wild type wt thp 1 cells  (ATCC)

Journal: Blood Cancer Journal

Article Title: The combination of CUDC-907 and gilteritinib shows promising in vitro and in vivo antileukemic activity against FLT3-ITD AML

doi: 10.1038/s41408-021-00502-7

Figure Lengend Snippet: A FLT3-ITD AML cell lines MOLM-13 and MV4-11 were treated with CUDC-907 for 4, 8, 12, or 24 h, at variable concentrations. Western blots were generated utilizing whole-cell lysates. Representative blots are shown. Densitometry measurements, normalized to β-actin and compared to vehicle control, are displayed below the corresponding blot. B FLT3-ITD AML cell lines MOLM-13 and MV4-11 were treated with CUDC-907 and/or Z-VAD-FMK (Z-VAD) for 24 h. Western blot analyses of FLT3 and cleaved-caspase 3 (cf-caspase 3) were performed. Densitometry measurements, normalized to β-actin and compared to vehicle control, are displayed below the corresponding blot. C Primary FLT3-ITD positive AML patient sample AML#208 was treated with CUDC-907 for 24 h at variable concentrations. Western blots were generated utilizing whole-cell lysates. Representative blots are shown. Densitometry measurements, normalized to β-actin and compared to vehicle control, are displayed below the corresponding blot. D MV4-11 xenograft models were treated once with CUDC-907 at doses of 100 or 150 mg/kg. After 24 h, mouse bone marrow cells were collected and human CD45+ cells were enriched. Whole-cell lysates were subjected to western blotting. Densitometry measurements, normalized to β-actin and compared to vehicle control, are displayed below the corresponding representative blot. E FLT3-ITD AML cell lines MOLM-13 and MV4-11, a primary FLT3-ITD positive AML patient sample, and the murine MV4-11 xenograft models were treated with CUDC-907 for 24 h. Total RNA was extracted, and real-time RT-PCR was performed. The relative changes in FLT3 transcripts, normalized to GAPDH, in comparison to control samples were quantified. The displayed results represent the mean of three independent experiments, with fold changes calculated via the comparative Ct method and normalized to GAPDH transcripts. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns indicates not significant compared to vehicle control. F MOLM-13 cells were treated with CUDC-907 for 12 h, washed, and then treated with cycloheximide for up to 3 h. Western blots were generated utilizing whole-cell lysates. The FLT3 densitometry fold changes were assessed (comparison to vehicle control and normalized to β-actin). Representative blots are shown on the left side, while densitometry measurements are graphed on the right. * p < 0.05 compared to vehicle control. G FLT3-ITD AML cell lines MOLM-13 and MV4-11 were treated with CUDC-907 alone or in combination with the proteasome inhibitor MG-132 for 24 h. Western blot analyses of FLT3 were performed, with densitometry results compared to vehicle control and normalized to β-actin.

Article Snippet: FLT3 wild-type (FLT3-wt) AML cell line THP-1 was purchased from ATCC (2014 and 2002, respectively), while OCI-AML3 was purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany; 2011).

Techniques: Western Blot, Generated, Control, Quantitative RT-PCR, Comparison

Journal: Blood Cancer Journal

Article Title: The combination of CUDC-907 and gilteritinib shows promising in vitro and in vivo antileukemic activity against FLT3-ITD AML

doi: 10.1038/s41408-021-00502-7

Figure Lengend Snippet: A FLT3-ITD AML cell lines MOLM-13 and MV4-11 were treated with or without CUDC-907, after 12 h gilteritinib was introduced to the indicated samples, for a total CUDC-907 treatment duration of 24 h. Samples were then stained using annexin V/PI, and analysis performed via flow cytometry. Representative experiments are displayed, with combination index (CI) calculated using CompuSyn software. ***p < 0.001 compared to single-drug treatments. Individual CI values are shown in Supplementary Table . B FLT3-ITD AML cell lines MOLM-13 and MV4-11 were treated with gilteritinib, CUDC-907, both, or neither, for 24 h concurrently, and then annexin V/PI staining and flow cytometry analysis were performed. Representative results from one experiment are displayed. C MOLM-13 and MV4-11 cells were treated with gilteritinib, CUDC-907, both, or neither, for 24 h concurrently, and then whole-cell lysates underwent western blot analysis of cleaved-caspase 3 (cf-caspase 3) and β-actin. D MV4-11 cells were treated with stepwise increasing concentrations of cytarabine to generate MV4-11 cells with acquired cytarabine resistance (designated MV4-11/AraC-R). MV4-11/AraC-R cells were cultured in the presence or absence of the AraC concentration used to maintain resistance (1100 nM) for 5 days. Then the AraC resistant cells and parental cells were treated with variable concentrations of AraC for 72 h. Viable cells were determined using the MTT assay. Representative curves are shown on the left. IC50s were calculated and are presented on the right. *** p < 0.001 compared to the parental cells. MV4-11/AraC-R cells were treated with vehicle control, CUDC-907, gilteritinib, or in combination for 24 h. Cells were stained with annexin V/PI and subjected to flow cytometry analyses. CI values were calculated using CompuSyn software. Individual CI values are shown in Supplementary Table . *** p < 0.001 compared to control and single-drug treatment. E Western blots of FLT3 were generated utilizing whole-cell lysates from AML cell lines MOLM-13, MV4-11, OCI-AML3, and THP-1. Densitometry results compared to MOLM-13 and normalized to β-actin are shown. F OCI-AML3 and THP-1 cells were treated with CUDC-907 for 24 h. Whole-cell lysates were subjected to western blotting. Densitometry results for FLT3 compared to vehicle control and normalized to β-actin are shown. G The FLT3-wt AML cell lines THP-1 and OCI-AML3 were treated with CUDC-907, gilteritinib, both, or neither, for 24 h concurrently, and then underwent annexin V/PI staining and flow cytometry analysis. CI values were calculated using CompuSyn software. Individual CI values are shown in Supplementary Table . * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to single-drug treatment. H , I NSGS mice were injected with 1 × 10 6 MV4-11 cells/mouse via the tail vein on day 0. Mice were then randomized to one of four treatment groups: vehicle control (3% 200 proof ethanol, 1% polyoxyethylene sorbitan monooleate, and USP water; n = 5), CUDC-907 (100 mg/kg; n = 5), gilteritinib (40 mg/kg; n = 5), or combination (100 mg/kg CUDC-907 plus 40 mg/kg gilteritinib; n = 6). Treatment was initiated on day 3, with daily gilteritinib dosing and 5 days of daily CUDC-907 dosing followed by 2 days off, for a total of 28 treatment days. Daily body weights were measured and graphed as the mean for each group ( H ). Overall proportion of mice in each treatment group surviving, estimated via Kaplan–Meier method, is shown in panel ( I ). One mouse in the CUDC-907 group was excluded due to a drug-independent technical issue. NR indicates not reached.

Article Snippet: FLT3 wild-type (FLT3-wt) AML cell line THP-1 was purchased from ATCC (2014 and 2002, respectively), while OCI-AML3 was purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany; 2011).

Techniques: Staining, Flow Cytometry, Software, Western Blot, Cell Culture, Concentration Assay, MTT Assay, Control, Generated, Injection

Journal: Blood Cancer Journal

Article Title: The combination of CUDC-907 and gilteritinib shows promising in vitro and in vivo antileukemic activity against FLT3-ITD AML

doi: 10.1038/s41408-021-00502-7

Figure Lengend Snippet: A Primary AML patient samples (FLT3-ITD positive) were treated with gilteritinib, CUDC-907, both, or neither, for 24 h concurrently, and then annexin V/PI staining and flow cytometry analysis was performed. CI values were calculated using CompuSyn software. Individual CI values are shown in Supplementary Table . B Three primary patient samples (FLT3-ITD positive) were treated with gilteritinib and/or CUDC-907 for 24 h and then whole-cell lysates were subjected to western blot analysis of cleaved-caspase 3 (cf-caspase 3) and β-actin. C , D Six FLT3-wt AML patient samples (panel C ) and two samples of normal peripheral blood mononuclear cells (panel D ) were treated with gilteritinib, CUDC-907, both, or neither, for 24 h concurrently, and then annexin V/PI staining and flow cytometry analysis were performed. CI values were calculated using CompuSyn software. Individual CI values are shown in Supplementary Table . * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to single-drug treatments. E , F Primary AML patient samples (panel E ) and normal human umbilical cord blood cells (panel F ) were treated with gilteritinib, CUDC-907, both, or vehicle control for 24 h concurrently, and then plated in methylcellulose. The number of leukemic colonies (AML-CFUs), erythroid, and myeloid colonies were counted 10–14 days later. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.0,1 and *** p < 0.001 compared to control. # p < 0.05 and ### p < 001 compared to single-drug treatments. Technical triplicates were performed.

Article Snippet: FLT3 wild-type (FLT3-wt) AML cell line THP-1 was purchased from ATCC (2014 and 2002, respectively), while OCI-AML3 was purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany; 2011).

Techniques: Staining, Flow Cytometry, Software, Western Blot, Control

Journal: Blood Cancer Journal

Article Title: The combination of CUDC-907 and gilteritinib shows promising in vitro and in vivo antileukemic activity against FLT3-ITD AML

doi: 10.1038/s41408-021-00502-7

Figure Lengend Snippet: A FLT3-ITD AML cell lines MOLM-13 and MV4-11 were treated with gilteritinib, CUDC-907, both, or neither for 4, 8, 12, or 24 h, at concentrations of 12.5 or 25 nM, as indicated. Western blots were generated using whole-cell lysates. One representative blot is shown. Densitometry measurements, compared to vehicle control and normalized to β-actin, are shown below the corresponding blots. B MOLM-13 and MV4-11 cells were treated as described in panel A and then stained with annexin V/PI and subjected to flow cytometry analysis. ** p < 0.01 and *** p < 0.001 compared to single-drug treatments at the same timepoint. C MOLM-13, MV4-11, and primary FLT3-ITD positive AML patient sample AML#207 were treated with gilteritinib, CUDC-907, both, or vehicle control for 24 h at the indicated concentrations. Total RNA was extracted, and FLT3 transcripts relative to GAPDH were determined by real-time RT-PCR. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to vehicle control. D , E MOLM-13 cells were treated with CUDC-907 and/or gilteritinib for 12 h, washed, and then treated with cycloheximide for up to 3 h. Western blots were generated utilizing whole-cell lysates. The fold changes for the FLT3 densitometry were assessed via comparison to vehicle control and normalized to β-actin. Representative blots are shown in panel ( D ), while densitometry measurements are graphed in panel ( E ). ** p < 0.01, *** p < 0.001, and ns indicates not significant.

Article Snippet: FLT3 wild-type (FLT3-wt) AML cell line THP-1 was purchased from ATCC (2014 and 2002, respectively), while OCI-AML3 was purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany; 2011).

Techniques: Western Blot, Generated, Control, Staining, Flow Cytometry, Quantitative RT-PCR, Comparison

Journal: Blood Cancer Journal

Article Title: The combination of CUDC-907 and gilteritinib shows promising in vitro and in vivo antileukemic activity against FLT3-ITD AML

doi: 10.1038/s41408-021-00502-7

Figure Lengend Snippet: A MV4-11 cells were treated with gilteritinib, CUDC-907, both, or neither for 4, 8, 12, or 24 h. Western blots were generated utilizing whole-cell lysates, with representative blots shown, and densitometry displayed below each blot. Densitometry was assessed via comparison to vehicle control and normalized to β-actin. B A primary FLT3-ITD positive AML patient sample was treated with gilteritinib and/or CUDC-907 for 24 h. Densitometry was assessed via comparison to vehicle control and normalized to β-actin. C , D MOLM-13 and MV4-11 cells were treated using CUDC-907 either with or without AZD1480, a selective JAK2 inhibitor, for 24 h. Flow cytometry analysis of annexin V/PI stained cells is shown in the upper panels and western blot analyses of phosphorylated STAT5 are shown in the lower panels. *** p < 0.001 compared to single-drug treatments. E MOLM-13, MV4-11, and primary patient sample AML#213 were treated with gilteritinib, CUDC-907, both, or vehicle control for 24 h. Western blotting was performed to analyze expression of members of the Bcl-2 family. Densitometry was measured via comparison to vehicle control and normalized to β-actin. F Mcl-1 overexpression and red fluorescent protein (RFP) control MV4-11 cells were generated using lentivirus particles as described in the “Methods” section. Whole-cell lysates were subjected to western blotting to confirm overexpression (upper panel). The cells were then treated with vehicle control, gilteritinib, CUDC-907, or in combination for 24 h, and then annexin V/PI staining and flow cytometry analysis were performed (lower panel). *** p < 0.001 compared to RFP under the same drug treatment. G , H shRNA knockdown of Bim, Bak, and Bax, or non-template control (NTC) was performed in MV4-11 cells. Whole-cell lysates were subjected to western blotting (upper panels). Cells were treated with vehicle control, gilteritinib, CUDC-907, or in combination for 24 h. Annexin V/PI staining and flow cytometry analysis results are shown (lower panels). *** p < 0.001 compared to NTC for the same drug treatment.

Article Snippet: FLT3 wild-type (FLT3-wt) AML cell line THP-1 was purchased from ATCC (2014 and 2002, respectively), while OCI-AML3 was purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany; 2011).

Techniques: Western Blot, Generated, Comparison, Control, Flow Cytometry, Staining, Expressing, Over Expression, shRNA, Knockdown