Left, representative histograms; Right, statistical data of 3 independent experiments, the vertical axis stands for the sum of all dead cells. Error bars represent 95% confidence intervals. **, P < 0.01; ***, P < 0.0001, one-way ANOVA, post hoc comparisons, Tukey’s test. (B) The indicated cells were treated with or without ponatinib (1 nM for EOL-1, 300 nM for BaF3-WT and -T674I FIP1L1-PDGFRα cells, respectively) for 24 h, washed with PBS and fixed with 2% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3). Representative photographs (9700×) were acquired under transmission electron microscopy. (C) The concentration- (for 24 h) and time-dependent (1 nM for EOL-1, 300 nM for BaF3-WT and -T674I FIP1L1-PDGFRα cells) cleavage of PARP and caspase-3 triggered by ponatinib was analyzed by immunoblotting. (D) Ponatinib elicited release of AIF and cytochrome c into the cytosol. Cells were treated with 1 nM ponatinib for the indicated durations and the cytosolic fraction was extracted with digitonin buffer. Levels of AIF and Cytochrome c (Cyto c) were detected by immunoblotting. (E) Immunoblotting of apoptosis-related proteins in CEL cells after treatment for 24 h.
Mentions: We next assessed whether ponatinib induced apoptosis. The FIP1L1-PDGFRα-expressing cells were exposed to increasing concentrations of ponatinib for 24 h, and apoptosis was measured by Annexin V binding. Ponatinib led to remarkable apoptotic cell death in all 3 cell lines (Figure 3A). Further, condensation of chromatin in the periphery of the nuclei was observed by transmission electron microscopy, and this was consistent with the induction of apoptosis by ponatinib (Figure 3B). Moreover, ponatinib induced concentration- and time-dependent specific cleavage of PARP and caspase-3 activation in all three FIP1L1-PDGFRα-expressing cell lines (Figure 3C). Therefore, ponatinib could efficiently induce apoptosis in CEL cells harboring WT or T674I FIP1L1-PDGFRα.

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