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The selective Trk inhibitor AZ623 inhibits brain-derived neurotrophic factor–mediated neuroblastoma cell proliferation and signaling and is synergistic with topotecan
Article first published online: 19 OCT 2010
Copyright © 2010 American Cancer Society
Volume 117, Issue 6, pages 1321–1391, 15 March 2011
How to Cite
Zage, P. E., Graham, T. C., Zeng, L., Fang, W., Pien, C., Thress, K., Omer, C., Brown, J. L. and Zweidler -McKay, P. A. (2011), The selective Trk inhibitor AZ623 inhibits brain-derived neurotrophic factor–mediated neuroblastoma cell proliferation and signaling and is synergistic with topotecan. Cancer, 117: 1321–1391. doi: 10.1002/cncr.25674
- Issue published online: 4 MAR 2011
- Article first published online: 19 OCT 2010
- Manuscript Accepted: 24 AUG 2010
- Manuscript Revised: 23 AUG 2010
- Manuscript Received: 15 JUL 2010
- brain-derived neurotrophic factor (BDNF);
TrkB expression is associated with poor prognosis for patients with neuroblastoma. AZ623 is a novel potent and selective inhibitor of the Trk family of tyrosine kinases. The authors hypothesized that AZ623 would inhibit TrkB-mediated signaling in neuroblastoma tumor cells and would be synergistic when combined with chemotherapy.
Neuroblastoma cell lines were screened for TrkB receptor mRNA expression and for their proliferation rates in response to brain-derived neurotrophic factor (BDNF). The effects of AZ623 on Trk receptor phosphorylation, signaling, and cell growth were evaluated in BDNF-treated neuroblastoma cells. Mice with human neuroblastoma xenograft tumors were treated with AZ623 alone and in combination with topotecan, and tumor growth rates were determined during and after treatment.
Neuroblastoma cell lines expressed various levels of the TrkB receptor and demonstrated increased proliferation in response to BDNF. BDNF treatment stimulated TrkB phosphorylation and downstream signaling that could be inhibited by AZ623. Neuroblastoma cells demonstrated in vitro sensitivity to AZ623, with concentration that inhibits 50% (IC50) values between 0.8 to 7 μM. AZ623 treatment was found to inhibit BDNF-mediated neuroblastoma cell proliferation. Mice with human neuroblastoma xenograft tumors demonstrated tumor growth inhibition when treated with AZ623 and with AZ623 combined with topotecan. Limited tumor regrowth was noted in mice with tumors treated with AZ623 combined with topotecan after treatment discontinuation.
AZ623 is a novel selective Trk inhibitor that inhibits BDNF-mediated signaling and neuroblastoma cell proliferation. AZ623 treatment inhibits the growth of human neuroblastoma xenograft tumors, and treatment with AZ623 combined with topotecan results in the prolonged inhibition of tumor regrowth. On the basis of these results, further preclinical development is warranted. Cancer 2011. © 2010 American Cancer Society.
Neuroblastoma is the most common extracranial solid tumor of childhood. However, overall survival rates for children with high-risk neuroblastoma are approximately 30% with current treatment regimens,1-3 despite intensive treatment that includes high-dose chemotherapy, surgery, radiotherapy, and retinoic acid.4 Furthermore, children with refractory or recurrent neuroblastoma have poor responses to salvage therapy and have a median survival of <6 months.5 Biologically targeted therapies may provide alternative treatments that are more easily administered and potentially less toxic options for these patients.
The TRK gene family encodes 3 growth factor receptors for the neurotrophin family of ligands, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). The 3 Trk receptor isoforms, TrkA, TrkB, and TrkC, have well-defined roles in neuronal development.6, 7 Trk receptors have also been shown to have roles in malignant transformation, chemotaxis, metastasis, and survival in human tumors.8-10 In neuroblastoma tumors, TrkB expression is a strong predictor of aggressive tumor growth and poor prognosis, while TrkA expression is associated with good prognosis.11 Furthermore, TrkB overexpression and signaling through the phosphatidylinositol-3-kinase (PI3K)/AKT signaling axis is associated with increased chemotherapy resistance in neuroblastoma tumor cells.12, 13
Based on the many roles of the Trk receptor family in tumorigenesis, both small molecule and monoclonal antibody inhibitors of Trk receptors have been developed for therapeutic purposes. Indolocarbazole analogs k252a and CEP-701 (lestaurtinib) are Trk kinase inhibitors that are currently available and that have been shown to be effective against neuroblastoma in preclinical studies.14, 15 However, these agents inhibit a variety of kinase targets, and their efficacy may be only in part related to their inhibition of Trk kinase activity. CEP-701 has been demonstrated to inhibit the platelet-derived growth factor receptor (PDGFR), the vascular endothelial growth factor receptor (VEGFR), the REarranged during Transfection (RET) and protein kinase C (PKC) kinases and has been studied in clinical trials for adults as a fms-related tyrosine kinase 3 (Flt3) inhibitor for the treatment of acute myeloid leukemia (AML) and as a Janus kinase 2 (JAK2) kinase inhibitor in myeloproliferative disorders.16-19
The linkage between Trk receptor expression and neuroblastoma pathophysiology suggests that Trk kinase inhibitors may be effective for the treatment of children with neuroblastoma. AZ623, a selective small molecule inhibitor of the Trk tyrosine kinase family, is a potent ATP-competitive inhibitor of the Trk receptor kinase family and blocks tumor growth in a preclinical neuroblastoma xenograft model.20 We hypothesized that AZ623 would inhibit BDNF-mediated signaling and proliferation in neuroblastoma tumor cells and would be synergistically effective with chemotherapy against neuroblastoma xenograft tumors.
MATERIALS AND METHODS
Cells and Culture Conditions
The characteristics of neuroblastoma cell lines SK-N-SH, SH-EP, SH-SY5Y, NGP, CHP-134, LA1-55N, SK-N-AS, SMS-KCN, SMS-KCNR, and IMR-32 used in this study have been previously described,21-24 and were generously provided by Susan Cohn (The University of Chicago Children's Hospital, Chicago, Ill) and John Maris (Children's Hospital of Philadelphia, Philadelphia, Penn). Neuroblastoma cell lines were grown at 37°C in 5% carbon dioxide in RPMI-1640 (Invitrogen, Carlsbad, Calif) supplemented with 10% heat-inactivated fetal bovine serum (USB, Minneapolis, Minn), L-glutamine, sodium pyruvate, nonessential amino acids, and penicillin/streptomycin (Sigma Chemical Company, St. Louis, Mo).
AZ623 was provided by AstraZeneca (Macclesfield, Cheshire, United Kingdom). A stock solution (100 mM) was generated in dimethyl sulfoxide (Sigma Chemical Company) and stored at −20°C. Before use, a 10-mm stock solution was created in phosphate-buffered saline (PBS) and 30% acetic acid. A 10-mm stock solution of topotecan (Alexis Biochemicals, San Diego, Calif) was prepared in PBS and stored. AZ623 and topotecan were diluted to final concentrations in fresh serum-free cell culture media immediately before cell culture use. Stock solutions of BDNF (Peprotech, Rochy Hill, NJ), NGF (ProSpec, Rehovot, Israel), and NT-3 (Sigma Chemical Company) at 100 mg/L were generated in water and stored at −20°C. Before use, BDNF, NGF, and NT-3 were diluted to final concentrations in fresh serum-free cell culture media.
Quantitative Reverse Transcriptase-Polymerase Chain Reaction
Neuroblastoma cells were grown to near-confluence on 10-cm tissue culture plates. Cells were harvested and total RNA was generated using Qiagen RNeasy Mini-kits following the manufacturer's instructions (Qiagen, Valencia, Calif). RNA was quantified using a Nanodrop 1000 spectrophotometer (Nanodrop, Wilmington, Del). cDNA was synthesized using the Omniscript reverse transcription kit (Qiagen). Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers for TrkA, TrkB, and TrkC (NTRK1, NTRK2, and NTRK3; Applied Biosystems, Foster City, Calif) was performed using a BioRad iCycler iQ real-time PCR detection system with relative levels normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Cell Proliferation/Viability Assays
Approximately 5000 neuroblastoma cells per well were plated in 96-well plates and allowed to adhere overnight. Cells were then treated with 10 ng/mL of BDNF, NGF, or NT-3 for 72 hours. KCN cells were subjected to additional studies at concentrations of BDNF from 0.1 ng/mL to 10 ng/mL. Ten μL of AlamarBlue reagent (Invitrogen) was added to each well and plates were incubated at 37°C for at least 2 hours. Plates were read on a Spectramax Gemini EM spectrophotometer (Molecular Devices, Sunnyvale, Calif) at 544 nanometers (nm) and 590 nm. For viability assays, cells were treated with 0 to 10 μM of AZ623 for 72 hours and AlamarBlue assays were performed as described. Additional wells were treated with 0.1, 1.0, or 10 ng/mL BDNF combined with 0.1, 1.0, or 10 μM AZ623 for 48 hours. Concentration that inhibits 50% (IC50) values were derived using best-fit trendlines and values calculated using the relevant curve-fit equations.
Neuroblastoma cells were plated in 10-cm tissue culture dishes and allowed to grow to approximately 70% confluence. Cells were then treated with 1.25 μM of AZ623, 15 nM of topotecan, or both for 72 hours. Cells were photographed at ×200 magnification with a Nikon Digital Sight DS-L1 camera attached to a Nikon Eclipse TS 100 Microscope (Nikon Inc, Melville, NY). Additional cells were treated as above, and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assays were then performed on the cells, following the manufacturer's instructions (Promega, Madison, Wis), followed by counterstaining with Hoescht 33342 stain (dilution of 1:10,000; Invitrogen) and the addition of an antifade reagent (SlowFade Gold; Invitrogen)
Cell Signaling Assays
Neuroblastoma cells were plated on 10-cm tissue culture plates and grown until approximately 70% confluent. The cells were then washed, placed in serum-free media overnight, treated with 0 to 100 ng/mL BDNF for 15 minutes, and then harvested. Additional cells were treated with 25 nM or 50 nM AZ623 for 50 minutes, followed by 0 to 100 ng/mL BDNF for an additional 10 minutes. Cells were collected and incubated for 5 minutes with PhosphoSafe Extraction Reagent (Novagen, Darmstadt, Germany). Fifty μg of total denatured protein from each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.22-μM polyvinylidene fluoride (PVDF) membranes using standard techniques. Membranes were blocked with 5% milk and incubated with primary antibodies to total (Cell Signaling, Danvers, Mass; #4606) and phosphorylated (LifeSpan Biosciences, Seattle, Wash; LS-C50093) TrkB, β-actin (Sigma Chemical Company; A5441), phosphorylated PI3K (Cell Signaling; 4228S), phosphorylated AKT (Cell Signaling; 9271S), phosphorylated BAD (Cell Signaling; 9291S), phosphorylated p70S6K (Cell Signaling; 9205), phosphorylated ribosomal protein S6 (RPS6) (Cell Signaling; 2211S), phosphorylated MEK1/2 (Cell Signaling; 9102), and phosphorylated ERK1/2 (Cell Signaling; 9154S), all diluted to appropriate concentrations in 2.5% milk. Membranes were washed and incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ; NA931V and NA934V) and then developed with the ECL Western Blotting Analysis System (Amersham, Piscataway, NJ) and a Kodak film developer (Eastman Kodak, Rochester, NY).
NOD/SCID/IL2Rγ mice (stock #005557; Jackson Laboratories, Bar Harbor, Me) at the age of 6 to 8 weeks were inoculated subcutaneously into the right flank with 7 × 107 neuroblastoma tumor cells. Once tumors were palpable (approximately 5 mm in diameter), mice were treated once daily, 5 days per week, by gavage feeding with either vehicle alone, AZ623 alone (100 mg/kg/day), topotecan alone (1.5 mg/kg/day),25 or AZ623 combined with topotecan. Drugs were diluted in 100 μL of 0.5% hydroxypropylmethylcellulose (HPMC)/0.1% Tween-80 (Sigma Chemical Company). Five to 6 mice were treated in each group. Tumor measurements were obtained 3 times per week by the same investigator (L.Z.) using Vernier calipers (Fisher Scientific, Pittsburgh, Penn) and tumor volume was calculated using the following formula: tumor volume = (length ×width2)/2. Animals were sacrificed 14 days after the initiation of treatment and tumors were subsequently harvested for further analysis. Tumors harvested from mice were immediately snap frozen in liquid nitrogen. Serial sections were used for immunofluorescence analysis. TUNEL assays were then performed on the sections, as described above, followed by counterstaining with Hoescht 33342 1:10,000 dilution; Invitrogen) and the addition of antifade reagent (SlowFade Gold; Invitrogen).
In a separate experiment, mice with subcutaneous xenograft tumors were treated as detailed above with AZ623, topotecan, or AZ623 plus topotecan. Three to 4 mice were treated in each group. After 14 days, treatment was discontinued and tumor regrowth was monitored for an additional 15 days to determine regrowth rates. All animals were treated according to National Institutes of Health (NIH) guidelines for animal care and use, and protocols were approved by the Institutional Animal Care and Use Committee at The University of Texas M. D. Anderson Cancer Center.
Experiments were performed in triplicate unless otherwise noted. Two-tailed Student t test and analysis of variance were used to compare the significance between mean values for in vitro experiments and between xenograft tumor sizes in vivo, respectively.
Expression of TrkB Receptor in Neuroblastoma Cell Lines
In order to determine the expression pattern of TrkB in neuroblastoma cell lines, a panel of neuroblastoma cell lines was evaluated for expression of TrkB receptor mRNA. RT-PCR demonstrated variable levels of mRNA, with the highest levels identified in KCN cells, which had approximately 10,000-fold higher expression levels than LA155N cells, which had the lowest TrkB mRNA expression level (Fig. 1).
Response of Neuroblastoma Tumor Cells to TrkB Stimulation With BDNF
In order to determine the responses of neuroblastoma tumor cells to the Trk receptor ligands NGF, BDNF, and NT-3, neuroblastoma cell lines were exposed to 10 ng/mL of each ligand and evaluated for cellular proliferation. BDNF resulted in the highest amount of cell proliferation in each of the tested cell lines (Fig. 2A). An expanded panel of neuroblastoma cell lines was then exposed to increasing concentrations of BDNF to determine the relative responsiveness of each cell line. KCN cells were the most sensitive, demonstrating significantly increased cell proliferation in response to as little as 0.1 ng/mL of BDNF. All other cell lines demonstrated increased cell proliferation at doses of BDNF >10 ng/mL (Fig. 2B; data not shown).
In order to evaluate which downstream signaling pathways were activated by BDNF stimulation, neuroblastoma tumor cells were treated with increasing concentrations of BDNF, and the activation status of potential downstream signaling targets was evaluated by immunoblot analysis for phosphorylation. BDNF stimulation with up to 20 ng/mL resulted in phosphorylation of the TrkB receptor in addition to the PI3K, AKT, BAD, p70S6K, and RPS6 proteins, but not the MEK or ERK kinase members (Fig. 3A), suggesting a role for multiple signaling pathways in the proliferative response of neuroblastoma tumor cells to BDNF.
Inhibition of Trk Receptor Phosphorylation and Downstream Signaling by AZ623
AZ623 has been demonstrated to specifically inhibit the phosphorylation of TrkA, TrkB, and TrkC in cell lines in vitro.20 In order to confirm that AZ623 could inhibit TrkB phosphorylation in our neuroblastoma model systems, neuroblastoma tumor cells were treated with BDNF to stimulate TrkB receptor phosphorylation. This TrkB phosphorylation could be inhibited by co-incubation with AZ623 (Fig. 3B).
In order to evaluate whether the downstream signaling pathways activated by BDNF would be inhibited by AZ623, neuroblastoma tumor cells were treated with increasing concentrations of BDNF, and the activation status of potential downstream signaling targets was evaluated by immunoblot analysis for phosphorylation. Both AKT and RPS6 were phosphorylated after BDNF treatment, and this phosphorylation could be inhibited by co-incubation with AZ623 (Fig. 3C).
Effects of AZ623 on Neuroblastoma Tumor Cell Viability and BDNF-Mediated Proliferation
In order to determine the effects of AZ623 on cell viability, neuroblastoma tumor cells were treated with increasing concentrations of AZ623, cell viability was measured, and IC50 values were calculated. The majority of neuroblastoma cell lines had calculated IC50 values between 1.9 and 7 μM, with the exception of KCN cells, which demonstrated significantly increased sensitivity and an IC50 value of 800 nM (Fig. 4).
In order to evaluate whether the neuroblastoma cell proliferation induced by BDNF could be inhibited by AZ623, neuroblastoma tumor cells were treated with increasing concentrations of BDNF with or without AZ623, and cell viability was measured and normalized to untreated cells. Neuroblastoma viability was increased after BDNF treatment, and this proliferation could be inhibited by AZ623 (Fig. 5).
Effects of AZ623 Combined With Topotecan on Neuroblastoma Tumor Cells
With the need for more effective combination therapies in children with neuroblastoma, we wanted to determine effective combinations of established treatments with AZ623. Topotecan has been demonstrated to be an effective agent against neuroblastoma tumor cells in preclinical models and in patients with neuroblastoma.25-28 Neuroblastoma tumor cells were treated with AZ623, topotecan, or the combination of AZ623 with topotecan. Treatment with either AZ623 or topotecan alone resulted in cell rounding and detachment from the cell surface, which was significantly increased when the 2 agents were combined (Fig. 6A-6D). Treatment with AZ623 did not induce significant apoptosis, as demonstrated by TUNEL staining (Fig. 6F), while treatment with the combination of AZ623 plus topotecan resulted in more TUNEL-positive cells compared with topotecan alone (Fig. 6G and 6H), suggesting a synergistic effect.
The combination of AZ623 and topotecan was then tested in immunocompromised mice with human neuroblastoma xenograft tumors. Treatment of mice with human neuroblastoma xenografts with AZ623 or topotecan alone resulted in significantly reduced growth. Treatment of these mice with the combination of AZ623 and topotecan resulted in nearly complete inhibition of tumor growth (Fig. 7A, 7C and 7D) and in an increase in TUNEL-positive tumor cells, suggesting that the tumor growth inhibition was secondary to induction of intratumoral apoptosis (Fig. 7B). Furthermore, after the cessation of treatment, mice previously treated with AZ623 or topotecan alone demonstrated significant regrowth of xenograft tumors, while mice treated with the combination of AZ623 and topotecan demonstrated almost complete inhibition of tumor regrowth (Fig. 7E), with final tumor volumes reduced by 89% and 86%, respectively, compared with AZ623 or topotecan alone.
New treatment strategies are needed for children with high-risk and recurrent neuroblastoma. We demonstrated that the novel Trk kinase inhibitor AZ623 effectively inhibits BDNF-mediated signaling and neuroblastoma cell proliferation. Furthermore, AZ623 combined with topotecan represents a novel synergistic combination that is effective at inhibiting neuroblastoma xenograft tumor growth, even after the discontinuation of therapy.
AZ623 is a novel and potent small molecule Trk inhibitor with a selectivity profile distinct from other reported agents. AZ623 inhibits the 3 Trk isoforms (TrkA, TrkB, and TrkC) at low nanomolar concentrations in cellular assays, but in vitro screening of AZ623 against a large panel of other kinases revealed only minimal activity.20 Our results with AZ623 suggest that selective Trk inhibition may be sufficient to inhibit neuroblastoma tumor growth in vivo, particularly when combined with topotecan, a topoisomerase inhibitor that has been shown to be effective against neuroblastoma in early phase clinical trials,26, 29 and is currently used for children with neuroblastoma in upfront treatment regimens and in cases of recurrent or refractory disease.27
In the current study, we have demonstrated variable levels of TrkB receptor mRNA expression in a panel of neuroblastoma cell lines. Each of the tested neuroblastoma cell lines demonstrated increased proliferation in response to BDNF, suggesting a critical role for TrkB signaling in the control of neuroblastoma cell proliferation. The mechanisms of the observed variability in the sensitivity of neuroblastoma cell lines to AZ623 is not clear. SMS-KCN neuroblastoma cells demonstrated the highest levels of TrkB expression and were the most sensitive to AZ623. Responses to AZ623 in the other tested neuroblastoma cell lines do not appear to be correlated with expression levels of TrkB or with other known biologic features of neuroblastoma tumor cells, such as N-myc amplification or chromosome 1p deletion.
Data accumulated over the past decade have clearly established Trk receptors as viable targets for anticancer therapy. Small molecular weight kinase inhibitors offer the most straightforward approach to the clinical targeting of the Trk receptors. For example, CEP-701 (lestaurtinib), a potent and orally available pan-Trk kinase inhibitor, has shown activity against a range of in vivo models, including prostate, pancreatic, and thyroid cancer xenografts.30-32 However, lestaurtinib inhibits several additional tyrosine kinase targets and remains active in trials in clinical settings in which Trk kinases are not believed to be pathogenic, such as acute myeloid leukemia and myeloproliferative disease.17-19 Therefore, the development of potent and selective Trk kinase inhibitors remains critical to evaluate the role of Trk signaling pathway inhibition in the treatment of patients with neuroblastoma.
AZ623 represents a novel agent with the potential for therapeutic utility in the treatment of children with neuroblastoma. We have demonstrated that AZ623 inhibits BDNF-mediated neuroblastoma cell signaling and proliferation and that the combination of AZ623 and topotecan is effective against neuroblastoma tumor cells in vitro and in vivo. The evidence for the role of Trk receptor signaling in stimulating neuroblastoma tumor cell growth and the demonstrated in vitro and in vivo efficacy of the combination of AZ623 and topotecan in these studies provide biological and clinical rationale for the further exploration of this combination and testing in children with recurrent or refractory neuroblastoma.
CONFLICT OF INTEREST DISCLOSURES
Supported by a grant from the Lorrie Olivier Neuroblastoma Research Fund (to P.E.Z. and P.A.Z.-M.). Christine Pien, Ken Thress, Charles Omer, and Jeffrey L Brown are employees of AstraZeneca R&D and provided the AZ623 used in this research. No financial support was provided.
- 1Multivariate analysis of risk factors in stage 4 neuroblastoma patients over the age of one year treated with megatherapy and stem-cell transplantation: a report from the European Bone Marrow Transplantation Solid Tumor Registry. J Clin Oncol. 1998; 16: 953-965., , , et al.
- 23Biological classification of cell lines derived from human extra-cranial neural tumors. Adv Neuroblastoma Res. 1988; 2: 291-306., , , et al.
- 27Ifosfamide/carboplatin/etoposide (ICE) as front-line, topotecan/cyclophosphamide as second-line and oral temozolomide as third-line treatment for advanced neuroblastoma over one year of age. Acta Paediatr Suppl. 2004; 93: 6-11., , , et al.