Neuroblastoma Differentiation Research Articles

M6Am sequesters PCF11 to suppress premature termination and drive neuroblastoma differentiation

N6,2'-O-dimethyladenosine (m6Am) is an abundant mRNA modification that impacts multiple diseases, but its function remains controversial because the m6Am reader is unknown. Using quantitative proteomics, we identified transcriptional terminator premature cleavage factor II (PCF11) as a m6Am-specific reader in human cells. Direct quantification of mature versus nascent RNAs reveals that m6Am does not regulate mRNA stability but promotes nascent transcription. Mechanistically, m6Am functions by sequestering PCF11 away from proximal RNA polymerase II (RNA Pol II). This suppresses PCF11 from dissociating RNA Pol II near transcription start sites, thereby promoting full-length transcription of m6Am-modified RNAs. m6Am's unique relationship with PCF11 means m6Am function is enhanced when PCF11 is reduced, which occurs during all-trans-retinoic-acid (ATRA)-induced neuroblastoma-differentiation therapy. Here, m6Am promotes expression of ATF3, which represses neuroblastoma biomarker MYCN. Depleting m6Am suppresses MYCN repression in ATRA-treated neuroblastoma and maintains their tumor-stem-like properties. Collectively, we characterize m6Am as an anti-terminator RNA modification that suppresses premature termination and modulates neuroblastoma's therapeutic response.

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.

Ferroptosis and Iron Metabolism During Neuroblastoma Differentiation

Ferroptosis and Iron Metabolism During Neuroblastoma Differentiation

KLF7 promotes neuroblastoma differentiation through the GTPase signaling pathway by upregulating neuroblast differentiation-associated protein AHNAKs and glycerophosphodiesterase GDPD5.

The arrest of neural crest-derived sympathoadrenal neuroblast differentiation contributes to neuroblastoma formation, and overriding this blocked differentiation is a clear strategy for treating high-risk neuroblastoma. A better understanding of neuroblast or neuroblastoma differentiation is essential for developing new therapeutic approaches. It has been proposed that Krueppel-like factor 7 (KLF7) is a neuroblastoma super-enhancer-associated transcription factor gene. Moreover, KLF7 was found to be intensely active in postmitotic neuroblasts of the developing nervous system during embryogenesis. However, the role of KLF7 in the differentiation of neuroblast or neuroblastoma is unknown. Here, we find a strong association between high KLF7 expression and favorable clinical outcomes in neuroblastoma. KLF7 induces differentiation of neuroblastoma cells independently of the retinoic acid (RA) pathway and acts cooperatively with RA to induce neuroblastoma differentiation. KLF7 alters the GTPase activity and multiple differentiation-related genes by binding directly to the promoters of neuroblast differentiation-associated protein (AHNAK and AHNAK2) and glycerophosphodiester phosphodiesterase domain-containing protein 5 (GDPD5) and regulating their expression. Furthermore, we also observe that silencing KLF7 in neuroblastoma cells promotes the adrenergic-to-mesenchymal transition accompanied by changes in enhancer-mediated gene expression. Our results reveal that KLF7 is an inducer of neuroblast or neuroblastoma differentiation with prognostic significance and potential therapeutic value.

ZMYND8 Is a Regulator of Sonic Hedgehog Signaling in ATRA-Mediated Differentiation of Neuroblastoma Cells.

Zinc Finger MYND (Myeloid, Nervy, and DEAF-1) type containing 8 (ZMYND8) is a crucial epigenetic regulator that plays a multifaceted role in governing a spectrum of vital cellular processes, encompassing proliferation, apoptosis, migration, tumor suppression, and differentiation. It has emerged as a key player in neuronal differentiation by orchestrating the expression of neuronal lineage-committed genes. The present study uncovers the role of ZMYND8 in regulating the Sonic Hedgehog (SHH) signaling axis, which is crucial for neuronal differentiation. Genetic deletion of ZMYND8 leads to a significant reduction in SHH pathway genes, GLI1, and PTCH1 expression during all-trans-retinoic acid (ATRA)-induced differentiation. ZMYND8 and RNA pol II S5P are found to co-occupy the GLI1 and PTCH1 gene promoters, positively impacting their gene transcription upon ATRA treatment. Interestingly, ZMYND8 is found to counteract the inhibitory effects of Cyclopamine that block the upstream SHH pathway protein SMO, resulting in enhanced neurite formation in neuroblastoma cells following their treatment with ATRA. These results indicate that ZMYND8 is an epigenetic regulator of the SHH signaling pathway and has tremendous therapeutic potential in ATRA-mediated differentiation of neuroblastoma.

Anlotinib induces neuronal-like differentiation of neuroblastoma by downregulating CRMP5.

The therapeutic effect of anlotinib on neuroblastoma is still not fully understood. This study aims to explore the differentiation therapeutic effects of anlotinib on neuroblastoma and its potential association with the neural development regulatory protein collapsin response mediator protein 5 (CRMP5), both in vivo and in vitro. A patient-derived xenograft (PDX) model was established to observe the therapeutic effect of anlotinib. Neuroblastoma cell lines SK-N-SH and SK-N-AS were cultured to observe the morphological impact of anlotinib. Transwell assay was used to evaluate the cell invasion, and Western blot analysis and immunohistochemistry were employed to detect the expressions of neuronal differentiation-related proteins. Results indicate that anlotinib effectively inhibited tumor growth in the PDX model, modulated the expressions of neuronal differentiation markers. In vitro, anlotinib treatment induced neurite outgrowth in neuroblastoma cells and inhibited their invasive ability, reflecting a change in neuronal marker expression patterns consistent with the PDX model. Similarly, in the SK-N-AS mouse xenograft model, anlotinib demonstrated comparable tumor-suppressing effects and promoted neuronal-like differentiation. Additionally, anlotinib significantly downregulated CRMP5 expression in neuroblastoma both in vivo and in vitro. Overexpression of CRMP5 significantly reversed the differentiation therapy effect of anlotinib, exacerbating the aggressiveness and reducing the differentiation level of neuroblastoma. These findings highlight the potential of anlotinib as an anti-neuroblastoma agent. It may suppress tumor proliferation and invasion by promoting the differentiation of tumor cells towards a neuronal-like state, and this differentiation therapy effect involves the inhibition of CRMP5 signaling.

Lineage specific transcription factor waves reprogram neuroblastoma from self-renewal to differentiation

Temporal regulation of super-enhancer (SE) driven transcription factors (TFs) underlies normal developmental programs. Neuroblastoma (NB) arises from an inability of sympathoadrenal progenitors to exit a self-renewal program and terminally differentiate. To identify SEs driving TF regulators, we use all-trans retinoic acid (ATRA) to induce NB growth arrest and differentiation. Time-course H3K27ac ChIP-seq and RNA-seq reveal ATRA coordinated SE waves. SEs that decrease with ATRA link to stem cell development (MYCN, GATA3, SOX11). CRISPR-Cas9 and siRNA verify SOX11 dependency, in vitro and in vivo. Silencing the SOX11 SE using dCAS9-KRAB decreases SOX11 mRNA and inhibits cell growth. Other TFs activate in sequential waves at 2, 4 and 8 days of ATRA treatment that regulate neural development (GATA2 and SOX4). Silencing the gained SOX4 SE using dCAS9-KRAB decreases SOX4 expression and attenuates ATRA-induced differentiation genes. Our study identifies oncogenic lineage drivers of NB self-renewal and TFs critical for implementing a differentiation program.

Neuroblastoma differentiation type diagnosis algorithm based on Dense-U-Net using whole slide images

Neuroblastoma differentiation type diagnosis algorithm based on Dense-U-Net using whole slide images

Stable knockdown of Drp1 improves retinoic acid-BDNF-induced neuronal differentiation through global transcriptomic changes and results in reduced phosphorylation of ERK1/2 independently of DUSP1 and 6.

Background: Dynamin-related protein Drp1 -a major mitochondrial fission protein- is widely distributed in the central nervous system and plays a crucial role in regulating mitochondrial dynamics, specifically mitochondrial fission and the organelle's shaping. Upregulated Drp1 function may contribute to the pathological progression of neurodegenerative diseases by dysregulating mitochondrial fission/ fusion. The study aims to investigate the effects of Drp1 on retinoic acid-BDNF-induced (RA-BDNF) neuronal differentiation and mitochondrial network reorganization in SH-SY5Y neuroblastoma cells. Methods: We generated an SH-SY5Y cell line with stably depleted Drp1 (shDrp1). We applied RNA sequencing and analysis to study changes in gene expression upon stable Drp1 knockdown. We visualized the mitochondria by transmission electron microscopy and used high-content confocal imaging to characterize and analyze cell morphology changes and mitochondrial network reorganization during neuronal differentiation. Results: shDrp1 cells exhibited fused mitochondrial ultrastructure with perinuclear clustering. Stable knockdown of Drp1 resulted in the upregulation of genes involved in nervous system development. High content analysis showed improved neurite outgrowth, segmentation, and extremities in differentiated shDrp1 cells. Neuronal differentiation was associated with a significant reduction in ERK1/2 phosphorylation, and ERK1/2 phosphorylation was independent of the dual specificity phosphatases DUSP1/6 in shDrp1 cells. Differentiated control underwent mitochondrial morphology remodeling, whereas differentiated shDrp1 cells retained the highly fused mitochondria and developed long, elongated structures. The shDrp1 cells responded to specific apoptotic stimuli like control in vitro, suggesting that Drp1 is not a prerequisite for apoptosis in SH-SY5Y cells. Moreover, Drp1 downregulation reduced the formation of toxic mHtt aggregates in vitro. Discussion: Our results indicate that Drp1 silencing enhances RA-BDNF-induced neuronal differentiation by promoting transcriptional and mitochondrial network changes in undifferentiated cells. We also demonstrate that the suppression of Drp1 reduces toxic mHtt aggregate formation in vitro, suggesting protection against neurotoxicity. Thus, Drp1 may be an attractive target for further investigation in future strategies to combat neurodegenerative diseases.

Ankrd26 is a retinoic acid-responsive plasma membrane-binding and -shaping protein critical for proper cell differentiation

Morphogens are important triggers for differentiation processes. Yet, downstream effectors that organize cell shape changes in response to morphogenic cues, such as retinoic acid, largely remain elusive. Additionally, derailed plasma membrane-derived signaling often is associated with cancer. We identify Ankrd26 as a critical player in cellular differentiation and as plasma membrane-localized protein able to self-associate and form clusters at the plasma membrane in response to retinoic acid. We show that Ankrd26 uses an N-terminal amphipathic structure for membrane binding and bending. Importantly, in an acute myeloid leukemia-associated Ankrd26 mutant, this critical structure was absent, and Ankrd26's membrane association and shaping abilities were impaired. In line with this, the mutation rendered Ankrd26 inactive in both gain-of-function and loss-of-function/rescue studies addressing retinoic acid/brain-derived neurotrophic factor (BDNF)-induced neuroblastoma differentiation. Our results highlight the importance and molecular details of Ankrd26-mediated organizational platforms for cellular differentiation at the plasma membrane and how impairment of these platforms leads to cancer-associated pathomechanisms involving these Ankrd26 properties.

Abstract B002: ALK signaling primes the DNA damage response sensitizing ALK-driven neuroblastoma to therapeutic ATR inhibition

Abstract High-risk neuroblastoma (NB) currently presents significant clinical challenges. MYCN and ALK, which are often involved in high-risk NB, lead to increased replication stress in cancer cells, providing options for therapeutic exploitation. We previously identified an ATR/ALK inhibitor combination as an effective therapeutic approach in two independent genetically modified mouse NB models. Here, we identify an underlying molecular mechanism, in which ALK signaling leads to phosphorylation of ATR and CHK1, supporting an effective DNA damage response. The importance of ALK inhibition is supported by mouse data, in which monotreatment with ATRi resulted in a robust initial response, but subsequent relapse, in contrast to a 14-day ALKi/ATRi combination treatment that resulted in robust and sustained responses. Finally, we show that the remarkable response to the 14-day combined ATR/ALK inhibition protocol reflects a robust differentiation response in the tumor, reprogramming tumor cells to a neuronal/Schwann cell lineage identity. Our results identify a unique ability of ATR inhibition to trigger neuroblastoma differentiation and underscore the importance of further exploring combined ALK/ATR inhibition in NB, particularly in high-risk patient groups with oncogene-induced replication stress. Citation Format: Ruth H. Palmer, Bengt Hallberg, Marcus Borenäs, Ganesh Umapathy, Dan E. Lind, Wei-Yun Lai, Jikui Guan, Joel Johansson, Agata Aniszewska. ALK signaling primes the DNA damage response sensitizing ALK-driven neuroblastoma to therapeutic ATR inhibition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B002.

Tiam1 regulates proliferation, invasion, and differentiation in neuroblastoma through the Tiam1/Rac1 signaling pathway.

Neuroblastoma is the most common extracranial solid tumor in children. The purpose of the present study is to detect the prognostic role and potential therapeutic efficacy of the T lymphoma invasion and metastasis 1 (Tiam1) in neuroblastoma. The overexpression of Tiam1 protein is frequently observed in neuroblastoma. Tiam1 expression is closely associated with adverse prognosis of neuroblastoma and risk group classification. Knockdown of TIAM1 by lentivirus expressing short hairpin RNA against TIAM1 (sh-TIAM1) inhibited the proliferation, invasion and cell-cycle progression, and promoted apoptosis of the neuroblastoma cell lines SH-SY5Y and SK-N-AS. Additionally, downregulation of the differentiation-related protein expression and decreased Rac1 expression was observed in the sh-TIAM1-transfected SH-SY5Y cells. In vivo, nude mice bearing TIAM1 knockdown SH-SY5Y cells showed improved overall survival and tumor growth suppression. The results demonstrate that inhibition of Tiam1 expression is a potential strategy for targeted therapy in neuroblastoma.

ALK signaling primes the DNA damage response sensitizing ALK-driven neuroblastoma to therapeutic ATR inhibition.

High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and Anaplastic Lymphoma Kinase (ALK), which are often involved in high-risk NB, lead to increased replication stress in cancer cells, suggesting therapeutic strategies. We previously identified an ATR (ataxia telangiectasia and Rad3-related)/ALK inhibitor (ATRi/ALKi) combination as such a strategy in two independent genetically modified mouse NB models. Here, we identify an underlying molecular mechanism, in which ALK signaling leads to phosphorylation of ATR and CHK1, supporting an effective DNA damage response. The importance of ALK inhibition is supported by mouse data, in which ATRi monotreatment resulted in a robust initial response, but subsequent relapse, in contrast to a 14-d ALKi/ATRi combination treatment that resulted in a robust and sustained response. Finally, we show that the remarkable response to the 14-d combined ATR/ALK inhibition protocol reflects a robust differentiation response, reprogramming tumor cells to a neuronal/Schwann cell lineage identity. Our results identify an ability of ATR inhibition to promote NB differentiation and underscore the importance of further exploring combined ALK/ATR inhibition in NB, particularly in high-risk patient groups with oncogene-induced replication stress.

N6-methyladenosine-mediated overexpression of long noncoding RNA ADAMTS9-AS2 triggers neuroblastoma differentiation via regulating LIN28B/let-7/MYCN signaling.

Neuroblastomas have shed light on the differentiation disorder that is associated with spontaneous regression or differentiation in the same tumor at the same time. Long noncoding RNAs (lncRNAs) actively participate in a broad spectrum of biological processes. However, the detailed molecular mechanisms underlying lncRNA regulation of differentiation in neuroblastomas remain largely unknown. Here, we sequenced clinical samples of ganglioneuroma, ganglioneuroblastoma, and neuroblastoma. We compared transcription profiles of neuroblastoma cells, ganglion cells, and intermediate state cells; verified the profiles in a retinoic acid-induced cell differentiation model and clinical samples; and screened out the lncRNA ADAMTS9 antisense RNA 2 (ADAMTS9-AS2), which contributed to neuroblastoma differentiation. ADAMTS9-AS2 upregulation in neuroblastoma cell lines inhibited proliferation and metastatic potential. Additional mechanistic studies illustrated that the interactions between ADAMTS9-AS2 and LIN28B inhibited the association between LIN28B and primary let-7 (pri-let-7) miRNA, then released pri-let-7 into cytoplasm to form mature let-7, resulting in the inhibition of oncogene MYCN activity that subsequently affected cancer stemness and differentiation. Furthermore, we showed that the observed differential expression of ADAMTS9-AS2 in neuroblastoma cells was due to N6-methyladenosine methylation. Finally, ADAMTS9-AS2 upregulation inhibited proliferation and cancer stem-like capabilities in vivo. Taken together, these results show that ADAMTS9-AS2 loss leads to malignant neuroblastoma by increasing metastasis and causing dysfunctional differentiation.

Aryl hydrocarbon receptor is a tumor promoter in MYCN-amplified neuroblastoma cells through suppression of differentiation

Neuroblastoma is the most common extracranial solid tumor in children. MYCN amplification is detected in almost half of high-risk cases and is associated with poorly differentiated tumors, poor patient prognosis and poor response to therapy, including retinoids. We identify the aryl hydrocarbon receptor (AhR) as a transcription factor promoting the growth and suppressing the differentiation of MYCN-amplified neuroblastoma. A neuroblastoma specific AhR transcriptional signature reveals an inverse correlation of AhR activity with patients' outcome, suggesting AhR activity is critical for disease progression. AhR modulates chromatin structures, reducing accessibility to regions responsive to retinoic acid. Genetic and pharmacological inhibition of AhR results in induction of differentiation. Importantly, AhR antagonism with clofazimine synergizes with retinoic acid in inducing differentiation both invitro and invivo. Thus, we propose AhR as a target for MYCN-amplified neuroblastoma and that its antagonism, combined with current standard-of-care, may result in a more durable response in patients.

Discovery of Novel Oxazepine Derivatives as Akt/ROCK Inhibitors for Growth Arrest and Differentiation Induction in Neuroblastoma Treatment.

Patients with high-risk neuroblastoma face limited treatment choices, typically involving a combination of cytotoxic and differentiation maintenance therapies due to a scarcity of drugs. Evidence suggests that targeted inhibitors may provide opportunities for inducing neuroblastoma differentiation while inhibiting proliferation. Here, we demonstrate the synergistic effect of inhibiting Akt and ROCK in antineuroblastoma and present the design and discovery of a new Akt/ROCK inhibitor, B12. It displays strong antiproliferative effects and excellent differentiation inducing activity against Neuro2a cells. Treatment with B12 results in the arrest of G0/G1 cell cycles, a significant decrease in N-myc protein level, and an increase in differentiation markers. The administration of B12 effectively suppresses xenograft tumor growth and promotes differentiation. Overall, the discovery of B12 based on the Akt/ROCK dual inhibition strategy may provide hope for the development of more effective and targeted therapies for this challenging disease.

Palbociclib releases the latent differentiation capacity of neuroblastoma cells

Neuroblastoma is the most common extracranial solid tumor in infants, arising from developmentally stalled neural crest-derived cells. Driving tumor differentiation is a promising therapeutic approach for this devastating disease. Here, we show that the CDK4/6 inhibitor palbociclib not only inhibits proliferation but induces extensive neuronal differentiation of adrenergic neuroblastoma cells. Palbociclib-mediated differentiation is manifested by extensive phenotypic and transcriptional changes accompanied by the establishment of an epigenetic program driving expression of mature neuronal features. Invivo palbociclib significantly inhibits tumor growth in mouse neuroblastoma models. Furthermore, dual treatment with retinoic acid resets the oncogenic adrenergic core regulatory circuit of neuroblastoma cells, further suppresses proliferation, and can enhance differentiation, altering gene expression in ways that significantly correlate with improved patient survival. We therefore identify palbociclib as a therapeutic approach to dramatically enhance neuroblastoma differentiation efficacy that could be used in combination with retinoic acid to improve patient outcomes.

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.

Discovery of 5-sulfonyltetrazoles as neuroblastoma differentiation agents

Previously, we developed an innovative high-content screening (HCS) approach to quantify neuroblastoma cell differentiation based on neurite outgrowth, a morphological differentiation marker of neuroblastoma cells. Here, we report the utilization of this platform to identify 1-methyl-5-(ethylsulfonyl)-1H-tetrazole (3a) as a new neuroblastoma differentiation agent using the ChemBridge DiversetTM commercial synthetic small molecule compound library. We show that this activity can be extended to a group of analogues, which can be accessed via a short two-step synthetic sequence. A new analogue, 5-(allylsulfonyl)-1-methyl-1H-tetrazole (3c) was identified in this synthetic effort as a compound that has even more pronounced differentiation and cytotoxic activities than the original hit compound 3a.

Targeting TRIM24 promotes neuroblastoma differentiation and decreases tumorigenicity via LSD1/CoREST complex.

High-risk neuroblastoma (NB) still has an unfavorable prognosis and inducing NB differentiation is a potential strategy in clinical treatment, yet underlying mechanisms are still elusive. Here we identify TRIM24 as an important regulator of NB differentiation. Multiple datasets and clinical specimens were analyzed to define the role of TRIM24 in NB. The effects of TRIM24 on differentiation and growth of NB were determined by cell morphology, spheres formation, soft agar assay, and subcutaneous xenograft in nude mice. RNA-Seq and qRT-PCR were used to identify genes and pathways involved. Mass spectrometry and co-immunoprecipitation were used to explore the interaction of proteins. Trim24 is highly expressed in spontaneous NB in TH-MYCN transgenic mice and clinical NB specimens. It is associated with poor NB differentiation and unfavorable prognostic. Knockout of TRIM24 in neuroblastoma cells promotes cell differentiation, reduces cell stemness, and inhibits colony formation in soft agar and subcutaneous xenograft tumor growth in nude mice. Mechanistically, TRIM24 knockout alters genes and pathways related to neural differentiation and development by suppressing LSD1/CoREST complex formation. Besides, TRIM24 knockout activates the retinoic acid pathway. Targeting TRIM24 in combination with retinoic acid (RA) synergistically promotes NB cell differentiation and inhibits cell viability. Our findings demonstrate that TRIM24 is critical for NB differentiation and suggest that TRIM24 is a promising therapeutic target in combination with RA in NB differentiation therapy.