Growth Of Neuroblastoma Cells Research Articles

Abstract A062 Therapeutic targeting of the SAGA KAT module impairs MYCN-amplified neuroblastoma growth through reduction of the MYCN oncogenic gene expression program

Abstract Pediatric cancers are frequently driven by genomic alterations that result in aberrant transcription factor activity. While direct targeting of transcription factors is difficult, one method to circumvent this problem is to target co-factors and chromatin complexes that are necessary for their oncogenic function. To evaluate protein complex dependencies enriched in MYCN-amplified neuroblastoma, a disease of dysregulated development driven by aberrant expression of an oncogenic expression program, we curated a list of protein complexes using the CORUM database and mined the Dependency Map using single sample gene set enrichment analysis. This analysis identified the KAT module of the transcriptional coactivator Spt-Ada-Gcn5-acetyltransferase (SAGA) complex as a selective dependency in MYCN-amplified neuroblastoma. Loss of SAGA complex activity in neuroblastoma through both genetic knockout and induced protein degradation of the lysine acetyltransferase (KAT) module subunit TADA2B resulted in a global reduction of histone lysine acetylation and impaired neuroblastoma cell growth. Genetic knockout of TADA2B did not impair the growth of pediatric sarcoma cell lines, suggesting that SAGA loss-of-function is not broadly deleterious. Furthermore, SAGA loss-of-function reduced MYCN binding to chromatin, resulting in suppression of the MYCN-driven gene expression program and impaired cell cycle progression. Importantly, the SAGA complex is pharmacologically targetable in vitro and in vivo with GSK-699, a PROTAC that induces proteolysis of both KAT2A and KAT2B paralogs. As observed with genetic knockout studies, loss of SAGA activity through KAT2A/B degradation reduced the MYCN-driven transcriptional signature and impaired cell cycle progression. Our findings expand our understanding of the chromatin complexes that maintain the oncogenic transcriptional state in MYCN-amplified neuroblastoma and suggest therapeutic potential for inhibitors of SAGA KAT activity in MYCN-amplified neuroblastoma. Citation Format: Nathaniel W. Mabe, Clare F. Malone, Alexandra B. Forman, Gabriela Alexe, Kathleen L. Engel, Ying-Jiun C. Chen, Melinda Soeung, Silvi Salhotra, Allen Basanthakumar, Bin Liu, Sharon YR Dent, Kimberly Stegmaier. Therapeutic targeting of the SAGA KAT module impairs MYCN-amplified neuroblastoma growth through reduction of the MYCN oncogenic gene expression program [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr A062.

MicroRNA-32-5p inhibits metastasis by directly targeting VPS4B and increases sensitivity to dihydroartemisinin in neuroblastoma

Abstract Background Neuroblastoma (NB) is a malignant pediatric tumor requiring new therapies. Accumulating evidence has confirmed that microRNAs play critical roles in NB metastasis. Dihydroartemisinin (DHA) is capable of inhibiting the growth of NB cells. The primary objective of the current investigation was to characterize a newly discovered microRNA, miR-32-5p, in terms of the functional role, underlying mechanism of action, and potential synergistic therapeutic impact in the context of NB metastasis. Materials and methods Real-time quantitative polymerase chain reaction and Western blotting were employed to assess the expression levels of miR-32-5p and its target, vacuolar protein sorting 4B (VPS4B). Furthermore, Transwell assay was utilized to evaluate in vitro cell migration and invasion, whereas a metastasis xenograft model was established in nude mice via caudal vein injections. Results Gene Expression Omnibus database and real-time quantitative polymerase chain reaction analysis showed that miR-32-5p was downregulated in human NB samples and NB cell lines, in comparison with the normal tissue and cell lines. Inhibiting miR-32-5p induced the migration and invasion of NB cells, whereas overexpression of miR-32-5p prevented the migration and invasion in NB cell lines. Furthermore, VPS4B was identified as the direct target of miR-32-5p and the miR-32-5p reduction associated with NB metastasis upregulated the expression of VPS4B. Conversely, overexpression of VPS4B reversed the suppressive effects of miR-32-5p on NB cells. Moreover, miR-32-5p increased the sensitivity to DHA both in NB cells and in the metastasis xenograft model of nude mice. Conclusions The downregulation of miR-32-5p in NB regulates NB metastasis by targeting VPS4B. Moreover, miR-32-5b can improve the sensitivity of DHA in the xenograft mouse model. Our findings have important implications for the combined application of miR-32-5p and DHA in the treatment of NB.

Super-enhancer-driven IRF2BP2 enhances ALK activity and promotes neuroblastoma cell proliferation.

Super-enhancers (SEs) typically govern the expression of critical oncogenes and play a fundamental role in the initiation and progression of cancer. Focusing on genes that are abnormally regulated by SE in cancer may be a new strategy for understanding pathogenesis. In the context of this investigation, we have identified a previously unreported SE-driven gene IRF2BP2 in neuroblastoma (NB). The expression and prognostic value of IRF2BP2 were detected in public databases and clinical samples. The effect of IRF2BP2 on NB cell growth and apoptosis was evaluated through in vivo and in vitro functional loss experiments. The molecular mechanism of IRF2BP2 was investigated by the study of chromatin regulatory regions and transcriptome sequencing. The sustained high expression of IRF2BP2 results from the activation of a novel SE established by NB master transcription factors MYCN, MEIS2 and HAND2, and they form a new complex that regulates the gene network associated with the proliferation of NB cell populations. We also observed a significant enrichment of the AP-1 family at the binding sites of IRF2BP2. Remarkably, within NB cells, AP-1 plays a pivotal role in shaping the chromatin accessibility landscape, thereby exposing the binding site for IRF2BP2. This orchestrated action enables AP-1 and IRF2BP2 to collaboratively stimulate the expression of the NB susceptibility gene ALK, thereby upholding the highly proliferative phenotype characteristic of NB. Our findings indicate that SE-driven IRF2BP2 can bind to AP-1 to maintain the survival of tumor cells via regulating chromatin accessibility of NB susceptibility gene ALK.

Antitumor Activity of a Pyrrolobenzodiazepine Antibody-Drug Conjugate Targeting LGR5 in Preclinical Models of Neuroblastoma.

Neuroblastoma (NB) is a cancer of the peripheral nervous system found in children under 15 years of age. It is the most frequently diagnosed cancer during infancy, accounting for ~12% of all cancer-related deaths in children. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a membrane receptor that is associated with the primary tumor formation and metastasis of cancers in the gastrointestinal system. Remarkably, high levels of LGR5 are found in NB tumor cells, and high LGR5 expression is strongly correlated with poor survival. Antibody-drug conjugates (ADCs) are monoclonal antibodies that are covalently linked to cell-killing cytotoxins to deliver the payloads into cancer cells. We generated an ADC with an anti-LGR5 antibody and pyrrolobenzodiazepine (PBD) dimer-based payload SG3199 using a chemoenzymatic conjugation method. The resulting anti-LGR5 ADC was able to inhibit the growth of NB cells expressing LGR5 with high potency and specificity. Importantly, the ADC was able to completely inhibit the growth of NB xenograft tumors in vivo at a clinically relevant dose for the PBD class of ADCs. The findings support the potential of targeting LGR5 using the PBD class of payload for the treatment of high-risk NBs.

MiR-1271-5p promotes the growth and migration of neuroblastoma cells by regulating ACY-1.

Aminoacylase 1 (ACY-1) has been found to be a tumor suppressor gene in neuroblastoma (NB). This study aimed to identify and verify the microRNAs (miRNAs) that may regulate ACY-1 through database prediction analysis, and verify the mutual regulatory effect of miRNA and ACY-1 in NB through cell experiments. The miRNAs that might bind ACY-1 were predicted and selected by TargetScan, miRTarBase and four other databases, the expression of the predicted miRNAs and ACY-1 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in four groups of clinical samples, and the differentially expressed miRNAs were screened. Then, luciferase vector was constructed according to the ACY-1 gene sequence to detect whether ACY-1 binds to the selected miRNA. Then, miR-1271-5p expression was silenced to detect miR-1271-5p function in the growth and migration of NB. Finally, ACY-1 and miR-1271-5p were silenced to change ACY-1 expression, and ACY-1 function in NB and the regulatory role of miR-1271-5p were explored. ACY-1 was downregulated in NB, miR-1271-5p was upregulated in NB, and miR-1271-5p could be targeted to ACY-1. Silencing miR-1271-5p expression can reduce cell viability and inhibit tumor progression. After interfering with ACY-1 expression in cells, cell viability was enhanced, apoptosis was significantly decreased, and migration and invasion were enhanced. After partially restoring ACY-1 expression, the effect of si-ACY-1 on cells was weakened. In SK-N-SH and SH-SY-5Y cells, the miR-1271-5p inhibitor restored ACY-1 expression and improved ACY-1 function. MiR-1271-5p can promote the growth and migration of tumor cells by inhibiting ACY-1 expression in NB.

Anaplastic Lymphoma Kinase signaling stabilizes SLC3A2 expression via MARCH11 to promote neuroblastoma cell growth

Solute Carrier Family 3, Member 2 (SLC3A2 or 4F2hc) is a multifunctional glycoprotein that mediates integrin-dependent signaling, acts as a trafficking chaperone for amino acid transporters, and is involved in polyamine transportation. We identified SLC3A2 as a potential Anaplastic Lymphoma Kinase (ALK) interacting partner in a BioID-proximity labeling screen in neuroblastoma (NB) cells. In this work we show that endogenous SLC3A2 and ALK interact in NB cells and that this SLC3A2:ALK interaction was abrogated upon treatment with the ALK inhibitor lorlatinib. We show here that loss of ALK activity leads to decreased SLC3A2 expression and reduced SLC3A2 protein stability in a panel of NB cell lines, while stimulation of ALK with ALKAL2 ligand resulted in increased SLC3A2 protein levels. We further identified MARCH11, an E3 ligase, as a regulator of SLC3A2 ubiquitination downstream of ALK. Further, knockdown of SLC3A2 resulted in inhibition of NB cell growth. To investigate the therapeutic potential of SLC3A2 targeting, we performed monotreatment of NB cells with AMXT-1501 (a polyamine transport inhibitor), which showed only moderate effects in NB cells. In contrast, a combination lorlatinib/AMXT-1501 treatment resulted in synergistic inhibition of cell growth in ALK-driven NB cell lines. Taken together, our results identify a novel role for the ALK receptor tyrosine kinase (RTK), working in concert with the MARCH11 E3 ligase, in regulating SLC3A2 protein stability and function in NB cells. The synergistic effect of combined ALK and polyamine transport inhibition shows that ALK/MARCH11/SLC3A2 regulation of amino acid transport is important for oncogenic growth and survival in NB cells.

Development of red blood cell-derived extracellular particles as a biocompatible nanocarrier of microRNA-204 (REP-204) to harness anti-neuroblastoma effect

Neuroblastoma (NB) is the most common extracranial solid tumor in the pediatric population with a high degree of heterogeneity in clinical outcomes. Upregulation of the tumor suppressor miR-204 in neuroblastoma is associated with good prognosis. Although miR-204 has been recognized as a potential therapeutic candidate, its delivery is unavailable. We hypothesized that REP-204, the red blood cell-derived extracellular particles (REP) with miR-204 loading, can suppress neuroblastoma cells in vitro. After miR-204 loading by electroporation, REP-204, but not REP carriers, inhibited the viability, migration, and 3D spheroid growth of neuroblastoma cells regardless of MYCN amplification status. SWATH-proteomics revealed that REP-204 treatment may trigger a negative regulation of mRNA splicing by the spliceosome, suppression of amino acid metabolism and protein production, and prevent SLIT/ROBO signaling-mediated cell migration, to halt neuroblastoma tumor growth and metastasis. The therapeutic efficacy of REP-204 should be further investigated in preclinical models and clinical studies.

Lysophosphatidic Acid Stimulates Mitogenic Activity and Signaling in Human Neuroblastoma Cells through a Crosstalk with Anaplastic Lymphoma Kinase.

Lysophosphatidic acid (LPA) is a well-documented pro-oncogenic factor in different cancers, but relatively little is known on its biological activity in neuroblastoma. The LPA effects and the participation of the tyrosine kinase receptor anaplastic lymphoma kinase (ALK) in LPA mitogenic signaling were studied in human neuroblastoma cell lines. We used light microscopy and [3H]-thymidine incorporation to determine cell proliferation, Western blot to study intracellular signaling, and pharmacological and molecular tools to examine the role of ALK. We found that LPA stimulated the growth of human neuroblastoma cells, as indicated by the enhanced cell number, clonogenic activity, and DNA synthesis. These effects were curtailed by the selective ALK inhibitors NPV-TAE684 and alectinib. In a panel of human neuroblastoma cell lines harboring different ALK genomic status, the ALK inhibitors suppressed LPA-induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), which are major regulators of cell proliferation. ALK depletion by siRNA treatment attenuated LPA-induced ERK1/2 activation. LPA enhanced ALK phosphorylation and potentiated ALK activation by the ALK ligand FAM150B. LPA enhanced the inhibitory phosphorylation of the tumor suppressor FoxO3a, and this response was impaired by the ALK inhibitors. These results indicate that LPA stimulates mitogenesis of human neuroblastoma cells through a crosstalk with ALK.

Opaganib Downregulates N-Myc Expression and Suppresses In Vitro and In Vivo Growth of Neuroblastoma Cells.

Neuroblastoma (NB), the most common cancer in infants and the most common solid tumor outside the brain in children, grows aggressively and responds poorly to current therapies. We have identified a new drug (opaganib, also known as ABC294640) that modulates sphingolipid metabolism by inhibiting the synthesis of sphingosine 1-phosphate (S1P) by sphingosine kinase-2 and elevating dihydroceramides by inhibition of dihydroceramide desaturase. The present studies sought to determine the potential therapeutic activity of opaganib in cell culture and xenograft models of NB. Cytotoxicity assays demonstrated that NB cells, including cells with amplified MYCN, are effectively killed by opaganib concentrations well below those that accumulate in tumors in vivo. Opaganib was shown to cause dose-dependent decreases in S1P and hexosylceramide levels in Neuro-2a cells, while concurrently elevating levels of dihydroceramides. As with other tumor cells, opaganib reduced c-Myc and Mcl-1 protein levels in Neuro-2a cells, and also reduced the expression of the N-Myc protein. The in vivo growth of xenografts of human SK-N-(BE)2 cells with amplified MYCN was suppressed by oral administration of opaganib at doses that are well tolerated in mice. Combining opaganib with temozolomide plus irinotecan, considered the backbone for therapy of relapsed or refractory NB, resulted in increased antitumor activity in vivo compared with temozolomide plus irinotecan or opaganib alone. Mice did not lose additional weight when opaganib was combined with temozolomide plus irinotecan, indicating that the combination is well tolerated. Opaganib has additive antitumor activity toward Neuro-2a tumors when combined with the checkpoint inhibitor anti-CTLA-4 antibody; however, the combination of opaganib with anti-PD-1 or anti-PD-L1 antibodies did not provide increased antitumor activity over that seen with opaganib alone. Overall, the data demonstrate that opaganib modulates sphingolipid metabolism and intracellular signaling in NB cells and inhibits NB tumor growth alone and in combination with other anticancer drugs. Amplified MYCN does not confer resistance to opaganib, and, in fact, the drug attenuates the expression of both c-Myc and N-Myc. The safety of opaganib has been established in clinical trials with adults with advanced cancer or severe COVID-19, and so opaganib has excellent potential for treating patients with NB, particularly in combination with temozolomide and irinotecan or anti-CTLA-4 antibody.

EGCG Disrupts the LIN28B/Let-7 Interaction and Reduces Neuroblastoma Aggressiveness.

Neuroblastoma (NB) is the most commonly diagnosed extracranial solid tumor in children, accounting for 15% of all childhood cancer deaths. Although the 5-year survival rate of patients with a high-risk disease has increased in recent decades, NB remains a challenge in pediatric oncology, and the identification of novel potential therapeutic targets and agents is an urgent clinical need. The RNA-binding protein LIN28B has been identified as an oncogene in NB and is associated with a poor prognosis. Given that LIN28B acts by negatively regulating the biogenesis of the tumor suppressor let-7 miRNAs, we reasoned that selective interference with the LIN28B/let-7 miRNA interaction would increase let-7 miRNA levels, ultimately leading to reduced NB aggressiveness. Here, we selected (-)-epigallocatechin 3-gallate (EGCG) out of 4959 molecules screened as the molecule with the best inhibitory activity on LIN28B/let-7 miRNA interaction and showed that treatment with PLC/PLGA-PEG nanoparticles containing EGCG (EGCG-NPs) led to an increase in mature let-7 miRNAs and a consequent inhibition of NB cell growth. In addition, EGCG-NP pretreatment reduced the tumorigenic potential of NB cells in vivo. These experiments suggest that the LIN28B/let-7 miRNA axis is a good therapeutic target in NB and that EGCG, which can interfere with this interaction, deserves further preclinical evaluation.

L-Glyceraldehyde Inhibits Neuroblastoma Cell Growth via a Multi-Modal Mechanism on Metabolism and Signaling.

Glyceraldehyde (GA) is a three-carbon monosaccharide that can be present in cells as a by-product of fructose metabolism. Bruno Mendel and Otto Warburg showed that the application of GA to cancer cells inhibits glycolysis and their growth. However, the molecular mechanism by which this occurred was not clarified. We describe a novel multi-modal mechanism by which the L-isomer of GA (L-GA) inhibits neuroblastoma cell growth. L-GA induces significant changes in the metabolic profile, promotes oxidative stress and hinders nucleotide biosynthesis. GC-MS and 13C-labeling was employed to measure the flow of carbon through glycolytic intermediates under L-GA treatment. It was found that L-GA is a potent inhibitor of glycolysis due to its proposed targeting of NAD(H)-dependent reactions. This results in growth inhibition, apoptosis and a redox crisis in neuroblastoma cells. It was confirmed that the redox mechanisms were modulated via L-GA by proteomic analysis. Analysis of nucleotide pools in L-GA-treated cells depicted a previously unreported observation, in which nucleotide biosynthesis is significantly inhibited. The inhibitory action of L-GA was partially relieved with the co-application of the antioxidant N-acetyl-cysteine. We present novel evidence for a simple sugar that inhibits cancer cell proliferation via dysregulating its fragile homeostatic environment.

Effects of Anionic Liposome Delivery of All-Trans-Retinoic Acid on Neuroblastoma Cell Differentiation.

All-trans-retinoic acid (ATRA) has long been known to affect cell growth and differentiation. To improve ATRA's therapeutic efficacy and pharmacodynamics, several delivery systems have been used. In this study, free ATRA and anionic-liposome-encapsulated ATRA were compared for their effects on SK-N-SH human neuroblastoma cell growth and differentiation. Anionic liposomes made of L-α-phosphatidylcholine (PC) and L-α-phosphatidic acid (PA), empty (PC-PA) and loaded with ATRA (PC-PA-ATRA), were characterized by dynamic light scattering (DLS) and electrophoretic mobility measurements, and drug entrapment efficiency (EE%) was measured to evaluate the applicability of the new colloidal formulation. The results of brightfield microscopy and cell growth curves indicated that ATRA, whether free or encapsulated, reduced growth and induced differentiation, resulting in SK-N-SH cells changing from epithelioid to neuronal-like morphologies, and producing a significant increase in neurite growth. To further characterize the neuro-differentiation of SK-N-SH cells, the expression of βIII-Tubulin and synaptophysin and mitochondria localization were analyzed via immunofluorescence. Increased expression of neuronal markers and a peculiar localization of mitochondria in the neuritic extensions were apparent both in ATRA- and PC-PA-ATRA-differentiated cells. As a whole, our results strongly indicate that ATRA treatment, by any means, can induce the differentiation of parent SK-N-SH, and they highlight that its encapsulation in anionic liposomes increases its differentiation ability in terms of the percentage of neurite-bearing cells. Interestingly, our data also suggest an unexpected differentiation capability of anionic liposomes per se. This work highlights the importance of developing and carefully testing novel delivery nanocarriers, which are a necessary first "step" in the development of new therapeutic settings.

CDK5RAP3 is a novel super-enhancer-driven gene activated by master TFs and regulates ER-Phagy in neuroblastoma

Super enhancers (SEs) are genomic regions comprising multiple closely spaced enhancers, typically occupied by a high density of cell-type-specific master transcription factors (TFs) and frequently enriched in key oncogenes in various tumors, including neuroblastoma (NB), one of the most prevalent malignant solid tumors in children originating from the neural crest. Cyclin-dependent kinase 5 regulatory subunit-associated protein 3 (CDK5RAP3) is a newly identified super-enhancer-driven gene regulated by master TFs in NB; however, its function in NB remains unclear. Through an integrated study of publicly available datasets and microarrays, we observed a significantly elevated CDK5RAP3 expression level in NB, associated with poor patient prognosis. Further research demonstrated that CDK5RAP3 promotes the growth of NB cells, both in vitro and in vivo. Mechanistically, defective CDK5RAP3 interfered with the UFMylation system, thereby triggering endoplasmic reticulum (ER) phagy. Additionally, we provide evidence that CDK5RAP3 maintains the stability of MEIS2, a master TF in NB, and in turn, contributes to the high expression of CDK5RAP3. Overall, our findings shed light on the molecular mechanisms by which CDK5RAP3 promotes tumor progression and suggest that its inhibition may represent a novel therapeutic strategy for NB.

Differential Competitive Growth of Transgenic Subclones of Neuroblastoma Cells Expressing Different Levels of Cathepsin D Co-Cultured in 2D and 3D in Response to EGF: Implications in Tumor Heterogeneity and Metastasis.

Neuroblastoma (NB) is an embryonal tumor arising from the sympathetic central nervous system. The epidermal growth factor (EGF) plays a role in NB growth and metastatic behavior. Recently, we have demonstrated that cathepsin D (CD) contrasts EGF-induced NB cell growth in 2D by downregulating EGFR/MAPK signaling. Aggressive NB is highly metastatic to the bone and the brain. In the metastatic process, adherent cells detach to form clusters of suspended cells that adhere once they reach the metastatic site and form secondary colonies. Whether CD is involved in the survival of metastatic NB clones is not known. Therefore, in this study, we addressed how CD differentially affects cell growth in suspension versus the adherent condition. To mimic tumor heterogeneity, we co-cultured transgenic clones silenced for or overexpressing CD. We compared the growth kinetics of such mixed clones in 2D and 3D models in response to EGF, and we found that the Over CD clone had an advantage for growth in suspension, while the CD knocked-down clone was favored for the adherent growth in 2D. Interestingly, on switching from 3D to 2D culture conditions, the expression of E-cadherin and of N-cadherin increased in the KD-CD and Over CD clones, respectively. The fact that CD plays a dual role in cancer cell growth in 2D and 3D conditions indicates that during clonal evolution, subclones expressing different level of CD may arise, which confers survival and growth advantages depending on the metastatic step. By searching the TCGA database, we found up to 38 miRNAs capable of downregulating CD. Interestingly, these miRNAs are associated with biological processes controlling cell adhesion and cell migration. The present findings support the view that during NB growth on a substrate or when spreading as floating neurospheres, CD expression is epigenetically modulated to confer survival advantage. Thus, epigenetic targeting of CD could represent an additional strategy to prevent NB metastases.

Abstract 2852: Novel combination therapy with a KAT6A/B inhibitor together with retinoids induces irreversible differentiation of neuroblastoma cells

Abstract Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system derived from migratory neural crest cells that have committed to become sympathetic neuroblasts, but their definitive differentiation is blocked. Neuroblastoma cell identity depends on the adrenergic core regulatory circuit (CRC) of transcription factors that collaborate with MYCN to drive cell proliferation and the oncogenic gene expression program. Retinoic acid is used as differentiation therapy for patients with high-risk neuroblastoma, with the goal to block proliferation and drive sympathetic neuronal differentiation. We recently showed that retinoic acid re-organizes the enhancer landscape of neuroblastoma and promotes a new retino-sympathetic cell state characterized by MYCN downregulation, proliferative arrest, and sympathetic neuronal differentiation. However, the effects of retinoic acid on epigenetic rewiring of the adrenergic CRC and suppression of MYCN expression are reversible in vitro and in vivo, leading to tumor re-growth when the retinoic acid is withdrawn. Therefore, we sought to determine if retinoid-induced differentiation can be forced to progress to irreversible neuronal maturation by the addition of inhibitors of epigenetic modifying enzymes. Here, we conducted a drug screen of epigenetic modifying drugs alone and in combination with retinoic acid to determine their effects on neuroblastoma cell growth and differentiation. In this screen, we found that an inhibitor of the histone H3K23 acetyltransferases KAT6A/B, PF-9363, synergistically inhibits neuroblastoma cell growth in combination with retinoic acid. Moreover, retinoid-resistant neuroblastoma cell lines can be sensitized to retinoic acid when combined with a KAT6 inhibitor. Importantly, this combination treatment renders the differentiated cell state irreversible, such that it is maintained after retinoic acid is withdrawn, with continued suppression of the adrenergic CRC and MYCN expression. In conclusion, the retino-sympathetic differentiated cell state induced by retinoic acid in neuroblastoma becomes irreversible when the cells are also treated with an inhibitor of the KAT6A/B histone H3K23 acetyltransferases, implicating the essential role of these enzymes in restoring the proliferative immature progenitor phenotype in adrenergic neuroblastoma cells. Citation Format: Nina Weichert-Leahey, Alla Berezovskaya, Mark Zimmerman, Brian J. Abraham, Adam W. Durbin, A Thomas Look. Novel combination therapy with a KAT6A/B inhibitor together with retinoids induces irreversible differentiation of neuroblastoma cells [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 2852.

Retracted] microRNA‑146b promotes neuroblastoma cell growth through targeting NUMB.

[This retracts the article DOI: 10.3892/etm.2020.8623.].

ADGRL4 Promotes Cell Growth, Aggressiveness, EMT, and Angiogenesis in Neuroblastoma via Activation of ERK/STAT3 Pathway.

Neuroblastoma (NB) is one of the most common pediatric solid tumors. Emerging evidence has indicated that ADGRL4 can act as a master regulator of tumor progression. In addition, it is well documented that the ERK/STAT3 signaling pathway can promote the proliferation, EMT, angiogenesis, and metastasis in tumors. The current study was formulated to elucidate the exact role of ADGRL4 in the malignant behaviors of NB cells and to investigate the intrinsic mechanism. In this work, expression differences of ADGRL4 in human NB cell lines and HUVECs were assessed via RT-qPCR and western blot analysis. For functional experiments, sh-ADGRL4 was transfected into SK-N-SH cells to generate ADGRL4 knockdown stable cell line. Moreover, ADGRL4 knockdown stable SK-N-SH cells were treated with LM22B-10 (an ERK activator) for rescue experiments. CCK-8 colony formation determined NB cells' growth, migration, invasion, wound healing, and transwell assays. Meanwhile, proliferation-, metastasis- and EMT- associated proteins were also detected. Additionally, a tube formation assay was employed to evaluate in vitro angiogenesis. VM-cadherin, the marker of angiogenesis, was assessed using immunofluorescence staining. Data showed notably upregulated ADGRL4 in NB cells, especially in SK-NSH cells. ADGRL4 knockdown inhibited NB cell growth, migration, invasion, EMT, and in vitro angiogenesis. ADGRL4 knockdown inactivated ERK/STAT3 signaling pathway. Activation of the ERK/STAT3 signaling pathway partially rescued the tumor suppression effects of ADGRL4 knockdown on NB cells. To conclude, the downregulation of ADGRL4 may inhibit cell growth, aggressiveness, EMT, and angiogenesis in NB by inactivating the ERK/STAT3 signaling pathway.

Developing targeted therapies for neuroblastoma by dissecting the effects of metabolic reprogramming on tumor microenvironments and progression.

Rationale: Synergic reprogramming of metabolic dominates neuroblastoma (NB) progression. It is of great clinical implications to develop an individualized risk prognostication approach with stratification-guided therapeutic options for NB based on elucidating molecular mechanisms of metabolic reprogramming. Methods: With a machine learning-based multi-step program, the synergic mechanisms of metabolic reprogramming-driven malignant progression of NB were elucidated at single-cell and metabolite flux dimensions. Subsequently, a promising metabolic reprogramming-associated prognostic signature (MPS) and individualized therapeutic approaches based on MPS-stratification were developed and further validated independently using pre-clinical models. Results: MPS-identified MPS-I NB showed significantly higher activity of metabolic reprogramming than MPS-II counterparts. MPS demonstrated improved accuracy compared to current clinical characteristics [AUC: 0.915 vs. 0.657 (MYCN), 0.713 (INSS-stage), and 0.808 (INRG-stratification)] in predicting prognosis. AZD7762 and etoposide were identified as potent therapeutics against MPS-I and II NB, respectively. Subsequent biological tests revealed AZD7762 substantially inhibited growth, migration, and invasion of MPS-I NB cells, more effectively than that of MPS-II cells. Conversely, etoposide had better therapeutic effects on MPS-II NB cells. More encouragingly, AZD7762 and etoposide significantly inhibited in-vivo subcutaneous tumorigenesis, proliferation, and pulmonary metastasis in MPS-I and MPS-II samples, respectively; thereby prolonging survival of tumor-bearing mice. Mechanistically, AZD7762 and etoposide-induced apoptosis of the MPS-I and MPS-II cells, respectively, through mitochondria-dependent pathways; and MPS-I NB resisted etoposide-induced apoptosis by addiction of glutamate metabolism and acetyl coenzyme A. MPS-I NB progression was fueled by multiple metabolic reprogramming-driven factors including multidrug resistance, immunosuppressive and tumor-promoting inflammatory microenvironments. Immunologically, MPS-I NB suppressed immune cells via MIF and THBS signaling pathways. Metabolically, the malignant proliferation of MPS-I NB cells was remarkably supported by reprogrammed glutamate metabolism, tricarboxylic acid cycle, urea cycle, etc. Furthermore, MPS-I NB cells manifested a distinct tumor-promoting developmental lineage and self-communication patterns, as evidenced by enhanced oncogenic signaling pathways activated with development and self-communications. Conclusions: This study provides deep insights into the molecular mechanisms underlying metabolic reprogramming-mediated malignant progression of NB. It also sheds light on developing targeted medications guided by the novel precise risk prognostication approaches, which could contribute to a significantly improved therapeutic strategy for NB.

Inhibition of Survivin Homodimerization Decreases Neuroblastoma Cell Growth.

Increased expression of BIRC5/survivin, a crucial regulator of the mitotic spindle checkpoint, is associated with poor prognosis in neuroblastoma (NB), the most common extracranial tumor of childhood. Transcriptional inhibitors of survivin have been tested in adult cancers and inhibitors of survivin homodimerization are emerging. We compared genetic inhibition of survivin transcription with the inhibition of survivin homodimerization by S12 and LQZ-7I, chosen from a larger panel of survivin dimerization inhibitors with activity against NB cells. Mice hemizygous for Birc5 were crossed with NB-prone TH-MYCN mice to generate Birc5+/-/MYCNtg/+ mice. The marked decrease of survivin transcription in these mice did not suffice to attenuate the aggressiveness of NB, even when tumors were transplanted into wild-type mice to assure that immune cell function was not compromised by the lack of survivin. In contrast, viability, clonogenicity and anchorage-independent growth of NB cells were markedly decreased by S12. S12 administered systemically to mice with subcutaneous NB xenotransplants decreased intratumoral hemorrhage, albeit not tumor growth. LQZ-7I, which directly targets the survivin dimerization interface, was efficacious in controlling NB cell growth in vitro at markedly lower concentrations compared to S12. LQZ-7I abrogated viability, clonogenicity and anchorage-independent growth, associated with massively distorted mitotic spindle formation. In vivo, LQZ-7I effectively reduced tumor size and cell proliferation of NB cells in CAM assays without apparent toxicity to the developing chick embryo. Collectively, these findings show that inhibiting survivin homodimerization with LQZ-7I holds promise for the treatment of NB and merits further investigation.

MYCN and SNRPD3 cooperate to maintain a balance of alternative splicing events that drives neuroblastoma progression

Many of the pro-tumorigenic functions of the oncogene MYCN are attributed to its regulation of global gene expression programs. Alternative splicing is another important regulator of gene expression and has been implicated in neuroblastoma development, however, the molecular mechanisms remain unknown. We found that MYCN up-regulated the expression of the core spliceosomal protein, SNRPD3, in models of neuroblastoma initiation and progression. High mRNA expression of SNRPD3 in human neuroblastoma tissues was a strong, independent prognostic factor for poor patient outcome. Repression of SNRPD3 expression correlated with loss of colony formation in vitro and reduced tumorigenicity in vivo. The effect of SNRPD3 on cell viability was in part dependent on MYCN as an oncogenic co-factor. RNA-sequencing revealed a global increase in the number of genes being differentially spliced when MYCN was overexpressed. Surprisingly, depletion of SNRPD3 in the presence of overexpressed MYCN further increased differential splicing, particularly of cell cycle regulators, such as BIRC5 and CDK10. MYCN directly bound SNRPD3, and the protein arginine methyltransferase, PRMT5, consequently increasing SNRPD3 methylation. Indeed, the PRMT5 inhibitor, JNJ-64619178, reduced cell viability and SNRPD3 methylation in neuroblastoma cells with high SNRPD3 and MYCN expression. Our findings demonstrate a functional relationship between MYCN and SNRPD3, which maintains the fidelity of MYCN-driven alternative splicing in the narrow range required for neuroblastoma cell growth. SNRPD3 methylation and its protein-protein interface with MYCN represent novel therapeutic targets.Hypothetical model for SNRPD3 as a co-factor for MYCN oncogenesis. SNRPD3 and MYCN participate in a regulatory loop to balance splicing fidelity in neuroblastoma cells. First MYCN transactivates SNRPD3 to lead to high-level expression. Second, SNRPD3 and MYCN form a protein complex involving PRMT5. Third, this leads to balanced alterative splicing (AS) activitiy that is favorable to neuroblastoma. Together this forms as a therapeutic vulnerability where SNRPD3 perturbation or PRMT5 inhibitors are selectively toxic to neuroblastoma by conditionally disturbing splicing activity.