Human Neuroblastoma-derived Cell Lines Research Articles

A sensitive cell-based assay for testing potency of botulinum neurotoxin type A

Botulinum neurotoxin type A (BoNT/A) is a biopharmaceutic widely used for the treatment of neu­rological diseases and in aesthetic medicine to achieve months-long paralysis of target muscles and glands. Large numbers of mice are used in the mouse bioassay (MBA) for various botulinum-related applications including batch release potency testing, antitoxin testing, countermeasure development, and basic research. BoNT/A intoxication causes severe suffering to the mice used for testing, and application-specific, non-animal alternatives are urgently needed. Cell-based assays (CBA) need to replicate all the physiological steps of botulinum intoxication, comprising neuronal binding, internali­zation, endosomal escape, and cleavage of synaptosomal-associated protein of 25 kDa (SNAP25). However, the CBA currently in use have limitations. In this study we show that LAN5 cells, a human neuroblastoma-derived cell line, are sensitive to BoNT/A and can be engineered to express a recom­binant NanoLuciferase (NanoLuc)-tagged SNAP25 reporter molecule. On exposure, the reporter molecule is cleaved and releases a NanoLuc-SNAP25 fragment that can be captured on a 96-well plate for quantitative luminometry using a cleavage-specific SNAP25 antibody. We demonstrate, using purified BoNT/A and a commercial BoNT/A product, that the sensitivity of this new cell-based assay is in the fM range, comparable to that of the MBA. The new assay could replace the MBA in research and commercial testing of BoNT/A, benefiting both scientific progress and animal welfare.

Caffeic acid recarbonization: A green chemistry, sustainable carbon nano material platform to intervene in neurodegeneration induced by emerging contaminants

Environmental agents such as pesticides, weedicides and herbicides (collectively referred to as pesticides) are associated with the onset and pathogenesis of neurodegenerative disorders such as Parkinson's (PD) and Alzheimer's (AD) diseases. The development of blood-brain barrier (BBB)-penetrating therapeutic candidates to both prevent and treat the aforementioned xenotoxicant-induced neurodegenerative disorders remains an unmet need. Here, we examine whether caffeic-acid based Carbon Quantum Dots (CACQDs) can intervene in pesticide-associated onset and progress of the PD phenotype. Pulse-chase fluorescence analyses revealed that CACQDs intervene in the soluble-to-toxic transformation of the amyloid-forming protein model Hen Egg White Lysozyme (HEWL). The sp2-rich CACQDs also scavenged free radicals, a milestone along the PD trajectory. In-vitro, CACQDs introduced into a human neuroblastoma-derived cell line (SH-SY5Y) demonstrated negligible cytotoxicity up to 5 mg/mL and protected the cell line against oxidative stress-induced neuronal injury induced by the pesticide and potent neurotoxin, paraquat. Our findings suggest that the potentially BBB-penetrating CACQDs derived from caffeic acid hold promise for mitigating neurodegenerative disorders associated with environmental pesticides and xenobiotic neurotoxicants. Importantly, CACQDs sourced from coffee, coupled with their facile synthesis, represent a sustainable, green chemistry platform for generating interventional candidates in neurodegeneration.

Sulfatide-Rich Liposome Uptake by a Human-Derived Neuroblastoma Cell Line

Liposomes are bilayer membrane vesicles that can serve as vehicles for drug delivery. They are a good alternative to free drug administration that provides cell-targeted delivery into tumors, limiting the systemic toxicity of chemotherapeutic agents. Previous results from our group showed that an astrocytoma cell line exhibits selective uptake of sulfatide-rich (SCB) liposomes, mediated by the low-density lipoprotein receptor (LDL-R). The goal of this study was to assess the uptake of liposomes in a neuroblastoma cell line. For this purpose, we used two types of liposomes, one representing a regular cell membrane (DOPC) and another rich in myelin components (SCB). An astrocytoma cell line was used as a control. Characterization of liposome uptake and distribution was conducted by flow cytometry and fluorescence microscopy. Similar levels of LDL-R expression were found in both cell lines. The uptake of SCB liposomes was higher than that of DOPC liposomes. No alterations in cell viability were found. SCB liposomes were located near the cell membrane and did not colocalize within the acidic cellular compartments. Two endocytic pathway inhibitors did not affect the liposome uptake. Neuroblastoma cells exhibited a similar uptake of SCB liposomes as astrocytoma cells; however, the pathway involved appeared to be different than the hypothesized pathway of LDL-R clathrin-mediated endocytosis.

YAP-Mediated Repression of HRK Regulates Tumor Growth, Therapy Response, and Survival Under Tumor Environmental Stress in Neuroblastoma.

Following chemotherapy and relapse, high-risk neuroblastoma tumors harbor more genomic alterations than at diagnosis, including increased transcriptional activity of the Yes-associated protein (YAP), a key downstream component of the Hippo signaling network. Although YAP has been implicated in many cancer types, its functional role in the aggressive pediatric cancer neuroblastoma is not well-characterized. In this study, we performed genetic manipulation of YAP in human-derived neuroblastoma cell lines to investigate YAP function in key aspects of the malignant phenotype, including mesenchymal properties, tumor growth, chemotherapy response, and MEK inhibitor response. Standard cytotoxic therapy induced YAP expression and transcriptional activity in patient-derived xenografts treated in vivo, which may contribute to neuroblastoma recurrence. Moreover, YAP promoted a mesenchymal phenotype in high-risk neuroblastoma that modulated tumor growth and therapy resistance in vivo. Finally, the BH3-only protein, Harakiri (HRK), was identified as a novel target inhibited by YAP, which, when suppressed, prevented apoptosis in response to nutrient deprivation in vitro and promoted tumor aggression, chemotherapy resistance, and MEK inhibitor resistance in vivo. Collectively, these findings suggest that YAP inhibition may improve chemotherapy response in patients with neuroblastoma via its regulation of HRK, thus providing a critical strategic complement to MEK inhibitor therapy. SIGNIFICANCE: This study identifies HRK as a novel tumor suppressor in neuroblastoma and suggests dual MEK and YAP inhibition as a potential therapeutic strategy in RAS-hyperactivated neuroblastomas.

Somatic structural variation targets neurodevelopmental genes and identifies SHANK2 as a tumor suppressor in neuroblastoma.

Neuroblastoma is a malignancy of the developing sympathetic nervous system that accounts for 12% of childhood cancer deaths. Like many childhood cancers, neuroblastoma shows a relative paucity of somatic single-nucleotide variants (SNVs) and small insertions and deletions (indels) compared to adult cancers. Here, we assessed the contribution of somatic structural variation (SV) in neuroblastoma using a combination of whole-genome sequencing (WGS) of tumor-normal pairs (n = 135) and single-nucleotide polymorphism (SNP) genotyping of primary tumors (n = 914). Our study design allowed for orthogonal validation and replication across platforms. SV frequency, type, and localization varied significantly among high-risk tumors. MYCN nonamplified high-risk tumors harbored an increased SV burden overall, including a significant excess of tandem duplication events across the genome. Genes disrupted by SV breakpoints were enriched in neuronal lineages and associated with phenotypes such as autism spectrum disorder (ASD). The postsynaptic adapter protein-coding gene, SHANK2, located on Chromosome 11q13, was disrupted by SVs in 14% of MYCN nonamplified high-risk tumors based on WGS and 10% in the SNP array cohort. Expression of SHANK2 was low across human-derived neuroblastoma cell lines and high-risk neuroblastoma tumors. Forced expression of SHANK2 in neuroblastoma cells resulted in significant growth inhibition (P = 2.6 × 10−2 to 3.4 × 10−5) and accelerated neuronal differentiation following treatment with all-trans retinoic acid (P = 3.1 × 10−13 to 2.4 × 10−30). These data further define the complex landscape of somatic structural variation in neuroblastoma and suggest that events leading to deregulation of neurodevelopmental processes, such as inactivation of SHANK2, are key mediators of tumorigenesis in this childhood cancer.

Epigenomic profiling of neuroblastoma cell lines

Understanding the aberrant transcriptional landscape of neuroblastoma is necessary to provide insight to the underlying influences of the initiation, progression and persistence of this developmental cancer. Here, we present chromatin immunoprecipitation sequencing (ChIP-Seq) data for the oncogenic transcription factors, MYCN and MYC, as well as regulatory histone marks H3K4me1, H3K4me3, H3K27Ac, and H3K27me3 in ten commonly used human neuroblastoma-derived cell line models. In addition, for all of the profiled cell lines we provide ATAC-Seq as a measure of open chromatin. We validate specificity of global MYCN occupancy in MYCN amplified cell lines and functional redundancy of MYC occupancy in MYCN non-amplified cell lines. Finally, we show with H3K27Ac ChIP-Seq that these cell lines retain expression of key neuroblastoma super-enhancers (SE). We anticipate this dataset, coupled with available transcriptomic profiling on the same cell lines, will enable the discovery of novel gene regulatory mechanisms in neuroblastoma.

Multiple epigenetic biomarkers for evaluation of students' academic performance.

Several reports have shown that methyl CpG-binding protein 2 (MeCP2), brain-derived neurotrophic factor (BDNF), phospho-cAMP response element-binding protein (p-CREB) and microRNAs may be important in regulating academic performance because of their roles in neuropsychiatry and cognitive diseases. The first goal of this study was to explore the associations among MeCP2, BDNF, CREB and academic performance. This study also examined the pathway responsible for the effects of MeCP2, BDNF, p-CREB and microRNAs on academic performance. Scores from the basic competency test, an annual national competitive entrance examination, were used to evaluate academic performance. Subjects' plasma RNA was extracted and analyzed. This study determined that participants in the higher academic performance group had a significant difference in MECP2 mRNA expression compared with the lower academic performance group. We then used neuronal human derived neuroblastoma cell line (SH-SY5Y) cells with inducible MeCP2 expression from a second copy of the gene as a gain-of-function model and found that MeCP2 overexpression positively affected p-CREB and BDNF expression initially. After negative feedback, the p-CREB and BDNF levels subsequently decreased. In the neuronal phenotype examination, we found a significant reduction in total outgrowth and branches in MeCP2-induced cells compared with noninduced cells. This work describes pathways that may be responsible for the effects of MeCP2, BDNF, p-CREB and microRNAs on academic performance. These results may shed light on the development of promising clinical treatment strategies in the area of neuropsychological adjustment.

Cu(TPMA)(Phen)](ClO4)2: Metallodrug Nanocontainer Delivery and Membrane Lipidomics of a Neuroblastoma Cell Line Coupled with a Liposome Biomimetic Model Focusing on Fatty Acid Reactivity.

The use of copper complexes for redox and oxidative-based mechanisms in therapeutic strategies is an important field of multidisciplinary research. Here, a novel Cu(II) complex [Cu(TPMA)(Phen)](ClO4)2 (Cu-TPMA-Phen, where TPMA = tris-(2-pyridylmethyl)amine and Phen = 1,10-phenanthroline) was studied using both the free and encapsulated forms. A hollow pH-sensitive drug-delivery system was synthesized, characterized, and used to encapsulate and release the copper complex, thus allowing for the comparison with the free drug. The human neuroblastoma-derived cell line NB100 was treated with 5 μM Cu-PMA-Phen for 24 h, pointing to the consequences on mono- and polyunsaturated fatty acids (MUFA and PUFA) present in the membrane lipidome, coupled with cell viability and death pathways (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium viability assay, flow cytometry, microscopy, caspase activation). In parallel, the Cu-TPMA-Phen reactivity with the fatty acid moieties of phospholipids was studied using the liposome model to work in a biomimetic environment. The main results concerned: (i) the membrane lipidome in treated cells, involving remodeling with a specific increase of saturated fatty acids (SFAs) and a decrease of MUFA, but not PUFA; (ii) cytotoxic events and lipidome changes did not occur for the encapsulated Cu-TPMA-Phen, showing the influence of such nanocarriers on drug activity; and (iii) the liposome behavior confirmed that MUFA and PUFA fatty acid moieties in membranes are not affected by oxidative and isomerization reactions, proving the different reactivities of thiyl radicals generated from amphiphilic and hydrophilic thiols and Cu-TPMA-Phen. This study gives preliminary but important elements of copper(II) complex reactivity in cellular and biomimetic models, pointing mainly to the effects on membrane reactivity and remodeling based on the balance between SFA and MUFA in cell membranes that are subjects of strong interest for chemotherapeutic activities as well as connected to nutritional strategies.

Abstract B42: The properties of ex vivo expanded γδ T cells provide for the rational use of combination therapies

Abstract Cell-based immunotherapies have relied mainly on the use of αβ T cells or dendritic cells, and technologies to expand and engineer these cells have resulted in potent antitumor products. Although the use of adaptive immunocompetent cells has advanced more rapidly than innate immune cells, such as natural killer or γδ T cells, these alternative cellular products can provide equivalent antitumor activity. However, the methods to expand clinically relevant innate cell products are not as advanced as those for other immunocompetent cells, such as αβ T cells. We have developed a safe and efficient method for expanding, storing, and genetically engineering γδ T cells, and have focused on the use of these ex vivo expanded cells to treat pediatric neuroblastoma. γδ T cells are an attractive candidate for cell-based products because their antitumor activity is directly cytolytic to tumor cells, in part, due to their receptors NKG2D and CD16. NKG2D facilitates the innate recognition of stress-induced ligands such as MICA/B and UL-16 binding proteins (ULBP), and CD16 enables mechanisms of antibody-dependent cellular cytotoxicity (ADCC). We have developed a serum-free expansion protocol using apheresis products from healthy donors and from neuroblastoma patients. γδ T cells from all starting products can be expanded to greater than 70% of the culture within 12-14 days. These γδ T cells are cytotoxic against K562 cells, which has become our standard for which to compare the various expansion products, and 5 human derived neuroblastoma cell lines. We have tested several freezing media and developed a method using serum-free conditions between days 12-14 of expansion, resulting in equivalent cytotoxicity by cells pre- and post-freezing while maintaining greater than 70% viability post-thaw. We also show that our expansion protocol results in γδ T cells that are greater than 90% positive for surface expression of the receptors NKG2D and CD16. Therefore, we are using these cells in combination treatments that include standard chemotherapy regimens for relapsed neuroblastoma patients, i.e., temozolomide (TMZ), and the monoclonal antibody dinutuximab. We have shown that stress antigens such as MICA/B and ULBP2/5/6 are upregulated in vitro on IMR5 cells for 6 hours after a 1-hour exposure to TMZ. Further, combining patient-expanded γδ T cells that express CD16 with a GD2-specific antibody, dinutuximab, induces 30% increased neuroblastoma cell death compared to γδ T cells alone. To test the in vivo effectiveness of 12-day expanded, frozen, and thawed cells, we injected NSG male and female mice with 5 x 106 IMR5 cells subcutaneously. After a palpable 125-mm3 tumor was established, we began treatment with various combinations of γδ T cells, dinutuximab, and TMZ. These ongoing studies show that γδ T cells alone do not provide an antitumor benefit, but tumor regression is achieved when incorporating γδ T cells into dinutuximab and TMZ treatment regimens. This advantage is only observed at lower TMZ doses, whereas at higher TMZ doses, >60 mg/kg, chemotherapy dominates the antitumor effect. Overall, these studies show that a safer, serum-free expanded γδ T cell product can be produced, which can be stored frozen without affecting the cytotoxic properties. The expression of NKG2D and CD16 on the expanded cells allows for the development of combination therapies using cytotoxic chemotherapy agents and antibody-based treatments. The combination of γδ T cells, TMZ, and dinutuximab can provide a rational advancement for treating pediatric neuroblastoma patients, and we hypothesize that this combination therapy is preferred over high-dose chemotherapy regimens because it can reduce treatment-related toxicities and provide a multifaceted immunotherapeutic approach to neuroblastoma treatment. Citation Format: Jaquelyn T. Zoine, Kathryn S. Sutton, Kristopher A. Knight, Kelly C. Goldsmith, Christopher B. Doering, H. Trent Spencer. The properties of ex vivo expanded γδ T cells provide for the rational use of combination therapies [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B42.

Amyloid-β Inhibits PDGFβ Receptor Activation and Prevents PDGF-BBInduced Neuroprotection.

PDGFβ receptors and their ligand, PDGF-BB, are upregulated in vivo after neuronal insults such as ischemia. When applied exogenously, PDGF-BB is neuroprotective against excitotoxicity and HIV proteins. Given this growth factor's neuroprotective ability, we sought to determine if PDGF-BB would be neuroprotective against amyloid-β (1-42), one of the pathological agents associated with Alzheimer's disease (AD). In both primary hippocampal neurons and the human-derived neuroblastoma cell line, SH-SY5Y, amyloid-β treatment for 24 h decreased surviving cell number in a concentrationdependent manner. Pretreatment with PDGF-BB failed to provide any neuroprotection against amyloid-β in primary neurons and only very limited protective effects in SH-SY5Y cells. In addition to its neuroprotective action, PDGF promotes cell growth and division in several systems, and the application of PDGFBB alone to serum-starved SH-SY5Y cells resulted in an increase in cell number. Amyloid-β attenuated the mitogenic effects of PDGF-BB, inhibited PDGF-BB-induced PDGFβ receptor phosphorylation, and attenuated the ability of PDGF-BB to protect neurons against NMDA-induced excitotoxicity. Despite the ability of amyloid-β to inhibit PDGFβ receptor activation, immunoprecipitation experiments failed to detect a physical interaction between amyloid-β and PDGF-BB or the PDGFβ receptor. However, G protein-coupled receptor transactivation of the PDGFβ receptor (an exclusively intracellular signaling pathway) remained unaffected by the presence of amyloid-β. As the PDGF system is upregulated upon neuronal damage, the ability of amyloid-β to inhibit this endogenous neuroprotective system should be further investigated in the context of AD pathophysiology.

New Application of an Atmospheric Pressure Plasma Jet as a Neuro-protective Agent Against Glucose Deprivation-induced Injury of SH-SY5Y Cells.

The atmospheric pressure plasma jet (APPJ) has attracted the attention of many researchers from multiple disciplines in recent years because its emissions include multiple types of reactive nitrogen species (RNS) and reactive oxygen species (ROS). Our previous study has shown the cytoprotective effect of the APPJ against oxidative stress-induced injuries. The aim of the present study is to provide a detailed in vitro treatment protocol regarding the neuroprotective applications of helium APPJs on glucose deprivation-induced injury in SH-SY5Y cells. The SH-SY5Y human neuroblastoma-derived cell line was maintained in RPMI 1640 medium supplemented with 15% fetal calf serum. The culture medium was then changed to RPMI 1640 without glucose before APPJ treatment. After a 1 h incubation in a cell incubator, cell viability was determined using Cell Counting Kit 8. The results showed that, compared to the glucose deprivation group, cells treated with APPJ exhibited significantly increased cell viability in a dose-dependent manner, with 8 s/well observed as an optimal dose. Meanwhile, helium flow had no effect on the glucose deprivation-induced cell impairment. Our results indicated that APPJ could be potentially used as a treatment method for the diseases in the central nervous system related to glucose deprivation. This protocol could also be used as a cytoprotective application for other cells with different impairments, but the cell culture and APPJ treatment conditions should be readjusted, and the treatment dose must be relatively low.

Dioxin induces expression of hsa-miR-146b-5p in human neuroblastoma cells

Dioxin can cause a series of neural toxicological effects. MicroRNAs (miRs) play important roles in regulating nervous system function and mediating cellular responses to environmental pollutants, such as dioxin. Hsa-miR-146b-5p appears to be involved in neurodegenerative diseases and brain tumors. However, little is known about effects of dioxin on the expression of hsa-miR-146b-5p. We found that the hsa-miR-146b-5p expression and its promoter activity were significantly increased in dioxin treated SK-N-SH cells, a human-derived neuroblastoma cell line. Potential roles of hsa-miR-146b-5p in mediating neural toxicological effects of dioxin may be due to the regulation of certain target genes. We further confirmed that hsa-miR-146b-5p significantly suppressed acetylcholinesterase (AChE) activity and targeted the 3′-untranslated region of the AChE T subunit, which has been down-regulated in dioxin treated SK-N-SH cells. Functional bioinformatic analysis showed that the known and predicted target genes of hsa-miR-146b-5p were involved in some brain functions or cyto-toxicities related to known dioxin effects, including synapse transmission, in which AChE may serve as a responsive gene for mediating the effect.

Ethanol-induced PGE2 up-regulates Aβ production through PKA/CREB signaling pathway

Ethanol abuse aggravates dementia-associated cognitive defects through the progression of Alzheimer's disease (AD) pathophysiology. Beta-site APP-cleaving enzyme 1 (BACE1) has been considered as a key regulator of AD pathogenesis by controlling amyloid beta peptide (Aβ) accumulation. In addition, previous studies reported that endoplasmic reticulum (ER) stress and neuroinflammation have been proposed in ethanol-induced neurodegeneration. Thus, we investigated the role of ER stress and PGE2, a neuroinflammation mediator, in the ethanol-stimulated BACE1 expression and Aβ production. Using the human-derived neuroblastoma cell line SK-N-MC, the results show that ethanol up-regulated BACE1 expression in a dose-dependent manner. Ethanol stimulated reactive oxygen species (ROS) production, which induced CHOP expression and eIF2α phosphorylation. PBA (an ER stress inhibitor) attenuated the ethanol-increased cyclooxygenase-2 (COX-2) expression and PGE2 production. By using salubrinal (an eIF2α dephosphorylation inhibitor) or EIF2A siRNA, we found that eIF2α phosphorylation mediated the ethanol-induced COX-2 expression. In addition, COX-2-induced BACE1 up-regulation was abolished by NS-398 (a selective COX-2 inhibitor). And, PF-04418948 (an EP-2 receptor inhibitor) pretreatment reduced ethanol-induced PKA activation and CREB phosphorylation as well as ethanol-stimulated Aβ production. Furthermore, 14-22 amide (a PKA inhibitor) pretreatment or CREB1 siRNA transfection suppressed the ethanol-induced BACE1 expression. In conclusion, ethanol-induced eIF2α phosphorylation stimulates COX-2 expression and PGE2 production which induces the BACE1 expression and Aβ production via EP-2 receptor-dependent PKA/CREB pathway.

Dual ALK and CDK4/6 Inhibition Demonstrates Synergy against Neuroblastoma.

Purpose: Anaplastic lymphoma kinase (ALK) is the most frequently mutated oncogene in the pediatric cancer neuroblastoma. We performed an in vitro screen for synergistic drug combinations that target neuroblastomas with mutations in ALK to determine whether drug combinations could enhance antitumor efficacy.Experimental Design: We screened combinations of eight molecularly targeted agents against 17 comprehensively characterized human neuroblastoma-derived cell lines. We investigated the combination of ceritinib and ribociclib on in vitro proliferation, cell cycle, viability, caspase activation, and the cyclin D/CDK4/CDK6/RB and pALK signaling networks in cell lines with representative ALK status. We performed in vivo trials in CB17 SCID mice bearing conventional and patient-derived xenograft models comparing ceritinib alone, ribociclib alone, and the combination, with plasma pharmacokinetics to evaluate for drug-drug interactions.Results: The combination of ribociclib, a dual inhibitor of cyclin-dependent kinase (CDK) 4 and 6, and the ALK inhibitor ceritinib demonstrated higher cytotoxicity (P = 0.008) and synergy scores (P = 0.006) in cell lines with ALK mutations as compared with cell lines lacking mutations or alterations in ALK Compared with either drug alone, combination therapy enhanced growth inhibition, cell-cycle arrest, and caspase-independent cell death. Combination therapy achieved complete regressions in neuroblastoma xenografts with ALK-F1174L and F1245C de novo resistance mutations and prevented the emergence of resistance. Murine ribociclib and ceritinib plasma concentrations were unaltered by combination therapy.Conclusions: This preclinical combination drug screen with in vivo validation has provided the rationale for a first-in-children trial of combination ceritinib and ribociclib in a molecularly selected pediatric population. Clin Cancer Res; 23(11); 2856-68. ©2016 AACR.

Preclinical Therapeutic Synergy of MEK1/2 and CDK4/6 Inhibition in Neuroblastoma.

Purpose: Neuroblastoma is treated with aggressive multimodal therapy, yet more than 50% of patients experience relapse. We recently showed that relapsed neuroblastomas frequently harbor mutations leading to hyperactivated ERK signaling and sensitivity to MEK inhibition therapy. Here we sought to define a synergistic therapeutic partner to potentiate MEK inhibition.Experimental Design: We first surveyed 22 genetically annotated human neuroblastoma-derived cell lines (from 20 unique patients) for sensitivity to the MEK inhibitor binimetinib. After noting an inverse correlation with sensitivity to ribociclib (CDK4/6 inhibitor), we studied the combinatorial effect of these two agents using proliferation assays, cell-cycle analysis, Ki67 immunostaining, time-lapse microscopy, and xenograft studies.Results: Sensitivity to binimetinib and ribociclib was inversely related (r = -0.58, P = 0.009). MYCN amplification status and expression were associated with ribociclib sensitivity and binimetinib resistance, whereas increased MAPK signaling was the main determinant of binimetinib sensitivity and ribociclib resistance. Treatment with both compounds resulted in synergistic or additive cellular growth inhibition in all lines tested and significant inhibition of tumor growth in three of four xenograft models of neuroblastoma. The augmented growth inhibition was attributed to diminished cell-cycle progression that was reversible upon removal of drugs.Conclusions: Here we demonstrate that combined binimetinib and ribociclib treatment shows therapeutic synergy across a broad panel of high-risk neuroblastoma preclinical models. These data support testing this combination therapy in relapsed high-risk neuroblastoma patients, with focus on cases with hyperactivated RAS-MAPK signaling. Clin Cancer Res; 23(7); 1785-96. ©2016 AACR.

Optimization of a Neural Stem-Cell-Mediated Carboxylesterase/Irinotecan Gene Therapy for Metastatic Neuroblastoma

Despite improved survival for children with newly diagnosed neuroblastoma (NB), recurrent disease is a significant problem, with treatment options limited by anti-tumor efficacy, patient drug tolerance, and cumulative toxicity. We previously demonstrated that neural stem cells (NSCs) expressing a modified rabbit carboxylesterase (rCE) can distribute to metastatic NB tumor foci in multiple organs in mice and convert the prodrug irinotecan (CPT-11) to the 1,000-fold more toxic topoisomerase-1 inhibitor SN-38, resulting in significant therapeutic efficacy. We sought to extend these studies by using a clinically relevant NSC line expressing a modified human CE (hCE1m6-NSCs) to establish proof of concept and identify an intravenous dose and treatment schedule that gave maximal efficacy. Human-derived NB cell lines were significantly more sensitive to treatment with hCE1m6-NSCs and irinotecan as compared with drug alone. This was supported by pharmacokinetic studies in subcutaneous NB mouse models demonstrating tumor-specific conversion of irinotecan to SN-38. Furthermore, NB-bearing mice that received repeat treatment with intravenous hCE1m6-NSCs and irinotecan showed significantly lower tumor burden (1.4-fold, p = 0.0093) and increased long-term survival compared with mice treated with drug alone. These studies support the continued development of NSC-mediated gene therapy for improved clinical outcome in NB patients.

Action of HMGB1 on miR-221/222 cluster in neuroblastoma cell lines.

microRNA (miR/miRNA) are small non-coding RNAs that control gene expression at the post-transcriptional level by targeting mRNAs. Aberrant expression of miRNAs is often observed in different types of cancer. Specific miRNAs function as tumor suppressors or oncogenes and interfere with various aspects of carcinogenesis, including differentiation, proliferation and invasion. Upregulation of miRNAs 221 and 222 has been shown to induce a malignant phenotype in numerous human cancers via inhibition of phosphatase and tensin homolog (PTEN) expression. Neuroblastoma is the most common extracranial solid malignancy in children, which is characterized by cellular heterogeneity that corresponds to different clinical outcomes. The different cellular phenotypes are associated with different gene mutations and miRs that control genetic and epigenetic factors. For this reason miRs are considered a potential therapeutic target in neuroblastoma. The aim of the present study was to investigate the mechanisms by which extracellular high mobility group box 1 (HMGB1) promotes cell growth in neuroblastoma. SK-N-BE(2) and SH-SY5Y neuroblastoma derived cell lines were transfected with the antisense oligonucleotides, anti-miR-221 and -222, followed by treatment with HMGB1 to investigate the expression of the oncosuppressor PTEN. In this study, it was demonstrated that HMGB1, which is released by damaged cells and tumor cells, upregulates miR-221/222 oncogenic clusters in the two human neuroblastoma derived cell lines. The results revealed that the oncogenic cluster miRs 221/222 were more highly expressed by the most undifferentiated cell line [SK-N-BE(2)] compared with the the less tumorigenic cell line (SH-SY5Y) and that exogenous HMGB1 increases this expression. In addition, HMGB1 modulates PTEN expression via miR-221/222, as demonstrated by transiently blocking miR-221/222 with anti-sense oligonucleotides. These results may lead to the development of novel therapeutic strategies for neuroblastoma.

Crizotinib Synergizes with Chemotherapy in Preclinical Models of Neuroblastoma.

The presence of an ALK aberration correlates with inferior survival for patients with high-risk neuroblastoma. The emergence of ALK inhibitors such as crizotinib has provided novel treatment opportunities. However, certain ALK mutations result in de novo crizotinib resistance, and a phase I trial of crizotinib showed a lack of response in patients harboring those ALK mutations. Thus, understanding mechanisms of resistance and defining circumvention strategies for the clinic is critical. The sensitivity of human neuroblastoma-derived cell lines, cell line-derived, and patient-derived xenograft (PDX) models with varying ALK statuses to crizotinib combined with topotecan and cyclophosphamide (topo/cyclo) was examined. Cultured cells and xenografts were evaluated for effects of these drugs on proliferation, signaling, and cell death, and assessment of synergy. In neuroblastoma murine xenografts harboring the most common ALK mutations, including those mutations associated with resistance to crizotinib (but not in those with wild-type ALK), crizotinib combined with topo/cyclo enhanced tumor responses and mouse event-free survival. Crizotinib + topo/cyclo showed synergistic cytotoxicity and higher caspase-dependent apoptosis than crizotinib or topo/cyclo alone in neuroblastoma cell lines with ALK aberrations (mutation or amplification). Combining crizotinib with chemotherapeutic agents commonly used in treating newly diagnosed patients with high-risk neuroblastoma restores sensitivity in preclinical models harboring both sensitive ALK aberrations and de novo-resistant ALK mutations. These data support clinical testing of crizotinib and conventional chemotherapy with the goal of integrating ALK inhibition into multiagent therapy for ALK-aberrant neuroblastoma patients.

Abstract 484: Fidelity of subclonal representation in human neuroblastoma-derived cell line and patient-derived xenograft models: A report from the NCI-TARGET project

Abstract Patient-derived xenografts and cell lines have been the underpinning of functional characterization and drug discovery efforts in cancer. The use of these models is often under the assumption that these systems are renewable, faithful representations of original progenitor tumor cell populations. To test this assumption, we performed whole exome (92X median coverage) and genome sequencing (36X median coverage) analysis of cell line and patient-derived mouse xenografts (PDXs) originating from 7 neuroblastoma patients. Data are from1 primary tumor, 3 PDXs, and 15 neuroblastoma cell lines cultured from tumor, bone marrow, or blood. The cell lines consisted of 4 pre-/post-therapy pairs and 3 pairs established and maintained in either hyperoxia (room air i.e. “standard” cell culture) or physiologic (bone marrow hypoxia = 5%) oxygen. 7 lymphoblastoid or fibroblast cell lines were used as matched normals to identify somatic mutations. Subclonal population structures were inferred from somatic mutation calls calibrated for copy number state and tumor purity. In all cell lines and xenografts, we observed 1-2 additional subclonal populations, primarily supported by deep coverage from exome sequencing. In nearly every case, we observed shifts in the proportional representation of genetic subclones and many subclones showed additional mutations not evident in the progenitor tissue or cancer line derived in parallel. Comparison of three cell line pairs established in bone marrow level hypoxia versus room air found only ∼40% of coding mutations in each line were shared (average 82 mutations per line), suggesting significant genetic impact of growing tumor cells in the two different culture conditions. Matched PDXs from these cases had only ∼17% of coding mutations shared across all three models. The greatest genetic similarity was seen between paired cell lines established from tissue obtained pre-/post-therapy from the same patient (36 coding mutations shared, 14 private to diagnosis and 13 private to progression). However, a second pre/post-therapy cell line pair did not share any coding mutations, although they did have 585 non-coding mutations in common (of 4,033 and 2,480 in each line), assuring that the relapse was derived from a diagnostic tumor clone. These results highlight a need for comprehensive subclonal analysis of human cancer laboratory models to better inform design and interpretation of biological and preclinical therapeutic studies. Citation Format: Maya Schonbach, Arnavaz Danesh, Jeff Bruce, Tito Woodburn, Tanja Davidsen, Leandro Hermida, Patee Gesuwan, Jaime Guidry Auvil, Oliver Hampton, David Wheeler, Julie Gastier-Foster, Malcolm Smith, Daniela Gerhard, John M. Maris, Patrick Reynolds, Trevor J. Pugh. Fidelity of subclonal representation in human neuroblastoma-derived cell line and patient-derived xenograft models: A report from the NCI-TARGET project. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 484. doi:10.1158/1538-7445.AM2015-484

Abstract 475: Identification of SHANK2 as a tumor suppressor disrupted by recurrent somatic structural variation (SV) in neuroblastoma

Abstract Background. Neuroblastoma is a malignancy of the developing sympathetic nervous system which exacts significant morbidity and mortality in children. Large sequencing efforts have revealed a relative paucity of somatic point mutations in this and other childhood cancers. Structural variations (SVs) including translocations, inversions, deletions, duplications, and other complex events can occur in germline DNA or be acquired somatically in tumors. Recurrent somatic SVs affecting known genes are likely to be functional and may elucidate novel tumor suppressors or oncogenes. Methods. We are performing whole-genome sequencing (WGS) of 187 matched tumor-normal pairs from high-risk patients as part of the NCI-TARGET project (http://www.ocg.cancer.gov/programs/target) using Complete Genomics technology. High confidence somatic SVs were identified and a subset subsequently validated with Sanger sequencing; in vitro functional studies were performed using human derived neuroblastoma cell line models. Results. To date, we have analyzed WGS data from 106 matched stage 4 tumor-normal pairs. Among 2,770 high-confidence somatic SVs (average 26.1 per tumor, range 1-158), we observed recurrent focal deletions in ZFHX3 (n = 5) and EZH2 (n = 2), in addition to ATRX (n = 2), ARID1B (n = 2), and PTPRD (n = 5), each of the latter genes previously reported in neuroblastoma. A total of 210 inter-chromosomal translocations disrupting known genes were detected (average 2.0 per tumor, range 0-12). Notably, 14.4% of MYCN non-amplified stage 4 tumors (11/76) harbored SV breakpoints within SHANK2 at 11q13; all SHANK2 breakpoints were validated by Sanger sequencing. With the exception of a single deletion at the c-terminus, all breakpoints mapped within the long isoform of SHANK2 (transcript variant 1: NM_012309), and clustered within the ankyrin repeat domain which is involved in protein-protein interactions. Low SHANK2 expression in primary tumors obtained at diagnosis was associated with worse overall survival (p = 6.3 × 10−5), suggesting SHANK2, known to regulate neuronal differentiation, may function as a tumor suppressor. Consistent with this hypothesis, mRNA expression of SHANK2 was low in a panel of 27 neuroblastoma cell lines and further silencing with siRNA did not produce an observable phenotype. In contrast, forced expression of the long isoform of SHANK2 in three independent neuroblastoma cell lines with low endogenous SHANK2 levels resulted in profound reduction in cell growth (p < 0.0001) and viability (p < 0.0001) as measured by RTCES and CellTiter-glo assays respectively. Conclusion. A plethora of SVs exist in neuroblastoma, and unlike somatic point mutations, many are recurrent. Studies are ongoing to determine the mechanism by which SHANK2 suppresses neuroblastoma tumorigenesis and to understand the biological relevance of other recurrent coding and non-coding SVs in this childhood malignancy. Citation Format: Karina Conkrite, Nicole Ferraro, Lee McDaniel, Derek A. Oldridge, Edward Attiyeh, Shahab Asgharzadeh, Maura Diamond, Jaime Guidry Auvil, Tanja Davidsen, Malcom Smith, Wendy B. London, Robert Seeger, Javed Khan, Daniela S. Gerhard, John M. Maris, Sharon J. Diskin. Identification of SHANK2 as a tumor suppressor disrupted by recurrent somatic structural variation (SV) in neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 475. doi:10.1158/1538-7445.AM2015-475