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FLT3-ITD confers resistance to the PI3K/Akt pathway inhibitors by protecting the mTOR/4EBP1/Mcl-1 pathway through STAT5 activation in acute myeloid leukemia.

Nogami A, Oshikawa G, Okada K, Fukutake S, Umezawa Y, Nagao T, Kurosu T, Miura O - Oncotarget (2015)

Bottom Line: GDC-0941 or MK-2206 induced dephosphorylation of 4EBP1 more conspicuously in 32D/TKD than in 32D/ITD, which was prevented or augmented by STAT5A1*6 or pimozide, respectively, and correlated with downregulation of the eIF4E/eIF4G complex formation and Mcl-1 expression.Furthermore, exogenous expression of Mcl-1 endowed resistance to GDC-0941 and MK-2206 on 32D/TKD cells.These data suggest that the robust STAT5 activation by FLT3-ITD protects cells treated with the PI3K/Akt pathway inhibitors from apoptosis by maintaining Mcl-1 expression through the mTORC1/4EBP1/eIF4E pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.

ABSTRACT
FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in acute myeloid leukemia (AML), with the former associated with poor prognosis. Here, we show that the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 induced apoptosis through the mitochondria-mediated intrinsic pathway more efficiently in hematopoietic 32D cells driven by FLT3-TKD (32D/TKD) than FLT3-ITD (32D/ITD), which robustly activated STAT5. The resistance to GDC-0941 and MK-2206 was gained by expression of the constitutively activated STAT5 mutant STAT5A1*6 in 32D/TKD cells, while it was abrogated by the STAT5 inhibitor pimozide in 32D/ITD cells or FLT3-ITD-expressing human leukemic MV4-11 cells. GDC-0941 or MK-2206 induced dephosphorylation of 4EBP1 more conspicuously in 32D/TKD than in 32D/ITD, which was prevented or augmented by STAT5A1*6 or pimozide, respectively, and correlated with downregulation of the eIF4E/eIF4G complex formation and Mcl-1 expression. Furthermore, exogenous expression of Mcl-1 endowed resistance to GDC-0941 and MK-2206 on 32D/TKD cells. Finally, it was confirmed in primary AML cells with FLT3-ITD that pimozide enhanced 4EBP1 dephosphorylation and Mcl-1 downregulation to augment cytotoxicity of GDC-0941. These data suggest that the robust STAT5 activation by FLT3-ITD protects cells treated with the PI3K/Akt pathway inhibitors from apoptosis by maintaining Mcl-1 expression through the mTORC1/4EBP1/eIF4E pathway.

No MeSH data available.


Related in: MedlinePlus

FLT3-ITD confers resistance to the PI3K/Akt pathway inhibitors through STAT5 activation by sustaining 4EBP1 phosphorylation and Mcl-1 expression to prevent Caspase-9 activationA. 32D/ITD (ITD) or 32D/TKD (TKD) cells were left untreated as control or treated with GDC-0941 (GDC) or MK-2206 (MK) at 3 μM for 30 h as indicated. Cells were lysed and subjected to Western blot analysis with antibodies against indicated proteins. Abbreviations used are: Casp-9, Caspase-9; Cl. Casp-9; Cleaved Caspase-9, 4EBP1-P, phospho-T37/49–4EBP1. B. 32D/ITD (ITD) or 32D/TKD (TKD) cells were treated with indicated concentrations (μM) of GDC-0941 for 4 h and subjected to Western blot analysis. Akt-PT, phospho-T308-Akt; Akt-PS, phospho-S473-Akt. C. 32D/ITD cells were treated with indicated concentrations (μM) of GDC-0941 in the presence or absence of 5 μM pimozide (PMZ), as indicated, for 24 h and analyzed. 4EBP1-nonP, non-phospho-T46–4EBP1. D. 32D/ITD (ITD) or 32D/TKD (TKD) cells were left untreated as control or treated with 1 μM GDC-0941 in the absence or presence of 5 μM pimozide (PMZ), as indicated, for 4 h. Cells were subjected to flow cytometric analysis with an antibody specific for unphosphorylated form of 4EBP1 (4EBP1-nonP). Cells treated with or without GDC-0941 are shown in a blue or red line, respectively, while a green line represents isotype control for the antibody used. E. MV4–11 cells were treated with indicated concentrations (μM) of GDC-0941 or MK-2206 in the presence or absence of 5 μM pimozide (PMZ), as indicated, for 20 h and analyzed. F. 32D/TKD cells transduced with STAT5A1*6 (STAT5*) or vector control cells (Cont.), as indicated, were treated with indicated concentrations (μM) of GDC-0941 (GDC) or MK-2206 (MK) for 20 h and analyzed. G. 32D/TKD cells transduced with STAT5A1*6 (STAT5*) or vector control cells (Cont.), as indicated, were treated with indicated concentrations (μM) of GDC-0941 for 4 h and analyzed.
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Figure 4: FLT3-ITD confers resistance to the PI3K/Akt pathway inhibitors through STAT5 activation by sustaining 4EBP1 phosphorylation and Mcl-1 expression to prevent Caspase-9 activationA. 32D/ITD (ITD) or 32D/TKD (TKD) cells were left untreated as control or treated with GDC-0941 (GDC) or MK-2206 (MK) at 3 μM for 30 h as indicated. Cells were lysed and subjected to Western blot analysis with antibodies against indicated proteins. Abbreviations used are: Casp-9, Caspase-9; Cl. Casp-9; Cleaved Caspase-9, 4EBP1-P, phospho-T37/49–4EBP1. B. 32D/ITD (ITD) or 32D/TKD (TKD) cells were treated with indicated concentrations (μM) of GDC-0941 for 4 h and subjected to Western blot analysis. Akt-PT, phospho-T308-Akt; Akt-PS, phospho-S473-Akt. C. 32D/ITD cells were treated with indicated concentrations (μM) of GDC-0941 in the presence or absence of 5 μM pimozide (PMZ), as indicated, for 24 h and analyzed. 4EBP1-nonP, non-phospho-T46–4EBP1. D. 32D/ITD (ITD) or 32D/TKD (TKD) cells were left untreated as control or treated with 1 μM GDC-0941 in the absence or presence of 5 μM pimozide (PMZ), as indicated, for 4 h. Cells were subjected to flow cytometric analysis with an antibody specific for unphosphorylated form of 4EBP1 (4EBP1-nonP). Cells treated with or without GDC-0941 are shown in a blue or red line, respectively, while a green line represents isotype control for the antibody used. E. MV4–11 cells were treated with indicated concentrations (μM) of GDC-0941 or MK-2206 in the presence or absence of 5 μM pimozide (PMZ), as indicated, for 20 h and analyzed. F. 32D/TKD cells transduced with STAT5A1*6 (STAT5*) or vector control cells (Cont.), as indicated, were treated with indicated concentrations (μM) of GDC-0941 (GDC) or MK-2206 (MK) for 20 h and analyzed. G. 32D/TKD cells transduced with STAT5A1*6 (STAT5*) or vector control cells (Cont.), as indicated, were treated with indicated concentrations (μM) of GDC-0941 for 4 h and analyzed.

Mentions: To explore the molecular mechanisms by which FLT3-ITD confers resistance to the PI3K/Akt pathway inhibitors, we examined the effects of GDC-0941 or MK-2206 on various factors involved in regulation of apoptosis and proliferation in these cells by Western blot analyses. Fig. 4A shows that Caspase-9 was cleaved specifically in 32D/TKD cells but not in 32D/ITD cells after treatment with GDC-0941 or MK-2206 for 30 h. Thus, under these conditions, the PI3K/Akt pathway inhibitors activated mitochondria-mediated apoptotic pathway selectively in 32D/TKD cells, as Caspase-9 is cleaved and activated just downstream of the mitochondria in this pathway [31]. Because activation of this pathway is protected by anti-apoptotic members of the Bcl-2 family proteins, we examined the expression level of Mcl-1, a critical member of this family in hematopoietic cells, including AML cells [23, 24]. As shown in Fig. 4A, the expression level of Mcl-1 was slightly lower and reduced more conspicuously by GDC-0941 or MK-2206 in 32D/TKD cells than in 32D/ITD cells. On the other hand, the expression level of Bcl-xL was not significantly reduced by these inhibitors in 32D/TKD cells. Because the expression level of Mcl-1 is regulated through translational as well as transcriptional and post-translational mechanisms [31], we examined 4EBP1, which is phosphorylated by mTORC1 downstream of the PI3K/Akt pathway and is known to play a critical role in cap-dependent translational of Mcl-1 mRNA [20, 21]. As shown in Fig. 4A, phosphorylation of 4EBP1 was reduced by GDC-0941 and MK-2206 to lower levels in 32D/TKD cells as compared with 32D/ITD cells, which correlated with the expression levels of Mcl-1 in these cells. To confirm and extend these observations, we treated these cells with increasing concentrations of GDC-0941 for a shorter period of time (4 h) and examined its effects on Mcl-1 and 4EBP1, because Mcl-1 has a short half-life and may be cleaved by activated caspases in cells undergoing apoptosis. As shown in Fig. 4B, GDC-0941 very efficiently inhibited the activation specific phosphorylation of Akt on T308 comparably in both 32D/ITD and 32D/TKD cells. However, the dose-dependent decline in Mcl-1 expression as well in 4EBP1 phosphorylation was more prominent in 32D/TKD cells than in 32D/ITD cells. Similar results were obtained with MK-2206 (data not shown). These results suggest the possibility that FLT3-ITD may maintain the 4EBP1/Mcl-1 axis downstream of the PI3K/Akt pathway to protect cells from activation of the mitochondrial apoptotic pathway leading to activation of Caspase-9 in these cells.


FLT3-ITD confers resistance to the PI3K/Akt pathway inhibitors by protecting the mTOR/4EBP1/Mcl-1 pathway through STAT5 activation in acute myeloid leukemia.

Nogami A, Oshikawa G, Okada K, Fukutake S, Umezawa Y, Nagao T, Kurosu T, Miura O - Oncotarget (2015)

FLT3-ITD confers resistance to the PI3K/Akt pathway inhibitors through STAT5 activation by sustaining 4EBP1 phosphorylation and Mcl-1 expression to prevent Caspase-9 activationA. 32D/ITD (ITD) or 32D/TKD (TKD) cells were left untreated as control or treated with GDC-0941 (GDC) or MK-2206 (MK) at 3 μM for 30 h as indicated. Cells were lysed and subjected to Western blot analysis with antibodies against indicated proteins. Abbreviations used are: Casp-9, Caspase-9; Cl. Casp-9; Cleaved Caspase-9, 4EBP1-P, phospho-T37/49–4EBP1. B. 32D/ITD (ITD) or 32D/TKD (TKD) cells were treated with indicated concentrations (μM) of GDC-0941 for 4 h and subjected to Western blot analysis. Akt-PT, phospho-T308-Akt; Akt-PS, phospho-S473-Akt. C. 32D/ITD cells were treated with indicated concentrations (μM) of GDC-0941 in the presence or absence of 5 μM pimozide (PMZ), as indicated, for 24 h and analyzed. 4EBP1-nonP, non-phospho-T46–4EBP1. D. 32D/ITD (ITD) or 32D/TKD (TKD) cells were left untreated as control or treated with 1 μM GDC-0941 in the absence or presence of 5 μM pimozide (PMZ), as indicated, for 4 h. Cells were subjected to flow cytometric analysis with an antibody specific for unphosphorylated form of 4EBP1 (4EBP1-nonP). Cells treated with or without GDC-0941 are shown in a blue or red line, respectively, while a green line represents isotype control for the antibody used. E. MV4–11 cells were treated with indicated concentrations (μM) of GDC-0941 or MK-2206 in the presence or absence of 5 μM pimozide (PMZ), as indicated, for 20 h and analyzed. F. 32D/TKD cells transduced with STAT5A1*6 (STAT5*) or vector control cells (Cont.), as indicated, were treated with indicated concentrations (μM) of GDC-0941 (GDC) or MK-2206 (MK) for 20 h and analyzed. G. 32D/TKD cells transduced with STAT5A1*6 (STAT5*) or vector control cells (Cont.), as indicated, were treated with indicated concentrations (μM) of GDC-0941 for 4 h and analyzed.
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Figure 4: FLT3-ITD confers resistance to the PI3K/Akt pathway inhibitors through STAT5 activation by sustaining 4EBP1 phosphorylation and Mcl-1 expression to prevent Caspase-9 activationA. 32D/ITD (ITD) or 32D/TKD (TKD) cells were left untreated as control or treated with GDC-0941 (GDC) or MK-2206 (MK) at 3 μM for 30 h as indicated. Cells were lysed and subjected to Western blot analysis with antibodies against indicated proteins. Abbreviations used are: Casp-9, Caspase-9; Cl. Casp-9; Cleaved Caspase-9, 4EBP1-P, phospho-T37/49–4EBP1. B. 32D/ITD (ITD) or 32D/TKD (TKD) cells were treated with indicated concentrations (μM) of GDC-0941 for 4 h and subjected to Western blot analysis. Akt-PT, phospho-T308-Akt; Akt-PS, phospho-S473-Akt. C. 32D/ITD cells were treated with indicated concentrations (μM) of GDC-0941 in the presence or absence of 5 μM pimozide (PMZ), as indicated, for 24 h and analyzed. 4EBP1-nonP, non-phospho-T46–4EBP1. D. 32D/ITD (ITD) or 32D/TKD (TKD) cells were left untreated as control or treated with 1 μM GDC-0941 in the absence or presence of 5 μM pimozide (PMZ), as indicated, for 4 h. Cells were subjected to flow cytometric analysis with an antibody specific for unphosphorylated form of 4EBP1 (4EBP1-nonP). Cells treated with or without GDC-0941 are shown in a blue or red line, respectively, while a green line represents isotype control for the antibody used. E. MV4–11 cells were treated with indicated concentrations (μM) of GDC-0941 or MK-2206 in the presence or absence of 5 μM pimozide (PMZ), as indicated, for 20 h and analyzed. F. 32D/TKD cells transduced with STAT5A1*6 (STAT5*) or vector control cells (Cont.), as indicated, were treated with indicated concentrations (μM) of GDC-0941 (GDC) or MK-2206 (MK) for 20 h and analyzed. G. 32D/TKD cells transduced with STAT5A1*6 (STAT5*) or vector control cells (Cont.), as indicated, were treated with indicated concentrations (μM) of GDC-0941 for 4 h and analyzed.
Mentions: To explore the molecular mechanisms by which FLT3-ITD confers resistance to the PI3K/Akt pathway inhibitors, we examined the effects of GDC-0941 or MK-2206 on various factors involved in regulation of apoptosis and proliferation in these cells by Western blot analyses. Fig. 4A shows that Caspase-9 was cleaved specifically in 32D/TKD cells but not in 32D/ITD cells after treatment with GDC-0941 or MK-2206 for 30 h. Thus, under these conditions, the PI3K/Akt pathway inhibitors activated mitochondria-mediated apoptotic pathway selectively in 32D/TKD cells, as Caspase-9 is cleaved and activated just downstream of the mitochondria in this pathway [31]. Because activation of this pathway is protected by anti-apoptotic members of the Bcl-2 family proteins, we examined the expression level of Mcl-1, a critical member of this family in hematopoietic cells, including AML cells [23, 24]. As shown in Fig. 4A, the expression level of Mcl-1 was slightly lower and reduced more conspicuously by GDC-0941 or MK-2206 in 32D/TKD cells than in 32D/ITD cells. On the other hand, the expression level of Bcl-xL was not significantly reduced by these inhibitors in 32D/TKD cells. Because the expression level of Mcl-1 is regulated through translational as well as transcriptional and post-translational mechanisms [31], we examined 4EBP1, which is phosphorylated by mTORC1 downstream of the PI3K/Akt pathway and is known to play a critical role in cap-dependent translational of Mcl-1 mRNA [20, 21]. As shown in Fig. 4A, phosphorylation of 4EBP1 was reduced by GDC-0941 and MK-2206 to lower levels in 32D/TKD cells as compared with 32D/ITD cells, which correlated with the expression levels of Mcl-1 in these cells. To confirm and extend these observations, we treated these cells with increasing concentrations of GDC-0941 for a shorter period of time (4 h) and examined its effects on Mcl-1 and 4EBP1, because Mcl-1 has a short half-life and may be cleaved by activated caspases in cells undergoing apoptosis. As shown in Fig. 4B, GDC-0941 very efficiently inhibited the activation specific phosphorylation of Akt on T308 comparably in both 32D/ITD and 32D/TKD cells. However, the dose-dependent decline in Mcl-1 expression as well in 4EBP1 phosphorylation was more prominent in 32D/TKD cells than in 32D/ITD cells. Similar results were obtained with MK-2206 (data not shown). These results suggest the possibility that FLT3-ITD may maintain the 4EBP1/Mcl-1 axis downstream of the PI3K/Akt pathway to protect cells from activation of the mitochondrial apoptotic pathway leading to activation of Caspase-9 in these cells.

Bottom Line: GDC-0941 or MK-2206 induced dephosphorylation of 4EBP1 more conspicuously in 32D/TKD than in 32D/ITD, which was prevented or augmented by STAT5A1*6 or pimozide, respectively, and correlated with downregulation of the eIF4E/eIF4G complex formation and Mcl-1 expression.Furthermore, exogenous expression of Mcl-1 endowed resistance to GDC-0941 and MK-2206 on 32D/TKD cells.These data suggest that the robust STAT5 activation by FLT3-ITD protects cells treated with the PI3K/Akt pathway inhibitors from apoptosis by maintaining Mcl-1 expression through the mTORC1/4EBP1/eIF4E pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.

ABSTRACT
FLT3-ITD and FLT3-TKD are the most frequent tyrosine kinase mutations in acute myeloid leukemia (AML), with the former associated with poor prognosis. Here, we show that the PI3K inhibitor GDC-0941 or the Akt inhibitor MK-2206 induced apoptosis through the mitochondria-mediated intrinsic pathway more efficiently in hematopoietic 32D cells driven by FLT3-TKD (32D/TKD) than FLT3-ITD (32D/ITD), which robustly activated STAT5. The resistance to GDC-0941 and MK-2206 was gained by expression of the constitutively activated STAT5 mutant STAT5A1*6 in 32D/TKD cells, while it was abrogated by the STAT5 inhibitor pimozide in 32D/ITD cells or FLT3-ITD-expressing human leukemic MV4-11 cells. GDC-0941 or MK-2206 induced dephosphorylation of 4EBP1 more conspicuously in 32D/TKD than in 32D/ITD, which was prevented or augmented by STAT5A1*6 or pimozide, respectively, and correlated with downregulation of the eIF4E/eIF4G complex formation and Mcl-1 expression. Furthermore, exogenous expression of Mcl-1 endowed resistance to GDC-0941 and MK-2206 on 32D/TKD cells. Finally, it was confirmed in primary AML cells with FLT3-ITD that pimozide enhanced 4EBP1 dephosphorylation and Mcl-1 downregulation to augment cytotoxicity of GDC-0941. These data suggest that the robust STAT5 activation by FLT3-ITD protects cells treated with the PI3K/Akt pathway inhibitors from apoptosis by maintaining Mcl-1 expression through the mTORC1/4EBP1/eIF4E pathway.

No MeSH data available.


Related in: MedlinePlus