MYCN-amplified Neuroblastoma Cell Lines Research Articles

Abstract 147: Identification of cell sub-populations in Neuroblastoma cell lines with single-cell-sequencing analysis

Abstract Neuroblastoma is the most frequent extracranial tumor in neonatal patients. Engineering SKNBE2 Myc-N amplified Neuroblastoma cell lines we generated individual clones expressing NDM29 non-coding-RNA at different levels. Using these cell models we documented a specific role of NDM29 in tumor cell differentiation leading to the identification of novel genes to be pharmacologically targeted for an efficient control of tumor malignancy (CA9 and MCM2 genes). Further studies taking advantage of the same models suggested the possible co-existence of different cell sub-populations in the apparently homogeneous cell culture with possible different roles in tumor maintenance and growth. Here we test this hypothesis by performing single-cell sequencing analysis and identified different sub-populations on the base of their gene expression patterns. The characterization of their specific profiles of membrane markers allowed an efficient separation of the individual populations by FACS analysis. Our preliminary results show the co-existance of cells with a marked neural phenotype with other two populations one VEGF secreting and the other VEGF recipient/responder. These preliminary findings suggest further detailed investigations of possible paracrine loops that might be at the base of tumor cell nodule growth and maintenance. Citation Format: Michele Tomanelli, Davide Ceresa, Adriana Bajetto, Paola Modesto, Paolo Malatesta, Tullio Florio, Aldo Pagano. Identification of cell sub-populations in Neuroblastoma cell lines with single-cell-sequencing analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 147.

Abstract 5122: Modulating anti-GD2 immunotherapy via dual DFMO and TGF-β inhibition in neuroblastoma

Abstract Neuroblastoma (NB) is an often-lethal childhood solid tumor. Anti-GD2 immunotherapy improves survival but not all responses are durable. Transforming growth factor-beta (TGFβ) has been implicated in solid tumor immunoresistance. High-risk NBs harbor activated MYC or MYCN to coordinately expand intratumoral polyamines (PA), oncometabolites with immunomodulatory roles, to support tumor progression. Difluoromethylornithine (DFMO) is an inhibitor of ornithine decarboxylase, the rate-limiting enzyme in PA synthesis, and has shown activity in preclinical NB models, including TH-MYCN+/+ transgenic mice, and is in clinical trials in children. We sought to augment anti-GD2 therapy using DFMO and the small molecule TGFβ-receptor inhibitor, galunisertib. We randomized TH-MYCN+/+ mice in two experiments (10 therapy arms; n=6-10 mice/arm) to receive combinations of DFMO, galunisertib, and anti-GD2 antibody m14G2a and to assess impact on survival. We measured 770 genes using nCounter Immuno-Oncology 360 (Nanostring), 36 cytokines using xMap bead-arrays (Luminex), and tumor-infiltrating leukocytes using flow cytometry in harvested tumors. Cell line TGFβ signaling was evaluated in tissue culture. In vitro, galunisertib consistently suppressed the upregulated TGFβ-Smad2/3 signaling cascade in MYCN-amplified neuroblastoma cell lines. In vivo, DFMO alone extends mouse survival and increases intra-tumoral neutrophils and natural killer (NK) cells. These NK cells increased surface expression of activation receptors like NKG2D (p<0.05), while tumor cells increased surface expression of NK-ligands. Tumor cells also expressed increased latency-associated peptide (LAP; p<0.05), a TGFβ precursor, suggesting NK cells were poised to kill but restrained by TGFβ. Cytotoxicity assays showed reversal of this inhibition with TGFβR1 inhibitor galunisertib in vitro. In a proof-of-concept trial, DFMO and galunisertib (DFMO/gal) demonstrated a clinical anti-tumor synergistic effect. DFMO/gal treated tumors had upregulated NK cell activity genes and downregulated TGFβ signaling genes. Cytokine analysis confirmed DFMO induced upregulation of TGFβ1 and TGFβ2 (p<0.01) that was modulated by galunisertib. IL6, GCSF, and GMCSF were markedly downregulated in tumors treated with both DFMO and galunisertib (p<0.05). An expanded trial showed DFMO/gal added to m14G2a showed no significant survival benefit over the DFMO and m14G2a combination. Ultimately, DFMO creates a pro-inflammatory cellular and cytokine milieu in TH-MYCN+/+ NBs. DFMO and TGFβR1 inhibition via galunisertib activate NK cells further and decrease the anti-inflammatory cytokine IL6. Ongoing studies are further characterizing the gene expression and cytokine levels of the NB TME under these treatment conditions. Citation Format: Om H. Gandhi, Christina S. Turn, Kangning Liu, Annette T. Vu, Hamid Bassiri, Michael D. Hogarty. Modulating anti-GD2 immunotherapy via dual DFMO and TGF-β inhibition in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5122.

Abstract 132: DNA-PK inhibition to enhance etoposide therapy in neuroblastoma

Abstract Introduction. Neuroblastoma is the most common extracranial solid tumor in children and accounts for ~15% of all pediatric cancer deaths. DNA-PK, a key regulator of repair responses to double-strand DNA breakages induced by radiation or induction chemotherapy is used in the treatment of high-risk neuroblastomas. Here, we have demonstrated that high DNA-PK expression is associated with poor overall survival in a patient population, DNA-PK is highly activated in neuroblastoma cell lines, and inhibition of DNA-PK enhances therapeutic effects of DNA damaging therapies. Methods. DNA double-strand break damage was induced with etoposide, a topoisomerase II inhibitor. DNA-PK activity was inhibited with peposertib or DNA-PK siRNA. DNA-PK Ser2056 phosphorylation was analyzed in MYCN amplified (SK-NDZ, BE(2)-C, LAN-1) and MYCN non-amplified (SK-N-AS, SK-N-SH) cell lines. Clonogenic and proliferation assays were used to evaluate the efficacy of peposertib in combination with etoposide in BE-2-C and SK-N-DZ neuroblastoma cell lines. DSB-induced DNA repair was visualized with confocal laser scanning microscopy using γ-H2AX immunofluorescence. Western blot was used to confirm inhibition of DNA-PKcs (pDNA-PKcs S2056) and analyze cleaved PARP expression in combination groups. Results. DNA-PK inhibition resulted in enhancement of etoposide cytotoxic therapy (BE-2-C IC50 ET = 2.77 ± 0.2, ET+Pep 0.76 ± 0.04; SK-N-DZ IC50 ET = 0.55 ± 0.06, IC50 ET+Pep = 0.11 ± 0.02) in both BE-2-C and SK-N-DZ cell lines. Immunofluorescence analysis detected γH2AX foci increase in the etoposide and peposertib combination group. DNA-PK inhibition in combination with etoposide treatment significantly increased PARP cleavage in BE-2-C and SK-N-DZ cell lines. The effect of DNA-PK knockdown on neuroblastoma cell survival was even more pronounced compared to DNA-PK inhibition with peposertib. DNA-PK knockdown by siRNA alone increased PARP cleavage to the levels comparable to 2 µM etoposide treatment. The combination of etoposide and DNA-PK knockdown enhanced PARP cleavage even further in neuroblastoma cells. Conclusions. High DNA repair activity is commonly associated with cancer drug resistance. We have shown that DNA-PK activation is continuously supported in both MYCN amplified and MYCN non-amplified neuroblastoma cell lines. The importance of DNA-PK activity in neuroblastoma cell lines is confirmed with DNA-PK knockdown that leads to apoptosis in MYCN amplifiedneuroblastoma cell lines. In addition, the combination of DNA-PK inhibition or DNA-PK knockdown with DNA-damaging therapies stalled DNA damage response and enhanced neuroblastoma cell apoptosis. These findings demonstrate the significance of high DNA-PK activity in neuroblastoma cells and provide a potent therapeutic strategy to enhance neuroblastoma cell apoptosis with selective DNA-PK inhibition. Citation Format: Mahnaz Norouzi, Beibei Zhu, Tadahide Izumi, Eric J. Rellinger, B. Mark Evers, Piotr G. Rychahou. DNA-PK inhibition to enhance etoposide therapy in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 132.

Triple Therapy with Metformin, Ketogenic Diet, and Metronomic Cyclophosphamide Reduced Tumor Growth in MYCN-Amplified Neuroblastoma Xenografts.

Neuroblastoma (NB) is a childhood cancer in which amplification of the MYCN gene is the most acknowledged marker of poor prognosis. MYCN-amplified NB cells rely on both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) for energy production. Previously, we demonstrated that a ketogenic diet (KD) combined with metronomic cyclophosphamide (CP) delayed tumor growth in MYCN-amplified NB xenografts. The anti-diabetic drug metformin (MET) also targets complex I of the OXPHOS system. Therefore, MET-induced disruptions of mitochondrial respiration may enhance the anti-tumor effect of CP when combined with a KD. In this study, we found that MET decreased cell proliferation and mitochondrial respiration in MYCN-amplified NB cell lines, while the combination of KD, MET, and low-dose CP (triple therapy) also reduced tumor growth and improved survival in vivo in MYCN-amplified NB xenografts. Gene ontology enrichment analysis revealed that this triple therapy had the greatest effect on the transcription of genes involved in fatty acid ß-oxidation, which was supported by the increased protein expression of CPT1A, a key mitochondrial fatty acid transporter. We suspect that alterations to ß-oxidation alongside the inhibition of complex I may hamper mitochondrial energy production, thus explaining these augmented anti-tumor effects, suggesting that the combination of MET and KD is an effective adjuvant therapy to CP in MYCN-amplified NB xenografts.

DNA Ligase 4 Contributes to Cell Proliferation against DNA-PK Inhibition in MYCN-Amplified Neuroblastoma IMR32 Cells.

Identifying the vulnerability of altered DNA repair machinery that displays synthetic lethality with MYCN amplification is a therapeutic rationale in unfavourable neuroblastoma. However, none of the inhibitors for DNA repair proteins are established as standard therapy in neuroblastoma. Here, we investigated whether DNA-PK inhibitor (DNA-PKi) could inhibit the proliferation of spheroids derived from neuroblastomas of MYCN transgenic mice and MYCN-amplified neuroblastoma cell lines. DNA-PKi exhibited an inhibitory effect on the proliferation of MYCN-driven neuroblastoma spheroids, whereas variable sensitivity was observed in those cell lines. Among them, the accelerated proliferation of IMR32 cells was dependent on DNA ligase 4 (LIG4), which comprises the canonical non-homologous end-joining pathway of DNA repair. Notably, LIG4 was identified as one of the worst prognostic factors in patients with MYCN-amplified neuroblastomas. It may play complementary roles in DNA-PK deficiency, suggesting the therapeutic potential of LIG4 inhibition in combination with DNA-PKi for MYCN-amplified neuroblastomas to overcome resistance to multimodal therapy.

MYCN Amplification Is Associated with Reduced Expression of Genes Encoding γ-Secretase Complex and NOTCH Signaling Components in Neuroblastoma.

Amplification of the MYCN oncogene is found in ~20% of neuroblastoma (NB) cases and correlates with high-risk disease and poor prognosis. Despite the plethora of studies describing the role of MYCN in NB, the exact molecular mechanisms underlying MYCN's contribution to high-risk disease are not completely understood. Herein, we implemented an integrative approach combining publicly available RNA-Seq and MYCN ChIP-Seq datasets derived from human NB cell lines to define biological processes directly regulated by MYCN in NB. Our approach revealed that MYCN-amplified NB cell lines, when compared to non-MYCN-amplified cell lines, are characterized by reduced expression of genes involved in NOTCH receptor processing, axoneme assembly, and membrane protein proteolysis. More specifically, we found genes encoding members of the γ-secretase complex, which is known for its ability to liberate several intracellular signaling molecules from membrane-bound proteins such as NOTCH receptors, to be down-regulated in MYCN-amplified NB cell lines. Analysis of MYCN ChIP-Seq data revealed an enrichment of MYCN binding at the transcription start sites of genes encoding γ-secretase complex subunits. Notably, using publicly available gene expression data from NB primary tumors, we revealed that the expression of γ-secretase subunits encoding genes and other components of the NOTCH signaling pathway was also reduced in MYCN-amplified tumors and correlated with worse overall survival in NB patients. Genetic or pharmacological depletion of MYCN in NB cell lines induced the expression of γ-secretase genes and NOTCH-target genes. Chemical inhibition of γ-secretase activity dampened the expression of NOTCH-target genes upon MYCN depletion in NB cells. In conclusion, this study defines a set of MYCN-regulated pathways that are specific to MYCN-amplified NB tumors, and it suggests a novel role for MYCN in the suppression of genes of the γ-secretase complex, with an impact on the NOTCH-target gene expression in MYCN-amplified NB.

Abstract 481: Investigating molecular glues as a new therapeutic approach to high-risk neuroblastoma

Abstract High-risk neuroblastoma (NB) is the most common extracranial solid tumor of childhood. Despite extensive study, treatments remain highly toxic and are often insufficient to cure. Thus, there is an urgent need to develop more effective and less toxic treatments for children with NB. NB commonly displays reliance on a relatively small cohort of transcription factors (TFs) and epigenetic proteins to drive cell growth, however, many of these targets are inaccessible with conventional small molecules. In recent years, new modalities such as targeted protein degradation (TPD) have emerged. TPD may permit small molecule degradation of traditionally “undruggable” targets, such as TFs. This makes TPD a particularly attractive modality for defining new therapeutic strategies for high-risk NB. One category of TPD agents are molecular glues (MGs). MGs are monovalent small molecules capable of simultaneously binding to an E3 ligase receptor such as CRBN (cereblon), and a target protein of interest. Importantly, in comparison to conventional protein inhibitors, MGs do not require a conventional binding pocket on the protein of interest to bind to it, and can be utilized in sub-stoichiometric concentrations. Thus, this may result in the recruitment, polyubiquitination, and subsequent proteasomal degradation of proteins previously thought to be “undruggable.” Here, we demonstrate that high-risk NB cells are critically sensitive to a cluster of previously unknown MGs. We performed high-throughput screening of a proprietary library (>4000 unique compounds) of MGs in two MYCN-amplified NB cell lines, Kelly and MHHNB11. These two cell lines demonstrate a strikingly close correlation in Cell-Titer Glo-based growth response to MGs, as compared with an outgroup of other MG-responsive cancer cell lines. We identified a unique subcluster of 112 common MGs that display >70% growth inhibition in both cell lines. To identify the mechanism of cell growth inhibition, we established Kelly cells with stable knockout of CRBN, and coupled these with screening to confirm that the majority of these MGs (84%; 94/112) require CRBN expression. To rule out common off-target effects of MGs, candidate compounds were then tested in cells transgenically expressing either wild-type or degradation-resistant GSPT1 (G575N) constructs, revealing a subset of MGs retaining cytotoxicity without effecting GSPT1 protein levels. Current studies are focused on determining the targets of these high confidence MGs, as a foundation for preclinical in vivo studies. These studies will identify new compounds with defined mechanisms of action, with an ultimate goal of developing new treatments that are more effective and less toxic for children with NB. Citation Format: Ian Delahunty, Gisele Nishiguchi, Yang Zhang, Abigail Fish, Anand Mayasundari, Jun Qi, Brian Abraham, Anang Shelat, Zoran Rankovic, Adam Durbin. Investigating molecular glues as a new therapeutic approach to high-risk neuroblastoma [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 481.

MYCN Amplification, along with Wild-Type RB1 Expression, Enhances CDK4/6 Inhibitors' Efficacy in Neuroblastoma Cells.

Neuroblastoma (NB) is one of the primary causes of death for pediatric malignancies. Given the high heterogeneity in NB's mutation landscape, optimizing individualized therapies is still challenging. In the context of genomic alterations, MYCN amplification is the most correlated event with poor outcomes. MYCN is involved in the regulation of several cellular mechanisms, including cell cycle. Thus, studying the influence of MYCN overexpression in the G1/S transition checkpoint of the cell cycle may unveil novel druggable targets for the development of personalized therapeutical approaches. Here, we show that high expression of E2F3 and MYCN correlate with poor prognosis in NB despite the RB1 mRNA levels. Moreover, we demonstrate through luciferase reporter assays that MYCN bypasses RB function by incrementing E2F3-responsive promoter activity. We showed that MYCN overexpression leads to RB inactivation by inducing RB hyperphosphorylation during the G1 phase through cell cycle synchronization experiments. Moreover, we generated two MYCN-amplified NB cell lines conditionally knockdown (cKD) for the RB1 gene through a CRISPRi approach. Indeed, RB KD did not affect cell proliferation, whereas cell proliferation was strongly influenced when a non-phosphorylatable RB mutant was expressed. This finding revealed the dispensable role of RB in regulating MYCN-amplified NB's cell cycle. The described genetic interaction between MYCN and RB1 provides the rationale for using cyclin/CDK complexes inhibitors in NBs carrying MYCN amplification and relatively high levels of RB1 expression.

P300 Interacted With N-Myc and Regulated Its Protein Stability via Altering Its Post-Translational Modifications in Neuroblastoma

MYCN amplification is an independent risk factor for poor prognosis in neuroblastoma (NB), but its protein product cannot be directly targeted because of protein structure. Thus, this study aimed to explore novel ways to indirectly target N-Myc by regulating its post-translational modifications (PTMs) and therefore protein stability. N-Myc coimmunoprecipitation combined with HPLC–MS/MS identified 16 PTM residues and 114 potential N-Myc-interacting proteins. Notably, both acetylation and ubiquitination were identified on lysine 199 of N-Myc. We then discovered that p300, which can interact with N-Myc, modulated the protein stability of N-Myc in MYCN-amplified NB cell lines and simultaneously regulated the acetylation level and ubiquitination level on lysine-199 of N-Myc protein in vitro. Furthermore, p300 correlated with poor prognosis in NB patients. Taken together, p300 can be considered as a potential therapeutic target to treat MYCN-amplified NB patients, and other identified PTMs and interacting proteins also provide potential targets for further study.

LncRNA SNHG1 regulates neuroblastoma cell fate via interactions with HDAC1/2

The small nucleolar RNA host gene 1 (SNHG1) is a novel oncogenic long non-coding RNA (lncRNA) aberrantly expressed in different tumor types. We previously found highly expressed SNHG1 was associated with poor prognosis and MYCN status in neuroblastoma (NB). However, the molecular mechanisms of SNHG1 in NB are still unclear. Here, we disrupted endogenous SNHG1 in the MYCN-amplified NB cell line SK-N-BE(2)C using the CRISPR/Cas9 system and demonstrated the proliferation and colony formation ability of SNHG1-knowndown cells were suppressed. The transcriptome analysis and functional assays of SNHG1-knockdown cells revealed SNHG1 was involved in various biological processes including cell growth, migration, apoptosis, cell cycle, and reactive oxygen species (ROS). Interestingly, the expression of core regulatory circuitry (CRC) transcription factors in MYCN-amplified NB, including PHOX2B, HAND2, GATA3, ISL1, TBX1, and MYCN, were decreased in SNHG1-knockdown cells. The chromatin-immunoprecipitation sequencing (ChIP-seq) and transposase-accessible chromatin using sequencing (ATAC-seq) analyses showed that chromatin status of these CRC members was altered, which might stem from interactions between SNHG1 and HDAC1/2. These findings demonstrate that SNHG1 plays a crucial role in maintaining NB identity via chromatin regulation and reveal the function of the lncRNA SNHG1 in NB.

Abstract 1823: Imipridones show pre-clinical efficacy in MYCN-amplified and MYCN non-amplified neuroblastoma cell lines

Abstract Neuroblastoma is the most common extracranial solid tumor of childhood, accounting for 15% of all pediatric cancer-related deaths, with around 63% survival for high-risk patients. Treatment remains limited for advanced disease, calling for the development of novel therapies. We investigated the anti-tumor effect of three imipridones (ONC201, ONC206, and ONC212), a promising new class of small molecules, in the treatment of neuroblastoma. Imipridones are potent anticancer drugs that have previously shown efficacy for a variety of adult and pediatric malignancies. ONC201 and ONC206 are antagonists of dopamine receptor D2 (DRD2), while ONC212 is an agonist of GPR132, both of which are overexpressed in many cancers. We performed drug treatment with ONC201, ONC206, and ONC212 on established MYCN-amplified SK-N-BE(2) and MYCN non-amplified SH-SY5Y pediatric neuroblastoma cell lines in vitro. Cell viability assays were performed 72 hours post-treatment to generate dose responses curves. The IC50s for ONC201, ONC206, and ONC212 were 17.82 uM, 547 nM, and 70 nM for SK-N-BE(2), and 997 nM, 314 nM, and 6 nM for SH-SY5Y. Imipridones demonstrated greater efficacy for the non-MYCN-amplified cell line. Using protein quantification studies and downstream target analysis on drug-treated cells, we investigated the mechanisms of how these therapies caused cell death. We showed that imipridones inactivate cell proliferation kinases Akt/ERK and induce cell death through the pro-apoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). They also induce tumor cell apoptosis by modifying the mitochondrial Clp protease complex, decreasing expression of chaperone subunit ClpX and activating mitochondrial proteolysis. Histone deacetylases (HDACs) play a role in controlling MYCN function, which is amplified in more aggressive neuroblastoma; HDAC activity and MYCN are also upregulated in chemotherapy-resistant neuroblastoma. Cell viability assays with HDAC inhibitors Vorinostat and Panobinostat demonstrated single-agent efficacy in vitro. The IC50s for Vorinostat and Panobinostat were 609 nM and 5.51 nM for SK-N-BE(2), and 884 nM and 6.13 nM for SH-SY5Y. However, further investigation is needed to determine synergy and mechanisms of synergy when histone deacetylase (HDAC) inhibitors are used in novel combinations with imipridones. Overall, our data reveals promise in imipridone therapy for neuroblastoma, and future studies are proposed to explore potential novel therapeutic combinations in this difficult-to-treat pediatric cancer. Citation Format: Claire Lin, Wen-I Chang, Joshua N. Honeyman, Lanlan Zhou, Varun V. Prabhu, Joshua Allen, Wafik El-Deiry. Imipridones show pre-clinical efficacy in MYCN-amplified and MYCN non-amplified neuroblastoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1823.

Extracellular Vesicle-Based Method for Detecting MYCN Amplification Status of Pediatric Neuroblastoma.

Simple SummaryMYCN gene amplification, the strongest prognostic marker of aggressive neuroblastoma, is detected on invasive biopsy tissues. This study aimed to establish a less invasive method to detect MYCN status based on MYCN mRNA contents in large extracellular vesicles or microvesicles. MYCN mRNA-containing microvesicles were detectable in three distinct MYCN-amplified neuroblastoma cell lines but absent in three neuroblastoma cells with MYCN-non-amplification. The feasibility of this EV-based workflow was successfully demonstrated by using the simulated samples (prepared by pulsing neuroblastoma MVs into the normal human serum) and bone marrow plasma specimens obtained from nine patients at various disease stages. Taken together, this study established the novel EV-based method for detecting MYCN status in pediatric neuroblastoma.MYCN amplification is the strongest predictor of high-risk neuroblastoma (NB). The standard procedure to detect MYCN status requires invasive procedures. Extracellular vesicles (EVs) contain molecular signatures of originated cells, present in biofluids, and serve as an invaluable source for cancer liquid biopsies. This study aimed to establish an EV-based method to detect the MYCN status of NB. Two EV subtypes, i.e., microvesicles (MVs) and exosomes, were sequentially isolated from the culture supernatant by step-wise centrifugation, ultrafiltration, and size-exclusion chromatography. Quantitative RT-PCR was performed to detect MYCN mRNA. As a result, MYCN mRNA was detectable in the MVs, but not exosomes, of MYCN-amplified NB cells. MYCN mRNA-containing MVs (MYCN-MV) were successfully detected in three distinct MYCN-amplified NB cell lines but absent in three MYCN non-amplification cells. The simulated samples were prepared by pulsing MVs into human serum. MYCN–MV detection in the simulated samples showed a less interfering effect from the human blood matrix. Validation using clinical specimens (2 mL bone marrow plasma) obtained from patients at various disease stages showed a promising result. Five out of six specimens of MYCN-amplified patients showed positive results, while there were no false positives in four plasma samples of the MYCN non-amplification group. This study communicated a novel EV-based method for detecting the MYCN status of pediatric NB based on MYCN mRNA contents in MVs. Future studies should be pursued in a prospective cohort to determine its true diagnostic performance.

MYCN upregulates the transsulfuration pathway to suppress the ferroptotic vulnerability in MYCN-amplified neuroblastoma.

Ferroptosis is an iron-dependent, oxidative form of cell death that is countered mainly by glutathione peroxidase 4 (GPX4) and the production of glutathione (GSH), which is formed from cysteine. The identification of the cancers that may benefit from pharmacological ferroptotic induction is just emerging. We recently demonstrated that inducing ferroptosis genetically or pharmacologically in MYCN-amplified neuroblastoma (NB) is a novel and effective way to kill these cells. MYCN increases iron metabolism and subsequent hydroxyl radicals through increased expression of the transferrin receptor 1 (TfR1) and low levels of the ferroportin receptor. To counter increased hydroxyl radicals, MYCN binds to the promoter of SLC3A2 (solute carrier family 3 member 2). SLC3A2 is a subunit of system Xc-, which is the cysteine-glutamate antiporter that exports glutamate and imports cystine. Cystine is converted to cysteine intracellularly. Here, we investigated other ways MYCN may increase cysteine levels. By performing metabolomics in a syngeneic NB cell line either expressing MYCN or GFP, we demonstrate that the transsulfuration pathway is activated by MYCN. Furthermore, we demonstrate that MYCN-amplified NB cell lines and tumors have higher levels of cystathionine beta-synthase (CBS), the rate-limiting enzyme in transsulfuration, which leads to higher levels of the thioether cystathionine (R-S-(2-amino-2-carboxyethyl)-l-homocysteine). In addition, MYCN-amplified NB tumors have high levels of methylthioadenosine phosphorylase (MTAP), an enzyme that helps salvage methionine following polyamine metabolism. MYCN directly binds to the promoter of MTAP. We propose that MYCN orchestrates both enhanced cystine uptake and enhanced activity of the transsulfuration pathway to counteract increased reactive oxygen species (ROS) from iron-induced Fenton reactions, ultimately contributing to a ferroptosis vulnerability in MYCN-amplified neuroblastoma.

Human embryonic stem cell-derived neural crest model unveils CD55 as a cancer stem cell regulator for therapeutic targeting in MYCN-amplified neuroblastoma.

BackgroundNeuroblastoma (NB) is a common childhood malignant tumor of neural crest (NC) origin with remarkable heterogeneity in outcomes. Amplification of the oncogene MYCN is strongly associated with highly malignant behaviour and poor prognosis.MethodsThis study aims to use a human embryonic stem cell (hESC)-derived NC model to identify novel downstream effectors of MYCN that can be potentially used as prognostic marker and/or therapeutic target.ResultsWe show that MYCN-driven NB derived from human neural crest cells (hNCCs) recapitulate the pathological and molecular features of MYCN-amplified neuroblastoma (MNA-NB). By using this platform, we identify a group of 14 surface protein-encoding genes that are associated with MYCN expression level in MNA-NB. Among these genes, high CD55 expression is correlated with poor survival in MNA-NB but not in non-MNA-NB. Furthermore, CD55 promotes tumorigenesis, tumor growth, and cancer stemness in MNA-NB cell lines (MNA-NBL) through regulating the JNK pathway. Mechanistically, MYCN binds to both canonical and noncanonical E-boxes on the promoter of CD55 to regulate its transcriptional expression. Finally, neutralizing antibody targeting CD55 significantly attenuates cancer stemness, suppresses tumor growth, and improves survival exclusively in MNA-NBL-inoculated mice.ConclusionMYCN shapes CD55 into a cancer stem cell regulator which represents a prognostic marker and therapeutic target of MNA-NB. The hESC-derived NC model serves as a valuable platform for investigating NB initiation and progression and developing potential therapeutic targets.

Therapeutic targeting of both dihydroorotate dehydrogenase and nucleoside transport in MYCN-amplified neuroblastoma

Metabolic reprogramming is an integral part of the growth-promoting program driven by the MYC family of oncogenes. However, this reprogramming also imposes metabolic dependencies that could be exploited therapeutically. Here we report that the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) is an attractive therapeutic target for MYCN-amplified neuroblastoma, a childhood cancer with poor prognosis. Gene expression profiling and metabolomic analysis reveal that MYCN promotes pyrimidine nucleotide production by transcriptional upregulation of DHODH and other enzymes of the pyrimidine-synthesis pathway. Genetic and pharmacological inhibition of DHODH suppresses the proliferation and tumorigenicity of MYCN-amplified neuroblastoma cell lines. Furthermore, we obtain evidence suggesting that serum uridine is a key factor in determining the efficacy of therapeutic agents that target DHODH. In the presence of physiological concentrations of uridine, neuroblastoma cell lines are highly resistant to DHODH inhibition. This uridine-dependent resistance to DHODH inhibitors can be abrogated by dipyridamole, an FDA-approved drug that blocks nucleoside transport. Importantly, dipyridamole synergizes with DHODH inhibition to suppress neuroblastoma growth in animal models. These findings suggest that a combination of targeting DHODH and nucleoside transport is a promising strategy to overcome intrinsic resistance to DHODH-based cancer therapeutics.

Abstract 3045: Investigating the influence of glycosylation using cellular models of neuroblastoma

Abstract Background: Neuroblastoma is a common childhood malignancy with approximately 800 new cases diagnosed each year in the United States. Treatment of neuroblastoma directly depends on patient risk categories and includes combinations of surgery, radiation therapy, chemotherapy, stem cell therapy, immunotherapy, and retinoid therapy. Various characteristics of cancer in general and some specific to neuroblastoma cell lines contribute to a difficulty in delivering clinically relevant concentrations of therapeutic agents to neuroblastoma masses. Amplification of the MYCN gene in certain neuroblastoma cell lines correlates with increased cellular surface glycosylation levels as well as a worse prognosis for patients. Overexpression of the MUC1 gene is also associated with increased glycosylation, specifically type-O, in various cancers, but the role of MUC1 in neuroblastoma has not been thoroughly defined. The objective of this study was to evaluate the influence of type-O glycosylation on therapeutic uptake in different neuroblastoma cell lines. Methods: The neuroblastoma cell line SK-N-DZ, a MYCN-amplified line, was compared to SK-N-SH, a non-MYCN-amplified line (negative control) and BT-20, a highly glycosylated breast cancer cell line (positive control). Benzyl-α-GalNAc, an O-glycosylation inhibitor, was introduced to each cell line. To determine glycosylation levels in the respective cell lines, fluorescence assays were performed using CD227 (anti-MUC1) antibody. In vitro cytotoxicity assays were performed to determine maximum non-toxic concentrations of the glycosylation inhibitor (benzyl-α-GalNAc) and antibody (CD227) and to subsequently evaluate the functional role of glycosylation using cellular models of neuroblastoma. Results: The maximum non-toxic concentrations per 10,000 cells for each neuroblastoma cell line was 0.05 mg/ml of benzyl-α-GalNAc, and 6 µl of CD227 stock preparation. Preliminary results from fluorescence detection studies showed an increase in CD227-FITC antibody uptake by the MYCN-amplified neuroblastoma cells (SK-N-DZ) following exposure to the O-glycosylation inhibitor (benzyl-α-GalNAc). Cellular uptake of CD227-FITC by non-MYCN-amplified cell line (SK-N-SH) was less by comparison. Conclusion: Preliminary findings suggest that O-glycosylation is a barrier capable of limiting cellular uptake of agents in MYCN-amplified neuroblastoma cell lines. Ongoing studies are focused on investigating the influence of glycosylation inhibition on cytotoxicity of FDA-approved therapeutic agents. Citation Format: Meghan Cook, Alma Garcia, Katie Konieczny, Meghan Ferguson, Robert Campbell. Investigating the influence of glycosylation using cellular models of neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3045.

Abstract 2481: Time-resolved transcriptome analysis of murine TH-MYCN driven neuroblastoma identifies MEIS2 as early initiating factor and novel core gene regulatory circuitry constituent

Abstract Introduction: Neuroblastoma (NB) is a pediatric malignancy arising from peripheral neuronal sympathoblasts and exhibiting remarkable clinical and genetic heterogeneity. Patients older than 18 months have a poor prognosis with tumors presenting with highly recurrent segmental copy number alterations and MYCN amplification in half of these high-risk cases. The mechanism by which MYCN contributes to the development of neuroblastoma is unresolved and direct targeting of this key oncogene is not currently possible. Experimental Procedures: Our discovery efforts focused on identifying cooperating interactors and vulnerabilities in the MYCN regulatory network. MYCN-driven NBs can be modeled in mice with morphologic and genomic features that recapitulate human MYCN amplified NBs. Thus, this model serves as a valid tool for cross-species genomic analysis. Using this model, we performed a time-resolved analysis of the dynamic transcriptional changes of protein coding genes during murine TH-MYCN driven neuroblastoma development, focusing on timepoints representing tumor initiation and early tumor growth. We triangulated expression changes of key genes with publicly available exome-wide CRISPR-cas9 knockout analyses on a panel of human neuroblastoma cell lines and patient survival data. This unique data resource uncovered the relevance of MEIS2 as putative early cooperating initiating factor for neuroblastoma. Analysis of the genome-wide binding profile of MEIS2 in MYCN-amplified NB cell lines showed a striking overlap with enhancer-driven gene expression in regions of open chromatin, providing evidence that MEIS2 is a novel member of the adrenergic neuroblastoma core-regulatory circuitry. CRISPR-Cas9 mediated deletion of MEIS2 in animal models suppresses establishment of neuroblastoma tumors, indicating its putative requirement for tumor initiation. MEIS2, as a member of the CRC binds to several master regulators of gene expression, including the FOXM1 locus. Summary and conclusion: In conclusion, we present an in-depth characterization of the dynamic transcriptome profiles of TH-MYCN driven murine premalignant and established tumors and integrate with both primary human neuroblastoma tumor expression data, epigenetic and functional genomics data to identify and validate candidate cooperating dependencies suitable for targeting as a precision medicine approach in neuroblastoma. Citation Format: Kaat Durinck, Mark Zimmerman, Nina Weichert-Leahey, Jolien Dewyn, Wouter Van Loocke, Carolina Nunes, Anneleen Beckers, Bieke Decaesteker, Pieter-Jan Volders, Christophe Van Neste, Belamy Cheung, Daniel Carter, Thomas A. Look, Glenn Marshall, Katleen De Preter, Adam Durbin, Franki Speleman. Time-resolved transcriptome analysis of murine TH-MYCN driven neuroblastoma identifies MEIS2 as early initiating factor and novel core gene regulatory circuitry constituent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2481.

The synergy of BET inhibitors with aurora A kinase inhibitors in MYCN-amplified neuroblastoma is heightened with functional TP53

Amplification of MYCN is a poor prognostic feature in neuroblastoma (NBL) indicating aggressive disease. We and others have shown BET bromodomain inhibitors (BETi) target MYCN indirectly by downregulating its transcription. Here we sought to identify agents that synergize with BETi and to identify biomarkers of resistance. We previously performed a viability screen of ∼1,900 oncology-focused compounds combined with BET bromodomain inhibitors against MYCN-amplified NBL cell lines. Reanalysis of our screening results prominently identified inhibitors of aurora kinase A (AURKAi) to be highly synergistic with BETi. We confirmed the anti-proliferative effects of several BETi+AURKAi combinations in MYCN-amplified NBL cell lines. Compared to single agents, these combinations cooperated to decrease levels of N-myc. We treated both TP53-wild type and mutant, MYCN-amplified cell lines with the BETi JQ1 and the AURKAi Alisertib. The combination had improved efficacy in the TP53-WT context, notably driving apoptosis in both genetic backgrounds. JQ1+Alisertib combination treatment of a MYCN-amplified, TP53-null or TP53-restored genetically engineered mouse model of NBL prolonged survival better than either single agent. This was most profound with TP53 restored, with marked tumor shrinkage and apoptosis induction in response to combination JQ1+Alisertib. BETi+AURKAi in MYCN-amplified NBL, particularly in the context of functional TP53, provided anti-tumor benefits in preclinical models. This combination should be studied more closely in a pediatric clinical trial.

Potential Role of miRNAs in the Acquisition of Chemoresistance in Neuroblastoma.

Neuroblastoma (NB) accounts for about 8–10% of pediatric cancers, and the main causes of death are the presence of metastases and the acquisition of chemoresistance. Metastatic NB is characterized by MYCN amplification that correlates with changes in the expression of miRNAs, which are small non-coding RNA sequences, playing a crucial role in NB development and chemoresistance. In the present study, miRNA expression was analyzed in two human MYCN-amplified NB cell lines, one sensitive (HTLA-230) and one resistant to Etoposide (ER-HTLA), by microarray and RT-qPCR techniques. These analyses showed that miRNA-15a, -16-1, -19b, -218, and -338 were down-regulated in ER-HTLA cells. In order to validate the presence of this down-regulation in vivo, the expression of these miRNAs was analyzed in primary tumors, metastases, and bone marrow of therapy responder and non-responder pediatric patients. Principal component analysis data showed that the expression of miRNA-19b, -218, and -338 influenced metastases, and that the expression levels of all miRNAs analyzed were higher in therapy responders in respect to non-responders. Collectively, these findings suggest that these miRNAs might be involved in the regulation of the drug response, and could be employed for therapeutic purposes.

TAS-119, a novel selective Aurora A and TRK inhibitor, exhibits antitumor efficacy in preclinical models with deregulated activation of the Myc, β-Catenin, and TRK pathways.

Aurora kinase A, a mitotic kinase that is overexpressed in various cancers, is a promising cancer drug target. Here, we performed preclinical characterization of TAS-119, a novel, orally active, and highly selective inhibitor of Aurora A. TAS-119 showed strong inhibitory effect against Aurora A, with an IC50 value of 1.04nmol/L. The compound was highly selective for Aurora A compared with 301 other protein kinases, including Aurora kinase B. TAS-119 induced the inhibition of Aurora A and accumulation of mitotic cells in vitro and in vivo. It suppressed the growth of various cancer cell lines harboring MYC family amplification and CTNNB1 mutation in vitro. In a xenograft model of human lung cancer cells harboring MYC amplification and CTNNB1 mutation, TAS-119 showed a strong antitumor activity at well-tolerated doses. TAS-119 induced N-Myc degradation and inhibited downstream transcriptional targets in MYCN-amplified neuroblastoma cell lines. It also demonstrated inhibitory effect against tropomyosin receptor kinase (TRK)A, TRKB, and TRKC, with an IC50 value of 1.46, 1.53, and 1.47nmol/L, respectively. TAS-119 inhibited TRK-fusion protein activity and exhibited robust growth inhibition of tumor cells via a deregulated TRK pathway in vitro and in vivo. Our study indicates the potential of TAS-119 as an anticancer drug, especially for patients harboring MYC amplification, CTNNB1 mutation, and NTRK fusion.