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