Journal of Pediatric Surgery

In vivo effects of short- and long-term MAPK pathway inhibition against neuroblastoma
Yuki Takeuchi, Tomoko Tanaka, Mayumi Higashi, Shigehisa Fumino, Tomoko Iehara, Hajime Hosoi, Toshiyuki Sakai, Tatsuro Tajiri

PII: S0022-3468(18)30532-3
DOI: doi:10.1016/j.jpedsurg.2018.08.026
Reference: YJPSU 58793
To appear in: Journal of Pediatric Surgery
Received date: 2 August 2018
Accepted date: 25 August 2018

Please cite this article as: Yuki Takeuchi, Tomoko Tanaka, Mayumi Higashi, Shigehisa Fumino, Tomoko Iehara, Hajime Hosoi, Toshiyuki Sakai, Tatsuro Tajiri , In vivo effects of short- and long-term MAPK pathway inhibition against neuroblastoma. Yjpsu (2018), doi:10.1016/j.jpedsurg.2018.08.026
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In vivo effects of short- and long-term MAPK pathway inhibition against neuroblastoma
Yuki Takeuchi*1), Tomoko Tanaka1), Mayumi Higashi1), Shigehisa Fumino1), Tomoko Iehara2), Hajime Hosoi2), Toshiyuki Sakai3), Tatsuro Tajiri1)

1) Department of Pediatric Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan
2) Department of Pediatrics, Kyoto Prefectural University of Medicine, Kyoto, Japan
3) Department of Molecular-Targeting Cancer Prevention, Kyoto Prefectural University of Medicine, Kyoto, Japan

*Corresponding Author:
Yuki Takeuchi, MD.
Department of Pediatric Surgery,
Kyoto Prefectural University of Medicine,
465 Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, 602-8566, Japan.
Tel: +81-75-251-5809
Fax: +81-75-251-5828
E-mail: [email protected]

Abstract Background/Purpose
It was reported that almost 80% of relapsed neuroblastomas showed MAPK pathway mutations. In our previous study, both trametinib (MEK inhibitor) and CH5126766 (RAF/MEK inhibitor) showed in vitro anti-tumor effects on neuroblastoma cells with ERK phosphorylation (pERK). In this study, we analyzed the in vivo effects of MAPK pathway inhibition in neuroblastoma xenografts.
Methods
Xenograft mice with IMR5, CHP-212, or SK-N-AS received daily oral administration of either trametinib or CH5126766 for two weeks (short-term) or eight weeks (long-term). The tumors were measured twice weekly and harvested after treatment for histopathological analyses, including pERK and Ki67 immunohistochemistry.
Results
In short-term treatment, both inhibitors showed significant growth inhibition in CHP-212 and SK-N-AS xenografts, which were pERK-positive before treatment. The number of pERK- and Ki67-positive cells decreased after treatment. Conversely, IMR5 xenografts, which were pERK-negative, were resistant to treatment. During long-term treatment, SK-N-AS xenografts started to regrow from about six weeks with partial differentiation. pERK-positive cells re-increased in these regrown tumors.
Conclusions
MAPK pathway inhibition was effective for treating pERK-positive neuroblastoma in vivo. Therefore, pERK immunohistochemistry might be a convenient biomarker for MAPK pathway inhibition in neuroblastoma treatment. However, neuroblastomas developed acquired drug resistance after long-term treatment. Further studies to overcome acquired resistance are needed.

Keywords: Neuroblastoma; MAPK pathway inhibition; MEK inhibitor; pERK IHC; Acquired drug resistance; Partial differentiation

 

Introduction
Despite major advances in the neuroblastoma treatment of low-risk patients during the past few decades, the cure rates of high-risk patients are still unsatisfyingly low. Therefore, it is urgent to find new and more effective therapies for children with high-risk neuroblastoma, especially recurrent disease. Recently, extensive studies have been performed to investigate small-molecule inhibitors targeting genetic alterations associated with neuroblastoma [1].
The mitogen-activated protein kinase (MAPK) pathway, also known as the RAS-RAF-MEK-ERK pathway, is an essential signal transduction cascade of cell proliferation. Various growth factors bind to cell surface receptors and activate RAS. This then leads to a series of phosphorylation events downstream in the MAPK pathway, resulting in the
phosphorylation and activation of ERK. Phosphorylated-ERK (pERK) translocates to the nucleus and induces the transcription of genes for the cell proliferation [2, 3].
Changes in the MAPK pathway have been reported in various cancers, mainly in the RAS or RAF genes [4]. Regarding neuroblastoma, Eleveld et al. reported that almost 80% of relapsed neuroblastoma show MAPK pathway mutations [5]. Therefore, the inhibition of the MAPK pathway may be an attractive therapy for high-risk neuroblastoma.
Trametinib is the first FDA approved MEK inhibitor, which selectively inhibits MEK kinase activities and inhibits MAPK pathway [2]. However, trametinib is reported to promote ERK-dependent negative feedback in MAPK pathway which leads to drug resistance [6, 7]. In contrast to trametinib, CH5126766, a dual RAF/MEK inhibitor, inhibits not only MEK but also RAF by the formation of a stable RAF/MEK complex [6] (Fig. 1). Recent studies reported that CH5126766 has superior anti-tumor effect in MAPK pathway-mutant cancers compared to other BRAF inhibitors or MEK inhibitors [7].
We previously reported that both trametinib and CH5126766 demonstrated in vitro growth inhibitory effects on neuroblastoma cell lines with MAPK pathway activation, which was detected by pERK immunocytochemistry (ICC). We also showed that CH5126766

demonstrated more effective tumor suppression than trametinib [8].
In this study, we analyzed the in vivo effects of MAPK pathway inhibition in neuroblastoma xenografts using trametinib or CH5126766 to verify the findings of our in vitro analyses. In addition, we investigated whether or not pERK immunohistochemistry (IHC) correlated with the drug response. Furthermore, we also analyzed the long-term effects of MAPK pathway inhibition to obtain new knowledge about drug resistance.

1. Materials and methods
1.1. Cell lines
Three human neuroblastoma cell lines were used in this study (IMR5, CHP-212, and SK-N-AS) (Table 1). Regarding MYCN amplification, we referred to the protocol of a previous report [9]. Regarding RAS/RAF mutation, a NRAS mutation in CHP-212 and SK-N-AS was detected in the Catalogue of Somatic Mutation in Cancer (http://cancer.sanger.ac.uk/cell_lines) database. In addition, we quoted the results of pERK ICC staining of IMR5 and SK-N-AS cells from our previous report [8]. ICC staining for pERK in CHP-212 cells was performed following the protocol we previously reported [8].
IMR5 and SK-N-AS cells were maintained in RPMI 1640 (Nacalai Tesque, Japan). CHP-212 cells were maintained in a 1:1 mixture of Minimum Essential Medium (Nacalai Tesque) and Ham’s Nutrient Mixture F12 (Nacalai Tesque). Culture media were supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin. Cells were incubated at 37 °C in 5% CO2 and 95% humidity.

1.2. Molecular inhibitors
Trametinib was purchased from LC Laboratories (Cat. #T-8123; United States). CH5126766 was kindly provided by Chugai Pharmaceutical (Japan). These inhibitors were dissolved in dimethyl sulfoxide (DMSO) and used for the administration to neuroblastoma xenograft mice.

1.3. In vivo xenograft study
Four-week-old female athymic nude mice (KSN/Slc) were purchased from Shimizu Laboratory (Japan) and maintained under sterile conditions. A total of 5×106 neuroblastoma cells (IMR5, CHP-212, and SK-N-AS) were injected subcutaneously into the right flank area of nude mice. When the tumor volume reached 100 mm3, the mice were randomized to 3 groups (n≥5), vehicle (10% DMSO), trametinib (3 mg/kg/day), or CH5126766 (3 mg/kg/day). Drugs were administered at the maximum tolerated dose which were validated in our preliminary experiment

(data not shown). The mice in each group were then orally treated once a day for two weeks (as short-term treatment for IMR5, CHP-212, and SK-N-AS xenografts) or eight weeks (as long-term treatment for SK-N-AS xenografts). Tumor diameters were measured with calipers twice weekly during treatment, and tumor volumes were calculated according to the formula V =
½ × A × B2 (V = tumor volume, A=largest diameter, B= smallest diameter). The mice were humanely sacrificed before or after treatment (2, 4, 6, and 8 weeks) or when the tumor volume exceeded 1,000 mm3.
All experimental procedures and protocols for the animals conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Committee for Animal Research of Kyoto Prefectural University of Medicine.

1.4. Tumor histological analyses
The tumor tissues were fixed in 4% paraformaldehyde for 48 h and embedded in paraffin before sectioning and staining. Tissue sections were stained with hematoxylin and eosin (H&E) according to the standard protocol. We also performed IHC staining with antibodies to pERK (1:400, #4370; Cell Signaling Technology, Japan) and Ki67 (1:100, Cat. #M724024-2; Dako, United States). The slides were then incubated with the appropriate secondary antibody followed by DAB Chromogen reagent (Dako), and counterstained with hematoxylin. The pERK- or Ki67-positive cells were manually counted as the proportion of positive-stained cells among the total cell number/high-powered field (HPF). Cells with strong brown nuclear staining were considered to be positively stained. The quantification was performed by an operator blinded to the treatment group represented in the images.

1.5. Statistical analyses
Data are expressed as the means ± standard error. All data were analyzed statistically using Student’s t-test. A value of p <0.05 was considered to be significantly different from controls.

2. Results
2.1. Short-term treatment
2.1.1. MAPK pathway inhibition suppressed tumor growth in neuroblastoma xenografts with pERK-positive IHC
First, we evaluated the short-term effects of both inhibitors with two-week administration. Regarding side effects, no animals died or gained or lost more than 10% of their body weight during treatment. Relative tumor growth curves showed that IMR5 (pERK-negative) xenografts were resistant to both inhibitors (Fig. 2A). In contrast, two-week treatment with both inhibitors significantly decelerated tumor growth in CHP-212 (pERK-positive) and SK-N-AS (pERK-positive) xenografts compared to controls (trametinib in CHP-212 and SK-N-AS: p<0.001; CH5126766 in CHP-212: p<0.0001; CH5126766 in SK-N-AS: p<0.001). However, the
efficacy of trametinib and CH5126766 did not significantly differ in pERK-positive xenografts (Fig. 2A and 2B).

2.1.2. pERK IHC correlated with MAPK pathway inhibition sensitivity
To further evaluate the effects of MAPK pathway inhibition, pERK and Ki67 (as a marker for cell proliferation) IHC analyses were performed. As shown in Fig. 2C, IMR5 was pERK-negative, while CHP-212 and SK-N-AS were pERK-positive before treatment, which correlated with the results of in vitro ICC staining [8]. The number of pERK-positive cells in CHP-212 xenografts significantly decreased after treatment with both inhibitors (trametinib: 0%, CH5126766: 0%, and DMSO: 65.5%; p<0.0001) (Fig. 2C and 2D). Similarly, in SK-N-AS
xenografts, the number of pERK-positive cells significantly decreased after treatment with both inhibitors (trametinib: 2.7%, CH5126766: 2.4%, and DMSO: 63.5%; p<0.0001). In addition, the number of Ki67-positive cells in CHP-212 (trametinib: 20.5%, CH5126766: 23.3%, and DMSO: 55.4%; p<0.0001) and SK-N-AS (trametinib: 33.6%, CH5126766: 31.1%, and DMSO: 55.4%;
p<0.0001) xenografts significantly decreased in both inhibitor-treated groups compared to the control group (Fig. 2E and 2F). Conversely, in IMR5 xenografts, which were not inhibited by

either inhibitor, the Ki67 expression showed no significant difference between either inhibitor-treated group and the control group (trametinib: 72.4%, CH5126766: 75.9%, and DMSO: 69.6%).

2.2. Long-term treatment
2.2.1 Neuroblastoma xenografts developed acquired resistance to MAPK pathway inhibition caused by MAPK pathway reactivation
We additionally examined the long-term effects of trametinib and CH5126766 with eight-week administration only to SK-N-AS xenografts. As shown in Fig. 3A, relative tumor growth curves showed that SK-N-AS xenografts were inhibited by both inhibitors during the first five weeks and subsequently started to regrow from about six weeks.
In IHC analyses, the number of pERK-positive cells increased again (trametinib: 53.2%, CH5126766: 50.1%, and DMSO: 71.3%) in these regrown SK-N-AS xenograft tumors (Fig. 3B). Similarly, the number of Ki67-positive cells also increased again (trametinib: 70.5%, CH5126766: 67.2%, and DMSO: 66.2%) (Fig. 3C).

2.2.2 Long-term MAPK pathway inhibition treatment partially induced neuroblastoma differentiation
We also histopathologically analyzed the sequential changes in xenograft tumors with CH5126766 treatment. H&E staining of SK-N-AS tumors at two and four weeks of CH5126766 treatment showed undifferentiated neuroblastoma histology (Fig. 4). In contrast, H&E staining of SK-N-AS tumors at six weeks of CH5126766 treatment showed partial areas of differentiated ganglion-like cells. These cells were negative for both pERK and Ki67 IHC, indicating that they had lost their proliferative ability due to the inhibition of MAPK pathway signaling. However, the proportion of differentiated ganglion-like cells lessened at eight weeks of treatment. Similar results were observed by H&E staining of SK-N-AS xenografts treated with trametinib (data not shown).

 

3. Discussion
In the present study, we demonstrated the in vivo anti-tumor effects of MAPK pathway inhibition against neuroblastoma xenografts with MAPK pathway activation. The short-term tumor inhibitory effects of MEK inhibitors in neuroblastoma have been already reported [5, 10, 11]. However, MEK inhibitors are well known to relieve ERK-dependent feedback inhibition of RAF and activate MAPK pathway signaling, subsequently causing drug resistance [6]. The dual RAF/MEK inhibitor CH5126766 inhibits MAPK pathway signaling more effectively than standard MEK inhibitors, such as trametinib, via the formation of a stable RAF-MEK complex [6, 7]. It has been also reported that CH5126766 showed greater anti-tumor activity in KRAS-mutant xenograft models (colon adenocarcinoma and lung adenocarcinoma) than standard MEK inhibitors [6]. Therefore, several clinical trials for CH5126766 in adult cancer are ongoing [3, 12].
In our previous study, we reported that CH5126766 showed more effective in vitro tumor suppression than trametinib [8]. However, no significant difference in the anti-tumor effects was found between trametinib and CH5126766 in the present in vivo study. Furthermore, to our knowledge, this is the first report on the in vivo effects of the RAF/MEK inhibitor CH5126766 on neuroblastoma.
We showed that pERK IHC correlated with sensitivity to MAPK pathway inhibition, similar to our findings with pERK ICC in the previous study. We also showed that the numbers of both pERK- and Ki67-positive cells decreased in response to tumor suppression by MAPK pathway inhibition. Our data on the short-term treatment suggested that MAPK pathway inhibition induced anti-tumor effects by blocking MAPK pathway signaling in pERK-positive xenografts, which was able to be detected by pre-treatment pERK IHC. Furthermore, pERK IHC positivity has been reported to correspond well with the MAPK pathway activity analyzed by RNA sequencing [13]. Although ERK phosphorylation reflects the activity of the MAPK pathway caused mainly by RAS or RAF mutation, it has been reported that alternative mechanisms, such as wild-type RAS overexpression and changes in numerous growth factors or

other pathways, activate the MAPK pathway [14]. Indeed, in the present study, neuroblastoma xenografts derived from NLF (a pERK-positive cell line), which had no RAS or RAF mutations [8], were sensitive to MAPK pathway inhibition (data not shown). Our results therefore suggest that RAS and RAF mutation analyses alone are not sufficient to predict the MAPK pathway activity, and pERK IHC is more convenient than a gene mutation analysis. In addition, in adult cancers, pERK IHC has been tested as a biomarker for MEK inhibitors [13, 14]. Therefore, pERK IHC may be a convenient biomarker for MAPK pathway inhibition in neuroblastoma treatment.
Although CH5126766 has shown promising therapeutic effects in other cancers, no marked difference in the efficacy between trametinib and CH5126766 was observed in the two-week short-term treatment of neuroblastoma. Therefore, we administered long-term treatment to investigate whether or not CH5126766 led to a longer progression-free survival. However, all SK-N-AS xenograft tumors in both inhibitor-treated groups showed regrowth at around the same time. Our data therefore suggest that long-term MAPK pathway inhibition results in acquired drug resistance. In addition, the numbers of pERK-positive cells increased again in the regrown tumors. Our result of IHC analyses confirmed that acquired drug resistance was mediated by MAPK pathway reactivation.
Intriguingly, H&E staining of neuroblastoma xenograft tumors at six weeks of MAPK pathway inhibition revealed partial areas of differentiated ganglion-like cells. This finding indicated that MAPK pathway inhibition potentially induces neuroblastoma differentiation. However, SK-N-AS xenograft tumors at eight weeks of MAPK pathway inhibition showed only a few differentiated cells, with rapid regrowth. Neuroblastoma pathology is well known to be an important determinant of the prognosis, and differentiating tumors tends to have a better prognosis than undifferentiated tumors [1, 15]. It has also been reported that the MEK inhibitor cobimetinib alone was able to induce differentiation, and the combination of cobimetinib and cis-retinoic acid (cis-RA) enhanced differentiation in an in vitro study [11]. We investigated whether or not the combination of MAPK pathway inhibition and cis-RA accelerated

differentiation and delayed the time to regrowth. However, the in vivo combination therapy did not enhance neuroblastoma differentiation (data not shown). Further experiments are needed to identify combination drug regimens to achieve a better prognosis.
In conclusion, consistent with our previous in vitro findings, MAPK pathway inhibition was effective in neuroblastoma with MAPK pathway activation detected by pERK IHC. Therefore, pERK IHC may be a practical biomarker for identifying MAPK pathway inhibition-sensitive neuroblastoma patients in a clinical setting. However, neuroblastoma induced drug resistance mediated by MAPK pathway reactivation after long-term treatment. Further studies to overcome the acquisition of drug resistance in neuroblastoma patients are needed.

Conflict of interest
The authors declare that they have no conflicts of interest.
Acknowledgments
This work was supported by a Grant-in-Aid for Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT KAKENHI grant number 17K11517) and by the Practical Research for Innovative Cancer Control from Japan Agency for Medical Research and Development, AMED.
We also thank Brian Quinn (Editor-in-Chief, Japan Medical Communication) for his review of the English used in this manuscript.

Figure Legends
Fig. 1. MAPK pathway inhibitors. Sequential phosphorylation and activation of the MAPK pathway lead to cell proliferation in many malignant tumors. Trametinib, a MEK inhibitor, inhibits the MAPK pathway by inhibiting MEK. CH5126766, a dual RAF/MEK inhibitor, inhibits both RAF and MEK.

Fig. 2. Short-term in vivo analyses of MAPK pathway inhibition in neuroblastoma. (A) Relative tumor growth curves of IMR5, CHP-212, and SK-N-AS xenografts treated with DMSO (as a control), trametinib (TR), and CH5126766 (CH) for two weeks as short-term treatment.
*TR vs. control, p < 0.001, **CH vs. control, p < 0.001. (B) Harvested SK-N-AS xenograft tumors of each treatment group after two-week administration. (C) Representative images of pERK IHC of pre- and post-treatment tumor sections of IMR5, CHP-212, and SK-N-AS xenografts (x40). (D) Quantification of pERK-positive cells/HPF in each group. *p < 0.001, **p
< 0.0001. (E) Representative images of Ki67 IHC of pre- and post-treatment tumor sections of IMR5, CHP-212, and SK-N-AS xenografts (x40). (F) Quantification of Ki67-positive cells/HPF in each group. *p < 0.001, **p < 0.0001

Fig. 3. Long-term in vivo analyses of MAPK pathway inhibition in neuroblastoma. (A) Relative tumor growth curves of SK-N-AS xenografts treated with DMSO, trametinib (TR), and CH5126766 (CH) for eight weeks as long-term treatment. (B) Representative images of pERK IHC of SK-N-AS xenografts after eight-week administration (x40). (C) Quantification of pERK-positive cells/HPF in each group. (D) Representative images of Ki67 IHC of SK-N-AS xenografts after eight-week administration (x40). (E) Quantification of pERK-positive cells/HPF in each group.

Fig. 4. Long-term MAPK pathway inhibition promotes partial differentiation. Top,

representative images of H&E staining of SK-N-AS xenografts treated with CH5126766 at each time point (x40). Bottom, representative images of pERK and Ki67 IHC after six-week administration (x40); black arrows indicate ganglion-like cells.

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