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. 2009 Mar 5;113(10):2302-11.
doi: 10.1182/blood-2008-07-167023. Epub 2008 Dec 8.

BH3-only protein Bim more critical than Puma in tyrosine kinase inhibitor-induced apoptosis of human leukemic cells and transduced hematopoietic progenitors carrying oncogenic FLT3

Amanda Nordigården  1 , Maria Kraft, Pernilla Eliasson, Verena Labi, Eric W-F Lam, Andreas Villunger, Jan-Ingvar Jönsson
Affiliations

BH3-only protein Bim more critical than Puma in tyrosine kinase inhibitor-induced apoptosis of human leukemic cells and transduced hematopoietic progenitors carrying oncogenic FLT3

Amanda Nordigården et al. Blood. .

Abstract

Constitutively activating internal tandem duplications (ITD) of FLT3 (FMS-like tyrosine kinase 3) are the most common mutations in acute myeloid leukemia (AML) and correlate with poor prognosis. Receptor tyrosine kinase inhibitors targeting FLT3 have developed as attractive treatment options. Because relapses occur after initial responses, identification of FLT3-ITD-mediated signaling events are important to facilitate novel therapeutic interventions. Here, we have determined the growth-inhibitory and proapoptotic mechanisms of 2 small molecule inhibitors of FLT3, AG1295 or PKC412, in hematopoietic progenitor cells, human leukemic cell lines, and primary AML cells expressing FLT3-ITD. Inactivation of the PI3-kinase pathway, but not of Ras-mitogen-activated protein (MAP) kinase signaling, was essential to elicit cytotoxic responses. Both compounds induced up-regulation of proapoptotic BH3-only proteins Bim and Puma, and subsequent cell death. However, only silencing of Bim, or its direct transcriptional activator FOXO3a, abrogated apoptosis efficiently. Similar findings were made in bone marrow cells from gene-targeted mice lacking Bim and/or Puma infected with FLT3-ITD and treated with inhibitor, where loss of Puma only provided transient protection from apoptosis, but loss of Bim preserved clonal survival upon FLT3-ITD inhibition.

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Figures

Figure 1
Figure 1. Growth stimulatory and antiapoptotic signals in FLT3-ITD–transduced FDC-P1 cells is mediated by the PI-3 kinase/AKT pathway
FDC-P1 cells were retrovirally transduced with human FLT3-ITD. (A) Expression was confirmed by immunoprecipitation and immunoblotting for human FLT3. FDC-P1 cells expressing wild-type FLT3 or FLT3-ITD were cytokine-deprived and analyzed for survival during a 9-day incubation period by annexin V–FITC/PI staining and flow cytometry. One representative experiment of 3 performed yielding similar results is shown. (B) Whole-cell lysates were prepared from FDC-P1/FLT3 and FDC-P1/FLT3-ITD cells after 16 hours of cytokine deprivation followed by stimulation with 50 ng/mL FL, 10 ng/mL IL-3, or no cytokine addition. Western blot analysis was performed for p-AKT and p-FOXO3a. All blots were reprobed with a GAPDH antibody to demonstrate equal loading. (C) FDC-P1/FLT3-ITD cells treated with either 20 μM LY294002 or 50 μM PD98059 for 24 hours were stained for apoptotic cells with annexin V–FITC/PI and analyzed by flow cytometry. (D) Cell-cycle analysis was performed on FDC-P1/FLT3-ITD cells after 16 hours of treatment with either 20 μM LY294002 or 50 μM PD98059. Numbers of viable cells in S-phase were determined by flow cytometry. Bim expression was determined by Western blot analysis. Data are mean (± standard deviation [SD]) from 3 individual experiments performed in duplicates. **P < .01 (SD over control without inhibitor); ***P < .001 (SD).
Figure 2
Figure 2. Inhibition of FLT3-ITD signaling by AG1295 or PKC412 leads to induction of apoptosis, cell-cycle arrest, and inhibition of AKT and ERK phosphorylation
FDC-P1/FLT3-ITD cells were treated for 24 hours with increasing concentrations of AG1295 (A) or PKC412 (B), then stained for FACS analysis with Annexin V–FITC/PI. (C) Cell-cycle analysis was performed on FDC-P1/FLT3-ITD cells after culturing with 10 μM AG1295 for 8 or 24 hours. Data from FACS analyses are mean (± SD) from 3 individual experiments. (D) Whole-cell lysates were prepared from FDC-P1/FLT3-ITD cells incubated for 24 hours in different concentrations of AG1295 and analyzed for p-AKT and p-ERK (1 representative blot is shown; n = 3).
Figure 3
Figure 3. Inhibition of FLT3-ITD signaling by AG1295 or PKC412 leads to dephosphorylation of FOXO3a and up-regulation of proapopotic Bim and Puma
FDC-P1/FLT3-ITD cells were treated for 24 hours with increasing concentrations of AG1295 (A) or PKC412 (B), then whole-cell lysates were prepared and analyzed for expression of Bim, Puma, and caspase-3. (C) FDC-P1/FLT3-ITD cells were treated with AG1295, and the amount of p-FOXO3a and expression of Bim, Puma, and caspase-3 was analyzed by Western blot analysis (n = 3). Anti-GAPDH antibody was used as a loading control. (D-F) FDC-P1/FLT3-ITD cells infected with pBabe:FOXO3(A3):ER were stimulated with 100 nM 4-OHT. After 8 and 24 hours, cells were analyzed for DNA content by PI staining and flow cytometry. Results are mean (± SD) from 2 experiments (D). Cells were also analyzed for apoptosis by flow cytometry after staining with annexin V–FITC/PI. Results are mean (± SD) from duplicates and repeated twice. **P < .01 (SD over control; E). Cell lysates were prepared at times indicated, and Western blot analysis was performed using Bim, Puma, or GAPDH antibodies (n = 2; F).
Figure 4
Figure 4. Treatment with AG1295 or PKC412 of human leukemic cell lines expressing mutated FLT3 leads to up-regulation of Bim and Puma and apoptosis induction
Leukemic cell lines expressing no FLT3 (THP1), wild-type FLT3 (NB4, RS4), or mutated FLT3 (MV4;11, MonoMac-6) were seeded at a density of 105-106 cells/mL in the absence or presence of AG1295 or PKC412. (A) Seventy-two hours later, cells were analyzed for apoptosis by flow cytometry after staining with annexin V–FITC/PI. Data shown are mean (± SD) from 3 experiments. *P < .05 (SD over control without inhibitor); **P < .01 (SD); ***P < .001 (SD). (B) Expression of Bim and Puma protein was analyzed by Western blot analysis after 48 hours of treatment. (C) Real-time PCR using Bim and Puma primers from cells after 24 hours of treatment with 30 μM AG1295 (□) or 50 nM PKC412 (formula image). The results are mean (± SD) from 2 experiments performed in triplicates and presented as relative expression compared with the control housekeeping gene GusB.
Figure 5
Figure 5. Cytotoxic effect of PKC412 on primary AML cells leads to up-regulation of Bim and Puma
Mononuclear cells were prepared from cryopreserved bone marrow or peripheral blood samples of 4 AML patients. (A) Apoptosis assay was performed with PKC412 at 2 high concentrations (100 and 300 nM) on AML cells cultured for 72 hours, then stained with PI, and analyzed by flow cytometry. Data are mean (± SD) from duplicates. Symbols indicate patients as: ■, AML no. 1; □, AML no. 3; ◆, AML no. 2; ◇, AML no. 4. (B) The relative expression levels of Bim (□) and Puma (formula image) mRNA from the same AML patients were measured by real-time PCR after 24 hours of treatment with 30 and 100 nM PKC412. Results are from triplicate reactions.
Figure 6
Figure 6. Gene silencing of Bim or FOXO3a by siRNA prevents apoptosis induced by RTK inhibitor
(A,B) FDC-P1/FLT3-ITD cells were transfected by nucleofection with siRNA specific for mouse Bim (0.7μg) or with a control siRNA (pmaxGFP; 1.5 μg). Four hours after transfection, AG1295 was added as indicated. Cells were harvested after 24 hours and analyzed for effects on Bim expression by Western blot analysis (A) or apoptosis after annexin V–FITC/PI staining and flow cytometry (B). Data shown are mean (+ SD) from 3 individual experiments. (C-F) FOXO3a siRNA oligonucleotides were introduced to FDC-P1/FLT3-ITD cells by nucleofection. Cell lysates were prepared after 24 hours for Western blot analysis (n = 2). In separate experiments, the expression of Bim was analyzed by Western blot analysis in cells transfected with pmaxGFP, 1.3 μg FOXO3a, or Bim siRNA (D). The numbers of viable cells were analyzed by annexin V–FITC/PI staining and flow cytometry after treatment with either AG1295 at 5 μM (E) or PKC412 at 5 nM (F) for 24 hours. The inhibitors were added to cells after 4, 24, or 48 hours after transfection. Data represent mean (± SD) from 2 experiments.
Figure 7
Figure 7. Bim is more critical than Puma in apoptotic induction of FLT3-ITD inhibition and is a direct transcriptional target of FOXO3a in FLT3-ITD cells treated with AG1295
(A,B) FDC-P1/FLT3-ITD cells were transfected with siRNA control pmaxGFP (■) or siRNA to Bim (□), Bim + Puma (formula image), or Puma (formula image). Cell lysates were prepared after 24 hours for Western blot analysis of Puma (A). In separate experiments, cells were transfected with siRNA as indicated, and 4 hours after transfection, AG1295 at 10 μM was added. Twenty-four hours later, cells were analyzed for apoptosis by flow cytometry after staining with annexin V–FITC and PI (B). Data shown are mean (± SD) from 1 representative experiment performed in duplicate and repeated twice. **P < .01 (SD over control siRNA); ***P < .001 (SD over control siRNA). (C,D) Wild-type (■), bim-/- (□), bim-/- puma-/- (formula image), or puma-/-(formula image) bone marrow–derived Lin− progenitor cells infected with FLT3-ITD were FACS-sorted based on expression of EYFP. After treatment with 10 and 20 nM PKC412, the viability was assessed by flow cytometry after 72 hours in culture and compared with cells cultured without treatment (C). The bone marrow cells were also analyzed for colony formation in the absence of supportive cytokine but treated with 10 nM PKC412. Colony numbers were assessed after 7 days of culture (D). Data are mean (± SD) from 3 experiments. *P < .03 (SD compared with bone marrow–derived colonies from bim-/- mice). (E,F) ChIP-quantitative PCR analysis for FOXO3a binding to the Bim promotor. Sonicated DNA from FDC-P1/FLT3-ITD cells treated with AG1295 at 10 μM for 4 and 10 hours was immunoprecipitated with anti-FOXO3a or control rabbit IgG and amplified by quantitative PCR using primers specific for the Bim promotor. Relative expression of Bim was normalized to the input value and then compared with the corresponding untreated samples. Error bars represent SEM of triplicate reactions from 1 representative analysis of 2 separate experiments performed (E). PCRs were also visualized on 1.5% agarose gels stained with ethidium bromide (F).
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