Journal: Clinical Cancer Research

Article Title: Reversal of Acquired Drug Resistance in FLT3-Mutated Acute Myeloid Leukemia Cells via Distinct Drug Combination Strategies

doi: 10.1158/1078-0432.ccr-13-2052

Figure Lengend Snippet: Figure 1. Acquired point mutations of FLT3 TKDs are associated with sorafenib resistance. A, cDNA- based mutation analysis was performed using cDNA sequencing with multiple primers in sorafenib- resistant cells (Ba/F3-ITD-Res) and parental cells Ba/F3-ITD. B, engineered cells with point mutations were exposed to varying concentrations of sorafenib for 48 hours, and apoptosis induction and cell-growth inhibition were assessed as the percentage of annexin V–positive cells by flow cytometry and by counting the numbers of viable cells using the Trypan blue dye exclusion method, respectively. Growth inhibition was expressed as percentage relative to that in the control group. Data are the mean of 3 independent determinations. C, resistant cells and their parental cells Ba/F3-ITD were treated with sorafenib for 2 hours, and phosphorylation levels of FLT3 and its downstream proteins were measured by immunoblot analysis. The ratios are semiquantitative analyses indicating the expression levels of phosphorylated proteins compared with their total proteins and normalized to the first control lane, which was defined as 1. Sora, sorafenib; p-, phosphorylated.

Article Snippet: Rabbit polyclonal antibodies were purchased from the following sources: phospho-FLT3 (Tyr589/591), phospho-ERK (Thr202/Tyr204), phospho-AKT (Ser473), phospho-S6K (Ser240/Ser244), phospho-PI3K, S6K, and cleaved caspase-3 all from Cell Signaling Technology; Bim from Calbiochem Co.; FLT3 from Santa Cruz Biotechnology; and Stat5A/B from R&D Systems.

Techniques: Mutagenesis, Sequencing, Inhibition, Cytometry, Control, Phospho-proteomics, Western Blot, Expressing

Journal: Clinical Cancer Research

Article Title: Reversal of Acquired Drug Resistance in FLT3-Mutated Acute Myeloid Leukemia Cells via Distinct Drug Combination Strategies

doi: 10.1158/1078-0432.ccr-13-2052

Figure Lengend Snippet: Figure 2. Type I kinase inhibitors retain sensitivity to sorafenib-resistant cells. A, sorafenib-resistant cells were exposed to the indicated concentrations of AC220 for 48 hours and apoptosis induction and cell-growth inhibition were measured by flow cytometry and Trypan blue dye exclusion method, respectively. B, sensitivity of resistant cells and their parental Ba/F3-ITD cells to the indicated inhibitor was assessed as described in Fig. 1. C, sensitivity of novel type I inhibitor crenolanib to sorafenib-resistant cells was assessed by exposing the cells in crenolanib for 72 hours as described in Fig. 1B. D, phosphorylation levels of FLT3 and its downstream proteins ERK and AKT were determined by immunoblot analysis after 24 hours of treatment with crenolanib. The ratios of semiquantitative analyses indicated the expression levels of phosphorylated proteins to their respective total proteins or housekeeping protein GAPDH.

Article Snippet: Rabbit polyclonal antibodies were purchased from the following sources: phospho-FLT3 (Tyr589/591), phospho-ERK (Thr202/Tyr204), phospho-AKT (Ser473), phospho-S6K (Ser240/Ser244), phospho-PI3K, S6K, and cleaved caspase-3 all from Cell Signaling Technology; Bim from Calbiochem Co.; FLT3 from Santa Cruz Biotechnology; and Stat5A/B from R&D Systems.

Techniques: Inhibition, Cytometry, Phospho-proteomics, Western Blot, Expressing

Journal: Clinical Cancer Research

Article Title: Reversal of Acquired Drug Resistance in FLT3-Mutated Acute Myeloid Leukemia Cells via Distinct Drug Combination Strategies

doi: 10.1158/1078-0432.ccr-13-2052

Figure Lengend Snippet: Figure 3. Combination of type I and type II kinase inhibitors or 2 type I inhibitors shows synergistic proapoptotic effects in sorafenib-resistant FLT3-mutant cell lines. A, the sorafenib-resistant and parental cells were treated with a combination of crenolanib and sorafenib at indicated concentrations for 48 hours. Apoptosis induction was determined as described in Fig. 1B. , P < 0.01, , P < 0.001. B, the modulation of correlative proteins was determined by using immunoblot analysis after 1 hour of single-agent/combination treatments. C, FLT3 with ITD plus Y842C mutant protein was isolated using immunoprecipitation (IP) with anti-FLT3 antibodies and in vitro kinase assay was performed in the presence/absence of crenolanib (0.5 mmol/L) and/or sorafenib (0.5 mmol/L). Phosphorylation levels of FLT3 were determined by anti- phospho-FLT3 antibodies using immunoblotting. D, sorafenib-resistant cells Ba/F3-ITDþ676/842 were treated with indicated single or multiple agents for 48 hours. Apoptosis induction was determined as described in Fig. 1B. creno, crenolanib; , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: Rabbit polyclonal antibodies were purchased from the following sources: phospho-FLT3 (Tyr589/591), phospho-ERK (Thr202/Tyr204), phospho-AKT (Ser473), phospho-S6K (Ser240/Ser244), phospho-PI3K, S6K, and cleaved caspase-3 all from Cell Signaling Technology; Bim from Calbiochem Co.; FLT3 from Santa Cruz Biotechnology; and Stat5A/B from R&D Systems.

Techniques: Mutagenesis, Western Blot, Isolation, Immunoprecipitation, In Vitro, Kinase Assay, Phospho-proteomics

Journal: Clinical Cancer Research

Article Title: Reversal of Acquired Drug Resistance in FLT3-Mutated Acute Myeloid Leukemia Cells via Distinct Drug Combination Strategies

doi: 10.1158/1078-0432.ccr-13-2052

Figure Lengend Snippet: Figure 5. Combination of crenolanib with sorafenib demonstrates synergistic/additive proapoptotic effects in primary human AML cells with FLT3 ITD mutations in vitro. A, primary AML mononuclear cells were exposed to the indicated agents for 48 hours and apoptosis induction was determined as described in Fig. 1B after gating on CD34 or CD33 positive populations. B, relevant proteins were analyzed by immunoblot analysis after treating the primary AML cells with indicated agents for 16 hours. The ratios of semiquantitative analyses indicated the expression levels of phosphorylated proteins to their respective total proteins or housekeeping protein GAPDH.

Article Snippet: Rabbit polyclonal antibodies were purchased from the following sources: phospho-FLT3 (Tyr589/591), phospho-ERK (Thr202/Tyr204), phospho-AKT (Ser473), phospho-S6K (Ser240/Ser244), phospho-PI3K, S6K, and cleaved caspase-3 all from Cell Signaling Technology; Bim from Calbiochem Co.; FLT3 from Santa Cruz Biotechnology; and Stat5A/B from R&D Systems.

Techniques: In Vitro, Western Blot, Expressing

Journal: Immunology and cell biology

Article Title: Constitutive Flt3 signaling impacts conventional dendritic cell function.

doi: 10.1111/imcb.12757

Figure Lengend Snippet: Figure 1. Flt3 expression by splenic DCs. (a) Representative histograms and quantification of surface Flt3 on splenocytes as determined by flow cytometry. Data are collated from two independent experiments, with cells isolated from two pooled spleens in each. The gMFI has been normalized to the maximum signal for each cell type. Bars are mean + SEM. (b) Purified cells were analyzed by immunoblotting and probed with anti-Flt3 (A2F10) or anti-Actin (A5060) antibodies. Blot is representative of three independent experiments. Band intensities of the higher molecular weight (MW) species and lower MW Flt3 were quantified relative to Actin. Bars are mean + SEM. (c) Splenic cDCs were isolated and stimulated with (+) or without () Flt3L (100 ng mL1) for 1 h at 37°C. Flt3 surface expression was determined by flow cytometry. Histograms are representative of three independent experiments. Symbols represent individual mice. Bars are mean SD, unpaired t-test. ****P < 0.0001 (d) Splenic cDCs were isolated and stimulated for 0, 3 or 16 h with CpG 1668 ODN (0.5 lM) at 37°C. Cell lysates were examined by immunoblotting and probing with anti-Flt3 (A2F10) or anti-tubulin (DM1A) antibodies. Blot is from one experiment. (e) Splenic cDCs were isolated and stimulated overnight with (+) or without () CpG 1668 ODN (0.5 lM) at 37°C. (f) Mice were injected intravenously with PBS (-CpG) or CPG 1668 ODN (0.2 lM) and spleens were harvested 1 day later or (g) at indicated times. (e–g) Surface expression of Flt3 was determined by flow cytometry. Histograms show representative surface expression. Symbols represent individual mice. (e, f) Data are from at least three independent experiments with the gMFI signal normalized to the maximum gMFI signal. Bars are mean SD, unpaired t-test, ****P < 0.0001. (g) Data are from one experiment. Bars are mean SD, one-way ANOVA with Dunnett’s multiple comparisons test. ***P < 0.001; **P < 0.01, ns not significant.

Article Snippet: Membranes were stained with primary antibodies against Flt3 (AF768, R&D Systems), phosphorylated tyrosine (4G10, Sigma-Aldrich), actin (A5060, Sigma-Aldrich) or tubulin (DM1A, Abacm) followed by HRP-conjugated secondary antibodies. cDC activation For in vitro activation assays, cDCs were isolated and incubated overnight in complete RPMI with or without 0.5 lM CpG 1668 ODN (Bioneer) at 37°C or 4°C.

Techniques: Expressing, Cytometry, Isolation, Western Blot, Molecular Weight, Injection

Journal: Immunology and cell biology

Article Title: Constitutive Flt3 signaling impacts conventional dendritic cell function.

doi: 10.1111/imcb.12757

Figure Lengend Snippet: Figure 2. Flt3-ITD expands splenic DC populations. (a) Representative gating used to identify Flt3+/+ and Flt3ITD/ITD splenic pDC (BST-2+ CD11cint), cDC1 (CD11c+ CD8+ CD11b), cDC2 (CD11c+ CD8 CD11b+), DP cDC1 (CD11c+ CD8+ CD11b CD103+ CD86+), NC cDC1 (CD11c+ CD8+

Article Snippet: Membranes were stained with primary antibodies against Flt3 (AF768, R&D Systems), phosphorylated tyrosine (4G10, Sigma-Aldrich), actin (A5060, Sigma-Aldrich) or tubulin (DM1A, Abacm) followed by HRP-conjugated secondary antibodies. cDC activation For in vitro activation assays, cDCs were isolated and incubated overnight in complete RPMI with or without 0.5 lM CpG 1668 ODN (Bioneer) at 37°C or 4°C.

Techniques: