Transcriptional Control and Signal Transduction

Synergistic antileukemic effects between ABT-869 and chemotherapy involve downregulation of cell cycle-regulated genes and c-Mos-mediated MAPK pathway

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Abstract

Internal tandem duplications (ITDs) of fms-like tyrosine kinase 3 (FLT3) receptor play an important role in the pathogenesis of acute myeloid leukemia (AML) and represent an attractive therapeutic target. ABT-869 has demonstrated potent effects in AML cells with FLT3-ITDs. Here, we provide further evidence that ABT-869 treatment significantly downregulates cyclins D and E but increases the expression of p21 and p27. ABT-869 induces apoptosis through downregulation of Bcl-xL and upregulation of BAK, BID and BAD. We also evaluate the combinations of ABT-869 and chemotherapy. ABT-869 demonstrates significant sequence-dependent synergism with cytarabine and doxorubicin in cell lines and primary leukemia samples. The optimal combination was validated in MV4-11 xenografts. Low-density array analysis revealed the synergistic interaction involved in downregulation of cell cycle and mitogen-activated protein kinase pathway genes. CCND1 and c-Mos were the most significantly inhibited targets on both transcriptional and translational levels. Treatment with short hairpin RNAs targeting either CCND1 or c-Mos further sensitized MV4-11 cells to ABT-869. These findings suggest that specific pathway genes were further targeted by adding chemotherapy and support the rationale of combination therapy. Thus, a clinical trial using sequence-dependent combination therapy with ABT-869 in AML is warranted.

Introduction

Internal tandem duplications (ITDs) of the fms-like tyrosine kinase 3 (FLT3), varying from 3 to 400 base pairs in the juxtamembrane domain, are found in 20–25% of adult acute myeloid leukemia (AML) cases.1, 2, 3 In addition, activating point mutations in the second kinase domain occur in about 7% of adult AML patients.4 FLT3 mutations therefore are the most common genetic alteration in AML. Clinically, FLT3-ITD is associated with poor outcome, but the prognosis of FLT3 activating point mutation remains inconclusive.5, 6, 7

FLT3-ITD mutations trigger strong autophosphorylation of the FLT3 kinase domain and constitutively activate several downstream effectors such as the PI3K-AKT pathway, RAS-MEK-mitogen-activated protein kinase (MAPK) pathway and the STAT5 pathway.8, 9 FLT3-ITD mutations also suppress transcription factors associated with myeloid differentiation and apoptosis, including PU.1, CCAAT/enhancer-binding protein α (C/EBPα),10 promyelocytic leukemia zinc finger protein,11 RUNX1/AML1,12 RSG2,13 and Foxo3a.14, 15, 16 In contrast, FLT3-ITDs upregulate proliferation-associated genes like PIM1.17 Taken together, FLT3-ITDs simultaneously bring on several hallmarks of leukemogenesis18 by blocking myeloid differentiation, inducing signaling for uncontrolled proliferation and producing resistance to apoptosis.

The mainstream chemotherapy regime for AML is a combination of cytosine arabinoside (Ara-C) and anthracyclines such as doxorubicin (Dox). Despite initial responses to chemotherapy, most adult AMLs eventually relapse. Long-term disease-free survival is only 20–30%. Thus, the development of novel therapeutic agents that target critical genetic aberrations holds promise for improving outcomes in patients with AML.

ABT-869, a novel ATP-competitive tyrosine kinase inhibitor (TKI), is active against FLT3 kinase (IC50=4 nM) and other platelet-derived growth factor receptor family members, as well as vascular endothelial growth factor (VEGF) receptors (IC50=4, 66 and 4 nM for kinase insert domain receptor (KDR), platelet-derived growth factor receptor-β and colony stimulating factor 1 receptor (CSF-1R), respectively), but less active against unrelated receptor tyrosine kinase (RTK)s.19, 20 Cellular assays and tumor xenograft models demonstrated that ABT-869 was effective in a broad range of cancers, including small-cell lung carcinoma, colon carcinoma, breast carcinoma and MV4-11 tumors in vitro and in vivo.19, 21 However, considering the complexity of the disease, monotherapy with ABT-869 is unlikely to deliver complete or lasting responses in AML. Furthermore, resistance to TKIs has been well described in patients treated with imatinib mesylate monotherapy for chronic myelogenous leukemia.22 Combination regimens, including ABT-869 and conventional chemotherapy, may potentially reduce resistance and achieve better outcomes for AML patients.

A combination approach has also been pursued with other TKIs. It has been reported that combination of SU11248 with Ara-C or Dox exerted synergistic effects23 and CEP-701 showed in vitro sequence-dependent synergistic cytotoxic effects on FLT3-ITD leukemia cells when combined with chemotherapy.24 In this study, the sequence-dependent synergism was attributed to CEP-701-induced cell cycle arrest, and it was speculated that the sequential treatment first induced pro-apoptotic signals, then withdrew pro-survival signals.25 Studies of the molecular mechanisms on synergistic interactions are needed for better understanding the full potential of combination therapy. The chemical structure of ABT-869 (N-(4-(3-amino-1H-indazol-4-yl)phenyl)-N1-(2-fluoro-5-methylphenyl) urea) is different from SU11248 (3-substituted indolinoneindolinone) and CEP-701 (indolocarbazole),19 suggesting that the therapeutic efficacy of ABT-869 cannot be extrapolated from the experience of related compounds. Hence, the clinical applications of ABT-869 will greatly benefit in better understanding the molecular mechanism of the compound in sole or combination therapies both in vitro and in vivo.

We, here, for the first time, present further characterization of molecular mechanism of G1-phase cell cycle arrest and apoptosis caused by ABT-869 as a single agent and the potential mechanism of synergism with the cytotoxic agents Ara-C and Dox in vitro and in vivo.

Materials and methods

Cell lines and primary patient samples

MV4-11 and MOLM-14 cells were cultured with RPMI1640 (Invitrogen, Carlsbad, CA, USA) supplemented with the addition of 10% fetal bovine serum (FBS; JRH Bioscience Inc., Lenexa, KS, USA) at a density of 2–10 × 105 cells ml−1 in a humid incubator with 5% CO2 at 37 °C.

Bone marrow blast cells (>90%) from newly diagnosed AML patients were obtained at National University Hospital in Singapore with informed consent. Three samples were confirmed to harbor a 36, 60/78 (two duplicated fragments detected), 62 bp ITDs of FLT3 gene, respectively, and one had D835Y (GAT → TAT at codon 835) point mutation. Thawed cells were cultured in EGM-2 medium (Cambrex, Walkersville, MD, USA) supplemented with SingleQuots (Cambrex) growth factors, cytokines (hFGF, hEGF, hydrocortisone, GA-1000, VEGF, R3-IGF-1) in the presence or absence of drug incubation.

ABT-869 and chemotherapy reagents

ABT-869 was provided by Abbott Laboratories (Chicago, IL, USA). For in vitro and in vivo experiments, ABT-869 was prepared as published previously.21 Clinical-grade Ara-C (100 mg ml−1, Pharmacia, Sydney, WA, Australia) and Dox (2 mg ml−1, Pharmacia) were diluted just before use. The MEK inhibitor U0126 was purchased from Promega and dissolved in dimethylsulfoxide at a concentration of 10 mM as stock. It was further diluted before use.

Cell viability assays

Leukemic cells were seeded in 96-well culture plates at a density of 2 × 104 viable cells per 100 μl per well in triplicates and were treated with ABT-869, chemotherapeutic agents or combination therapy. Colorimetric CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS assay, Promega, Madison, WI, USA) was used to determine the cytotoxicity. The absorbance of each well was recorded at 490 nm using an Ultramark 96-well plate reader (Bio-Rad, Hercules, CA, USA). The percentage of viable cell was reported as the mean of optical density of the treated wells divided by the mean of optical density of dimethylsulfoxide control wells after normalization to the signal from wells without cells. IC50 was determined by MTS assay and calculated by CalcuSyn software (Biosoft, Cambridge, UK). Each experiment was triplicated.

Combination index and isobologram analysis

The calculation of combination index (CI) and isobolograms with the CalcuSyn software was described previously.26 Briefly, the CI values were calculated according to the levels of growth inhibition (fraction affected) by each agent individually and combination of ABT-869 with Ara-C or Dox or U0126. Isobolograms, which indicate the equipotent combinations of different dose (ED50, ED75 and ED90, etc.), were used to illustrate synergism (CI<1), antagonism (CI>1) and additivity (CI=1). Constant ratio combinations of the two drugs at 0.25 × , 0.5 × , 1 × , 2 × and 4 × of their ED50 was used. Three independent studies were conducted for each combination.

Immunoblot analysis

Preparation of the cell lysate and immunoblotting were performed as described previously.26 Antibodies used were as follows: anti-cyclins D and E, anti-Bcl-xL, anti-Bcl2, anti-BAD, anti-BAX, anti-BAK, anti-poly (ADP-ribose) polymerase (PARP), anti-cleaved PARP, anti-caspase-3, anti-cleaved caspase-3, anti-caspase-7 and anti-cleaved caspase-7 from Cell Signaling Technology (CST, Danvers, MA, USA); anti-Actin, anti-p21, anti-p27, anti-p53, anti-cyclin-dependent kinase-2 (CDK2) and anti-CDK4 from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-human c-Mos oncoprotein polyclonal antibody was purchased from Chemicon (Temecula, CA, USA).

Low-density array

Gene expression profiling was investigated with custom PCR-based analysis using TaqMan Low Density Arrays (Applied Biosystems, Foster City, CA, USA).27 RNA was extracted from cells using Purescript RNA isolation kit (Genetra systems, Minneapolis, MN, USA). First-strand cDNA was synthesized with SuperScript III First-Strand Synthesis SuperMix (Invitrogen). PCR amplification was performed in the 7900HT Fast Real-time System (Applied Biosystems). The low-density array was custom-made with TaqMan Gene Expression Assays, which allows the simultaneous measurement of expression of 384 genes in a single sample. Each sample was duplicated. The target genes include anti- and pro-apoptotic genes, cell cycle-regulated genes, DNA-damage genes, stress gene, PI3K/AKT pathway, MAPK pathway, JAK/STAT pathway, mTOR pathway, VEGF pathway, NOTCH pathway, WNT pathway, NFκB pathway, invasion- and metastasis-related genes, oncogenes, as well as housekeeping genes. Sequence Detection System (SDS) 2.2.1 software (Applied Biosystems) was used to perform relative quantitation of target genes using the comparative CT (ΔΔCT) method.

Short-hairpin RNA studies

Expression Arrest Human retroviral pSM2 shRNAmir individual constructs CCND1 (clone ID: V2HS_88365) and c-Mos (clone ID: V2HS_36817) short-hairpin RNA (shRNA), as well as non-silencing shRNA control (RHS1707), were purchased from Open Biosystems (Huntsville, AL, USA). The Expression Arrest Human retroviral shRNAmir individual constructs are from the laboratory of Dr Greg Hannon at Cold Spring Harbor Laboratory, which created an RNAi Library comprised of multiple shRNAs specifically targeting annotated human genes. RetroPack PT67 cells (Clontech, Mountain View, CA, USA) were seeded into a six-well plate at 60–80% confluence (4 × 105 cells per well) 24 h before transfection; 5 μg of each shRNA vector and 10 μl of lipofectamine 2000 (Invitrogen) were used for transfection. PT67 cells were diluted and plated after transfection 24 h in culture medium with 2 μg ml−1 of puromycin (Clontech). After 1 week of selection, the large, healthy colonies were isolated and transferred into individual plates. Filtered medium containing viral particles together with 6 μg ml−1 of polybrene were used for infecting MV4-11 cells (2 × 106), respectively. Cultures were replaced with fresh medium post-infection 24 h and then subjected to immunoblot and cell viability assay.

Xenograft mouse model

Female severe combined immunodeficiency mice (17–20 g, 4–6 weeks old) were purchased from Animal Resources Centre (Canning Vale, WA, Australia). Exponentially growing MV4-11 cells (5 × 106) were subcutaneously injected into loose skin between the shoulder blades and left front leg of recipient mice. All treatment was started 25 days after the injection; when the mice had palpable tumor of 300–400 mm3 average size, Ara-C was intraperitoneally (i.p.) injected at 10 mg kg−1 day−1 for four consecutive days. ABT-869 was administrated at 15 mg kg−1 day−1 by oral gavage daily. In the combination group, Ara-C was given for 4 days, followed by ABT-869 daily for 26 days. Each group comprised of 10 mice.

The length (L) and width (W) of the tumor were measured with callipers, and tumor volume (TV) was calculated as TV=(L × W2)/2. The protocol was reviewed and approved by Institutional Animal Care and Use Committee in compliance with the guidelines on the care and use of animals for scientific purpose.

Immunohistochemistry

Tissue fixation and procedure of hematoxylin and eosin staining were processed as described previously.26 The sources and conditions of the primary antibodies were as follows: p-STAT5 (Tyr694, 1:50, Epitomics, Burlingame, CA, USA), p-AKT (Ser473, 1:200, CST), p-ERK1/2 (Tyr204, 1:50, Santa Cruz), VEGF (1:100, Lab Vision, CA, USA), cleaved PARP (1:50, CST). The anti-PIM1 antibody (clone 19F7) has been described previously.28 The slides were counterstained in hematoxylin for 30 s and mounted with cover slides. The images were analyzed by a Zeiss Axioplan 2 imaging system with AxioVision 4 software (Zeiss, Germany).

Statistical analysis

Number of viable cells, TV and survival time were expressed in mean±s.d. TV reduction of the treatment groups was compared to the untreated control group by Student’s t-test, and P-values of <0.05 were considered to be significant. Survival analysis was performed by Kaplan–Meier analysis (statistical package for social science (SPSS), ver.12). Survival curves of the treatment groups were compared to those of the untreated control group, and statistical significance were given in log-rank test (P<0.05).

Results

Molecular signaling pathways of cell cycle arrest and apoptosis induced by ABT-869 treatment

ABT-869 profoundly inhibited FTL3-ITD AML cell proliferation (MV4-11, MOLM-14 and TF1-ITD) (Supplementary Figure 1). ABT-869 induced G1 cell cycle arrest and apoptosis in both MV4-11 and MOLM-14 (Supplementary Figure 2). We further analyzed the molecular mechanisms of ABT-869-induced cell cycle arrest and apoptosis. Key cell cycle-regulated proteins were analyzed by immunoblotting. In MV4-11 and MOLM-14 cells, ABT-869 modulated the G1/S transition regulators in a time-dependent manner, as it entirely downregulated cyclins D and E by 16 h and induced the expression of p21waf1/Cip progressively. The increasing expression of cyclin E in MV4-11 cells at 4 h and in MOLM-14 cells at 1 h and of cyclin D in MOLM-14 cells at 8 h after drug exposure could be due to the fact that cells intended to progress to S-phase at the early time points.29 The expression of CDK2 and CDK4 was relatively stable. p27kip1 was increased and maximal in MV4-11 cells at 16 h and in MOLM-14 cells at 8 h after treatment (Figure 1a). These data suggested that simultaneous terminal reduction of cyclins D and E, the key G1/S cyclins and progressive increases in CDK inhibitors p21waf1/Cip and p27kip1 contributed to the blockage of G1/S progression induced by ABT-869.

Figure 1
figure1

The molecular mechanisms of cycle arrest and apoptosis induced by ABT-869 treatment in MV4-11 and MOLM-14 cells. MV4-11 and MOLM-14 cells were exposed with ABT-869 6 and 9 nM respectively for 0, 1, 4, 8 and 16 h, then washed, lysed and subjected to 12% SDS–polyacrylamide gel electrophoresis (SDS-PAGE). Western blots were detected with indicated antibodies for the assessment of the expression level changes in (a) cell cycle-regulated proteins and (b) apoptosis-regulated proteins. β-actin was used to confirm equal loading protein of each sample. C-BID and C-PARP referred to cleaved-BID and cleaved-PARP, respectively.

To elucidate the mechanisms of ABT-869-induced apoptosis of FLT3-ITD-AML cells, the expression of several apoptosis-associated proteins was examined. Proapoptotic BAD was gradually increased in MV4-11 cells and intensively increased after exposure to ABT-869 for 8 h in MOLM-14 cells. In both cell lines, ABT-869 augmented the expression of proapoptotic proteins BAK and BID and decreased the expression of anti-apoptotic Bcl-xL protein in a time-dependent manner. Cleaved BID could be visualized as early as 1 h after ABT-869 treatment. Another anti-apoptotic protein Bcl2 was not altered. ABT-869 also transiently induced the expression of p53 immediately after 1 h drug exposure. The protein level of BAX was increased only in MV4-11 cells at 16 h post-treatment, not in MOLM-14 cells (Figure 1b). After incubation with ABT-869, cleavage of effector caspase 7 was detected in MV4-11 at 1 h and in MOLM-14 at 4 h and increased in a time-dependent manner thereafter. However, cleaved caspase 3 was more prominently observed in MV4-11 cells than in MOLM-14 cells. Cleavage of PARP was also observed in both cells (Figure 1b).

Simultaneous treatment with ABT-869 and chemotherapeutic agents

Prior to studying the combination effect, the efficacy of Ara-C and Dox as single agent was first confirmed. The IC50 of Ara-C on MV4-11 and MOLM-14 cells at 48 h were 450 and 250 nM, respectively, and the IC50 of Dox for these two cell lines were 350 and180 nM, respectively (data not shown). MV4-11 and MOLM-14 cells were treated with ABT-869 and in combination with either Dox or Ara-C, then assayed for cell survival by MTS assay. As shown in the Figure 2a, the effect of combining ABT-869 and Ara-C at their ED50 or ED75 approximated the respective theoretic additive values indicated by the diagonals. In contrast, combining ABT-869 and Ara-C at their ED90 concentrations resulted in a value that fell far to the right of the diagonal in MV4-11 cells, but not in MOLM-14 cells. These data suggest that at lower doses there is an additive or mildly synergistic interaction, while at higher doses the two agents might interact in an antagonistic manner.26 All of the combination results of ABT-869 and Dox were to the lower left of the diagonals, indicating synergistic effects (Figure 2b).

Figure 2
figure2

Conservative isobolograms showing the interactions among three different models of combination with ABT-869 and chemotherapeutic agents on the proliferation of MV4-11 and MOLM-14 cells. The drug concentration unit is nM. The diagonal lines linking up the ED50, ED75 and ED90 values of two drugs represent the theoretic additive lines. Synergism is indicated by the ED points located on the lower left of the diagonal. Antagonism is implied by ED points located on the upper right above the diagonal. Additive effects are indicated when the ED points fall on the diagonal. These results were generated by CalcuSyn software for (a) simultaneous combination of ABT-869 with Ara-C, (b) simultaneous combination of ABT-869 and Dox, (c) pretreatment with ABT-869 first followed by Ara-C, (d) pretreatment with ABT-868 first followed by Dox, (e) pretreatment with Ara-C first in addition of ABT-869, (f) pretreatment with Dox first in addition of ABT-869. The results are from three representative independent experiments.

Sequence-dependent interactions between ABT-869 and chemotherapy

We next employed a sequence-dependent method as described by Levis et al.24 MV4-11 and MOLM-14 cells were treated with ABT-869 at various doses for 24 h, and washing was followed by addition of Ara-C or Dox incubation for 48 h. Isobologram analysis for both cell lines showed that the combination values were located on the diagonal (ED50) and far right of the diagonals (ED75 and ED90) (Figure 2c). This indicated that pretreatment with ABT-869 antagonized the cytotoxicity of Ara-C. But pretreatment with ABT-869 followed by Dox appeared to have both antagonistic (ED50) and synergistic (ED75 and ED90) effects on MV4-11 cells (Figure 2d, left isobologram) and have essentially antagonism in MOLM-14 cells (Figure 2d, right isobologram).

Lastly, chemotherapy was followed by ABT-869. MV4-11 and MOLM-14 cells were exposed to Ara-C or Dox for 24 h and washed out and then transferred into medium containing ABT-869 for an additional 48 h. Synergistic effect of pretreatment with Ara-C or Dox, followed by ABT-869, was consistently identified at ED50, ED75 and ED90 points (Figures 2e and f). The CI values obtained for ABT-869 in combination with Ara-C and Dox employing three sequences are shown in Table 1. To determine whether the combination therapy produces synergism in induction of apoptosis, the Annexin-V/PI double staining was used to assess MV4-11 cells treated with Ara-C followed by ABT-869. The CI values at ED50, ED75 and ED90 were 0.56, 0.50 and 0.38 respectively, which indicated synergism. These data illustrated that pretreatment with chemotherapy followed by ABT-869 produced synergistic effects on inhibition of proliferation and induction of apoptosis.

Table 1 CI values in three models of ABT-869 and chemotherapeutic agents

To further validate findings in cell lines, patient samples with either FLT3-ITD (patient numbers 1–3), FLT3-D835Y point mutation (patient number 4) or wild-type FLT3 (patient number 5–7) were treated with Ara-C 24 h first, followed by ABT-869. Primary cells were incubated with either ABT-869 (20, 40, 80, 160, 320 nM) or Ara-C (100, 200, 400, 800, 1600 nM) alone and in combination. The CI values of these patient samples with FLT-ITD and D835Y mutations ranged from 0.67 to 0.08, indicative of synergism between the two agents on a primary AML specimen with FLT3-ITD or D835Y point mutation. In contrast, the combination of Ara-C and ABT-869 on three patient samples with wild-type FLT3 did not produce a synergistic effect (CI values between 0.9 and 1.2).

Inhibition of cell cycle-related genes and MAPK pathway played an important role in the synergistic mechanism

To address the underlying molecular mechanism of the synergism between ABT-869 and chemotherapy, we utilized a real-time PCR-based approach to profile the gene expression between MV4-11 cells treated with combination therapy (Ara-C followed by ABT-869) and single-agent therapy. The significantly downregulated gene clusters in combination therapy contained probes for genes involved in cell cycle regulation and the MAPK pathway as compared to Ara-C or to ABT-869 treatment alone (Table 2). Among all the affected genes, CCND1 and Moloney murine sarcoma viral oncogene homolog (c-Mos) were the two most significantly downregulated genes. To examine their functional roles in the synergistic manifestation, western blot analysis confirmed that combination treatment also significantly decreased CCND1 and c-Mos at the protein level, as well as blockage of the MAPK pathways, indicated by reduced phosphorylation of ERK protein (Figure 3a). Specific inhibition of CCND1 (approximately 80% reduction) and c-Mos (approximately 60%) by shRNAs was confirmed by immunoblot analysis (Figure 3b, right panel). Essentially, silencing either CCND1 or c-Mos remarkably potentiated ABT-869-induced inhibition to a similar degree as combination therapy (Ara-C 100 nM followed by ABT-869) when compared to control shRNA treatment (P<0.01) (Figure 3b). To further validate the importance of MAPK pathway, we used an ERK inhibitor U0126 in combination with -869 in three different sequences. The IC50 of U0126 on MV4-11 is 14 μM. Both sequence-dependent combinations (ABT-869 first or U0126 first) produced synergism (Figure 3c, middle and right isobolograms). When the two drugs were given simultaneously, it achieved synergistic effect at IC50 and IC75 and additive effect at IC90 (Figure 3c, left isobologram). These data provide further evidences that MAPK signaling transduction pathway, specifically via MEK/ERK pathway, is critical for the synergism.

Table 2 LDA analysis revealed that combination therapy further downregulated genes involved in cell cycle regulation and MAPK pathway
Figure 3
figure3

CCND1 and c-Mos played important roles in the molecular mechanisms of synergistic effect by combination therapy. (a) MV4-11 cells were treated with dimethylsulfoxide control, ABT-869, Ara-C and combination therapy (Ara-C followed by ABT-869), then subjected to immunoblot analysis. (b) Silencing either c-Mos or CCND1 by shRNA augments the cell proliferation inhibition with ABT-869. MV4-11 cells were treated with control, c-Mos or CCND1 shRNA separately, then exposed to ABT-869 at various dosage or Ara-C 100 nM followed by ABT-869. MTS assay was used to assess the growth inhibition. (c) Conservative isobolograms of ABT-869 in combination with U0126 in three different sequences. MV4-11 cells were treated with ATB-869 at concentrations of 1.5, 3, 6, 12, 24 nM or U0126 at concentrations of 3.5, 7, 14, 28, 56 μM simultaneously or sequentially (ABT-869 first or U0126 first) in the same manner as ABT-869 in combination with chemotherapy. CalcuSyn software was used to generate the isobologram for simultaneous treatment (left panel), ABT-869 first followed by U0126 (middle panel) and U0126 first followed by ABT-869 (right panel). All CI values at IC50, IC75 and IC90 of the three combinations were shown in the table at the bottom. The results are from three representative independent experiments.

In addition, we investigated whether PI3K/AKT, another important pro-survival signaling pathway, was involved in combination therapy or not. Western blot revealed that the reduction of p-AKT was more obvious in ABT-869 alone than the combination (data not shown), suggesting that this pathway is not the mechanism for the synergistic effect in combination studies.

In Vivo efficacy of ABT-869, alone or in combination with cytotoxic drugs, for treatment in MV4-11 mice xenografts

Based on the in vitro results, the optimal combination sequence (chemotherapy followed by ABT-869) was studied in vivo. Tumors in mice treated with Ara-C alone showed an initial growth delay during chemotherapy treatment, then grew at the same rate as those in the vehicle control group (Figure 4a). In the ABT-869 monotherapy group, a complete response (no palpable tumor) was observed in 2/10 mice by day 35 and in all mice by day 39. In the combination group, a complete response was observed in 6/10 mice at day 35 and in all mice by day 39. All treatments were stopped at day 54. The anti-tumor effects of ABT-869 or the combination were significantly better when compared to Ara-C alone or control (P<0.001). The combination therapy resulted in faster reduction of tumor burden compared to ABT-869 treatment alone (P=0.03) and more complete responders as compared to ABT-869 treatment alone. We did not observe any adverse side effects in the treatment groups in terms of behavior or body weight changes.

Figure 4
figure4

Combination therapy achieved a faster reduction of established TV than ABT-869 single agent or Ara-C treatment.

Molecular events following in vivo treatment of MV4-11 tumors with ABT-869

In addition to reduction of TV, ABT-689 demonstrated significant biochemical effects on MV4-11 xenografts tumor. Histological examination of tumor specimens showed treated samples to be less cellular, compared to samples from mice treated with vehicle only (Figure 5a). A 15 mg kg−1 day−1 dose of ABT-869 effectively reduced p-STAT5 (Figure 5b), p-AKT (Figure 5c), p-ERK1/2 (Figure 5d) and PIM1 (Figure 5e), all of which are reported to be important FLT3 downstream effectors. In addition, the expression of VEGF was profoundly reduced in the treated tissue (Figure 5f). Cleavage of PARP was increased after the treatment (Figure 5g). Together, these data supported that the in vivo biological effect of ABT-869 is associated with the inhibition of multiple pathways including FLT3, STAT5, AKT, MAPK and angiogenesis.

Figure 5
figure5

In vivo effect of ABT-869 on MV4-11 tumor xenograft model. Severe combined immunodeficiency mice with established MV4-11 xenograft were treated with vehicle or ABT-869. Excised tumor pieces were embedded in paraffin and stained with either (a) hematoxylin and eosin or immunostained with (b) p-STAT5, (c) p-AKT, (d) p-ERK1/2, (e) PIM-1, (f) VEGF and (g) cleaved (C)-PARP. The magnification of all pictures is 400 × . Arrows indicate necrosis with fat replacement in this area.

Discussion

Multitargeted TKIs, including FLT3 inhibitors, are promising targeted therapeutics for leukemia-harboring FLT3 mutations. In this study, we further dissected the molecular mechanisms for ABT-869 on proliferation and apoptosis. We then demonstrated the importance of sequence-specific synergistic effect in combining targeted therapy such as ABT-869 with chemotherapy in cell lines and primary AML cells containing either FLT3-ITD or FLT3-D835Y. Our findings highlighted the ‘sequence specific’ feature of TKIs that has been suggested with other TKIs.24 The greatest synergism occurs when the cytotoxic agents were administered first, followed by ABT-869.

We observed cleaved caspase 3 mainly in MV4-11 cells. It has recently been reported that caspase 3 is responsible for DNA fragmentation and morphologic changes, while caspase 7 is responsible for the loss of cellular viability.30 MV4-11, which has both alleles with mutated FLT3, is more sensitive to ABT-869 than MOLM-14, which has one allele with FLT3-ITD and the other allele with wild type.

Furthermore, this study, for the first time, demonstrates that the synergism of combination therapy is due to downregulation of cell cycle-regulated genes and genes in MAPK pathway. Combination treatment not only completely inhibits phosphor-ERK1/2, but also results in decreased expression of wild-type ERK1, which likely also contributes to inhibition of MAPK pathway. In addition to its well-described function in G1- to S-phase progression, CCND1 overexpression has been found in a variety of cancers including B-cell lymphoma, multiple myeloma and breast cancer; thus, CCND1 is also regarded as an oncogene.31 The c-Mos proto-oncogene product, a serine/threonine kinase, is a strong activator of the MAPK pathway, which is important for oocyte maturation.32, 33 In somatic cells, constitutive expression of c-Mos in mouse fibroblasts leads to neoplastic transformation.34 Deregulated expression of c-Mos has been discovered in various human cancer cell lines and primary patient samples, including neuroblastoma,35 thyroid medullary carcinoma,36 and non-small-cell lung carcinomas.37 It is noteworthy that increased levels of CCND1 is found in both c-Mos-transformed cells and c-Mos-transgenic mice.34 The MAPK pathway is a major regulator of cell survival and proliferation, and its activation is well documented in leukemia.38 These observations are in line with our results of low-density array, immunoblot and shRNA analysis and U0126 inhibitor. Most interestingly, our data suggest that targeting cell cycle genes, notably CCND1 and c-Mos-mediated MAPK/MEK/ERK pathway, could be the main mechanism of the synergistic interactions between chemotherapy and ABT-869.

For simultaneous combinations, only ABT-869 and Ara-C together achieved an additive effect, while ABT-869 and Dox together produced synergism. SU11248, another FLT3 inhibitor, also was found to synergistically interact with Ara-C or Dox in vitro when given concurrently.23 In contrast, pretreatment with ABT-869 followed by chemotherapy yielded an undesirable antagonistic effect. The antagonism observed could result from G1-phase cell cycle arrest and the removal of cells in the S-G2/M boundary by ABT-869, resulting in more cells under quiescent condition. Ara-C is a phase-specific agent that is most active against cells in S-phase. In contrast, Dox is active against cells during multiple phases of the cell cycle.39 Collectively, pretreatment with ABT-869 would make subsequent chemotherapy less efficacious. In agreement with our data, antagonism has been reported with pretreatment with CEP-701, another FLT3 inhibitor, followed by Ara-C or etoposide.24

The animal experiment provided further evidence to support that chemotherapy followed by ABT-869 is the sequence of choice for combination. The in vivo immunohistochemistry study showed that ABT-869 has vigorous biological activities against FLT3 signaling pathways, demonstrated by the pronounced inhibition of several main FLT3 downstream targets. ABT-869 also reduced the expression of VEGF in the MV4-11 tumors. VEGF specifically promotes the proliferation of endothelial cells and is a major regulator of tumor angiogenesis in vivo. Because ABT-869 is a multitarget kinase inhibitor, the inhibitory effect of non-FLT3 targets such as VEGF could also contribute to the reduction of MV4-11 tumor in vivo. These findings highlight the critical role of in vivo animal models in the preclinical development of TKIs.

Our data demonstrate the ability of ABT-869 to interact synergistically with chemotherapy in a sequence-dependent manner and reveal the mechanism of synergy as further suppression of cell cycle-regulated genes and the c-Mos-mediated MAPK/MEK/ERK pathway. These observations will help to define the optimal combination therapy for future clinical trials in AML.

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Acknowledgements

We specially thank Professor Evelyn SC Koay for scientific discussion and Ms Khng Jiaying for administrative help, as well as Dr Richie Soong and Ms Baidah Binte Ahmad for excellent assistance in low-density array analysis. This work was supported by Singapore Cancer Syndicate Grant—TN0031, AN0038 and Terry Fox Run Cancer Research Grant 2004 (C-S Chen).

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Correspondence to C-S Chen.

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Declaration of commercial interest: Three of the authors (K.B.G., D.H.A. and S.K.D.) are employees of Abbott Laboratories, whose potential product was studied in the present work.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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Zhou, J., Pan, M., Xie, Z. et al. Synergistic antileukemic effects between ABT-869 and chemotherapy involve downregulation of cell cycle-regulated genes and c-Mos-mediated MAPK pathway. Leukemia 22, 138–146 (2008) doi:10.1038/sj.leu.2404960

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Keywords

  • AML
  • FLT3
  • kinase inhibitor
  • cytotoxic drugs
  • cell cycle
  • MAPK pathway

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