Activity In Neuroblastoma Cells Research Articles

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.

Development of Shortened miR-506-3p Mimics Exhibiting Strong Differentiation-Inducing Activity in Neuroblastoma Cells.

microRNA mimics are synthetic RNA molecules that imitate the mature miRNA duplexes and their functions. These mimics have shown promise in treating cancers. Nucleotide chemical modifications of microRNA mimics have been investigated and have improved the stability of miRNA mimics. However, the potential therapeutic benefit of mimic analogs based on sequence modifications has not been explored. miR-506-3p was identified as a differentiation-inducing microRNA in neuroblastoma cells, suggesting the potential of applying the miR-506-3p mimic in neuroblastoma differentiation therapy. In this study, we explored the possibility of developing shortened miR-506-3p analogs that can maintain differentiation-inducing activities comparable to the wild-type miR-506-3p mimic. We found that deleting up to two nucleotides at either the 3' end or within the middle region of the miR-506-3p sequence fully maintained the differentiation-inducing activity when compared to the wild-type mimic. Deleting up to four nucleotides from the 3' end or deleting three nucleotides in the middle positions diminished the differentiation-inducing activity, but the analogs still maintained differentiation-inducing activities that were significantly higher than the negative control oligo. The shortened analog designs potentially benefit patients from two perspectives: (1) the reduced cost of manufacturing shortened analogs, and (2) the reduced non-specific toxicity due to their smaller molecular sizes.

Synthesis and biological evaluation of simplified ajudazol derivatives reveal potent 5-lipoxygenase inhibition and considerable apoptotic activity in neuroblastoma cells

Simplified analogues of the myxobacterial polyketide ajudazol were obtained by synthesis and evaluated for their biological activities. Potent simplified 5-lipoxygenase inhibitors were identified. Moreover, strong antiproliferative and apoptotic activities were observed in brain cancer cell lines at low nano- to micromolar concentrations.

Naxitamab Activity in Neuroblastoma Cells Is Enhanced by Nanofenretinide and Nanospermidine

Neuroblastoma cells highly express the disialoganglioside GD2, a tumor-associated carbohydrate antigen, which is also expressed in neurons, skin melanocytes, and peripheral nerve fibers. Immunotherapy with monoclonal anti-GD2 antibodies has a proven efficacy in clinical trials and is included in the standard treatment for children with high-risk neuroblastoma. However, the strong neuro-toxicity associated with anti-GD2 antibodies administration has hindered, until now, the possibility for dose-escalation and protracted use, thus restraining their therapeutic potential. Strategies to increase the efficacy of anti-GD2 antibodies are actively sought, with the aim to enable chronic treatments that could eradicate minimal residual disease and subsequent relapses, often occurring after treatment. Here, we report that Nanofenretinide and Nanospermidine improved the expression of GD2 in neuroblastoma cells (CHP-134) and provided different effects in combination with the anti-GD2 antibody naxitamab. In particular, Nanofenretinide significantly increased the cytotoxic effect of naxitamab while Nanospermidine inhibited cell motility at extents proportional to naxitamab concentration. In neuroblastoma cells characterized by a low and heterogeneous basal expression of GD2, such as SH-SY5Y, which may represent the cell heterogeneity in tumors after chemotherapy, both Nanofenretinide and Nanospermidine increased GD2 expression in approximately 50% of cells, thus shifting the tumor population towards improved sensitivity to anti-GD2 antibodies.

Inhibition of PI3 kinase isoform p110α suppresses neuroblastoma growth and induces the reduction of Anaplastic Lymphoma Kinase

BackgroundIn neuroblastoma, hyperactivation of the PI3K signaling pathway has been correlated with aggressive neuroblastomas, suggesting PI3Ks as promising targets for the treatment of neuroblastoma. However, the oncogenic roles of individual PI3K isoforms in neuroblastoma remain elusive.ResultsWe found that PI3K isoform p110α was expressed at higher levels in neuroblastoma tissues compared with normal tissues, and its high expression was correlated with an unfavorable prognosis of neuroblastoma. Accordingly, PI3K activation in neuroblastoma cells was predominantly mediated by p110α but not by p110β or p110δ. Suppression of p110α inhibited the growth of neuroblastoma cells both in vitro and in vivo, suggesting a crucial role of p110α in the tumorigenesis of neuroblastoma. Mechanistically, inhibition of p110α decreased anaplastic lymphoma kinase (ALK) in neuroblastoma cells by decreasing its protein stability.ConclusionsIn this study, we investigated the oncogenic roles of PI3K isoforms in neuroblastoma. Our data shed light on PI3K isoform p110α in the tumorigenesis of neuroblastoma, and strongly suggest the p110α inhibitors as potential drugs in treating neuroblastoma.

Inhibiting Lysine Demethylase 1A Improves L1CAM-Specific CAR T Cell Therapy by Unleashing Antigen-Independent Killing via the FAS-FASL Axis.

Simple SummarySolid tumor cells can lose or heterogeneously express antigens to become resistant to chimeric antigen receptor (CAR) T cell therapy. Here, we explore whether epigenetic manipulation to unleash antigen-independent killing mechanisms can overcome this hurdle. KDM1A is overexpressed in many cancers and removes lysine methylation on histones that keeps the DNA firmly packed to selectively activate or repress gene activity, depending on the specific lysine target. KDM1A also regulates the expression of nonhistone proteins. We inhibited KDM1A in the childhood tumor, neuroblastoma, to increase FAS expression on tumor cells. The FAS receptor can be triggered to induce cell death when bound by the FAS ligand on CAR and other activated T cells present in the tumor environment, even if the tumor cells lack the target antigen. FAS upregulation via KDM1A inhibition sensitized neuroblastoma cells to FAS-FASL-mediated killing and augmented CAR T cell therapy against antigen-poor or even antigen-negative neuroblastoma. Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising treatment strategy, however, therapeutic success against solid tumors such as neuroblastoma remains modest. Recurrence of antigen-poor tumor variants often ultimately results in treatment failure. Using antigen-independent killing mechanisms such as the FAS receptor (FAS)-FAS ligand (FASL) axis through epigenetic manipulation may be a way to counteract the escape achieved by antigen downregulation. Analysis of public RNA-sequencing data from primary neuroblastomas revealed that a particular epigenetic modifier, the histone lysine demethylase 1A (KDM1A), correlated negatively with FAS expression. KDM1A is known to interact with TP53 to repress TP53-mediated transcriptional activation of genes, including FAS. We showed that pharmacologically blocking KDM1A activity in neuroblastoma cells with the small molecule inhibitor, SP-2509, increased FAS cell-surface expression in a strictly TP53-dependent manner. FAS upregulation sensitized neuroblastoma cells to FAS-FASL-dependent killing and augmented L1CAM-directed CAR T cell therapy against antigen-poor or even antigen-negative tumor cells in vitro. The improved therapeutic response was abrogated when the FAS-FASL interaction was abolished with an antagonistic FAS antibody. Our results show that KDM1A inhibition unleashes an antigen-independent killing mechanism via the FAS-FASL axis to make tumor cell variants that partially or totally suppress antigen expression susceptible to CAR T cell therapy.

Extending the Applicability of In Ovo and Ex Ovo Chicken Chorioallantoic Membrane Assays to Study Cytostatic Activity in Neuroblastoma Cells.

PurposeThe chick chorioallantoic membrane (CAM) assay can provide an alternative versatile, cost-effective, and ethically less controversial in vivo model for reliable screening of drugs. In the presented work, we demonstrate that CAM assay (in ovo and ex ovo) can be simply employed to delineate the effects of cisplatin (CDDP) and ellipticine (Elli) on neuroblastoma (Nbl) cells in terms of their growth and metastatic potential.MethodsThe Nbl UKF-NB-4 cell line was established from recurrent bone marrow metastases of high-risk Nbl (stage IV, MYCN amplification, 7q21 gain). Ex ovo and in ovo CAM assays were optimized to evaluate the antimetastatic activity of CDDP and Elli. Immunohistochemistry, qRT-PCR, and DNA isolation were performed.ResultsEx ovo CAM assay was employed to study whether CDDP and Elli exhibit any inhibitory effects on growth of Nbl xenograft in ex ovo CAM assay. Under the optimal conditions, Elli and CDDP exhibited significant inhibition of the size of the primary tumor. To study the efficiency of CDDP and Elli to inhibit primary Nbl tumor growth, intravasation, and extravasation in the organs, we adapted the in ovo CAM assay protocol. In in ovo CAM assay, both studied compounds (CDDP and Elli) exhibited significant (p < 0.001) inhibitory activity against extravasation to all investigated organs including distal CAM.ConclusionsTaken together, CAM assay could be a helpful and highly efficient in vivo approach for high-throughput screening of libraries of compounds with expected anticancer activities.

Discovery of neuroprotective agents that inhibit human prolyl hydroxylase PHD2

Prolyl hydroxylase (PHD) enzymes play a critical role in the cellular responses to hypoxia through their regulation of the hypoxia inducible factor α (HIF-α) transcription factors. PHD inhibitors show promise for the treatment of diseases including anaemia, cardiovascular disease and stroke. In this work, a pharmacophore-based virtual high throughput screen was used to identify novel potential inhibitors of human PHD2. Two moderately potent new inhibitors were discovered, with IC50 values of 4 μM and 23 μM respectively. Cell-based studies demonstrate that these compounds exhibit protective activity in neuroblastoma cells, suggesting that they have the potential to be developed into clinically useful neuroprotective agents.

Neuroinflammation induces synaptic scaling through IL-1β-mediated activation of the transcriptional repressor REST/NRSF

Neuroinflammation is associated with synapse dysfunction and cognitive decline in patients and animal models. One candidate for translating the inflammatory stress into structural and functional changes in neural networks is the transcriptional repressor RE1-silencing transcription factor (REST) that regulates the expression of a wide cluster of neuron-specific genes during neurogenesis and in mature neurons. To study the cellular and molecular pathways activated under inflammatory conditions mimicking the experimental autoimmune encephalomyelitis (EAE) environment, we analyzed REST activity in neuroblastoma cells and mouse cortical neurons treated with activated T cell or microglia supernatant and distinct pro-inflammatory cytokines. We found that REST is activated by a variety of neuroinflammatory stimuli in both neuroblastoma cells and primary neurons, indicating that a vast transcriptional change is triggered during neuroinflammation. While a dual activation of REST and its dominant-negative splicing isoform REST4 was observed in N2a neuroblastoma cells, primary neurons responded with a pure full-length REST upregulation in the absence of changes in REST4 expression. In both cases, REST upregulation was associated with activation of Wnt signaling and increased nuclear translocation of β-catenin, a well-known intracellular transduction pathway in neuroinflammation. Among single cytokines, IL-1β caused a potent and prompt increase in REST transcription and translation in neurons, which promoted a delayed and strong synaptic downscaling specific for excitatory synapses, with decreased frequency and amplitude of spontaneous synaptic currents, decreased density of excitatory synaptic connections, and decreased frequency of action potential-evoked Ca2+ transients. Most important, the IL-1β effects on excitatory transmission were strictly REST dependent, as conditional deletion of REST completely occluded the effects of IL-1β activation on synaptic transmission and network excitability. Our results demonstrate that REST upregulation represents a new pathogenic mechanism for the synaptic dysfunctions observed under neuroinflammatory conditions and identify the REST pathway as therapeutic target for EAE and, potentially, for multiple sclerosis.

Prosaposin and its receptors GRP37 and GPR37L1 show increased immunoreactivity in the facial nucleus following facial nerve transection.

Neurotrophic factor prosaposin (PS) is a precursor for saposins A, B, C, and D, which are activators for specific sphingolipid hydrolases in lysosomes. Both saposins and PS are widely contained in various tissues. The brain, skeletal muscle, and heart cells predominantly contain unprocessed PS rather than saposins. PS and PS-derived peptides stimulate neuritogenesis and increase choline acetyltransferase activity in neuroblastoma cells and prevent programmed cell death in neurons. We previously detected increases in PS immunoactivity and its mRNA in the rat facial nucleus following facial nerve transection. PS mRNA expression increased not only in facial motoneurons, but also in microglia during facial nerve regeneration. In the present study, we examined the changes in immunoreactivity of the PS receptors GPR37 and GPR37L1 in the rat facial nucleus following facial nerve transection. Following facial nerve transection, many small Iba1- and glial fibrillary acidic protein (GFAP)-positive cells with strong GPR37L1 immunoreactivity, including microglia and astrocytes, were observed predominately on the operated side. These results indicate that GPR37 mainly works in neurons, whereas GPR37L1 is predominant in microglia or astrocytes, and suggest that increased PS in damaged neurons stimulates microglia or astrocytes via PS receptor GPR37L1 to produce neurotrophic factors for neuronal recovery.

The effects of MgS nanoparticles-Cisplatin-bio-conjugate on SH-SY5Y neuroblastoma cell line.

Magnesium sulfide nanoparticles (MgS NPs) is a nanomaterial that has an important place in diagnosis, treatment, diagnosis, and drug delivery systems. Neuroblastoma, a type of brain cancer, is an extremely difficult cancer to treat with today's treatment options. This study was carried out to determine the cytotoxic, oxidant, and antioxidant effects on the neuroblastoma cancer line (SH-SY5Y cell line) along with the green synthesis and characterization of MgS NPs structures. MgS NPs were synthesized by green synthesis using Na2S and Punica granatum, a cleaner method for toxic effects, and characterized using Scanning Electron Microscopy, Fourier Transform Infrared spectroscopy, X-Ray diffraction methods. In cell culture, SH-SY5Y cells were grown in a suitable nutrient medium under favorable conditions. Five different doses of MgS NPs (10, 25, 50, 75, and 100µg/mL) were applied to the cell line for 24h. The analysis of the MgS NPs applications was performed with MTT cytotoxicity test and total oxidant and total antioxidant tests. According to the data obtained, 75μg/mL MgS NPs application decreased cancer cell viability up to 48.54%. MgS NPs exhibited a dose-dependent effect on the SH-SY5Y cell line. Also, it was determined that MgS NPs increased oxidant activity in neuroblastoma cells, which was compatible with the cytotoxicity test. As a result, MgS NPs exhibited an effective activity on the neuroblastoma cell line. It was clearly seen that NPs obtained by green synthesis prevented the related cancer line from proliferating.

The Potential Functional Roles of NME1 Histidine Kinase Activity in Neuroblastoma Pathogenesis.

Neuroblastoma is the most common extracranial solid tumor in childhood. Gain of chromosome 17q material is found in >60% of neuroblastoma tumors and is associated with poor patient prognosis. The NME1 gene is located in the 17q21.3 region, and high NME1 expression is correlated with poor neuroblastoma patient outcomes. However, the functional roles and signaling activity of NME1 in neuroblastoma cells and tumors are unknown. NME1 and NME2 have been shown to possess histidine (His) kinase activity. Using anti-1- and 3-pHis specific monoclonal antibodies and polyclonal anti-pH118 NME1/2 antibodies, we demonstrated the presence of pH118-NME1/2 and multiple additional pHis-containing proteins in all tested neuroblastoma cell lines and in xenograft neuroblastoma tumors, supporting the presence of histidine kinase activity in neuroblastoma cells and demonstrating the potential significance of histidine kinase signaling in neuroblastoma pathogenesis. We have also demonstrated associations between NME1 expression and neuroblastoma cell migration and differentiation. Our demonstration of NME1 histidine phosphorylation in neuroblastoma and of the potential role of NME1 in neuroblastoma cell migration and differentiation suggest a functional role for NME1 in neuroblastoma pathogenesis and open the possibility of identifying new therapeutic targets and developing novel approaches to neuroblastoma therapy.

Ginkgo biloba Prevents Oxidative Stress-Induced Apoptosis Blocking p53 Activation in Neuroblastoma Cells.

Oxidative stress has been associated to neuronal cell loss in neurodegenerative diseases. Neurons are post-mitotic cells that are very sensitive to oxidative stress—especially considering their limited capacity to be replaced. Therefore, reduction of oxidative stress, and inhibiting apoptosis, will potentially prevent neurodegeneration. In this study, we investigated the neuroprotective effect of Ginkgo biloba extract (EGb 761) against H2O2 induced apoptosis in SK-N-BE neuroblastoma cells. We analysed the molecular signalling pathway involved in the apoptotic cell death. H2O2 induced an increased acetylation of p53 lysine 382, a reduction in mitochondrial membrane potential, an increased BAX/Bcl-2 ratio and consequently increased Poly (ADP-ribose) polymerase (PARP) cleavage. All these effects were blocked by EGb 761 treatment. Thus, EGb 761, acting as intracellular antioxidant, protects neuroblastoma cells against activation of p53 mediated pathway and intrinsic mitochondrial apoptosis. Our results suggest that EGb 761, protecting against oxidative-stress induced apoptotic cell death, could potentially be used as nutraceutical for the prevention and treatment of neurodegenerative diseases.

Combination of Rapamycin and MK-2206 Induced Cell Death via Autophagy and Necroptosis in MYCN-Amplified Neuroblastoma Cell Lines.

Neuroblastoma (NB) is the most common pediatric malignant extracranial solid tumor. Despite multi-modality therapies, the emergence of drug resistance is an obstacle in the treatment of high-risk NB patients (with MYCN amplification). In our previous study, we found that rapamycin and MK-2206 synergistically induced cell death in MYCN-amplified cell lines but the mechanisms remained unclear. In our present study, either 3-MA or necroatatin-1 blocked the cell death induced by rapamycin and MK-2206, but z-VAD-fmk did not block this cell death. The expressions of autophagy markers (ATG5, ATG7, Beclin-1, LC3 B) and the necroptosis marker RIPK3 increased and another necroptosis marker RIPK1 decreased after the combination treatment of rapamycin and MK-2206, and were accompanied by the morphological characteristics of autophagy and necroptosis. In NB xenograft tumor tissues, the expressions of autophagy and necroptosis markers were consistent with observations in vitro. These data suggested that autophagy and necroptosis contributed to the cell death induced by rapamycin and MK-2206 in NB cells. To understand the role of MYCN in this process, MYCN expression was downregulated in MYCN-amplified cell lines (NGP, BE2) using siRNAs and was upregulated in MYCN non-amplified cell lines (AS, SY5Y) using plasmid. We found the cell death induced by rapamycin and MK-2206 was MYCN-dependent. We also found that the metabolic activity in NB cells was correlated with the expression level of MYCN. This study delineates the role of MYCN in the cell death induced by combination treatment of rapamycin and MK-2206 in MYCN-amplified NB cells.

SIRT1 and AROS suppress doxorubicin-induced apoptosis via inhibition of GSK3β activity in neuroblastoma cells

ABSTRACT SIRT1, the best-characterized member of the sirtuin family of deacetylases, is involved in cancer, apoptosis, inflammation, and metabolism. Active regulator of SIRT1 (AROS) was the first identified direct regulator of SIRT1. An increasing number of reports have indicated that SIRT1 plays an important role in controlling brain tumors. Here, we demonstrated that depletion of SIRT1 and AROS increases doxorubicin-mediated apoptosis in human neuroblastoma SH-SY5Y cells. Glycogen synthase kinase 3β (GSK3β) promoted doxorubicin-mediated apoptosis, but this effect was abolished by overexpression of SIRT1 and AROS. Interestingly, SIRT1 and AROS interacted with GSK3β and increased inhibitory phosphorylation of GSK3β on Ser9. Finally, we determined that AROS cooperates with SIRT1 to suppress GSK3β acetylation. Taken together, our results suggest that SIRT1 and AROS inhibit GSK3β activity and provide additional insight into drug resistance in the treatment of neuroblastoma.

Reduction of aluminum ion neurotoxicity through a small peptide application – NAP treatment of Alzheimer's disease

Alzheimer's disease (AD) is the most common cause of dementia in late life. It is difficult to precisely diagnose AD at early stages, making biomarker search essential for further developments. The objective of this study was to identify protein biomarkers associated with aluminum ions toxicity (AD-like toxicity) in a human neuroblastoma cell model, SH-SY5Y and assess potential prevention by NAP (NAPVSIPQ). Complete proteomic techniques were implemented. Four proteins were identified as up-regulated with aluminum ion treatment, CBP80/20-dependent translation initiation factor (CTIF), Early endosome antigen 1 (EEA1), Leucine-rich repeat neuronal protein 4 (LRRN4) and Phosphatidylinositol 3-kinase regulatory subunit beta (PI3KR2). Of these four proteins, EEA1 and PI3KR2 were down-regulated after NAP-induced neuroprotective activity in neuroblastoma cells. Thus, aluminum ions may increase the risk for neurotoxicity in AD, and the use of NAP is suggested as a treatment to provide additional protection against the effects of aluminum ions, via EEA1 and PI3KR2, associated with sorting and processing of the AD amyloid precursor protein (APP) through the endosomal system.

PO-124 PTTG and PBF functionally interact with P53 and predict overall survival in head and neck cancer

IntroductionReceptor tyrosine kinases (RTKs) play important roles in cellular proliferation and differentiation. In addition, RTKs reveal oncogenic potential when their kinase activities are constitutively enhanced by point mutation, amplification, or rearrangement of the corresponding genes. We are investigating ALK (Anaplastic Lymphoma Kinase) tyrosine kinase in neuroblastoma (NB) - the most frequently occurring extracranial tumour in children, where ALK activating mutations are found.In this study we performed phosphoproteomic and a gene signature profiling of ALK activity from several different neuroblastoma cells comparing treatment with the first and third generations ALK inhibitors.Material and methodsCrizotinib was one of the first ALK TKIs to be described; however the response in patients with ALK positive NB was poor. In contrast, robust and sustained clinical responses we seen in ALK rearranged IMT. Lorlatinib is a third generation ALK TKI with very positive responses in NSCLC. We treated two ALK addicted (CLB-BAR, CLB-GE) and one ALK non-addicted (SK-N-AS) NB cell lines with either crizotinib or lorlatinib and performed phosphoproteomic and RNAseq profiling to identify common targets for future combinational treatment for patients.Results and discussionsProfiling of ALK addicted CLB-BAR and CLB-GE cell lines led to the identification of 3345 and 2252 phosphoproteins, respectively. Phosphorylation of the ALK receptor itself was decreased in both lines upon treatment with crizotinib or lorlatinib. Parallel RNAseq profiling was performed at 24 hours. Comparison with non-treated controls revealed 19 232 differently expressed genes. In ALK addicted cells phosphoproteomic analysis revealed more than 50 proteins that were dephosphorylated upon treatment with ALK TKIs. More than 400 genes were downregulated, and more than 600 were upregulated, upon treatment with either crizotinib or lorlatinib. As an outcome we obtained a list of predicted targets for further analysis. We focused on validation of downstream signalling molecules such as: FOXO3a/4, DUSP4 and ETV3/4.ConclusionIn this study we established and compared phosphoproteomic and gene signature profiles of ALK activity in NB cells employing first and third generation ALK TKIs. This analysis has unveiled a number of leads for novel combinatorial treatment strategies for NB patients as well as an increased understanding ALK dependant signalling processes.

Sphingadienes show therapeutic efficacy in neuroblastoma in vitro and in vivo by targeting the AKT signaling pathway.

Neuroblastoma is a childhood malignancy that accounts for approximately 15% of childhood cancer deaths. Only 20-35% of children with metastatic neuroblastoma survive with standard therapy. Identification of more effective therapies is essential to improving the outcome of children with high-stage disease. Sphingadienes (SD) are growth-inhibitory sphingolipids found in natural sources including soy. They exhibit chemopreventive activity in mouse models of colon cancer, where they mediate cytotoxicity by inhibiting key pro-carcinogenic signaling pathways. In this study, the effect of SD on neuroblastoma was analyzed. Low micromolar concentrations of SD were cytotoxic to transformed and primary neuroblastoma cells independently of N-Myc amplification status. SD induced both caspase-dependent apoptosis and autophagy in neuroblastoma cells. However, only inhibition of caspase-dependent apoptosis protected neuroblastoma cells from SD-mediated cytotoxicity. SD also inhibited AKT activation in neuroblastoma cells as shown by reduced phosphorylated AKT levels. Pre-treatment with insulin attenuated SD-mediated cytotoxicity in vitro. SD-loaded nanoparticles (NP) administered parenterally to immunodeficient mice carrying neuroblastoma xenografts resulted in cytotoxic levels of SD in the circulation and significantly reduced tumor growth compared to vehicle-treated controls. Analysis of tumor extracts demonstrated reduced AKT activation in tumors of mice treated with SD-NP compared to controls treated with empty NP. Our findings indicate SD are novel potential chemotherapeutic agents that promote neuroblastoma cell death and reduce tumorigenicity in vivo.

Inability of DNAzymes to cleave RNA in vivo is due to limited Mg[Formula: see text] concentration in cells.

Sequence specific cleavage of RNA can be achieved by hammerhead ribozymes as well as DNAzymes. They comprise a catalytic core sequence flanked by regions that form double strands with complementary RNA. While different types of ribozymes have been discovered in natural organisms, DNAzymes derive from in vitro selection. Both have been used for therapeutic down-regulation of harmful proteins by reducing drastically the corresponding mRNA concentration. A priori DNAzymes appear advantageous because of the higher haemolytic stability and better cost effectiveness when compared to RNA. In the present work the 10-23 DNAzyme was applied to knockdown expression of the prion protein (PrP), the sole causative agent of transmissible spongiform encephalopathies. We selected accessible target sequences on the PrP mRNA based on a sequential folding algorithm. Very high effectivity of DNAzymes was found for cleavage of RNA in vitro, but activity in neuroblastoma cells was very low. However, siRNA directed to the identical target sequences reduced expression of PrP in the same cell type. According to our analysis, three Mg[Formula: see text] bind cooperatively to the DNAzyme to exert full activity. However, free ATP binds the Mg[Formula: see text] ions more strongly and already stoichiometric amounts of Mg[Formula: see text] and ATP inhibited the activity of DNAzymes drastically. In contrast, natural ribozymes form three-dimensional structures close to the cleavage site that stabilize the active conformation at much lower Mg[Formula: see text] concentrations. For DNAzymes, however, a similar stabilization is not known and therefore DNAzymes need higher free Mg[Formula: see text] concentrations than that available inside the cell.

Tumor-associated macrophages promote neuroblastoma via STAT3 phosphorylation and up-regulation of c-MYC.

Tumor-associated macrophages (TAMs) are strongly associated with poor survival in neuroblastomas that lack MYCN amplification. To study TAM action in neuroblastomas, we used a novel murine model of spontaneous neuroblastoma lacking MYCN amplification, and observed recruitment and polarization of TAMs, which in turn enhanced neuroblastoma proliferation and growth. In both murine and human neuroblastoma cells, we found that TAMs increased STAT3 activation in neuroblastoma cells and transcriptionally up-regulated the MYC oncogene. Analysis of human neuroblastoma tumor specimens revealed that MYC up-regulation correlates with markers of TAM infiltration. In an IL6ko neuroblastoma model, the absence of IL-6 protein had no effect on tumor development and prevented neither STAT3 activation nor MYC up-regulation. In contrast, inhibition of JAK-STAT activation using AZD1480 or the clinically admissible inhibitor ruxolitinib significantly reduced TAM-mediated growth of neuroblastomas implanted subcutaneously in NOD scid gamma mice. Our results point to a unique mechanism in which TAMs promote tumor cells that lack amplification of an oncogene common to the malignancy by up-regulating transcriptional expression of a distinct oncogene from the same gene family, and underscore the role of IL-6-independent activation of STAT3 in this mechanism. Amplification of MYCN or constitutive up-regulation of MYC protein is observed in approximately half of high-risk tumors; our findings indicate a novel role of TAMs as inducers of MYC expression in neuroblastomas lacking independent oncogene activation.