staurosporine derivative and protein kinase c inhibitor additionally affecting vegfr-2, pdgfra/b, kit and flt-3, called midostaurin  (Novartis)

Journal: Translational Oncology

Article Title: Inhibition of the STAT5/Pim Kinase Axis Enhances Cytotoxic Effects of Proteasome Inhibitors on FLT3-ITD–Positive AML Cells by Cooperatively Inhibiting the mTORC1/4EBP1/S6K/Mcl-1 Pathway 1

doi: 10.1016/j.tranon.2018.11.001

Figure Lengend Snippet: FLT3-ITD confers resistance to bortezomib by inhibiting activation of the mitochondria-mediated intrinsic apoptotic pathway involving activation of Bax and caspases. (A) 32D/ITD (ITD) or 32D/TKD (TKD) cells were cultured for 48 hours with or without 50 nM sorafenib or 3 ng/ml bortezomib, as indicated. The means of relative viable cell numbers from triplicate measurements are plotted as percentages of control cells without inhibitors, with error bars indicating standard errors. The asterisks indicate a statistically significant difference determined by Student's t test (* P < .05, * P < .005). (B) Cells indicated were cultured with 3 ng/ml bortezomib for indicated times, and viable cell numbers were counted after trypan blue staining. The means of percentages of viable cells from triplicate measurements are shown, with error bars indicating standard errors (* P < .001, * P < .0001). (C) Cells indicated were treated for 48 hours with or without 2 ng/ml bortezomib (BZM), as indicated, and analyzed for the cellular DNA content by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content are indicated. (D) Cells indicated were cultured for 24 hours with BZM, as indicated, and analyzed for the mitochondrial membrane potential (∆ψm) using DiOC6 by flow cytometry. Percentages of cells with reduced ∆ψm stained with or without PI are indicated. (E, F) Cells indicated were cultured for 24 hours with BZM, as indicated, and analyzed for activated Bax (E) and cleaved Caspase-3 (F) by flow cytometry. Percentages of cells with activated Bax or cleaved Caspase-3 are indicated. FSC: forward scatter. (G) Cells indicated were treated with indicated concentrations (ng/ml) of BZM for 42 hours and subjected to Western blot analysis with antibodies against indicated proteins. Abbreviations: Casp-9, Caspase-9; Cl.Casp-9, cleaved Caspase-9. HSP90 was used for the loading control. (H) Cells indicated were treated for 48 hours with indicated concentrations of carfilzomib (CZM) and analyzed. (I) 32D/ITD cells were treated for 48 hours with or without 1 ng/ml BZM in the presence or absence of 0.5 nM quizartinib (QZT) or 5 nM gilteritinib (GRT), as indicated, and analyzed.

Article Snippet: The STAT5 inhibitor pimozide and antibodies against FLT3 (SC-479), STAT5A (SC-1081), and HSP-90 (SC-13119) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Cell Culture, Control, Staining, Flow Cytometry, Membrane, Western Blot

Journal: Translational Oncology

Article Title: Inhibition of the STAT5/Pim Kinase Axis Enhances Cytotoxic Effects of Proteasome Inhibitors on FLT3-ITD–Positive AML Cells by Cooperatively Inhibiting the mTORC1/4EBP1/S6K/Mcl-1 Pathway 1

doi: 10.1016/j.tranon.2018.11.001

Figure Lengend Snippet: Activation of STAT5 mediates the resistance of FLT3-ITD–expressing cells including primary AML cells to bortezomib. (A) 32D/TKD cells transduced with STAT5A1*6 or vector control cells, as indicated, were cultured for 48 hours with indicated concentrations of bortezomib (BZM) and analyzed for the cellular DNA content by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content are indicated. (B) 32D/ITD cells in the absence (ITD) or presence (ITD/PMZ) of 2.5 μM pimozide as well as 32D/TKD (TKD) cells were cultured with indicated concentrations of bortezomib for 48 hours, and viable cell numbers were counted after trypan blue staining. The means of percentages of viable cells from triplicate measurements are shown, with error bars indicating standard errors. The asterisks indicate a statistically significant difference determined by Student's t test (* P < .0001, ** P < .0005). (C) 32D/ITD cells were cultured for 48 hours with or without BZM or 5 μM pimozide, as indicated, and analyzed. (D) MV4-11 cells were cultured for 48 hours with or without 0.1 ng/ml BZM or 1 μM pimozide (PMZ), as indicated. The means of relative viable cell numbers from triplicate measurements expressed as percentages of control cells are plotted, with error bars indicating standard errors (* P < .01). (E) MV4-11 cells were cultured for 48 hours with BZM or 10 μM pimozide, as indicated, and analyzed. (F) Primary AML cells from a FLT3-ITD–positive patient (ITD+ AML #1) were culture for 48 hours with 0.5 ng/ml BZM or 2 μM pimozide PMZ, as indicated, and analyzed (* P < .01). (G) Primary AML cells from a FLT3-ITD–positive patient (ITD+ AML #2) were culture for 24 hours with 1 ng/ml BZM or 10 μM PMZ, as indicated, and analyzed (* P < .05). (H) Cells indicated were cultured for 24 hours with 0.5 ng/ml BZM or 2 μM PMZ, as indicated, and subjected to Western blot analysis with antibodies against indicated proteins. Abbreviations: Cl.Casp-3, cleaved Caspase-3; STAT5-PY, phospho-Y694-STAT5. (I) 32D/ITD (ITD) or 32D/TKD (TKD) cells were cultured with indicated concentrations of carfilzomib (CZM) with or without 10 μM pimozide for 48 hours and analyzed.

Article Snippet: The STAT5 inhibitor pimozide and antibodies against FLT3 (SC-479), STAT5A (SC-1081), and HSP-90 (SC-13119) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Expressing, Transduction, Plasmid Preparation, Control, Cell Culture, Flow Cytometry, Staining, Western Blot

Journal: Translational Oncology

Article Title: Inhibition of the STAT5/Pim Kinase Axis Enhances Cytotoxic Effects of Proteasome Inhibitors on FLT3-ITD–Positive AML Cells by Cooperatively Inhibiting the mTORC1/4EBP1/S6K/Mcl-1 Pathway 1

doi: 10.1016/j.tranon.2018.11.001

Figure Lengend Snippet: FLT3-ITD protects the mTORC1 pathway through the robust STAT5 activation in bortezomib-treated cells to enhance cell survival. (A) 32D/TKD (TKD) cells were treated with indicated concentrations (ng/ml) of bortezomib (BZM) for 6 hours and subjected to Western blot analysis. Abbreviations: Akt-PT, phospho-T308-Akt; S6K-PT, phospho-T389-S6K; 4EBP1-P, phospho-T37/46-4EBP1; 4EBP1-nonP, non-phospho-T46-4EBP1. (B) 32D/TKD cells transduced with STAT5A1*6 (ST5*) or vector control cells, as indicated, were treated with or without 5 ng/ml BZM for 6 hours and analyzed. (C) 32D/ITD (ITD) or 32D/TKD (TKD) cells were treated with indicated concentrations (ng/ml) of BZM with or without 5 μM pimozide (PMZ), as indicated, for 6 hours and analyzed. STAT5-PY: phospho-Y694-STAT5. (D) MV4-11 cells were cultured for 3 hours with or without 10 μM PMZ, as indicated, and then treated with indicated concentrations (ng/ml) of BZM for 6 hours and analyzed. 4EBP1-S65P: phospho-S65-4EBP1. (E) MV4-11 cells were treated with indicated concentrations of carfilzomib (CZM) for 6 hours and analyzed. (F) 32D/ITD (ITD) cells were cultured with indicated concentrations of BZM with or without 50 nM rapamycin or 0.2 mM PP242, as indicated, for 48 hours and analyzed for the cellular DNA content by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content are indicated.

Article Snippet: The STAT5 inhibitor pimozide and antibodies against FLT3 (SC-479), STAT5A (SC-1081), and HSP-90 (SC-13119) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Western Blot, Transduction, Plasmid Preparation, Control, Cell Culture, Flow Cytometry

Journal: Translational Oncology

Article Title: Inhibition of the STAT5/Pim Kinase Axis Enhances Cytotoxic Effects of Proteasome Inhibitors on FLT3-ITD–Positive AML Cells by Cooperatively Inhibiting the mTORC1/4EBP1/S6K/Mcl-1 Pathway 1

doi: 10.1016/j.tranon.2018.11.001

Figure Lengend Snippet: Bortezomib induces REDD1 expression to downregulate the mTORC1 pathway and subsequently the FLT3-ITD expression. (A) MV4-11 cells were treated with indicated concentrations (ng/ml) of bortezomib (BZM) for 6 hours and subjected to Western blot analysis with antibodies against indicated proteins. Abbreviations: S6K-PT, phospho-T389-S6K; 4EBP1-P, phospho-T37/46-4EBP1; 4EBP1-nonP, non-phospho-T46-4EBP1. (B) Primary AML cells from FLT3-ITD–positive patients (ITD+ AML #1) were treated with indicated concentrations (ng/ml) of BZM for 6 hours and lysed. Cell lysates were run on duplicate gels and analyzed. Relative expression levels of REDD1 analyzed by densitometry and normalized by that of HSP90 (RED/HSP) are indicated. S6RP-SP: phospho-S240/244-S6RP. (C) BaF3/ITD cells inducibly overexpressing REDD1 (REDD1) or vector control cells (Cont.) cultured with or without doxycycline (DOX), as indicated, were treated with indicated concentrations (ng/ml) of BZM for 6 hours and analyzed. Akt-PT: phospho-T308-Akt. (D) Cells indicated were treated with 2 ng/ml BZM for 48 hours and analyzed for the cellular DNA content by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content are indicated. (E) MV4-11 cells were treated with or without 5 ng/ml BZM and 50 μM chloroquine (CQ), as indicated, for 8 hours and analyzed. Positions of LC3B-I and -II are indicated. Cl.Casp-3: cleaved Caspase-3. (F) MV4-11 cells were treated with indicated concentrations (ng/ml) of BZM for 8 hours simultaneously with 100 μM Boc-d-FMK (B-d-F) or with 5 μM MG132 added for the last 2 hours, as indicated, and analyzed. (G) MV4-11 cells were left untreated as control (Cont.) or treated with 5 ng/ml BZM, as indicated, for 5 hours. Cells were then treated with 50 μg/ml cycloheximide (CHX) for indicated times along with 100 μM CQ where indicated or treated with 5 μg/ml actinomycin D (ACD) for 4 hours, as indicated, and lysed for Western blot analysis. (H) MV4-11 cells were cultured for 4 hours with 50 μg/ml CHX in the absence (Cont.) or presence of 5 ng/ml of BZM. Cells were then washed three times and cultured with or without BZM, as indicated, for indicated times and analyzed. (I) MV4-11 cells were treated for 6 hours with 4 ng/ml BZM or left untreated as control (Cont.), as indicated, and subjected to flow cytometric analysis with an antibody against FLT3 or phospho-S240/244-S6RP (S6RP-SP), as indicated. Gray lines represent isotype controls. MFIR: mean fluorescent intensity ratio. (J) MV4-11 cells were treated with indicated concentrations of PP242 for 8 hours and analyzed.

Article Snippet: The STAT5 inhibitor pimozide and antibodies against FLT3 (SC-479), STAT5A (SC-1081), and HSP-90 (SC-13119) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Western Blot, Plasmid Preparation, Control, Cell Culture, Flow Cytometry

Journal: Translational Oncology

Article Title: Inhibition of the STAT5/Pim Kinase Axis Enhances Cytotoxic Effects of Proteasome Inhibitors on FLT3-ITD–Positive AML Cells by Cooperatively Inhibiting the mTORC1/4EBP1/S6K/Mcl-1 Pathway 1

doi: 10.1016/j.tranon.2018.11.001

Figure Lengend Snippet: Pim kinases play a key role in STAT5-mediated acquisition of bortezomib resistance by sustaining the mTORC1 pathway in FLT3-ITD–expressing cells. (A) 32D/ITD cells were treated for 48 hours with or without 1 ng/ml bortezomib (BZM) and 1 μM AZD1208 (AZD), as indicated, and analyzed for the cellular DNA content by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content are indicated. (B) 32D/TKD cells transduced with STAT5A1*6 or vector control cells (Vect. Cont.) were cultured for 48 hours with 2 ng/ml BZM and 1 μM AZD, as indicated, and analyzed. (C) MV4-11 cells were treated for 48 hours with or without 0.75 ng/ml BZM and 2 μM PIM447, as indicated, and analyzed. (D) Primary AML cells from a FLT3-ITD–positive patient (ITD+ AML #1) were cultured for 48 hours with 0.5 ng/ml BZM and 0.5 μM AZD, as indicated. The means of relative viable cell numbers from triplicate measurements expressed as percentages of control cells are plotted, with error bars indicating standard errors. The asterisks indicate statistically significant differences determined by Student's t test (** P < .01). (E) Primary AML cells from FLT3-ITD–positive patients (ITD+ AML #2) were cultured for 24 hours with 0.3 ng/ml BZM and 0.25 μM AZD, as indicated, and analyzed. (F) 32D/ITD or MV4-11 cells were treated with indicated concentrations (ng/ml) of BZM with or without 0.5 μM AZD, as indicated, for 6 hours and subjected to Western blot analysis with antibodies against indicated proteins. Abbreviations: S6K-PT, phospho-T389-S6K; 4EBP1-P, phospho-T37/46-4EBP1; 4EBP1-nonP, non-phospho-T46-4EBP1. (G) Cells indicated were treated for 8 hours with 3 ng/ml BZM and 0.5 μM AZD, as indicated, and analyzed. 4EBP1-S65P: phospho-S65-4EBP1. (H) Cells indicated were treated for 8 hours with 4 ng/ml BZM and 0.3 μM AZD, as indicated, and analyzed. (I) MV4-11 cells were treated with indicated concentrations (ng/ml) of BZM and 1 μM AZD for 6 hours and subjected to the cap-binding assay to analyze eIF4E-eIF4G complex formation. Proteins bound to m 7 -GTP-sepharose (m 7 -GTP) as well as total cell lysates (TCL) were analyzed by immunoblotting with indicated antibodies. STAT5-PY: phospho-Y694-STAT5. (J) 32D/TKD cells transduced with Pim-1 or vector control cells, as indicated, were treated with indicated concentrations of BZM for 48 hours and analyzed. (K) Cells indicated were treated with or without 4 ng/ml BZM, as indicated, for 6 hours and analyzed.

Article Snippet: The STAT5 inhibitor pimozide and antibodies against FLT3 (SC-479), STAT5A (SC-1081), and HSP-90 (SC-13119) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Flow Cytometry, Transduction, Plasmid Preparation, Control, Cell Culture, Western Blot, Binding Assay

Journal: Translational Oncology

Article Title: Inhibition of the STAT5/Pim Kinase Axis Enhances Cytotoxic Effects of Proteasome Inhibitors on FLT3-ITD–Positive AML Cells by Cooperatively Inhibiting the mTORC1/4EBP1/S6K/Mcl-1 Pathway 1

doi: 10.1016/j.tranon.2018.11.001

Figure Lengend Snippet: S6K also plays an essential role in acquisition of the bortezomib resistance by FLT3-ITD–expressing leukemic cells. (A) MV4-11 cells were cultured for 48 hours with or without 0.75 ng/ml bortezomib (BZM) and indicated concentrations of PF-4708671. The means of relative viable cell numbers from triplicate measurements expressed as percentages of cells cultured without PF-4708671 are plotted, with error bars indicating standard errors. The asterisks indicate statistically significant differences determined by Student's t test (* P < .05). (B) MV4-11 cells were treated for 48 hours with or without 0.75 ng/ml BZM and 10 μM PF-4708671, as indicated, and analyzed for the cellular DNA content by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content are indicated. (C) MV4-11 cells were treated for 36 hours with or without 0.75 ng/ml BZM and 5 μM PF-4708671 (PF) or 3 μM PIM447 (PIM), as indicated, and analyzed. (D) MV4-11 cells were treated as described for C and analyzed for activation of Bak and Bax by flow cytometry. Red and black lines represent cells treated with or without inhibitors. (E) 32D/ITD cells transduced with Mcl-1 or vector control cells were treated with or without 1.5 ng/ml bortezomib BZM and 10 μM PF, as indicated, for 48 hours and analyzed. (F) MV4-11 cells were treated with or without 2 ng/ml BZM and 10 μM PF, as indicated, for 8 hours and subjected to Western blot analysis with antibodies against indicated proteins. Abbreviations: eIF4B-S422P, phospho-S422-eIF4B; eIF4B-S406P, phospho-S406-eIF4B; 4EBP1-S65P, phospho-S65-4EBP1; S6RP-PS: phospho-S240/244-S6RP. (G) MV4-11 cells were treated for 48 hours with or without 0.75 ng/ml BZM, 7.5 μM PF, or 2 μM PIM, as indicated, and analyzed. (H) MV4-11 cells were treated for 6 hours with or without 3 ng/ml BZM, 10 μM PF, or 3 μM PIM, as indicated, and analyzed.

Article Snippet: The STAT5 inhibitor pimozide and antibodies against FLT3 (SC-479), STAT5A (SC-1081), and HSP-90 (SC-13119) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Cell Culture, Flow Cytometry, Activation Assay, Transduction, Plasmid Preparation, Control, Western Blot

Journal: Translational Oncology

Article Title: Inhibition of the STAT5/Pim Kinase Axis Enhances Cytotoxic Effects of Proteasome Inhibitors on FLT3-ITD–Positive AML Cells by Cooperatively Inhibiting the mTORC1/4EBP1/S6K/Mcl-1 Pathway 1

doi: 10.1016/j.tranon.2018.11.001

Figure Lengend Snippet: A schematic model of intracellular signaling mechanisms regulating proteasome inhibitor-induced apoptosis by FLT3-ITD involving the STAT5/Pim axis and the mTORC1/eIF4F/Mcl-1 pathway.

Article Snippet: The STAT5 inhibitor pimozide and antibodies against FLT3 (SC-479), STAT5A (SC-1081), and HSP-90 (SC-13119) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: