Abstract
Aurora kinases play an essential role in orchestrating chromosome alignment, segregation, and cytokinesis during mitotic progression and both aurora-A and B are frequently overexpressed in a variety of human malignancies. In this study, we report the effects of AZD1152-HQPA, a highly selective inhibitor of aurora-B kinase, in acute myeloid leukemia (AML) cell lines and primary samples. We show that AZD1152-HQPA inhibits the phosphorylation of Histone H3 (pHH3) on serine 10 resulting in polyploid cells, apoptosis, and loss of viability in a panel of AML cell lines. We also show that AZD1152-HQPA sensitivity in our cell lines is irrespective of p53 status and the FLT3-ITD–expressing MOLM-13 and MV4-11 cell lines are particularly sensitive to AZD1152-HQPA. Internal tandem duplications (ITD) within the FLT3 tyrosine kinase receptor are found in ∼25% of AML patients and are associated with a poor prognosis. Here, we report that AZD1152-HQPA directly targets phosphorylated FLT3 along with inhibiting its downstream target phospho–signal transducer and activator of transcription 5 (STAT5) in the FLT3-ITD cell lines. We show pHH3 expression in primary AML blasts and its inhibition by AZD1152-HQPA at low doses in all of our primary samples tested. AZD1152-HQPA inhibits the clonogenic potential of primary AML samples, with FLT3-ITD samples being the most sensitive (P = 0.029). FLT3-ITD primary samples are also more sensitive to pHH3 inhibition (P = 0.022) and are particularly sensitive to pSTAT5 downregulation after treatment with AZD1152-HQPA compared with FLT3 wild-type samples (P = 0.007). We conclude that mutant FLT3 is a secondary target of AZD1152-HQPA and that FLT3-ITD primary samples are particularly sensitive to the drug. Mol Cancer Ther; 9(3); 661–72
- Myeloid leukemia
- AZD1152
- aurora B
- FLT3
Introduction
The mammalian aurora kinases aurora-A, aurora-B, and aurora-C comprise a family of serine/threonine kinases that are essential for mitotic progression (1). Interest in the auroras has intensified since the observation that aurora-A and aurora-B are overexpressed in human tumors. Despite having high sequence homologies in their kinase domains, the three members have very distinct localizations and functions (2). Aurora-B is a chromosomal passenger protein that undergoes dynamic localization during mitosis, associating first to the inner centromeric region during prometaphase, and then to the spindle midzone and midbody from anaphase through cytokinesis (1). Aurora-B is the catalytic component of the chromosomal passenger complex, which is composed of three additional noncatalytic subunits that direct its activity: survivin, INCEP, and borealin. The chromosomal passenger complex orchestrates the accurate segregation of the chromatids at mitosis and cytokinesis (3). Aurora-B is also known to phosphorylate Histone H3 (pHH3) at the serine 10 position during mitosis (4, 5). Inhibition of pHH3 has been reported to prevent initiation of chromosome condensation and entry into mitosis (6). Both aurora-A and B are overexpressed in a wide variety of tumor types (7–10) including those of hematological origin (11, 12). The implication of the auroras in tumorigenesis and the fact that that they are kinases, amenable to small-molecule inhibition, makes them attractive targets for anticancer drug development. The increase in confidence that small-molecule inhibitors of specific kinases may prove to be highly effective anticancer agents comes from the success of agents such as imatinib in the treatment of chronic myelogenous leukemia (13).
A growing number of aurora kinase inhibitors have been described that show antitumor activity in vivo. Three nonselective aurora kinase inhibitors ZM447439, Hesperadin, and VX-680 all induce similar phenotypes when tested in cell-based assays (14–16). Specifically, all three inhibit pHH3 on serine 10 and induce DNA endoreduplication in the absence of cytokinesis, results that suggest that their cellular effects are largely due to the inhibition of aurora-B (17).
AZD1152 is an aurora kinase inhibitor that is selective for aurora-B (IC50s: aurora-A, 1,369 nmol/L; aurora-B, 0.36 nmol/L; aurora-C, 17.0 nmol/L). It is a quinazoline prodrug that is converted in plasma to the active moiety AZD1152-HQPA and it is the active AZD1152-HQPA that has been supplied by Astrazeneca for the purpose of this study. AZD1152 has been shown to significantly inhibit the growth of human colon, lung, and hematologic tumor xenografts in immunodeficient mice and as such has been selected for clinical evaluation (18, 19). It has also shown tumoricidal activity against a panel of tumor cell lines including those of acute myeloid leukemia (AML) origin (20–22).
AML is a heterogeneous clonal disorder of hemaopoietic progenitor cells in which both failure to differentiate and overproliferation results in the accumulation of nonfunctional cells termed myeloblasts (23). Intrinsic resistance or treatment-induced acquired resistance is one of the major obstacles to the effective treatment of patients with AML. Although ∼80% of younger AML patients may initially achieve complete remission with current therapy, most will relapse with resistant disease (24). Clinical outcomes in the elderly have been even more modest as these patients do not tend to tolerate intensive chemotherapy regimens and frequently have poor cytogenetics (25). Less than 10% of older patients with AML will achieve long-term disease-free survival with conventional chemotherapy (26). This inability to successfully treat AML patients, particularly the elderly, underlies the continuing need to develop new treatments for AML.
In this study, we will investigate the mode of cell death following treatment with AZD1152-HQPA in AML cells and also whether cellular FLT3 and p53 status affects cellular response. The downregulation of the biomarker pHH3 can be used to show aurora-B inhibition and we investigate the expression of pHH3 in primary AML samples and its response to AZD1152-HQPA.
Materials and Methods
Materials
Materials were from Sigma unless otherwise stated below.
Cell Lines
OCI-AML3, MOLM-13, and M-07e myeloid leukemia cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). U937 cell lines were from the European Collection of Animal Cell Cultures. MV4.11 cell lines were obtained from the American Type Culture Collection. U937, OCI-AML3, MOLM-13, and MV4-11 cell lines were maintained in RPMI 1640 with 10% fetal calf serum (FCS; First Link), 2 mmol/L l-glutamine, 100 U/mL penicillin, and 10 μg/mL streptomycin. The M-O7e cell line was maintained as above with 20% FCS and 10 ng/mL granulocyte macrophage colony-stimulating factor (Novartis). All cultures were kept at 37°C in 5% CO2 and all experiments were done with cell lines in log phase. Continued testing to authenticate these cell lines was done using a panel of monoclonal antibodies and FLT3 mutational analysis toward the final passage of each batch thawed.
Patient Samples
Presentation blood or bone marrow samples were obtained at diagnosis after acquiring written consent from patients with AML. Use of these samples was approved by the Local Research Ethics Committee. Mononuclear cells were isolated using a standard density gradient/centrifugation method with Histopaque and cryopreserved in liquid nitrogen. For analysis, cryopreserved samples were thawed and rested in culture medium enriched to 20% FCS for 90 min before experimental procedures. Only samples with >85% postrest viability were used. Primary AML samples were cultured at 106/mL in RPMI 1640 with 10% FCS, 2 mmol/L l-glutamine, supplemented with 20 ng/mL interleukin (IL)-3, 20 ng/mL SCF, 20 ng/mL IL-6 + 25 ng/mL granulocyte colony-stimulating factor (R+D Systems) + 0.07 μL/mL β-mercaptoethanol.
Determination of Cell Viability
This was done by a previously published in-house method that allows rapid evaluation of viable cells by flow cytometric counting of cells labeled with 7-amino Actinomycin D (7-AAD; ref. 27).
pHH3 Status and Cell Cycle Analysis by Intracellular Flow Cytometry
Cells were harvested by centrifugation washed in PBSAA (1% bovine serum albumin/0.5% sodium azide in PBS) and 3 × 105 cells treated with fixation medium (Abd Serotec) at room temperature (RT) for 15 min. Ice-cold methanol was added and after 10 min of incubation on ice, cells were washed in PBSAA, resuspended in permeabilisation medium (Abd Serotec), and 5 μg/mL of anti–phospho-histone H3 (Ser 10) mouse monoclonal antibody (Upstate; now part of Millipore) or mouse isotype control (DAKO) was added. After 2 h of incubation at RT, cells were washed twice in PBSAA, resuspended with 3 μL of secondary antibody (goat anti-mouse IgG FITC F(ab')2; DAKO), and incubated for 1 h in the dark. Cells were then washed twice in PBSAA and resuspended in 25 μg/mL 7-AAD (Sigma) for 20 min in the dark. Samples were collected using a FACSCalibur. Isotype controls were used for cytometer setup and doublets were gated out before fluorescence data were collected. Data were analyzed using the Cellquest software with the phosphorylation status expressed as percentage of total cells gated.
Apoptosis Assays
Detection of apoptosis was determined using Annexin V-FITC, active caspase-3, and Apop2.7 (7A6) antibodies. The Annexin V-FITC apoptosis detection kit was used according to the manufacturer's instructions. To measure active caspase-3 expression, treated cells were harvested by centrifugation and washed in PBSAA and 5 ×105 cells treated with fixation medium for 15 min at RT. Cells were then washed in PBSAA and resuspended in a permeabilization solution (Abd Serotec) with 20% normal rabbit serum. After 15 min at RT, 20 μL PE-conjugated polyclonal Rabbit anti–active caspase-3 (BD Pharmingen) were added and cells were incubated for 1 h at RT. Cells were washed and data were collected on a FACSCalibur (Becton Dickinson) using the Cellquest software for analysis. Cells for 7A6 analysis were counted and washed in PBSAA before resuspending in 25 μg/mL digitonin and incubation on ice for 20 min. The cells were then pelleted, and either 20 μL of anti–APO2.7-PE (Beckman Coulter) or mouse IgG1 PE isotype control (BD Pharmingen) were added before incubation for 20 min at RT. Cells were then washed in PBSAA before collection on a FACSCalibur and analysis using the Cellquest software.
p53 Sequencing
Cell line cDNA was prepared using QIAamp blood isolation kits (Qiagen) according to the manufacturer's instructions. Exons 2 to 11 of p53 were amplified using four overlapping sets of primers p53: 1F-gacacgcttccctggat tggc, 1R-gcaaaacatcttgttgagggca, 2F-gtttccg tctgggcttcttgca, 2R-ggtacagtcagagccaacctc, 3F-tggcccctcctcagcatctta, 3R-caaggcctcattcagctctc, 4F-cggcgcacagaggaagagaatc, 4R-cgcacacctattgcaa gcaaggg. Approximately 50 ng of cDNA were used as template in each of the PCR amplifications. The 50-μL reaction also included 150 μmol/L of each deoxynucleotide triphosphate (Amersham Biosciences), 1 μmol/L of each primer, 1.0 mmol/L MgCl2 for fragments 1 and 2 or 1.5 mmol/L MgCl2 for fragments 3 and 4, and 2 units of Amplitaq Gold (PE Applied Biosystems) in the manufacturer's buffer. Following an initial heat activation step at 95°C for 10 min, amplification was done in a PTC-100TM Programmable Thermal Controller (MJ Research) using the following conditions: denaturation at 95°C for 1 min, annealing for 1 min at 55°C for fragments 1 and 2 or 60°C for fragments 3 and 4, and extension at 72°C for 1 min for a total of 35 cycles ending with a final extension at 72°C for 10 min. PCR products were purified using a QIAquick PCR purification kit (Qiagen) before sequencing. Sequencing reactions were set up with ∼200 ng of purified PCR product and 3.2 pmol primer using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems). The primers used for sequencing were the same as those used for the PCR amplifications. The reactions were electrophoresed using an ABI 3130 Genetic Analyser (Applied Biosystems) and results were analyzed using the Sequencing Analysis 5.2 software (Applied Biosystems).
FLT3 Mutation Analysis
Genomic DNA was extracted from samples using a QIAamp blood DNA isolation kit (Qiagen) according to the manufacturer's protocol. Approximately 100 ng of genomic DNA were used as template in fluorescent PCR amplification (24 cycles) to amplify exons 14 and 15 and the intervening intron of the FLT3 gene using previously described primers with the FOR primer FAM labeled (28). Analysis was done on a 3130 Genetic Analyser using the GeneMapper ID software v3.2 (Applied Biosystems). This allowed for the determination of length of FLT3-ITD and proportion of DNA containing the length mutation.
Detection of Phosphorylated FLT3
Cell lines were washed in ice-cold PBS and resuspended in lysis buffer [50 nmol/L Tris (pH 7.4)], 150 mmol/L NaCl (Fisher Scientific), 1% NP40 (BDH Laboratory Supplies), 0.25% Na-deoxycholate, 1 mmol/L EDTA, 2 μg/mL leupeptin, 5 μg/mL aprotinin, 1 μg/mL pepstatin, 20 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, and 3 mmol/L sodium orthovanadate for 30 min. Samples were then sonicated before addition of 200 mmol/L phenylmethylsulfonyl fluoride and incubation for 30 min on ice. The samples were clarified by centrifugation at 100,000 g and the supernatant was assayed for protein content using Bio-Rad dye reagent. Rabbit polyclonal FLT3 (Santa Cruz) was added to the extract and incubated overnight with mixing. Protein A agarose beads (Upstate) were added for an additional 2 h. After sodium SDS-PAGE and transfer to nitrocellulose membranes, immunoblotting was done with mouse monoclonal anti-phophotyrosine antibody (Upstate) to detect phosphorylated FLT3 and then the blots were stripped and reprobed with rabbit polyclonal Flt-3/Flk-2 (Santa Cruz) antibody to measure total FLT3. Proteins were visualized using chemiluminescence (Hyperfilm ECL; Amersham), scanned using a Syngene densitometer, and analyzed using the GeneSnap software (Syngene).
Phosphorylated Signal Transducer and Activator of Transcription 5 Measurement
Phosphorylated signal transducer and activator of transcription 5 (STAT5) was measured on a semiquantitative scale using flow cytometry. The method used has previously been explained in detail by Pallis et al. (29).
Real-time PCR for Aurora Kinase B mRNA Levels
RNA from CD2-depleted AML blasts and the MV4-11 cell line was prepared using QIAamp RNA kits with DNase treatment according to the manufacturer's instructions (Qiagen). Up to 2 μg of RNA were used in a reverse transcription reaction with Moloney murine leukemia virus reverse transcriptase (Invitrogen) and random hexamers (Amersham Pharmacia). Quantitative PCR was done on an ABI Prism 7700 (Applied Biosystems) using the Excite Real-Time Mastermix with sybr green (Biogene). Each reaction consisted of 1× Excite mastermix, sybr green (1:60,000 final concentration), 2 μL of aurora kinase B forward and reverse primer mix (Qiagen; QT00067403), 1.6 μL cDNA (or dH2O), and H2O to a final volume of 20 μL. Thermal cycler conditions included an incubation at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 minute. Following the 40 cycles, the products were heated from 60°C to 95°C over 20 min during which a melting curve analysis was done. This allowed the specificity of the products to be determined (indicated by the presence of a single melting peak) and confirmed the absence of product generated by primer-dimer association. To enable the levels of transcripts to be quantified, standard curves were generated using serial dilutions of MV4-11 cDNA. The housekeeping gene β-2-microglobulin (β2M) was used to standardize the samples and the relative expression levels of ABCG2 transcripts were therefore calculated as the ratio between the level of ABCG2 and the level of β2M. The sequences of the primers used to quantify β2M have been previously published (30). Negative controls (no template) were included in each experiment and all reactions were run in triplicate.
Blast Cell Proliferation
To measure stimulated proliferation rate, blast cells were cultured in RPMI with the addition of 10% FCS, 2 mmol/L l-glutamine, 20 ng/mL IL-3, 20 ng/mL SCF, 20 ng/mL IL-6, 25 ng/mL granulocyte colony-stimulating factor (R+D Systems), and 0.07% β-mercaptoethanol. Cells were plated onto 96-microwell plates at a concentration of 1 × 106/mL. After 48 h of culture, cells were exposed to tritiated thymidine (3H-Tdr, 5 μCi per well; Amersham) for 6 h. Cells were harvested onto a 96-well filter plate using a Mash harvester and left overnight to dry. Incorporation of 3H-Tdr was then evaluated by β-scintillation counting. Basal proliferation was measured similarly without the 48 h of cytokine incubation.
Primary Cell Colony Formation Assay
Freshly isolated mononuclear cells were washed, resuspended in methylcellulose-based medium H4534 (Stem Cell Technologies) containing 0 to 30 nmol/L AZD1152-HQPA (Astra Zeneca), and plated in triplicates onto 96-well plates (100 μL containing 2 × 104 cells). The plates were incubated for 14 d at 37°c in 5% CO2 and the number of colonies formed were then counted under the microscope. Growth was defined as >12 colonies present in nontreated wells.
Statistical Analysis
Statistical analysis was carried out using the Statistical Package for Social Sciences, version 12 (SPSS). P values of ≤0.05 were considered to represent significance.
Results
AZD1152-HQPA Inhibits pHH3 on Ser10 and Leads to the Loss of Viability in AML Cell Lines
The effects of AZD1152-HQPA were examined in logarithmically growing MV4-11, MOLM-13, OCI-AML3, U937, and M-07e leukemic cell lines. An in-house flow cytometry protocol for measuring phosphorylated proteins was used, and the fixation and permeabilization technique previously developed for measuring phosphorylated STAT5 was used (29). The pHH3 antibody stained with extreme brightness and we wanted to be able to counter stain with propidium iodide to measure cellular DNA content. However, compensation was impossible to set with any confidence, so the counter stain was changed to 7-AAD, which has minimal spectral overlap with FITC (Fig. 1A; ref. 31). Back gating solely on the pHH3-positive population (Fig. 1A(iii)) showed pHH3 expression only in the G2-M phase of the cell cycle as determined by 7-AAD staining. Inhibition of aurora-B activity by AZD1152-HQPA was confirmed by a decrease in pHH3 on Ser10. The basal range of pHH3 expression in the cell lines was 2.5% to 4.8% of total cells. At concentrations of 0 to 100 nmol/L after 24 hours, inhibition of pHH3 was achieved in all five cell lines with complete inhibition achieved at 100 nmol/L in all but one of the lines (Fig. 1B). Of the cell lines tested, the FLT3-ITD–expressing MV4-11 and MOLM-13 cell lines were the most sensitive to pHH3 inhibition. Seventy-two hour incubation with AZD1152-HQPA caused loss of viability in all the cell lines, with an IC50 achieved at <30 nmol/L (Fig. 1C). Again, the FLT3-ITD MV4-11 and MOLM-13 cell lines were the most sensitive. After 24 hours of incubation with 30 nmol/L AZD1152-HQPA, all the cell lines developed polyploid (8N) DNA (Fig. 1D).
pHH3 inhibition and loss of viability in AZD1152-HQPA treated cells. A, flow cytometric measurement of pHH3 expression (FL1-H) and 7-AAD (DNA) content (FL3-A) in log phase U937 cell line (i) and (ii). The histogram in plot iii is gated solely on the pHH3-positive population (R3) and confirms 7AAD content/pHH3 expression only in the G2-M phase of the cell cycle. B, flow cytometric measurement of pHH3 expression in AML cell lines after 24 h of AZD1152-HQPA exposure. Columns, mean of three experiments; bars, SD. C, flow cytometric measurement of viable cells after 72 h of exposure to AZD1152. Columns, mean of three experiments; bars, SD. D, flow cytometric DNA content histograms of AML cell lines exposed to 30 nmol/L AZD1152-HQPA for 0 and 24 h. AZD1152-HQPA–treated cells fail to divide and develop polyploid (8N) DNA.
AZD1152-HQPA Induces Apoptotic Cell Death Occurring from the Endoreduplicated Subset of Cells
Annexin staining of AZD1152-HQPA–treated cells shows that cell death is by apoptosis (Fig. 2A). By back gating on the polyploid population (Fig. 2A(iii)), we can see that this population contains apoptotic cells. This agrees with previous findings that treatment with aurora-B inhibitors results in the accumulation of polyploid cells and subsequent apoptosis (12, 19). To investigate this further, we used two more markers of apoptosis, namely active caspase-3 and 7A6 antigen. Caspase-3 is a key protease that is activated in the early stages of apoptosis (32). 7A6 is a mitochondrial membrane protein also expressed in early apoptosis, which is recognized by the Apop2.7 antibody (33). We measured active caspase-3 and 7A6 expression in U937 cells after 48 hours of culture with 0 to 300 nmol/L AZD1152-HQPA (Fig. 2B). Both apoptotic markers were increased in a dose-dependent manner to AZD1152-HQPA and back gating on the polyploid population showed that both active caspase-3 and 7A6 antigen are only expressed in this population (data not shown).
AZD1152-HQPA induces cell death in AML cell lines by apoptosis occurring from the endoreduplicated subset of cells. A, apoptosis in U937 cells measured using Annexin V FITC. Nontreated control cells (i) with cells treated for 48 h with 30 nmol/L AZD1152-HQPA gated on the whole-cell population (ii) and treated cells gated on the SSC-H/FSC-H high (polyploid) population (iii). Gating on the whole population of treated cells (ii) shows a clear apoptotic (FL1 high/FL2 low) and necrotic (FL1 high/FL2 high) population. Gating only on the polyploid population (ii) shows apoptotic cells within this population. B, flow cytometric detection of active caspase-3 (i) and 7A6 expression (ii) in U937 cells after 48 h of exposure to AZD1152-HQPA. Experiments were repeated thrice.
AZD1152-HQPA–Induced Endoreduplication Is Paused but not Stopped in p53wt Cell Lines
VX-680 is an aurora kinase inhibitor that is selective for aurora-A, B, and C and has been shown to preferentially induce endoreduplication in cells with compromised p53 (34). With this in mind, we sequenced our cell lines for p53 status and examined its affect on the cell line response to AZD1152-HQPA by measuring cellular DNA content (Fig. 3A). p53 compromised HL60 and U937 cells acquired 8N DNA 24 hours after AZD1152-HQPA treatment (data not shown) and continued DNA synthesis to a point in which, at 72 hours, 8N peaks are not detected. The p53 wt cell lines also acquire 8N at 24 hours at which point DNA synthesis seems to be partially checked because the 8N peaks remain visible for up to 72 hours. This seems to agree with results seen with VX-680 in which differences in cellular DNA content after AZD1152-HQPA treatment depends on p53 status (34). However, if we look at the same AZD1152-HQPA–treated p53wt cells and analyze by flow cytometry with decreased voltage in the FL2 channel, we can detect a further 16N peak (Fig. 3B). So although the p53wt cells are checked compared with p53-compromised cells, p53 is not saving them from massive endoreduplication and subsequent loss of viability. A 16N peak was not detected in the p53-compromised cells lines after decreasing the FL2 voltage (data not shown), indicating that these cells apoptose before reaching this ploidy.
AZD1152-HQPA–induced endoreduplication is paused but not stopped in p53wt cell lines. A, cell line DNA content histograms (measured using 7-AAD) after treatment with 30 nmol/L AZD1152-HQPA. B, p53 wt cell line DNA content after 72 h of treatment with 30 nmol/L AZD1152-HQPA. In these plots, the cells were acquired at a lower voltage in the FL-2 channel, which allows us to see a further 16N peak compared with the plots in 3A.
AZD1152-HQPA Inhibits FLT3 Phosphorylation in FLT3-ITD Cell Lines Along with Its Downstream Target pSTAT5
Of the AML cell lines tested, the FLT3-ITD–expressing MV4-11 and MOLM-13 cell lines were particularly sensitive to AZD1152-HQPA, with viability and pHH3 IC50s below 10 nmol/L (Fig. 1). The FLT-ITD gene mutation is found in 24% of AML and is a poor prognostic factor. Internal tandem duplication (ITD) mutation of FLT3 induces activating phosphorylation of the receptor in the absence of ligand (35). With this in mind, we wanted to measure pFLT3 levels in the ITD cell lines after treatment with AZD1152-HQPA. Downregulation of pFLT3 was confirmed using immunoblotting with downregulation seen in the ITD cell lines at concentrations as low as 30 nmol/L AZD1152-HQPA after 1 h of incubation (Fig. 4A). In addition, oncogenic activation by ITD mutation is known to activate aberrant signaling, including direct STAT5 activation (36). We measured pSTAT5 by flow cytometry; this assay is fairly rapid and requires less material than for immunoblotting, which makes it potentially useful for patient material. Experiments confirmed the downregulation of pSTAT5 after 1 h of treatment with AZD1152-HQPA in the FLT3-ITD cell lines (Fig. 4C).
AZD1152-HQPA inhibits FLT3 phosphorylation in FLT3-ITD cell lines along with its downstream target pSTAT5. A, immunoblots showing pFLT3 downregulation in FLT3-ITD cells treated for 1 h with AZD1152-HQPA. Data represents one of three independent experiments. Full-length blots are presented in Supplementary Fig. S1. B, effect of 1 h of AZD1152-HQPA exposure on pFLT3 expression measured by immunoblotting and normalized to total FLT3 expression. Columns, mean of three experiments; bars, SD. C, effect of 1 h of AZD1152-HQPA exposure on pSTAT5 expression measured by flow cytometry. Columns, mean of three experiments; bars, SD.
pHH3 Expression in Primary AML Samples and Inhibition with AZD1152-HQPA
The level of pHH3 detectable in nontreated cell lines was low (2.5–4.8% of total cells) and expression in primary cells was expected to be even lower, as a very small population of cells are actively dividing at the time of sampling. With this in mind, we decided to preincubate primary samples with a cytokine cocktail to drive the cells into cycle before treatment with AZD1152-HQPA. Cellular proliferation in primary samples was confirmed by 3H-Tdr uptake with or without 48 hours of preincubation with cytokine cocktail (Fig. 5A). pHH3 expression correlated extremely well with the amount of proliferation shown by 3H-Tdr uptake and was comparable with the levels seen in cell lines. Mean basal pHH3 expression in the primary samples was 3.01% (range, 0.04–13.14%) of total cells. As expected, basal pHH3 expression in primary samples compared well with aurora-B mRNA levels (P = 0.015; Fig. 5B). Our next aim was to try to inhibit pHH3 with AZD1152-HQPA in these primary samples. We achieved significant pHH3 inhibition in preincubated primary samples at short time periods with 300 nmol/L AZD1152-HQPA (Fig. 5C). pHH3 expression was measured in 37 primary samples after 1 hour of treatment with 300 nmol/L AZD1152-HQPA (Fig. 5D). IC50 was achieved in all but three samples (91.9%) with a mean downregulation of 78% (range, 9.1–100%).
pHH3 is expressed in primary samples comparable with cell lines and can be inhibited with short-term AZD1152-HQPA treatment. A, [3H]-Tdr uptake in three primary AML samples after 6 h and after 48 h of preincubation with cytokine cocktail (20 ng/mL IL-3, SCF, IL-6, and 25 ng/mL granulocyte colony-stimulating factor). The histograms show pHH3 expression in the corresponding samples after 48 h of preincubation with cytokine cocktail. B, scatter plot for basal pHH3 expression and aurora-B mRNA levels in 35 primary AML samples. C, primary AML samples were preincubated with cytokine cocktail for 48 h and then treated with 300 nmol/L AZD1152-HQPA for 15 or 60 min followed by pHH3 measurement by flow cytometry. Percent total pHH3 expression is shown. D, primary AML samples were preincubated with cytokine cocktail for 48 h and then treated with 300 nmol/L AZD1152-HQPA for 60 min followed by pHH3 measurement by flow cytometry. Expression was calculated as a percentage of nontreated controls.
Primary FLT3-ITD Samples Are More Sensitive to AZD1152-HQPA–Induced Growth Inhibition, pHH3 Downregulation, and pSTAT5 Downregulation Compared with FLT3-WT Samples
Fourteen FLT3-ITD and 23 FLT3-WT primary AML samples were preincubated with cytokine cocktail for 48 hours and then treated with 300 nmol/L AZD1152-HQPA for 1 hour. pHH3 expression was measured by flow cytometry and inhibition was calculated as a percentage of total pHH3 in nontreated controls. The FLT3-ITD samples were more sensitive to AZD1152-HQPA–induced pHH3 inhibition (P = 0.022; Fig. 6A).
Primary FLT3-ITD samples are more sensitive than FLT3-WT samples to AZD1152-HQPA–induced pHH3 inhibition and pSTAT5 downregulation. A, 14 FLT3-ITD and 23 FLT3-WT primary AML samples were preincubated with cytokine cocktail for 48 h and then treated with 300 nmol/L AZD1152-HQPA for 1 h. pHH3 expression was measured by flow cytometry and inhibition was calculated as a percentage of total pHH3 in nontreated controls. B, 31 primary AML samples were preincubated with cytokine cocktail for 48 h and then treated with 300 nmol/L AZD1152-HQPA for 1 h. pSTAT5 expression was measured by flow cytometry and inhibition was calculated as a percentage of total pSTAT5 in nontreated controls.
Thirty-one primary AML samples were preincubated with cytokine cocktail for 48 hours and then treated with 300 nmol/L AZD1152-HQPA for 1 hour. pSTAT5 expression was measured by flow cytometry and inhibition was calculated as a percentage of total pSTAT5 in nontreated controls. The FLT3-ITD samples were significantly more sensitive to AZD1152-HQPA–induced pSTAT5 downregulation (P = 0.007; Fig. 6B).
Consecutive fresh primary AML samples (20 FLT3-WT and 8 FLT3-ITD) were grown for 14 days in a methylcellulose-based medium containing 0 to 30 nmol/L AZD1152-HQPA, and IC50s were calculated compared with nontreated controls. Colonies were detected in nontreated samples from 11 FLT3-WT but only 2 FLT3-ITD cases. The two FLT3-ITD samples were both more sensitive to AZD1152-HQPA–induced growth inhibition than any of the wild-type samples (P = 0.029).
Discussion
Drugs that target the aurora kinases are emerging as potential new strategies for the treatment of cancer. Several aurora kinase inhibitors have been described that show antitumor activity in vivo. Three nonspecific aurora kinase inhibitors have been shown to induce similar phenotypes when tested in cell-based assays (14–16). All three inhibited pHH3 on serine 10 and induced DNA endoreduplication in the absence of cytokinesis, results that suggest that their cellular effects are largely due to the inhibition of aurora-B (17). With this in mind, we investigated the activity of AZD1152-HQPA, a selective inhibitor of aurora-B, in AML cell lines and primary blasts.
pHH3 on serine 10 is a direct downstream target of aurora-B activity and previous reports have shown inhibition of pHH3 following treatment with aurora kinase inhibitors (14, 17). When pHH3 is inhibited, chromosome condensation is prevented and entry into mitosis is blocked. Here, we show that treatment with AZD1152-HQPA results in inhibition of pHH3, accumulation of cells with >4N DNA content, and subsequent loss of viability in a panel of leukemic cell lines. By using Annexin binding and active caspase-3 and 7A6 antigen expression we show that AZD1152-HQPA–treated cell lines undergo apoptosis and that the polyploid population contains apoptotic cells. This agrees with previous studies that showed that the aurora-B disruption in tumor cells forces cells through a catastrophic mitotic exit leading to polyploid cells that subsequently lose viability (14).
Through the course of our studies, we noticed the particular sensitivity of the FLT3-ITD–expressing MV4-11 and MOLM-13 cell lines to AZD1152-HQPA. Both had IC50s for viability and pHH3 inhibition of <10 nmol/L, which was the most sensitive of any of the lines tested. The FLT3 gene is one of the most commonly mutated genes in AML either as FLT3-ITD (24%) or FLT3 activation loop mutation, (7%) and is associated with poor prognosis (35, 37). Interestingly, VX-680 a selective inhibitor of all three aurora kinases also exhibits cross-inhibitory activity against the receptor tyrosine kinase FLT3 and ablated colony formation in primary AML cells with FLT3-ITD (15, 38). VX-680 has also been shown to preferentially induce endoreduplication in cells with compromised p53 (34). With this in mind, we sequenced our cell lines for p53 expression and measured their DNA content after treatment with AZD1152-HQPA. Although the p53wt cell lines are checked compared with p53-compromised cells, p53 does not save our AML cell lines from massive endoreduplication and ultimately loss of viability.
Because AZD1152-HQPA has been designed to specifically target aurora-B kinase, one of the questions that we wanted to answer was whether AZD1152-HQPA is also directly targeting FLT3 in the sensitive FLT3-ITD cell lines. ITD mutation of FLT3 induces activating phosphorylation of the receptor in the absence of ligand (35). Here, we show FLT3 dephosphorylation after 1 hour of incubation at doses as low as 30 nmol/L AZD1152-HQPA. Oncogenic activation by ITD mutation is also known to activate aberrant signaling, including direct STAT5 activation (36). Again, we show the downregulation of pSTAT5 at low doses of AZD1152-HQPA after 1 hour of treatment. This is the first demonstration that AZD1152-HQPA directly targets pFLT3 in ITD cell lines along with its downstream target pSTAT5.
In the next phase of our study, we investigated the effect of AZD1152-HQPA on primary AML blasts, starting with pHH3 expression. The level of pHH3 detectable in nontreated cell lines was low and expression in primary cells was expected to be even lower as a very small population of cells is actively dividing at the time of sampling. With this in mind, we preincubated the AML blasts with a cytokine cocktail to drive the cells into cycle before treatment with AZD1152-HQPA. We found levels of pHH3 comparable with those found in cell lines and were also able to inhibit pHH3 at short time periods after treatment with AZD1152-HQPA in all of the primary samples tested. Real-time PCR confirmed that aurora-B mRNA levels correlated to basal pHH3 expression in primary AML blasts. We also found that the FLT3-ITD samples were the most sensitive to AZD1152-HQPA–induced pHH3 inhibition, which is intriguing as pHH3 is widely reported to be a direct substrate of aurora kinase B. One possible explanation for this could be the role of survivin, which has recently been shown to lie downstream of FLT3-ITD signaling (39). Survivin along with aurora-B, INCENP, and borealin makes up the chromosomal passenger complex that plays a key role in mitotic progression. It has recently been reported that treatment of FLT3-ITD leukemia cells with the FLT3-ITD inhibitor SU5416 resulted in reduced survivin expression and inhibited cell proliferation (39). AZD1152-HQPA caused the growth inhibition of primary AML blasts in our colony formation assays and again the FLT3-ITD samples were the most sensitive of those tested. FLT3-ITD primary samples are also particularly sensitive to pSTAT5 downregulation after treatment with AZD1152-HQPA, compared with FLT3 WT samples.
In conclusion, AZD1152-HQPA seems to be a promising new agent for treatment of individuals with AML, particularly those who have an ITD mutation in the FLT3 gene.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Astrazeneca for a financial contribution toward reagent costs and the National Cancer Research Network AML Working Group for permitting access to stored samples from AML patients.
Grant Support: Research funding from Astra Zeneca.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Footnotes
Note: Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).
- Received December 10, 2009.
- Accepted January 7, 2010.
- ©2010 American Association for Cancer Research.
References
Volume 9, Issue 3
