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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 potently abrogates the growth of imatinib-resistant neoplastic cells expressing T674I FIP1L1-PDGFRα in nude mouse xenografts. (A) BALB/c nu/nu nude mice were subcutaneously inoculated with BaF3-T674I FIP1L1-PDGFRα cells, then randomized into 3 groups (10 animals each) for daily oral administration of vehicle [30% Cremophor EL/ethanol (4:1), 70% PBS], imatinib or ponatinib during days 5–21 after inoculation of cells. The tumor growth curves are plotted. Error bars represent 95% confidence intervals. (B) Dissected tumor xenografts were measured on day 21. ***, P < 0.0001, one-way ANOVA, post hoc comparisons, Tukey’s test. Columns, mean; error bars, 95% confidence intervals. Representative tumors removed from mice of each group are shown (upper). (C) Immnunohistochemical analysis with anti-Ki67 and H & E staining of xenograft tissues from mice sacrificed 21 days after tumor inoculation. (D) The signaling of PDGFRα in tumor tissue was inhibited by ponatinib. Whole cell lysates prepared from xenografts of each group were detected by immunoblotting with the indicated antibodies.
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Figure 6: Ponatinib potently abrogates the growth of imatinib-resistant neoplastic cells expressing T674I FIP1L1-PDGFRα in nude mouse xenografts. (A) BALB/c nu/nu nude mice were subcutaneously inoculated with BaF3-T674I FIP1L1-PDGFRα cells, then randomized into 3 groups (10 animals each) for daily oral administration of vehicle [30% Cremophor EL/ethanol (4:1), 70% PBS], imatinib or ponatinib during days 5–21 after inoculation of cells. The tumor growth curves are plotted. Error bars represent 95% confidence intervals. (B) Dissected tumor xenografts were measured on day 21. ***, P < 0.0001, one-way ANOVA, post hoc comparisons, Tukey’s test. Columns, mean; error bars, 95% confidence intervals. Representative tumors removed from mice of each group are shown (upper). (C) Immnunohistochemical analysis with anti-Ki67 and H & E staining of xenograft tissues from mice sacrificed 21 days after tumor inoculation. (D) The signaling of PDGFRα in tumor tissue was inhibited by ponatinib. Whole cell lysates prepared from xenografts of each group were detected by immunoblotting with the indicated antibodies.

Mentions: The in vivo antineoplastic activity of ponatinib as a single agent against imatinib-resistant T674I FIP1L1-PDGFRα-expressing cells was evaluated in the nude mouse xenograft model. Thirty nu/nu BALB/c mice were subcutaneously injected with BaF3-T674I PDGFRα cells. Four days later, when tumor sizes were ~50 mm3, the mice were randomized to receive treatment with vehicle, imatinib (50 mg/kg/d) or ponatinib (30 mg/kg/d) for 15 days (n = 10). The tumor growth curve (the estimated tumor size calculated from the tumor dimension versus time) with imatinib was almost the same as with vehicle (Figure 6A), which indicates in vivo resistance of BaF3-T674I PDGFRα cells to imatinib. In contrast, ponatinib treatment abrogated the growth of tumors (Figure 6A). Tumor weight did not differ between imatinib- and vehicle-treated tumors but was lower in ponatinib-treated than control tumors (Figure 6B). Cell proliferation, as reflected by Ki67 immunohistochemistry, was inhibited by ponatinib (Figure 6C) as compared with the two controls. Immunoblotting of cell lysates from tumors from ponatinib-treated mice showed pronounced decreases in phosphorylated PDGFRα, Stat5, Akt, and Erk1/2 but not their total counterparts, so ponatinib blocked PDGFRα signaling in xenografts (Figure 6D). In addition, the level of β-catenin was decreased by ponatinib (Figure 6D). The body weights of the mice remained stable, with no significant differences between drug-treated and control mice (data not shown). Motor activity and feeding behavior of the mice were all normal. No mice died until the scheduled sacrifice. Overall, ponatinib was well tolerated at the dosage used.


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 potently abrogates the growth of imatinib-resistant neoplastic cells expressing T674I FIP1L1-PDGFRα in nude mouse xenografts. (A) BALB/c nu/nu nude mice were subcutaneously inoculated with BaF3-T674I FIP1L1-PDGFRα cells, then randomized into 3 groups (10 animals each) for daily oral administration of vehicle [30% Cremophor EL/ethanol (4:1), 70% PBS], imatinib or ponatinib during days 5–21 after inoculation of cells. The tumor growth curves are plotted. Error bars represent 95% confidence intervals. (B) Dissected tumor xenografts were measured on day 21. ***, P < 0.0001, one-way ANOVA, post hoc comparisons, Tukey’s test. Columns, mean; error bars, 95% confidence intervals. Representative tumors removed from mice of each group are shown (upper). (C) Immnunohistochemical analysis with anti-Ki67 and H & E staining of xenograft tissues from mice sacrificed 21 days after tumor inoculation. (D) The signaling of PDGFRα in tumor tissue was inhibited by ponatinib. Whole cell lysates prepared from xenografts of each group were detected by immunoblotting with the indicated antibodies.
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Related In: Results  -  Collection

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Figure 6: Ponatinib potently abrogates the growth of imatinib-resistant neoplastic cells expressing T674I FIP1L1-PDGFRα in nude mouse xenografts. (A) BALB/c nu/nu nude mice were subcutaneously inoculated with BaF3-T674I FIP1L1-PDGFRα cells, then randomized into 3 groups (10 animals each) for daily oral administration of vehicle [30% Cremophor EL/ethanol (4:1), 70% PBS], imatinib or ponatinib during days 5–21 after inoculation of cells. The tumor growth curves are plotted. Error bars represent 95% confidence intervals. (B) Dissected tumor xenografts were measured on day 21. ***, P < 0.0001, one-way ANOVA, post hoc comparisons, Tukey’s test. Columns, mean; error bars, 95% confidence intervals. Representative tumors removed from mice of each group are shown (upper). (C) Immnunohistochemical analysis with anti-Ki67 and H & E staining of xenograft tissues from mice sacrificed 21 days after tumor inoculation. (D) The signaling of PDGFRα in tumor tissue was inhibited by ponatinib. Whole cell lysates prepared from xenografts of each group were detected by immunoblotting with the indicated antibodies.
Mentions: The in vivo antineoplastic activity of ponatinib as a single agent against imatinib-resistant T674I FIP1L1-PDGFRα-expressing cells was evaluated in the nude mouse xenograft model. Thirty nu/nu BALB/c mice were subcutaneously injected with BaF3-T674I PDGFRα cells. Four days later, when tumor sizes were ~50 mm3, the mice were randomized to receive treatment with vehicle, imatinib (50 mg/kg/d) or ponatinib (30 mg/kg/d) for 15 days (n = 10). The tumor growth curve (the estimated tumor size calculated from the tumor dimension versus time) with imatinib was almost the same as with vehicle (Figure 6A), which indicates in vivo resistance of BaF3-T674I PDGFRα cells to imatinib. In contrast, ponatinib treatment abrogated the growth of tumors (Figure 6A). Tumor weight did not differ between imatinib- and vehicle-treated tumors but was lower in ponatinib-treated than control tumors (Figure 6B). Cell proliferation, as reflected by Ki67 immunohistochemistry, was inhibited by ponatinib (Figure 6C) as compared with the two controls. Immunoblotting of cell lysates from tumors from ponatinib-treated mice showed pronounced decreases in phosphorylated PDGFRα, Stat5, Akt, and Erk1/2 but not their total counterparts, so ponatinib blocked PDGFRα signaling in xenografts (Figure 6D). In addition, the level of β-catenin was decreased by ponatinib (Figure 6D). The body weights of the mice remained stable, with no significant differences between drug-treated and control mice (data not shown). Motor activity and feeding behavior of the mice were all normal. No mice died until the scheduled sacrifice. Overall, ponatinib was well tolerated at the dosage used.

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