1. Pim-1 is up-regulated by constitutively activated FLT3 and plays a role in FLT3-mediated cell survival
by Kyu-Tae Kim, Kristin Baird, Joon-Young Ahn, Paul Meltzer, Michael Lilly, Mark Levis, and Donald Small
Blood
Volume 105(4):
February 15, 2005
©2005 by American Society of Hematology

2. CEP-701 inhibits FLT3 autophosphorylation reversibly and induces cytotoxicity in EOL-1 cells.
CEP-701 inhibits FLT3 autophosphorylation reversibly and induces cytotoxicity in EOL-1 cells. (A) EOL-1 cells were incubated with increasing concentrations of CEP-701. The graph shows cytotoxicity assessed by MTT assay after 48 hours. The inset shows whole-cell extracts (500 μg/sample) that were prepared after 1 hour of exposure to CEP-701 and immunoprecipitated with anti-human FLT3 antibody, followed by separation in 8% polyacrylamide gel electrophoresis and immunoblotting with antiphosphotyrosine antibody. The membrane was stripped and reprobed with anti-FLT3 antibody to demonstrate equal loading of FLT3 in each lane. (B) Aliquots of EOL-1 cells were incubated with 50 nM CEP-701 for 4 hours and 8 hours. Additional cell aliquots were then washed by PBS to remove the inhibitor and incubated for an additional 4 and 8 hours. Most of the cells from each of these time points were harvested for RNA preparation for microarray analysis. A small fraction was harvested for analysis of FLT3 phosphorylation status as shown.
Kyu-Tae Kim et al. Blood 2005;105:
©2005 by American Society of Hematology

3. Gene expression changes in EOL-1, MV4-11, and MOLM-14 cells treated with CEP-701.
Gene expression changes in EOL-1, MV4-11, and MOLM-14 cells treated with CEP-701. RNAs harvested from cell cultures treated as shown in Figure 1B were hybridized to cDNA microarray slides to examine changes in gene expression. RNA from uninhibited cells was labeled with Cy-5 dUTPs (green fluorescence signal). RNAs from inhibited cells or cells after inhibitor washout were labeled with Cy-3 dUTPs (red fluorescence signal). The uninhibited cell RNA was used as the control to examine changes in inhibited cell RNAs after 4 and 8 hours of inhibition or 4 hours and 8 hours release after inhibition. The inhibited RNA after 4 and 8 hours of inhibition or 4 hours and 8 hours of release after inhibition were represented as 4 hours inhibited, 8 hours inhibited, 4 hours released, and 8 hours released, respectively. The figure shows a pseudocolor representation of the intensity ratios of the raw data from the 2-channel fluorescent images. These data are quality filtered, and the top 15 genes ordered by degree of decreased expression in the EOL-1 cells inhibited by CEP-701 for 8 hours are shown.
Kyu-Tae Kim et al. Blood 2005;105:
©2005 by American Society of Hematology

4. Pim-1 decreases in response to FLT3 inhibition in EOL-1, MV4-11, TF1/ITD, BaF3/ITD, and 32D/ITD cells.
Pim-1 decreases in response to FLT3 inhibition in EOL-1, MV4-11, TF1/ITD, BaF3/ITD, and 32D/ITD cells. Cell lines were incubated for increasing times or with increasing concentrations of CEP-701 or CEP RNA was then harvested and used for Pim-1 expression analysis by QPCR. GAPDH or ribosomal S16 gene expression were used as internal controls to normalize the level of Pim-1 expression. Inhibition of Pim-1 expression by 50 nM CEP-701 with increasing time of treatment in (A) the constitutively activated FLT3-expressing human leukemia-derived cell lines, EOL-1, MV4-11, TF1/ITD; and (B) the constitutively activated FLT3/ITD-transformed murine cell lines, BaF3/ITD and 32D/ITD. (C) Changes in Pim-1 expression in EOL-1 cells after treatment and release from 50 nM CEP-5214 inhibition. EOL-1/C indicates control; EOL-1/4h, 4-hour inhibition; EOL-1/4R, 4-hour release after 4 hours of inhibition; EOL-1/8h, 8-hour inhibition; EOL-1/8R, 8-hour release after 8 hours of inhibition. (D) Pim-1 response in BaF3/ITD cells to increasing concentrations of CEP-701 treatment for 2 hours.
Kyu-Tae Kim et al. Blood 2005;105:
©2005 by American Society of Hematology

5. Pim-1 protein expression decreases in response to FLT3 inhibition.
Pim-1 protein expression decreases in response to FLT3 inhibition. EOL-1, TF1/ITD, and BaF3/ITD cells were incubated with 50 nM CEP-701 or CEP-5214 for 0 to 6 hours or incubated with an increasing concentration of the inhibitors for 2 hours. Cell extracts were immunoprecipitated with anti-Pim-1 antibody followed by immunoblotting with the same antibody. (A) Time course of treatment with 50 nM CEP-701 or CEP-5214 in EOL-1, TF1/ITD, and TF1 (treated with GM-CSF) cells. (B) Dose response to increasing concentrations of CEP-701 or CEP-5214 treatment of EOL-1 and TF1/ITD cells for 2 hours. (C) Changes in Pim-1 expression, FLT3 phosphorylation, and expression in BaF3/ITD cells in response to treatment with 50 nM CEP-701 for increasing times. Note that mouse Pim-1 also expresses a 44-kDa isoform. (D) Pim-1 expression in BaF3 cells treated with IL-3 plus 50 nM CEP-701 for increasing times.
Kyu-Tae Kim et al. Blood 2005;105:
©2005 by American Society of Hematology

6. Pim-1 is expressed in BaF3/ITD cells but not by FL stimulation of BaF3/FLT3 cells.
Pim-1 is expressed in BaF3/ITD cells but not by FL stimulation of BaF3/FLT3 cells. Comparison of Pim-1 expression in BaF3 cells, FLT3-transformed BaF3 cells, and FLT3/ITD-transformed BaF3 cells treated with or without IL-3, FL, or CEP-701. Cell extracts (100 μg/sample) were resolved by 12% polyacrylamide gel electrophoresis and immunoblotted with anti-Pim-1 antibody. Lanes 1, 5, and 9 have no cytokines or CEP-701 treatment; lanes 2, 6, and 10, IL-3 (1 ng/mL) for 2 hours; lanes 3, 7, 11, FL (10 ng/mL) for 2 hours; lanes 4, 8, 12, IL-3 (1 ng/mL) and 50 nM CEP-701 for 2 hours; and lane 13, 50 nM CEP-701 for 2 hours.
Kyu-Tae Kim et al. Blood 2005;105:
©2005 by American Society of Hematology

7. Expression of Pim-1 constructs in stably transfected BaF3 cells.
Expression of Pim-1 constructs in stably transfected BaF3 cells. (A) Expression of Pim-1 constructs was detected by monoclonal anti-Pim-1 antibody from abstracts of transfected cell lines. Extracts (100 μg/sample) were resolved by separation in 12% polyacrylamide gels and immunoblotted with anti-Pim-1 antibody. Lane 1 shows BaF3 cells deprived of IL-3 for 8 hours; lane 2, BaF3/ITD; lane 3, Pim-1/44 (44 kDa); lane 4, Pim-1/33 (33 kDa); lane 5, Pim-1/NT81 (27.5 kDa). (B) Expression of Pim-1, FLT3, and Actin after treatment with or without CEP-701 for 1 hour. Pim-1 and Actin were directly assessed by immunoblotting with the anti-Pim-1 and anti-Actin antibodies. FLT/ITD expression and phosphorylation were detected by immunoprecipitation with FLT3 antibody (S-18) followed by immunoblotting with 4G10.
Kyu-Tae Kim et al. Blood 2005;105:
©2005 by American Society of Hematology

8. Enforced Pim-1 expression renders BaF3/ITD cells more resistant to cytotoxicity induced by CEP-701 while dominant-negative Pim-1 expression inhibits colony growth.
Enforced Pim-1 expression renders BaF3/ITD cells more resistant to cytotoxicity induced by CEP-701 while dominant-negative Pim-1 expression inhibits colony growth. (A) Triplicate samples of cells were incubated with increasing concentration of CEP-701 for 24 hours, and cytotoxicity was assessed by MTT assay. The graph displays the MTT results for each cell line normalized to the untreated controls. Error bars represent standard deviation from 3 independent experiments. (B) Pim-1/NT81 inhibits colony formation by FLT3/ITD. BaF3/ITD cells were electroporated with 15 μg of the expression vectors for the N-terminal deletion form of Pim-1 (Pim-1/NT81) or control vector (pLXSN). One day after electroporation, 100, 500, and 2500 cells were seeded on triplicate dishes containing fetal calf serum (FCS), IMDM, 1% methylcellulose, and G418. The colonies were grown at 37°C in 5% CO2 and counted on day 7. The mean colony numbers are counted from triplicate dishes of 100, 500, and 2500 plated cells. The graph shows the relative mean colony number difference and standard deviation of Pim-1/NT81 transfectants compared with control vector transfectants from each group from 3 independent experiments. The inset shows one each of the 10-mm culture dishes from control vector and Pim-1/NT81 electroporations. (C) Cells were incubated with increasing concentrations of CEP-701 for 24 hours and assayed for viability by annexin V and 7-AAD binding by fluorescence activated cell sorting (FACS) analysis. The graph displays the percentage of cell death induced in each cell line by the treatment normalized to the untreated controls. Error bars represent standard deviation from 3 independent experiments.
Kyu-Tae Kim et al. Blood 2005;105:
©2005 by American Society of Hematology

9. Pim-1 is overexpressed in FLT3/ITD-positive samples from patients with AML, and its expression is decreased after treatment with CEP-701.
Pim-1 is overexpressed in FLT3/ITD-positive samples from patients with AML, and its expression is decreased after treatment with CEP-701. (A) Samples from patients with AML were analyzed for Pim-1 expression by QPCR and compared with expression in normal bone marrow. The graph shows the relative expression levels of Pim-1 normalized to GAPDH in FLT3/ITD, FLT3/WT (wild-type) AML samples and normal bone marrow samples. The results show the mean and standard deviation for duplicate QPCR results from 3 independent experiments. The data were analyzed by Student t test and P values are shown. (B) Two FLT3/ITD-positive AML patient blast samples (patient 1 and patient 2) were incubated with 50 nM CEP-701 for 6 hours, and total RNA was harvested for QPCR. The results show the mean and standard deviation for triplicate QPCR results from 3 independent experiments.
Kyu-Tae Kim et al. Blood 2005;105:
©2005 by American Society of Hematology