Pediatr Blood Cancer

Sorafenib Inhibits Neuroblastoma Cell Proliferation and Signaling, Blocks Angiogenesis, and Impairs Tumor Growth Nisha C. Kakodkar, MD,1 Radhika R. Peddinti, MD,3 Yufeng Tian, BS,1 Lisa J. Guerrero, BS,1 Alexandre Chlenski, PhD,1 Qiwei Yang, PhD,1 Helen R. Salwen, BS,1 Michael L. Maitland, MD, PhD,2 and Susan L. Cohn, MD1* Background. More effective therapy for children with high-risk neuroblastoma is desperately needed. Preclinical studies have shown that neuroblastoma tumor growth can be inhibited by agents that block angiogenesis. We hypothesized that drugs which target both neuroblastoma cells and tumor angiogenesis would have potent anti-tumor activity. In this study we tested the effects of sorafenib, a multi-kinase inhibitor, on neuroblastoma cell proliferation and signaling, and in mice with subcutaneous human neuroblastoma xenografts or orthotopic adrenal tumors. Procedure. Mice with subcutaneous neuroblastoma xenografts or orthotopic adrenal tumors were treated with sorafenib, and tumor growth rates were measured. Blood vessel architecture and vascular density were evaluated histologically in treated and control neuroblastoma tumors. The in vitro effects of sorafenib on neuroblastoma

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proliferation, cell cycle, and signaling were also evaluated. Results. Sorafenib inhibited tumor growth in mice with subcutaneous and orthotopic adrenal tumors. Decreased numbers of cycling neuroblastoma cells and tumor blood vessels were seen in treated versus control tumors, and the blood vessels in the treated tumors had more normal architecture. Sorafenib treatment also decreased neuroblastoma cell proliferation, attenuated ERK signaling, and enhanced G1/G0 cell cycle arrest in vitro. Conclusions. Our results demonstrate that sorafenib inhibits the growth of neuroblastoma tumors by targeting both neuroblastoma cells and tumor blood vessels. Single agent sorafenib should be evaluated in future phase II neuroblastoma studies. Pediatr Blood Cancer ß 2011 Wiley Periodicals, Inc.

angiogenesis; extracellular signal-regulated MAP kinases; neuroblastoma; sorafenib

INTRODUCTION Neuroblastoma is a clinically heterogeneous pediatric malignancy. Approximately half of the 650 new cases of neuroblastoma diagnosed each year in the United States meet the Children’s Oncology Group criteria for high-risk disease [1]. Although the majority of high-risk tumors are initially chemotherapy responsive, relapse rates, and therapy-related toxicities are high, rendering long-term survival dismal [2]. Thus, novel therapies are desperately needed for this group of patients. Angiogenesis has been shown to play a critical role in the growth and metastasis of all malignant tumors [3], and increased vascular density and microvascular proliferation are associated with clinically aggressive disease and poor prognosis in neuroblastoma [4–6]. Anti-angiogenic agents have been validated as a therapeutic option in adult malignancies [7], and anti-tumor effects have been observed in preclinical neuroblastoma models [8–10]. We hypothesized that the multikinase inhibitor, sorafenib, would target both neoplastic cells and endothelial cells, leading to increased therapeutic benefit when compared to agents that target angiogenesis alone. Sorafenib inhibits autophosphorylation of vascular endothelial growth factor (VEGF), platelet-derived growth factor b (PDGFb), c-Kit, and RET receptors, which are critical for pro-angiogenic signaling [11]. Recently, sorafenib has been shown to inhibit HIF-1a upregulation in neuroblastoma cell lines [12], which may further amplify its anti-angiogenic activity in neuroblastoma tumors. Sorafenib also competitively inhibits B-Raf and C-Raf activation, resulting in attenuation of the prooncogenic mitogen activated protein kinase (MAPK) signaling pathway [13]. In this study we analyzed the effects of sorafenib on neuroblastoma angiogenesis and tumor growth in subcutaneous xenograft and orthotopic adrenal neuroblastoma preclinical models. We also investigated the direct effects of sorafenib on neuroblastoma cell signaling and proliferation. Our studies show that sorafenib potently inhibits angiogenesis and neuroblastoma tumor

ß 2011 Wiley Periodicals, Inc. DOI 10.1002/pbc.24004 Published online in Wiley Online Library (wileyonlinelibrary.com).

growth in both preclinical models. We also confirm that sorafenib mediates direct changes in neuroblastoma cell signaling, resulting in inhibited cell cycle and proliferation.

METHODS Cell Lines MYCN-amplified, human neuroblastoma cell lines NBL-W-N, SMS-KCNR, and LA1-55n were grown at 5% CO2 in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Invitrogen), and 1% penicillin/streptomycin. NBL-W-N was established in our laboratory [14], SMS-KCNR was a kind gift from Dr. Carol Thiele, and LA1-55n a kind gift from Dr. June Biedler. Cell lines were authenticated by short tandem repeat (STR) profiling using the AmpF/STR Identifiler PCR Amplification Kit (Applied Biosystems, Carlsbad, CA). Analysis was performed at The Johns Hopkins University Fragment Analysis

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Departments of Pediatrics, University of Chicago, Chicago, IL; Departments of Medicine, University of Chicago, Chicago, IL; 3 Department of Pediatrics, Stroger Hospital, Chicago, IL 2

Grant sponsor: Little Heroes Cancer Research Foundation; Grant sponsor: Neuroblastoma Children’s Cancer Society; Grant sponsor: Super Jake Foundation; Grant sponsor: Elise Anderson Neuroblastoma Research Fund; Grant sponsor: St. Baldrick’s Foundation PostDoctoral Fellowship for Childhood Cancer Research. Dr. Maitland is a co-inventor on a patent application for use of sorafenib in the treatment of pulmonary hypertension. The authors declare no additional conflicting interests in relation to the described work. *Correspondence to: Susan L. Cohn, MD, Department of Pediatrics, University of Chicago, 900 East 57th Street KCBD Rm. 5100 Chicago, IL 60637. E-mail: [email protected] Received 19 August 2011; Accepted 18 October 2011

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Facility (Baltimore, MD) and STR profiles were found to be identical to known profiles for the cell lines.

In Vivo Neuroblastoma Models To establish neuroblastoma subcutaneous xenografts, female nude mice (Harlan, Madison, WI) underwent subcutaneous injection of 1
107 SMS-KCNR neuroblastoma cells as previously described [15]. Once tumors reached a palpable size of approximately 70 mm3, mice were treated by oral gavage with vehicle control (5% Cremophor EL/5% ethanol/90% ddH2O) or 45 mg/kg sorafenib tosylate (30 mg/kg free base equivalent), a dosage found to be effective and well tolerated in multiple preclinical models of cancer [13]. Sorafenib was prepared as previously described [16], and formulated fresh daily at 4X the highest dose in a Cremophor EL/ethanol (50:50) solution. Final dosing solutions were prepared on the day of use by dilution to 1X with ddH2O and mixed immediately before dosing. Mice were treated 1X/day, 5 days/week for a total of 8 doses over 10 days and sacrificed when control tumors reached terminal size. Tumor size was determined every 2–3 days by external measurements with a caliper and volumes were calculated using the formula: Tumor volume ¼ ðlength
width2 Þ=2. To generate orthotopic tumors, SMS-KCNR cells were engrafted into the adrenal gland of nude mice. A left side paracostal approach was taken and the spleen was displaced cranially allowing exposure of the left adrenal gland. 2
106 SMS-KCNR cells in 20 ml of RPMI with 10% FBS were injected through a 27 G needle into the adrenal gland. Flank musculature was closed with a single 4–0 absorbable suture and the skin closed with 3 or 4 staples. Due to the complexity of the procedure, injections were performed over 2 days. Animals were randomized 7 days after injection and treatment was started at that time. The animals were all sacrificed on the same day, after 16 or 17 doses. In the test group, 7 animals received 16 doses; 6 received 17 doses. In the control group, 2 received 16 doses; 5 received 17. Animals were weighed 3X/week and sacrificed when mice in the control group showed signs of distress due to tumor burden, such as stooped posture, inability to stand, and loss of skin turgor. Tumors were removed, measured, weighed, and photographed. Tissue was fixed with 10% buffered formalin, embedded in paraffin and 5 mmthick sections were prepared for histologic evaluation. Animals were treated according to NIH guidelines for animal care and use, and protocols approved by the University of Chicago Institutional Animal Care and Use Committee.

Histological Analysis and Immunofluorescence Neuroblastoma xenografts and orthotopic tumors were processed for histologic evaluation, stained with hematoxylin and eosin (H&E), and endothelial cells were detected with anti-human CD31 antibody as described [6]. For immunofluorescence analysis, endothelial cells were stained with anti-CD31 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at a 1:50 dilution and FITC-labeled secondary antibody. Pericytes were visualized with anti-a-SMA (Sigma, St. Louis, MO) at 1:100 dilution and Texas Red-labeled secondary antibody as described [17]. For analysis of proliferation rate, slides were incubated with Ki-67 mouse antihuman monoclonal antibody (clone MIB-1, 1:200; DakoCytomation, Carpinteria CA) as previously described [6]. Pediatr Blood Cancer DOI 10.1002/pbc

Quantification of Vascular Density and Architecture Tumor sections stained for CD31 were examined at x400 magnification. The number of CD31 positive cells per high power field were counted in triplicate by two blinded investigators and then averaged. Two-dimensional shape descriptors of Image J software were used to quantitatively assess blood vessel architecture, and the abnormality of blood vessel structure was graded using the formula 1  (circularity
roundness
solidity) as described [18].

Proliferation Assay The CellTiter 96 AQueous Non-radioactive Proliferation Assay kit (Promega, Madison, WI) was used as previously described [15]. Briefly, cells were seeded and after 24 hours sorafenib was added to triplicate wells at concentrations from 0 to 100 mM. Following a 96 hours incubation, 3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was added and absorbance measured using a Synergy 2 microplate reader (Bio-Tek Instruments, Winooski, VT).

Measurement of Cell Cycle Phase Distribution Neuroblastoma cell lines were cultured with 10 mM sorafenib or vehicle control for 24 hours. Following treatment, cells were washed with phosphate buffered saline (PBS), fixed in 70% ethanol, and stored at 48C. Cells were hypotonically lysed in 1 ml of 0.05 mg/ml propidium iodide (Sigma) and 0.1% Triton X-100, and data analyzed on a FACSCanto flow cytometer (BD Biosciences, San Jose, CA) using Flowjo software (Tree Star, Ashland, OR).

Western Blot Analysis for Extracellular Signal-Regulated Kinase (ERK), Phospho-ERK (pERK), and Cyclins D1, D2, D3, and E NBL-W-N, SMS-KCNR, and LA1-55n neuroblastoma cells were treated with 5 and 10 mM sorafenib for 24 hours. Lysates were prepared by boiling cell pellets in buffer containing 50 mM TRIS-HCl (pH 6.8), 2% SDS, and protease inhibitor cocktail (Sigma) for 10 min. Total protein (10 mg) was electrophoresed on 4–20% SDS-PAGE gradient gels and transferred to nitrocellulose membranes. Antibodies against cyclins D1, D2, D3, and E were obtained from BD Pharmingen, San Diego, CA, and antibodies against p44/42 MAPK (Erk1/2), and phospho-p44/42 MAPK (pErk1/2, Thr202/Tyr204) were obtained from Cell Signaling Technologies, Beverly, MA. Membranes were blocked in Tris-buffered saline (TBS) with 0.1% Tween-20 and 5% nonfat dry milk (ERK and cyclins) or TBS with 0.1% Tween-20 and 5% bovine serum albumin (BSA) for pERK. Blots were developed with anti-mouse or anti-rabbit horseradish peroxidase secondary antibodies (KPL, Gaithersburg, MD) and Immun-Star Western C Detection Kit (Bio-Rad Laboratories, Hercules, CA).

Statistical Analysis All in vitro experiments were repeated at least in triplicate and standard deviations were calculated. At least five mice per group were included in each animal experiment and mean values of the tumor volumes, weights, and vessel densities were compared. All

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the quantitative values obtained in the experiments were evaluated using paired Student’s t-test. A P-value of 0.05 was considered statistically significant. A nonlinear regression, sigmoidal fourparameter dose-response model was used to determine IC50 by plotting log(inhibitor concentration) versus relative survival after 96-hours treatment with sorafenib. Prism software (GraphPad Software, Inc., La Jolla, CA) was utilized to determine the IC50.

RESULTS Sorafenib Inhibits Neuroblastoma Tumor Growth In Vivo After 4 days of sorafenib treatment, the average size of subcutaneous xenografts in the animals receiving sorafenib was significantly smaller compared to tumors in control mice (376.3  199.5 mm3 vs. 685.9  207.2 mm3, respectively; P ¼ 0.033). The mice were sacrificed after receiving eight doses of sorafenib over 10 days, when control tumors reached terminal size. As shown in Figure 1A, after 10 days of treatment, the average size of the tumors in the sorafenib treated-mice was approximately 25% of the size of the tumors in controls (541.0 þ 320.6 mm3 vs. 2317.1 þ 773.1 mm3, respectively, P < 0.001) Because orthotopic tumors have been shown to be superior models of tumor angiogenesis [19], we also tested the effects of sorafenib in mice with adrenal orthotopic neuroblastomas. Treatment was initiated 7 days after tumor cell inoculation, and continued for 16–17 doses. The animals were sacrificed when signs of tumor burden distress appeared in the control group. We again found that tumors in the animals treated with sorafenib were significantly smaller than tumors in the controls (538.6  376.3 mm3 vs. 1615.6  753.4 mm3, respectively; P < 0.001, Fig. 1B). Similarly, the average tumor weight in treated versus control animals was significantly less (0.5  0.2 g vs. 1.6  0.7 g, respectively; P < 0.0001, Fig. 1C).

Sorafenib Inhibits Neuroblastoma Tumor Angiogenesis To investigate the effects of sorafenib treatment on angiogenesis, we counted the number of endothelial cells in xenograft tumor sections immunostained with anti-CD31 antibody. As shown in Figure 2A and B, the average number of CD31 positive cells per high power field was significantly lower in the sorafenib-treated tumors compared to controls (79.4  9.9 vs. 106.8  10.1, respectively; P ¼ 0.0008). We used immunofluorescent staining and Image J software to further characterize blood vessel number and architecture, and to assign quantitative values relevant to blood vessel architecture as previously described [18]. Circularity is a function of shape, roundness measures elongation, and solidity approximates the density. Blood vessels in sorafenib-treated tumors displayed increased circularity (P ¼ 0.003) and solidity (P ¼ 0.007) compared to controls. Although the roundness was also higher in the treated tumors, the difference did not reach statistical significance (P ¼ 0.13). The calculated vessel abnormality was significantly lower in the sorafenib-treated xenografts (P ¼ 0.01, Fig. 2C). Similarly, Image J analysis of the sorafenib-treated orthotopic tumors demonstrated increased blood vessel circularity (P < 0.001) and solidity (P < 0.001), and calculated blood vessel abnormality was reduced (P ¼ 0.01, Fig. 2D and E). Pediatr Blood Cancer DOI 10.1002/pbc

Fig. 1. Sorafenib inhibits neuroblastoma tumor growth. A: Growth curves of neuroblastoma subcutaneous xenografts treated 1 time per day, 5 days per week for a total of eight doses of 45 mg/kg sorafenib (n ¼ 6) or vehicle control (n ¼ 5). A statistically significant decrease in tumor size was seen after 4 days of treatment with sorafenib (P ¼ 0.033 on day 4 and < 0.001 on days 7 and 10 (SD)). B: Bar graph showing the average final tumor volume (SD) of the control and sorafenib-treated adrenal orthotopic tumors (P < 0.001). C: Bar graph showing the average final tumor weight (SD) of the control and sorafenib-treated orthotopic neuroblastoma tumors (P < 0.001). Mice were treated with 16–17 doses of 45 mg/kg sorafenib (n ¼ 13) or vehicle control (n ¼ 7). Statistically significant differences from the control group are marked with an asterisk.

Sorafenib Inhibits Neuroblastoma Tumor Cell Proliferation To assess the effects of sorafenib on tumor cell proliferation in vivo, neuroblastoma xenograft tumor sections were immunohistochemically stained for the cell proliferation marker Ki-67. A significant increase in the percentage of G0 (non-proliferating) cells was noted in treated versus control tumors (45.7  8.7 vs. 27.2  2.8%, respectively; P ¼ 0.01, Fig. 3).

Sorafenib Inhibits Human Neuroblastoma Cell Proliferation In Vitro and Causes G1 Arrest To investigate whether sorafenib had direct effects on neuroblastoma cell proliferation, we conducted proliferation assays. Treatment with increasing concentrations of sorafenib for 96 hours caused inhibition of neuroblastoma cell proliferation, with an IC50 of 1.6 mM in SMS-KCNR, 1.2 mM in NBL-W-N, and 4.3 mM in LA1-55n, cells (Fig. 4A). Flow cytometric analysis

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Fig. 2. Sorafenib inhibits angiogenesis in neuroblastoma tumors. A: To quantify tumor blood vessels, histologic sections of subcutaneous neuroblastoma xenografts were stained with anti-CD31 antibody to highlight endothelial cells. The average number of CD31-positive cells per high power field was significantly reduced in the sorafenib-treated versus control xenografts (P < 0.001). B: Representative photographs of CD31-stained xenograft tumors. C: Image J quantitative analyses of the blood vessel structure in the sorafenib-treated and control xenografts. Blood vessels in sorafenib-treated tumors had higher circularity (P ¼ 0.003) and solidity (P ¼ 0.007) compared to controls. Vessel abnormality was determined to be reduced in tumors treated with sorafenib (P ¼ 0.01), indicating that treatment induced normalization of tumor vasculature. D: Representative photographs of green CD31- and red SMA-stained orthotopic adrenal tumor sections. E: Image J quantitative analyses demonstrated that blood vessels in the sorafenib-treated orthotopic tumors had higher circularity (P < 0.001) and solidity (P < 0.001) than controls. Reduced blood vessel abnormality (P ¼ 0.01) was seen in the sorafenib-treated tumors. Error bars show SD, statistically significant differences from the control group are marked with an asterisk.

of SMS-KCNR cells revealed an increase in G1 arrested cells from 50% to 70% with 10 mM sorafenib treatment for 24 hours (P ¼ 0.03). Sorafenib also induced an increase in G1 arrested NBL-W-N (60–80%; P ¼ 0.04). However, no significant increase in the percentage of G1 arrested cells was seen in experiments with LA1-55n cells (Fig. 4B).

Sorafenib Attenuates ERK Signaling and Cyclin D1 Expression in Human Neuroblastoma Cell Lines To examine the effects of sorafenib on MAPK pathway signaling in neuroblastoma cells, we performed western blot analyses using anti-ERK and anti-pERK antibodies. ERK phosphorylation was inhibited by 5 and 10 mM sorafenib after 24 hours of treatment in all three neuroblastoma cell lines (Fig. 5). We also found that sorafenib treatment profoundly decreased expression of the downstream protein cyclin D1 in all three human neuroblastoma cell lines. Levels of cyclins D2 and E were also decreased, however, total ERK expression was unchanged (Fig. 5). Fig. 3. Sorafenib inhibits neuroblastoma tumor cell proliferation in vivo. A: The average number of Ki-67-negative cells per high power field (SD) was increased in sorafenib-treated versus control neuroblastoma xenografts (P ¼ 0.01, marked with an asterisk). B: Representative tumor sections stained with Ki-67. Pediatr Blood Cancer DOI 10.1002/pbc

DISCUSSION In this study, we investigated the anti-tumor activity of the multi-kinase inhibitor sorafenib, utilizing two preclinical

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Fig. 4. Sorafenib induces G1 arrest in neuroblastoma cell lines. A: Treatment with increasing concentrations of sorafenib reduced the survival of three MYCN-amplified human neuroblastoma cell lines, as measured by the MTS assay. The IC50 of each cell line is indicated. B: Propidium iodide staining and flow cytometry were utilized to determine cell cycle distribution. Sorafenib increased the percentage of G1-arrested SMSKCNR (P < 0.001) and NBL-W-N (P < 0.001) cells. No significant increase in the number of G1-arrested LA1-55n cells was seen with sorafenib treatment (P ¼ 0.329). Bars represent percentage of cells in indicated cell cycle phase after 24 hours of treatment with 10 mM sorafenib. Statistically significant differences from the control group are marked with an asterisk.

neuroblastoma models. Consistent with previous studies, we showed that sorafenib significantly inhibits the growth of subcutaneous neuroblastoma xenografts [20]. In addition, we demonstrated for the first time that sorafenib inhibits the growth of orthotopic adrenal neuroblastomas, a biologically more relevant preclinical model for testing the efficacy of anti-angiogenic agents than subcutaneous neuroblastoma xenografts [19]. Sorafenibtreated tumors had fewer blood vessels than controls, and the blood vessel architecture was more normal. Decreased neuroblastoma cell proliferation was also observed in the treated tumors, suggesting that sorafenib also directly affects neuroblastoma cell signaling and cell cycle. To test this hypothesis, we examined the effects of sorafenib on neuroblastoma cells in vitro. G1 arrest was detected with 10 mM of drug in two of the three neuroblastoma cell lines tested, and a reduction in pERK was seen with doses of 5 and 10 mM. We also demonstrated a dose-dependent decrease in the viability of the three neuroblastoma cell lines. Although the sensitivity varied with IC50 ranging from 1.2 to 4.3 mM, this concentration is achievable clinically [21]. Of the three cell lines tested, LA1-55n was more resistant, requiring a higher IC50 and showing no evidence of G1 arrest at 10 mM. Interestingly, ERK signaling was inhibited in LA1-55n cells with sorafenib treatment indicating that activation of alternative signaling pathways likely contributes to its resistance. Sorafenib is a multikinase inhibitor which was recently approved by the Food and Drug Administration (FDA) for treatment of renal cell carcinoma and hepatocellular carcinoma [13], and a Phase I pediatric trial has recently been completed by the Children’s Oncology Group (COG) in patients with refractory solid tumors and leukemia [22]. In the pediatric study, no grade 3 hypertension was seen at the maximal tolerated dose of 200 mg/m2, and only 1/6 patients experienced a grade 3 dose limiting toxicity of elevated liver enzymes. However, clinical data on the effect of sorafenib in neuroblastoma are not yet Pediatr Blood Cancer DOI 10.1002/pbc

available as no reference to neuroblastoma patients was included in this trial. Others have also shown that sorafenib has anti-neuroblastoma activity in preclinical studies. Roy et al. [23] showed that the growth of two neuroblastoma cell lines is inhibited in vitro after treatment with sorafenib. Further, Chai et al. [24] demonstrated that sorafenib decreases ERK, Akt1/2/3, AMPK, and STAT3 phosphorylation in a single neuroblastoma cell line. More recently, Keir et al. [20] reported results from the Pediatric Preclinical Testing Program (PPTP) demonstrating that sorafenib inhibited the proliferation of a number of pediatric cancer cell lines including three of four human neuroblastoma cell lines. Although no mice were cured by sorafenib, decreased tumor growth and prolonged event-free survival were seen with sorafenib treatment [20]. Interestingly, the IC50 for the more resistant LA1-55n cells used in our study was equal to the median of the entire panel analyzed by the PPTP. Thus, SMS-KCNR and NBL-W-N appear to be more sensitive than the other cell lines tested in this panel. It is well established that significant differences in tumor blood vessel density are seen in subcutaneous versus orthotopic preclinical tumor models [19]. The orthotopic adrenal neuroblastoma model used in our studies more closely mimics the blood supply of clinical tumors, and the activity of sorafenib in this model further emphasizes its potential clinical utility. By competitively inhibiting B-Raf and C-Raf activation, sorafenib inhibits the pro-oncogenic MAPK signaling pathway [25]. Insulin growth factor 1 (IGF-1) activates this pathway in neuroblastoma, and Misawa et al. [26] showed that following IGF-1 treatment of KP-N-RT human neuroblastoma cells, cell cycle progression was stimulated and MYCN expression was up-regulated. Further, experiments demonstrated that MAPK pathway activation was required for the induction of MYCN expression and cell proliferation, as treatment with a MEK1 inhibitor blocked these effects. Sandoval et al. [27] similarly demonstrated the anti-

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Kakodkar et al. disease [8,9]. The promising results seen in the preclinical studies conducted by our group and others, together with the low toxicity profile observed in early phase clinical trials, indicate that sorafenib may have therapeutic utility in children with neuroblastoma. Further preclinical studies testing the efficacy of combination therapy and the optimal sequence of administration of the combination therapies are warranted.

ACKNOWLEDGMENT The authors thank Little Heroes Cancer Research Foundation, the Neuroblastoma Children’s Cancer Society, the Super Jake Foundation, and the Elise Anderson Neuroblastoma Research Fund for supporting this research. This research was supported in part by St. Baldrick’s Foundation Post-Doctoral Fellowship for Childhood Cancer Research. Sorafenib tosylate was kindly provided by Bayer HealthCare Pharmaceuticals (Wayne, NJ).

REFERENCES

Fig. 5. Sorafenib attenuates ERK signaling and decreases cyclin D1 expression in a concentration-dependent fashion. Western blots showing decreased expression of phosphorylated ERK and cyclins D1, D2, and E in NBL-W-N, SMS-KCNR, and LA1-55n human neuroblastoma cells following treatment with 0, 5, and 10 mM sorafenib for 24 hours. No significant change in total ERK was seen.

proliferative effects of the MAPK kinase inhibitor U0126 in neuroblastoma cells, although the sensitivity to this agent was shown to vary in different cell lines. Thus, the decrease in MAPK pathway signaling induced with sorafenib in neuroblastoma cells combined with its ability to inhibit angiogenesis is likely to drive the potent anti-neuroblastoma activity we observed in our preclinical models. Although the goal of this study was to examine the antineuroblastoma activity of sorafenib as monotherapy, subsequent combination studies with conventional chemotherapeutic agents are planned. Similar to other inhibitors of angiogenesis, sorafenib is likely to have increased clinical efficacy when combined with cytotoxic agents or other signaling inhibitors. Because sorafenib induces cell-cycle arrest, combination with non-cell cycle dependent agents may be optimal. In addition, administration of chemotherapy agents before sorafenib rather than after may lead to better response. Sorafenib may also be effective in postconsolidation maintenance therapy, as we and others have shown that in preclinical models, anti-angiogenic agents can inhibit tumor re-growth when given in the setting of minimal residual Pediatr Blood Cancer DOI 10.1002/pbc

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