ABSTRACT Background Group 3 (MYC-driven) medulloblastoma (MB) is a highly aggressive brain tumor with poor-prognosis and limited treatment options. We previously identified protein-arginine methyltransferase-5 (PRMT5) as a promising target in Group 3 MB with its control on MYC protein stability. In this follow up study, we further mechanistically investigated PRMT5 control on MYC transcription and targeted it pharmacologically for therapeutic proof-of-concept. Methods Using pharmacogenetic inhibition approaches against PRMT5 in MYC-amplified (Group 3) MB cell line and neurosphere models in vitro and in vivo , we investigated molecular mechanism(s) and anti-cancer efficacy of PRMT5 inhibition. Results Our experiments demonstrated that PRMT5 epigenetically regulates MYC transcription in MYC-amplified MB cells by binding to the proximal-promoter region of the MYC gene and contributing to the enriched symmetric-dimethylation of histone H4R3 in the same region. We further showed that PRMT5 is recruited to the MYC promoter by its interaction with BRD4, the major BET-protein responsible for MYC transcription. PRMT5 inhibition caused the suppression of MYC-induced transcriptional programs and target genes, with widespread disruption of splicing across the transcriptome, particularly affecting metabolism-related gene products. Pharmacologic inhibition of PRMT5 using a panel of selective small-molecule inhibitors demonstrates suppression of cell growth/survival in a MYC-dependent manner in MB cells. Moreover, our in vivo analyses of PRMT5 inhibition, in mice treated with one of the potent pharmacologic inhibitors, particularly a lipid-decorated form of it, demonstrated reduced cerebellar tumor growth with suppressed MYC expression and prolonged survival of mice with MYC-amplified MB xenografts. Conclusions Our findings establish a functional link between PRMT5 and MYC-mediated transcriptional regulation, suggesting a promising therapeutic approach targeting the PRMT5-MYC axis for MYC-driven MB. Key Points PRMT5 acts as an epigenetic regulator of MYC transcription, RNA splicing and associated energy metabolism in MYC-driven MB. PRMT5 inhibition selectively suppresses cell growth/survival in MYC-driven MB. PRMT5 inhibition reduces tumor burden and prolongs survival in a MYC-driven MB mouse model. Importance of the Study Group 3 medulloblastoma is a highly aggressive pediatric brain tumor marked by MYC amplification, malignant clinical behavior, and poor survival outcomes despite intensive multimodal therapy. Because MYC remains largely undruggable, there is an urgent need for effective and less toxic treatment options for affected children. This study identifies protein arginine methyltransferase 5 (PRMT5) as a key epigenetic regulator of MYC transcription and MYC-dependent oncogenic programs in Group 3 MB. We show that PRMT5 is recruited to the MYC promoter via BRD4, sustains MYC-driven transcription and RNA splicing networks associated with metabolism, and supports MB tumor growth. Importantly, pharmacologic inhibition of PRMT5 using a selective brain-penetrant inhibitor suppresses MYC expression, reduces cerebellar tumor burden, and prolongs survival in MYC-amplified MB models. These findings provide a strong translational rationale for PRMT5 inhibition as a targeted therapeutic strategy for high-risk MB, with the potential to improve outcomes while reducing treatment-related toxicity.

Neuroblastoma (NB), a devastating pediatric cancer originating from neural crest cells crucial for nervous system development, poses a significant therapeutic challenge. Despite chemotherapy being the primary treatment, approximately 70% of high-risk NB cases develop resistance. Autophagy is vital for neuronal development, balance, and differentiation of neural stem cells into mature neurons. However, the intricate mechanisms governing autophagy and the pivotal genes orchestrating its regulation in NB remain largely elusive. In this study, we first identified Sin3A Associated Protein 30 (SAP30) as a novel regulator of autophagy in NB. Silencing SAP30 inhibits autophagy and disrupts starvation-induced physiological autophagy in NB cells. Conversely, ectopic expression of SAP30 induces autophagy in NB cells under normal or starvation conditions. Mechanistically, SAP30 transcriptionally regulates STX17, a crucial protein involved in autophagosome-lysosome fusion during autophagy. Reduction of SAP30 decreases STX17 expression, hindering its translocation to the autophagic membrane and inhibiting autophagosome-lysosome fusion. SAP30-mediated autophagy enhances cell growth and provides protection in NB cells treated with chemotherapy drugs. Notably, suppressing SAP30 in vivo increases LC3B accumulation, an autophagy marker, along with reduced proliferation markers, both in vivo and in PDX tumors. Therefore, SAP30 emerges as a potential target to enhance NB responsiveness to chemotherapy drugs.

Amyloid precursor-like protein 2 (APLP2) has been previously associated with pro-tumor phenotypes in cancer cells, and in this current study we investigated the expression and functions of this protein in macrophages. Our findings showed that APLP2 expression was increased in monocyte-like U937 cells after cytokine-induced differentiation to macrophage-like cells. Evaluation of human mRNA data revealed that APLP2 is more highly expressed in human M2/anti-inflammatory (pro-tumor) macrophages than in M1 macrophages (which have a pro-inflammatory, anti-tumor phenotype). Consistent with the mRNA data, by immunoblotting we identified increased APLP2 protein expression in mouse M2/anti-inflammatory macrophages. Intratumoral infiltration of M2/anti-inflammatory macrophages has been reported in several cancers, including neuroblastoma (NB). We observed that treatment of macrophages with NB-conditioned media induced M2/anti-inflammatory and mixed phenotypes. Through comparison of macrophages from wild-type and APLP2-knockout mice, we correlated alterations in inflammation-associated markers with the presence of APLP2. This suggests that APLP2 influences macrophage polarization dynamics between M0/unpolarized and pro- and anti-inflammatory states, and populations altered by APLP2 KO resemble the macrophage profiles altered with NB-conditioned media treatment. In total, our work implicates APLP2 as a mediator of macrophage status, namely in the M0/unpolarized macrophage and the M1/pro-inflammatory and M2/anti-inflammatory axis.

The 4th Symposium on Childhood Cancer Health Disparities was held at Texas Children's Hospital in Houston, Texas, on September 26, 2023. The symposium registered 94 attendees from different backgrounds (e.g. clinicians, epidemiologists, exposure assessment scientists, geospatial experts) with an interest in environmental health disparities of pediatric cancer susceptibility and treatment outcomes. The focus of the symposium was to provide an overview of the role of environmental risk factors in studies of pediatric cancer, introduce novel exposure assessment tools that can be applied to the field, and highlight opportunities to study the impact of environmental health disparities in pediatric cancer susceptibility and outcomes. This report summarizes the scientific content of the symposium and highlights priorities to advance the field.

Background: Cancer is the leading cause of death by disease among children in the United States. Residing in rural areas may impact cancer outcomes as rural areas tend to have fewer available healthcare resources. Few population-based studies have investigated rural/urban disparities in pediatric cancer outcomes. The objective of this study was to examine rural/urban differences in (1) five-year relative survival and (2) cancer-specific survival among children in the United States. Methods: The study is a population-based longitudinal study using data from the Surveillance, Epidemiology, and End Results (SEER) 17 registries database (2000-2021). We included data for individuals aged 0-19 with a first primary malignant cancer diagnosed from 2000 to 2016. Rurality was measured by Rural-Urban Continuum Codes (RUCCs). Five-year relative survival rates, Kaplan-Meier curves, and Cox regression analysis were used to determine the differences in pediatric cancer survivorship between rural and urban areas. Results: Both five-year relative survival rates and log-rank tests of survival probabilities over time by rurality showed no statistically significant difference between individuals living in urban versus rural counties. However, after adjusting for age, sex, race/ethnicity, cancer type, median household income, and region, children diagnosed in rural counties had a 9 % higher risk of death compared to children diagnosed in urban counties (95 % CI 1.02-1.17), with the highest increased risk observed for children with retinoblastoma (aHR: 6.12, 95 % CI 2.01-18.59). A higher increased risk of death was observed for children living in the most rural counties (aHR: 1.18, 95 % CI 1.07-1.32). Conclusion: In this study, residing in rural areas was associated with an increased risk of death from pediatric cancer, especially for children residing in rural areas not adjacent to urban areas. Our findings warrant further investigation to determine the rural/urban disparities in pediatric cancer outcomes and to develop interventions to deliver high-quality cancer care to rural children.

Neuroblastoma (NB) poses a significant challenge in pediatric cancer care due to its aggressive nature and poor prognosis. While advances have been made in clinical treatments, therapy resistance remains a tough hurdle in NB treatment. While much research has focused on identifying oncogenes in NB, there has been less emphasis on understanding tumor suppressors. This study aimed to discover a new transcription factor that could address patient stage, risk level, and MYCN amplifi- cation status while exhibiting tumor-suppressive properties in NB patients. Using advanced bioinformatics techniques, we identified unique transcription factor signature that corresponded to patient characteristics. By analyzing regulon specifi city scores, we prioritized Forkhead Box J3 (FOXJ3) as potential novel driver transcription factor with tumor-suppressive functions in NB. Validation experiments on NB patients and patient-derived xenograft (PDX) tumors confirmed higher FOXJ3 expression in low-risk versus high-risk patients and in PDXs from diagnostic tumors versus relapse-specific tumors. Notably, the overexpression of FOXJ3 was associated with reduced cell density, proliferation, cells in S phase, colony-formation ability, transwell migration, neurosphere formation, spheroid diameter, and inhibition of AKT signaling in NB cells. Overall, these fi ndings suggest that FOXJ3 functions as a novel tumor suppressor in NB, holding promise for potential therapeutic interventions.

Neuroblastoma (NB) is an aggressive pediatric cancer, with high-risk patients facing a five-year survival rate of ~50%. Standard therapies, including surgery, chemotherapy, radiation, and immunotherapy, are associated with significant long-term toxicities and frequent relapse. Histone deacetylase (HDAC) inhibitors have emerged as promising agents for cancer therapy, given their role in modulating gene expression and tumor phenotypes. This study evaluated M344 [4-(dimethylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)benzamide], an HDAC inhibitor, for its efficacy and mechanisms of action against NB. Analysis of clinical NB Gene Expression Omnibus data revealed advanced-stage tumors exhibit higher HDAC expression relative to early-stage samples. M344 treatment effectively increased histone acetylation, induced G0/G1 cell cycle arrest, and activated caspase-mediated cell death. Relative to vorinostat, an HDAC inhibitor in clinical use for lymphoma and clinical trials for NB, M344 displayed superior cytostatic, cytotoxic, and migration-inhibitory effects. In vivo, metronomic M344 dosing suppressed tumor growth and extended survival. Combination therapy with M344 and topotecan improved topotecan tolerability, while M344 co-administration with cyclophosphamide reduced tumor rebound post-therapy. In total, M344 demonstrated strong therapeutic potential for NB, offering improved tumor suppression, reduced off-target toxicities, and enhanced control of tumor growth post-therapy. These findings support further investigation of HDAC inhibitors, such as M344, for clinical application in NB treatment.

Cyclophosphamide (CPX) is an alkylating agent commonly used for various hematological and solid malignancies. In addition to its use as a cytotoxic agent to directly kill tumor cells, numerous immunomodulatory properties of CPX in the tumor microenvironment (TME) of several cancer types have also been documented. These properties include the selective depletion of immune-suppressive regulatory T cells (Tregs), triggering of immunogenic cell death (ICD) and enhanced antigen presentation, and release of type I interferons (IFNs). Moreover, preclinical models as well as human clinical trials have investigated the efficacy of the low-dose “metronomic” scheduling of CPX in combination with immunotherapies such as immune checkpoint inhibitors, dendritic cell tumor vaccines, and tumor antigen peptide vaccines. The metronomic dosing schedule involves administering a continuous (or frequent, such as daily) low dose of chemotherapy rather than using the canonical approach of administering the maximum tolerated dose. Despite the approval of immune checkpoint inhibitors for clinical usage against an increasing number of cancers, many malignancies simply do not respond to checkpoint inhibition, in part due to the heterogeneous intratumoral network of immune-suppressive cell populations. The immunomodulatory effects of cyclophosphamide have strong translational applicability and could serve to enhance and bolster anti-tumor immunity, potentially synergizing with immune checkpoint inhibitors and other existing immunotherapy agents.

Neuroblastoma (NB) is a highly aggressive pediatric cancer that originates from immature nerve cells, presenting significant treatment challenges due to therapy resistance. Despite intensive treatment, approximately 50% of high-risk NB cases exhibit therapy resistance or experience relapse, resulting in poor outcomes often associated with tumor immune evasion. B7-H3 is an immune checkpoint protein known to inhibit immune responses. MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation. Our study aims to explore the impact of miRNAs on B7-H3 regulation, the anti-tumor immune response, and tumorigenicity in NB. Analysis of NB patients and patient-derived xenograft tumors revealed a correlation between higher B7-H3 expression and poorer patient survival. Notably, deceased patients exhibited a depletion of miR-29 family members (miR-29a, miR-29b, and miR-29c), which displayed an inverse association with B7-H3 expression in NB patients. Overexpression and knockdown experiments demonstrated that these miRNAs degrade B7-H3 mRNA, resulting in enhanced NK cell activation and cytotoxicity. In vivo, experiments provided further evidence that miR-29 family members reduce tumorigenicity, macrophage infiltration, and microvessel density, promote infiltration and activation of NK cells, and induce tumor cell apoptosis. These findings offer a rationale for developing more effective combination treatments that leverage miRNAs to target B7-H3 in NB patients.

Abstract Background Pediatric cancer is the leading cause of death by disease among children. With advanced treatments, survivors are living longer. However, they often experience multiple symptoms even after the treatment completion. Little is known about whether rural-urban residences are associated with multiple symptoms occurrence in pediatric cancer survivors. Methods We conducted a cross-sectional study of 130 pediatric cancer survivors aged 10-25 who completed treatment within eight years from 2 major hospitals in Nebraska. We used a survey to collect data during their hospital visit and the validated Memorial Symptom Assessment Scale tool to collect information on 30 symptoms. We used the PedsQoL instrument to measure the quality of life. We conducted a multiple linear regression model to find the association between rurality and multiple symptoms and to identify factors associated with multiple symptoms at a level of significance with a p-value of less than 0.05 and an adjusted beta coefficient. Results The mean age of participants was 15.00, the mean year since diagnosis and treatment completion was 4.52 and 2.92, respectively, and the mean number of symptoms was 6.18.Difficulty concentrating (49.23%), lack of energy (46.92%), and feeling drowsy (46.92%) were the most common symptoms. The mean total quality of life score was 82.16 out of 100. In the unadjusted model, the association of residence and multiple symptoms was not found to be significant, but when adjusted for possible confounders such as age, race, ethnicity, education, diagnosis, body mass index, health-related quality of life, the association was found to be significant indicating there are a higher number of symptoms in pediatric cancer survivors residing in rural areas compared to urban areas. Quality of life is also poorer when the number of symptoms is higher. Conclusion Even after the treatment completion, multiple symptoms occur in pediatric cancer survivors, and the number of symptoms is higher for those residing in rural areas.Further exploration is needed because of the higher number of symptoms in those residing in rural areas. The association of multiple symptoms with other neighborhood-level socio-demographic indices, such as the area deprivation index, should also be assessed. Citation Format: Krishtee Napit, Don Coulter, Katrina Cordts, Daisy Dai, Evi Farazi, Shinobu Watanabe-Galloway. Rural-urban differences in multiple symptoms occurrence in pediatric cancer survivors after treatment completion in Nebraska, United States [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 B040.

The cell cycle comprises sequential events during which a cell duplicates its genome and divides it into two daughter cells. This process is tightly regulated to ensure that the daughter cell receives identical copied chromosomal DNA and that any errors in the DNA during replication are correctly repaired. Cyclins and their enzyme partners, cyclin-dependent kinases (CDKs), are critical regulators of G to M phase transitions during the cell cycle. Mitogenic signals induce the formation of the cyclin/CDK complexes, resulting in phosphorylation and activation of the CDKs. Once activated, cyclin/CDK complexes phosphorylate specific substrates that drive the cell cycle forward. The sequential activation and inactivation of cyclin-CDK complexes are tightly controlled by activating and inactivating phosphorylation events induced by cell cycle proteins. The noncoding RNAs (ncRNAs), which do not code for proteins, regulate cell cycle proteins at the transcriptional and translational levels, thereby controlling their expression at different cell cycle phases. Deregulation of ncRNAs can cause abnormal expression patterns of cell cycle regulating proteins, resulting in abnormalities in cell cycle regulation and cancer development. This review explores how ncRNA dysregulation can disrupt cell division balance and discusses potential therapeutic approaches targeting these ncRNAs to control cell cycle events in cancer treatment.

Background Medulloblastoma (MB) patients with MYC oncogene amplification or overexpression exhibit extremely poor prognoses and therapy resistance. However, MYC itself has been one of the most challenging targets for cancer treatment. Here, we identify a novel marinopyrrole natural derivative, MP1, that shows desirable anti-MYC and anti-cancer activities in MB. Methods In this study, using MYC-amplified (Group 3) and non-MYC amplified MB cell lines in vitro and in vivo, we evaluated anti-cancer efficacies and molecular mechanism(s) of MP1. Results MP1 significantly suppressed MB cell growth and sphere counts and induced G2 cell cycle arrest and apoptosis in a MYC-dependent manner. Mechanistically, MP1 strongly downregulated the expression of MYC protein. Our results with RNA-seq revealed that MP1 significantly modulated global gene expression and inhibited MYC-associated transcriptional targets including translation/mTOR targets. In addition, MP1 inhibited MYC-target metabolism, leading to declined energy levels. The combination of MP1 with an FDA-approved mTOR inhibitor temsirolimus synergistically inhibited MB cell growth/survival by downregulating the expression of MYC and mTOR signaling components. Our results further showed that as single agents, both MP1 and temsirolimus, were able to significantly inhibit tumor growth and MYC expression in subcutaneously or orthotopically MYC-amplified MB bearing mice. In combination, there were further anti-MB effects on the tumor growth and MYC expression in mice. Conclusion These preclinical findings highlight the promise of marinopyrrole MP1 as a novel MYC inhibition approach for MYC-amplified MB.

Background: Medulloblastoma (MB) patients with MYC oncogene amplification or overexpression exhibit extremely poor clinical outcomes and respond poorly to current therapies. Epigenetic deregulation is very common in MYC- driven MB. The bromodomain extra-terminal (BET) proteins and histone deacetylases (HDACs) are epigenetic regulators of MYC transcription and its associated tumorigenic programs. This study aimed to investigate the therapeutic potential of inhibiting the BET proteins and HDACs together in MB. Aim: The study explores the potential of dual-inhibition targeting BET proteins and HDACs, both of which play crucial roles in MYC transcription and tumorigenic programs. Methods: Using clinically relevant BET inhibitors (JQ1 or OTX015) and a pan-HDAC inhibitor (panobinostat), we evaluated the effects of combined inhibition on cell growth/survival in MYC-amplified MB cell lines and xenografts and examined underlying molecular mechanism(s). Single agent and combined efficacies of these inhibitors on MB cell growth, apoptosis and cell cycle were determined using MTT, Annexin-V and propidium-iodide staining assays, respectively. Underlying mechanisms of these efficacies were investigated using RNA-seq, siRNA-transfection, RT-PCR and western blot analyses. In vivo antitumor efficacies were evaluated using subcutaneously MB-bearing xenografts. Results: Co-treatment of JQ1 or OTX015 with panobinostat synergistically suppressed growth/survival of MYC- amplified MB cells by inducing G2 cell cycle arrest and apoptosis. Mechanistic investigation using RNA-seq revealed that co-treatment of JQ1 with panobinostat synergistically modulated global gene expression including MYC/HDAC targets. SYK and MSI1 oncogenes were among the top 50 genes synergistically downregulated by JQ1 and panobi- nostat. RT-PCR and western blot analyses confirmed that JQ1 and panobinostat synergistically inhibited the mRNA and protein expression of MSI1/SYK along with MYC expression. Reduced SYK/MSI expression after BET (specifically, BRD4) gene-knockdown further confirmed the epigenetic regulation of SYK and MSI1 genes. In addition, the combi- nation of OTX015 and panobinostat significantly inhibited tumor growth in MYC-amplified MB xenografted mice by downregulating expression of MYC, compared to single-agent therapy. Conclusions: Together, our findings demonstrated that dual-inhibition of BET and HDAC proteins of the epigenetic pathway can be a novel therapeutic approach against MYC-driven MB. The findings demonstrate the synergistic effects of this combination therapy in suppressing MB cell growth and survival, providing valuable insights into potential treatment strategies for MYC-driven MB and highlighting the importance of epigenetic regulation in this context. On the other hand, this is a significant finding that could lead to the development of new treatments for this devastating disease.

Abstract Medulloblastoma (MB) is the most common type of childhood brain cancer worldwide. Although current treatment with surgery and extensive chemoradiation has led to increased survival rates, many MB patients still die from the disease. Moreover, surviving patients suffer severe long-term side effects as a consequence of treatment. It is therefore crucial to develop more effective and less toxic therapies. The most aggressive subtype of MB tumors often exhibits amplification or overexpression of the MYC oncogene. Patients with MYC-amplified MB exhibit a high frequency of cerebrospinal tumor dissemination, often experience treatment resistance and have extremely poor prognoses. While the MYC oncogene is established as the oncogenic driver in Group 3 MB, it has remained undruggable. Thus, targeting regulatory components of MYC and the signaling pathways regulated by it is of great potential therapeutic value. Studies have revealed that MB has very few germline mutations in cancer predisposition genes, suggesting that dysregulated epigenetic pathways might be critical in MB pathogenesis. Particularly, dysregulation of epigenetic modifiers, including histone methyltransferases and histone demethylases, is very common in Group 3 MB, compared to other MB subgroups. Therefore, it is important to identify such epigenetic modifiers that may have controls on MYC and its tumorigenic activities and explore these as the epigenetic drug-candidate targets in Group 3 (MYC-driven) MB. In this regard, we found that protein arginine demethylase Jumonji C domain-containing protein 6 (JMJD6), an emerging key epigenetic enzyme in cancers, is a novel regulator of MYC expression in MYC-driven MB. We observed high levels of JMJD6 that not only mirror MYC expression in the most aggressive MB but also correlate with poor outcomes in these patients. Knockdown of JMJD6 decreased MYC expression, cellular proliferation/survival and stemness in MYC-amplified MB cells. Mechanistically, our results revealed that JMJD6 forms complexes with proteins involved in maintaining and regulating the promoter-pause at super-enhancers, suggesting that JMJD6 can regulate MYC at the transcription level. Moreover, our in vivo analyses of JMJD6 inhibition, either with inducible gene knockdown or a pharmacologic small molecule inhibitor, demonstrated anti-MB potential with suppressed MYC expression. Based on this background and preliminary observations, we hypothesize that JMJD6 plays crucial roles in the most aggressive MB by regulating MYC expression and hence MYC-driven tumorigenesis. Accordingly, we hypothesize that targeting the JMJD6-MYC axis by JMJD6 inhibition can serve as a powerful therapeutic strategy for MYC-driven MB. Citation Format: Matthew Kling, Devendra Kumar, Sutapa Ray, Shantaram Joshi, Don Coulter, Nagendra K. Chaturvedi. JMJD6 as a novel tumorigenic factor and therapeutic target in group 3 (MYC-driven) medulloblastoma [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 3346.

Table S1. The list of genes whose expression were positively and strongly correlated with MYC expression in Group 3 MB.

MYC amplification or overexpression is most common in Group 3 medulloblastomas and is positively associated with poor clinical outcomes. Recently, protein arginine methyltransferase 5 (PRMT5) overexpression has been shown to be associated with tumorigenic MYC functions in cancers, particularly in brain cancers such as glioblastoma and medulloblastoma. PRMT5 regulates oncogenes, including MYC, that are often deregulated in medulloblastomas. However, the role of PRMT5-mediated post-translational modification in the stabilization of these oncoproteins remains poorly understood. The potential impact of PRMT5 inhibition on MYC makes it an attractive target in various cancers. PRMT5 inhibitors are a promising class of anti-cancer drugs demonstrating preclinical and preliminary clinical efficacies. Here, we review the publicly available preclinical and clinical studies on PRMT5 targeting using small molecule inhibitors and discuss the prospects of using them in medulloblastoma therapy.

Fig. S1. RNA-seq analysis between MYC vs non-MYC amplified MB cell lines. Fig. S2. Synergistic effects of OTX and mTOR inhibitors on MYC-amplified MB cell growth. Fig. S3. JQ1 chemosensitizes the MB cells. Fig. S4. Flow cytometry plots for cell cycle distribution (as summarized in main Fig. 2A and 2D) in MB cell lines-treated with inhibitors alone or combined as indicated. Fig. S5. The representative Annexin-V flow plots for apoptosis (as summarized in main Fig. 2C and 2E) in HD-MB03 MB cell lines-treated with inhibitors alone or combined as indicated. Fig. S6. Combination effects of BET protein inhibitor OTX with mTOR inhibitors (BEZ/TEM) on cell cycle and apoptosis in MYC-driven HD-MB03 cells. Fig. S7. Combination effects of JQ1/BEZ or JQ1/TEM on associated pathways/molecules. Fig. S8. Effects of inhibitors on body weight and histology of the MB xenograft mice. Fig. S9. Combined in vivo effects of JQ1 and BEZ on the expression levels of target key proteins using subcutaneous MYC-amplified MB xenografts.

While neuroblastoma accounts for an estimated 8

MB is a common childhood malignancy of the central nervous system, with significant morbidity and mortality. Among the four molecular subgroups, MYC-amplified Group 3 MB is the most aggressive type and has the worst prognosis due to therapy resistance. The present study aimed to investigate the role of activated STAT3 in promoting MB pathogenesis and chemoresistance via inducing the cancer hallmark MYC oncogene. Targeting STAT3 function either by inducible genetic knockdown (KD) or with a clinically relevant small molecule inhibitor reduced tumorigenic attributes in MB cells, including survival, proliferation, anti-apoptosis, migration, stemness and expression of MYC and its targets. STAT3 inhibition attenuates MYC expression by affecting recruitment of histone acetyltransferase p300, thereby reducing enrichment of H3K27 acetylation in the MYC promoter. Concomitantly, it also decreases the occupancy of the bromodomain containing protein-4 (BRD4) and phosphoSer2-RNA Pol II (pSer2-RNAPol II) on MYC, resulting in reduced transcription. Importantly, inhibition of STAT3 signaling significantly attenuated MB tumor growth in subcutaneous and intracranial orthotopic xenografts, increased the sensitivity of MB tumors to cisplatin, and improved the survival of mice bearing high-risk MYC-amplified tumors. Together, the results of our study demonstrate that targeting STAT3 may be a promising adjuvant therapy and chemo-sensitizer to augment treatment efficacy, reduce therapy-related toxicity and improve quality of life in high-risk pediatric patients.

Background: Emerging data indicate that BCHE, a gene encoding the enzyme butyrylcholinesterase, is a negative prognostic marker in MYCN-amplified neuroblastoma. Levels of butyrylcholinesterase in children newly diagnosed with neuroblastoma are proportional to MYCN amplification and the response to therapy. To better understand the functions of butyrylcholinesterase in neuroblastoma, we examine interactions of this enzyme with several neuroblastoma-associated kinases and provide in depth review of known associations. Methods: BCHE-deleted cells (KO) were produced from MYCN-amplified BE(2)-C cells (WT) by the CRISPR-Cas9 targeted disruption of the BCHE locus. Activation levels of several oncoproteins and the expression of N-Myc in KO were compared to WT cells. N-Myc protein expression, multiplexed detection of relative protein expression and phosphorylation of 71 tyrosine kinases and 17 proteins in the MAPK pathway were assessed using Western immunoblotting and microarrays in exponentially growing untreated cells and in cells exposed to the genotoxic stress. Results: BCHE locus disruption and butyrylcholinesterase deficiency result in the loss of N-Myc protein and a significant deactivation of several kinases associated with the aggressive neuroblastoma phenotype as well as major changes in the phosphorylation of upstream and downstream partners of these kinases Conclusions: Butyrylcholinesterase appears to contribute to the activation of several pathways in MYCN-amplified cells including FGF-R1, Ltk, TrkB, and Ros1. Deletion of BCHE and ensuing butyrylcholinesterase deficit deactivate these pathways suggesting the role of BChE as a novel druggable target in neuroblastoma therapy.