Journal of the National Cancer Institute Advance Access originally published online on January 29, 2008
JNCI Journal of the National Cancer Institute 2008 100(3):184-198; doi:10.1093/jnci/djm328
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© The Author 2008. Published by Oxford University Press.
ARTICLES |
Mutant FLT3: A Direct Target of Sorafenib in Acute Myelogenous Leukemia
Affiliations of authors: Section of Molecular Hematology and Therapy, Department of Stem Cell Transplantation and Cellular Therapy (WZ, MK, YxS, TM, XL, MA), and Leukemia Department (DH, ZE, AQC, JC, MA), The University of Texas M. D. Anderson Cancer Center, Houston, TX; Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD (DS)
Correspondence to: Michael Andreeff, MD, PhD, Section of Molecular Hematology and Therapy, Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Unit 448, Houston, TX 77030 (e-mail: mandreef{at}mdanderson.org).
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ABSTRACT |
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Background: Internal tandem duplication (ITD) mutations in the juxtamembrane domain–coding sequence of the Fms-like tyrosine kinase 3 (FLT3) gene have been identified in 30% of acute myeloid leukemia (AML) patients and are associated with a poor prognosis. The kinase inhibitor sorafenib induces growth arrest and apoptosis at much lower concentrations in AML cell lines that harbor FLT3-ITD mutations than in AML cell lines with wild-type FLT3.
Methods: The antileukemic activity of sorafenib was investigated in isogenic murine Ba/F3 AML cell lines that expressed mutant (ITD, D835G, and D835Y) or wild-type human FLT3, in primary human AML cells, and in a mouse leukemia xenograft model. Effects of sorafenib on apoptosis and signaling in AML cell lines were investigated by flow cytometry and immunoblot analysis, respectively, and the in vivo effects were determined by monitoring the survival of leukemia xenograft–bearing mice treated with sorafenib (groups of 15 mice). In a phase 1 clinical trial, 16 patients with refractory or relapsed AML were treated with sorafenib on different dose schedules. We determined their FLT3 mutation status by a polymerase chain reaction assay and analyzed clinical responses by standard criteria. All statistical tests were two-sided.
Results: Sorafenib was 1000- to 3000-fold more effective in inducing growth arrest and apoptosis in Ba/F3 cells with FLT3-ITD or D835G mutations than in Ba/F3 cells with FLT3-D835Y mutant or wild-type FLT3 and inhibited the phosphorylation of tyrosine residues in ITD mutant but not wild-type FLT3 protein. In a mouse model, sorafenib decreased the leukemia burden and prolonged survival (median survival in the sorafenib-treated group vs the vehicle-treated group = 36.5 vs 16 days, difference = 20.5 days, 95% confidence interval = 20.3 to 21.3 days; P = .0018). Sorafenib reduced the percentage of leukemia blasts in the peripheral blood and the bone marrow of AML patients with FLT3-ITD (median percentages before and after sorafenib: 81% vs 7.5% [P = .016] and 75.5% vs 34% [P = .05], respectively) but not in patients without this mutation.
Conclusion: Sorafenib may have therapeutic efficacy in AML patients whose cells harbor FLT3-ITD mutations.
Prior knowledge The kinase inhibitor sorafenib induces growth arrest and apoptosis at much lower concentrations in acute myeloid leukemia (AML) cell lines that harbor Fms-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) mutations than in AML cell lines with wild-type FLT3. Study design In vitro assays in isogenic murine AML cell lines that expressed mutant (ITD, D835G, or D835Y) or wild-type human FLT3 and in primary human AML cells, in vivo mouse leukemia xenograft model, and correlative studies in an ongoing phase 1 trial of the therapeutic efficacy of sorafenib in 16 AML patients with known FLT3 gene mutation status. Contribution Sorafenib preferentially induced growth arrest and apoptosis of FLT3-ITD mutant murine AML cells, prolonged survival of mice bearing FLT3-ITD xenografts, and reduced the percentage of leukemia blasts in the peripheral blood and bone marrow of AML patients harboring FLT3-ITD mutations. Implications Sorafenib may have therapeutic efficacy in AML patients whose cells harbor FLT3-ITD mutations. Limitations Discontinuation of sorafenib administration led to AML recurrence. Long-term culture in vitro with low doses of sorafenib might induce resistance to this compound. The bone marrow microenvironment might reduce the proapoptotic efficacy of sorafenib in AML cells.
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The Fms-like tyrosine kinase 3 (FLT3) gene encodes a receptor that, when activated by its ligand (FLT3 ligand), supports the survival, proliferation, and differentiation of primitive hematopoietic progenitor cells. Activating mutations in the FLT3 gene, including internal tandem duplications (ITDs) and missense point mutations in the tyrosine kinase domain, are the most frequently observed molecular abnormalities in blood cells of patients with acute myeloid leukemia (AML); these mutations lead to overexpression or constitutive activation of the tyrosine kinase (1). Internal tandem duplications of the juxtamembrane domain in the FLT3 gene are associated with a poor prognosis (2). Recently, activating point mutations that involve the D835 residue in the second tyrosine kinase domain were found to be associated with worse disease-free survival in young AML patients (3). One of the downstream targets of activating FLT3 mutations is the Raf/MEK/ERK signaling pathway (4). We have previously shown that extracellular signal-regulated kinase (ERK) is constitutively phosphorylated in the majority of primary AML samples (5) and that phosphorylated ERK is associated with poor prognosis in AML, even in the absence of FLT3 gene abnormalities (6). These observations suggest that activation of ERK itself, either by FLT3 gene mutation or by other factors, such as RAS mutations, may contribute to the survival of AML blast cells.
Inhibitors of several signaling pathways have been developed and are currently in different stages of clinical development, including the FLT3 tyrosine kinase inhibitors lestaurtinib [CEP-701 (7)], PKC412, SU11248, and AG1296 (8–10); the MEK1/2 inhibitor CI1040 (PD184352) (11); and PTK787/ZK 222584, which target all vascular endothelial growth factor receptor (VEGFR) tyrosine kinases (12). However, these agents have not been particularly successful in the treatment of AML when used alone (7,9,12). It is conceivable that a compound that concomitantly affects several target may be more efficacious than one that targets a single protein.
One such multifunctional kinase inhibitor is sorafenib. Sorafenib was initially designed as a small-molecule inhibitor of c-Raf kinase but has also been shown to inhibit the activities of FLT3, VEGFR-2, VEGFR-3, and members of the platelet-derived growth factor receptor family (ie, PDGFR-beta and Kit) (13,14). Sorafenib was recently approved by the US Food and Drug Administration for the treatment of renal cell and hepatocellular carcinomas. In this study, we investigated the efficacy and mechanisms of action of sorafenib in AML cells harboring FLT3 mutations.
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Materials and Methods |
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Chemicals and Cell Lines
Sorafenib (Bay 43-9006) was provided by Dr Mark Lynch (Bayer Pharmaceuticals Co, West Haven, CT). AG1296 (an inhibitor of FLT3) and MAZ51 (an inhibitor of VEGFR-3) were purchased from Sigma-Aldrich (St Louis, MO), and the MEK inhibitor CI1040 was kindly provided by Dr J. S. Sebolt-Leopold (Cancer Molecular Sciences, Pfizer Global Research and Development, Ann Arbor, MI). All other chemicals and solvents used were of the highest grade commercially available.
Murine pro-B lymphocyte lines stably transfected with expression vectors that encoded wild-type human FLT3 (Ba/F3-FLT3 cells) or human FLT3 containing an ITD mutation (Ba/F3-ITD cells) or the D835G (Ba/F3-D835G cells) or D835Y (Ba/F3-D835Y cells) point mutations, which lead to constitutive functional activation of the protein, were generated by Dr Small's Laboratory (Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD) as described (15). Ba/F3-FLT3 cells were maintained in RPMI-1640 medium containing 10% fetal calf serum (FCS, HyClone, Logan, UT) that was supplemented with murine interleukin 3 (IL-3, PeproTech, Inc, Rocky Hill, NJ) at a concentration of 2 ng/mL. In some experiments, Ba/F3-FLT3 cells were cultured in serum-containing medium that was supplemented with recombinant FLT3 ligand (R&D Systems, Minneapolis, MN) at a concentration of 25–50 ng/mL. Ba/F3-ITD, Ba/F3-D835Y, and Ba/F3-D835G cells were maintained in RPMI-1640 medium supplemented with 10% FCS only.
Antibodies
The following antibodies were used for immunoblotting: rabbit polyclonal phospho-FLT3 (Tyr591) (Cell Signaling Technology, Beverly, MA), rabbit polyclonal Raf and FLT3 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal phospho-MEK1/2 (Ser218/222) (Upstate USA, Inc, Charlottesville, VA), and mouse monoclonal phospho-ERK (Thr202/Tyr204) and phospho-tyrosine (p-Tyr-100) (Cell Signaling Technology). Horseradish peroxidase (HRP)–conjugated goat anti-rabbit and anti-mouse IgG secondary antibodies were purchased from Santa Cruz Biotechnology. Rabbit polyclonal anti–green fluorescent protein (GFP) antibody and fluorescein isothiocyanate (FITC)–conjugated goat anti-rabbit IgG (both from Santa Cruz Biotechnology) were used for immunofluorescence microscopy. Allophycocyanin (APC)-conjugated anti-CD34 or anti-CD33 and phycoerythrin (PE)-conjugated anti–phospho-ERK (pT202/pY204) IgG (BD Biosciences, San Jose, CA), annexin-V-FLUOS (Roche Diagnostics Co, Indianapolis, IN), and Alexa 488–conjugated phospho-FLT3 (Tyr591) antibodies (Cell Signaling Technology) were used for flow cytometry analyses.
Cell Viability and Apoptosis Assays
Ba/F3-FLT3, Ba/F3-ITD, Ba/F3-D835G, and Ba/F3-D835Y cells were plated in 24-well plates at 2 x 105 cells/mL (1–2 mL per well), and sorafenib (at various concentrations in dimethyl sulfoxide [DMSO]) was added to the culture medium. Ba/F3-FLT3 cells were grown in the presence of IL-3 (2 ng/mL) and/or FLT3 ligand (25 ng/mL). In rescue experiments, IL-3 (2 ng/mL) was added to Ba/F3-ITD cells that had been cultured in the presence of sorafenib for 6 hours and the cells were incubated for 72 hours. Cell viability was assessed using the trypan blue dye exclusion method, and apoptosis was determined by annexin V positivity detected by flow cytometry, as previously described (16). All experiments were performed in triplicate.
Immunoblot Analysis
Ba/F3-ITD and Ba/F3-FLT3 cells (3 x 106/10 mL in T25 flasks) were serum starved by culturing them for 20 hours in RPMI-1640 medium containing 1% FCS and then treated with various concentrations of sorafenib or DMSO (control) for 4 hours, followed by the addition of FCS to a final concentration of 15% and 50 ng/mL FLT3 ligand (for Ba/F3-FLT3 cells only). After a 30-minute incubation, the cells were collected and lysed in a lysis buffer (1% Triton X-100, 50 mM HEPES [pH 7.4], 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM Na3VO4, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/mL leupeptin). The levels of phosphorylated and total MEK1/2, FLT3, and ERK in cell lysate were determined by immunoblotting with the corresponding antibodies, as previously described (17). Briefly, cell lysates (30–60 µg protein per well) were resolved by electrophoresis on 10%–12% precast sodium dodecyl sulfate-polyacrylamide gels (Bio-Rad, Hercules, CA) and transferred to Hybond-P membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membranes were first incubated in TBST (50 mM Tris–HCl, 150 mM NaCl, 0.1% Tween-20) containing 5% nonfat dry milk for 1 hour to block nonspecific protein binding, then with primary antibody overnight at 4°C, washed with TBST three times, and incubated with HRP-conjugated secondary antibody (1:3000 dilution) for 1 hour at room temperature. The membranes were then washed, and antibody binding was visualized with the use of an enhanced chemiluminescence detection system (ECL-plus; Amersham Pharmacia Biotech). The semiquantitative immunoblotting data were generated by Scion Imaging software (Beta 4.03; Scion Corporation, Frederick, MD).
In Vitro Kinase Assay
We performed an in vitro kinase assay to examine the effect of sorafenib on the phosphorylation status of wild-type and FLT3-ITD mutant proteins. Ba/F3-FLT3 and Ba/F3-ITD cells were cultured for 20 hours in RPMI-1640 medium containing 0.5% FCS and were then used to prepare cell lysates with nondenaturing lysis buffer (Epitomics, Inc, Burlingame, CA). The lysates (500 µg protein for each sample) were incubated by gentle rocking overnight at 4°C with an anti-FLT3 antibody and protein A/G agarose beads (30 µL of 50% bead slurry, Santa Cruz Biotechnology) and then centrifuged (1000g) for 30 seconds at 4°C, and the pellets were washed five times with 500 µL of lysis buffer. The pellets were incubated for 30 minutes at 30°C in magnesium/ATP-containing reaction buffer (20 mM 3-[N-morpholino]propanesulfonic acid [pH 7.2], 25 mM β-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 500 µM ATP, and 75 mM magnesium chloride) that contained various concentrations of sorafenib. The reaction mixtures were boiled for 5 minutes and subjected to immunoblot analysis with a rabbit anti-phosphotyrosine antibody to detect autophosphorylation of wild-type FLT3 and FLT3-ITD proteins as described above.
Leukemia Xenografts in Severe Combined Immunodeficient Mice
Ba/F3-ITD cells were infected with a lentivirus-based construct to establish a stable cell line that expressed Renilla luciferase and GFP (ie, Ba/F3-ITD-GFP/Luc cells), as previously described (18). Briefly, the lentiviral plasmid was created by inserting the 2-kb Renilla luciferase cDNA fragment from the pMOD-LucSH plasmid (InvivoGen, San Diego, CA) into the lentiviral transfer vector pWIP/GFP (a generous gift from Dr Didier Trono, Geneva University, Geneva, Switzerland). Luciferase and GFP genes were introduced into Ba/F3-ITD cells by lentiviral infection, and GFP-positive Ba/F3-ITD cells were sorted by flow cytometry and expanded for xenograft experiments. The animal experiments were approved by the institutional animal care and use committee. CB.17 severe combined immunodeficient (SCID) female mice (5–6 weeks old; purchased from Harlan Sprague-Dawley, Madison, WI) were injected intravenously with 1 x 106 Ba/F3-ITD-GFP/Luc cells. The mice were randomly assigned to receive sorafenib (n = 8 mice per group) at 10 mg/kg of body weight or vehicle (solvent only control, n= 7 mice per group) by oral gavage once per day on days 9–24 after Ba/F3-ITD-GFP/Luc cell injection. The mice were subjected to noninvasive imaging to detect luciferase signal twice per week with the use of a Xenogen In Vivo Imaging System (Hopkinton, MA) 5 minutes after intraperitoneal injection with the luciferase substrate colenterazine (20 µg per mouse) (Biotium, Hayward, CA). Total body bioluminescence was quantified for a region of interest that included each mouse in its entirety. Mice were monitored daily until they showed signs of terminal illness (ie, paralysis), at which point they were humanely killed by CO2 asphyxiation.
Immunofluorescence Analysis of Leukemia Infiltration
To examine the effects of sorafenib on the dissemination of leukemia cells (ie, leukemia burden), mice injected with Ba/F3-ITD-GFP/Luc cells (two mice per group) were subjected to CO2 asphyxiation on day 24 for the sorafenib-treated group (the end of sorafenib therapy) or on day 16 for the vehicle-treated group (the day these mice started dying) after cell injection and their organs (ie, spleen, liver, and bone marrow) were harvested, fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned. The sections (4-µm thick) were deparaffinized in xylene, rehydrated using a graded ethanol series and distilled water, and subjected to antigen retrieval by heating them in 10 mM sodium citrate buffer (pH 6.5) for 20 minutes on the auto defrost setting of a microwave oven. The sections were incubated overnight at 4°C with a rabbit polyclonal anti-GFP antibody (1:50), then washed with phosphate-buffered saline (PBS) and incubated with an FITC-conjugated goat anti-rabbit IgG (1:250). The sections were washed three times with PBS, mounted under coverslips in mounting medium containing 4',6-diamidino-2-phenylindole, and examined with the use of a fluorescence microscope.
Polymerase Chain Reaction Assay of FLT3 Mutation Status
Genomic DNA was assessed for FLT3 gene mutations representing either internal tandem duplications or point mutations involving codon 835 by polymerase chain reaction (PCR) followed by capillary electrophoretic separation on an Applied Biosystems genetic analyzer, as previously reported (19,20). Briefly, the forward primers for each site were labeled fluorescently with 6-carboxyfluorescein (FAM). The forward and reverse primers used to assess internal tandem duplications were 5'-FAM-GCAATTTAG GTATGAAAGCCAGC-3' and 5'-CTTTCAGCATTTTGACG GCAACC-3', respectively. The forward and reverse primers used to assess point mutations in codon 835 were 5'-FAM-CCGCCA GGAACGTGCTTG-3' and 5'-GCAGCCTCACATTGCCCC-3', respectively. For the codon 835 mutation determination, the PCR products were subsequently digested with EcoRV. In the unmutated FLT3 sequence EcoRV digestion creates two fragments, of which the smaller one is detected by the fluorochrome label, in contrast to the large undigested labeled fragment seen with the mutant versions of FLT3. The sensitivity of the FLT3-ITD and codon 835 assays was such that they could detect 1% mutation-bearing cells, as established by dilution studies included in every experiment.
AML Blast Colony Formation Assay
Primary bone marrow cells were collected from patients at M. D. Anderson Cancer Center with newly diagnosed or recurrent AML and a high (>40%) blast count, and normal bone marrow cells were collected from healthy donors. Acute myeloid leukemia blast colony formation was assayed by a previously described method (21). Briefly, the bone marrow cells were cultured at 2 x 105 cells/mL in 0.8% methylcellulose (Fluka Chemical Corp, Ronkonkoma, NY) containing 10% FCS, RPMI-1640 medium, recombinant human granulocyte-macrophage (GM) colony-stimulating factor (0.1 ng/mL) and stem cell factor (5 ng/mL; both from Invitrogen Co, Carlsbad, CA), and increasing concentrations of sorafenib. The culture mixture was placed in 35-mm petri dishes (Nunc Inc, Naperville, IL) in triplicate and incubated for 7 days at 37°C with 5% CO2 in air in a humidified atmosphere. Colonies were then counted with the use of an inverted microscope. A colony was defined as a cluster of more than 40 cells.
Phase 1 Clinical Trial of Sorafenib in Patients With AML
To evaluate the clinical efficacy of sorafenib in AML patients, a National Cancer Institute (NCI) Cancer Therapy Evaluation Program (CTEP)–sponsored phase 1 clinical trial (CTEP 25XS068 01) has been initiated at M. D. Anderson Cancer Center. Thus far, 16 patients have been randomly assigned to receive sorafenib in 21-day cycles of 5 days per week (n = 7 patients; arm A) or of 14 days (n = 9 patients; arm B). In both arms, the starting dose level was 200 mg twice daily. Subsequent dose levels were 600, 800, and 1200 mg daily in cohorts of three subjects at each dose level.
Whole-blood samples were collected into 10-mL sodium heparin tubes (Becton Dickinson, Sparks, MD) at baseline (day 0, before sorafenib administration), at 2 hours, 24 hours (day 1), 120 hours (day 5), and on day 14 during administration of the first course of sorafenib. Red cells were lysed in hypotonic RBC lysis buffer (0.15 M NH4Cl, 0.02 M Tris–HCl), and mononuclear cells were resuspended and washed once with PBS. For evaluation of apoptosis, the mononuclear cells from patient blood samples were stained with APC-conjugated anti-CD34 or anti-CD33 antibodies and annexin-V-FLUOS, and the changes in cellular mitochondrial inner transmembrane potential were determined by staining with chloromethyl-X-rosamine (CMXRos, Invitrogen Co). The samples were analyzed by three-color flow cytometry as previously described (16,22). For analyses of the effects of sorafenib on ERK and FLT3 phosphorylation, the mononuclear cells were fixed in 2% paraformaldehyde in PBS for 10 minutes at room temperature, permeabilized in cold 90% methanol for 20 minutes, and stained with PE-conjugated phospho-ERK and Alexa 488–conjugated phospho-FLT3 antibodies. The levels of phosphorylated ERK and FLT3 were determined by flow cytometry and expressed as mean fluorescence intensity (23). For immunoblot analyses, the mononuclear cells were lysed in cell lysis buffer, and protein expression and phosphorylation levels were determined using the respective antibodies as described above. The percentage of blasts in bone marrow and peripheral blood was determined, and cytogenetic analyses of patients before enrollment in this trial were performed in certified laboratories at M. D. Anderson Cancer Center. All studies involving human material were approved by the M. D. Anderson Institutional Review Board. All patients provided written informed consent. Response criteria were defined according to the International Working Group (24). Adverse event monitoring and reporting was conducted per NCI CTEP guidelines (http://ctep.cancer.gov/reporting/adeers.html).
Simultaneous Targeting of Multiple Signaling Pathways
Ba/F3-ITD cells were resuspended in RPMI-1640 medium at 3 x 105/mL and treated with varying concentrations of the signaling inhibitors AG1296 and either CI1040 or MAZ51 for 48 hours. The induction of apoptosis was estimated by determining the percentage of annexin V–positive cells by using flow cytometry. The isobologram and combination index (CIN) analyses were performed using CalcuSyn (BioSoft, Ferguson, MO) software, a widely used method for evaluating combinatorial synergy between cancer therapeutics (25).
Statistical Analyses
Student t test was used to analyze the immunoblot, cell growth, and apoptosis data. P values less than .05 were considered statistically significant. The Kolmogorov-Smirnov test was used to evaluate flow cytometric data for phospho-ERK and phospho-FLT3 positivity by comparing binding of each antibody with the binding of its matched isotype control. D values ranged from 0 (identical distribution histograms) to 1.0 (no overlap in distribution histograms), and D values greater than or equal to 0.2 were considered statistically significant (26). The effects of sorafenib on mouse survival were estimated by the Kaplan-Meier method (27), and log-rank statistics were used to test for differences in survival. For evaluating the drug-induced synergy of apoptosis induction, the CIN values were determined by the Chou and Talalay method (28). A CIN equal to 1 indicates an additive effect, a CIN less than 1 indicates synergy, and a CIN greater than 1 indicates antagonism. The average CIN values were calculated at different effect levels (EC50, EC75, and EC90 [ie, concentration lethal to 50%, 75%, and 90% of the cells, respectively]) based on triplicate experiments. All statistical tests were two-sided.
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Results |
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Effect of Sorafenib on Cell Growth and Survival in Mouse Ba/F3 Cells Expressing Mutant Human FLT3
We first examined the effects of sorafenib on the growth and viability of mouse Ba/F3 cells that stably expressed wild-type human FLT3 or one of three mutant versions of FLT3 (ie, FLT3-ITD, FLT3-D835G, and FLT3-D835Y). Cells with FLT3-ITD or D835G mutations were 1000- to 3000-fold more sensitive to sorafenib than FLT3-D835Y mutant or FLT3 wild-type cells (the mean IC50 values [ie, the mean concentrations that inhibited the growth of 50% of the cells] were 1.2 nM [95% confidence interval {CI} = 0.13 to 17.14 nM] and 14.3 nM [95% CI = 2.6 to 28.68 nM], respectively, vs 1593.9 nM [95% CI = 599.2 to 2006.5 nM] and 3302.9 nM [95% CI = 2059 to 5246 nM], respectively) (Fig. 1, A). The higher sensitivity to sorafenib of cells with FLT3-ITD or FLT3-D835G compared with cells with wild-type FLT3 or FLT3-D835Y suggested that sorafenib may inhibit growth through mechanisms other than blockade of ERK signaling because ERK was inhibited in all cell types. We next examined the effect of sorafenib on the growth of Ba/F3-FLT3 cells cultured in the presence of FLT3 ligand, which stimulates wild-type FLT3, and/or IL-3, which stimulates cytokine-dependent growth via the IL-3 receptor. Notably, regardless of the addition of IL-3, FLT3 ligand, or IL-3 plus FLT3 ligand to the culture medium, sorafenib had no effect on Ba/F3-FLT3 cell growth or induction of apoptosis until the sorafenib concentration exceeded 1 µM (Fig. 1, B). In addition, culture of Ba/F3-ITD cells in the presence of IL-3 rescued cells from sorafenib-induced apoptosis and growth arrest (mean IC50s were 1786.5 nM [95% CI = 1162.7 to 2744.9 nM] for cells growing with IL-3 vs 1.33 nM [95% CI = 0.03 to 52.6 nM] for cells supplemented with 10% FCS only) (Fig. 1, C). Immunoblot analysis of these cells revealed that the addition of IL-3 did not abrogate sorafenib-modulated inhibition of FLT3 phosphorylation in Ba/F3-ITD cells but partially restored sorafenib-induced inhibition of ERK phosphorylation (Fig. 1, D). Taken together, these results suggest that sorafenib selectively inhibits the growth of hematopoietic cells that depend on FLT3 kinase activity for survival.
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Effects of Sorafenib on Phosphorylation of FLT3, MEK, and ERK Proteins in Ba/F3-FLT3 and Ba/F3-ITD Mutant Cells and on FLT3 Kinase Activity
Next, we investigated the effects of sorafenib on phosphorylation of FLT3 protein and Raf/MEK/ERK signaling in mouse Ba/F3-FLT3 and Ba/F3-ITD cells by treating the cells with sorafenib for 4.5 hours and measuring phosphorylation levels by immunoblotting. In Ba/F3-ITD cells, sorafenib inhibited the phosphorylation of both endogenous FLT3 and FLT3-ITD mutant as well as of ERK. By contrast, sorafenib inhibited the phosphorylation of ERK but only slightly reduced the level of phosphorylated FLT3 in Ba/F3-FLT3 cells cultured in the presence of FLT3 ligand (Fig. 2, A). Furthermore, an in vitro kinase assay revealed that sorafenib at 5–10 nM inhibited autophosphorylation of tyrosine residues in FLT3-ITD protein but not in wild-type FLT3 protein and that inhibition of phosphorylation was specific for the 143-kDa nonglycosylated form of FLT3-ITD (the main form of mutated FLT3 protein) versus the 158-kDa glycosylated form (29) (Fig. 2, B). These data indicate that sorafenib inhibits the function of mutant FLT3-ITD protein directly, by decreasing its autophosphorylation. However, sorafenib at concentrations of up to 10 nM had essentially no effect on the phosphorylation of the downstream Raf target MEK in Ba/F3 cells (Fig. 2, C), suggesting that phospho-ERK levels are suppressed through pathways different from those that block Raf kinase activity.
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Effect of Sorafenib in a Mouse Xenograft Model of FLT3-ITD Mutant Leukemia
To examine the in vivo effects of sorafenib in a murine leukemia model, mice bearing xenografts derived from Ba/F3-ITD-GFP/Luc cells were treated with sorafenib (n = 8) or vehicle (n = 7) on days 9–24 after tumor cell implantation and their survival was monitored. The median survival of mice in the sorafenib-treated group was 36.5 versus 16 days for mice in the vehicle-treated group (difference = 20.5 days, 95% CI = 20.3 to 21.3 days; P = .0018) (Fig. 3, A). Bioluminescence imaging of individual whole live mice revealed that after 7 days of treatment (ie, by day 16 after tumor cell injection), Ba/F3-ITD cells were broadly disseminated in the vehicle-treated mice whereas bioluminescence was barely detectable in the sorafenib-treated mice (mean luminescence at day 16, vehicle vs sorafenib treatment = 1.76 x 107 vs 2.25 x 105 luminescence counts; difference = 1.7 x 107 luminescence counts, 95% CI = 0.4 x 107 to 3.7 x 107 luminescence counts; P = .04) (Fig. 3, B and C). At necropsy, the spleens from vehicle-treated control mice were substantially larger than those from sorafenib-treated mice; the spleens of sorafenib-treated mice were approximately the same size as those from untreated healthy SCID mice (Fig. 3, D). Histologic analysis revealed that the spleen, liver, and bone marrow of sorafenib-treated mice were infiltrated by substantially fewer GFP-positive leukemic cells than those of vehicle-treated mice (Fig. 3, E).
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Effect of Sorafenib in Patients With Primary AML
Next, we examined the effects of sorafenib on the ex vivo clonogenic growth of primary blasts from AML patients who carried the FLT3-ITD gene mutation (n = 2) or wild-type FLT3 (n = 3) (Fig. 4, A). Sorafenib strongly inhibited colony formation in FLT3-ITD mutant primary cells at statistically significantly lower concentrations (mean IC50 = 10 nM, 95% CI = 3.9 to 18.8 nM) than in wild-type FLT3 AML cells (mean IC50 = 2680 nM, 95% CI = 1704 to 3174.9 nM, P = .02). At these concentrations, sorafenib had no inhibitory effect on the ex vivo clonogenic growth of bone marrow cells from healthy bone marrow donors (n = 3) (Fig. 4, B): normal GM, erythroid, and mixed colony-forming cells were remarkably resistant to sorafenib compared with both FLT3-ITD mutation or FLT3 wild-type leukemic cells shown in Fig. 4, A.
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We also investigated the therapeutic efficacy of sorafenib in 16 AML patients who were enrolled in a phase 1 clinical trial. The median age of the patients was 61.5 years (range = 48–81 years), the median number of prior therapies was 2 (range = 1–5), and the median time from diagnosis to initiation of sorafenib therapy was 9 months (range = 5–46 months). Analyses of cytogenetics and FLT3 mutation status in blasts from bone marrow or peripheral blood before enrollment revealed that eight patients had a normal diploid karyotype and eight patients had karyotypes with complex cytogenetics. Mutational analysis demonstrated the presence of wild-type FLT3 in seven patients, FLT3-D835 point mutations in two patients, FLT3-D835 plus FLT3-ITD mutations in one patient, and FLT3-ITD in six patients (Table 1).
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Analysis of apoptosis markers (annexin V and mitochondrial membrane potential loss measured by CMXRos) indicated that sorafenib induced apoptosis in AML blasts (Table 2; Fig. 5, A). Sorafenib therapy also suppressed ERK phosphorylation in peripheral blood blasts in all six (100%) AML patients who carried only an FLT3-ITD mutation and suppressed FLT3 phosphorylation in three (50%) of these six patients. By contrast, among the seven AML patients with wild-type FLT3 who were treated with sorafenib, only two (29%) had a decrease in levels of phosphorylated ERK whereas four (57%) had a detectable decrease in FLT3 phosphorylation as measured by flow cytometry. However, no phosphorylated FLT3 signal was detectable by immunoblot analysis, indicating a lower basal level of wild-type FLT3 in this group of patients (Table 2; Fig. 5, B and C).
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A clinical response was observed in nine (56%) of 16 patients (three patients in arm A and six patients in arm B). It is notable that all six patients who carried solely an FLT3-ITD mutation (three patients in each arm) had a clinical response and that three of the patients with wild-type FLT3 (all in arm B) had a modest response in peripheral blood. However, two patients who carried the D835 point mutation, and one patient with dual mutations (D835 and ITD) showed no response, although the levels of phosphorylated ERK and phosphorylated FLT3 were suppressed (Tables 1 and 2).
Among patients who carried solely an FLT3-ITD mutation (n = 6), the median percentages of blasts in circulation at study entry and after sorafenib administration were 81% and 7.5%, respectively (mean difference = 50.5%, 95% CI = 43.2% to 57.8%; P = .016), and the median percentages of blasts in the bone marrow were 75.5% and 34%, respectively (mean difference = 27.3%, 95% CI = 21.8% to 32.8%; P = .05). The lowest percentage of blasts after sorafenib treatment was 0% in the peripheral blood (patients 1 and 12) and 12% in the bone marrow (patients 5 and 15). Examples of changes in blast counts after sorafenib administration are shown for patients 12 and 13 (Fig. 5, D). Figure 5, E, shows the variation in the mean percentage of circulating blasts over one treatment cycle for patients who did and did not carry an FLT3-ITD mutation. Sorafenib treatment resulted in a strong decrease in the numbers of both circulating and bone marrow blasts in patients with FLT3-ITD mutation and essentially no change in patients without the mutation (Table 2).
We also evaluated the clinical tolerability of sorafenib among the 16 patients in the phase 1 trial. Sorafenib was well tolerated, and no dose-limiting toxic effects have been observed as of December, 2007, at doses up to 400 mg daily in either arm. Only one grade 3 toxic effect was observed, a pleural effusion at the daily dose of 600 mg in arm A that occurred during cycle 2 of therapy. No grade 4 toxic effects were observed. All patients received at least one cycle of sorafenib and remained on therapy for a median of 4 weeks (range = 1–14 weeks). Five patients (31%) discontinued sorafenib therapy (three in arm A, two in arm B) due to disease progression.
Overall, sorafenib showed promising antileukemic activity among AML patients who carried an FLT3-ITD mutation. Patient accrual for this phase 1 trial is ongoing so that the optimal sorafenib dose schedule can be determined.
Effect of Simultaneous Targeting of FLT3 and the MEK or VEGFR Pathways in Ba/F3-ITD Cells
Finally, to determine whether the efficacy of sorafenib in FLT3-ITD mutant AML might be attributable to its multiple targets in addition to the specific suppression of ITD mutant protein, we examined the cytotoxic effects of the FLT3 kinase inhibitor AG1296 alone and in combination with the MEK kinase inhibitor CI1040 or the VEGFR-3 kinase inhibitor MAZ51 in mouse Ba/F3-ITD cells. We found that combinations of these pathway inhibitors synergistically induced Ba/F3-ITD cells to undergo apoptosis (Fig. 6, A). That is, the mean CIN values for AG1296 + CI1040 at EC50, EC75, and EC90 were 0.42 (95% CI = 0.35 to 0.49), 0.36 (95% CI = 0.29 to 0.42), and 0.30 (95% CI = 0.24 to 0.36), respectively. The mean CIN values for AG1296 + MAZ51 at EC50, EC75, and EC90 were 0.87 (95% CI = 0.77 to 0.96), 0.74 (95% CI = 0.68 to 0.81), and 0.63 (95% CI = 0.53 to 0.72), respectively (Fig. 6, B).
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Discussion |
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Sorafenib was designed as a small-molecule inhibitor of c-Raf kinase; it is capable of inhibiting Raf kinase activity at concentrations of 2–12 nM in cell-free systems (31), but micromolar concentrations of sorafenib are required to inhibit the growth of AML cell lines and primary AML cells (32,33). Recent studies (34–36) have shown that sorafenib directly targets mutant FLT3 kinase, thereby inhibiting the growth and survival of mouse and human FLT3-ITD AML cells at low nanomolar concentrations. In this study, we examined the effects of sorafenib on the growth and survival of isogenic mouse Ba/F3 cells that expressed mutant (ITD, D835G, or D835Y) or wild-type human FLT3, which were cultured in the absence or presence of FLT3 ligand and/or IL-3. Sorafenib induced death of cells that expressed the FLT3-ITD or FLT3-D835G but not cells that expressed the FLT3-D835Y point mutant or wild-type FLT3 receptor. These FLT3 mutant cells have constitutively active kinase activity, whereas cells with wild-type FLT3 require ligation of the receptor by FLT3 ligand for kinase activity. We further found that the addition of IL-3 to the culture medium reversed the sorafenib-mediated growth arrest of Ba/F3-ITD cells. IL-3 induces activation of several prosurvival signaling pathways, including the MEK/ERK (37,38), phosphatidylinositol 3-kinase/AKT (39,40), and STAT5 (41) pathways. Our results showed that IL-3 could partially reverse the sorafenib-induced decrease of ERK phosphorylation but not the decrease of FLT3 phosphorylation in Ba/F3-ITD cells. These findings suggest that sorafenib does not interfere with IL-3–driven hematopoietic cell proliferation but is cytotoxic to Ba/F3-ITD cells when their growth depends on FLT3 kinase activity, implying that sorafenib selectively kills FLT3-ITD mutant–harboring leukemic cells in vivo. Although the sorafenib-induced growth arrest and apoptosis in AML cells could result from the suppression of phosphorylation of both FLT3 and its downstream target ERK or from ERK activity modulated by the Raf-MEK-ERK axis, the efficacy of low concentrations of sorafenib in FLT3-driven Ba/F3 cells seems to be independent of the inhibitory effects of sorafenib on Raf kinase because no blockade of the Raf downstream target MEK was observed at nanomolar concentrations that were efficacious in suppressing Ba/F3-ITD mutant cells.
To address the mechanism by which sorafenib targets FLT3, we examined its ability to inhibit FLT3 kinase activity in vitro. Our results demonstrated that 10 nM sorafenib directly inhibits the phosphorylation of tyrosine residues in mutated FLT3-ITD protein but not in wild-type FLT3 protein. These results indicate that sorafenib targets FLT3 directly and that it is a more potent inhibitor of FLT3-ITD than of wild-type FLT3. Although additional studies are required to dissect this differential effect of sorafenib on mutant and wild-type FLT3, molecular modeling of the structure of the FLT3 protein reveals that the amino-terminal region of the juxtamembrane binding motif of FLT3 changes conformation in the ITD protein, which results in an increased opening of the binding pocket and allows for more optimal interactions between FT3-ITD protein and an inhibitor such as sorafenib (D. Maxwell, W. Bornmann, personal communication). Studies are currently under way at M. D. Anderson Cancer Center to develop three-dimensional structure models of FLT3-D835G and -D835Y mutants that might clarify the mechanism by which sorafenib interacts with these point mutations (D. Maxwell and W. Bornmann, unpublished data).
Our in vivo studies in a leukemia xenograft model of Ba/F3-ITD further confirmed the potency of sorafenib for FLT3-ITD. Mice treated with sorafenib at 10 mg/kg of body weight per day on days 9–24 after leukemia cell injection lived approximately 2.3 times longer than mice treated with vehicle. By contrast, mice in the same xenograft model that were continuously treated with lestaurtinib, a specific inhibitor of FLT3 kinase, at 10 mg/kg of body weight either twice or three times per day starting on the day of leukemia cell injection lived only 1.2–1.5 times longer than mice treated with vehicle (42). In addition, we found that at 23 days after injection with luciferase-transduced Ba/F3-ITD cells, the sorafenib-treated mice were still alive and leukemia cells were barely detectable by bioluminescence imaging (data not shown), whereas all vehicle-treated mice had succumbed to leukemia dissemination and died (median survival was 16 days). The leukemia cell dissemination was confirmed by our finding that sorafenib-treated mice had normal-sized spleens on day 23 after leukemia cell injection and that only scattered GFP-positive leukemia cells were observed in the spleens and livers of sorafenib-treated mice. It is notable that sorafenib was not as effective in preventing leukemia cell infiltration of the bone marrow, suggesting that the bone marrow microenvironment that supplies growth factors and cytokines might protect leukemia cells from sorafenib-mediated proapoptotic effects in vivo. This finding is consistent with our observation that IL-3 rescued Ba/F3-ITD cells from sorafenib-induced death in vitro. Further studies are warranted to understand the mechanisms that might be responsible for the protection afforded by the bone marrow microenvironment.
Further evidence of the antileukemic activity of sorafenib in primary human AML cells was demonstrated by clonogenic assays, in which sorafenib showed greater efficacy in inhibiting colony formation by primary AML cells bearing the FLT3-ITD mutation (IC50 = 10 nM) than by wild-type FLT3 AML cells (IC50 = 2680 nM). It is important to note that, at the low nanomolar concentrations that inhibited clonogenic growth of FLT3-ITD primary AML cells, sorafenib had no effect on the clonogenic growth of normal bone marrow cells, demonstrating an excellent therapeutic window for the selective killing of AML progenitor cells. This finding has potential clinical significance given that Levis et al. (8) have demonstrated the presence of FLT3-ITD mutations in leukemic stem cells.
Results of the correlative studies in the phase 1 trial further confirmed the preclinical data: sorafenib induced apoptosis and/or the loss of mitochondrial membrane potential in peripheral blood blasts of AML patients who carried an FLT3-ITD mutation. These effects were associated with decreases in the levels of phosphorylated ERK and FLT3 as early as 2 hours after drug administration in patients who carried an FLT3-ITD mutation. Importantly, the median blast percentage dropped dramatically (from 81% to 7.5% in peripheral blood and from 75.5% to 34% in the bone marrow) after oral administration of sorafenib in patients who had an FLT3-ITD mutation. By contrast, sorafenib had no statistically significant effects on circulating or bone marrow blasts in AML patients with either wild-type FLT3 or an FLT3-D835 mutation. The antileukemia efficacy of sorafenib appears to be superior to that reported for several specific FLT3 inhibitors that are currently under development as therapeutic agents for AML. For example, in a phase 1 trial of lestaurtinib (7), only five of 17 patients with refractory AML who harbored an FLT3-ITD mutation showed reductions in the percentages of peripheral blood and bone marrow blasts. In a phase 1 study of SU11248 (9), only a partial decrease in the percentage of circulating blasts was achieved in all four patients harboring FLT3-ITD or FLT3-D835 mutations and morphologic or partial responses were achieved in two of seven evaluable patients with wild-type FLT3. In a phase 2 trial (43), PKC412 achieved a greater than 50% reduction in the number of peripheral blasts in 14 (70%) of 20 patients harboring an FLT3 mutation. In comparison, sorafenib caused a greater than 50% reduction in the percentage of circulating blasts in 100% of patients harboring an FLT3-ITD mutation. This finding implies that treatment with sorafenib might preferentially benefit patients with FLT3-ITD AML.
The molecular mechanisms underlying the preferential efficacy of sorafenib in FLT3-ITD mutant AML require further investigation. Our data showed that not all patients who had a clinical response to sorafenib had suppression of FLT3 phosphorylation. Thus, the clinical efficacy of sorafenib might also result from its ability to suppress the activity of multiple kinases, including Raf-MEK and VEGFR. Our studies using a specific FLT3 inhibitor in combination with MEK kinase or VEGFR inhibitors further confirmed the synergistic effects of targeting multiple kinases simultaneously in FLT3-ITD mutant AML cells in vitro, which implied a crucial role for targeting several kinases in FLT3-ITD–mutated AML.
Although sorafenib showed potential efficacy in targeting FLT3-ITD mutant–harboring AML, this study has several limitations. First, we observed that discontinuation of sorafenib administration in the murine model and in the clinical trial leads to recurrence of AML, as indicated by rising blast counts. It is unclear if AML recurrence can be prevented by extending the treatment duration, which should be feasible given the lack of toxicity (data not shown). Second, long-term culture in vitro with low doses of sorafenib might induce resistance to this compound by mechanisms that need clarification. Third, the bone marrow microenvironment might reduce the proapoptotic efficacy of sorafenib in AML cells, as shown in the clinical trial, where circulating leukemia cells were more sensitive to sorafenib than bone marrow–resident cells. Therefore, further studies are needed to fully assess the therapeutic potential of sorafenib by investigating the efficacy of sorafenib in vitro in coculture systems of AML cells and bone marrow–derived stroma cells and by optimizing the therapeutic regimens by extending the period of drug administration while increasing the dose of sorafenib to determine the maximal tolerated dose. Another limitation is the absence of data showing effects on leukemic stem cells.
In summary, our results showed that the primary target of sorafenib is the mutant protein FLT3-ITD and that sorafenib induces cell growth arrest and apoptosis in Ba/F3-ITD cells, inhibits the clonogenic growth of primary AML cells, suppresses leukemia cell growth in a mouse xenograft model, and, in a phase 1 trial, reduced the number of circulating and bone marrow blasts in all FLT3-ITD mutant AML patients. The multiple targets of sorafenib may explain its superior efficacy compared with that of other FLT3-ITD–specific inhibitors. Notably, the plasma concentrations of sorafenib achieved in clinical trials (7.1 mg/L at an oral dose of 400 mg twice daily) exceeded the minimum concentration that we found was required to inhibit the growth of leukemia cells with FLT-3 mutations. Sorafenib was well tolerated clinically, and the maximum tolerated dose has not yet been reached. Taken together, our findings imply that sorafenib is a potent antileukemic agent in patients with FLT3-ITD mutant AML, a form of AML that responds poorly to traditional chemotherapy (44). A phase 2 clinical trial at M. D. Anderson Cancer Center (protocol No. 2006-0977) is currently recruiting patients to investigate the efficacy of sorafenib in combination with idarubicin and cytosine arabinoside in AML patients with and without mutant FLT3.
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TopAbstractContext and CaveatsMaterials and MethodsResultsDiscussionFundingReferencesNotes |
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National Institutes of Health (CA55164 to M.A., CA100632 [GenBank] to M.K.); Leukemia SPORE Career Development Award (CA100632 [GenBank] to W.Z.), Cancer Therapy Evaluation Program (25XS068 01 to J.C.).
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NOTES |
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W. Zhang, M. Konopleva, and M. Andreeff designed the experiments; W. Zhang, Y.-x. Shi, T. McQueen, and D. Harris performed the research; D. Small and X. Ling contributed new reagents and/or analytic tools; J. Cortes and A. Quintás-Cardama conducted the phase 1 clinical trial and analyzed the data; and W. Zhang, M. Konopleva, Z. Estrov, and M. Andreeff analyzed and interpreted the data.
We thank Wenjing Chen, Ellen Jackson, and Sheela V. Mathews for valued assistance in the collection of the patient clinical information and Betty L. Notzon and Vickie J. Williams for critical review of the manuscript.
The study sponsor had no role in the design of the study; the collection, analysis, interpretation of the data; the writing of the manuscript; or the decision to submit the manuscript for publication.
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Manuscript received May 30, 2007; revised November 27, 2007; accepted December 26, 2007.
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