Early evolutionary branching across spatial domains predisposes to clonal replacement under chemotherapy in neuroblastoma.

Neuroblastoma (NB) is one of the most lethal childhood cancers due to its propensity to become treatment resistant. By spatial mapping of subclone geographies before and after chemotherapy across 89 tumor regions from 12 NBs, we find that densely packed territories of closely related subclones present at diagnosis are replaced under effective treatment by islands of distantly related survivor subclones, originating from a different most recent ancestor compared to lineages dominating before treatment. Conversely, in tumors that progressed under treatment, ancestors of subclones dominating later in disease are present already at diagnosis. Chemotherapy treated xenografts and cell culture models replicate these two contrasting scenarios and show branching evolution to be a constant feature of proliferating NB cells. Phylogenies based on whole genome sequencing of 505 individual NB cells indicate that a rich repertoire of parallel subclones emerges already with the first oncogenic mutations and lays the foundation for clonal replacement under treatment.

Role of Genetic Polymorphisms -238 G>A and -308 G>A, and Serum TNF-α Levels in a Cohort of Mexican Pediatric Neuroblastoma Patients: Preliminary Study.

The results of in vitro and in vivo studies have shown the pro-tumor effects of TNF-α, and this cytokine's increased expression is associated with poor prognosis in patients with some types of cancer. Our study objective was to evaluate the possible association of TNF-α genetic polymorphisms and serum levels with susceptibility and prognosis in a cohort of Mexican patients with NB. We performed PCR-RFLP and ELISA methods to analyze the genetics of these SNPs and determine serum concentrations, respectively. The distribution of the -308 G>A and -238 G>A polymorphisms TNFα genotypes was considerably different between patients with NB and the control group. The SNP rs1800629 GG/GA genotypes were associated with a decreased risk of NB (OR = 0.1, 95% CI = 0.03-0.393, p = 0.001) compared with the AA genotype, which was associated with susceptibility to NB (OR = 2.89, 95% CI = 1.45-5.76, p = 0.003) and related to unfavorable histology and high-risk NB. The rs361525 polymorphism GG genotype was associated with a lower risk of developing NB compared with the GA and AA genotypes (OR = 0.2, 95% CI = 0.068-0.63, p = 0.006). Circulating TNF-α serum concentrations were significantly different (p < 0.001) between patients with NB and healthy controls; however, we found no relationship between the analyzed TNF-α serum levels and SNP genotypes. We found associations between the rs1800629AA genotype and lower event-free survival (p = 0.026); SNP rs361525 and TNF-α levels were not associated with survival in patients with NB. Our results suggest the TNF-α SNP rs1800629 as a probable factor of NB susceptibility. The -308 G/A polymorphism AA genotype has a probable role in promoting NB development and poor prognosis associated with unfavorable histology, high-risk tumors, and lower EFS in Mexican patients with NB. It should be noted that it is important to conduct research on a larger scale, through inter-institutional studies, to further evaluate the contribution of TNF-α genetic polymorphisms to the risk and prognosis of NB.

Event-free survival in neuroblastoma with MYCN amplification and deletion of 1p or 11q may be associated with altered immune status.

BACKGROUND: Neuroblastoma exhibits substantial heterogeneity, which is intricately linked to various genetic alterations. We aimed to explore immune status in the peripheral blood and prognosis of patients with neuroblastoma with different genetic characteristics. METHODS: We enrolled 31 patients with neuroblastoma and collected samples to detect three genetic characteristics. Peripheral blood samples were tested for immune cells and cytokines by fluorescent microspheres conjugated with antibodies and flow cytometry. Event-free survival (EFS) was analyzed using the Kaplan‒Meier method. RESULTS: Twenty-two patients had genetic aberrations, including MYCN amplification in 6 patients, chromosome 1p deletion in 9 patients, and chromosome 11q deletion in 14 patients. Two genetic alterations were present in seven patients. The EFS was worse in patients with MYCN amplification or 1p deletion than in the corresponding group, whereas 11q deletion was a prognostic factor only in patients with unamplified MYCN. Changes in immune status revealed a decrease in the proportion of T cells in blood, and an increase in regulatory T cells and immunosuppression-related cytokines such as interleukin (IL)-10. The EFS of the IL-10 high-level group was lower than that of the low-level group. Patients with concomitant genetic alterations and a high level of IL-10 had worse EFS than other patients. CONCLUSIONS: Patients with neuroblastoma characterized by these genetic characteristics often have suppressed T cell response and an overabundance of immunosuppressive cells and cytokines in the peripheral blood. This imbalance is significantly associated with poor EFS. Moreover, if these patients show an elevated levels of immunosuppressive cytokines such as IL-10, the prognosis will be worse.

Mechanism of Drug Resistance to First-Line Chemotherapeutics Mediated by TXNDC17 in Neuroblastomas.

BACKGROUND: The prognosis of high-risk neuroblastomas (NB) that are resistant to first-line induction chemotherapy is relatively poor. This study explored the mechanism of resistance to first-line chemotherapeutics mediated by TXNDC17 and its potential solutions in NB. METHODS: The genetic and clinical data of patients with NB were obtained from the Therapeutically Applicable Research to Generate Effective Treatments dataset. TXNDC17 and BECN1 expressions in NB cells were up- and downregulated by transfection with plasmids and shRNA, respectively. Autophagy-related proteins were detected by western blot. Cell viability was determined using cell proliferation and toxicity experiments. Apoptotic cells were detected using flow cytometry. RESULTS: Overall, 1076 pediatric and adolescent patients with NB were enrolled in this study. The 10-year overall survival (OS) rates and event-free survival (EFS) rates for the patients with a mutation of BECN1 were 37.4 ± 9.1% and 34.5 ± 8.8%, respectively. For patients with a mutation of TXNDC17, the 10-year OS and EFS were 41.4 ± 5.9% and 24.3 ± 5.1%, respectively, which were significantly lower than those in the unaltered group. The overexpression of BECN1 and TXNDC17 reduced NB sensitivity to cisplatin (DDP), etoposide (VP16), and cyclophosphamide (CTX). Autophagy mediated by BECN1 was regulated by TXNDC17, and this process was involved in the resistance to DDP, VP16, and CTX in NB. Suberoylanilide hydroxamic acid (SAHA) can enhance the sensitivity and apoptosis of NB cells to chemotherapeutics by inhibiting TXNDC17, ultimately decreasing autophagy-mediated chemoresistance. CONCLUSIONS: Acquired resistance to first-line chemotherapeutics was associated with autophagy mediated by BECN1 and regulated by TXNDC17, which can be reversed by SAHA.

Golgi-localized Ring Finger Protein 121 is necessary for MYCN-driven neuroblastoma tumorigenesis.

MYCN amplification predicts poor prognosis in childhood neuroblastoma. To identify MYCN oncogenic signal dependencies we performed N-ethyl-N-nitrosourea (ENU) mutagenesis on the germline of neuroblastoma-prone TH-MYCN transgenic mice to generate founders which had lost tumorigenesis. Sequencing of the mutant mouse genomes identified the Ring Finger Protein 121 (RNF121WT) gene mutated to RNFM158R associated with heritable loss of tumorigenicity. While the RNF121WT protein localised predominantly to the cis-Golgi Complex, the RNF121M158R mutation in Helix 4 of its transmembrane domain caused reduced RNF121 protein stability and absent Golgi localisation. RNF121WT expression markedly increased during TH-MYCN tumorigenesis, whereas hemizygous RNF121WT gene deletion reduced TH-MYCN tumorigenicity. The RNF121WT-enhanced growth of MYCN-amplified neuroblastoma cells depended on RNF121WT transmembrane Helix 5. RNF121WT directly bound MYCN protein and enhanced its stability. High RNF121 mRNA expression associated with poor prognosis in human neuroblastoma tissues and another MYC-driven malignancy, laryngeal cancer. RNF121 is thus an essential oncogenic cofactor for MYCN and a target for drug development.

Combining ERAP1 silencing and entinostat therapy to overcome resistance to cancer immunotherapy in neuroblastoma.

BACKGROUND: Checkpoint immunotherapy unleashes tumor control by T cells, but it is undermined in non-immunogenic tumors, e.g. with low MHC class I expression and low neoantigen burden, such as neuroblastoma (NB). Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that trims peptides before loading on MHC class I molecules. Inhibition of ERAP1 results in the generation of new antigens able of inducing potent anti-tumor immune responses. Here, we identify a novel non-toxic combinatorial strategy based on genetic inhibition of ERAP1 and administration of the HDAC inhibitor (HDACi) entinostat that increase the immunogenicity of NB, making it responsive to PD-1 therapy. METHODS: CRISPR/Cas9-mediated gene editing was used to knockout (KO) the ERAP1 gene in 9464D NB cells derived from spontaneous tumors of TH-MYCN transgenic mice. The expression of MHC class I and PD-L1 was evaluated by flow cytometry (FC). The immunopeptidome of these cells was studied by mass spectrometry. Cocultures of splenocytes derived from 9464D bearing mice and tumor cells allowed the assessment of the effect of ERAP1 inhibition on the secretion of inflammatory cytokines and activation and migration of immune cells towards ERAP1 KO cells by FC. Tumor cell killing was evaluated by Caspase 3/7 assay and flow cytometry analysis. The effect of ERAP1 inhibition on the immune content of tumors was analyzed by FC, immunohistochemistry and multiple immunofluorescence. RESULTS: We found that inhibition of ERAP1 makes 9464D cells more susceptible to immune cell-mediated killing by increasing both the recall and activation of CD4+ and CD8+ T cells and NK cells. Treatment with entinostat induces the expression of MHC class I and PD-L1 molecules in 9464D both in vitro and in vivo. This results in pronounced changes in the immunopeptidome induced by ERAP1 inhibition, but also restrains the growth of ERAP1 KO tumors in vivo by remodelling the tumor-infiltrating T-cell compartment. Interestingly, the absence of ERAP1 in combination with entinostat and PD-1 blockade overcomes resistance to PD-1 immunotherapy and increases host survival. CONCLUSIONS: These findings demonstrate that ERAP1 inhibition combined with HDACi entinostat treatment and PD-1 blockade remodels the immune landscape of a non-immunogenic tumor such as NB, making it responsive to checkpoint immunotherapy.

Systematic Analysis of miR-506-3p Target Genes Identified Key Mediators of Its Differentiation-Inducing Function.

Background/Objectives: miR-506-3p has been demonstrated to be a strong inducer of neuroblastoma cell differentiation, highlighting the potential of applying miR-506-3p mimics to neuroblastoma differentiation therapy. However, the target genes of miR-506-3p that mediate its differentiation-inducing function have not been fully defined. This study aims to comprehensively investigate the targetome of miR-506-3p regarding its role in regulating neuroblastoma cell differentiation. Methods: We combined gene expression profiling and functional high-content screening (HCS) to identify miR-506-3p target genes that have differentiation-modulating functions. For evaluating the potential clinical relevance of the identified genes, we analyzed the correlations of gene expressions with neuroblastoma patient survival. Results: We identified a group of 19 target genes with their knockdown significantly inducing cell differentiation, suggesting that these genes play a key role in mediating the differentiation-inducing activity of miR-506-3p. We observed significant correlations of higher mRNA levels with lower patient survival with 13 of the 19 genes, suggesting that overexpression of these 13 genes plays important roles in promoting neuroblastoma development by disrupting the cell differentiation pathways. Conclusions: Through this study, we identified novel target genes of miR-506-3p that function as strong modulators of neuroblastoma cell differentiation. Our findings represent a significant advancement in understanding the mechanisms by which miR-506-3p induces neuroblastoma cell differentiation. Future investigations of the identified 13 genes are needed to fully define their functions and mechanisms in controlling neuroblastoma cell differentiation, the understanding of which may reveal additional targets for developing novel differentiation therapeutic agents.

NBAtlas: A harmonized single-cell transcriptomic reference atlas of human neuroblastoma tumors.

Neuroblastoma, a rare embryonic tumor arising from neural crest development, is responsible for 15% of pediatric cancer-related deaths. Recently, several single-cell transcriptome studies were performed on neuroblastoma patient samples to investigate the cell of origin and tumor heterogeneity. However, these individual studies involved a small number of tumors and cells, limiting the conclusions that could be drawn. To overcome this limitation, we integrated seven single-cell or single-nucleus datasets into a harmonized cell atlas covering 362,991 cells across 61 patients. We use this atlas to decipher the transcriptional landscape of neuroblastoma at single-cell resolution, revealing associations between transcriptomic profiles and clinical outcomes within the tumor compartment. In addition, we characterize the complex immune-cell landscape and uncover considerable heterogeneity among tumor-associated macrophages. Finally, we showcase the utility of our atlas as a resource by expanding it with additional data and using it as a reference for data-driven cell-type annotation.

Spliceosomal vulnerability of MYCN-amplified neuroblastoma is contingent on PRMT5-mediated regulation of epitranscriptomic and metabolomic pathways.

Approximately 50 % of poor prognosis neuroblastomas arise due to MYCN over-expression. We previously demonstrated that MYCN and PRMT5 proteins interact and PRMT5 knockdown led to apoptosis of MYCN-amplified (MNA) neuroblastoma. Here we evaluate the highly selective first-in-class PRMT5 inhibitor GSK3203591 and its in vivo analogue GSK3326593 as targeted therapeutics for MNA neuroblastoma. Cell-line analyses show MYCN-dependent growth inhibition and apoptosis, with approximately 200-fold greater sensitivity of MNA neuroblastoma lines. RNA sequencing of three MNA neuroblastoma lines treated with GSK3203591 reveal deregulated MYCN transcriptional programmes and altered mRNA splicing, converging on key regulatory pathways such as DNA damage response, epitranscriptomics and cellular metabolism. Stable isotope labelling experiments in the same cell lines demonstrate that glutamine metabolism is impeded following GSK3203591 treatment, linking with disruption of the MLX/Mondo nutrient sensors via intron retention of MLX mRNA. Interestingly, glutaminase (GLS) protein decreases after GSK3203591 treatment despite unchanged transcript levels. We demonstrate that the RNA methyltransferase METTL3 and cognate reader YTHDF3 proteins are lowered following their mRNAs undergoing GSK3203591-induced splicing alterations, indicating epitranscriptomic regulation of GLS; accordingly, we observe decreases of GLS mRNA m6A methylation following GSK3203591 treatment, and decreased GLS protein following YTHDF3 knockdown. In vivo efficacy of GSK3326593 is confirmed by increased survival of Th-MYCN mice, with drug treatment triggering splicing events and protein decreases consistent with in vitro data. Together our study demonstrates the PRMT5-dependent spliceosomal vulnerability of MNA neuroblastoma and identifies the epitranscriptome and glutamine metabolism as critical determinants of this sensitivity.

Low Expression of CASP8 Could be a Prognostic Biomarker in Neuroblastoma Patients.

The aim of study was to investigate whether CASP8 (CASPASE8) could be a biomarker for prognosis in neuroblastoma. The prognostic value of CASP8 was determined by analyzing CASP8 methylation status and gene expressions in the tumor tissues of 37 neuroblastoma patients. Bisulfite and quantitative multiplex-methylation-specific polymerase chain reaction (PCR) were used to identify the methylation status. CASP8 messenger ribonucleic acid (RNA) expression levels were determined using reverse transcriptase-quantitative PCR. CASP8 expression levels associated with prognostic value were also analyzed using the TARGET NBL (141 cases) database through PDX for Childhood Cancer Therapeutics (PCAT) and SEQC (498 cases) via the R2 platform. CASP8 methylation status was associated with risk groups, MYCN amplification, and 17q gain status. CASP8 expression was found to be statistically different between high- and low-risk neuroblastoma groups. Low expression of CASP8 was associated with MYCN amplification status. Low expression of CASP8 has shown statistically significant prognostic value through TARGET NBL and SEQC-498 data sets. CASP8 messenger RNA expressions and methylation status were associated with the MYCN amplified high-risk group in neuroblastoma. CASP8 messenger RNA expressions may be considered as a clinical prognostic marker in neuroblastoma.

Targeted inhibition of glycogen synthase kinase-3 using 9-ING-41 (elraglusib) enhances CD8 T-cell-reactivity against neuroblastoma cells.

The prognosis of patients with high-risk neuroblastoma remains poor, partly due to inadequate immune recognition of the tumor. Neuroblastomas display extremely low surface MHC-I, preventing recognition by cytotoxic T lymphocytes (CTLs) and contributing to an immunosuppressive tumor microenvironment. Glycogen synthase kinase-3 beta (GSK-3ß) is involved in pathways that may affect the MHC-I antigen processing and presentation pathway. We proposed that therapeutic inhibition of GSK-3ß might improve the surface display of MHC-I molecules on neuroblastoma cells, and therefore tested if targeting of GSK-3ß using the inhibitor 9-ING-41 (Elraglusib) improves MHC-I-mediated CTL recognition. We analyzed mRNA expression data of neuroblastoma tumor datasets and found that non-MYCN-amplified neuroblastomas express higher GSK-3ß levels than MYCN-amplified tumors. In non-MYCN-amplified cells SH-SY5Y, SK-N-AS and SK-N-SH 9-ING-41 treatment enhanced MHC-I surface display and the expression levels of a subset of genes involved in MHC-I antigen processing and presentation. Further, 9-ING-41 treatment triggered increased STAT1 pathway activation, upstream of antigen presentation pathways in two of the three non-MYCN-amplified cell lines. Finally, in co-culture experiments with CD8 + T cells, 9-ING-41 improved immune recognition of the neuroblastoma cells, as evidenced by augmented T-cell activation marker levels and T-cell proliferation, which was further enhanced by PD-1 immune checkpoint inhibition. Our preclinical study provides experimental support to further explore the GSK-3ß inhibitor 9-ING-41 as an immunomodulatory agent to increase tumor immune recognition in neuroblastoma.

Correlation Between the Expression of DNA Damage Repair Protein OGG1 and Ubiquitination Pathway Protein STUB1 in Pediatric Neuroblastoma.

BACKGROUND: Neuroblastoma, a pediatric malignancy, is significantly influenced by genetic factors. Prior research indicates that the OGG1 rs1052133 G > C polymorphism correlates with a decreased risk of neuroblastoma. METHODS: We analyzed 57 neuroblastoma and 21 adrenal samples, using immunohistochemistry to measure OGG1 and STUB1 expression levels. We conducted a survival analysis to explore relationship between the expressions and neuroblastoma prognosis. RESULTS: Notably higher OGG1 expression and significantly lower STUB1 expression in neuroblastoma. OGG1 levels were significantly correlated with patient age, tumor location, histological grade, Shimada classification, INSS stage, and risk category. A negative association was observed between OGG1 and STUB1 expressions. Higher OGG1 expression was linked to reduced PFS and OS. Lower STUB1 expression was associated with unfavorable PFS. Additionally, OGG1 expression and risk category emerged as independent predictors of prognosis. CONCLUSION: OGG1 potentially functions as an oncogene in NB, with its activity possibly modulated by STUB1 through the ubiquitination pathway.

Enhancing Retinoic Acid-mediated Effects Through Inhibition of CYP26A1, CYP26B1 and HGF Signaling in Neuroblastoma Cells.

BACKGROUND/AIM: Retinoic acid (RA) induces tumor cell differentiation in diseases like acute promyelocytic leukemia or high-risk neuroblastoma. However, the formation of resistant cells, which results from dysregulation of different signaling pathways, limits therapy success. The present study aimed to characterize basic regulatory processes induced by the application of RA in human neuroblastoma cells, to identify therapeutic targets independent of the often amplified oncogene MYCN. MATERIALS AND METHODS: In MYCN-amplified Kelly and MYCN non-amplified SH-SY5Y cells, different assays were employed to quantify the viability and cytotoxicity, while RA-mediated expression changes were examined using genome-wide gene expression analysis followed by quantitative PCR. Enzyme-linked immunoabsorbent assays (ELISA) and western blots were used to determine the levels or activation of the examined proteins. RESULTS: In Kelly cells, treatment with 5 µM RA for 3 days significantly reduced the cell number due to attenuated proliferation, while SH-SY5Y cells were less responsive. An up-regulation of the RA-metabolizing enzymes CYP26A1 and CYP26B1 was observed in both cell lines, and co-treatment with the selective CYP26 inhibitor talarozole markedly decreased cell viability. When RA and ketoconazole, which inhibits CYP26 as well as RA-degrading CYP3A enzymes, were co-administered, not only cell survival was impaired in both cell lines, but also the release of hepatocyte growth factor (HGF). Accordingly, co-application of the c-Met inhibitor tepotinib and RA or ketoconazole substantially decreased cell viability. CONCLUSION: Independent of MYCN amplification, inhibitors of RA metabolism or HGF signaling might prevent the emergence of RA-resistant neuroblastoma cells when co-applied with RA.

Germline functional variants contribute to somatic mutation and outcomes in neuroblastoma.

Germline genetic context may play a significant role in the development and evolution of cancer, particularly in childhood cancers such as neuroblastoma. This study investigates the role of putatively functional germline variants in neuroblastoma, even if they do not directly increase disease risk. Our whole-exome sequencing analysis of 125 patients with neuroblastoma reveals a positive correlation between germline variant burden and somatic mutations. Moreover, patients with higher germline variant burden exhibit worse outcomes. Similar findings are observed in the independent neuroblastoma cohort where a higher germline variant burden correlates with a higher somatic mutational burden and a worse overall survival outcome. However, contrasting results emerge in adult-onset cancer, emphasizing the importance of germline genetics in neuroblastoma. The enrichment of putatively functional germline variants in cancer predisposition genes is borderline significant when compared to healthy populations (P = 0.077; Odds Ratio, 1.45; 95% confidence intervals, 0.94-2.21) and significantly more pronounced against adult-onset cancers (P = 0.016; Odds Ratio, 2.13; 95% confidence intervals, 1.10-3.91). Additionally, the presence of these variants proves to have prognostic significance in neuroblastoma (log-rank P < 0.001), and combining germline with clinical risk factors notably improves survival predictions.

Intercellular extrachromosomal DNA copy-number heterogeneity drives neuroblastoma cell state diversity.

Neuroblastoma exhibits significant inter- and intra-tumor genetic heterogeneity and varying clinical outcomes. Extrachromosomal DNAs (ecDNAs) may drive this heterogeneity by independently segregating during cell division, leading to rapid oncogene amplification. While ecDNA-mediated oncogene amplification is linked to poor prognosis in various cancers, the effects of ecDNA copy-number heterogeneity on intermediate phenotypes are poorly understood. Here, we leverage DNA and RNA sequencing from the same single cells in cell lines and neuroblastoma patients to investigate these effects. By analyzing ecDNA amplicon structures, we reveal extensive intercellular ecDNA copy-number heterogeneity. We also provide direct evidence of how this heterogeneity influences the expression of cargo genes, including MYCN and its downstream targets, and the overall transcriptional state of neuroblastoma cells. Our findings highlight the role of ecDNA copy number in promoting rapid adaptability of cellular states within tumors, underscoring the need for ecDNA-specific treatment strategies to address tumor formation and adaptation.

LncRNA BASP1-AS1 is a positive regulator of stemness and pluripotency in human SH-SY5Y neuroblastoma cells.

Neuroblastoma is the most common extra-cranial solid tumor diagnosed mostly in children below the age of five years and comprises of about 15 % of all paediatric cancer deaths. Tumor initiating cancer stem cells (CSCs) can be targeted for better treatment approaches. BASP1-AS1 is a long non coding (Lnc) RNA that is a divergent LncRNA for its coding gene brain abundant membrane attached signal protein 1 (BASP1). We had earlier demonstrated it to be expressed in foetus derived human neural progenitor cells (hNPCs), where it was a positive regulator of BASP1 and was critical for neural differentiation. In this study, we have investigated the role of BASP1-AS1 in CSCs derived from the human neuroblastoma cell line SH-SY5Y. We cultured SH-SY5Y cells on Poly-d-Lysine coated flasks in serum free media supplemented with growth factors, which led to the enrichment of CSCs as determined by marker expression. When grown on ultra-low attachment flasks, these cells formed CSCs enriched neurospheres. We examined the effects of BASP1-AS1 siRNA mediated knockdown on CSCs enriched SH-SY5Y cells and SH-SY5Y derived neurospheres. BASP1-AS1 knockdown decreased the levels of the corresponding gene BASP1 and the rate of cell proliferation of CSCs enriched cells along with low expression of Ki67. It also reduced the mRNA levels of stem cell and pluripotency gene markers (CD133, CD44, c-KIT, SOX2, OCT4 and NANOG), as also Wnt 2 and the Wnt pathway effector ß catenin. It also abrogated the formation of neurospheres in ultra-low attachment flasks. A similar effect on proliferation and stemness related properties was seen on BASP1 knockdown. BASP1-AS1 and its related pathways may provide a point of intervention for the CSCs population in neuroblastoma.

Expansion of a neural crest gene signature following ectopic MYCN expression in sympathoadrenal lineage cells in vivo.

Neural crest cells (NCC) are multipotent migratory stem cells that originate from the neural tube during early vertebrate embryogenesis. NCCs give rise to a variety of cell types within the developing organism, including neurons and glia of the sympathetic nervous system. It has been suggested that failure in correct NCC differentiation leads to several diseases, including neuroblastoma (NB). During normal NCC development, MYCN is transiently expressed to promote NCC migration, and its downregulation precedes neuronal differentiation. Overexpression of MYCN has been linked to high-risk and aggressive NB progression. For this reason, understanding the effect overexpression of this oncogene has on the development of NCC-derived sympathoadrenal progenitors (SAP), which later give rise to sympathetic nerves, will help elucidate the developmental mechanisms that may prime the onset of NB. Here, we found that overexpressing human EGFP-MYCN within SAP lineage cells in zebrafish led to the transient formation of an abnormal SAP population, which displayed expanded and elevated expression of NCC markers while paradoxically also co-expressing SAP and neuronal differentiation markers. The aberrant NCC signature was corroborated with in vivo time-lapse confocal imaging in zebrafish larvae, which revealed transient expansion of sox10 reporter expression in MYCN overexpressing SAPs during the early stages of SAP development. In these aberrant MYCN overexpressing SAP cells, we also found evidence of dampened BMP signaling activity, indicating that BMP signaling disruption occurs following elevated MYCN expression. Furthermore, we discovered that pharmacological inhibition of BMP signaling was sufficient to create an aberrant NCC gene signature in SAP cells, phenocopying MYCN overexpression. Together, our results suggest that MYCN overexpression in SAPs disrupts their differentiation by eliciting abnormal NCC gene expression programs, and dampening BMP signaling response, having developmental implications for the priming of NB in vivo.

Epigenetic regulation of cell state by H2AFY governs immunogenicity in high-risk neuroblastoma.

Childhood neuroblastoma with MYCN amplification is classified as high risk and often relapses after intensive treatments. Immune checkpoint blockade therapy against the PD-1/L1 axis shows limited efficacy in patients with neuroblastoma, and the cancer intrinsic immune regulatory network is poorly understood. Here, we leverage genome-wide CRISPR/Cas9 screens and identify H2AFY as a resistance gene to the clinically approved PD-1 blocking antibody nivolumab. Analysis of single-cell RNA-Seq datasets reveals that H2AFY mRNA is enriched in adrenergic cancer cells and is associated with worse patient survival. Genetic deletion of H2afy in MYCN-driven neuroblastoma cells reverts in vivo resistance to PD-1 blockade by eliciting activation of the adaptive and innate immunity. Mapping of the epigenetic and translational landscape demonstrates that H2afy deletion promotes cell transition to a mesenchymal-like state. With a multiomics approach, we uncovered H2AFY-associated genes that are functionally relevant and prognostic in patients. Altogether, our study elucidates the role of H2AFY as an epigenetic gatekeeper for cell states and immunogenicity in high-risk neuroblastoma.

Targeting SWI/SNF ATPases reduces neuroblastoma cell plasticity.

Tumor cell heterogeneity defines therapy responsiveness in neuroblastoma (NB), a cancer derived from neural crest cells. NB consists of two primary subtypes: adrenergic and mesenchymal. Adrenergic traits predominate in NB tumors, while mesenchymal features becomes enriched post-chemotherapy or after relapse. The interconversion between these subtypes contributes to NB lineage plasticity, but the underlying mechanisms driving this phenotypic switching remain unclear. Here, we demonstrate that SWI/SNF chromatin remodeling complex ATPases are essential in establishing an mesenchymal gene-permissive chromatin state in adrenergic-type NB, facilitating lineage plasticity. Targeting SWI/SNF ATPases with SMARCA2/4 dual degraders effectively inhibits NB cell proliferation, invasion, and notably, cellular plasticity, thereby preventing chemotherapy resistance. Mechanistically, depletion of SWI/SNF ATPases compacts cis-regulatory elements, diminishes enhancer activity, and displaces core transcription factors (MYCN, HAND2, PHOX2B, and GATA3) from DNA, thereby suppressing transcriptional programs associated with plasticity. These findings underscore the pivotal role of SWI/SNF ATPases in driving intrinsic plasticity and therapy resistance in neuroblastoma, highlighting an epigenetic target for combinational treatments in this cancer.