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Published in final edited form as: J Pediatr Surg. 2023 Feb 17:S0022-3468(23)00138-0. doi: 10.1016/j.jpedsurg.2023.02.018

Inhibition of PIM kinases promotes neuroblastoma cell differentiation to a neuronal phenotype

Janet R Julson 1, Colin H Quinn 1, Laura V Bownes 1, Sara C Hutchins 2, Jerry E Stewart 1, Jamie Aye 2, Karina J Yoon 3, Elizabeth A Beierle 1,†
PMCID: PMC10198809  NIHMSID: NIHMS1877256  PMID: 36907773

Abstract

Background:

Neuroblastoma arises from aberrancies in neural stem cell differentiation. PIM kinases contribute to cancer formation, but their precise role in neuroblastoma tumorigenesis is poorly understood. In the current study, we evaluated the effects of PIM kinase inhibition on neuroblastoma differentiation.

Methods:

Versteeg database query assessed the correlation between PIM gene expression and the expression of neuronal stemness markers and relapse free survival. PIM kinases were inhibited with AZD1208. Viability, proliferation, motility were measured in established neuroblastoma cells lines and high-risk neuroblastoma patient-derived xenografts (PDXs). qPCR and flow cytometry detected changes in neuronal stemness marker expression after AZD1208 treatment.

Results:

Database query showed increased levels of PIM1, PIM2, or PIM3 gene expression were associated with higher risk of recurrent or progressive neuroblastoma. Increased levels of PIM1 were associated with lower relapse free survival rates. Higher levels of PIM1 correlated with lower levels of neuronal stemness markers OCT4, NANOG, and SOX2. Treatment with AZD1208 resulted in increased expression of neuronal stemness markers.

Conclusions:

Inhibition of PIM kinases differentiated neuroblastoma cancer cells toward a neuronal phenotype. Differentiation is a key component of preventing neuroblastoma relapse or recurrence and PIM kinase inhibition provides a potential new therapeutic strategy for this disease.

Keywords: neuroblastoma, PIM kinase, stem cell, AZD1208

1. Introduction

Neuroblastoma is the most common extracranial solid tumor in children. Children with high-risk disease, defined as the presence of MYCN amplification or widespread metastasis in children older than 18 months, experience 5 year survival rates as low as 50% [1]. The treatment of high-risk disease involves an intensive, multimodal approach including chemotherapy, surgery, myeloablative therapy for stem cell transplantation, radiation, immunotherapy, and ultimately retinoic acid to serve as a differentiating agent [2]. For those children that do survive, they remain at risk for secondary malignancies and may suffer from long-term, life-altering side effects secondary to these aggressive treatment regimens [3, 4]. Clearly, novel therapeutic approaches are needed.

One potential therapeutic target is the family of Provirus Integrate site for Moloney murine leukemia virus (PIM) kinases. PIM kinases belong to a family of serine/threonine kinases consisting of PIM1, PIM2, and PIM3. They have been implicated as drivers of cancer through numerous oncogenic roles including inhibition of apoptosis, dysregulation of cell cycle progression, increasing cellular motility, and cancer cell stemness maintenance [5]. Through a preliminary kinome assay, we found that PIM1 kinase was activated in neuroblastoma cells.

In pediatric malignancies, the role of PIM kinases in cancer cell stemness is poorly understood. The majority of our understanding comes from research conducted by Stafman et al. in hepatoblastoma which showed mRNA abundance of cancer stemness markers decreased in PIM3 KO cells [6] and that small molecule inhibition of PIM kinases could inhibit the cancer stem cell phenotype [7, 8]. Currently, limited data exists regarding the role of PIM kinases in neuroblastoma. It has been shown that children with less differentiated neuroblastoma tumors have worse outcomes [9]. Because neuroblastoma arises from an error in the differentiation of neural crest tissue [10, 11], investigating the mechanisms behind cellular differentiation and stemness in neuroblastoma is crucial to preventing disease relapse. In the current study, we investigate the utility of small molecule inhibition of PIM kinases to promote neural stem cell differentiation in neuroblastoma.

2. Materials & Methods

2.1. Patient database

R2 (http://r2.amc.nl, accessed on April 3, 2022), a publicly available genomic analysis platform, was utilized to interrogate microarray and RNA-seq data from the Versteeg neuroblastoma database [12] a to investigate PIM1, PIM2, and PIM3 expression and the potential correlation between PIM1, PIM2, PIM3, NANOG, and SOX2 in human samples.

2.2. Cells and cell culture

The MYCN non-amplified neuroblastoma cell line, SK-N-AS (female), and the MYCN amplified cell line, SK-N-BE(2) (male), were utilized for this study. The cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained with standard cell culture conditions at 5 % carbon dioxide (CO2) and 37 °C. SK-N-AS cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Corning Inc., Corning, NY, USA) with 10 % fetal bovine serum (FBS, Hyclone, Suwanee, GA, USA), 1 μg/mL penicillin/streptomycin (Sigma Aldrich, Burlington, MA, USA), 4 mM L-glutamine (Thermo Fisher Scientific Inc., Waltham, MA, USA), and 1 μM non-essential amino acids (Life Technologies, Carlsbad, CA, USA). SK-N-BE(2) cells were maintained in a 1:1 mixture of minimum Eagle medium and Ham F-12 medium (Corning) with 10% FBS (Hyclone), 1 μg/mL penicillin/streptomycin (Sigma Aldrich), 2 mM L-glutamine (Thermo Fisher Scientific Inc.), and 1 μM non-essential amino acids (Life Technologies). All cell lines were verified within the last 12 months using short tandem repeat analysis (Genomics Core, University of Alabama at Birmingham (UAB), Birmingham, AL, USA) and were deemed free of Mycoplasma infection by the Universal Mycoplasma Detection Kit (30–1012K, ATCC).

Patient-derived xenografts (PDXs), COA6 and COA129, have been previously described [13, 14]. These PDXs were generated at our institution under UAB Institutional Review Board (IRB) and Institutional Animal Care and Use Committee (IACUC) approved protocols (IRB-130627006, IACUC-009186, respectively). After obtaining written informed consent of each patient’s guardian and assent from each patient as appropriate, a fresh tumor specimen was obtained and temporarily placed in serum-free Roswell Park Memorial Institute 1640 (RPMI, 30–2001, ATCC) medium. To implant the tumor, 3 % inhalational isoflurane was utilized to anesthetize mice and 16 mm3 pieces dissociated tumor were implanted into the subcutaneous space of the flank. Animals resided in a pathogen free environment and were routinely monitored for overall health and tumor growth measured weekly. Tumor volumes were calculated using the formula (width2 × length)/2. The PDXs were propagated in this fashion. For experimentation, tumors were harvested when the volumes met IACUC parameters and were dissociated into single cell suspensions using a Tumor Dissociation Kit (Miltenyi Biotec, San Diego, CA, USA) per manufacturer’s protocol. The dissociated tumor cells were cultured in neurobasal (NB) medium (Life Technologies) with the addition of B-27 without Vitamin A (Life Technologies), N2 (Life Technologies), L-glutamine (2 mM, Thermo Fisher Scientific Inc.), gentamicin (50 μg/mL, Thermo Fisher Scientific Inc.), amphotericin B (250 μg/mL, Thermo Fisher Scientific Inc.), epidermal growth factor (10 ng/mL, Miltenyi Biotec), and fibroblast growth factor (10 ng/mL, Miltenyi Biotec) and maintained at 37 °C with 5 % CO2 overnight prior to utilizing for in vitro experiments. Routine real-time qPCR was performed to assess the percentage of human and mouse DNA contained in the PDXs to ensure that the tumors did not harbor murine contamination (TRENDD RNA/DNA Isolation and TaqMan QPCR/Genotyping Core Facility, UAB). All PDXs were verified within the last 12 months using short tandem repeat analysis (Genomics Core, UAB).

2.3. Antibodies and reagents

Primary antibodies utilized included rabbit monoclonal anti-PIM1 (3247S) from Cell Signaling Technology (Beverly, MA, USA) and mouse monoclonal anti-β-actin (A1978) from Sigma Aldrich (St. Louis, MO, USA). The pan-PIM inhibitor AZD1208 was purchased from Selleckchem (Houston, TX, USA).

2.4. Cell viability and proliferation

The alamarBlue Cell Viability Assay (Thermo Fisher Scientific) was used to measure cell viability. SK-N-AS or SK-N-BE(2) cells (1.5 × 103 per well) were plated in 96-well plates under adherent conditions, allowed to attach overnight, and treated with AZD1208 at increasing concentrations (0 to 50 μM). Similarly, COA6 (3.0 × 104 per well) or COA129 cells (5 × 104 per well) were plated in 96-well plates under non-adherent conditions and treated at increasing concentrations of AZD1208 (0 to 50 μM). Following 72 hours of treatment, 10 μL of alamarBlue reagent was added to each well and the absorbance was measured at excitation wavelength of 562 nm and emission wavelength of 595 nm using a microplate reader (BioTek Gen5, BioTek, Winooski, VT, USA).

The CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA) was used to measure proliferation. SK-N-AS or SK-N-BE(2) cells (5 × 103 per well) were plated in 96-well plates under adherent conditions, allowed to attach overnight, and treated with AZD1208 at increasing concentrations (0 to 50 μM). Similarly, COA6 cells (1 × 104 per well) or COA129 cells (3 × 104 per well) were plated in 96-well plates under non-adherent conditions and treated at increasing concentrations of AZD1208 (0 to 50 μM). After 72 hours of treatment, 10 μL of CellTiter 96 reagent was added to each well and the absorbance was measured at 490 nm using a microplate reader (BioTek Gen5).

2.5. Immunoblotting

Cells were lysed using radio-immunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors (Sigma Aldrich), phosphatase inhibitors (Sigma Aldrich), and phenyl-methane-sulfonyl-fluoride (Sigma Aldrich). Immunoblotting, gel transfer, and immunodetection were performed as previously described [15]. The Precision Plus Protein Kaleidoscope molecular weight marker (Bio-Rad) confirmed the expected size of target proteins. Antibodies were utilized according to the manufacturers’ recommendations. β-actin expression was used as an internal control to confirm equal protein loading.

2.6. Cell growth

Cell growth was assessed utilizing growth curve. SK-N-AS or SK-N-BE(2) cells (5 × 104 per well) were plated in 12-well plates, allowed to attach for 4 hours, and then treated with increasing concentrations of AZD1208 (0 to 30 μM). At 24, 48, and 72 hour time points, cells were lifted, stained with 18 μL 0.4 % trypan blue, and counted.

2.7. Motility

Motility was assessed using monolayer wound healing (scratch) assay in the adherent SK-N-AS and SK-N-BE(2) cell lines. Cells (5 × 105) were plated in 12-well plates and treated with increasing doses of AZD1208 (0, 15, and 30 μM). Once cells reached confluence, a sterile 200 μL pipette tip was used to make a standard wound in the cell layer. Photographs of the plates were obtained at 0, 12, 24, and 36 hours. ImageJ MRI Wound Healing Tool (http://imagej.nih.gov/ij/, accessed August 1, 2021) [16] was employed to quantify the open wound area. Data were reported as fold change in wound healing area.

2.8. PCR

As previously described [17], quantitative real-time PCR (qPCR), assessed the mRNA abundance of neuronal stemness markers homeobox protein (NANOG), sex determining region Y-box2 (SOX2), and octamer-binding transcription factor 4 (OCT4). Briefly, cDNA was synthesized using an iScript cDNA Synthesis kit (Bio-Rad) according to supplier’s instructions. SsoAdvanced SYBR Green Supermix (Bio-Rad) was utilized according to manufacturer’s protocol. Probes specific for OCT4, NANOG, SOX2 and β-ACTIN were obtained from Applied Biosystems (Foster City, CA, USA) [6]. Probes specific for PIM1 (Forward: 5’-TGTAAAACGACGGCCAGT-3’, Reverse: 5’-CAGGAAACAGCTATGACC-3’) were designed using Primer3 web version 4.1.0 and checked for non-specific binding using the basic local alignment search tool (BLAST, NCBI, https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed June 7, 2022). qPCR was performed with 10 ng cDNA in 20 μL reaction volume. An Applied Biosystems 7900HT cycler (Applied Biosystems) performed the amplification with cycling conditions of 95 °C for 2 min, followed by 39-cycle amplification at 95 °C for 5 s and 60 °C for 30 s. Samples were analyzed in triplicate with β-actin utilized as an internal control. The ΔΔCt method [18] calculated the gene expression, which was reported as mean fold change ± standard error of the mean (SEM).

2.9. CD133 and CXCR4 expression

Cell surface expression of CD133 and C-X-C chemokine receptor type 4 (CXCR4) were evaluated using flow cytometry. After treatment with increasing doses of AZD1208, SK-N-AS, SK-N-BE(2), COA6, or COA129 cells (1 × 106) were labeled with either allophycocyanin (APC)-conjugated mouse immunoglobulin G1 (IgG1) anti-human CD133/1 (clone AC133, Miltenyi Biotec) or CXCR4 phycoerythrin (PE)-conjugated mouse immunoglobulin G2aκ (IgG2aκ) anti-human CXCR4 antibody (Miltenyi Biotec), according to the manufacturer’s instructions. Unlabeled cells served as negative controls. The percent of cells positive for APC (CD133/1) or PE (CXCR4) was determined via flow cytometry using the Attune NxT Flow Cytometer (Invitrogen, Thermo Fisher). All flow cytometry analyses were performed using FlowJo software (FlowJo, LLC, Ashland, OR, USA).

2.10. Statistical analysis

We generated data with a minimum of three biologic replicates unless otherwise stated and reported the results as mean ± SEM of separate experiments [19]. Student’s t-test or analysis of variance (ANOVA) was used to compare means between groups and the chi-square test was used for categorical variables as appropriate, with p<0.05 considered statistically significant.

3. Results

3.1. PIM1 expression is associated with recurrent or progressive disease and disease relapse.

We utilized the publicly available Versteeg database to evaluate whether PIM gene expression was associated with recurrent or progressive disease. Of the 88 patients, 87 had levels of individual PIM gene expression determined. Those with recurrent or progressive disease (n=36) had significantly higher PIM1 gene expression compared to those without recurrent or progressive disease (n=51, p≤0.01, Fig. 1A). Similarly, patients with recurrent or progressive disease had significantly higher PIM2 (n=51, p≤0.001) (Fig. 1B) or PIM3 (n=51, p≤0.001) (Fig. 1C) gene expression compared to those without recurrent or progressive disease. Patients with high PIM1 expression (n=71) were significantly less likely to have relapse free survival than those with low PIM1 expression (n=17, p≤0.01, Fig. 1D). There was no association between level of PIM2 (n=88, p=0.074) or PIM3 (n=88, p=0.203) gene expression and relapse free survival (Supplemental Fig. S1A, B, respectively). Since PIM1 gene expression was significantly associated with a lower probability of relapse free survival, we evaluated PIM1 protein expression. PIM1 protein has been confirmed by others [20] in SK-N-AS and SK-N-BE(2) cell lines. We detected PIM1 protein in these two cell lines and in in our unique PDX models, COA6 and COA129, using immunoblotting (Fig. 1E).

Fig. 1. PIM1 expression is associated with recurrent, progressive, or relapsed disease.

Fig. 1.

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(A) Versteeg database query (primary neuroblastoma patients, n=87) demonstrated higher PIM1 gene expression in individuals with recurrent or progressive disease (Yes, n=36) compared to patients without (No, n=51). (B) There was significantly greater PIM2 expression in individuals with recurrent or progressive disease compared to patients without. (C) Similarly, PIM3 gene expression was also significantly greater in patients with recurrent or progressive disease. (A, B, C) The bars on either end of the box plots represent the maximum and minimum 2log expression of each PIM gene with the median denoted by the solid line in the box. (D) Versteeg database query (primary neuroblastoma patients, n=88) demonstrated patients with high PIM1 expression (n=71) had significantly worse relapse free survival compared to those with low PIM1 expression (n=17). (E) Immunoblotting of whole cell lysates of SK-N-AS and SK-N-BE(2) cell lines and COA6 and COA129 human neuroblastoma PDXs for expression of PIM1 protein. PIM1 kinase protein was detected in all four cell types. β-actin served as a loading control.

3.2. PIM kinases affect neuroblastoma viability, proliferation, and growth

Due to the prevalence of PIM1 in relapsed disease, we sought to evaluate the effect of PIM1 inhibition on viability, proliferation, and growth in established neuroblastoma cell lines as well as in human neuroblastoma PDX cells. Treatment with the pan-PIM inhibitor, AZD1208, significantly decreased viability in SK-N-AS (lethal dose 50 % (LD50) = 41.5 μM) (Fig. 2A) and SK-N-BE(2) (LD50 = 37.7 μM) (Fig. 2B) at 72 hours. Similar results were seen in the PDX cells. COA6 cells demonstrated significantly decreased viability (LD50 = 28.5 μM) after treatment with AZD1208 (Fig. 2C). Viability of COA129 cells was also decreased with AZD1208 treatment (LD50 = 11.3 μM) (Fig. 2D).

Fig. 2. AZD1208 decreases neuroblastoma viability.

Fig. 2.

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AlamarBlue Cell Viability Assay assessed viability after treatment with pan-PIM inhibitor, AZD1208, for 72 hours. (A) SK-N-AS cells (1.5 × 103 per well) were plated in 96-well plates and treated with AZD1208 at increasing concentrations (0 to 50 μM). AZD1208 significantly decreased viability with a calculated lethal dose 50% (LD50) of 41.5 μM. (B) SK-N-BE(2) cells (1.5 × 103 per well) were plated in 96-well plates and treated with AZD1208 at increasing concentrations (0 to 50 μM). AZD1208 significantly decreased viability with a calculated LD50 of 37.7 μM. (C) COA6 cells (3.0 × 104 per well) were plated in 96-well plates and treated with increasing concentrations of AZD1208 (0 to 50 μM). Treatment with AZD1208 significantly decreased viability with a calculated LD50 of 28.5 μM. (D) COA129 cells (5 × 104 per well) were plated in 96-well plates and treated with increasing concentrations of AZD1208 (0 to 40 μM). AZD1208 significantly decreased viability with a calculated LD50 of 11.3 μM. COA129 viability assay was completed in biologic duplicate, all other experiments were completed with at least three biologic replicates. Data reported as mean ± SEM and evaluated with two-tailed t-test. *p≤0.05, **p≤0.01, *** p≤0.001.

Since PIM kinases have been shown to affect proliferation in cancer [5], we examined the effects of PIM inhibition on proliferation. SK-N-AS cells had significantly decreased proliferation after AZD1208 treatment (half-inhibitory concentration (IC50) = 43.1 μM) (Fig. S2A) with similar results seen in SK-N-BE(2) cells (IC50 = 47.1 μM) (Fig. S2B). AZD1208 treatment significantly decreased proliferation in COA6 (IC50 = 56.5 μM) and COA129 PDX cells (IC50 = 31.7 μM) (Fig. S2C, D, respectively).

Next, we evaluated cell growth in the established neuroblastoma cell lines as PDX cells do not propagate well in culture. After pharmacologic inhibition of PIM kinases with AZD1208 at doses lower than both the LD50 and IC50, there was a statistically significant decrease in growth at 72 hours in both cell lines. SK-N-AS cells exhibited decreased growth after 30 μM of AZD1208 treatment (p≤0.01) at 72 hours (Fig. 3A), and SK-N-BE(2) cells had decreased growth with 15 μM (p≤0.01) and 30 μM (p≤0.01) AZD1208 treatment at 72 hours (Fig. 3B). We also examined the cellular morphology of SK-N-AS and SK-N-BE(2) after AZD1208 treatment. AZD1208 treated cells had elongated cell bodies and neurite outgrowths (Fig. 3C, lower panels, closed arrows) compared to rounded morphology without neurite outgrowths seen in untreated cells (Fig. 3C, upper panels, open arrows), demonstrating AZD1208 induced properties of differentiation rather than necroptosis, which is characterized by swollen organelles and lysis of the cell membrane.

Fig. 3. AZD1208 decreased neuroblastoma growth but did not affect motility.

Fig. 3.

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Cell growth was assessed using growth curves. (A) SK-N-AS cells (5 × 104 per well) were plated in 12-well plates, allowed to attach for 4 hours, and then treated with increasing concentrations of AZD1208 (0 to 30 μM). After 24, 48, and 72 hours cells were lifted, stained with trypan blue, and counted. AZD1208 (30 μM) significantly decreased cell growth. (B) SK-N-BE(2) cells (5 × 104 per well) were plated in 12-well plates, allowed to attach for 4 hours, and then treated with increasing concentrations of AZD1208 (0 to 30 μM). After 24, 48, and 72 hours cells were lifted, stained with trypan blue, and counted. AZD1208 (15 and 30 μM) significantly decreased cell growth. (C) Representative photomicrographs (10x) of cell morphology of SK-N-AS and SK-N-BE(2) (control, no AZD1208) and AZD1208 treated (30 μL) cells. Cells treated with AZD1208 (30 μM) developed elongated cell bodies and neurite outgrowths (lower panel, closed arrows) compared to rounded morphology (upper panel, open arrows) seen in untreated cells. (D, E) Motility was evaluated with monolayer wounding (scratch) assays. (D) SK-N-AS cells (5 × 105) were plated in 12-well plates and treated with increasing doses of AZD1208 (0, 15, and 30 μM). Once cells reached confluence, a sterile 200 μL pipette tip was used to make a standard scratch in the cell layer. Photographs of the plates were obtained at 0, 12, 24, and 36 hours. ImageJ MRI Wound Healing Tool (http://imagej.nih.gov/ij/Accessed August 1, 2021) quantified the open wound area. AZD1208 had no effect on motility. (E) SK-N-BE(2) cells (5 × 105) were plated in 12-well plates, treated with increasing doses of AZD1208 (0, 15, and 30 μM), and a standard scratch was made when cells reached confluence. Photographs of the plates were obtained at 0, 12, 24, and 36 hours and the open area quantified using ImageJ software. AZD1208 treatment had no effect on motility. Experiments were completed with at least three biologic replicates. Data reported as mean ± SEM and evaluated with two-tailed t-test. Scale bars represent 300 μm. *p≤0.05, **p≤0.01.

To further document the effects of AZD1208 on neuroblastoma phenotype, we evaluated cell motility in the established cell lines. AZD1208 did not affect motility of either SK-N-AS or SK-N-BE(2) cells (Fig. 3D, E).

3.3. Role of PIM1 in neuronal stemness

Since AZD1208 had noticeable effects on cell growth and morphology, but no effect on motility, we hypothesized these findings may be due to PIM kinase inhibition-induced differentiation. To further investigate this hypothesis, we began exploring the effects of PIM1 inhibition on neuronal stemness since neuroblastoma arises through a defect in neural crest cell differentiation [10]. First, using the Versteeg database [12], we examined the relation between PIM genes and known neuronal stemness markers NANOG and SOX2. The database did not contain information for OCT4, another neuronal stemness marker. There was a statistically significant negative correlation between PIM1 and NANOG (r2 = (−)0.287, p=0.007, Fig. 4A), and SOX2 (r2 = (−)0.260, p=0.014, Fig. 4B) gene expression, indicating that as PIM1 increases, expression of neuronal stemness genes decreases. There was no correlation between PIM2 or PIM3 gene expression and the expression of the stemness markers, NANOG or SOX2 (Fig. S1C–F).

Fig. 4. Correlation between PIM1 and neuron stemness markers.

Fig. 4.

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(A) Versteeg database query (primary neuroblastoma patients, n=88) demonstrated an inverse correlation between PIM1 gene expression and expression of the neuronal stemness marker NANOG (r2 = (−)0.287, p=0.007). (B) Versteeg database query (primary neuroblastoma patients, n=88) demonstrated an inverse correlation between PIM1 gene expression and expression of the neuronal stemness marker SOX2 (r2 = (−0.260), p=0.014).

To corroborate the database findings, we investigated neuronal stemness in the neuroblastoma cell lines following AZD1208 treatment. Initially, we evaluated the mRNA abundance of known neuronal stemness markers, OCT4, SOX2, and NANOG [21–24], utilizing qPCR. AZD1208 treatment increased the abundance of mRNA of SOX2 and NANOG stemness markers in SK-N-BE(2) (Fig. 5A). Abundance of mRNA of all three markers were significantly increased in COA6 (Fig. 5B) and COA129 cells (Fig. 5C), but decreased in SK-N-AS cells (Fig. 5A). Next, we examined the cell surface expression of CD133 [25, 26] and CXCR4 [21, 27], both of which are associated with stemness in neural crest cells. After AZD1208 treatment, cell surface expression of CD133 was increased significantly in all four cell lines (Fig. 5D). Expression of CXCR4 was also increased, but only reached statistical significance in SK-N-AS and SK-N-BE(2) cell lines.

Fig. 5. PIM kinase inhibition of increased neuronal stemness.

Fig. 5.

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(A) SK-N-AS MYCN non-amplified and SK-N-BE(2) MYCN amplified neuroblastoma cells were treated with AZD1208 (30 μM) and qRT-PCR detected abundance of neuronal stemness markers OCT4, SOX2, and NANOG. In SK-N-AS the mRNA abundance of these neuronal stemness markers was decreased, but SOX2 and NANOG were significantly increased in SK-N-BE(2) following AZD1208 treatment. (B) COA6 MYCN amplified neuroblastoma PDX cells were treated with AZD1208 (15 μM) and qRT-PCR detected abundance of neuronal stemness markers OCT4, SOX2, and NANOG. The abundance of mRNA of these neuronal stemness markers was increased with AZD1208 treatment. (C) COA129 MYCN amplified neuroblastoma PDX cells were treated with AZD1208 (2.5 μM) and qRT-PCR detected abundance of neuronal stemness markers OCT4, SOX2, and NANOG. The abundance of mRNA of these neuronal stemness markers was significantly increased with AZD1208 treatment. (D) SK-N-AS, SK-N-BE(2), COA6 and COA129 cells were treated with AZD1208 at doses below their LD50 and flow cytometry was used to detect the cell surface expression of the neural crest stemness markers CD133 and CXCR4. The cell surface expression of CD133 was significantly increased in all four cell lines. CXCR4 was increased in all four cell lines, but reached statistical significance only in SK-N-AS and SK-N-BE(2) cells. Experiments were completed with at least three biologic replicates. Data reported as mean ± SEM and evaluated with two-tailed t-test. *p≤0.05, **p≤0.01.

Collectively, these data suggest PIM inhibition with AZD1208 drives neuroblastoma tumor cells toward cells with a more neuronal phenotype expressing markers of neuronal stemness rather than cancer stemness (Fig. 6).

Fig. 6. Cartoon.

Fig. 6.

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Neuroblastoma arises from an error in neural crest stem cell differentiation. PIM1 kinase inhibition directs the neuroblastoma cancer cell to have characteristics of a neuronal stem cell. This consequence of PIM inhibition may be exploited in the clinical arena to promote differentiation of neuroblastoma tumor cells into more benign-behaving neuronal stem cell-like cells. Figure created with BioRender.com.

4. Discussion

Brunen et al. were the first to publish data on the role of PIM kinases in neuroblastoma. They demonstrated significantly lower overall survival in neuroblastoma patients who had increased individual PIM1, 2, or 3, or total PIM expression [20]. Additionally, there was significantly higher PIM1 or PIM2 expression in stage IV tumors. They also found that knockdown of PIM1, specifically, was sufficient to reduce cell viability [20]. Similarly, Trigg et al. found PIM1 to be a relevant target in neuroblastoma and described its role in promoting resistance to ALK inhibitors [28]. In the current study, analysis of the Versteeg database demonstrated no correlation between PIM2 and NANOG (p=0.845) (Supplemental Fig. S1C) or PIM3 and NANOG (p=0.979) (Supplemental Fig. S1D). Similarly, there was no correlation between PIM2 and SOX2 (p=0.714) (Supplemental Fig. S1E) or PIM3 and SOX2 (p=0.134) (Supplemental Fig. S1F). These data led us to concentrate on PIM1 kinase in the current studies. Further motivation for exploring PIM1 inhibition was the ability to exploit this target clinically, so we chose to employ pharmacologic PIM1 inhibition over genetic manipulations such as siRNA knockdown or CRISPR-Cas9 knockout. There are currently no pharmacologic agents that specifically target PIM1, so we utilized the pan-PIM inhibitor, AZD1208, as it has been previously tested in humans and was well tolerated in several Phase I clinical trials [29].

Some researchers have previously described neuroblastoma cell lines as being resistant to AZD1208 due to the inability to target downstream pathways of cell signaling [20]. Similar to the findings of Brunen et al. [20], we did not find a correlation between level of PIM1 protein expression and sensitivity to PIM kinase inhibition. Although our data show that targeting PIM kinase activity is cytotoxic, fairly high concentrations were required to see these effects in the neuroblastoma cell lines. Further, there was no change in cell growth or proliferation until 72 hours after treatment, all of which led us to postulate that the most important function for PIM1 kinase in neuroblastoma may be related to other entities besides growth, such as stemness and differentiation.

Human neuroblastoma cancer cells are highly mobile, and motility plays an important role in invasion and metastases [30]. Zaizen et al. demonstrated a high degree of motility in tumors with MYCN amplification [30]. Others have demonstrated that MYCN plays a role in the mesenchymal to epithelial transition (MET), lending support to the higher degree of motility in these cancer cells [10]. PIM kinases have been shown to play a role in cellular motility in other malignancies [31–33], prompting us to investigate the effect of PIM inhibition on neuroblastoma motility. However, in the current study, pharmacologic inhibition with AZD1208 did not alter motility, again supporting alternate roles for PIM kinases in neuroblastoma oncogenesis.

While conducting growth and motility assays, we noticed obvious changes in neuroblastoma cell morphology following AZD1208 treatment. Treated cells demonstrated a more neuron-like morphology with elongated cell bodies and neurite outgrowths. Such findings in neuroblastoma are suggestive of differentiation and a phenotypic change from one of an aggressive cancer to a benign neural entity [34]; explaining the observed decrease in cell proliferation and growth. There are no data in the current literature regarding the role of PIM kinases in neuroblastoma differentiation, a critical determining factor of patient outcomes [9] and a primary focus of treatment for high-risk tumors. The cells did not exhibit features of necroptosis include swelling of organelles and lysis of the cell membrane which would serves as an etiology for decreased growth.

The signaling pathways whereby pluripotent embryonic stem cells differentiate into distinct cell lineages are complex. It has been previously demonstrated that differentiating embryonic stem cells have upregulation of Oct4, Sox2, and Nanog proteins [35]. Additionally, knockdown of SOX2 [36] or CXCR4 [21] been shown to inhibit neural differentiation, and that a delicate balance exists between SOX2, OCT4, and NANOG expression in determining neuroectodermal cell fate [35]. Both the genetic and phenotypic changes demonstrated in the current study suggest a change towards a more differentiated neuronal cell type following PIM kinase inhibition.

Histologically, neuroblastoma is categorized based on differentiation by the Shimada classification including: neuroblastoma (Schwannian stroma poor (least differentiated)), nodular ganglioneuroblastoma, intermixed ganglioneuroblastoma, and ganglioneuroma (Schwannian stroma dominant (most differentiated)) [37]. There are also four clinical stages as defined by the Children’s Oncology Group (COG) [1]. Importantly, MS tumors, found in children less than 18 months of age with favorable histology and non-amplified MYCN with metastasis limited to liver, skin, and bone marrow (< 10% marrow involvement), are unique in that they have been shown to spontaneously regress. The mechanisms by which regression occurs are not well understood, but MS tumors have an upregulation of genes and proteins involved in differentiation [38–40]. Retinoic acid, used to promote cellular differentiation, is a main component of the current neuroblastoma treatment strategy for high-risk disease [41]. However, retinoic acid has been associated with significant hyperlipidemia and mucositis which limit its tolerability in some children [42–43]. Therefore, the ability to promote differentiation through an alternate or an additional mechanism such as PIM kinases inhibition, may provide increased retinoic acid efficacy with lower dosing concentrations resulting in decreased toxicities. This concept will certainly be an area of future investigation.

Cisplatin is a primary agent utilized to treat children with high-risk neuroblastoma. Similar to retinoic acid, cisplatin has a number of adverse effects including nephrotoxicity, ototoxicity, and gonadotoxicity which limit its use children [44–46]. The development of cancer cell resistance to cisplatin may also limit its utility in these aggressive tumors. PIM kinases are upregulated in cisplatin resistant tumor cells [47], and have been shown to potentiate cisplatin resistance in pediatric malignancies such as hepatoblastoma [8, 47, 48]. Therefore, the concept of PIM inhibition as an adjunct to potentiate standard neuroblastoma chemotherapeutic agents will be the subject of future investigations.

This study does not go without limitations. The use of PDXs as a pre-clinical model help to better recapitulate the human cancer condition as they more closely display the heterogeneity present within the original tumor. However, PDXs require extended time periods for engraftment and tumor establishment, in the case of COA6 or COA129 cells up to 6 months which limits their feasibility for use as in vivo models. A further limitation was the use of a single pharmacologic agent. There are other PIM kinase inhibitors in development which may have improved PIM1 specificity, and it will be critical to evaluate them when they become available.

Conclusion

While PIM kinases have been studied in a number of malignancies, their role in neuroblastoma has yet to be fully delineated. In the current study, we examined how pharmacologic inhibition of PIM kinases may play a role in differentiation of neuroblastoma cancer cells to neuronal stem cells. Promoting cellular differentiation is an essential part of the current algorithm of treating high-risk neuroblastoma. These results warrant further exploration of the role of PIM kinase inhibition as a therapeutic adjunct for high-risk neuroblastoma to aid in preventing disease recurrence or relapse.

Supplementary Material

1

Highlights:

  • PIM kinases are oncogenes implicated in numerous malignancies, though knowledge about their role in neuroblastoma is lacking.

  • Differentiation is a key component of neuroblastoma therapy to prevent relapse or recurrence and PIM kinases serve as a novel potential therapeutic target to promote differentiation.

Acknowledgements

The authors would like to thank the laboratory of Dr. Namasivayam Ambalavanan for their assistance with qPCR. We would also like to thank Sagar Hanumanthu and UAB Comprehensive Flow Cytometry Core for their assistance with flow cytometry.

Funding

This project was made possible by funding from the National Cancer Institute of the National Institutes of Health under award numbers T32 CA229102 (JRJ and LVB) and 5T32GM008361 (CHQ). and P30AR048311. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Other funding sources include Hyundai Hope on Wheels, Rally Foundation for Childhood Cancer Research, Sid Strong Foundation, Elaine Roberts Foundation, Open Hands Overflowing Hearts, Kaul Pediatric Research Foundation (EAB).

Footnotes

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