Neuroblastoma Model Research Articles

A phase 1b study of crizotinib in combination with temsirolimus in pediatric ALK- or MET-aberrated relapsed or refractory neuroblastoma (ITCC-053): Results of the phase 1 part.

10036 Background: Preclinical data in neuroblastoma (NBL) models provided evidence that the addition of an mTOR inhibitor with crizotinib may overcome the relative resistance to ALK inhibitors (Berry et al., 2012, Cancer Cell). The primary objective of this phase 1b trial was to establish the recommended phase 2 dose (RP2D) of crizotinib in combination with the mTOR inhibitor temsirolimus in pediatric NBL patients. Methods: Patients aged 1-21 years with relapsed/refractory ALK/MET aberrated NBL were eligible to enroll onto stratum 2 of the CRISP trial (EudraCT: 2015-005437-53, ITCC-053), sponsored by Erasmus MC. This was an open-label, non-randomized, two-stage phase 1b dose finding study. Crizotinib was dose escalated using the EWOC method in four dose levels (DL) ranging from 100 mg/m2 to 280 mg/m2 twice daily (BID) in 28 day cycles. Temsirolimus was administered intravenously in a fixed dose of 60 mg/m2/week. Both starting doses were reduced compared to the single agent RP2D because of an expected CYP3A4 interaction. Primary endpoint was dose limiting toxicity (DLT) during cycle 1. Radiological disease assessments and blood sampling for pharmacokinetics were scheduled per protocol. Results: Nine NBL patients were enrolled onto stratum 2 between 06-2018 and 12-2022. Eight patients with a median age of 7 years (range: 1-12) received trial treatment. The temsirolimus dose was reduced to 40 mg/m2 after the first 3 patients based on unexpected toxicity. DLTs occurred in 2/3 patients treated with crizotinib at DL2 (200 mg/m2 BID); grade 3 elevated ALT (alanine aminotransferase) and grade 3 gastritis (temsirolimus dose; see table). No DLT occurred in 5 patients treated with crizotinib DL1 (150 mg/m2 BID). Dose re-escalation of crizotinib to DL2 was considered not feasible. The RP2D was established at 150 mg/m2 BID crizotinib with 40 mg/m2 temsirolimus. Frequent grade 3 treatment-related adverse events were anorexia, gastritis, oral mucositis (n = 2) and anemia (n = 3). Treatment, toxicity and response are summarized in table. Conclusions: We established a RP2D of 150 mg/m2 BID crizotinib in combination with 40 mg/m2 temsirolimus. The expansion cohort is currently open for enrollment. Clinical trial information: EUCTR2015-005437-53 . [Table: see text]

Effects of Monensin and Rapamycin Combination Therapy on Tumor Growth and Apoptosis in a Xenograft Mouse Model of Neuroblastoma.

Neuroblastoma is the most common pediatric solid tumor originating from the neural crest. New treatment options are needed to improve treatment outcomes and the survival of patients with neuroblastoma. Monensin is an ionophore antibiotic with antiparasitic, antibacterial, and anticancer properties isolated from Streptomyces cinnamonensis. The aim of this study was to investigate the therapeutic effects of single and combined monensin and rapamycin treatments on mTOR (mammalian target of rapamycin) signaling pathway-mediated apoptosis and tumor growth in an SH-SY5Y neuroblastoma cell xenograft model. Control, monensin, rapamycin, and monensin + rapamycin groups were formed in the xenograft neuroblastoma model obtained from CD1 nude mice, and tumor volumes and animal weights were recorded throughout the treatment. In xenograft neuroblastoma tumor tissues, apoptosis was determined by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) and cleaved-caspase 3 immunohistochemistry, and PI3K (phosphoinositide-3-kinase)/AKT/mTOR expression was determined by the immunohistochemistry and immunofluorescence methods. The combination of monensin and rapamycin was to reduce the growth of xenograft neuroblastoma tumor tissues, trigger apoptosis, and suppress the expression of PI3K/AKT/mTOR. A significant increase in apoptotic cell rate was demonstrated in the combination group, supported by cleaved-caspase 3 immunohistochemistry results. In addition, it was reported that the combination treatment regime triggered apoptosis by reducing the expression of phosphorylated PI3K/AKT/mTOR. Our preclinical results may be a precursor to develop new therapeutic approaches to treat neuroblastoma.

Development of anti-GD2 Antibody-producing Mesenchymal Stem Cells as Cellular Immunotherapy.

Using the tyrosine hydroxylase (TH)-MYCN mouse neuroblastoma (NB) model, we have previously reported the accumulation of mouse mesenchymal stem cells (mMSCs) on tumors in vivo and the antitumor effect of mMSCs transfected with a small molecule (IFN-β) expression gene. In this study, we have developed novel MSCs secreting anti-disialoganglioside GD2 antibody (anti-GD2-MSCs) and evaluated their antitumor effects in vitro. We generated an anti-GD2 antibody construct (14.G2a-Fcx2-GFP) incorporating FLAG-tagged single-chain fragment variable against GD2 fused to a linker sequence, a fragment of the constant portion of human IgG1, and GFP protein. The construct was lentivirally transduced into mMSCs and the transduction efficiency was assessed by GFP expression. The secretion of FLAG-tagged anti-GD2 antibody was detected by Western blotting using anti-FLAG antibody. Antibody binding capacity was confirmed by flow cytometry. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using human NB cells and human natural killer (NK) cells to assess whether the antitumor activity was enhanced in the presence of the produced antibodies. The transduction efficiency of anti-GD2-MSCs was more than 90%. anti-GD2-MSCs secreted antibodies extracellularly and these antibodies had high affinity to GD2-expressing human NB cells. ADCC assays showed that the addition of antibodies secreted from anti-GD2-MSCs significantly increased the cytotoxic activity of NK cells against NB cells. Newly developed anti-GD2-MSCs produced functional antibodies that have affinity to the GD2 antigen on NB cells and can induce ADCC-mediated cytotoxicity. Anti-GD2-MSCs based cellular immunotherapy has the potential to be a novel therapeutic option for intractable NB.

Reversible transitions between noradrenergic and mesenchymal tumor identities define cell plasticity in neuroblastoma

Noradrenergic and mesenchymal identities have been characterized in neuroblastoma cell lines according to their epigenetic landscapes and core regulatory circuitries. However, their relationship and relative contribution in patient tumors remain poorly defined. We now document spontaneous and reversible plasticity between the two identities, associated with epigenetic reprogramming, in several neuroblastoma models. Interestingly, xenografts with cells from each identity eventually harbor a noradrenergic phenotype suggesting that the microenvironment provides a powerful pressure towards this phenotype. Accordingly, such a noradrenergic cell identity is systematically observed in single-cell RNA-seq of 18 tumor biopsies and 15 PDX models. Yet, a subpopulation of these noradrenergic tumor cells presents with mesenchymal features that are shared with plasticity models, indicating that the plasticity described in these models has relevance in neuroblastoma patients. This work therefore emphasizes that intrinsic plasticity properties of neuroblastoma cells are dependent upon external cues of the environment to drive cell identity.

Abstract LB226: FGFR2 modulates ALK inhibition response in high-risk neuroblastoma

Abstract High-risk neuroblastoma (NB) has a poor prognosis despite multimodal treatment. To improve survival and minimise treatment side-effects, research has focused on developing more effective therapeutic strategies. Anaplastic lymphoma kinase (ALK) is a promising druggable target as its expression rapidly decreases in healthy tissues postnatally, and it is expressed as a hyperactivated mutant form in ~14% of high-risk NB. The ALK tyrosine kinase inhibitor lorlatinib is a promising treatment, but resistance has been reported, hindering long-term benefits. With a trimodal approach, consisting of genome-wide CRISPR-Cas9-overexpression (CRISPRa) screens, RNA sequencing and high-throughput drug screens, we have identified genes whose expression is associated with decreased sensitivity to lorlatinib creating novel therapeutic vulnerabilities. In particular, we validate the CRISPRa screen hit FGFR2 as a bypass signalling mechanism desensitising mutant ALK-expressing NB cells to lorlatinib; overexpression of FGFR2 increased, while silencing decreased resistance of NB cells to lorlatinib. Furthermore, RNA sequencing of lorlatinib resistant (LR) NB cells developed in our lab, compared to parental cell lines, showed FGFR2 to be expressed to a higher level in the LR cells. High-throughput drug screens exposing LR and parental NB cell lines to an FDA-approved drug library of 1430 compounds showed that drugs targeting receptor tyrosine kinases, including FGFR2, were amongst the compounds most significantly effective in reducing the viability of LR NB cells compared to parental cells. The multi-kinase inhibitor ponatinib and the selective FGFR inhibitor erdafitinib acted synergistically with lorlatinib in treating both parental and LR NB cells although both inhibitors were more effective in inhibiting the growth of LR compared to parental cells when given alone, suggesting that FGFR2 may represent a novel vulnerability to treat lorlatinib resistant NB. In vivo studies using patient-derived xenograft (PDX) models of high-risk NB (MYCN-amplified and ALKF1174L mutant) showed that combinations of either ponatinib or erdafitinib with lorlatinib decreased tumour growth and increased survival compared to PDXs treated with vehicle or either agent alone. In conclusion, these findings suggest that FGFR2 alters NB sensitivity to lorlatinib and modulation of this pathway in combination with ALK inhibition is a promising approach to improve NB treatment response and ultimately patient survival. Citation Format: Perla Pucci, Charlotte Barrett, Ricky Trigg, Leila Jahangiri, Jamie D. Matthews, Luca Mologni, G. A. Amos Burke, Suzanne D Turner. FGFR2 modulates ALK inhibition response in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB226.

Targeting B7-H3-A Novel Strategy for the Design of Anticancer Agents for Extracranial Pediatric Solid Tumors Treatment.

Recent scientific data recognize the B7-H3 checkpoint molecule as a potential target for immunotherapy of pediatric solid tumors (PSTs). B7-H3 is highly expressed in extracranial PSTs such as neuroblastoma, rhabdomyosarcoma, nephroblastoma, osteosarcoma, and Ewing sarcoma, whereas its expression is absent or very low in normal tissues and organs. The influence of B7-H3 on the biological behavior of malignant solid neoplasms of childhood is expressed through different molecular mechanisms, including stimulation of immune evasion and tumor invasion, and cell-cycle disruption. It has been shown that B7-H3 knockdown decreased tumor cell proliferation and migration, suppressed tumor growth, and enhanced anti-tumor immune response in some pediatric solid cancers. Antibody-drug conjugates targeting B7-H3 exhibited profound anti-tumor effects against preclinical models of pediatric solid malignancies. Moreover, B7-H3-targeting chimeric antigen receptor (CAR)-T cells demonstrated significant in vivo activity against different xenograft models of neuroblastoma, Ewing sarcoma, and osteosarcoma. Finally, clinical studies demonstrated the potent anti-tumor activity of B7-H3-targeting antibody-radioimmunoconjugates in metastatic neuroblastoma. This review summarizes the established data from various PST-related studies, including in vitro, in vivo, and clinical research, and explains all the benefits and potential obstacles of targeting B7-H3 by novel immunotherapeutic agents designed to treat malignant extracranial solid tumors of childhood.

Abstract 4998: Selective PLK4 inhibition demonstrates synthetic lethality in TRIM37 amplified neuroblastoma and breast cancer models while less selective inhibitors do not

Abstract Amplification and copy number gains of the 17q23 amplicon are common in breast cancer and neuroblastoma and have been associated with early relapse and poor prognosis (Ganesan et al 2012; Takita et al. 2017). A synthetic lethal interaction of PLK4 with 17q23 amplicon-driven overexpression of TRIM37 was discovered in these tumor types (Meitinger et al. 2020; Yeow et al. 2020). High levels of TRIM37 prevented acentrosomal spindle assembly and rendered cells mitotically vulnerable to inhibition of polo-like kinase 4 (PLK4), a serine/threonine protein kinase that controls centriole duplication. We have discovered that exquisitely selective small molecule inhibitors of PLK4, which are highly selective against the kinome including against the closely related aurora kinases and PLK1-3, display this synthetic lethal interaction with TRIM37, while less selective inhibitors do not. PLK4 protein levels are regulated through proteasomal degradation induced by PLK4 trans-autophosphorylation of the phosphodegron. PLK4 inhibition results in blocked trans-autophosphorylation leading to stabilization of PLK4, thus directly demonstrating target engagement in cells. Importantly, PLK4 protein stabilization correlated with cell viability for selective PLK4 inhibitors but not for less selective compounds, providing a quantifiable pharmacodynamic (PD) association with antitumor activity. Cell viability assessment in cancer cell lines revealed that highly selective PLK4 inhibitors showed greater potency in TRIM37 high cancer cell lines as compared to TRIM37 low cell lines. In contrast, less selective compounds, including from the clinical literature, did not display differential potency in TRIM37 high versus low cancer cell lines. Additionally, selective PLK4 inhibition induced significantly greater apoptosis in TRIM37 high versus low cancer cell lines as measured with a caspase 3/7 assay. We confirmed that only selective PLK4 inhibitors are synthetic lethal with TRIM37 amplification using an engineered cell line system of PLK4 G95L, in which the Leucine mutation blocks compound binding but allows the PLK4 enzyme to function. In cell viability assays, selective PLK4 inhibitors were potent in the parental G95 cells and lost activity in L95 cells, unlike less selective inhibitors whose potency did not depend on PLK4. Oral dosing of a selective PLK4 inhibitor resulted in tumor growth inhibition in TRIM37 high xenograft tumors with no body weight loss. In summary, we have discovered that highly selective small molecule inhibitors of PLK4 confirm the potential of the synthetic lethal impact in treating tumors with high levels of TRIM37. Citation Format: Siobhan K. McRee, Chelsea Chen, Christophe Colas, Wie Fang, Wayne Kong, Fang Liu, Jason Long, Jared Moore, Alex Pankov, Dan Shore, Joanne Tan, Robert Warne, Rakesh Vekariya, Amy Young, Anneleen Daemen, Anthony Romero, Melissa R. Junttila, Lori S. Friedman, Kyle A. Edgar. Selective PLK4 inhibition demonstrates synthetic lethality in TRIM37 amplified neuroblastoma and breast cancer models while less selective inhibitors do not. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4998.

Abstract 4871: Dysregulation of MYC-family proteins sensitizes cancers to NMT inhibition: identification of NMTi sensitivity and mechanism

Abstract N-myristoyltransferases (NMTs) catalyze the protein N-terminal modification N-myristoylation, a lipidation event which affects >150 proteins and is involved in protein localization, stability, and function. NMT has been suggested as a target in cancers, but there has been a lack of rationale to identify patients who may respond to NMT inhibition. Additionally, the mechanism of action of NMT inhibitors (NMTi) is difficult to dissect, as NMT substrates feature in multiple biological pathways, and many studies have focused only on a single substrate. Here, a combination of bioinformatic, biochemical, proteomic, and cellular biology techniques have been combined to show that deregulation of MYC-family proteins sensitizes cells to NMT inhibition. Cell lines sensitive to NMTi were identified through screening of hundreds of cancer cell lines, and the transcriptome of sensitive and insensitive lines compared to obtain a “Sensitive to NMTi” gene set. This gene set correlated well with MYC-related gene sets and mutation, amplification, or chromosomal rearrangement in MYC(N) were predictive for NMTi sensitivity, suggesting that MYC deregulated cancer cells are sensitive to NMTi. This was verified by cytotoxicity assays in B-cell lymphoma and neuroblastoma lines, where highly MYC(N) expressing lines exhibited increased NMTi sensitivity, including in the SHEP21N and P493-6 lines, in which MYC(N) levels can be regulated. Furthermore, proteomic profiling identified multiple pathways which are affected by NMTi, and these were further validated. In particular, an impact on Complex I formation was seen, and mitochondrial dysfunction in high MYC(N) cells upon NMTi was seen through the loss of basal and maximal respiration, ATP production and spare respiratory capacity. Loss of Complex I formation was shown to be caused by the loss of the NMT substrate NDUFAF4 upon NMTi treatment. Furthermore, application of an orally bioavailable NMTi eliminated tumors in mouse models of both diffuse large B-cell lymphoma and neuroblastoma and were well tolerated with no changed in body weight, suggesting that NMT inhibitors are well tolerated and efficacious in vivo. Citation Format: James Zhang, Gregor A. Lueg, Monica Faronato, Evon Poon, Andrii Gorelik, Andrea G. Grocin, Eva Caamano-Gutierrez, Francesco Falciani, Roberto Solari, Robin Carr, Andrew S. Bell, Edward Bartlett, Jennie Hutton, Miriam Llorian-Sopena, Probir Chakravarty, Bernadette Brzezicha, Martin Janz, Matther J. Garnett, Louis Chesler, Dinis P. Calado, Edward W. Tate. Dysregulation of MYC-family proteins sensitizes cancers to NMT inhibition: identification of NMTi sensitivity and mechanism. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4871.

Abstract 2981: Anti-tumor activity of vobramitamab duocarmazine (MGC018), an investigational duocarmycin-based anti-B7-H3 antibody-drug conjugate (ADC), in preclinical neuroblastoma models

Abstract Introduction: MGC018 is an ADC composed of a humanized anti-B7-H3 IgG1 monoclonal antibody conjugated via a cleavable linker to vc-seco-duocarmycin-hydroxybenzamide azaindole (DUBA), a synthetic DNA alkylating agent whose cytotoxic activity is cell-cycle independent and retained in multidrug-resistant cells. MGC018 has shown preliminary clinical activity in B7-H3-expressing tumors, including castration-resistant metastatic prostate cancer. Owing to its expression in several childhood tumors, B7-H3 is a potential target for pediatric cancers, including neuroblastoma (NB). Methods: B7-H3 expression was evaluated by flow-cytometry (FCM) in a panel of human NB cell lines. Cytotoxicity was evaluated in monolayer and in multicellular tumor spheroid (MCTS) models by MTS assay and Cell Titer Glo 3D cell viability assay, respectively. Apoptotic cell death was investigated by the Annexin V staining. Pseudometastatic, orthotopic, and resected mouse NB models were developed via tumor cells tail vein injection, implantation in the adrenal gland, or implantation followed by surgical resection of the primary tumor mass, respectively, to mimic disease conditions related to circulating tumor cells and metastases, primary tumor growth, and minimal residual disease. Results: All cell lines expressed cell surface B7-H3 in a unimodal fashion, ranging between 200 and 400 in mean ratio fluorescence intensity over unstained cells. MGC018 was cytotoxic in a dose- and time-dependent manner against all NB cell lines (IC50 range 5.1-53.9 ng/mL) and NB MCTS (IC50 range 17.8-364 ng/mL). MGC018 was inactive against a murine NB cell line (NX-S2) that did not express human B7-H3; however, NX-S2 cells were killed in the presence of MGC018 when co-cultured with human B7-H3-expressing cells, demonstrating by-stander activity. In the pseudometastatic model and in five orthotopic NB mouse models, weekly iv treatments with 1 mg/kg MGC018 for 3 weeks resulted in delayed tumor growth and increased survival rates compared to animals treated with an irrelevant (anti-CD20) duocarmycin-ADC or an anti-GD2 antibody (dinutuximab beta). A 4-week course of treatment further ameliorated MGC018 antitumor effect in both the orthotopic and resected NB models and increased the survival of NB-bearing mice co-treated with TOpotecan-TEMozolomide (TOTEM), the standard-of-care therapy for relapsed disease. In the orthotopic model, tumor relapse was temporarily or completely arrested by 2 or 3 courses of MGC018, respectively. MGC018 treatment was not associated with body weight loss, hematological toxicity, or clinical chemistry abnormalities. Conclusion: MGC018 exerts relevant antitumor activity in pre-clinical NB models and may represent a potential candidate for future NB clinical translation. Citation Format: Chiara Brignole, Veronica Bensa, Enzo Calarco, Elena Giusto, Eleonora Ciampi, Patrizia Perri, Maria Valeria Corrias, Simonetta Astigiano, Michele Cilli, Deryk Loo, Ezio Bonvini, Fabio Pastorino, Mirco Ponzoni. Anti-tumor activity of vobramitamab duocarmazine (MGC018), an investigational duocarmycin-based anti-B7-H3 antibody-drug conjugate (ADC), in preclinical neuroblastoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2981.

Verteporfin-induced proteotoxicity impairs cell homeostasis and survival in neuroblastoma subtypes independent of YAP/TAZ expression

Neuroblastoma (NB) is a highly aggressive extracranial solid tumor in children. Due to its heterogeneity, NB remains a therapeutic challenge. Several oncogenic factors, including the Hippo effectors YAP/TAZ, are associated with NB tumorigenesis. Verteporfin (VPF) is an FDA-approved drug shown to directly inhibit YAP/TAZ activity. Our study aimed to investigate VPF’s potential as a therapeutic agent in NB. We show that VPF selectively and efficiently impairs the viability of YAP/TAZ-expressing NB GI-ME-N and SK-N-AS cells, but not of non-malignant fibroblasts. To investigate whether VPF-mediated NB cell killing is YAP-dependent, we tested VPF potency in CRISPR-mediated YAP/TAZ knock-out GI-ME-N cells, and BE(2)-M17 NB cells (a MYCN-amplified, predominantly YAP-negative NB subtype). Our data shows that VPF-mediated NB cell killing is not dependent on YAP expression. Moreover, we determined that the formation of higher molecular weight (HMW) complexes is an early and shared VPF-induced cytotoxic mechanism in both YAP-positive and YAP-negative NB models. The accumulation of HMW complexes, involving STAT3, GM130 and COX IV proteins, impaired cell homeostasis and triggered cell stress and cell death mechanisms. Altogether, our study shows significant in vitro and in vivo VPF-induced suppression of NB growth, making VPF a potential therapeutic candidate against NB.

β3-adrenergic receptor on tumor-infiltrating lymphocytes sustains IFN-γ-dependent PD-L1 expression and impairs anti-tumor immunity in neuroblastoma

Neuroblastoma (NB) is a heterogeneous extracranial tumor occurring in childhood. A distinctive feature of NB tumors is their neuroendocrine ability to secrete catecholamines, which in turn, via β-adrenergic receptors ligation, may affect different signaling pathways in tumor microenvironment (TME). It was previously demonstrated that specific antagonism of β3-adrenergic receptor (β3-AR) on NB tumor cells affected tumor growth and progression. Here, in a murine syngeneic model of NB, we aimed to investigate whether the β3-AR modulation influenced the host immune system response against tumor. Results demonstrated that β3-AR antagonism lead to an immune response reactivation, partially dependent on the PD-1/PD-L1 signaling axis involvement. Indeed, β3-AR blockade on tumor-infiltrating lymphocytes (TILs) dampened their ability to secrete IFN-γ, which in turn reduced the PD-L1 expression, caused by TILs infiltration, on NB tumor cells. Further investigations, through a genomic analysis on NB patients, showed that high ADRB3 gene expression correlates with worse clinical outcome compared to the low expression group, and that ADRB3 gene expression affects different immune-related pathways. Overall, results indicate that β3-AR in NB TME is able to modulate the interaction between tumor and host immune system, and that its antagonism hits multiple pro-tumoral signaling pathways.

GLUT inhibitor WZB117 induces cytotoxicity with increased production of amyloid-beta peptide in SH-SY5Y cells preventable by beta-hydroxybutyrate: implications in Alzheimer's disease.

Inhibitors of glucose transporters are being explored as potential anti-cancer drugs. Decreased cerebral glucose utilization with reduced levels of several glucose transporters is also an important pathogenic signature of neurodegeneration of Alzheimer's disease, but its exact role in the pathogenesis of this disease is not established. We explored in an experimental model if inhibitors of glucose transporters could lead to altered amyloid-beta homeostasis, mitochondrial dysfunction, and neuronal death, which are relevant in the pathogenesis of Alzheimer's disease. SH-SY5Y cells (human neuroblastoma cell line) were exposed to an inhibitor (WZB117) of several types of glucose transporters. We examined the effects of glucose hypometabolism on SH-SY5Y cells in terms of mitochondrial functions, production of reactive oxygen species, amyloid-beta homeostasis, and neural cell death. The effect of β-hydroxybutyrate in ameliorating the effects of WZB117 on SH-SY5Y cells was also examined. We observed that exposure of SH-SY5Y cells to WZB117 caused mitochondrial dysfunction, increased production of reactive oxygen species, loss of cell viability, increased expression of BACE 1, and intracellular accumulation of amyloid β peptide (Aβ42). All the effects of WZB117 could be markedly prevented by co-treatment with β-hydroxybutyrate. Cyclosporine A, a blocker of mitochondrial permeability transition pore (mPTP) activation, could not prevent cell death caused by WZB117. Results in this neuroblastoma model have implications for the pathogenesis of Alzheimer's disease and warrant further explorations of WZB117 in primary cultures of neurons and experimental animal models.

Combining multiple cell death pathway-related risk scores to develop neuroblastoma cell death signature.

Cell death plays an important role in tumourigenesis and progression; nevertheless, the clinical significance of cell death-related genes in neuroblastoma remains incompletely understood. We separately constructed the corresponding risk scores for each of the eight cell death pathways separately and assessed their predictive performance. Through Cox regression analysis, these eight risk scores were integrated to obtain final cell death risk scores (CDRS). We evaluated the predictive performance of CDRS in multiple datasets and compared its accuracy with the clinical characteristics of patients and some existing prognostic models for neuroblastoma. We then explored the differences in immune infiltration between the high and low CDRS groups, and the significance of CDRS on EFS and disease progression. All eight risk scores have high predictive accuracy, with the Immunogenic-RS being the most accurate and the cuproptosis-RS the least accurate. Model genes are mainly enriched in a variety of cancer-related pathways and are closely related to the clinical characteristics. CDRS showed superior and robust predictive performance in multiple datasets and was more accurate than the clinical characteristics of patients and some existing prognostic models for neuroblastoma. High CDRS group featured distinct immune cold tumor profiles and may have poorer immune checkpoint inhibitor efficacy. CDRS had significance in predicting EFS and disease progression. We integrated risk scores associated with multiple cell death pathways to develop a high-performing and robust neuroblastoma signature. CDRS was a promising tool that may help with risk assessment and prediction of overall prognosis, and thus improve clinical outcomes.

Diglycolic acid inhibits succinate dehydrogenase activity, depletes mitochondrial membrane potential, and induces inflammation in an SH-SY5Y neuroblastoma model of neurotoxicity in vitro

Diethylene glycol is a toxic industrial solvent resulting in a well-defined toxidrome. Diglycolic acid (DGA) has been identified as the metabolite responsible for the nephrotoxicity and hepatotoxicity. These studies assess the mechanism of DGA-induced neurotoxicity, specifically addressing the known ability of DGA to chelate calcium (Ca2+) in solution and inhibit mitochondrial complex II. SH-SY5Y cells were seeded into 96-well plates to assess intracellular Ca2+ chelation, complex II activity, mitochondrial membrane potential (ΔΨm), ATP production, and release of inflammatory cytokines TNF-α and IL-1β with 2-, 4-, 6-, 24-, and 48-h DGA exposure. Peak Ca2+ chelation occurred at 4 h in cells treated with 6.25–50 mM DGA; however, effects were transient. Complex II activity was significantly decreased at all DGA concentrations tested, with 12.5 mM DGA causing 80% inhibition and 25 and 50 mM DGA causing 97 and 100% inhibition, respectively. Subsequently, 12.5–50 mM DGA concentrations significantly decreased ΔΨm at all time points. 50 mM DGA significantly increased release of TNF-α and IL-1β after 24 and 48 h with significantly decreased ATP production observed at the same time points and concentration. These studies demonstrate that the DGA-induced mechanism of SH-SY5Y cell death involves complex II inhibition leading to mitochondrial depolarization, and subsequent ATP depletion with accompanying inflammatory cytokine release. These results indicate a direct mechanism of DGA-induced neurotoxicity in vitro, similarly observed in other DEG-affected target organs.

Patient-derived models: Advanced tools for precision medicine in neuroblastoma.

Neuroblastoma is a childhood cancer derived from the sympathetic nervous system. High-risk neuroblastoma patients have a poor overall survival and account for ~15% of childhood cancer deaths. There is thus a need for clinically relevant and authentic models of neuroblastoma that closely resemble the human disease to further interrogate underlying mechanisms and to develop novel therapeutic strategies. Here we review recent developments in patient-derived neuroblastoma xenograft models and in vitro cultures. These models can be used to decipher mechanisms of metastasis and treatment resistance, for drug screening, and preclinical drug testing. Patient-derived neuroblastoma models may also provide useful information about clonal evolution, phenotypic plasticity, and cell states in relation to neuroblastoma progression. We summarize current opportunities for, but also barriers to, future model development and application. Integration of patient-derived models with patient data holds promise for the development of precision medicine treatment strategies for children with high-risk neuroblastoma.

Green Tea Catechin Potentiating Doxorubicine Effects against BE(2)C Neuroblastoma Cells In Vitro

Background: Neuroblastoma (NB), a malignant sympathetic nervous system cancer, is the second most common type of pediatric tumor. Increasing the number of NB death emerges to design a new strategy for NB treatment. Nowadays, the development of natural compounds has gradually increased due to their ability to apoptosis induction. Tea catechin, a flavonoid compound, is one of the natural combinations which inhibit tumor growth and enhance tumor cell apoptosis. In the current study, the effects of pure catechin, doxorubicin (DOX), and their combination on a cellular model of NB [BE(2)C cells] are perused. (NB) a malignant sympathetic nervous system cancer is the second most common type of pediatric tumor. Increasing the number of NB death emerges to design a new strategy for NB treatment. Nowadays, the development of natural compounds has gradually increased due to their ability to apoptosis induction. Tea catechin, a flavonoid compound, is one of the natural combinations which inhibit tumor growth and enhance tumor cell apoptosis. Objectives: In the current study, the effects of pure catechin, doxorubicin (DOX), and their combination on a cellular model of NB [BE(2)C cells] are perused. Methods: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was done to assess a response dose for each drug. Fluorescent Microscopic and cell cycle analyses were performed for apoptosis detection. Finally, Colony formation was performed to examine cell migration and invasion. Results: The MTT assay showed that catechin and DOX treatment inhibited the viability of the cells while the combination of their ineffective doses had more cytotoxic effects. However, these treatments could not inhibit the cell growth of the normal human fibroblast. Moreover, this combination reduced cell attachment, chromatin fragmentation, and G/S arrest in the cell cycle. The clonogenic assay demonstrated that colony size and numbers obviously reduced after ten days; therefore, Catchin and its combination with DOX suppressed cell capacities of clone formation and migration. Conclusions: These results suggest that catechin, DOX, and their combination may inhibit the proliferation, invasion, and migration of BE(2)C Neuroblastoma cells in vitro while inducing cell apoptosis by arresting the cell cycle.

Analysis of Serial Neuroblastoma PDX Passages in Mice Allows the Identification of New Mediators of Neuroblastoma Aggressiveness.

Neuroblastoma is a neural crest cell-derived pediatric tumor characterized by high inter- and intra-tumor heterogeneity, and by a poor outcome in advanced stages. Patient-derived xenografts (PDXs) have been shown to be useful models for preserving and expanding original patient biopsies in vivo, and for studying neuroblastoma biology in a more physiological setting. The maintenance of genetic, histologic, and phenotypic characteristics of the original biopsy along serial PDX passages in mice is a major concern regarding this model. Here we analyze consecutive PDX passages in mice, at both transcriptomic and histological levels, in order to identify potential changes or highlight similarities to the primary sample. We studied temporal changes using mRNA and miRNA expression and correlate those with neuroblastoma aggressiveness using patient-derived databases. We observed a shortening of tumor onset and an increase in proliferative potential in the PDXs along serial passages. This behavior correlates with changes in the expression of genes related to cell proliferation and neuronal differentiation, including signaling pathways described as relevant for neuroblastoma malignancy. We also identified new genes and miRNAs that can be used to stratify patients according to survival, and which could be potential new players in neuroblastoma aggressiveness. Our results highlight the usefulness of the PDX neuroblastoma model and reflect phenotypic changes that might be occurring in the mouse environment. These findings could be useful for understanding the progression of tumor aggressiveness in this pathology.

Combination Therapies Targeting ALK-aberrant Neuroblastoma in Preclinical Models.

ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX). Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma.

Development of a Mitochondrial Targeting Lipid Nanoparticle Encapsulating Berberine.

Delivering drugs to mitochondria, the main source of energy in neurons, can be a useful therapeutic strategy for the treatment of neurodegenerative diseases. Berberine (BBR), an isoquinoline alkaloid, acts on mitochondria and is involved in mechanisms associated with the normalization and regulation of intracellular metabolism. Therefore, BBR has attracted considerable interest as a possible therapeutic drug for neurodegenerative diseases. While BBR has been reported to act on mitochondria, there are few reports on the efficient delivery of BBR into mitochondria. This paper reports on the mitochondrial delivery of BBR using a lipid nanoparticle (LNP), a "MITO-Porter" that targets mitochondria, and its pharmacological action in Neuro2a cells, a model neuroblastoma. A MITO-Porter containing encapsulated BBR (MITO-Porter (BBR)) was prepared. Treatment with MITO-Porter (BBR) increased the amount of BBR that accumulated in mitochondria compared with a treatment with naked BBR. Treatment with MITO-Porter (BBR) resulted in increased ATP production in Neuro2a cells, which are important for maintaining life phenomena, compared with treatment with naked BBR. Treatment with MITO-Porter (BBR) also increased the level of expression of mitochondrial ubiquitin ligase (MITOL), which is involved in mitochondrial quality control. Our findings indicate that increasing the accumulation of BBR into mitochondria is important for inducing enhanced pharmacological actions. The use of this system has the potential for being important in terms of the regulation of the metabolic mechanism of mitochondria in nerve cells.

Non-viral nitric oxide-based gene therapy improves perfusion and liposomal doxorubicin sonopermeation in neuroblastoma models.

Neuroblastoma (NB) is a pediatric malignancy that accounts for 15% of cancer-related childhood mortality. High-risk NB requires an aggressive chemoradiotherapy regimen that causes significant off-target toxicity. Despite this invasive treatment, many patients either relapse or do not respond adequately. Recent studies suggest that improving tumor perfusion can enhance drug accumulation and distribution within the tumor tissue, potentially augmenting treatment effects without inflicting systemic toxicity. Accordingly, methods that transiently increase tumor perfusion prior to treatment may help combat this disease. Here, we show the use of gene therapy to confer inducible nitric oxide synthase (iNOS) expression solely in the tumor space, using focused ultrasound targeting. NOS catalyzes the reaction that generates nitric oxide (NO), a potent endogenous vasodilator. This study reports the development of a targeted non-viral image-guided platform to deliver iNOS-expressing plasmid DNA (pDNA) to vascular endothelial cells encasing tumor blood vessels. Following transfection, longitudinal quantitative contrast-enhanced ultrasound (qCEUS) imaging revealed an increase in tumor perfusion over 72 h, attributed to elevated intratumoral iNOS expression. Methods: To construct a gene delivery vector, cationic ultrasound-responsive agents (known as "microbubbles") were employed to carry pDNA in circulation and transfect tumor vascular endothelial cells in vivo using focused ultrasound (FUS) energy. This was followed by liposomal doxorubicin (L-DOX) treatment. The post-transfection tumor response was monitored longitudinally using qCEUS imaging to determine relative changes in blood volumes and perfusion rates. After therapy, ex vivo analysis of tumors was performed to examine the bioeffects associated with iNOS expression. Results: By combining FUS therapy with cationic ultrasound contrast agents (UCAs), we achieved selective intratumoral transfection of pDNA encoding the iNOS enzyme. While transitory, the degree of expression was sufficient to induce significant increases in tumoral perfusion, to appreciably enhance the chemotherapeutic payload and to extend survival time in an orthotopic xenograft model. Conclusion: We have demonstrated the ability of a novel targeted non-viral gene therapy strategy to enhance tumor perfusion and improve L-DOX delivery to NB xenografts. While our results demonstrate that transiently increasing tumor perfusion improves liposome-encapsulated chemotherapeutic uptake and distribution, we expect that our iNOS gene delivery paradigm can also significantly improve radio and immunotherapies by increasing the delivery of radiosensitizers and immunomodulators, potentially improving upon current NB treatment without concomitant adverse effects. Our findings further suggest that qCEUS imaging can effectively monitor changes in tumor perfusion in vivo, allowing the identification of an ideal time-point to administer therapy.