Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2018 Aug 1.
Published in final edited form as: Cancer Lett. 2017 Apr 26;400:61–68. doi: 10.1016/j.canlet.2017.04.022

The second-generation ALK inhibitor alectinib effectively induces apoptosis in human neuroblastoma cells and inhibits tumor growth in a TH-MYCN transgenic neuroblastoma mouse model

Jiaxiong Lu 1,2, Shan Guan 2, Yanlin Zhao 2, Yang Yu 3, Sarah E Woodfield 3, Huiyuan Zhang 2, Kristine L Yang 2, Shayahati Bieerkehazhi 2, Lin Qi 4, Xiaonan Li 4, Jerry Gu 2, Xin Xu 2, Jinling Jin 2, Jodi A Muscal 5,*, Tianshu Yang 1,*, Guo-Tong Xu 1,*, Jianhua Yang 2,*
PMCID: PMC5502736  NIHMSID: NIHMS871497  PMID: 28455243

Abstract

Activating germline mutations of anaplastic lymphoma kinase (ALK) occur in most cases of hereditary neuroblastoma (NB) and the constitutively active kinase activity of ALK promotes cell proliferation and survival in NB. Therefore, ALK kinase is a potential therapeutic target for NB. In this study, we show that the novel ALK inhibitor alectinib effectively suppressed cell proliferation and induces apoptosis in NB cell lines with either wild-type ALK or mutated ALK (F1174L and D1091N) by blocking ALK-mediated PI3K/Akt/mTOR signaling. In addition, alectinib enhanced doxorubicin-induced cytotoxicity and apoptosis in NB cells. Furthermore, alectinib induced apoptosis in an orthotopic xenograft NB mouse model. Also, in the TH-MYCN transgenic mouse model, alectinib resulted in decreased tumor growth and prolonged survival time. These results indicate that alectinib may be a promising therapeutic agent for the treatment of NB.

Keywords: Neuroblastoma, ALK inhibitor, alectinib, PI3K/Akt/mTOR, apoptosis

1. Introduction

Neuroblastoma (NB) is the most common childhood extracranial malignant tumor [2, 31]. With current treatments, the outcomes for low- and intermediate-risk NB patients have improved. However, the prognosis for patients with high-risk NB remains dismal. Due to the lack of effective therapies, identification of novel therapeutic targets for high-risk NB is urgently needed [20, 36].

Anaplastic lymphoma kinase (ALK), known as ALK tyrosine kinase receptor or cluster of differentiation 246 (CD246), has been identified as one of the major oncogenes in tumor pathogenesis [8, 11, 12, 16, 21, 26, 51]. In NB, high expression levels of ALK closely correlates with poor outcomes, especially in high-risk NB [22, 42, 49]. It is reported that ALK with activating mutations in its tyrosine kinase domain occur in most cases of hereditary NB [17, 33]. In addition, ALK-activating point mutations have been identified in approximately 8% of investigated NB tumors [1]. In prior studies, inhibition of ALK led to a significant decrease in cell proliferation in ALK-positive cancers, including non-small cell lung cancer (NSCLC) [26, 27, 29], anaplastic large cell lymphoma (ALCL) [6, 34] and NB [15, 44, 50, 52]. ALK-targeted chemotherapies have been shown to downregulate PI3K/Akt signaling, leading to cell apoptosis and tumor regression [4, 15, 51, 52].

Alectinib (CH5424802) is an orally available, highly selective, potent second-generation inhibitor of ALK [13, 43, 46, 53]. It exhibits ten-fold greater potency in kinase assays than the first generation of ALK inhibitor crizotinib [23, 39]. Alectinib is derived from a carbonitrile in physical structure and shows potent inhibitory effects against tumors addicted to ALK activity, such as NSCLC which expresses the EML4-ALK fusion protein [30, 32, 38, 39, 54]. Unlike other ALK inhibitors, the crystal structural analysis of alectinib exhibits only one hinge hydrogen bond with kinase, indicating that this compound may achieve higher selectivity for ALK [39]. In prior studies, alectinib has shown substantial inhibitory effects against tumors with ALK mutations, including ALKL1196M, ALKL1152R, ALKF1174L, and ALKR1275Q [28, 35, 40, 48, 55]. Furthermore, chemoresistant cells harboring crizotinib-mediated ALK mutations ALK F1174L [10, 47] are sensitive to alectinib. Alectinib demonstrated a favorable safety profile and clinically meaningful response in patients with ALK-positive metastatic NSCLC who progressed on crizotinib and, therefore, was granted accelerated approval by the United States Food and Drug Administration (FDA) on December 11, 2015 [25]. Because of the established oncogenic role of ALK in NB [3], and the potent inhibitory efficacy of alectinib against advanced/recurrent ALK-mutated tumors, we hypothesize that alectinib is a promising treatment for NB.

In this study, we evaluate the inhibitory effects of alectinib in vitro in NB cell lines and in vivo in two mouse models of the disease. Our results demonstrated that ALK inhibitor alectinib suppresses ALK-induced PI3K/Akt/mTOR signaling and induces apoptosis in both ALK wild-type (WT) and ALK mutant NB cells. These results suggest that alectinib is a potential therapeutic compound for NB patients and should be investigated further in this devastating pediatric malignancy.

2. MATERIALS AND METHODS

2.1 Cell lines

IMR-32, SH-SY5Y and LA-N-6 cell lines were cultured in RPMI 1640 medium (Lonza, Walkersville, MD, USA), 20% (v/v) heat-inactivated Fetal Bovine Serum (FBS) (SAFC Biosciences, Lenexa, KS, USA), and 100 units/mL penicillin/streptomycin. Kelly, NB-19 and SK-N-AS were grown in RPMI 1640 medium (Lonza, Walkersville, MD, USA), 10% (v/v) heat-inactivated FBS (SAFC Biosciences, Lenexa, KS, USA), and 100 units/mL penicillin/streptomycin. All cells were maintained in a humidified incubator at a constant temperature of 37 °C and 5% CO2.

2.2 Antibodies and Reagents

Alectinib (HY-13011) was purchased from MedChem Express (NJ 08852, USA). Doxorubicin (Dox, D1515) and anti-β-Actin antibody (A2228) were purchased from Sigma (Sigma-Aldrich Corp, St. Louis, MO, USA). Anti-PARP (9532 S), anti-Caspase-3 (9662S), anti-phospho-Akt (4060S), anti-Akt (9272), anti-phospho-S6 (4858S), anti-S6 (2217S), anti-Mouse (7076S) and anti-Rabbit (7074S) antibodies were purchased from Cell Signaling Technology (Cell Signaling Technology, Danvers, MA, USA).

2.3 Cell viability assay

Cell viability experiments were performed with the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Rockville, MA, USA) according to the manufacturer’s instructions. All assays were conducted in replicates of six in 96-well plates. The absorbance at 450 nm of each well was measured by the Synergy™ 4 Hybrid Microplate Reader (Biotek). The IC50 value based on the cell viability data was calculated with Prism 5 (Graphpad Software Inc., La Jolla, CA).

2.4 Flow Cytometry and Propidium Iodide (PI) Staining Assay

Cells were seeded into 6-cm dishes with 1 × 106 cells/dish. Twenty-four hours later, cells were treated with 10 μM alectinib for 0 or 24 hours. Cells were then trypsinized and resuspended in RPMI-1640 medium followed by centrifugation at 450 g for 5 min (4 °C). Cells were then washed with cold 1× PBS with 1% BSA twice, and resuspended at a density of 1 × 106 cells/ml in 1× binding buffer (51-66121E; BD Biosciences, San Jose, CA, USA). Afterwards, 100 μl of cell suspension was transferred into a new tube and stained with 10 μl of 10 μg/mL Propidium Iodide solution (40017; Biotium, Inc.). The cells in the tubes were gently vortexed and incubated for 15 min at RT (25 °C) in the dark. Unstained cells were used as a negative control, and untreated cells were used as a control for treated cells. Then flow cytometry analyses were performed on a LSR-II flow cytometer (BD Biosciences) using BD FACDiva software v.6.0.

2.5 Colony Formation Assay

A 5% (w/v) base agar layer was prepared by adding agar (214220, Difco Laboratories, Detroit, MI, USA) into distilled water and then autoclaving the mixture for 50 min before cooling in a 56 °C water bath. The 2 mL bottom agar layer was made of 0.5% agar and media solution, and added to each well until semi-solid. The 1.5 ml top agar layer was made of 0.3% agar and media solution, and each NB cell line was counted and added to the mixture at 1 × 104 cells/well along with the indicated concentrations of alectinib. Cells were grown at 37 °C for 2 to 3 weeks, and subsequently stained with 500 μL of 5 mg/mL Thiazolyl Blue Tetrazolium Bromide (MTT, M5655, Sigma). After 4 hours, images were captured by the microscope and colonies were counted. Each assay was performed in triplicate.

2.6 Western blot analysis

2×106 cells were plated in 60 × 15 mm tissue culture dishes and cultivated in a humidified incubator for 24 h. Cells were then treated and harvested. Pellets were washed twice with ice cold PBS and then lysed on a rotator at 4 °C for 30 min in cooled RIPA buffer (150 mM NaCl, 50 mM Tris-HCl at pH 7.4, 50 mM sodium fluoride, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.1 mM sodium orthovanadate, 10 μg/mL leupeptin, 1% NP-40, 0.25% sodium deoxycholate, and phosphatase inhibitor cocktail 2 and 3 (p5726 and p0044, Sigma)). Lysates were then centrifuged at 13,000 rpm for 15 min, and supernatants were collected. Protein concentration of each sample was quantified by the Bradford reagent (Bio-Rad Laboratories, Hercules, CA, USA). Lysates (100 μg protein) were separated by the SDS-PAGE, and then transferred to PVDF (polyvinylidence fluoride) membranes (BioRad). Membranes were blocked in 5% milk for 1 h at room temperature, and then incubated with the indicated primary antibodies overnight at 4°C. The membranes were then incubated with anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase at room temperature for 1 h. Chemiluminescent visualization was detected by The ECL-Plus Western detection system (GE Health Care, Buckinghamshire, UK).

2.7 Orthotopic mouse model of NB

All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Baylor College of Medicine. Female athymic NCR nude mice were purchased from Taconic (Taconic, Hudson, NY, USA) and maintained under pathogen-free conditions. The preclinical mouse model of NB was established using orthotopic (intrarenal) implantation of the NB cells as described previously [9]. 1.5 × 106 human luciferase-transduced NGP cells in 0.1 ml of PBS were surgically injected into the left renal capsule and toward the superior pole of the left kidney of the animal. Several weeks later, mice bearing similar-sized tumors were randomly divided into two groups and treated with either DMSO or alectinib (25 mg/kg, intraperitoneal injection once daily). Three days later, the mice were sacrificed and the tumors were harvested and lysed for immunoblotting.

2.8 TH-MYCN transgenic Mouse Model of NB

All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Baylor College of Medicine. Homozygous TH-MYCN transgenic mice were identified via PCR genotyping. At four weeks of age, these mice were randomly divided into groups and treated with 25 mg/kg alectinib or an equal volume of DMSO every other day by i.p. injection for three weeks. Mice were then sacrificed when they were 7 weeks old or kept until death. The survival time of each group was recorded, and tumor and corresponding kidneys were photographed and weighed. The inhibitory effects of alectinib on the tumor of TH-MYCN transgenic mice was conducted as followings: seven-week-old homozygous TH-MYCN transgenic mice were treated with 25 mg/kg alectinib or an equal volume of DMSO i.p. once daily for two days. At the end of treatment, the mice were sacrificed and the tumors were harvested. The tumor tissues were then lysed for protein immunoblotting with the indicated antibodies.

2.9 Statistical Analysis

The results were presented as mean ± standard deviation (SD). A two-tailed Student’s t-test was used to determine the statistical significance among control and drug treatment groups. P <0.05 was considered as statistically significant.

3. Results

3.1 ALK inhibitor alectinib suppresses cell proliferation in both ALK-WT and ALK-mutant NB cells

First, we hypothesized that treatment of NB cells in vitro with the ALK inhibitor alectinib would result in decreases in cell viability. To test this hypothesis, we examined relative cell viability in NB cells treated with alectinib with CCK-8 assays. We selected six NB cell lines, including three ALK-WT cell lines (IMR-32, NB-19, and SK-N-AS) and three ALK-mutant cell lines (Kelly, SH-SY5Y, and LA-N-6). The results show that cell viability was greatly reduced by alectinib treatment in all six cell lines tested (Figure 1A), and there were morphological changes in the NB cells (Figure 1C). The IC50s of the cell lines tested ranges from 3.181 μM to 9.6 μM. ALK mutated cells Kelly and SH-SY5Y were relatively more sensitive to the treatment of alectinib and showed greater inhibition of cell growth. In addition, alectinib showed potent inhibitory effects on ALK-WT cells (Figure 1B) with IC50s ranging from 4 to 9.4 μM. These results indicated that alectinib significantly inhibits cell growth in both ALK-WT and ALK-mutant NB cells.

Figure 1.

Figure 1

Open in a new tab

Alectinib shows anti-proliferation effects on NB cell lines. (A) Six NB cell lines were treated with increasing concentrations of alectinib for 72 hours. Cell viability was assessed by CCK-8 assay. Data was represented as % vehicle ± S.D. with P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***) indicated. (B) The IC50 values of alectinib in each cell line listed were calculated based on the data collected in the cell viability assays in (A). ALK status in NB cell lines was also shown. (C) Morphological changes of the six different NB cell lines treated with concentrations (0, 1, 5 μM) of alectinib were shown.

3.2 ALK inhibitor alectinib inhibits colony formation abilities of NB cells

The ability to form colonies in an anchorage-independent manner in soft agar is regarded as one of the characteristics of cancer cells [7]. To evaluate if alectinib inhibits anchorage-independent colony formation of NB cells, we conducted soft agar assays with the same six NB cell lines, including ALK-WT and ALK-mutant cells. Our results show that the number of colonies were significantly reduced in all alectinib-treated groups, indicating that alectinib inhibited colony formation in all NB cell lines tested (Figure 2A, 2B).

Figure 2.

Figure 2

Open in a new tab

Alectinib suppresses anchorage-independent growth of NB cells. (A) A panel of six NB cell lines were grown on soft agar with 0, 1, or 5 μM of alectinib for 2 to 3 weeks. Cells were stained with crystal violet to visualize colonies. (B) Colonies were counted and colony numbers were represented as % vehicle ± S.D. with P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) indicated.

3.3 Alectinib inhibits ALK-mediated PI3K/Akt/mTOR signaling and induces apoptosis in NB cells

In prior studies of NB, gain-of-function mutations in ALK primarily signal through the activation of the PI3K/Akt/mTOR pathways [1]. We aimed to determine if alectinib inhibits the activation of PI3K/Akt/mTOR signaling by blocking ALK in NB. We measured the phosphorylation levels of Akt in six NB cell lines including three N-myc amplified cells, NB-19, IMR-32 and Kelly, and three N-myc non-amplified cells, SH-SY5Y, LA-N-6 and SK-N-AS. Our results show that alectinib effectively suppresses the phosphorylation levels of Akt at S473 and S6 at S235/236 in all cell lines tested (Figure 3). We observed that the phosphorylation of Akt and S6 was inhibited in Kelly and SH-SY5Y cells (Figure 3), indicating that ALK-mutant cell lines are relatively more sensitive to alectinib compared to ALK-WT cell lines. In addition, treatment with alectinib greatly induced apoptosis in all NB cells tested, as shown by caspase 3 and PARP cleavages (Figure 3). Consistent with these results, the flow cytometry analysis showed that alectinib could induce cell death in Kelly, NB-19, SH-SY5Y and SK-N-AS cell lines (Supplement 1A). These findings demonstrate that alectinib induced apoptosis by blocking PI3K/Akt/mTOR pathways in both N-myc amplified and N-myc non-amplified NB cells.

Figure 3.

Figure 3

Open in a new tab

Alectinib inhibits PI3K/Akt/mTOR signaling and induces apoptosis in NB cells. NB-19, Kelly, IMR-32, SH-SY5Y, SK-N-AS and LA-N-6 cells were treated with 10 μM alectinib for various time points as indicated. The anti-β-Actin antibody was used as a loading control for whole cell extracts.

3.4 ALK inhibitor alectinib enhances dox-induced apoptosis in NB

Because of the genomic instability and heterogeneity, cancer cells can rapidly recover from oncogene addiction, resulting in failure to achieve long-lasting efficacy with monotherapies. Therefore, combined cancer therapies were designed and demonstrated better efficacy compared to single agent therapies [18]. Thus, we evaluated the combined effect of alectinib with the traditional chemotherapeutic drug doxorubicin (dox) on cell viability in a panel of six NB cell lines, including ALK-WT cell lines, ALK-mutant cell lines, and the chemoresistant LA-N-6 cell line. Our results showed that alectinib enhanced dox-induced cytotoxicity in all cell lines tested, including in LA-N-6 cells, which are resistant to dox alone (Figure 4A). In addition, we observed that alectinib greatly increased dox-induced PARP and Caspase 3 cleavages in all cells tested including LA-N-6 (Figure 4B). These results show that alectinib enhances dox-induced apoptosis and even overcomes acquired chemoresistance to dox in NB cells.

Figure 4.

Figure 4

Open in a new tab

Alectinib enhances the cytotoxic effect of dox on NB cell lines. (A) Six NB cells were seeded and incubated with the indicated concentrations (0, 0.1, 0.5, 1, 2 μM) of dox or 5 μM alectinib for 24 hrs. Cell viability was then measured by CCK-8 assay. Cell viabilities were represented as % vehicle ± S.D. with P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***) as indicated. (B) NB-19 and SH-SY5Y cells were treated with either Dox (0.2 μM), alectinib (5 μM), or a combination of both for 12 hrs; IMR-32, Kelly and SK-N-AS cells were treated with either dox (0.5 μM), alectinib (5 μM), or a combination of both for 12 hrs; LA-N-6 cells were treated with either dox (2 μM), alectinib (5 μM), or a combination of both for 12 hrs. PARP and Caspase 3 cleavages were examined.

3.5 ALK inhibitor alectinib induces apoptosis in xenograft NB mouse model

Having shown the inhibitory effects of alectinib on NB cells in vitro, we evaluated the in vivo effects of the drug on apoptosis in orthotopic xenograft mouse models of NB. Mice bearing xenograft tumors generated with NGP cells were randomly divided into two groups and treated with either alectinib or dimethylsulfoxide (DMSO) (carrier control) for 3 days at a dose of 25 mg/kg intraperitoneally daily, tumors were harvested and analyzed for PI3K/Akt/mTOR signaling and apoptotic effectors. Our results show that, in contrast to tumors treated with DMSO, tumors treated with alectinib exhibited significantly decreased levels of p-Akt and p-S6. Furthermore, we observed increased PARP and Caspase 3 cleavages in tumors treated with alectinib compared to controls (Figure 5A). These results suggest that alectinb inhibites the PI3K/Akt/mTOR pathways and induces apoptosis in a xenograft mouse model of NB.

Figure 5.

Figure 5

Open in a new tab

Alectinib induces apoptosis in an orthotopic mouse model of NB and the TH-MYCN transgenic mouse model. (A–B) NGP-luciferase xenograft mouse bearing tumors (A) and the TH-MYCN transgenic mice bearing tumors (B) were treated with DMSO or 25 mg/kg of alectinib daily for 3 days. Then tumors were harvested and analyzed for apoptotic marker and for Akt and S6 phosphorylation. (C–D) Four-week-old TH-MYCN transgenic mouse were treated with DMSO or 25 mg/kg of alectinib every other day until the end of seven-week-old (11 times). Then mice were sacrificed, tumors and corresponding kidneys in each group were photographed (C) and weighted (D). (E) Four-week old TH-MYCN transgenic mouse were treated with DMSO or 25 mg/kg of alectinib every other day for another three weeks (11 times). Mice were kept until natural death, the survival time points of control and treatment groups were recorded.

3.6 ALK inhibitor alectinib suppresses of tumor growth in the TH-MYCN transgenic mouse model

To determine if alectinib could successfully inhibit NB tumor development, we assessed the efficacy of alectinib in the TH-MYCN transgenic mouse model, which is the most widely used murine NB model [37]. We observed that treatment with alectinib resulted in decreased tumor growth in TH-MYCN transgenic mice when compared with the control animals treated with placebo (Figure 5C, 5D). In addition, alectinib induced PARP and Caspase 3 cleavages in TH-MYCN tumor tissue and blocked PI3K/Akt/mTOR signaling as shown by the loss of phosphorylation of Akt at S473 and S6 at S235/236 (Figure 5B). Furthermore, compared with DMSO treatment, alectinib exposure prolonged the survival time of TH-MYCN transgenic mice (Figure 5E, Supplement 1B). These data suggest that alectinib inhibits NB tumor growth, as well as induces ALK inhibition-mediated apoptosis and blocks PI3K/Akt/mTOR in the TH-MYCN transgenic mouse model.

4. Discussion

Constitutively activated ALK plays an oncogenic role in a variety of tumors, including NSCLC, ALCL, and NB [5, 45]. In NB, ALK-related aberrations are closely linked with poor clinical outcome, especially in high-risk NB [24]. Herein, we evaluated the effects of the ALK inhibitor alectinib in NB. Our results indicate that alectinib shows significant inhibitory activity against NB cells by suppressing cell proliferation and inducing apoptosis through the inhibition of PI3K/Akt/mTOR signaling. In addition, alectinib enhances dox-induced apoptosis and increases sensitivity to dox treatment in vitro. Furthermore, treatment of alectinib blocks PI3K/Akt/mTOR pathways and, in turn induces apoptosis in two murine models of NB, a xenograft mouse model and the TH-MYCN transgenic model. Most notably, alectinib successfully inhibited tumor development in the TH-MYCN transgenic NB mouse model, resulting in slowed or regressed tumor growth as well as prolonged lifespan of the animals. Since we observed potent efficacy of alectinib combined with dox in vitro, we hypothesize that combining alectinib with current therapeutic agents would enhance efficacy and improve outcomes.

Inhibition of ALK signaling is an effective therapeutic strategy for cancer. The clinical efficacy of crizotinib, a first generation of ALK inhibitor, has been demonstrated in several cancer types, including ALK-mutated NB, ALK-rearranged NSCLC and ALK-positive ALCL [39]. On August 26, 2011, crizotinib was approved by the FDA for treating ALK-rearranged NSCLC, which is regarded as one of the milestones for clinical development of small molecular inhibitors in cancer therapy [14]. However, with prolonged exposure, crizotinib treatment might lead to the development of mutations within the ALK tyrosine kinase domain [41]. In prior studies, crizotinib-induced ALK activations have been detected in approximately 10% of cases of NB, most commonly as ALKR1275Q and ALKF1174L [41]. Therefore, to overcome acquired drug resistance generated by treatment with crizotinib, and to effectively treat tumors with crizotinib-induced ALK mutations, the second and third generation of ALK inhibitors have been developed.

Based on our experiments, the ALK inhibitor alectinib shows significant inhibitory effects on both ALK-WT and ALK-mutant NB cells. Importantly, ALK-mutant cells Kelly (N-myc amplified, ALKF1174L mutation) and SH-SY5Y (N-myc non-amplified, ALKF1174L mutation) were more sensitive to alectinib. These results are encouraging since ALKR1275, ALKF1174 and ALKF1245 account for more than 85% of ALK mutations in NB [19, 52]. In addition, MYCN amplification is reported as a crucial factor driving NB tumor development [31]. However, our results show that there were no obvious differences in the effects of alectinib between N-myc amplified and N-myc non-amplified NB cells. These results suggest that the cytotoxicity and sensitivity of alectinib are dependent more on ALK activation status, rather than on the N-myc amplification status in NB.

Our findings may have important clinical implications for NB patients who develop acquired resistance for crizotinib. The crizotinib-resistant ALKF1174L and ALKR1275Q mutations that arise de novo during crizotinib treatment in NB could result in acquired chemoresistance [10]. Thus alectinib may be very effective treatment option since it induces apoptosis in Kelly and SH-SY5Y, both of which harbor the ALKF1174L mutation. This result indicates that alectinib is able to overcome crizotinib-induced chemoresistance. Our study supports further work evaluating the efficacy of alectinib in NB patients, especially in patients whose tumors have become resistant to crizotinib. The efficacy of alectinib as a single agent therapy or in combination with conventional agents should be further investigated.

Supplementary Material

supplement

Supplement 1. (A) Two ALK-WT cell lines NB-19 and SK-N-AS, two ALK-mutated cell lines Kelly and SH-SY5Y were treated with 10 μM alectinib for 0 or 24 hours. The percentage of cell death in the treated cells was detected by flow cytometry analysis. (B) Four-week old TH-MYCN transgenic mouse were treated with DMSO or 25 mg/kg of alectinib every other day for three weeks (11 times). Mice were kept until natural death, the Kaplan Meier survival curve of control and treatment groups were shown.

Highlights.

  • Alectinib treatment strongly induces apoptosis in human neuroblastoma cells.

  • Alectinib inhibits neuroblastoma tumorigenesis.

  • Alectinib is able to overcome crizotinib-induced chemoresistance.

Acknowledgments

FUNDING

We appreciate Dr. Andrew Davidoff (St. Jude Children’s Research Hospital, Memphis, TN, USA) and Dr. Robert Seeger (Children’s Hospital of Los Angeles, Los Angeles, CA, USA) for providing the NB cell lines described in this paper. This work was supported by the Ministry of Science and Technology of China (2015CB964600 (G.X.), 2013CB967501 (G.X.)), National Natural Science Foundation of China (31171419, 81000383) (G.X.), the Fundamental Research Funds for the Central Universities (T.Y.), Foundation from State Key Laboratory of Ophthalmology, Zhong Shan Ophthalmic Center, Sun Yat-sen University (T.Y.), Foundation from Shanghai Municipal Health Bureau (20134y013) (T.Y.), and the NIH/NINDS grants 1R01NS072420 (J.Y.), 1R21NS085467 (J.Y.) and 5R21NS094654 (J.Y.).

Abbreviations

NB

neuroblastoma

ALK

anaplastic lymphoma kinase

WT

wild type

dox

doxorubicin

DMSO

dimethyl sulfoxide

FDA

Food and Drug Administration

ALCL

anaplastic large cell lymphoma

NSCLC

non-small cell lung cancer

FBS

Fetal Bovine Serum

Footnotes

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Azarova AM, Gautam G, George RE. Emerging importance of ALK in neuroblastoma. Seminars in cancer biology. 2011;21:267–275. doi: 10.1016/j.semcancer.2011.09.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Bosse KR, Maris JM. Advances in the translational genomics of neuroblastoma: From improving risk stratification and revealing novel biology to identifying actionable genomic alterations. Cancer. 2016;122:20–33. doi: 10.1002/cncr.29706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Bresler SC, Weiser DA, Huwe PJ, Park JH, Krytska K, Ryles H, Laudenslager M, Rappaport EF, Wood AC, McGrady PW, Hogarty MD, London WB, Radhakrishnan R, Lemmon MA, Mosse YP. ALK mutations confer differential oncogenic activation and sensitivity to ALK inhibition therapy in neuroblastoma. Cancer cell. 2014;26:682–694. doi: 10.1016/j.ccell.2014.09.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Broutin S, Stewart A, Thavasu P, Paci A, Bidart JM, Banerji U. Insights into significance of combined inhibition of MEK and m-TOR signalling output in KRAS mutant non-small-cell lung cancer. British journal of cancer. 2016;115:549–552. doi: 10.1038/bjc.2016.220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Caccese M, Ferrara R, Pilotto S, Carbognin L, Grizzi G, Calio A, Brunelli M, Cuppone F, Petraglia S, Scarpa A, Tortora G, Bria E. Current and developing therapies for the treatment of non-small cell lung cancer with ALK abnormalities: update and perspectives for clinical practice. Expert opinion on pharmacotherapy. 2016 doi: 10.1080/14656566.2016.1242578. [DOI] [PubMed] [Google Scholar]
  • 6.Cao Q, Liu F, Li S, Liu N, Li L, Li C, Peng T. Primary rare anaplastic large cell lymphoma, ALK positive in small intestine: case report and review of the literature. Diagnostic pathology. 2016;11:83. doi: 10.1186/s13000-016-0539-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Castillo V, Valenzuela R, Huidobro C, Contreras HR, Castellon EA. Functional characteristics of cancer stem cells and their role in drug resistance of prostate cancer. International journal of oncology. 2014;45:985–994. doi: 10.3892/ijo.2014.2529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Chen Y, Takita J, Choi YL, Kato M, Ohira M, Sanada M, Wang L, Soda M, Kikuchi A, Igarashi T, Nakagawara A, Hayashi Y, Mano H, Ogawa S. Oncogenic mutations of ALK kinase in neuroblastoma. Nature. 2008;455:971–974. doi: 10.1038/nature07399. [DOI] [PubMed] [Google Scholar]
  • 9.Chen Z, Wang Z, Pang JC, Yu Y, Bieerkehazhi S, Lu J, Hu T, Zhao Y, Xu X, Zhang H, Yi JS, Liu S, Yang J. Multiple CDK inhibitor dinaciclib suppresses neuroblastoma growth via inhibiting CDK2 and CDK9 activity. Scientific reports. 2016;6:29090. doi: 10.1038/srep29090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Debruyne DN, Bhatnagar N, Sharma B, Luther W, Moore NF, Cheung NK, Gray NS, George RE. ALK inhibitor resistance in ALK(F1174L)-driven neuroblastoma is associated with AXL activation and induction of EMT. Oncogene. 2016;35:3681–3691. doi: 10.1038/onc.2015.434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Deeks ED. Ceritinib: a Review in ALK-Positive Advanced NSCLC. Targeted oncology. 2016 doi: 10.1007/s11523-016-0460-7. [DOI] [PubMed] [Google Scholar]
  • 12.Facchinetti F, Tiseo M, Di Maio M, Graziano P, Bria E, Rossi G, Novello S. Tackling ALK in non-small cell lung cancer: the role of novel inhibitors. Translational lung cancer research. 2016;5:301–321. doi: 10.21037/tlcr.2016.06.10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Gainor JF. Alectinib-a new chapter in the management of ALK-positive lung cancer. Translational lung cancer research. 2016;5:343–346. doi: 10.21037/tlcr.2016.03.05. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Gandhi L, Janne PA. Crizotinib for ALK-rearranged non-small cell lung cancer: a new targeted therapy for a new target. Clinical cancer research: an official journal of the American Association for Cancer Research. 2012;18:3737–3742. doi: 10.1158/1078-0432.CCR-11-2393. [DOI] [PubMed] [Google Scholar]
  • 15.Guan J, Tucker ER, Wan H, Chand D, Danielson LS, Ruuth K, El Wakil A, Witek B, Jamin Y, Umapathy G, Robinson SP, Johnson TW, Smeal T, Martinsson T, Chesler L, Palmer RH, Hallberg B. The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN. Disease models & mechanisms. 2016;9:941–952. doi: 10.1242/dmm.024448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Hallberg B, Palmer RH. The role of the ALK receptor in cancer biology. Annals of oncology: official journal of the European Society for Medical Oncology/ESMO. 2016;27(Suppl 3):iii4–iii15. doi: 10.1093/annonc/mdw301. [DOI] [PubMed] [Google Scholar]
  • 17.Hara J. Development of treatment strategies for advanced neuroblastoma. International journal of clinical oncology. 2012;17:196–203. doi: 10.1007/s10147-012-0417-5. [DOI] [PubMed] [Google Scholar]
  • 18.Huang H, Weng H, Zhou H, Qu L. Attacking c-Myc: targeted and combined therapies for cancer. Current pharmaceutical design. 2014;20:6543–6554. doi: 10.2174/1381612820666140826153203. [DOI] [PubMed] [Google Scholar]
  • 19.Infarinato NR, Park JH, Krytska K, Ryles HT, Sano R, Szigety KM, Li Y, Zou HY, Lee NV, Smeal T, Lemmon MA, Mosse YP. The ALK/ROS1 Inhibitor PF-06463922 Overcomes Primary Resistance to Crizotinib in ALK-Driven Neuroblastoma. Cancer discovery. 2016;6:96–107. doi: 10.1158/2159-8290.CD-15-1056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Kalaskar RR, Kalaskar AR. Neuroblastoma in early childhood: A rare case report and review of literature. Contemporary clinical dentistry. 2016;7:401–404. doi: 10.4103/0976-237X.188579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Kelleher FC, McDermott R. The emerging pathogenic and therapeutic importance of the anaplastic lymphoma kinase gene. European journal of cancer. 2010;46:2357–2368. doi: 10.1016/j.ejca.2010.04.006. [DOI] [PubMed] [Google Scholar]
  • 22.Kim EK, Kim S. ALK gene copy number gain and immunohistochemical expression status using three antibodies in neuroblastoma. Pediatric and developmental pathology: the official journal of the Society for Pediatric Pathology and the Paediatric Pathology Society. 2016 doi: 10.1177/1093526616686445. [DOI] [PubMed] [Google Scholar]
  • 23.Kinoshita K, Asoh K, Furuichi N, Ito T, Kawada H, Hara S, Ohwada J, Miyagi T, Kobayashi T, Takanashi K, Tsukaguchi T, Sakamoto H, Tsukuda T, Oikawa N. Design and synthesis of a highly selective, orally active and potent anaplastic lymphoma kinase inhibitor (CH5424802) Bioorganic & medicinal chemistry. 2012;20:1271–1280. doi: 10.1016/j.bmc.2011.12.021. [DOI] [PubMed] [Google Scholar]
  • 24.Kwon MJ, Choi YL, Sung KW, Kang SY, Park SM, Choi SY, Kim JS, Suh YL. Oncogenic anaplastic lymphoma kinase (ALK) mutation in neuroblastomas and other pediatric tumors. Pathology, research and practice. 2011;207:634–639. doi: 10.1016/j.prp.2011.07.011. [DOI] [PubMed] [Google Scholar]
  • 25.Larkins E, Blumenthal GM, Chen H, He K, Agarwal R, Gieser G, Stephens O, Zahalka E, Ringgold K, Helms W, Shord S, Yu J, Zhao H, Davis G, McKee AE, Keegan P, Pazdur R. FDA Approval: Alectinib for the Treatment of Metastatic ALK-Positive Non-Small Cell Lung Cancer Following Crizotinib. Clinical cancer research: an official journal of the American Association for Cancer Research. 2016 doi: 10.1158/1078-0432.CCR-16-1293. [DOI] [PubMed] [Google Scholar]
  • 26.Le T, Gerber DE. ALK alterations and inhibition in lung cancer. Seminars in cancer biology. 2016 doi: 10.1016/j.semcancer.2016.08.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Leal T, Wakelee H, Infante J, Blumenschein G, Reckamp K, Carter C, Waqar S, Gockerman J, Lovly C, Dukart G, Harrow K, Liang C, Gibbons J, Horn L. P1.44 (also presented as PD2.02): Phase I/II Trial of X-396, A Novel ALK Inhibitor, in Patients With ALK+ NSCLC: Track: Advanced NSCLC. Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer. 2016;11:S209–S210. [Google Scholar]
  • 28.Li J, Liu W, Luo H, Bao J. Insight into drug resistance mechanisms and discovery of potential inhibitors against wild-type and L1196M mutant ALK from FDA-approved drugs. Journal of molecular modeling. 2016;22:231. doi: 10.1007/s00894-016-3099-5. [DOI] [PubMed] [Google Scholar]
  • 29.Li W, Zhang J, Guo L, Chuai S, Shan L, Ying J. Combinational analysis of FISH and immunohistochemistry reveals rare genomic events in ALK fusion patterns in NSCLC and responds to crizotinib treatment. Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer. 2016 doi: 10.1016/j.jtho.2016.08.145. [DOI] [PubMed] [Google Scholar]
  • 30.Lin JJ, Kennedy E, Sequist LV, Brastianos PK, Goodwin KE, Stevens S, Wanat AC, Stober LL, Digumarthy SR, Engelman JA, Shaw AT, Gainor JF. Clinical Activity of Alectinib in Advanced RET-Rearranged Non-Small Cell Lung Cancer. Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer. 2016 doi: 10.1016/j.jtho.2016.08.126. [DOI] [PubMed] [Google Scholar]
  • 31.Maris JM. Recent advances in neuroblastoma. The New England journal of medicine. 2010;362:2202–2211. doi: 10.1056/NEJMra0804577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Metro G, Lunardi G, Bennati C, Chiarini P, Sperduti I, Ricciuti B, Marcomigni L, Costa C, Crino L, Floridi P, Gori S, Chiari R. Alectinib’s activity against CNS metastases from ALK-positive non-small cell lung cancer: a single institution case series. Journal of neuro-oncology. 2016;129:355–361. doi: 10.1007/s11060-016-2184-z. [DOI] [PubMed] [Google Scholar]
  • 33.Mosse YP, Laudenslager M, Longo L, Cole KA, Wood A, Attiyeh EF, Laquaglia MJ, Sennett R, Lynch JE, Perri P, Laureys G, Speleman F, Kim C, Hou C, Hakonarson H, Torkamani A, Schork NJ, Brodeur GM, Tonini GP, Rappaport E, Devoto M, Maris JM. Identification of ALK as a major familial neuroblastoma predisposition gene. Nature. 2008;455:930–935. doi: 10.1038/nature07261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Murga-Zamalloa CA, Mendoza-Reinoso V, Sahasrabuddhe AA, Rolland D, Hwang SR, McDonnell SR, Sciallis AP, Wilcox RA, Bashur V, Elenitoba-Johnson K, Lim MS. NPM-ALK phosphorylates WASp Y102 and contributes to oncogenesis of anaplastic large cell lymphoma. Oncogene. 2016 doi: 10.1038/onc.2016.366. [DOI] [PubMed] [Google Scholar]
  • 35.Ou SH, Milliken JC, Azada MC, Miller VA, Ali SM, Klempner SJ. ALK F1174V mutation confers sensitivity while ALK I1171 mutation confers resistance to alectinib. The importance of serial biopsy post progression. Lung cancer. 2016;91:70–72. doi: 10.1016/j.lungcan.2015.09.006. [DOI] [PubMed] [Google Scholar]
  • 36.Pinto NR, Applebaum MA, Volchenboum SL, Matthay KK, London WB, Ambros PF, Nakagawara A, Berthold F, Schleiermacher G, Park JR, Valteau-Couanet D, Pearson AD, Cohn SL. Advances in Risk Classification and Treatment Strategies for Neuroblastoma. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2015;33:3008–3017. doi: 10.1200/JCO.2014.59.4648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Quarta C, Cantelli E, Nanni C, Ambrosini V, D’Ambrosio D, Di Leo K, Angelucci S, Zagni F, Lodi F, Marengo M, Weiss WA, Pession A, Tonelli R, Fanti S. Molecular imaging of neuroblastoma progression in TH-MYCN transgenic mice. Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging. 2013;15:194–202. doi: 10.1007/s11307-012-0576-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Rossi A. Alectinib for ALK-positive non-small-cell lung cancer. Expert review of clinical pharmacology. 2016;9:1005–1013. doi: 10.1080/17512433.2016.1195262. [DOI] [PubMed] [Google Scholar]
  • 39.Sakamoto H, Tsukaguchi T, Hiroshima S, Kodama T, Kobayashi T, Fukami TA, Oikawa N, Tsukuda T, Ishii N, Aoki Y. CH5424802, a selective ALK inhibitor capable of blocking the resistant gatekeeper mutant. Cancer cell. 2011;19:679–690. doi: 10.1016/j.ccr.2011.04.004. [DOI] [PubMed] [Google Scholar]
  • 40.Santarpia M, Altavilla G, Rosell R. Alectinib: a selective, next-generation ALK inhibitor for treatment of ALK-rearranged non-small-cell lung cancer. Expert review of respiratory medicine. 2015;9:255–268. doi: 10.1586/17476348.2015.1009040. [DOI] [PubMed] [Google Scholar]
  • 41.Sasaki T, Okuda K, Zheng W, Butrynski J, Capelletti M, Wang L, Gray NS, Wilner K, Christensen JG, Demetri G, Shapiro GI, Rodig SJ, Eck MJ, Janne PA. The neuroblastoma-associated F1174L ALK mutation causes resistance to an ALK kinase inhibitor in ALK-translocated cancers. Cancer research. 2010;70:10038–10043. doi: 10.1158/0008-5472.CAN-10-2956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Satoh S, Takatori A, Ogura A, Kohashi K, Souzaki R, Kinoshita Y, Taguchi T, Hossain MS, Ohira M, Nakamura Y, Nakagawara A. Neuronal leucine-rich repeat 1 negatively regulates anaplastic lymphoma kinase in neuroblastoma. Scientific reports. 2016;6:32682. doi: 10.1038/srep32682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Shaw AT, Gandhi L, Gadgeel S, Riely GJ, Cetnar J, West H, Camidge DR, Socinski MA, Chiappori A, Mekhail T, Chao BH, Borghaei H, Gold KA, Zeaiter A, Bordogna W, Balas B, Puig O, Henschel V, Ou SH i study. Alectinib in ALK-positive, crizotinib-resistant, non-small-cell lung cancer: a single-group, multicentre, phase 2 trial. The Lancet Oncology. 2016;17:234–242. doi: 10.1016/S1470-2045(15)00488-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Siaw JT, Wan H, Pfeifer K, Rivera VM, Guan J, Palmer RH, Hallberg B. Brigatinib, an anaplastic lymphoma kinase inhibitor, abrogates activity and growth in ALK-positive neuroblastoma cells, Drosophila and mice. Oncotarget. 2016;7:29011–29022. doi: 10.18632/oncotarget.8508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Simionato F, Frizziero M, Carbone C, Tortora G, Melisi D. Current Strategies to Overcome Resistance to ALK-Inhibitor Agents. Current drug metabolism. 2015;16:585–596. doi: 10.2174/1389200216666150812142059. [DOI] [PubMed] [Google Scholar]
  • 46.Skoulidis F, Papadimitrakopoulou VA. Personalized Medicine Tackles Clinical Resistance: Alectinib in ALK-positive NSCLC Progressing on First Generation ALK Inhibitor. Clinical cancer research: an official journal of the American Association for Cancer Research. 2016 doi: 10.1158/1078-0432.CCR-16-1415. [DOI] [PubMed] [Google Scholar]
  • 47.Song Z, Wang M, Zhang A. Alectinib: a novel second generation anaplastic lymphoma kinase (ALK) inhibitor for overcoming clinically-acquired resistance. Acta pharmaceutica Sinica B. 2015;5:34–37. doi: 10.1016/j.apsb.2014.12.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Tchekmedyian N, Ali SM, Miller VA, Haura EB. Acquired ALK L1152R Mutation Confers Resistance to Ceritinib and Predicts Response to Alectinib. Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer. 2016;11:e87–88. doi: 10.1016/j.jtho.2016.03.018. [DOI] [PubMed] [Google Scholar]
  • 49.Theruvath J, Russo A, Kron B, Paret C, Wingerter A, El Malki K, Neu MA, Alt F, Staatz G, Stein R, Seidmann L, Prawitt D, Faber J. Next-generation sequencing reveals germline mutations in an infant with synchronous occurrence of nephro- and neuroblastoma. Pediatric hematology and oncology. 2016;33:264–275. doi: 10.1080/08880018.2016.1184362. [DOI] [PubMed] [Google Scholar]
  • 50.Ueda T, Nakata Y, Yamasaki N, Oda H, Sentani K, Kanai A, Onishi N, Ikeda K, Sera Y, Honda ZI, Tanaka K, Sata M, Ogawa S, Yasui W, Saya H, Takita J, Honda H. ALK(R1275Q) perturbs extracellular matrix, enhances cell invasion and leads to the development of neuroblastoma in cooperation with MYCN. Oncogene. 2016;35:4447–4458. doi: 10.1038/onc.2015.519. [DOI] [PubMed] [Google Scholar]
  • 51.Versteeg R, George RE. Targeting ALK: The Ten Lives of a Tumor. Cancer discovery. 2016;6:20–21. doi: 10.1158/2159-8290.CD-15-1411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Wang Y, Wang L, Guan S, Cao W, Wang H, Chen Z, Zhao Y, Yu Y, Zhang H, Pang JC, Huang SL, Akiyama Y, Yang Y, Sun W, Xu X, Shi Y, Zhang H, Kim ES, Muscal JA, Lu F, Yang J. Novel ALK inhibitor AZD3463 inhibits neuroblastoma growth by overcoming crizotinib resistance and inducing apoptosis. Scientific reports. 2016;6:19423. doi: 10.1038/srep19423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Wu J, Savooji J, Liu D. Second- and third-generation ALK inhibitors for non-small cell lung cancer. Journal of hematology & oncology. 2016;9:19. doi: 10.1186/s13045-016-0251-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Yoshida T, Hida T, Yatabe Y. Rapid and dramatic response to alectinib in an anaplastic lymphoma kinase rearranged non-small-cell lung cancer patient who is critically ill. Anti-cancer drugs. 2016;27:573–575. doi: 10.1097/CAD.0000000000000356. [DOI] [PubMed] [Google Scholar]
  • 55.Yoshimura Y, Kurasawa M, Yorozu K, Puig O, Bordogna W, Harada N. Antitumor activity of alectinib, a selective ALK inhibitor, in an ALK-positive NSCLC cell line harboring G1269A mutation: Efficacy of alectinib against ALK G1269A mutated cells. Cancer chemotherapy and pharmacology. 2016;77:623–628. doi: 10.1007/s00280-016-2977-y. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supplement

Supplement 1. (A) Two ALK-WT cell lines NB-19 and SK-N-AS, two ALK-mutated cell lines Kelly and SH-SY5Y were treated with 10 μM alectinib for 0 or 24 hours. The percentage of cell death in the treated cells was detected by flow cytometry analysis. (B) Four-week old TH-MYCN transgenic mouse were treated with DMSO or 25 mg/kg of alectinib every other day for three weeks (11 times). Mice were kept until natural death, the Kaplan Meier survival curve of control and treatment groups were shown.

NIHMS871497-supplement.jpg (514.8KB, jpg)

RESOURCES