Neuroblastoma Cell Death Research Articles

Evaluation of TRPA1 as a Therapeutic Target in MYCN-Amplified Neuroblastoma.

Neuroblastoma (NB) is a childhood cancer with a high relapse rate despite intensive treatment. TRPA1 is a pain-sensing ion channel with downstream impacts on proliferative and pro-apoptotic pathways. Here, we evaluated TRPA1 expression in NB and performed pharmacological inhibition in preclinical models to assess its potential as a therapeutic target in NB. TRPA1 protein levels were assessed in NB patient tumors on tissue microarrays. Bulk and single-cell gene expression data were retrieved from publicly available databases. The effects of three TRPA1 inhibitors (AP-18, A967079, and Bay 390) on NB cell viability and cell death were evaluated using NB patient-derived xenograft (PDX)-derived organoids. In vivo testing was performed in a MYCN-amplified NB PDX model. Drug combination testing was performed using combination or sequential treatments and evaluated using drug synergy scores. TRPA1 is widely expressed in NB patient tumors and preclinical patient-derived NB models. Pharmacological TRPA1 inhibition decreased NB cell viability and increased cell death. In vivo TRPA1 inhibition alone did not significantly affect NB tumor growth. Pretreatment with TRPA1 inhibition prior to chemotherapy resulted in synergistic effects in vitro. TRPA1 is expressed in NB tumors, and pharmacological TRPA1 inhibition can be effective in vitro and synergistic when used as pretreatment to chemotherapy. However, the tested inhibitors did not show in vivo efficacy, at least as monotherapy.

Selective targeting of genome amplifications and repeat elements by CRISPR-Cas9 nickases to promote cancer cell death

Focal gene amplification serves as an oncogenic driver during tumorigenesis and is a hallmark of many forms of cancer. Oncogene amplifications promote genomic instability, which is integral to cancer cell survival and evolution. However, focal gene amplification potentially affords an opportunity for therapeutic exploitation. As a proof-of-concept, we leverage CRISPR-Cas9 nickase to selectively promote cancer cell death in MYCN-amplified neuroblastoma in a gene amplification-dependent manner. Our analysis demonstrates that CRISPR-Cas9 nickase can generate a lethal number of highly toxic, replication-dependent double-strand breaks in cells harboring amplified loci. Furthermore, we demonstrate that Cas9 nickase—mediated toxicity can be modulated in combination with small molecule inhibitors targeting key regulators of the DNA-damage response or cell death pathways. Importantly, our findings in MYCN-amplified neuroblastoma translate to other cancer types with distinct oncogene amplifications.

In Vivo Antitumor Activity of Allicin in a Pediatric Neuroblastoma Patient‐derived Xenograft (PDX) Mouse Model

Background/AimAllicin is a small-molecule natural product found in garlic (Allium sativum). We previously showed that allicin inhibits ornithine decarboxylase (ODC) in vitro and induces apoptotic cell death in pediatric neuroblastoma (NB) cancer cell cultures. However, its potency as an anticancer agent in vivo has not been sufficiently explored.Materials and MethodsIn this study, we used cell proliferation assays, immunoblotting techniques, and light microscopy to study NB tumor cell cultures and human primary neonatal skin fibroblast control cells as well as a MYCN-amplified NB patient-derived xenograft (PDX) mouse tumor model to study the efficacy of allicin in vivo.ResultsAllicin strongly inhibits NB tumor cell proliferation in a dose-dependent manner while non-cancerous human primary neonatal skin fibroblast control cells were largely unaffected. Importantly, two intra-tumoral injections of allicin over a two-week trial period significantly reduced the NB tumor burden in mice compared to controls (N=4-9 mice/group). Excised tumor tissues revealed that allicin treatment increased the cyclin-dependent kinase inhibitor p27Kip1 protein levels, suggesting that in vivo, allicin increases p27Kip1-mediated G1/S cell cycle arrest.ConclusionOur findings warrant further preclinical development of allicin as a potential anticancer agent, especially for those types of cancers that are treatable by intra-tumoral injections, including neuroblastoma, glioblastoma, and medulloblastoma.

Effects of herbaceous bioflavonoid herbacetin on oxidative stress, and alpha-synuclein regulation, programmed cell death in a Parkinson illness

Background Herbacetin, a flavonoid present in many types of herbs, which include linaceae, ephedraceae, and crassulaceae, exhibits a range of medicinal properties. 1-methyl-4-phenylpyridinium (MPP+) is one of the neurotoxins used in cell-based Parkinson’s disease (PI) models. Whereas the precise chemical mechanism of iron association with free radical cell damage and apoptosis is yet unknown, intracellular irons are a key factor for MPP+-derived apoptosis. Methods We examine whether the antiapoptotic properties of flaxseed bioflavonoid herbacetin (HB) are associated with the stimulation of the intrinsic caspase-dependent pathway and exposing of MPP+ caused neuronal death in the human dopaminergic neuroblastoma cells. Four groups were created out of the cells. Groups I, II, III, and IV are the control, HB+MPP+, MPP+, and HB, respectively. Following a 24-hour incubation period, the cells were subjected to several parameters. Results We discovered in neuroblastoma cells that HB dramatically reduced the cell death induced by MPP+. Additionally, HB significantly reduced the formation of ROS and counteracted the reduction in MMP resulting from MPP+ treatment. HB reduces the stimulation of the intrinsic caspase-dependent apoptotic mechanism and suppresses the MPP+-mediated apoptotic signalling pathway. Furthermore, HB predicted a better binding interaction with alpha-synuclein and drastically decreased alpha-synuclein expression and accumulation in neuroblastoma cells. Conclusion Consequently, our findings imply that HB shields neurons by reducing oxidative stress, alpha-synuclein misfolding in neuroblastoma, and apoptosis prompts the death of neuroblastoma cells.

Abstract 1634: The role of the deubiquitinase USP7 and the E3/E4 ubiquitin ligase complex ITCH/UBE4B in the regulation of cell death in neuroblastoma

Abstract Dysregulation of the Ubiquitin Proteasome System has been linked to many human diseases, including cancer. Expression of UBE4B ubiquitin ligase is associated with neuroblastoma patient outcomes and its functional roles in neuroblastoma pathogenesis are not known. We have recently identified an ubiquitin ligase complex ITCH/UBE4B which mediates ubiquitination of Ku70 and c-FLIPL proteins for proteasomal degradation allowing HDAC inhibitor-mediated caspase-8 dependent apoptosis. We also confirmed that the deubiquitinase USP7, critical player in tumor suppression and DNA repair, can be a potential therapeutic target for neuroblastoma. Highly selective USP7 inhibitors have demonstrated significant antitumor activity in preclinical models of adult cancer, and we reported that these inhibitors alone can be more effective against neuroblastoma tumor growth. Our hypothesis is that USP7 inhibition will be more effective against neuroblastoma tumors through destabilization of ITCH/UBE4B complex protein targets allowing a stronger induction of cell death in response to treatment for the children. To evaluate efficacy of USP7 inhibitors in combination with HDAC inhibitors or chemotherapy against neuroblastoma tumor growth, neuroblastoma cell lines depleted for UBE4B or USP7 were treated with increasing concentrations of USP7 inhibitors alone or in combination with chemotherapy or with HDAC inhibitors. Cell proliferation was measured using continuous live cell imaging and apoptosis by caspase cleavage and PARP cleavage detection by western blot. We have evaluated also, whether ubiquitination/deubiquitination and degradation rates of p53, Ku70 and c-FLIPL proteins could modulate induction of apoptosis and necroptosis. USP7 inhibition resulted in decreases in cell viability in p53 wild-type neuroblastoma cell lines via the induction of apoptosis and necroptosis by increasing phosphorylation of MLKL, RIPK3 and RIPK1. In UBE4B or USP7 depleted cells, a huge induction of necroptosis and a small rate of apoptosis were observed. We also identified USP7 as a deubiquitinase of Ku70 and c-FLIPL, stabilizing the Ku70/c-FLIPL/Bax “FLIPosome” complex. USP7 inhibition destabilizes ku70 and c-FLIPL for protein degradation leading to induction of apoptosis as well as necroptosis. UBE4B depletion in neuroblastoma inhibits HDAC inhibitors mediated caspase-8 mediated apoptosis but enhances necroptosis, notably because the stabilization of the FLIPosome Ku70-c-FLIPL can allow the blockade of caspase-8 activation and can trigger the activation of the necroptosome. Our data suggests that USP7 and ITCH/UBE4B can control the switch between apoptosis and necroptosis in response to USP7 and HDAC inhibitors. USP7 inhibition and ITCH/UBE4B activation by HDAC inhibitors could be a promising therapeutic strategy for children with high-risk and relapsed neuroblastoma. Citation Format: Christophe Le Clorennec, Carla Sampaio, Peter E. Zage. The role of the deubiquitinase USP7 and the E3/E4 ubiquitin ligase complex ITCH/UBE4B in the regulation of cell death in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1634.

Abstract 7038: Direct targeting of DNMT1 inhibits high-risk neuroblastoma growth through epigenetic reprogramming

Abstract DNA Methyltransferase 1 (DNMT1) mediated hypermethylation represents a crucial epigenetic modification implicated in neuroblastoma (NB), a devastating pediatric cancer with a 60% relapse rate. DNMT1, a prominent member of the DNA methyltransferase family of enzymes, catalyzes the transfer of methyl groups to the 5-carbon position of cytosine residues within DNA sequences. While extensive research has established that hypermethylation of CpG islands leads to transcriptional silencing of tumor suppressor genes across various cancer types, the specific molecular mechanisms and therapeutic implications of DNMT1-mediated hypermethylation in NB remained largely unexplored. In this study, we analyzed the publicly available R2 genomic dataset, which revealed the direct correlation between DNMT1 overexpression and decreased overall NB patient survival rates. Further, we performed a small molecule screening and identified a highly potent and selective DNMT1 inhibitor. Therapeutic targeting of DNMT1 with this inhibitor resulted in significant dose-dependent reductions in multiple hallmarks of cancer progression, including overall cell proliferation, colony formation capacity, and 3D spheroid development. Furthermore, the DNMT1 inhibitor induced cell cycle arrest in the G2/M phase checkpoint and significantly enhanced apoptotic cell death in NB cells. These promising in vitro findings were validated through in vivo tumor xenograft studies, where the DNMT1 inhibitor significantly inhibited NB tumor growth without any observable toxicity in preclinical models. Further, we employed MethylFlash technology to quantify 5-methylcytosine (5-mc) levels, which verified a significant reduction (p < 0.0001) in global DNA methylation status following exposure to increasing concentrations (5, 10, and 20 µM) of the DNMT1 inhibitor. Further molecular analyses revealed profound effects on vital gene and protein expression patterns alongside an enhancement of NB cell sensitivity to conventional chemotherapeutic agents. These findings collectively establish DNMT1 inhibition as a promising therapeutic strategy for NB patients. Our data demonstrates the role of DNMT1-mediated hypermethylation in NB tumorigenesis while establishing that targeted DNMT1 inhibition effectively reduces hypermethylation, thereby suppressing malignant proliferation, inhibiting colony formation, and preventing both 3D spheroid development and in vivo tumor growth. Our data also highlights that the direct DNMT1 targeting approach is a promising epigenetic-based approach for NB treatment. Further mechanistic research of DNMT1 inhibition in NB progression is essential to facilitate the clinical translation of this promising therapeutic strategy. Citation Format: Danielle C. Rouse, Saurabh Agarwal. Direct targeting of DNMT1 inhibits high-risk neuroblastoma growth through epigenetic reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7038.

Abstract 4238: DNMT1 inhibition by SGI-1027 promotes neuroblastoma cell death through a p53-independent pathway

Abstract Introduction: Neuroblastoma is an aggressive childhood cancer originating from neural crest cells, accounting for 700-800 new cases annually and cuases15% of pediatric cancer deaths in the U.S. Treatment for high-risk neuroblastoma includes chemotherapy, stem cell transplants, radiation, and immunotherapy. Recent evidence suggests that DNMTs inhibitors such as SGI-1027 and CM-272 can potentiate apoptosis via epigenetic modulation of critical regulatory pathways in cancers. Hence, we hypothesized that SGI-1027 can induce apoptosis in SK-N-SH neuroblastoma cells by upregulating p21 through DNMT1 inhibition. Our study aimed to elucidate the underlying mechanisms and assess the potential of using SGI-1027 for treating high-risk neuroblastoma. Methods: SK-N-SH cells were treated with 2 µM of SGI -1027 for 24 hrs. The cell viability was evaluated using Trypan Blue Dye Exclusion method, while apoptotic activity was quantified through a fluorescence assay using DEVD-amc substrate for measuring caspase-3/7 activation. qRT-PCR and Western Blot analyses were conducted to assess expression levels of the cell cycle, apoptotic, and epigenetic pathway-related markers such as p53, p21, PARP, BAX, BCL-XL, and DNMT1. Apoptotic changes in cells were further confirmed through Immunofluorescence studies. Results: Our study results confirmed that SK-N-SH cells treated with SGI-1027 showed significant cell death and enhanced fluorescence in the DEVD-amc, indicating activation of apoptotic activity. Consistent with our hypothesis, we observed an elevation in p21 levels, an increased BAX/BCL-XL ratio, and a downregulation in the levels of DNMT1, suggesting that SGI-1027 may be facilitating neuroblastoma cell death through a p21-driven mechanism that is independent of p53 pathway. Conclusion: This study highlights the potential of DNMT1 inhibitor SGI-1027 to induce apoptosis in neuroblastoma cells through upregulating p21 and modulating the BAX/BCL-XL ratio. Our findings suggest that targeting DNMT1 using SGI-1027 may offer an effective pathway for inducing cell death and controlling neuroblastoma growth. We are in the process of validating DNMT1 targeted therapies as a powerful addition for treating aggressive neuroblastoma. Acknowledgments: This project was supported by the National Pediatric Cancer Foundation (NPCF) and the Royal Dames of Cancer Research Inc., Fort Lauderdale, Florida. Citation Format: Farah Naz, Shyam Sundar Jaganathan, Umamaheswari Natarajan, Appu Rathinavelu. DNMT1 inhibition by SGI-1027 promotes neuroblastoma cell death through a p53-independent pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4238.

Suppression of necroptosis-driven cell death and inflammation in hypoxic neuroblastoma (SH-SY5Y) cells by necrostatin-1.

Neuroblastoma (NB) is the most typical malignant extracranial solid tumor in the pediatric population. The advent of drug resistance is an essential deterrent in treating high-risk NB patients despite a multi-modality remedy. Inflammation-induced early neuronal degeneration plays a leading part in the pathogenesis of NB via necroptosis; however, the mechanisms remained cryptic. Our current investigation determines the anti-inflammatory and neuroprotective effect of necroptosis inhibitor necrostatin-1 (Nec-1) in receptor-interacting proteins 1 and 3 (RIP1/3)-induced cell death pathway and inflammation caused by hypoxia mimetic agent cobalt chloride (CoCl2). Our biomolecular study illustrates that necroptosis marker RIP1/3 and mixed-lineage kinase domain-like pseudokinase (MLKL) protein expression was increased after treatment with CoCl2 in SH-SY5Y cells. Subsequently, elevated expression levels of RIP1/3 and MLKL further contributed to the inflammation by activating transcription factors extracellular signal-regulated protein kinase (ERK1/2), nuclear factor kappa-B (NF-κB), and releasing high levels of proinflammatory cytokines, such as vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1/CCL2). At the same time, Nec-1 treatment reduced the RIP1/3 and MLKL, phospho-ERK1/2, p65 subunit of NF-κB expression, and VEGF and MCP-1 levels. Molecular docking analysis of RIP1/3-necrostatin-1 complex highlights a significant interaction between necrostatin-1 and specific amino acid residues within the protein. Based on our promising results, necrostatin-1 could be exploited as a therapeutic agent during neuroblastoma's pathogenesis and its molecular therapy.

Viral-Porphyrin Combo: Photodynamic and Oncolytic Viral Therapy for Potent Glioblastoma Treatment.

Combined viral and photodynamic therapy for oncological diseases has great potential to treat aggressive tumors such as glioblastomas. A conjugate of vesicular stomatitis virus (VSV) with protoporphyrin IX was prepared, and its oncolytic effects were studied and compared to the effects of the individual components. The VSV showed an oncolytic effect on glioblastoma cell lines T98G and LN229 at a virus titer of 105 TCID50/mL. A VSV titer of 104 TCID50/mL was sufficient for neuroblastoma cell death. A study of the effect of VSV in tumor 3D cell modeling found that VSV had a clear viral cytopathic effect on spheroids of T98G and LN229 cells. Conjugation with the porphyrin significantly reduced the viral titer, but when irradiated, lysis of cells was observed. Photodynamic treatment of T98G and LN229 cells and spheroids with protoporphyrin IX as a photosensitizer also had a cytotoxic effect on cells and, to a lesser extent, on the tumoroids, as complete cell death was not achieved for the tumoroids. The combination therapy, which involved sequential photodynamic therapy using protoporphyrin IX as a photosensitizer and treatment with VSV, was shown to significantly enhance efficacy, resulting in complete cell death of both T98G and LN229 cells and tumoroids. The combination treatment allowed for the use of a lower viral titer (103-104 TCID50/mL) and a lower porphyrin concentration (0.5 μg/mL) to achieve a significant cytotoxic effect. As a result, the implementation of this combination therapy would likely lead to fewer side effects from the treatment. This study clearly demonstrated the excellent perspectives of combination therapy for the treatment of highly aggressive tumors such as glioblastomas.

Multi-omics revealed that ELAVL3 regulates MYCN in neuroblastoma via immunogenic cell death: Risk stratification and experimental research

Multi-omics revealed that ELAVL3 regulates MYCN in neuroblastoma via immunogenic cell death: Risk stratification and experimental research

Oncolytic virotherapy augments self-maintaining natural killer cell line cytotoxicity against neuroblastoma

BackgroundNeuroblastoma is the most common extracranial solid tumor in children and accounts for 15% of pediatric cancer related deaths. Targeting neuroblastoma with immunotherapies has proven challenging due to a paucity of immune cells in the tumor microenvironment and the release of immunosuppressive cytokines by neuroblastoma tumor cells. We hypothesized that combining an oncolytic Herpes Simplex Virus (oHSV) with natural killer (NK) cells might overcome these barriers and incite tumor cell death.MethodsWe utilized MYCN amplified and non-amplified neuroblastoma cell lines, the IL-12 expressing oHSV, M002, and the human NK cell line, NK-92 MI. We assessed the cytotoxicity of NK cells against neuroblastoma with and without M002 infection, the effects of M002 on NK cell priming, and the impact of M002 and priming on the migratory capacity and CD107a expression of NK cells. To test clinical applicability, we then investigated the effects of M002 and NK cells on neuroblastoma in vivo.ResultsNK cells were more attracted to neuroblastoma cells that were infected with M002. There was an increase in neuroblastoma cell death with the combination treatment of M002 and NK cells both in vitro and in vivo. Priming the NK cells enhanced their cytotoxicity, migratory capacity and CD107a expression.ConclusionsTo the best of our knowledge, these investigations are the first to demonstrate the effects of an oncolytic virus combined with self-maintaining NK cells in neuroblastoma and the priming effect of neuroblastoma on NK cells. The current studies provide a deeper understanding of the relation between NK cells and neuroblastoma and these data suggest that oHSV increases NK cell cytotoxicity towards neuroblastoma.

Nordihydroguaiaretic Acid Affects Undifferentiated and Differentiated Neuroblastoma Cells Differently through Mechanisms that Impact on Cell Viability.

We aimed to investigate the mechanisms involved in the neurotoxic effects of NDGA on differentiated and undifferentiated human neuroblastoma cells (MSN), assessing cell viability, changes in the actin cytoskeleton, cell migration and the expression of the 5-LOX enzyme and the inhibitor of cell cycle progression p21WAF1/CIP1. High expression and activity of the lipoxygenase enzyme (LOX) have been detected in several tumors, including neuroblastoma samples, suggesting the use of LOX inhibitors as potential therapy molecules. Among these, the natural compound nordihydroguaiaretic acid (NDGA) has been extensively tested as an antiproliferative drug against diverse types of cancer cells. In this study, we analyzed the toxic effect of NDGA on neuroblastoma cells at a dose that did not affect cell survival when they differentiated to a neuron-like phenotype and the potential mechanisms involved in the anticancer properties. We exposed human neuroblastoma cells (MSN) to different concentrations of NDGA before and after a differentiation protocol with retinoic acid and nerve growth factor and analyzed cell viability, cell migration, actin cytoskeleton morphology and the levels of the cell cycle inhibitor p21WAF1/CIP1 and 5-LOX. We found that differentiated human neuroblastoma cells are more resistant to NDGA than undifferentiated cells. The toxic effects of NDGA were accompanied by reduced cell migration, changes in actin cytoskeleton morphology, induction of p21WAF1/CIP1 and decreased levels of the 5-LOX enzyme. This study provides new evidence regarding the potential use of NDGA to induce cell death in human neuroblastoma.

Two new compounds isolated from the aerial parts of gastrodia elata blume

Two new aromatic compounds, namely gastupdin A (1), and gastupdin B (2), together with three known compounds, arundin(3), phomosines B (4) and monocillin IV (5), were isolated from the aerial parts of Gastrodia elata Blume. The structures of the new compounds were confirmed through spectral analyses including NMR, HR-ESI-MS, ECD, UV, and IR. All isolated compounds were evaluated for their neuroprotective effects against 6-hydroxydopamine-induced cell death in Human Neuroblastoma Cells, with curcumin as the positive control, however, the activity of all compounds was weaker than the positive control, showing no significant activity.

Abstract 5988: The role of the deubiquitinase USP7 and the E3/E4 ubiquitin ligase complex ITCH/UBE4B in the regulation of cell death in neuroblastoma

Abstract Dysregulation of the Ubiquitin Proteasome System has been linked to many human diseases, including cancer. Expression of UBE4B ubiquitin ligase is associated with neuroblastoma patient outcomes and its functional roles in neuroblastoma pathogenesis are not known. We have recently identified a ubiquitin complex ITCH/UBE4B which mediates ubiquitination of Ku70 and c-FLIPL proteins for proteasomal degradation allowing HDAC inhibitor-mediated caspase-8 dependent apoptosis. We also confirmed that the deubiquitinase USP7, critical player in tumor suppression and DNA repair, can be a potential therapeutic target for neuroblastoma. Highly selective USP7 inhibitors have demonstrated significant antitumor activity in preclinical models of adult cancer, and we reported that these inhibitors alone can be more effective against neuroblastoma tumor growth. Our hypothesis is that USP7 inhibition will be more effective against neuroblastoma tumors through destabilization of ITCH/UBE4B complex protein targets allowing a stronger induction of cell death in response to treatment for the children. To evaluate efficacy of USP7 inhibitors in combination with HDAC inhibitors or chemotherapy against neuroblastoma tumor growth, neuroblastoma cell lines depleted for UBE4B or USP7 were treated with increasing concentrations of USP7 inhibitors alone or in combination with chemotherapy or with HDAC inhibitors. Cell proliferation was measured using continuous live cell imaging and apoptosis by caspase cleavage and PARP cleavage detection by western blot. We have evaluated also, whether ubiquitination/deubiquitination and degradation rates of p53, Ku70 and c-FLIPL proteins could modulate induction of apoptosis and necroptosis. USP7 inhibition resulted in decreases in cell viability in p53 wild-type neuroblastoma cell lines. USP7 inhibition induced apoptosis and necroptosis by increasing phosphorylation of MLKL, RIPK3 and RIPK1. In UBE4B or USP7 depleted cells, a huge induction of necroptosis and a small rate of apoptosis were observed. We also identified USP7 as a deubiquitinase of Ku70, stabilizing Ku70 at the basal level, leading to stabilization of Ku70/c-FLIPL/Bax complex. USP7 inhibition destabilizes ku70 and c-FLIPL for protein degradation leading to induction of apoptosis as well as necroptosis. UBE4B depletion in neuroblastoma inhibits HDAC inhibitors mediated caspase-8 mediated apoptosis but enhances necroptosis, due to the inhibition of Ku70 and c-FLIPL proteins degradation leading to the blockade of caspase-8 activation and the activation of the necroptosome. Our data suggests that USP7 and ITCH/UBE4B can control the switch between apoptosis and necroptosis in response to USP7 and HDAC inhibitors. USP7 inhibition and ITCH/UBE4B activation by HDAC inhibitors could be a promising therapeutic strategy for children with high-risk and relapsed neuroblastoma. Citation Format: Christophe Le Clorennec, CARLA SAMPAIO, PETER E. ZAGE. The role of the deubiquitinase USP7 and the E3/E4 ubiquitin ligase complex ITCH/UBE4B in the regulation of cell death 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 5988.

Abstract 5472: Direct cytotoxicity of hu14.18K.322A in high-risk neuroblastoma

Abstract Background: Neuroblastoma is a childhood cancer of the sympathetic nervous system and is associated with disialoganglioside GD2 overexpression. The main goal of this study is to investigate the involvement of GD2 targeting humanized antidisialoganglioside monoclonal antibody hu14.18K.322A (hu14) in high-risk neuroblastoma direct cell cytotoxicity, assess for synergy with induction chemotherapy agents, and delineate the mechanisms involved in cell death. Methods: For this study, we used a panel of GD2 expressing cell lines [SK-N-BE (2), SK-N-BE (1), LAN5, CHLA15] and a GD2 non-expressing cell line LAN6. We measured surface expression of GD2 and performed cell cycle analysis using flow cytometry. The role of hu14 in neuroblastoma cell death was assessed using cell viability assays, proliferation assays, real time PCR and western blot analysis. We measured synergy between hu14 and induction phase chemotherapy drugs. Further, we employed next generation sequencing (NGS) to analyze the transcriptomic changes in human neuroblastoma cell lines in response to hu14 treatment to understand the pathways involved and the molecular mechanisms of hu14-mediated cellular response. Results: Using a cell viability assay, we found that CHLA15 and SK-N-BE (1) cell lines were sensitive to hu14 treatment whereas SK-N-BE (2), LAN5, LAN6 were resistant. Hu14 treatment resulted in a dose dependent reduction in cell proliferation in these cell lines. We also found that hu14 treatment resulted in increased expression of apoptotic and autophagy pathway proteins, but not ferroptosis or necroptosis pathways proteins. Hu14 induced cell death was partially inhibited by a pan-caspase inhibitor, confirming that caspase-dependent pathways are involved. Western blot analysis also showed decreased levels of p53 phosphorylation in both sensitive cell lines upon hu14 treatment suggesting an important control mechanism centered on the p53 pathway. Hu14 treatment reduced the surface expression of GD2 in the sensitive cells suggesting direct inhibition of the GD2 synthesis pathway. However, PCR analysis of GD2 synthesis genes did not show any significant difference upon treatment suggesting posttranscriptional control of these genes. Furthermore, NGS analysis revealed alterations in several pathways, but these changes were distinct between the cell lines reconfirming the complexity of therapy response in high-risk Neuroblastoma. Conclusion: Taken together, our findings suggest that hu14.18K322A induces direct cell cytotoxicity in a subset of high-risk Neuroblastoma and synergizes well with standard chemotherapy drugs. Furthermore, our research highlights the complex nature of signaling pathways involved in hu14-mediated cytotoxicity that can be harnessed towards targeted therapy. Citation Format: Manu Gnanamony, Maria Thomas, Thu Hien Nguyen, Amber M. D'Souza, Pedro de Alarcon. Direct cytotoxicity of hu14.18K.322A in high-risk 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 5472.

Inhibition of OCT4 binding at the MYCN locus induces neuroblastoma cell death accompanied by downregulation of transcripts with high-open reading frame dominance.

Amplification of MYCN is observed in high-risk neuroblastomas (NBs) and is associated with a poor prognosis. MYCN expression is directly regulated by multiple transcription factors, including OCT4, MYCN, CTCF, and p53 in NB. Our previous study showed that inhibition of p53 binding at the MYCN locus induces NB cell death. However, it remains unclear whether inhibition of alternative transcription factor induces NB cell death. In this study, we revealed that the inhibition of OCT4 binding at the MYCN locus, a critical site for the human-specific OCT4-MYCN positive feedback loop, induces caspase-2-mediated cell death in MYCN-amplified NB. We used the CRISPR/deactivated Cas9 (dCas9) technology to specifically inhibit transcription factors from binding to the MYCN locus in the MYCN-amplified NB cell lines CHP134 and IMR32. In both cell lines, the inhibition of OCT4 binding at the MYCN locus reduced MYCN expression, thereby suppressing MYCN-target genes. After inhibition of OCT4 binding, differentially downregulated transcripts were associated with high-open reading frame (ORF) dominance score, which is associated with the translation efficiency of transcripts. These transcripts were enriched in splicing factors, including MYCN-target genes such as HNRNPA1 and PTBP1. Furthermore, transcripts with a high-ORF dominance score were significantly associated with genes whose high expression is associated with a poor prognosis in NB. Because the ORF dominance score correlates with the translation efficiency of transcripts, our findings suggest that MYCN maintains the expression of transcripts with high translation efficiency, contributing to a poor prognosis in NB. In conclusion, the inhibition of OCT4 binding at the MYCN locus resulted in reduced MYCN activity, which in turn led to the downregulation of high-ORF dominance transcripts and subsequently induced caspase-2-mediated cell death in MYCN-amplified NB cells. Therefore, disruption of the OCT4 binding at the MYCN locus may serve as an effective therapeutic strategy for MYCN-amplified NB.

IP3R-Mediated Calcium Release Promotes Ferroptotic Death in SH-SY5Y Neuroblastoma Cells.

Ferroptosis is an iron-dependent cell death pathway that involves the depletion of intracellular glutathione (GSH) levels and iron-mediated lipid peroxidation. Ferroptosis is experimentally caused by the inhibition of the cystine/glutamate antiporter xCT, which depletes cells of GSH, or by inhibition of glutathione peroxidase 4 (GPx4), a key regulator of lipid peroxidation. The events that occur between GPx4 inhibition and the execution of ferroptotic cell death are currently a matter of active research. Previous work has shown that calcium release from the endoplasmic reticulum (ER) mediated by ryanodine receptor (RyR) channels contributes to ferroptosis-induced cell death in primary hippocampal neurons. Here, we used SH-SY5Y neuroblastoma cells, which do not express RyR channels, to test if calcium release mediated by the inositol 1,4,5-trisphosphate receptor (IP3R) channel plays a role in this process. We show that treatment with RAS Selective Lethal Compound 3 (RSL3), a GPx4 inhibitor, enhanced reactive oxygen species (ROS) generation, increased cytoplasmic and mitochondrial calcium levels, increased lipid peroxidation, and caused cell death. The RSL3-induced calcium signals were inhibited by Xestospongin B, a specific inhibitor of the ER-resident IP3R calcium channel, by decreasing IP3R levels with carbachol and by IP3R1 knockdown, which also prevented the changes in cell morphology toward roundness induced by RSL3. Intracellular calcium chelation by incubation with BAPTA-AM inhibited RSL3-induced calcium signals, which were not affected by extracellular calcium depletion. We propose that GPx4 inhibition activates IP3R-mediated calcium release in SH-SY5Y cells, leading to increased cytoplasmic and mitochondrial calcium levels, which, in turn, stimulate ROS production and induce lipid peroxidation and cell death in a noxious positive feedback cycle.

Neuroprotective Properties of Coriander-Derived Compounds on Neuronal Cell Damage under Oxidative Stress-Induced SH-SY5Y Neuroblastoma and in Silico ADMET Analysis

An imbalance between reactive oxygen species (ROS) production and antioxidant defense driven by oxidative stress and inflammation is a critical factor in the progression of neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Coriander (Coriandrum sativum L.), a culinary plant in the Apiaceae family, displays various biological activities, including anticancer, antimicrobial, and antioxidant effects. Herein, neuroprotective properties of three major bioactive compounds derived from coriander (i.e., linalool, linalyl acetate, and geranyl acetate) were investigated on hydrogen peroxide-induced SH-SY5Y neuroblastoma cell death by examining cell viability, ROS production, mitochondrial membrane potential, and apoptotic profiles. Moreover, underlying mechanisms of the compounds were determined by measuring intracellular sirtuin 1 (SIRT1) enzyme activity incorporated with molecular docking. The results showed that linalool, linalyl acetate, and geranyl acetate elicited their neuroprotection against oxidative stress via protecting cell death, reducing ROS production, preventing cell apoptosis, and modulating SIRT1 longevity. Additionally, in silico pharmacokinetic predictions indicated that these three compounds are drug-like agents with a high probability of absorption and distribution, as well as minimal potential toxicities. These findings highlighted the potential neuroprotective linalool, linalyl acetate, and geranyl acetate for developing alternative natural compound-based neurodegenerative therapeutics and prevention.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11064-024-04239-0.

Multi-Target Actions of Flavonoid Derivatives from Mesua ferrea Linn Flower against Alzheimer’s disease Pathogenesis

OBJECTIVE Kaempferol-3-O-rhamnoside (compound 1) and quercetin- 3-O-rhamnoside (compound 2), two flavonoids isolated from Mesua ferrea L. flowers, were examined for their activities related Alzheimer’s disease (AD) pathogenesis including antioxidant, acetylcholinesterase (AChE) inhibition, anti-beta amyloid (Ab) aggregation and neuroprotection. METHODS The two flavonoids were isolated from M. ferrea L. flowers using the column chromatography technique. Both compounds were evaluated for their effects on AD pathogenesis, including antioxidant action by ABTS assay, AChE inhibition by Ellman’s method, and anti-Ab aggregation by thioflavin T (ThT) assay and neuroprotection by cell base assay. To explain the mechanism of AChE inhibition and anti-Ab aggregation, binding interactions between the test compounds and AChE and Ab were studied in-silico. RESULTS Compounds 1 and 2 showed an ability to scavenge ABTS radicals, with IC50 values of 424.57±2.97 and 308.67±9.90 µM, respectively, and to inhibit AChE function with IC50 values of 769.23±6.23 and 520.64±5.94, respectively. ThT assay indicated that both compounds inhibited Ab aggregation with IC50 values of 406.43±9.95 and 300.69 ±1.18 µM, respectively. The neuroprotection study revealed that the two flavonoids could reduce human neuroblastoma (SH-SY5Y) cell death induced by H2O2. The in-silico study showed that both compounds bound AChE at catalytic anionic and peripheral anionic sites. In addition, the test compounds prevented Ab aggregation by interacting at the central hydrophobic core, the C-terminal hydrophobic region, and the important residues of Ile41. CONCLUSIONS Together, the results showed that kaempferol-3-O- rhamnoside and quercetin-3-O- rhamnoside exhibit multiple mechanisms of action that are involved in the pathogenesis of AD including antioxidant, AChE inhibition, anti-Ab aggregation, and neuroprotection. KEYWORDS flavonoid rhamnosides, Alzheimer’s disease, oxidation, beta amyloid, acetylcholinesterase, molecular docking

Targeted Degradation of XIAP is Sufficient and Specific to Induce Apoptosis in MYCN-overexpressing High-risk Neuroblastoma.

XIAP degradation is sufficient to kill MYCN-amplified neuroblastoma which overexpresses and relies on XIAP as a brake against cell death, without affecting normal cells.