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EIF4EBP1 is transcriptionally upregulated by MYCN and associates with poor prognosis in neuroblastoma

EIF4EBP1 is transcriptionally upregulated by MYCN and associates with poor prognosis in... www.nature.com/cddiscovery ARTICLE OPEN EIF4EBP1 is transcriptionally upregulated by MYCN and associates with poor prognosis in neuroblastoma 1 1,2 3,4,5 1 1,2,6 1 3 Kai Voeltzke , Katerina Scharov , Cornelius Maximilian Funk , Alisa Kahler , Daniel Picard , Laura Hauffe , Martin F. Orth , 1,2,6 7 8,9 10 11,12 3,4,5,13 Marc Remke , Irene Esposito , Thomas Kirchner , Alexander Schramm , Barak Rotblat , Thomas G. P. Grünewald , 1,6 1✉ Guido Reifenberger and Gabriel Leprivier © The Author(s) 2022 Neuroblastoma (NB) accounts for 15% of cancer-related deaths in childhood despite considerable therapeutic improvements. While several risk factors, including MYCN amplification and alterations in RAS and p53 pathway genes, have been defined in NB, the clinical outcome is very variable and difficult to predict. Since genes of the mechanistic target of rapamycin (mTOR) pathway are upregulated in MYCN-amplified NB, we aimed to define the predictive value of the mTOR substrate-encoding gene eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1) expression in NB patients. Using publicly available data sets, we found that EIF4EBP1 mRNA expression is positively correlated with MYCN expression and elevated in stage 4 and high-risk NB patients. In addition, high EIF4EBP1 mRNA expression is associated with reduced overall and event-free survival in the entire group of NB patients in three cohorts, as well as in stage 4 and high-risk patients. This was confirmed by monitoring the clinical value of 4EBP1 protein expression, which revealed that high levels of 4EBP1 are significantly associated with prognostically unfavorable NB histology. Finally, functional analyses revealed that EIF4EBP1 expression is transcriptionally controlled by MYCN binding to the EIF4EBP1 promoter in NB cells. Our data highlight that EIF4EBP1 is a direct transcriptional target of MYCN whose high expression is associated with poor prognosis in NB patients. Therefore, EIF4EBP1 may serve to better stratify patients with NB. Cell Death Discovery (2022) 8:157 ; https://doi.org/10.1038/s41420-022-00963-0 INTRODUCTION may spontaneously regress, high-risk patients have an increased Neuroblastoma (NB) is a pediatric malignant tumor that develops likelihood of relapse and available treatment options for relapsed from progenitor cells of the sympathetic nervous system and the patients are rarely successful. Indeed, the 5-year overall survival adrenal glands [1, 2]. NB is the most commonly occurring rate for high-risk patients is ranging from 31 to 86%, in contrast to extracranial solid tumor in childhood and the major cause of 97–100% for low-risk patients [6]. In addition, success rates of cancer-related mortality in infants [2]. NB tumors are classified into second line treatment in relapsed patients remain poor [5, 7]. five stages (1, 2, 3, 4, and 4S) according to tumor size, the presence Therefore, it is critical to define novel stratification factors for NB of metastasis, and the outcome of surgical resection [1]. patients to better predict individual risk and to facilitate Noteworthy, stage 4S represents a special form of NB in infants administration of the most appropriate therapeutic option. that is associated with a high chance of spontaneous regression NB is rarely familial (1–2%) and only few predisposition genes, despite metastatic spread [1]. Apart from surgical resection, such as PHOX2B and ALK, have been reported [4, 8–10]. treatment options may include response-adjusted chemotherapy Genetically, several acquired alterations have been detected in for low to intermediate risk groups or a mix of surgery, high-dose NB and linked to patient outcome. These include gain-of-function chemotherapy, immunotherapy, and radiation for patients mutations in ALK, gain of chromosome arm 17q, loss of belonging to the high-risk group. The risk level is determined chromosome arm 11q, amplification of MYCN [4, 11], and, more based on the tumor stage combined with age at diagnosis, tumor recently reported, alterations in genes related to the RAS and p53 ploidy, genetic alterations, and tumor histology [1, 3]. However, NB pathways [12]. MYCN amplification is found in about 20% of NB represents a particularly heterogeneous type of cancer, posing and is associated with aggressive tumors, therapy resistance and challenges to precisely predict therapeutic response and clinical poor survival [13]. MYCN is a member of the MYC oncogene family outcome in the individual patient [4, 5]. While some NB tumors and encodes a transcription factor that recognizes a specific DNA 1 2 Institute of Neuropathology, University Hospital Düsseldorf, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany. Department of Pediatric Oncology, Hematology, and Clinical Immunology, University Hospital Düsseldorf, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany. Faculty of Medicine, Max-Eder Research Group for Pediatric Sarcoma Biology, Institute of Pathology, LMU Munich, Munich, Germany. Division of Translational Pediatric Sarcoma Research, German Cancer Research Center (DKFZ), 5 6 Heidelberg, Germany. Hopp Children’s Cancer Center (KiTZ), Heidelberg, Germany. German cancer consortium (DKTK), partner site Essen/Düsseldorf, Düsseldorf, Germany. 7 8 Institute of Pathology, Heinrich Heine University, Medical Faculty, and University Hospital Düsseldorf, Düsseldorf, Germany. Institute of Pathology, Faculty of Medicine, LMU 9 10 Munich, Munich, Germany. German cancer consortium (DKTK) partner site Munich, Munich, Germany. Department of Medical Oncology, West German Cancer Center, 11 12 University of Duisburg-Essen, Essen, Germany. Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel. The National Institute for Biotechnology in the Negev, Beer Sheva, Israel. Institute of Pathology, Heidelberg University Hospital, Heidelberg, Germany. email: gabriel.leprivier@med.uni-duesseldorf.de Received: 19 January 2022 Revised: 10 March 2022 Accepted: 18 March 2022 Official journal of CDDpress 1234567890();,: K. Voeltzke et al. element referred to as E-box [14, 15]. This allows MYCN to regulate a potential MYCN target gene [41, 42]. However, how MYCN the transcription of genes involved in cell cycle progression, exactly controls the EIF4EBP1 promoter is still poorly understood. proliferation, differentiation, and survival [13]. MYCN is a strong In this study, we analyzed publicly available NB patient data sets driver of NB tumorigenesis, as tissue-specific overexpression of and revealed that EIF4EBP1 is overexpressed in NB compared to MYCN is sufficient to induce NB tumor development in mouse normal tissues, is significantly co-expressed with MYCN, and is models [16]. Mechanistically, MYCN is proposed to rewire elevated in high-risk relatively to low-risk tumor groups. High metabolism to enable NB tumor cells to proliferate, in turn EIF4EBP1 levels were found to be significantly linked to poor preserving the intracellular redox balance while producing overall survival in all NB patients, as well as in the more aggressive enough energy by inducing a glycolytic switch [17–19]. In stage 4 and high-risk groups. In addition, immunohistochemistry particular, MYCN actively augments the transcription of multiple staining of NB tissues confirmed the mRNA-based associations genes whose products are involved in the protein synthesis and showed that high 4EBP1 protein expression associates with machinery [18]. Even though MYCN represents a highly attractive unfavorable histology in NB. Finally, by applying gene reporter therapeutic target in NB, as a transcription factor that lacks assays and by modulating MYCN expression in vitro, we found hydrophobic pockets which can be targeted by drug-like small that MYCN upregulates the EIF4EBP1 promoter activity by binding molecules, it is still considered as being “undruggable” [20, 21]. to three distinct E-boxes. Thus, identification of downstream effectors involved in MYCN- driven NB progression is a promising approach to uncover novel targets for molecularly guided therapeutic approaches. MATERIALS AND METHODS To better delineate the molecular basis of MYCN-amplified NB Databases The RNA-seq, microarray, and ChIP-seq data were retrieved from ‘R2: aggressiveness, several approaches have been undertaken. In Genomics Analysis and Visualization Platform’ (http://r2.amc.nl). Data were particular, RNA-sequencing (RNA-seq) has been used to uncover visualized with IGV or Affinity Designer. For the MYCN occupancy profile in the set of genes induced in MYCN-amplified compared to MYCN- BE(2)-C cells, the ChIP-seq data by Durbin et al. (GSE94824) were accessed non-amplified NB [22]. Strikingly, this analysis identified regula- using the human genome GRCh 38/hg 38. For the initial across dataset tors of protein synthesis which are components of the mechan- analysis, “Normal Adrenal gland” dataset from R2 (corresponding to istic target of rapamycin (mTOR) pathway, including the mTOR samples taken from multiple data sets [GSE3526, GSE7307, GSE8514] and target eukaryotic initiation factor 4E binding protein 1 (EIF4EBP1). combined into a single data set) and four publicly available and The corresponding protein, 4EBP1, is inhibited through mTOR- independent cohorts, namely the Versteeg et al. (GSE16476), Lastowska mediated phosphorylation when nutrients are available, leading et al. (GSE13136), Hiyama et al. (GSE16237), and Delattre et al. (GSE14880) datasets were used. The normalization was done automatically by R2 using to active mRNA translation initiation [23]. Under nutrient- MAS5.0. The remaining expression, amplification, and survival data deprived conditions, when mTOR is inhibited, 4EBP1 gets consisted of the independent SEQC/ MAQC-III Consortium (GSE49710), activated and thus binds to the translation initiation factor eIF4E, Kocak et al. study (GSE45547) and Neuroblastoma Research Consortium in turn blocking cap-dependent mRNA translation initiation [23]. [NRC] (GSE85047) cohorts. For the expression analysis of TH-MYCN At the cellular level, 4EBP1 is negatively regulating proliferation transgenic NB model, the dataset from Balamuth et al. (GSE17740) was and mitochondrial activity [24, 25]. The exact role of 4EBP1 in used. For the expression analysis of SH-SY5Y cells treated with all-trans cancer is still debated. 4EBP1 was found to exert a tumor retinoic acid (RA), the dataset from Takeda et al. (GSE9169) was used. suppressive function in vivo, as 4EBP1 knock-out leads to enhanced tumor formation in mouse models of head and neck Immunohistochemistry squamous cell carcinoma [26], and prostate cancer [27]. In For immunohistochemistry, deparaffinated tissue sections were pretreated contrast, 4EBP1 was shown to mediate angiogenesis and facilitate with citrate buffer at 98 °C for 20 min, cooled down to room temperature, tumor growth in a breast cancer model in vivo, highlighting a and blocked with 2% horse serum, avidin blocking solution, and biotin cancer type-specific function of 4EBP1 [28]. In keeping with that, blocking solution (Avidin/Biotin Blocking Kit, SP-2001, Vector Laboratories, the clinical relevance of EIF4EBP1 expression depends on the Burlingame, CA, USA) for 10 min each. Staining for 4EBP1 was carried out tumor type. EIF4EBP1 was reported to be overexpressed in a with monoclonal anti-4EBP1 raised in rabbit (1:200; ab32024, Abcam, number of tumor entities in adults [29], including breast cancer Cambridge, UK) for 2 h at 37 °C. Detection was carried out using the Dako REAL detection system, alkaline phosphatase/RED, rabbit/mouse following [30], in which EIF4EBP1 is amplified as part of the 8p11–12 manufacturer´s instructions (Detection Kit #K5005, Agilent Technologies, amplicon, as well as in ovarian and prostate cancer [31, 32]. In Santa Clara, CA, USA). Immunostained tissue sections were counterstained breast and liver cancer, high EIF4EBP1 expression has been with hematoxylin solution according to Mayer (T865.1, Roth, Karlsruhe, associated with poor survival [30, 33]. In contrast, EIF4EBP1 Germany). expression was found to be reduced in head and neck cancer, in Evaluation of immunoreactivity of 4EBP1 was carried out in analogy to which low expression is correlated with poor prognosis [26]. In scoring of hormone receptor Immune Reactive Score (IRS) ranging from NB, the expression of EIF4EBP1 is deregulated, even though 0–12. The percentage of cells with expression of the given antigen was contradictory findings have been reported. While EIF4EBP1 was scored and classified in five grades (grade 0 = 0–19%, grade 1 = 20–39%, characterized as a gene upregulated in MYCN-amplified versus grade 2 = 40–59%, grade 3 = 60–79%, and grade 4 = 80 − 100%). In addition, the intensity of marker immunoreactivity was determined MYCN-non-amplified NB tissues and cells [22], another study (grade 0 = none, grade 1 = low, grade 2 = moderate and grade 3 = reported that EIF4EBP1 levels were higher in favorable stages of strong). The product of these two grades defined the final IRS. IRS 0–6 NB as compared to advanced stage 4 tumors [34]. In addition, was considered as “low” staining level while IRS 7–12 was categorized as Meng et al. showed that EIF4EBP1 is part of a gene signature that “high” staining level. predicts poor overall survival [35]. However, it was not Tissue microarrays (TMAs) were constructed by taking three representa- investigated whether EIF4EBP1 expression alone can predict NB tive cores (each 1 mm in diameter) from respective blocks exhibiting at patient prognosis. Thus, the clinical relevance of EIF4EBP1 least 80% viable tumor tissue. Tumor blocks were retrieved from the expression in NB needs further evaluation. Overexpression of archives of the Institutes of Pathology of the LMU Munich or the University EIF4EBP1 in cancer is mediated by certain transcription factors, Hospital Düsseldorf with IRB approval (study numbers 550-16 UE for LMU Munich and 2018-174 for the University Hospital Düsseldorf). Informed such as MYC [36, 37], androgen receptor [38], and the stress consent was obtained from all patients. regulators ATF4 [39] and HIF-1α [40], which all bind to and thereby modulate the activity of the EIF4EBP1 promoter. More specifically, ChIP-sequencing (ChIP-seq) revealed binding of Statistics MYCN to the EIF4EBP1 promoter in NB cells, and MYCN was All experiments were, if not otherwise stated, independently carried out at reported to impact EIF4EBP1 transcription, pointing to EIF4EBP1 as least three times. Statistical significance was calculated using Student’s t Cell Death Discovery (2022) 8:157 K. Voeltzke et al. test or Mann–Whitney U-test in GraphPad Prism 8. For survival analysis, the luciferase expressing pGL4.22 plasmid (Promega, Madison, WI, USA). Each cohorts were stratified based on relative expression of EIF4EBP1. The of the three identified MYCN binding site was subsequently mutated alone median was chosen as expression cutoff to determine high and low or in a combination of two sites. Each of the E-box sequence has been EIF4EBP1 level. Statistical significance was determined by the logrank test. mutated to CAAGGC. All cloning was performed by GENEWIZ Germany Multivariate analysis was performed using the Cox Regression method in GmbH (Leipzig, Germany). SPSS v21 (IBM, Armonk, NY, USA). To calculate significance of the scoring of immunohistochemistry staining, the Chi-square test was used. The data are Luciferase Reporter Assay represented as means ± standard deviation. A p-value of less than 0.05 was For the promoter reporter assay, HEK-293-T cells were seeded into 12- considered significant. well plates and co-transfected the following day with 500 ng of the EIF4EBP1 WT or mutant promoter pGL4.22 plasmids, 50 ng of the Cell culture MYCN overexpressing pcDNA3.1 plasmid or empty pcDNA3.1 plasmid, Cells were maintained using standard tissue culture procedures in a and 3 ng of the Renilla Luciferase expressing pRL-SV40 plasmid humidified incubator at 37 °C with 5% CO and atmospheric oxygen. NB cell (Promega) for normalization. For transfection, plasmids were incubated lines IMR-32 and Kelly, and HEK-293-T cells were obtained from American with 3 µl CalFectin (SignaGen laboratories, Rockville, MD, USA) in Opti- Type Culture Collections (ATCC, Manassas, VA, USA). SHEP-TR-MYCN MEM (Thermo Fisher Scientific) for 20 min before adding the mix engineered NB cell lines have been previously described [19]. NB cell lines dropwise onto the cells. 48 h post-transfection, cells were passively were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium lysed and processed according to the protocol of the Dual-Luciferase® (Thermo Fisher Scientific, Waltham, MA, USA), while HEK-293-T cells were Reporter Assay System (Promega), besides using only half the maintained in Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher recommended volume of detection buffers. Fireflyand Renilla Scientific). All cell culture media were supplemented with 10% (volume/ luciferase activities were sequentially measured using a Tecan Spark volume) fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MI, USA) and 1% plate reader and the ratio of fireflyluciferaseto Renilla luciferase penicillin/streptomycin (Thermo Fisher Scientific). Cells were treated with luminescence was calculated. The experiments were repeated inde- 3 µg/ml plasmocin (Invivogen, San Diego, CA, USA) to prevent mycoplasma pendently for three times. contamination. To induce MYCN expression, SHEP-TR-MYCN cells were treated with 1 µg/ml doxycycline. All cell lines were routinely confirmed to be mycoplasma-free using Venor GeM Classic kit (Minerva Biolabs, Berlin, Germany). Cell lines were authenticated by STR-profiling (Genomics and RESULTS Transcriptomics Laboratory, Heinrich Heine University, Dusseldorf, Germany). EIF4EBP1 expression is increased in NB and correlates with MYCN expression To assess the clinical significance of EIF4EBP1 expression, we first RNA extraction, cDNA synthesis, and quantitative real-time examined EIF4EBP1 mRNA levels in NB tumor tissue samples and PCR normal tissues. We pooled microarray data of four different NB Total RNA was purified from cells using the RNeasy plus mini kit (QIAgen, Hilden, Germany) according to the manufacturer’s handbook. RNA cohorts and retrieved expression data from adrenal tissue used concentration and purity were assessed by spectrophotometry using the as the corresponding normal tissue (Fig. 1a). This indicated that NanoDrop2000 (Thermo Fisher Scientific). Subsequently, each sample was EIF4EBP1 expression is significantly elevated in NB compared to diluted to a concentration of 100 ng/µl in nuclease-free water. For cDNA adrenal gland (p < 0.0001, Fig. 1a). We then determined whether synthesis, 1 µg RNA was processed in a total reaction volume of 20 µl using EIF4EBP1 expression is related to the MYCN amplification status. the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, By comparing the level of EIF4EBP1 in MYCN-amplified versus Waltham, MA, USA), following the manufacturer’s protocol. Quantitative MYCN-non-amplified NB samples, we found that EIF4EBP1 is real-time reverse transcription (qRT) PCR was performed using SYBR green expressed at higher levels in MYCN-amplified compared to PCR master mix (Applied Biosystems) and the CFX384 Touch Real-Time PCR MYCN-non-amplified NB in the SEQC and Kocak cohorts [43, 44] Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Relative expression levels of MYCN and EIF4EBP1 were normalized to internal (p < 0.0001, Fig. 1b; p < 0.0001, Fig. 1c). This further supports and housekeeping genes GUSB and PPIA. The primer list can be found in extends previous observations made in a limited number of NB supplementary table 1. samples (n = 20) showing EIF4EBP1 overexpression in MYCN- amplified versus MYCN-non-amplified NB tumors [22]. Since MYCN amplification may result in different levels of MYCN,we Immunoblot analysis of protein expression Cells were washed with phosphate-buffered saline (PBS) and lysed in next investigated whether expression levels of MYCN and radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris- EIF4EBP1 in NB correlate with each other. Our analyses high- HCl, pH 8, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS) lighted a significant coexpression between MYCN and EIF4EBP1 supplemented with protease inhibitors (Sigma-Aldrich) and phosphatase in the SEQC (correlation coefficient [r]=0.564, p < 0.0001, Fig. 1d) inhibitors mix (PhosphoSTOP, Roche, Penzberg, Germany). Cell lysates and Kocak ([r]=0.532, p < 0.0001, Fig. 1e) cohorts. These findings were centrifuged at 21,000 rpm for 15 min at 4 °C to separate cell debris are in line with the reports that EIF4EBP1 is a potential MYCN and DNA from protein lysates. Protein concentration was measured with target gene in NB [41, 42]. We also assessed whether the the BCA protein assay kit (Thermo Fisher Scientific), according to expression of EIF4EBP1 is determined by NB stages or risk groups, manufacturer’s protocol. Protein lysates were separated by SDS-PAGE andfoundthat EIF4EBP1 levels are increased according to NB and transferred onto a nylon membrane. The membrane was incubated for 1 h in Tris-buffered saline Tween (TBST) (50 mM Tris-Cl, 150 mM NaCl, pH tumor aggressiveness in two cohorts (Fig. 1f, g). In particular, 7.5, 0.1% Tween-20) containing 5% bovine serum albumin (BSA), to EIF4EBP1 is expressed at higher levels in stage 4 NB tumors as prevent non-specific antibody binding, followed by an overnight incuba- compared to stage 1 and stage 2 tumors (stage 4 versus stage 1, tion at 4 °C with the following primary antibodies: 4EBP1 (1:1000; #9644, p < 0.0001, Fig. 1f; p < 0.0001 Fig. 1g). Interestingly, samples from Cell Signaling Technology, Cambridge, UK), MYCN (1:1,000; #9405, Cell stage 4S NB showed significantly lower EIF4EBP1 levels Signaling Technology), GAPDH (1:1000; #2118, Cell Signaling Technology), compared to stage 4 tumors (stage 4S versus stage 4, p <0.01, and β-Actin (1:5,000; #A2228, Sigma-Aldrich). The secondary antibodies Fig. 1f; p <0.001, Fig. 1g). In support of this finding, we observed IRDye 800CW Goat anti-Rabbit (1:10,000; #926-32211, LI-COR Biosciences, that in the SEQC cohort EIF4EBP1 expression is higher in high-risk Bad Homburg, Germany) or IRDye 800CW Goat anti-Mouse (1:10,000; #926- compared to low-risk NB, as based on the Children’sOncology 32210, LI-COR Biosciences) were incubated at room temperature for 1 h, followed by detection of the fluorescent signal with the Odyssey CLx Group (COG) classification (p < 0.0001, Fig. 1h). Such clinical imager (LI-COR Biosciences). information was not available in any other publicly available cohorts with mRNA expression data. Taken together, we present evidence that EIF4EBP1 is commonly overexpressed in NB tumors Plasmid construction and that EIF4EBP1 level is increased in MYCN-amplified NB and The promoter region of the human EIF4EBP1 gene, spanning from −192 to +1372, was inserted into the SacI and BglII restriction sites of the firefly advanced NB stages. Cell Death Discovery (2022) 8:157 K. Voeltzke et al. Fig. 1 EIF4EBP1 mRNA expression is associated with MYCN mRNA expression and is increased in more advanced and aggressive NB subsets. a Expression levels of EIF4EBP1 mRNA in a pool of four different NB cohorts (total n = 203), compared to healthy control tissues (adrenal gland, n = 13). b, c Expression levels of EIF4EBP1 mRNA in MYCN-amplified (n = 92, SEQC [b] and n = 93, Kocak [c]) compared to MYCN- non-amplified (n= 401, SEQC [b] and n = 550, Kocak [c]) NB patients of the SEQC (b) and Kocak (c) cohorts. d, e Expression levels of EIF4EBP1 mRNA plotted against expression levels of MYCN mRNA in SEQC (r = 0.5637, d) and Kocak (r = 0.5321, e) cohorts. f, g Expression levels of EIF4EBP1 mRNA per NB stage in SEQC (f) and Kocak (g) cohorts. h Expression levels of EIF4EBP1 mRNA in high-risk (n = 176) compared to non- high-risk (n = 322) NB in the SEQC cohort. Data were retrieved from the R2: Genomics Analysis and Visualization Platform. Statistics were determined using Mann–Whitney U-test. Exact p-values are presented. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Cell Death Discovery (2022) 8:157 K. Voeltzke et al. Fig. 2 EIF4EBP1 mRNA expression correlates with overall survival in NB patients. a–c Kaplan–Meier survival estimates of overall survival of NB patients stratified by their EIF4EBP1 mRNA expression levels (median cut off) in the SEQC (a), Kocak (b), and NRC (c) cohorts. d–h Kaplan–Meier survival estimates of overall survival of patients with MYCN-non-amplified NB (d, e), high-risk NB (f), or stage 4 NB (g, h) stratified by their EIF4EBP1 mRNA expression levels in the indicated NB cohorts. Significance was determined by log-rank test. Data were obtained from the R2: Genomics Analysis and Visualization Platform. Cell Death Discovery (2022) 8:157 K. Voeltzke et al. s- Table 1. Multivariate analysis for overall survival of NB patients in the i- SEQC cohort. Variables HR 95.0% CI p value MYCN amplification 22.373 8.89–56.306 0 High EIF4EBP1 mRNA 2.16 1.255–3.717 0.005 expression MYCN amplification*high 0.222 0.08–0.614 0.004 EIF4EBP1 mRNA expression Stage 4 17.618 6.694–46.366 0 High EIF4EBP1 mRNA 5.457 2.026–14.697 0.001 expression Stage 4*high EIF4EBP1 0.292 0.097–0.879 0.029 mRNA expression Age at diagnosis 33.018 7.835–139.139 0 High EIF4EBP1 mRNA 12.204 2.832–52.598 0.001 expression Age at diagnosis*high 0.16 0.035–0.74 0.019 EIF4EBP1 mRNA expression Table 2. Multivariate analysis for overall survival of NB patients in the NRC cohort. Variables HR 95.0% CI p value Fig. 3 4EBP1 protein expression is associated with histological MYCN amplification 4.967 1.118–22.066 0.035 subtype of NB. a Representative images at 40X magnification of low High EIF4EBP1 mRNA 3.031 1.543–5.954 0.001 (left panel) and high (right panel) 4EBP1 immunohistochemical expression staining levels of selected NB samples represented on the NB TMAs. MYCN amplification*high 0.656 0.135–3.181 0.601 b Distribution of NB cases showing low (IRS 0–6) versus high (IRS EIF4EBP1 mRNA expression 7–12) 4EBP1 protein expression in prognostically favorable versus unfavorable histological subtypes according to International Neuro- Stage 4 15.050 4.239–53.432 0.018 blastoma Pathology Classification (INPC). Fisher’s exact test was used High EIF4EBP1 mRNA 5.144 1.330–19.895 0 to calculate significance. *P < 0.05. expression gnificant for event-free survival only in the SEQC cohort Stage 4*high EIF4EBP1 0.36 0.081–1.598 0.179 (supplementary Fig. 1d, e). On the other hand, Kaplan–Meier mRNA expression survival estimates in high-risk NB patients (SEQC cohort) revealed Age at diagnosis 0.27 0.036–2.056 0 that high EIF4EBP1 levels were correlated with poor overall survival High EIF4EBP1 mRNA 0.364 0.048–2.772 0.002 (p = 7.4e-03, Fig. 2f), as well as with reduced event-free survival expression (supplementary Fig. 1f), suggesting that EIF4EBP1 expression can Age at diagnosis*high 55.427 3.258–942.88 0.005 stratify patients within the most aggressive NB subset. We EIF4EBP1 mRNA expression additionally analyzed the prognostic value of EIF4EBP1 expression in stage 4 NB patients. We found high EIF4EBP1 expression to EIF4EBP1 expression is a factor of poor prognosis in NB significantly predict decreased overall and event-free survival of Since we found EIF4EBP1 mRNA levels to be elevated in aggressive stage 4 patients in two independent cohorts (SEQC and NRC NB subsets, we examined whether EIF4EBP1 expression is linked to cohorts) (p = 3.2e-04, Fig. 2g; p = 3.8e-03, Fig. 2h and supplemen- prognosis in NB patients. Kaplan-Meier estimates univocally tary Fig. 1g, h). This highlights that EIF4EBP1 expression robustly showed that high EIF4EBP1 levels (using median expression level stratifies patients within the advanced NB subgroups. Altogether, as cut off) were significantly associated with reduced overall and our analyses support that EIF4EBP1 expression is a factor of poor event-free survival in three independent cohorts, namely SEQC, prognosis in all NB, as well as in high-risk and stage 4 NB. Kocak, and NRC cohorts [45](p = 3.1e-08, Fig. 2a; p = 4.2e-11, Fig. 2b; p = 1.7e-06, Fig. 2c, and supplementary Fig. 1a–c). To test High 4EBP1 protein expression is associated with dependence of EIF4EBP1 expression as prognostic factor on prognostically unfavorable histology of NB established factors of poor prognosis, we performed multivariate To independently confirm the prognostic value of EIF4EBP1/4EBP1 in analysis to determine the statistical interaction between high NB and to determine the biomarker potential of 4EBP1 protein EIF4EBP1 expression and MYCN amplification status, tumor stage expression in NB, we immunohistochemically analyzed NB TMAs or age at diagnosis. This indicated that MYCN amplification status, consisting of 69 patient samples. Staining of the TMAs with a 4EBP1- tumor stage and age at diagnosis each influenced the prognostic specific antibody revealed a cytoplasmic staining (Fig. 3a), consistent value of high EIF4EBP1 expression in the SEQC and NRC cohorts with the expected cellular localization of 4EBP1 [46]. We semi- (Tables 1 and 2). Therefore, high EIF4EBP1 expression is not an quantitatively evaluated 4EBP1 staining intensity and correlated independent factor of poor prognosis in NB. However, we 4EBP1 immunoreactivity with the NB histological subtypes accord- uncovered that EIF4EBP1 expression can predict overall survival ing to the International Neuroblastoma Pathology Classification in clinically relevant NB subsets, including more advanced and (INPC), which distinguishes patients with favorable or unfavorable aggressive NB subgroups. Indeed, our analyses highlighted that histology based on grade of neuroblastic differentiation and mitosis- high EIF4EBP1 expression significantly predicted reduced overall karyorrhexis index. We found that tumors with unfavorable survival in MYCN-non-amplified patients of the SEQC and NRC histology more frequently exhibited a high 4EBP1 staining score cohorts (p = 3.8e-03, Fig. 2d; p = 0.04, Fig. 2e), while it was (IRS 7–12) as compared to tumors with favorable histology (Fig. 3b), Cell Death Discovery (2022) 8:157 K. Voeltzke et al. Fig. 4 EIF4EBP1 promoter activity is regulated by MYCN. a ChIP peaks of MYCN in the EIF4EBP1 promoter region in Kelly NB cell line. b Scheme of the EIF4EBP1 promoter reporter highlighting the three E-boxes corresponding to MYCN binding sites. c HEK-293-T cells were transfected with the wildtype EIF4EBP1 promoter firefly luciferase construct and with the indicated amounts of MYCN expressing plasmid (pMYCN). A Renilla Luciferase vector was used as an internal control. d MYCN and 4EBP1 protein expression were monitored in cell lysates from c by immunoblot analyses using the indicated antibodies. e HEK-293-T were transfected with wildtype or different E-box mutants EIF4EBP1 promoter firefly luciferase constructs with or without a MYCN expressing plasmid (pMYCN). A Renilla Luciferase vector was used as an internal control. Statistics were determined using Student’s t test or Mann–Whitney U-test. Exact p-values are presented. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. indicating that high 4EBP1 protein expression is associated with MYCN-amplified NB cell line, Kelly. This revealed that MYCN more aggressive NB subsets. binds the endogenous EIF4EBP1 promoter region (which encompasses exon 1 and a part of intron 1) at three distinct EIF4EBP1 promoter activity and transcription is controlled by positions, indicating three potential MYCN binding sites (Fig. 4a). MYCN In silico analysis of the promoter region sequence confirmed the To delineate how elevated EIF4EBP1 expression is mechanisti- presence of structural E-boxes at the three occupied locations cally connected to MYCN amplification and overexpression in (Fig. 4b). To evaluate the impact of MYCN on the regulation of NB, we investigated the transcriptional regulation of EIF4EBP1 by EIF4EBP1 promoter activity, we designed a luciferase-based gene MYCN. A previous report detected the presence of MYCN on reporter assay by cloning the EIF4EBP1 promoter region (−192 EIF4EBP1 promoter by ChIP-seq in BE(2)-C, a MYCN-amplified NB to +1372) in front of a firefly luciferase gene (Fig. 4b). The cell line [41, 42]. We validated and further extended this finding activity of the wildtype EIF4EBP1 promoter was dose- by analyzing ChIP-seq data available from an additional dependently increased upon forced expression of MYCN in Cell Death Discovery (2022) 8:157 K. Voeltzke et al. HEK-293-T cells (Fig. 4c), which was accompanied by an the group of patients with aggressive stage 4 NB. Of note, less upregulation of endogenous 4EBP1 protein level (Fig. 4d). To than a third of stage 4 patients carry a MYCN amplification. Thus, it investigate which E-boxes are necessary for the transcriptional may be worth considering that, in addition to MYCN amplification activation of the EIF4EBP1 promoter by MYCN, either a single or a status, levels of EIF4EBP1 expression could help identifying combination of two of the three potential binding sites were patients carrying clinically more aggressive tumors within the mutated. Mutation of either of the three binding sites alone was stage 4 NB patients group. EIF4EBP1 expression was also linked to sufficient to significantly reduce MYCN-induced promoter worse outcome among high-risk NB patients. Given that MYCN activity (Fig. 4e). Any combinations of two mutated binding amplification is not able of predicting outcome within high-risk NB sites further reduced promoter activity driven by MYCN over- patients [50], it appears that EIF4EBP1 expression has a prognostic expression (Fig. 4e), suggesting that two binding sites, without a power beyond MYCN amplification in this patient subset. Thus, specific preference of one over another, are needed for full EIF4EBP1 expression may represent a promising biomarker for induction of EIF4EBP1 promoter activity by MYCN. We next prognostic stratification of high-risk NB patients, in addition to the intended to confirm whether MYCN directly regulates EIF4EBP1 recently reported genetic alterations in the RAS and p53 pathways transcription in NB cell lines. To do so, we chose two MYCN- [12]. This is further supported by the association we observed amplified NB cell lines, IMR-32 and Kelly, in which we knocked between high 4EBP1 protein expression and unfavorable NB down MYCN expression by siRNA and examined the impact on histological subtype. Together, our findings highlight a previously EIF4EBP1 mRNA levels by qPCR. The depletion of MYCN caused a underappreciated prognostic factor, i.e., EIF4EBP1/4EBP1, which significant reduction of EIF4EBP1 transcript levels in both cell may help refining risk stratification of NB patients, including lines (Fig. 5a, b). To further support these observations, we MYCN-non-amplified, stage 4 and high-risk patients, and could assessed the impact of forced MYCN expression on EIF4EBP1 potentially assist in tailoring more personalized treatment options. transcript and protein levels by using SHEP-TR-MYCN cells, Beyond NB, EIF4EBP1 expression was reported to be a factor of which are MYCN-non-amplified NB cells engineered to express poor prognosis in breast and liver cancers [30, 33], as well as in all exogenous MYCN with a tetracycline inducible system [19]. TCGA tumor types combined [29]. While our data indicate that Doxycycline treatment markedly increased EIF4EBP1 mRNA level EIF4EBP1 expression has prognostic power in pediatric cancer, over time (Fig. 5c), in parallel with progressive upregulation of together this supports that EIF4EBP1 expression represents a factor MYCN expression (Fig. 5d). This was accompanied by a net of poor prognosis in a large number of different tumor types. increase in the 4EBP1 protein level (Fig. 5d), supporting that Our study also extends previous knowledge by providing MYCN positively controls EIF4EBP1 mRNA and protein expression further experimental evidence to explain the association between in NB cells. To determine whether MYCN regulation of EIF4EBP1 EIF4EBP1 and MYCN expression in NB and the overexpression of has relevance during NB differentiation, we analyzed expression EIF4EBP1 in MYCN-amplified NB. Our data revealed that MYCN data of MYCN-non-amplified SH-SY5Y cells treated with RA. This induces transcription of EIF4EBP1 by regulating its promoter indicated that both MYCN and EIF4EBP1 expression are through multiple binding sites, which was originally suggested by decreased over time upon treatment, and that levels of both detection of MYCN binding to the EIF4EBP1 promoter by ChIP genes are correlated during NB differentiation (Fig. 5e–g). analysis [41, 42]. However, whether MYCN could transcriptionally Finally, analyses of expression data from a transgenic mouse regulate the EIF4EBP1 promoter was still elusive. We demonstrate model of MYCN-driven NB (TH-MYCN; [47]) revealed that that MYCN activates the EIF4EBP1 promoter through binding at EIF4EBP1 expression is upregulated in NB tumors as compared three distinct E-boxes, which in turn leads to transcriptional increase of EIF4EBP1 even with low to medium MYCN expression, to the corresponding normal tissue, i.e. the ganglia (Fig. 5h). Taken together, our data provide further evidence that EIF4EBP1 suggesting a threshold for MYCN level. Together with the previous is a transcriptional target of MYCN, potentially providing a ChIP analysis, this supports that EIF4EBP1 is a direct target gene of mechanistic basis for the observed overexpression of EIF4EBP1 in MYCN in NB cells. These findings are in line with previous studies MYCN-amplified NB patients. reporting that MYC controls EIF4EBP1 by binding its endogenous promoter in colorectal and prostate cancer cells [36, 37], as demonstrated by ChIP, highlighting a general regulation of DISCUSSION EIF4EBP1 by MYC family members in cancer cells. MYCN-amplification is accountable for aggressive NB subsets as it Expression levels of EIF4EBP1 appear not only elevated in MYCN- has been associated with increased risk of relapse and reduced amplified versus MYCN-non-amplified NB but are also upregulated overall survival of patients [13]. Since MYCN is considered in MYCN-non-amplified tumors relative to control tissue. It might “undruggable”, there is a demand for identifying targetable be speculated that in MYCN-non-amplified NB, EIF4EBP1 expres- downstream effectors of MYCN [20, 21]. In addition, since NB is sion may be regulated by transcription factors other than MYCN. a clinically heterogenous disease, ranging from spontaneous In particular, ATF4, which is critical for the metabolic response of regression to progression despite aggressive therapies, novel NB cells to glutamine starvation [51, 52], has been shown to markers that improve patient risk stratification and hence allow for control EIF4EBP1 promoter and transcription in pancreatic beta optimal treatment allocation are warranted [4, 48, 49]. Here, we cells [39]. This transcription factor is highly expressed in NB, and in report that EIF4EBP1 expression levels are significantly elevated in particular in advanced stage 4 [52]. In addition, another NB compared to corresponding non-tumor tissues and positively transcription factor that is commonly overexpressed in NB is correlate with both MYCN expression and MYCN amplification OCT4 [53]. Of note, this transcription factor has been identified by status in at least two independent NB patient cohorts. Further- ChIP-seq to bind the promoter region of EIF4EBP1 in human more, using three independent NB cohorts, we report that high embryonic stem cells [54, 55], thus OCT4 may also activate EIF4EBP1 expression is a strong predictor of poor overall and EIF4EBP1 transcription in NB cells. Together, these data suggest event-free survival across all NB patients. This was not indepen- potential mechanisms underlying the MYCN independent regula- dent of MYCN amplification status, tumor stage or age at tion of EIF4EBP1 expression in MYCN-non-amplified NB patients. diagnosis, which can be explained in part by the regulation of Given the prognostic significance of EIF4EBP1/4EBP1 in NB, it is EIF4EBP1 promoter by MYCN which we characterized. However, possible that 4EBP1 confers advantages to NB tumor growth or EIF4EBP1 expression can predict prognosis within distinct patient tumor cell survival. As evidenced by the presence of necrotic areas groups like the MYCN-non-amplified patients subset, for which flanked by HIF-1α positive staining [56], NB experience metabolic few biomarkers have been identified. Moreover, we observed that stress, corresponding to nutrient deprivation and hypoxia, as a high EIF4EBP1 expression was associated with poor prognosis in consequence of abnormal and immature vascularization [57, 58]. Cell Death Discovery (2022) 8:157 K. Voeltzke et al. One important mechanism for cancer cells to adapt to metabolic hypoxia by stimulating the synthesis of pro-angiogenic factors, stress is through reprogramming of mRNA translation [59]. As a like HIF-1α and VEGF, to facilitate tumor angiogenesis in vivo [28]. major regulator of mRNA translation, 4EBP1 may aid NB cells to In addition, the control of mRNA translation was shown to be cope with hypoxia and nutrient deprivation. This is supported by critical to prevent the deleterious effects of MYCN and MYC the report that 4EBP1 promotes survival of breast tumors under overexpression, as we and others previously reported [37]. In fact, Cell Death Discovery (2022) 8:157 K. Voeltzke et al. Fig. 5 EIF4EBP1 expression is regulated by MYCN in NB. a, b Relative MYCN and EIF4EBP1 mRNA levels upon siRNA-mediated knockdown of MYCN in the MYCN-amplified IMR-32 (a) and Kelly (b) cell lines, as measured by qRT-PCR. c, d SHEP-TR-MYCN cells were treated with doxycycline (1 µg/ml) for the indicated times; EIF4EBP1 mRNA levels were determined by qRT-PCR (c) and levels of MYCN and 4EBP1 proteins were monitored by immunoblot using the indicated antibodies (d). The different 4EBP1 bands correspond to different phosphorylated forms of 4EBP1. e, f Expression levels of MYCN (e)or EIF4EBP1 (f) mRNA in SH-SY5Y cells treated with RA for the indicated times (Takeda’s dataset, n = 2 for each time point; [60]). Statistics were calculated for each time point compared to the control 0 h time point. g Expression levels of EIF4EBP1 mRNA plotted against expression levels of MYCN mRNA in SH-SY5Y cells treated with RA (Takeda’s dataset, n= 2 for each time point; [60]). h Relative EIF4EBP1 mRNA expression in healthy control tissues (ganglia, n= 9) and NB tumors (n = 26) of a TH-MYCN transgenic mouse model of NB (Balamuth’s dataset; [47]). Data were retrieved from the R2: Genomics Analysis and Visualization Platform. Statistics were determined using Student’s t test or Mann–Whitney U-test. Exact p-values are presented. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 4EBP1, by reducing overall protein synthesis, was reported to 15. Liu R, Shi P, Wang Z, Yuan C, Cui H. Molecular mechanisms of MYCN dysregu- lation in cancers. Front Oncol. 2020;10:625332. https://doi.org/10.3389/ prevent cell death induced upon MYC overexpression, likely by fonc.2020.625332 blunting accumulation of misfolded proteins and proteotoxic ER 16. Weiss WA, Aldape K, Mohapatra G, Feuerstein BG, Bishop JM. Targeted expression stress [37]. It is possible that in a similar manner 4EBP1 contributes of MYCN causes neuroblastoma in transgenic mice. EMBO J. 1997;16:2985–95. to inhibit cell death induced by MYCN overexpression in MYCN- https://doi.org/10.1093/emboj/16.11.2985 amplified NB. 17. Oliynyk G, Ruiz-Pérez MV, Sainero-Alcolado L, Dzieran J, Zirath H, Gallart-Ayala H, In summary, the findings reported here indicate that EIF4EBP1 is et al. MYCN-enhanced oxidative and glycolytic metabolism reveals vulnerabilities a direct target gene of MYCN in NB, explaining the observed high for targeting neuroblastoma. iScience. 2019;21:188–204. https://doi.org/10.1016/ expression of EIF4EBP1 in NB, and that EIF4EBP1 mRNA and protein j.isci.2019.10.020 expression have prognostic values in NB patients, especially for 18. Boon K, Caron HN, van Asperen R, Valentijn L, Hermus MC, van Sluis P, et al. N-myc enhances the expression of a large set of genes functioning in ribosome stratifying high-risk NB patients. biogenesis and protein synthesis. EMBO J. 2001;20:1383–93. https://doi.org/ 10.1093/emboj/20.6.1383 19. Tjaden B, Baum K, Marquardt V, Simon M, Trajkovic-Arsic M, Kouril T, et al. N-Myc- DATA AVAILABILITY induced metabolic rewiring creates novel therapeutic vulnerabilities in neuro- The data that support the findings of this study are available from the corresponding blastoma. Sci Rep. 2020;10:7157. https://doi.org/10.1038/s41598-020-64040-1 author upon reasonable request. 20. Bell E, Chen L, Liu T, Marshall GM, Lunec J, Tweddle DA. MYCN oncoprotein targets and their therapeutic potential. Cancer Lett. 2010;293:144–57. https://doi. org/10.1016/j.canlet.2010.01.015 REFERENCES 21. Wolpaw AJ, Bayliss R, Büchel G, Dang CV, Eilers M, Gustafson WC, et al. Drugging 1. Maris JM, Hogarty MD, Bagatell R, Cohn SL. Neuroblastoma. Lancet. the “undruggable” MYCN oncogenic transcription factor: overcoming previous 2007;369:2106–20. https://doi.org/10.1016/S0140-6736(07)60983-0 obstacles to impact childhood cancers. Cancer Res. 2021;81:1627–32. https://doi. 2. van Arendonk KJ, Chung DH. Neuroblastoma: tumor biology and its implications org/10.1158/0008-5472.CAN-20-3108 for staging and treatment. Children (Basel) 2019;6. https://doi.org/10.3390/ 22. Schramm A, Köster J, Marschall T, Martin M, Schwermer M, Fielitz K, et al. Next- children6010012 generation RNA sequencing reveals differential expression of MYCN target genes 3. Tolbert VP, Matthay KK. Neuroblastoma: clinical and biological approach to risk and suggests the mTOR pathway as a promising therapy target in MYCN- stratification and treatment. Cell Tissue Res. 2018;372:195–209. https://doi.org/ amplified neuroblastoma. Int. J. Cancer. 2013;132:E106–15. https://doi.org/ 10.1007/s00441-018-2821-2 10.1002/ijc.27787 4. Maris JM. The biologic basis for neuroblastoma heterogeneity and risk stratifi- 23. Musa J, Orth MF, Dallmayer M, Baldauf M, Pardo C, Rotblat B, et al. Eukaryotic cation. Curr Opin Pediatrics. 2005;17:7–13. https://doi.org/10.1097/01. initiation factor 4E-binding protein 1 (4E-BP1): a master regulator of mRNA mop.0000150631.60571.89 translation involved in tumorigenesis. Oncogene. 2016;35:4675–88. https://doi. 5. London WB, Castel V, Monclair T, Ambros PF, Pearson ADJ, Cohn SL, et al. Clinical org/10.1038/onc.2015.515 and biologic features predictive of survival after relapse of neuroblastoma: a 24. Morita M, Gravel S-P, Chénard V, Sikström K, Zheng L, Alain T, et al. mTORC1 report from the International Neuroblastoma Risk Group project. J Clin Oncol. controls mitochondrial activity and biogenesis through 4E-BP-dependent trans- 2011;29:3286–92. https://doi.org/10.1200/JCO.2010.34.3392 lational regulation. Cell Metab. 2013;18:698–711. https://doi.org/10.1016/j. 6. Pinto NR, Applebaum MA, Volchenboum SL, Matthay KK, London WB, Ambros PF, cmet.2013.10.001 et al. Advances in risk classification and treatment strategies for neuroblastoma. J 25. Dowling RJO, Topisirovic I, Alain T, Bidinosti M, Fonseca BD, Petroulakis E, et al. Clin Oncol. 2015;33:3008–17. https://doi.org/10.1200/JCO.2014.59.4648 mTORC1-mediated cell proliferation, but not cell growth, controlled by the 4E- 7. Simon T, Berthold F, Borkhardt A, Kremens B, Carolis B, de, Hero B. Treatment BPs. Science. 2010;328:1172–6. https://doi.org/10.1126/science.1187532 and outcomes of patients with relapsed, high-risk neuroblastoma: results of 26. Wang Z, Feng X, Molinolo AA, Martin D, Vitale-Cross L, Nohata N, et al. 4E-BP1 is a German trials. Pediatr Blood Cancer. 2011;56:578–83. https://doi.org/10.1002/ tumor suppressor protein reactivated by mTOR inhibition in head and neck pbc.22693 cancer. Cancer Res. 2019;79:1438–50. https://doi.org/10.1158/0008-5472.CAN-18- 8. Chen Y, Takita J, Choi YL, Kato M, Ohira M, Sanada M, et al. Oncogenic mutations of ALK kinase in neuroblastoma. Nature. 2008;455:971–4. https://doi.org/10.1038/ 27. Ding M, van der Kwast TH, Vellanki RN, Foltz WD, McKee TD, Sonenberg N, et al. nature07399 The mTOR targets 4E-BP1/2 restrain tumor growth and promote hypoxia toler- 9. Mossé YP, Laudenslager M, Longo L, Cole KA, Wood A, Attiyeh EF, et al. Identi- ance in PTEN-driven prostate cancer. Mol Cancer Res. 2018;16:682–95. https://doi. fication of ALK as a major familial neuroblastoma predisposition gene. Nature. org/10.1158/1541-7786.MCR-17-0696 2008;455:930–5. https://doi.org/10.1038/nature07261 28. Braunstein S, Karpisheva K, Pola C, Goldberg J, Hochman T, Yee H, et al. A hypoxia- 10. Janoueix-Lerosey I, Lequin D, Brugières L, Ribeiro A, Pontual L, de, Combaret V, controlled cap-dependent to cap-independent translation switch in breast cancer. et al. Somatic and germline activating mutations of the ALK kinase receptor in Mol Cell. 2007;28:501–12. https://doi.org/10.1016/j.molcel.2007.10.019 neuroblastoma. Nature. 2008;455:967–70. https://doi.org/10.1038/nature07398 29. Wu S & Wagner G. Deep computational analysis of human cancer and non-cancer 11. Amelio I, Bertolo R, Bove P, Candi E, Chiocchi M, Cipriani C, et al. Cancer predictive tissues details dysregulation of eIF4F components and their interactions in studies. Biol Direct. 2020;15:18. https://doi.org/10.1186/s13062-020-00274-3 human cancers. bioRxiv 2020. https://doi.org/10.1101/2020.10.12.336263 12. Ackermann S, Cartolano M, Hero B, Welte A, Kahlert Y, Roderwieser A, et al. A 30. Karlsson E, Pérez-Tenorio G, Amin R, Bostner J, Skoog L, Fornander T, et al. The mechanistic classification of clinical phenotypes in neuroblastoma. Science. mTOR effectors 4EBP1 and S6K2 are frequently coexpressed, and associated with 2018;362:1165–70. https://doi.org/10.1126/science.aat6768 a poor prognosis and endocrine resistance in breast cancer: a retrospective study 13. Huang M, Weiss WA. Neuroblastoma and MYCN. Cold Spring Harb Perspect Med. including patients from the randomised Stockholm tamoxifen trials. Breast 2013;3:a014415. https://doi.org/10.1101/cshperspect.a014415 Cancer Res. 2013;15:R96. https://doi.org/10.1186/bcr3557 14. Zeid R, Lawlor MA, Poon E, Reyes JM, Fulciniti M, Lopez MA, et al. Enhancer 31. Kremer CL, Klein RR, Mendelson J, Browne W, Samadzedeh LK, Vanpatten K, et al. invasion shapes MYCN-dependent transcriptional amplification in neuro- Expression of mTOR signaling pathway markers in prostate cancer progression. blastoma. Nat Genet. 2018;50:515–23. https://doi.org/10.1038/s41588-018-0044-9 Prostate. 2006;66:1203–12. https://doi.org/10.1002/pros.20410 Cell Death Discovery (2022) 8:157 K. Voeltzke et al. 32. Lee M, Kim EJ, Jeon MJ. MicroRNAs 125a and 125b inhibit ovarian cancer cells 53. Yang S, Zheng J, Ma Y, Zhu H, Xu T, Dong K, et al. Oct4 and Sox2 are over- through post-transcriptional inactivation of EIF4EBP1. Oncotarget. expressed in human neuroblastoma and inhibited by chemotherapy. Oncol Rep. 2016;7:8726–42. https://doi.org/10.18632/oncotarget.6474 2012;28:186–92. https://doi.org/10.3892/or.2012.1765 33. Cha Y-L, Li P-D, Yuan L-J, Zhang M-Y, Zhang Y-J, Rao H-L, et al. EIF4EBP1 over- 54. Gifford CA, Ziller MJ, Gu H, Trapnell C, Donaghey J, Tsankov A, et al. Transcrip- expression is associated with poor survival and disease progression in patients tional and epigenetic dynamics during specification of human embryonic stem with hepatocellular carcinoma. PLoS ONE. 2015;10:e0117493. https://doi.org/ cells. Cell. 2013;153:1149–63. https://doi.org/10.1016/j.cell.2013.04.037 10.1371/journal.pone.0117493 55. Čančer M, Hutter S, Holmberg KO, Rosén G, Sundström A, Tailor J, et al. Huma- 34. Fransson S, Abel F, Kogner P, Martinsson T, Ejeskär K. Stage-dependent expres- nized stem cell models of pediatric medulloblastoma reveal an Oct4/mTOR axis sion of PI3K/Akt‑pathway genes in neuroblastoma. Int J Oncol. 2013;42:609–16. that promotes malignancy. Cell Stem Cell. 2019;25:855–870.e11. https://doi.org/ https://doi.org/10.3892/ijo.2012.1732 10.1016/j.stem.2019.10.005 35. Meng X, Li H, Fang E, Feng J, Zhao X. Comparison of stage 4 and stage 4s 56. Påhlman S, Mohlin S. Hypoxia and hypoxia-inducible factors in neuroblastoma. neuroblastoma identifies autophagy-related gene and LncRNA signatures asso- Cell Tissue Res. 2018;372:269–75. https://doi.org/10.1007/s00441-017-2701-1 ciated with prognosis. Front Oncol. 2020;10:1411. https://doi.org/10.3389/ 57. Schaaf MB, Garg AD, Agostinis P. Defining the role of the tumor vasculature in fonc.2020.01411 antitumor immunity and immunotherapy. Cell Death Dis. 2018;9:115. https://doi. 36. Balakumaran BS, Porrello A, Hsu DS, Glover W, Foye A, Leung JY, et al. MYC org/10.1038/s41419-017-0061-0 activity mitigates response to rapamycin in prostate cancer through eukaryotic 58. Lugano R, Ramachandran M, Dimberg A. Tumor angiogenesis: causes, con- initiation factor 4E-binding protein 1-mediated inhibition of autophagy. Cancer sequences, challenges and opportunities. Cell Mol Life Sci. 2020;77:1745–70. Res. 2009;69:7803–10. https://doi.org/10.1158/0008-5472.CAN-09-0910 https://doi.org/10.1007/s00018-019-03351-7 37. Tameire F, Verginadis II, Leli NM, Polte C, Conn CS, Ojha R, et al. ATF4 couples 59. Leprivier G, Rotblat B, Khan D, Jan E, Sorensen PH. Stress-mediated translational MYC-dependent translational activity to bioenergetic demands during tumour control in cancer cells. Biochim. Biophys. Acta. 2015;1849:845–60. https://doi.org/ progression. Nat Cell Biol. 2019;21:889–99. https://doi.org/10.1038/s41556-019- 10.1016/j.bbagrm.2014.11.002 0347-9 60. Nishida Y, Adati N, Ozawa R, Maeda A, Sakaki Y, Takeda T. Identification and 38. Liu Y, Horn JL, Banda K, Goodman AZ, Lim Y, Jana S et al. The androgen receptor classification of genes regulated by phosphatidylinositol 3-kinase- and TRKB- regulates a druggable translational regulon in advanced prostate cancer. Sci mediated signalling pathways during neuronal differentiation in two subtypes of Transl Med. 2019;11. https://doi.org/10.1126/scitranslmed.aaw4993 the human neuroblastoma cell line SH-SY5Y. BMC Res Notes. 2008;1:95. https:// 39. Yamaguchi S, Ishihara H, Yamada T, Tamura A, Usui M, Tominaga R, et al. ATF4- doi.org/10.1186/1756-0500-1-95 mediated induction of 4E-BP1 contributes to pancreatic beta cell survival under endoplasmic reticulum stress. Cell Metab. 2008;7:269–76. https://doi.org/10.1016/ j.cmet.2008.01.008 40. Azar R, Lasfargues C, Bousquet C, Pyronnet S. Contribution of HIF-1α in 4E-BP1 ACKNOWLEDGEMENTS gene expression. Mol. Cancer Res. 2013;11:54–61. https://doi.org/10.1158/1541- We would like to thank Dr. Bastian Malzkorn (Institute of Neuropathology, Heinrich 7786.MCR-12-0095 Heine University Düsseldorf) for helpful discussions. GL was supported by funding 41. Cheung CHY, Hsu C-L, Tsuei C-Y, Kuo T-T, Huang C-T, Hsu W-M, et al. Combina- from the Elterninitiative Düsseldorf e.V., the Research Commission of the Medical torial targeting of MTHFD2 and PAICS in purine synthesis as a novel therapeutic Faculty of Heinrich Heine University, the Deutsche Forschungsgemeinschaft (Grant LE strategy. Cell Death Dis. 2019;10:786. https://doi.org/10.1038/s41419-019-2033-z 3751/2-1), and the German Cancer Aid (Grant 70112624). The laboratory of TGPG is 42. Hsu C-L, Chang H-Y, Chang J-Y, Hsu W-M, Huang H-C, Juan H-F. Unveiling supported by the Barbara und Wilfried Mohr Foundation. BR is supported by the MYCN regulatory networks in neuroblastoma via integrative analysis of het- Israel Science Foundation (grant No. 1436/19). erogeneous genomics data. Oncotarget. 2016;7:36293–310. https://doi.org/ 10.18632/oncotarget.9202 43. SEQC/MAQC consortium. SEQC/MAQC consortium: a comprehensive assessment of RNA-seq accuracy, reproducibility and information. Nat Biotechnol. AUTHOR CONTRIBUTIONS 2014;32:903–14. https://doi.org/10.1038/nbt.2957 Conception and design: KV and GL. Provision of study material and patients: IE and 44. Kocak H, Ackermann S, Hero B, Kahlert Y, Oberthuer A, Juraeva D, et al. Hox-C9 TK. Financial and administrative support: GR. Data analysis and interpretation: KV, activates the intrinsic pathway of apoptosis and is associated with spontaneous TGPG, AS, and GL. Critical review and discussion: BR, MR, AS, GR, and GL. Experimental regression in neuroblastoma. Cell Death Dis. 2013;4:e586. https://doi.org/ support: KV, KS, CF, AK, DP, LH, and MFO. Manuscript writing: KV, GR, and GL. Final 10.1038/cddis.2013.84 approval of the manuscript: all authors. 45. Rajbhandari P, Lopez G, Capdevila C, Salvatori B, Yu J, Rodriguez-Barrueco R, et al. Cross-cohort analysis identifies a TEAD4-MYCN positive feedback loop as the core regulatory element of high-risk neuroblastoma. Cancer Discov. 2018;8:582–99. FUNDING https://doi.org/10.1158/2159-8290.CD-16-0861 Open Access funding enabled and organized by Projekt DEAL. 46. Armengol G, Rojo F, Castellví J, Iglesias C, Cuatrecasas M, Pons B, et al. 4E-binding protein 1: a key molecular “funnel factor” in human cancer with clinical implica- tions. Cancer Res. 2007;67:7551–5. https://doi.org/10.1158/0008-5472.CAN-07-0881 47. Balamuth NJ, Wood A, Wang Q, Jagannathan J, Mayes P, Zhang Z, et al. Serial COMPETING INTERESTS transcriptome analysis and cross-species integration identifies centromere- TK received honoraria for Consulting/Advisory by Amgen, AstraZeneca, BMS, Merck associated protein E as a novel neuroblastoma target. Cancer Res. KGaA, MSD, Novartis, Pfizer, Roche, for Research Funding by Merck KGaA and Roche; 2010;70:2749–58. https://doi.org/10.1158/0008-5472.CAN-09-3844 for talks by Merck KGaA, AstraZeneca. The other authors declare no conflict of 48. Rugolo F, Bazan NG, Calandria J, Jun B, Raschellà G, Melino G, et al. The interest. expression of ELOVL4, repressed by MYCN, defines neuroblastoma patients with good outcome. Oncogene. 2021;40:5741–51. https://doi.org/10.1038/s41388-021- 01959-3 49. Pieraccioli M, Nicolai S, Pitolli C, Agostini M, Antonov A, Malewicz M, et al. ZNF281 ADDITIONAL INFORMATION inhibits neuronal differentiation and is a prognostic marker for neuroblastoma. Proc. Natl Acad. Sci. USA. 2018;115:7356–61. https://doi.org/10.1073/pnas.1801435115 Supplementary information The online version contains supplementary material 50. Lee JW, Son MH, Cho HW, Ma YE, Yoo KH, Sung KW, et al. Clinical significance of available at https://doi.org/10.1038/s41420-022-00963-0. MYCN amplification in patients with high-risk neuroblastoma. Pediatr Blood Cancer. 2018;65:e27257. https://doi.org/10.1002/pbc.27257 Correspondence and requests for materials should be addressed to Gabriel Leprivier. 51. Qing G, Li B, Vu A, Skuli N, Walton ZE, Liu X, et al. ATF4 regulates MYC-mediated neuroblastoma cell death upon glutamine deprivation. Cancer Cell. Reprints and permission information is available at http://www.nature.com/ 2012;22:631–44. https://doi.org/10.1016/j.ccr.2012.09.021 reprints 52. Ren P, Yue M, Xiao D, Xiu R, Gan L, Liu H, et al. ATF4 and N-Myc coordinate glutamine metabolism in MYCN-amplified neuroblastoma cells through ASCT2 Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims activation. J Pathol. 2015;235:90–100. https://doi.org/10.1002/path.4429 in published maps and institutional affiliations. Cell Death Discovery (2022) 8:157 K. Voeltzke et al. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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www.nature.com/cddiscovery ARTICLE OPEN EIF4EBP1 is transcriptionally upregulated by MYCN and associates with poor prognosis in neuroblastoma 1 1,2 3,4,5 1 1,2,6 1 3 Kai Voeltzke , Katerina Scharov , Cornelius Maximilian Funk , Alisa Kahler , Daniel Picard , Laura Hauffe , Martin F. Orth , 1,2,6 7 8,9 10 11,12 3,4,5,13 Marc Remke , Irene Esposito , Thomas Kirchner , Alexander Schramm , Barak Rotblat , Thomas G. P. Grünewald , 1,6 1✉ Guido Reifenberger and Gabriel Leprivier © The Author(s) 2022 Neuroblastoma (NB) accounts for 15% of cancer-related deaths in childhood despite considerable therapeutic improvements. While several risk factors, including MYCN amplification and alterations in RAS and p53 pathway genes, have been defined in NB, the clinical outcome is very variable and difficult to predict. Since genes of the mechanistic target of rapamycin (mTOR) pathway are upregulated in MYCN-amplified NB, we aimed to define the predictive value of the mTOR substrate-encoding gene eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1) expression in NB patients. Using publicly available data sets, we found that EIF4EBP1 mRNA expression is positively correlated with MYCN expression and elevated in stage 4 and high-risk NB patients. In addition, high EIF4EBP1 mRNA expression is associated with reduced overall and event-free survival in the entire group of NB patients in three cohorts, as well as in stage 4 and high-risk patients. This was confirmed by monitoring the clinical value of 4EBP1 protein expression, which revealed that high levels of 4EBP1 are significantly associated with prognostically unfavorable NB histology. Finally, functional analyses revealed that EIF4EBP1 expression is transcriptionally controlled by MYCN binding to the EIF4EBP1 promoter in NB cells. Our data highlight that EIF4EBP1 is a direct transcriptional target of MYCN whose high expression is associated with poor prognosis in NB patients. Therefore, EIF4EBP1 may serve to better stratify patients with NB. Cell Death Discovery (2022) 8:157 ; https://doi.org/10.1038/s41420-022-00963-0 INTRODUCTION may spontaneously regress, high-risk patients have an increased Neuroblastoma (NB) is a pediatric malignant tumor that develops likelihood of relapse and available treatment options for relapsed from progenitor cells of the sympathetic nervous system and the patients are rarely successful. Indeed, the 5-year overall survival adrenal glands [1, 2]. NB is the most commonly occurring rate for high-risk patients is ranging from 31 to 86%, in contrast to extracranial solid tumor in childhood and the major cause of 97–100% for low-risk patients [6]. In addition, success rates of cancer-related mortality in infants [2]. NB tumors are classified into second line treatment in relapsed patients remain poor [5, 7]. five stages (1, 2, 3, 4, and 4S) according to tumor size, the presence Therefore, it is critical to define novel stratification factors for NB of metastasis, and the outcome of surgical resection [1]. patients to better predict individual risk and to facilitate Noteworthy, stage 4S represents a special form of NB in infants administration of the most appropriate therapeutic option. that is associated with a high chance of spontaneous regression NB is rarely familial (1–2%) and only few predisposition genes, despite metastatic spread [1]. Apart from surgical resection, such as PHOX2B and ALK, have been reported [4, 8–10]. treatment options may include response-adjusted chemotherapy Genetically, several acquired alterations have been detected in for low to intermediate risk groups or a mix of surgery, high-dose NB and linked to patient outcome. These include gain-of-function chemotherapy, immunotherapy, and radiation for patients mutations in ALK, gain of chromosome arm 17q, loss of belonging to the high-risk group. The risk level is determined chromosome arm 11q, amplification of MYCN [4, 11], and, more based on the tumor stage combined with age at diagnosis, tumor recently reported, alterations in genes related to the RAS and p53 ploidy, genetic alterations, and tumor histology [1, 3]. However, NB pathways [12]. MYCN amplification is found in about 20% of NB represents a particularly heterogeneous type of cancer, posing and is associated with aggressive tumors, therapy resistance and challenges to precisely predict therapeutic response and clinical poor survival [13]. MYCN is a member of the MYC oncogene family outcome in the individual patient [4, 5]. While some NB tumors and encodes a transcription factor that recognizes a specific DNA 1 2 Institute of Neuropathology, University Hospital Düsseldorf, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany. Department of Pediatric Oncology, Hematology, and Clinical Immunology, University Hospital Düsseldorf, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany. Faculty of Medicine, Max-Eder Research Group for Pediatric Sarcoma Biology, Institute of Pathology, LMU Munich, Munich, Germany. Division of Translational Pediatric Sarcoma Research, German Cancer Research Center (DKFZ), 5 6 Heidelberg, Germany. Hopp Children’s Cancer Center (KiTZ), Heidelberg, Germany. German cancer consortium (DKTK), partner site Essen/Düsseldorf, Düsseldorf, Germany. 7 8 Institute of Pathology, Heinrich Heine University, Medical Faculty, and University Hospital Düsseldorf, Düsseldorf, Germany. Institute of Pathology, Faculty of Medicine, LMU 9 10 Munich, Munich, Germany. German cancer consortium (DKTK) partner site Munich, Munich, Germany. Department of Medical Oncology, West German Cancer Center, 11 12 University of Duisburg-Essen, Essen, Germany. Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, Israel. The National Institute for Biotechnology in the Negev, Beer Sheva, Israel. Institute of Pathology, Heidelberg University Hospital, Heidelberg, Germany. email: gabriel.leprivier@med.uni-duesseldorf.de Received: 19 January 2022 Revised: 10 March 2022 Accepted: 18 March 2022 Official journal of CDDpress 1234567890();,: K. Voeltzke et al. element referred to as E-box [14, 15]. This allows MYCN to regulate a potential MYCN target gene [41, 42]. However, how MYCN the transcription of genes involved in cell cycle progression, exactly controls the EIF4EBP1 promoter is still poorly understood. proliferation, differentiation, and survival [13]. MYCN is a strong In this study, we analyzed publicly available NB patient data sets driver of NB tumorigenesis, as tissue-specific overexpression of and revealed that EIF4EBP1 is overexpressed in NB compared to MYCN is sufficient to induce NB tumor development in mouse normal tissues, is significantly co-expressed with MYCN, and is models [16]. Mechanistically, MYCN is proposed to rewire elevated in high-risk relatively to low-risk tumor groups. High metabolism to enable NB tumor cells to proliferate, in turn EIF4EBP1 levels were found to be significantly linked to poor preserving the intracellular redox balance while producing overall survival in all NB patients, as well as in the more aggressive enough energy by inducing a glycolytic switch [17–19]. In stage 4 and high-risk groups. In addition, immunohistochemistry particular, MYCN actively augments the transcription of multiple staining of NB tissues confirmed the mRNA-based associations genes whose products are involved in the protein synthesis and showed that high 4EBP1 protein expression associates with machinery [18]. Even though MYCN represents a highly attractive unfavorable histology in NB. Finally, by applying gene reporter therapeutic target in NB, as a transcription factor that lacks assays and by modulating MYCN expression in vitro, we found hydrophobic pockets which can be targeted by drug-like small that MYCN upregulates the EIF4EBP1 promoter activity by binding molecules, it is still considered as being “undruggable” [20, 21]. to three distinct E-boxes. Thus, identification of downstream effectors involved in MYCN- driven NB progression is a promising approach to uncover novel targets for molecularly guided therapeutic approaches. MATERIALS AND METHODS To better delineate the molecular basis of MYCN-amplified NB Databases The RNA-seq, microarray, and ChIP-seq data were retrieved from ‘R2: aggressiveness, several approaches have been undertaken. In Genomics Analysis and Visualization Platform’ (http://r2.amc.nl). Data were particular, RNA-sequencing (RNA-seq) has been used to uncover visualized with IGV or Affinity Designer. For the MYCN occupancy profile in the set of genes induced in MYCN-amplified compared to MYCN- BE(2)-C cells, the ChIP-seq data by Durbin et al. (GSE94824) were accessed non-amplified NB [22]. Strikingly, this analysis identified regula- using the human genome GRCh 38/hg 38. For the initial across dataset tors of protein synthesis which are components of the mechan- analysis, “Normal Adrenal gland” dataset from R2 (corresponding to istic target of rapamycin (mTOR) pathway, including the mTOR samples taken from multiple data sets [GSE3526, GSE7307, GSE8514] and target eukaryotic initiation factor 4E binding protein 1 (EIF4EBP1). combined into a single data set) and four publicly available and The corresponding protein, 4EBP1, is inhibited through mTOR- independent cohorts, namely the Versteeg et al. (GSE16476), Lastowska mediated phosphorylation when nutrients are available, leading et al. (GSE13136), Hiyama et al. (GSE16237), and Delattre et al. (GSE14880) datasets were used. The normalization was done automatically by R2 using to active mRNA translation initiation [23]. Under nutrient- MAS5.0. The remaining expression, amplification, and survival data deprived conditions, when mTOR is inhibited, 4EBP1 gets consisted of the independent SEQC/ MAQC-III Consortium (GSE49710), activated and thus binds to the translation initiation factor eIF4E, Kocak et al. study (GSE45547) and Neuroblastoma Research Consortium in turn blocking cap-dependent mRNA translation initiation [23]. [NRC] (GSE85047) cohorts. For the expression analysis of TH-MYCN At the cellular level, 4EBP1 is negatively regulating proliferation transgenic NB model, the dataset from Balamuth et al. (GSE17740) was and mitochondrial activity [24, 25]. The exact role of 4EBP1 in used. For the expression analysis of SH-SY5Y cells treated with all-trans cancer is still debated. 4EBP1 was found to exert a tumor retinoic acid (RA), the dataset from Takeda et al. (GSE9169) was used. suppressive function in vivo, as 4EBP1 knock-out leads to enhanced tumor formation in mouse models of head and neck Immunohistochemistry squamous cell carcinoma [26], and prostate cancer [27]. In For immunohistochemistry, deparaffinated tissue sections were pretreated contrast, 4EBP1 was shown to mediate angiogenesis and facilitate with citrate buffer at 98 °C for 20 min, cooled down to room temperature, tumor growth in a breast cancer model in vivo, highlighting a and blocked with 2% horse serum, avidin blocking solution, and biotin cancer type-specific function of 4EBP1 [28]. In keeping with that, blocking solution (Avidin/Biotin Blocking Kit, SP-2001, Vector Laboratories, the clinical relevance of EIF4EBP1 expression depends on the Burlingame, CA, USA) for 10 min each. Staining for 4EBP1 was carried out tumor type. EIF4EBP1 was reported to be overexpressed in a with monoclonal anti-4EBP1 raised in rabbit (1:200; ab32024, Abcam, number of tumor entities in adults [29], including breast cancer Cambridge, UK) for 2 h at 37 °C. Detection was carried out using the Dako REAL detection system, alkaline phosphatase/RED, rabbit/mouse following [30], in which EIF4EBP1 is amplified as part of the 8p11–12 manufacturer´s instructions (Detection Kit #K5005, Agilent Technologies, amplicon, as well as in ovarian and prostate cancer [31, 32]. In Santa Clara, CA, USA). Immunostained tissue sections were counterstained breast and liver cancer, high EIF4EBP1 expression has been with hematoxylin solution according to Mayer (T865.1, Roth, Karlsruhe, associated with poor survival [30, 33]. In contrast, EIF4EBP1 Germany). expression was found to be reduced in head and neck cancer, in Evaluation of immunoreactivity of 4EBP1 was carried out in analogy to which low expression is correlated with poor prognosis [26]. In scoring of hormone receptor Immune Reactive Score (IRS) ranging from NB, the expression of EIF4EBP1 is deregulated, even though 0–12. The percentage of cells with expression of the given antigen was contradictory findings have been reported. While EIF4EBP1 was scored and classified in five grades (grade 0 = 0–19%, grade 1 = 20–39%, characterized as a gene upregulated in MYCN-amplified versus grade 2 = 40–59%, grade 3 = 60–79%, and grade 4 = 80 − 100%). In addition, the intensity of marker immunoreactivity was determined MYCN-non-amplified NB tissues and cells [22], another study (grade 0 = none, grade 1 = low, grade 2 = moderate and grade 3 = reported that EIF4EBP1 levels were higher in favorable stages of strong). The product of these two grades defined the final IRS. IRS 0–6 NB as compared to advanced stage 4 tumors [34]. In addition, was considered as “low” staining level while IRS 7–12 was categorized as Meng et al. showed that EIF4EBP1 is part of a gene signature that “high” staining level. predicts poor overall survival [35]. However, it was not Tissue microarrays (TMAs) were constructed by taking three representa- investigated whether EIF4EBP1 expression alone can predict NB tive cores (each 1 mm in diameter) from respective blocks exhibiting at patient prognosis. Thus, the clinical relevance of EIF4EBP1 least 80% viable tumor tissue. Tumor blocks were retrieved from the expression in NB needs further evaluation. Overexpression of archives of the Institutes of Pathology of the LMU Munich or the University EIF4EBP1 in cancer is mediated by certain transcription factors, Hospital Düsseldorf with IRB approval (study numbers 550-16 UE for LMU Munich and 2018-174 for the University Hospital Düsseldorf). Informed such as MYC [36, 37], androgen receptor [38], and the stress consent was obtained from all patients. regulators ATF4 [39] and HIF-1α [40], which all bind to and thereby modulate the activity of the EIF4EBP1 promoter. More specifically, ChIP-sequencing (ChIP-seq) revealed binding of Statistics MYCN to the EIF4EBP1 promoter in NB cells, and MYCN was All experiments were, if not otherwise stated, independently carried out at reported to impact EIF4EBP1 transcription, pointing to EIF4EBP1 as least three times. Statistical significance was calculated using Student’s t Cell Death Discovery (2022) 8:157 K. Voeltzke et al. test or Mann–Whitney U-test in GraphPad Prism 8. For survival analysis, the luciferase expressing pGL4.22 plasmid (Promega, Madison, WI, USA). Each cohorts were stratified based on relative expression of EIF4EBP1. The of the three identified MYCN binding site was subsequently mutated alone median was chosen as expression cutoff to determine high and low or in a combination of two sites. Each of the E-box sequence has been EIF4EBP1 level. Statistical significance was determined by the logrank test. mutated to CAAGGC. All cloning was performed by GENEWIZ Germany Multivariate analysis was performed using the Cox Regression method in GmbH (Leipzig, Germany). SPSS v21 (IBM, Armonk, NY, USA). To calculate significance of the scoring of immunohistochemistry staining, the Chi-square test was used. The data are Luciferase Reporter Assay represented as means ± standard deviation. A p-value of less than 0.05 was For the promoter reporter assay, HEK-293-T cells were seeded into 12- considered significant. well plates and co-transfected the following day with 500 ng of the EIF4EBP1 WT or mutant promoter pGL4.22 plasmids, 50 ng of the Cell culture MYCN overexpressing pcDNA3.1 plasmid or empty pcDNA3.1 plasmid, Cells were maintained using standard tissue culture procedures in a and 3 ng of the Renilla Luciferase expressing pRL-SV40 plasmid humidified incubator at 37 °C with 5% CO and atmospheric oxygen. NB cell (Promega) for normalization. For transfection, plasmids were incubated lines IMR-32 and Kelly, and HEK-293-T cells were obtained from American with 3 µl CalFectin (SignaGen laboratories, Rockville, MD, USA) in Opti- Type Culture Collections (ATCC, Manassas, VA, USA). SHEP-TR-MYCN MEM (Thermo Fisher Scientific) for 20 min before adding the mix engineered NB cell lines have been previously described [19]. NB cell lines dropwise onto the cells. 48 h post-transfection, cells were passively were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium lysed and processed according to the protocol of the Dual-Luciferase® (Thermo Fisher Scientific, Waltham, MA, USA), while HEK-293-T cells were Reporter Assay System (Promega), besides using only half the maintained in Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher recommended volume of detection buffers. Fireflyand Renilla Scientific). All cell culture media were supplemented with 10% (volume/ luciferase activities were sequentially measured using a Tecan Spark volume) fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MI, USA) and 1% plate reader and the ratio of fireflyluciferaseto Renilla luciferase penicillin/streptomycin (Thermo Fisher Scientific). Cells were treated with luminescence was calculated. The experiments were repeated inde- 3 µg/ml plasmocin (Invivogen, San Diego, CA, USA) to prevent mycoplasma pendently for three times. contamination. To induce MYCN expression, SHEP-TR-MYCN cells were treated with 1 µg/ml doxycycline. All cell lines were routinely confirmed to be mycoplasma-free using Venor GeM Classic kit (Minerva Biolabs, Berlin, Germany). Cell lines were authenticated by STR-profiling (Genomics and RESULTS Transcriptomics Laboratory, Heinrich Heine University, Dusseldorf, Germany). EIF4EBP1 expression is increased in NB and correlates with MYCN expression To assess the clinical significance of EIF4EBP1 expression, we first RNA extraction, cDNA synthesis, and quantitative real-time examined EIF4EBP1 mRNA levels in NB tumor tissue samples and PCR normal tissues. We pooled microarray data of four different NB Total RNA was purified from cells using the RNeasy plus mini kit (QIAgen, Hilden, Germany) according to the manufacturer’s handbook. RNA cohorts and retrieved expression data from adrenal tissue used concentration and purity were assessed by spectrophotometry using the as the corresponding normal tissue (Fig. 1a). This indicated that NanoDrop2000 (Thermo Fisher Scientific). Subsequently, each sample was EIF4EBP1 expression is significantly elevated in NB compared to diluted to a concentration of 100 ng/µl in nuclease-free water. For cDNA adrenal gland (p < 0.0001, Fig. 1a). We then determined whether synthesis, 1 µg RNA was processed in a total reaction volume of 20 µl using EIF4EBP1 expression is related to the MYCN amplification status. the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, By comparing the level of EIF4EBP1 in MYCN-amplified versus Waltham, MA, USA), following the manufacturer’s protocol. Quantitative MYCN-non-amplified NB samples, we found that EIF4EBP1 is real-time reverse transcription (qRT) PCR was performed using SYBR green expressed at higher levels in MYCN-amplified compared to PCR master mix (Applied Biosystems) and the CFX384 Touch Real-Time PCR MYCN-non-amplified NB in the SEQC and Kocak cohorts [43, 44] Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Relative expression levels of MYCN and EIF4EBP1 were normalized to internal (p < 0.0001, Fig. 1b; p < 0.0001, Fig. 1c). This further supports and housekeeping genes GUSB and PPIA. The primer list can be found in extends previous observations made in a limited number of NB supplementary table 1. samples (n = 20) showing EIF4EBP1 overexpression in MYCN- amplified versus MYCN-non-amplified NB tumors [22]. Since MYCN amplification may result in different levels of MYCN,we Immunoblot analysis of protein expression Cells were washed with phosphate-buffered saline (PBS) and lysed in next investigated whether expression levels of MYCN and radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris- EIF4EBP1 in NB correlate with each other. Our analyses high- HCl, pH 8, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS) lighted a significant coexpression between MYCN and EIF4EBP1 supplemented with protease inhibitors (Sigma-Aldrich) and phosphatase in the SEQC (correlation coefficient [r]=0.564, p < 0.0001, Fig. 1d) inhibitors mix (PhosphoSTOP, Roche, Penzberg, Germany). Cell lysates and Kocak ([r]=0.532, p < 0.0001, Fig. 1e) cohorts. These findings were centrifuged at 21,000 rpm for 15 min at 4 °C to separate cell debris are in line with the reports that EIF4EBP1 is a potential MYCN and DNA from protein lysates. Protein concentration was measured with target gene in NB [41, 42]. We also assessed whether the the BCA protein assay kit (Thermo Fisher Scientific), according to expression of EIF4EBP1 is determined by NB stages or risk groups, manufacturer’s protocol. Protein lysates were separated by SDS-PAGE andfoundthat EIF4EBP1 levels are increased according to NB and transferred onto a nylon membrane. The membrane was incubated for 1 h in Tris-buffered saline Tween (TBST) (50 mM Tris-Cl, 150 mM NaCl, pH tumor aggressiveness in two cohorts (Fig. 1f, g). In particular, 7.5, 0.1% Tween-20) containing 5% bovine serum albumin (BSA), to EIF4EBP1 is expressed at higher levels in stage 4 NB tumors as prevent non-specific antibody binding, followed by an overnight incuba- compared to stage 1 and stage 2 tumors (stage 4 versus stage 1, tion at 4 °C with the following primary antibodies: 4EBP1 (1:1000; #9644, p < 0.0001, Fig. 1f; p < 0.0001 Fig. 1g). Interestingly, samples from Cell Signaling Technology, Cambridge, UK), MYCN (1:1,000; #9405, Cell stage 4S NB showed significantly lower EIF4EBP1 levels Signaling Technology), GAPDH (1:1000; #2118, Cell Signaling Technology), compared to stage 4 tumors (stage 4S versus stage 4, p <0.01, and β-Actin (1:5,000; #A2228, Sigma-Aldrich). The secondary antibodies Fig. 1f; p <0.001, Fig. 1g). In support of this finding, we observed IRDye 800CW Goat anti-Rabbit (1:10,000; #926-32211, LI-COR Biosciences, that in the SEQC cohort EIF4EBP1 expression is higher in high-risk Bad Homburg, Germany) or IRDye 800CW Goat anti-Mouse (1:10,000; #926- compared to low-risk NB, as based on the Children’sOncology 32210, LI-COR Biosciences) were incubated at room temperature for 1 h, followed by detection of the fluorescent signal with the Odyssey CLx Group (COG) classification (p < 0.0001, Fig. 1h). Such clinical imager (LI-COR Biosciences). information was not available in any other publicly available cohorts with mRNA expression data. Taken together, we present evidence that EIF4EBP1 is commonly overexpressed in NB tumors Plasmid construction and that EIF4EBP1 level is increased in MYCN-amplified NB and The promoter region of the human EIF4EBP1 gene, spanning from −192 to +1372, was inserted into the SacI and BglII restriction sites of the firefly advanced NB stages. Cell Death Discovery (2022) 8:157 K. Voeltzke et al. Fig. 1 EIF4EBP1 mRNA expression is associated with MYCN mRNA expression and is increased in more advanced and aggressive NB subsets. a Expression levels of EIF4EBP1 mRNA in a pool of four different NB cohorts (total n = 203), compared to healthy control tissues (adrenal gland, n = 13). b, c Expression levels of EIF4EBP1 mRNA in MYCN-amplified (n = 92, SEQC [b] and n = 93, Kocak [c]) compared to MYCN- non-amplified (n= 401, SEQC [b] and n = 550, Kocak [c]) NB patients of the SEQC (b) and Kocak (c) cohorts. d, e Expression levels of EIF4EBP1 mRNA plotted against expression levels of MYCN mRNA in SEQC (r = 0.5637, d) and Kocak (r = 0.5321, e) cohorts. f, g Expression levels of EIF4EBP1 mRNA per NB stage in SEQC (f) and Kocak (g) cohorts. h Expression levels of EIF4EBP1 mRNA in high-risk (n = 176) compared to non- high-risk (n = 322) NB in the SEQC cohort. Data were retrieved from the R2: Genomics Analysis and Visualization Platform. Statistics were determined using Mann–Whitney U-test. Exact p-values are presented. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Cell Death Discovery (2022) 8:157 K. Voeltzke et al. Fig. 2 EIF4EBP1 mRNA expression correlates with overall survival in NB patients. a–c Kaplan–Meier survival estimates of overall survival of NB patients stratified by their EIF4EBP1 mRNA expression levels (median cut off) in the SEQC (a), Kocak (b), and NRC (c) cohorts. d–h Kaplan–Meier survival estimates of overall survival of patients with MYCN-non-amplified NB (d, e), high-risk NB (f), or stage 4 NB (g, h) stratified by their EIF4EBP1 mRNA expression levels in the indicated NB cohorts. Significance was determined by log-rank test. Data were obtained from the R2: Genomics Analysis and Visualization Platform. Cell Death Discovery (2022) 8:157 K. Voeltzke et al. s- Table 1. Multivariate analysis for overall survival of NB patients in the i- SEQC cohort. Variables HR 95.0% CI p value MYCN amplification 22.373 8.89–56.306 0 High EIF4EBP1 mRNA 2.16 1.255–3.717 0.005 expression MYCN amplification*high 0.222 0.08–0.614 0.004 EIF4EBP1 mRNA expression Stage 4 17.618 6.694–46.366 0 High EIF4EBP1 mRNA 5.457 2.026–14.697 0.001 expression Stage 4*high EIF4EBP1 0.292 0.097–0.879 0.029 mRNA expression Age at diagnosis 33.018 7.835–139.139 0 High EIF4EBP1 mRNA 12.204 2.832–52.598 0.001 expression Age at diagnosis*high 0.16 0.035–0.74 0.019 EIF4EBP1 mRNA expression Table 2. Multivariate analysis for overall survival of NB patients in the NRC cohort. Variables HR 95.0% CI p value Fig. 3 4EBP1 protein expression is associated with histological MYCN amplification 4.967 1.118–22.066 0.035 subtype of NB. a Representative images at 40X magnification of low High EIF4EBP1 mRNA 3.031 1.543–5.954 0.001 (left panel) and high (right panel) 4EBP1 immunohistochemical expression staining levels of selected NB samples represented on the NB TMAs. MYCN amplification*high 0.656 0.135–3.181 0.601 b Distribution of NB cases showing low (IRS 0–6) versus high (IRS EIF4EBP1 mRNA expression 7–12) 4EBP1 protein expression in prognostically favorable versus unfavorable histological subtypes according to International Neuro- Stage 4 15.050 4.239–53.432 0.018 blastoma Pathology Classification (INPC). Fisher’s exact test was used High EIF4EBP1 mRNA 5.144 1.330–19.895 0 to calculate significance. *P < 0.05. expression gnificant for event-free survival only in the SEQC cohort Stage 4*high EIF4EBP1 0.36 0.081–1.598 0.179 (supplementary Fig. 1d, e). On the other hand, Kaplan–Meier mRNA expression survival estimates in high-risk NB patients (SEQC cohort) revealed Age at diagnosis 0.27 0.036–2.056 0 that high EIF4EBP1 levels were correlated with poor overall survival High EIF4EBP1 mRNA 0.364 0.048–2.772 0.002 (p = 7.4e-03, Fig. 2f), as well as with reduced event-free survival expression (supplementary Fig. 1f), suggesting that EIF4EBP1 expression can Age at diagnosis*high 55.427 3.258–942.88 0.005 stratify patients within the most aggressive NB subset. We EIF4EBP1 mRNA expression additionally analyzed the prognostic value of EIF4EBP1 expression in stage 4 NB patients. We found high EIF4EBP1 expression to EIF4EBP1 expression is a factor of poor prognosis in NB significantly predict decreased overall and event-free survival of Since we found EIF4EBP1 mRNA levels to be elevated in aggressive stage 4 patients in two independent cohorts (SEQC and NRC NB subsets, we examined whether EIF4EBP1 expression is linked to cohorts) (p = 3.2e-04, Fig. 2g; p = 3.8e-03, Fig. 2h and supplemen- prognosis in NB patients. Kaplan-Meier estimates univocally tary Fig. 1g, h). This highlights that EIF4EBP1 expression robustly showed that high EIF4EBP1 levels (using median expression level stratifies patients within the advanced NB subgroups. Altogether, as cut off) were significantly associated with reduced overall and our analyses support that EIF4EBP1 expression is a factor of poor event-free survival in three independent cohorts, namely SEQC, prognosis in all NB, as well as in high-risk and stage 4 NB. Kocak, and NRC cohorts [45](p = 3.1e-08, Fig. 2a; p = 4.2e-11, Fig. 2b; p = 1.7e-06, Fig. 2c, and supplementary Fig. 1a–c). To test High 4EBP1 protein expression is associated with dependence of EIF4EBP1 expression as prognostic factor on prognostically unfavorable histology of NB established factors of poor prognosis, we performed multivariate To independently confirm the prognostic value of EIF4EBP1/4EBP1 in analysis to determine the statistical interaction between high NB and to determine the biomarker potential of 4EBP1 protein EIF4EBP1 expression and MYCN amplification status, tumor stage expression in NB, we immunohistochemically analyzed NB TMAs or age at diagnosis. This indicated that MYCN amplification status, consisting of 69 patient samples. Staining of the TMAs with a 4EBP1- tumor stage and age at diagnosis each influenced the prognostic specific antibody revealed a cytoplasmic staining (Fig. 3a), consistent value of high EIF4EBP1 expression in the SEQC and NRC cohorts with the expected cellular localization of 4EBP1 [46]. We semi- (Tables 1 and 2). Therefore, high EIF4EBP1 expression is not an quantitatively evaluated 4EBP1 staining intensity and correlated independent factor of poor prognosis in NB. However, we 4EBP1 immunoreactivity with the NB histological subtypes accord- uncovered that EIF4EBP1 expression can predict overall survival ing to the International Neuroblastoma Pathology Classification in clinically relevant NB subsets, including more advanced and (INPC), which distinguishes patients with favorable or unfavorable aggressive NB subgroups. Indeed, our analyses highlighted that histology based on grade of neuroblastic differentiation and mitosis- high EIF4EBP1 expression significantly predicted reduced overall karyorrhexis index. We found that tumors with unfavorable survival in MYCN-non-amplified patients of the SEQC and NRC histology more frequently exhibited a high 4EBP1 staining score cohorts (p = 3.8e-03, Fig. 2d; p = 0.04, Fig. 2e), while it was (IRS 7–12) as compared to tumors with favorable histology (Fig. 3b), Cell Death Discovery (2022) 8:157 K. Voeltzke et al. Fig. 4 EIF4EBP1 promoter activity is regulated by MYCN. a ChIP peaks of MYCN in the EIF4EBP1 promoter region in Kelly NB cell line. b Scheme of the EIF4EBP1 promoter reporter highlighting the three E-boxes corresponding to MYCN binding sites. c HEK-293-T cells were transfected with the wildtype EIF4EBP1 promoter firefly luciferase construct and with the indicated amounts of MYCN expressing plasmid (pMYCN). A Renilla Luciferase vector was used as an internal control. d MYCN and 4EBP1 protein expression were monitored in cell lysates from c by immunoblot analyses using the indicated antibodies. e HEK-293-T were transfected with wildtype or different E-box mutants EIF4EBP1 promoter firefly luciferase constructs with or without a MYCN expressing plasmid (pMYCN). A Renilla Luciferase vector was used as an internal control. Statistics were determined using Student’s t test or Mann–Whitney U-test. Exact p-values are presented. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. indicating that high 4EBP1 protein expression is associated with MYCN-amplified NB cell line, Kelly. This revealed that MYCN more aggressive NB subsets. binds the endogenous EIF4EBP1 promoter region (which encompasses exon 1 and a part of intron 1) at three distinct EIF4EBP1 promoter activity and transcription is controlled by positions, indicating three potential MYCN binding sites (Fig. 4a). MYCN In silico analysis of the promoter region sequence confirmed the To delineate how elevated EIF4EBP1 expression is mechanisti- presence of structural E-boxes at the three occupied locations cally connected to MYCN amplification and overexpression in (Fig. 4b). To evaluate the impact of MYCN on the regulation of NB, we investigated the transcriptional regulation of EIF4EBP1 by EIF4EBP1 promoter activity, we designed a luciferase-based gene MYCN. A previous report detected the presence of MYCN on reporter assay by cloning the EIF4EBP1 promoter region (−192 EIF4EBP1 promoter by ChIP-seq in BE(2)-C, a MYCN-amplified NB to +1372) in front of a firefly luciferase gene (Fig. 4b). The cell line [41, 42]. We validated and further extended this finding activity of the wildtype EIF4EBP1 promoter was dose- by analyzing ChIP-seq data available from an additional dependently increased upon forced expression of MYCN in Cell Death Discovery (2022) 8:157 K. Voeltzke et al. HEK-293-T cells (Fig. 4c), which was accompanied by an the group of patients with aggressive stage 4 NB. Of note, less upregulation of endogenous 4EBP1 protein level (Fig. 4d). To than a third of stage 4 patients carry a MYCN amplification. Thus, it investigate which E-boxes are necessary for the transcriptional may be worth considering that, in addition to MYCN amplification activation of the EIF4EBP1 promoter by MYCN, either a single or a status, levels of EIF4EBP1 expression could help identifying combination of two of the three potential binding sites were patients carrying clinically more aggressive tumors within the mutated. Mutation of either of the three binding sites alone was stage 4 NB patients group. EIF4EBP1 expression was also linked to sufficient to significantly reduce MYCN-induced promoter worse outcome among high-risk NB patients. Given that MYCN activity (Fig. 4e). Any combinations of two mutated binding amplification is not able of predicting outcome within high-risk NB sites further reduced promoter activity driven by MYCN over- patients [50], it appears that EIF4EBP1 expression has a prognostic expression (Fig. 4e), suggesting that two binding sites, without a power beyond MYCN amplification in this patient subset. Thus, specific preference of one over another, are needed for full EIF4EBP1 expression may represent a promising biomarker for induction of EIF4EBP1 promoter activity by MYCN. We next prognostic stratification of high-risk NB patients, in addition to the intended to confirm whether MYCN directly regulates EIF4EBP1 recently reported genetic alterations in the RAS and p53 pathways transcription in NB cell lines. To do so, we chose two MYCN- [12]. This is further supported by the association we observed amplified NB cell lines, IMR-32 and Kelly, in which we knocked between high 4EBP1 protein expression and unfavorable NB down MYCN expression by siRNA and examined the impact on histological subtype. Together, our findings highlight a previously EIF4EBP1 mRNA levels by qPCR. The depletion of MYCN caused a underappreciated prognostic factor, i.e., EIF4EBP1/4EBP1, which significant reduction of EIF4EBP1 transcript levels in both cell may help refining risk stratification of NB patients, including lines (Fig. 5a, b). To further support these observations, we MYCN-non-amplified, stage 4 and high-risk patients, and could assessed the impact of forced MYCN expression on EIF4EBP1 potentially assist in tailoring more personalized treatment options. transcript and protein levels by using SHEP-TR-MYCN cells, Beyond NB, EIF4EBP1 expression was reported to be a factor of which are MYCN-non-amplified NB cells engineered to express poor prognosis in breast and liver cancers [30, 33], as well as in all exogenous MYCN with a tetracycline inducible system [19]. TCGA tumor types combined [29]. While our data indicate that Doxycycline treatment markedly increased EIF4EBP1 mRNA level EIF4EBP1 expression has prognostic power in pediatric cancer, over time (Fig. 5c), in parallel with progressive upregulation of together this supports that EIF4EBP1 expression represents a factor MYCN expression (Fig. 5d). This was accompanied by a net of poor prognosis in a large number of different tumor types. increase in the 4EBP1 protein level (Fig. 5d), supporting that Our study also extends previous knowledge by providing MYCN positively controls EIF4EBP1 mRNA and protein expression further experimental evidence to explain the association between in NB cells. To determine whether MYCN regulation of EIF4EBP1 EIF4EBP1 and MYCN expression in NB and the overexpression of has relevance during NB differentiation, we analyzed expression EIF4EBP1 in MYCN-amplified NB. Our data revealed that MYCN data of MYCN-non-amplified SH-SY5Y cells treated with RA. This induces transcription of EIF4EBP1 by regulating its promoter indicated that both MYCN and EIF4EBP1 expression are through multiple binding sites, which was originally suggested by decreased over time upon treatment, and that levels of both detection of MYCN binding to the EIF4EBP1 promoter by ChIP genes are correlated during NB differentiation (Fig. 5e–g). analysis [41, 42]. However, whether MYCN could transcriptionally Finally, analyses of expression data from a transgenic mouse regulate the EIF4EBP1 promoter was still elusive. We demonstrate model of MYCN-driven NB (TH-MYCN; [47]) revealed that that MYCN activates the EIF4EBP1 promoter through binding at EIF4EBP1 expression is upregulated in NB tumors as compared three distinct E-boxes, which in turn leads to transcriptional increase of EIF4EBP1 even with low to medium MYCN expression, to the corresponding normal tissue, i.e. the ganglia (Fig. 5h). Taken together, our data provide further evidence that EIF4EBP1 suggesting a threshold for MYCN level. Together with the previous is a transcriptional target of MYCN, potentially providing a ChIP analysis, this supports that EIF4EBP1 is a direct target gene of mechanistic basis for the observed overexpression of EIF4EBP1 in MYCN in NB cells. These findings are in line with previous studies MYCN-amplified NB patients. reporting that MYC controls EIF4EBP1 by binding its endogenous promoter in colorectal and prostate cancer cells [36, 37], as demonstrated by ChIP, highlighting a general regulation of DISCUSSION EIF4EBP1 by MYC family members in cancer cells. MYCN-amplification is accountable for aggressive NB subsets as it Expression levels of EIF4EBP1 appear not only elevated in MYCN- has been associated with increased risk of relapse and reduced amplified versus MYCN-non-amplified NB but are also upregulated overall survival of patients [13]. Since MYCN is considered in MYCN-non-amplified tumors relative to control tissue. It might “undruggable”, there is a demand for identifying targetable be speculated that in MYCN-non-amplified NB, EIF4EBP1 expres- downstream effectors of MYCN [20, 21]. In addition, since NB is sion may be regulated by transcription factors other than MYCN. a clinically heterogenous disease, ranging from spontaneous In particular, ATF4, which is critical for the metabolic response of regression to progression despite aggressive therapies, novel NB cells to glutamine starvation [51, 52], has been shown to markers that improve patient risk stratification and hence allow for control EIF4EBP1 promoter and transcription in pancreatic beta optimal treatment allocation are warranted [4, 48, 49]. Here, we cells [39]. This transcription factor is highly expressed in NB, and in report that EIF4EBP1 expression levels are significantly elevated in particular in advanced stage 4 [52]. In addition, another NB compared to corresponding non-tumor tissues and positively transcription factor that is commonly overexpressed in NB is correlate with both MYCN expression and MYCN amplification OCT4 [53]. Of note, this transcription factor has been identified by status in at least two independent NB patient cohorts. Further- ChIP-seq to bind the promoter region of EIF4EBP1 in human more, using three independent NB cohorts, we report that high embryonic stem cells [54, 55], thus OCT4 may also activate EIF4EBP1 expression is a strong predictor of poor overall and EIF4EBP1 transcription in NB cells. Together, these data suggest event-free survival across all NB patients. This was not indepen- potential mechanisms underlying the MYCN independent regula- dent of MYCN amplification status, tumor stage or age at tion of EIF4EBP1 expression in MYCN-non-amplified NB patients. diagnosis, which can be explained in part by the regulation of Given the prognostic significance of EIF4EBP1/4EBP1 in NB, it is EIF4EBP1 promoter by MYCN which we characterized. However, possible that 4EBP1 confers advantages to NB tumor growth or EIF4EBP1 expression can predict prognosis within distinct patient tumor cell survival. As evidenced by the presence of necrotic areas groups like the MYCN-non-amplified patients subset, for which flanked by HIF-1α positive staining [56], NB experience metabolic few biomarkers have been identified. Moreover, we observed that stress, corresponding to nutrient deprivation and hypoxia, as a high EIF4EBP1 expression was associated with poor prognosis in consequence of abnormal and immature vascularization [57, 58]. Cell Death Discovery (2022) 8:157 K. Voeltzke et al. One important mechanism for cancer cells to adapt to metabolic hypoxia by stimulating the synthesis of pro-angiogenic factors, stress is through reprogramming of mRNA translation [59]. As a like HIF-1α and VEGF, to facilitate tumor angiogenesis in vivo [28]. major regulator of mRNA translation, 4EBP1 may aid NB cells to In addition, the control of mRNA translation was shown to be cope with hypoxia and nutrient deprivation. This is supported by critical to prevent the deleterious effects of MYCN and MYC the report that 4EBP1 promotes survival of breast tumors under overexpression, as we and others previously reported [37]. In fact, Cell Death Discovery (2022) 8:157 K. Voeltzke et al. Fig. 5 EIF4EBP1 expression is regulated by MYCN in NB. a, b Relative MYCN and EIF4EBP1 mRNA levels upon siRNA-mediated knockdown of MYCN in the MYCN-amplified IMR-32 (a) and Kelly (b) cell lines, as measured by qRT-PCR. c, d SHEP-TR-MYCN cells were treated with doxycycline (1 µg/ml) for the indicated times; EIF4EBP1 mRNA levels were determined by qRT-PCR (c) and levels of MYCN and 4EBP1 proteins were monitored by immunoblot using the indicated antibodies (d). The different 4EBP1 bands correspond to different phosphorylated forms of 4EBP1. e, f Expression levels of MYCN (e)or EIF4EBP1 (f) mRNA in SH-SY5Y cells treated with RA for the indicated times (Takeda’s dataset, n = 2 for each time point; [60]). Statistics were calculated for each time point compared to the control 0 h time point. g Expression levels of EIF4EBP1 mRNA plotted against expression levels of MYCN mRNA in SH-SY5Y cells treated with RA (Takeda’s dataset, n= 2 for each time point; [60]). h Relative EIF4EBP1 mRNA expression in healthy control tissues (ganglia, n= 9) and NB tumors (n = 26) of a TH-MYCN transgenic mouse model of NB (Balamuth’s dataset; [47]). Data were retrieved from the R2: Genomics Analysis and Visualization Platform. Statistics were determined using Student’s t test or Mann–Whitney U-test. Exact p-values are presented. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 4EBP1, by reducing overall protein synthesis, was reported to 15. Liu R, Shi P, Wang Z, Yuan C, Cui H. Molecular mechanisms of MYCN dysregu- lation in cancers. Front Oncol. 2020;10:625332. https://doi.org/10.3389/ prevent cell death induced upon MYC overexpression, likely by fonc.2020.625332 blunting accumulation of misfolded proteins and proteotoxic ER 16. Weiss WA, Aldape K, Mohapatra G, Feuerstein BG, Bishop JM. Targeted expression stress [37]. It is possible that in a similar manner 4EBP1 contributes of MYCN causes neuroblastoma in transgenic mice. EMBO J. 1997;16:2985–95. to inhibit cell death induced by MYCN overexpression in MYCN- https://doi.org/10.1093/emboj/16.11.2985 amplified NB. 17. Oliynyk G, Ruiz-Pérez MV, Sainero-Alcolado L, Dzieran J, Zirath H, Gallart-Ayala H, In summary, the findings reported here indicate that EIF4EBP1 is et al. MYCN-enhanced oxidative and glycolytic metabolism reveals vulnerabilities a direct target gene of MYCN in NB, explaining the observed high for targeting neuroblastoma. iScience. 2019;21:188–204. https://doi.org/10.1016/ expression of EIF4EBP1 in NB, and that EIF4EBP1 mRNA and protein j.isci.2019.10.020 expression have prognostic values in NB patients, especially for 18. Boon K, Caron HN, van Asperen R, Valentijn L, Hermus MC, van Sluis P, et al. N-myc enhances the expression of a large set of genes functioning in ribosome stratifying high-risk NB patients. biogenesis and protein synthesis. EMBO J. 2001;20:1383–93. https://doi.org/ 10.1093/emboj/20.6.1383 19. Tjaden B, Baum K, Marquardt V, Simon M, Trajkovic-Arsic M, Kouril T, et al. N-Myc- DATA AVAILABILITY induced metabolic rewiring creates novel therapeutic vulnerabilities in neuro- The data that support the findings of this study are available from the corresponding blastoma. Sci Rep. 2020;10:7157. https://doi.org/10.1038/s41598-020-64040-1 author upon reasonable request. 20. Bell E, Chen L, Liu T, Marshall GM, Lunec J, Tweddle DA. MYCN oncoprotein targets and their therapeutic potential. Cancer Lett. 2010;293:144–57. https://doi. org/10.1016/j.canlet.2010.01.015 REFERENCES 21. Wolpaw AJ, Bayliss R, Büchel G, Dang CV, Eilers M, Gustafson WC, et al. Drugging 1. Maris JM, Hogarty MD, Bagatell R, Cohn SL. Neuroblastoma. Lancet. the “undruggable” MYCN oncogenic transcription factor: overcoming previous 2007;369:2106–20. https://doi.org/10.1016/S0140-6736(07)60983-0 obstacles to impact childhood cancers. Cancer Res. 2021;81:1627–32. https://doi. 2. van Arendonk KJ, Chung DH. Neuroblastoma: tumor biology and its implications org/10.1158/0008-5472.CAN-20-3108 for staging and treatment. Children (Basel) 2019;6. https://doi.org/10.3390/ 22. Schramm A, Köster J, Marschall T, Martin M, Schwermer M, Fielitz K, et al. Next- children6010012 generation RNA sequencing reveals differential expression of MYCN target genes 3. Tolbert VP, Matthay KK. Neuroblastoma: clinical and biological approach to risk and suggests the mTOR pathway as a promising therapy target in MYCN- stratification and treatment. Cell Tissue Res. 2018;372:195–209. https://doi.org/ amplified neuroblastoma. Int. J. Cancer. 2013;132:E106–15. https://doi.org/ 10.1007/s00441-018-2821-2 10.1002/ijc.27787 4. Maris JM. The biologic basis for neuroblastoma heterogeneity and risk stratifi- 23. Musa J, Orth MF, Dallmayer M, Baldauf M, Pardo C, Rotblat B, et al. Eukaryotic cation. Curr Opin Pediatrics. 2005;17:7–13. https://doi.org/10.1097/01. initiation factor 4E-binding protein 1 (4E-BP1): a master regulator of mRNA mop.0000150631.60571.89 translation involved in tumorigenesis. Oncogene. 2016;35:4675–88. https://doi. 5. London WB, Castel V, Monclair T, Ambros PF, Pearson ADJ, Cohn SL, et al. Clinical org/10.1038/onc.2015.515 and biologic features predictive of survival after relapse of neuroblastoma: a 24. Morita M, Gravel S-P, Chénard V, Sikström K, Zheng L, Alain T, et al. mTORC1 report from the International Neuroblastoma Risk Group project. J Clin Oncol. controls mitochondrial activity and biogenesis through 4E-BP-dependent trans- 2011;29:3286–92. https://doi.org/10.1200/JCO.2010.34.3392 lational regulation. Cell Metab. 2013;18:698–711. https://doi.org/10.1016/j. 6. Pinto NR, Applebaum MA, Volchenboum SL, Matthay KK, London WB, Ambros PF, cmet.2013.10.001 et al. Advances in risk classification and treatment strategies for neuroblastoma. J 25. Dowling RJO, Topisirovic I, Alain T, Bidinosti M, Fonseca BD, Petroulakis E, et al. Clin Oncol. 2015;33:3008–17. https://doi.org/10.1200/JCO.2014.59.4648 mTORC1-mediated cell proliferation, but not cell growth, controlled by the 4E- 7. Simon T, Berthold F, Borkhardt A, Kremens B, Carolis B, de, Hero B. Treatment BPs. Science. 2010;328:1172–6. https://doi.org/10.1126/science.1187532 and outcomes of patients with relapsed, high-risk neuroblastoma: results of 26. Wang Z, Feng X, Molinolo AA, Martin D, Vitale-Cross L, Nohata N, et al. 4E-BP1 is a German trials. Pediatr Blood Cancer. 2011;56:578–83. https://doi.org/10.1002/ tumor suppressor protein reactivated by mTOR inhibition in head and neck pbc.22693 cancer. Cancer Res. 2019;79:1438–50. https://doi.org/10.1158/0008-5472.CAN-18- 8. Chen Y, Takita J, Choi YL, Kato M, Ohira M, Sanada M, et al. Oncogenic mutations of ALK kinase in neuroblastoma. Nature. 2008;455:971–4. https://doi.org/10.1038/ 27. Ding M, van der Kwast TH, Vellanki RN, Foltz WD, McKee TD, Sonenberg N, et al. nature07399 The mTOR targets 4E-BP1/2 restrain tumor growth and promote hypoxia toler- 9. Mossé YP, Laudenslager M, Longo L, Cole KA, Wood A, Attiyeh EF, et al. Identi- ance in PTEN-driven prostate cancer. Mol Cancer Res. 2018;16:682–95. https://doi. fication of ALK as a major familial neuroblastoma predisposition gene. Nature. org/10.1158/1541-7786.MCR-17-0696 2008;455:930–5. https://doi.org/10.1038/nature07261 28. Braunstein S, Karpisheva K, Pola C, Goldberg J, Hochman T, Yee H, et al. A hypoxia- 10. Janoueix-Lerosey I, Lequin D, Brugières L, Ribeiro A, Pontual L, de, Combaret V, controlled cap-dependent to cap-independent translation switch in breast cancer. et al. Somatic and germline activating mutations of the ALK kinase receptor in Mol Cell. 2007;28:501–12. https://doi.org/10.1016/j.molcel.2007.10.019 neuroblastoma. Nature. 2008;455:967–70. https://doi.org/10.1038/nature07398 29. Wu S & Wagner G. Deep computational analysis of human cancer and non-cancer 11. Amelio I, Bertolo R, Bove P, Candi E, Chiocchi M, Cipriani C, et al. Cancer predictive tissues details dysregulation of eIF4F components and their interactions in studies. Biol Direct. 2020;15:18. https://doi.org/10.1186/s13062-020-00274-3 human cancers. bioRxiv 2020. https://doi.org/10.1101/2020.10.12.336263 12. Ackermann S, Cartolano M, Hero B, Welte A, Kahlert Y, Roderwieser A, et al. A 30. Karlsson E, Pérez-Tenorio G, Amin R, Bostner J, Skoog L, Fornander T, et al. The mechanistic classification of clinical phenotypes in neuroblastoma. Science. mTOR effectors 4EBP1 and S6K2 are frequently coexpressed, and associated with 2018;362:1165–70. https://doi.org/10.1126/science.aat6768 a poor prognosis and endocrine resistance in breast cancer: a retrospective study 13. Huang M, Weiss WA. Neuroblastoma and MYCN. Cold Spring Harb Perspect Med. including patients from the randomised Stockholm tamoxifen trials. Breast 2013;3:a014415. https://doi.org/10.1101/cshperspect.a014415 Cancer Res. 2013;15:R96. https://doi.org/10.1186/bcr3557 14. Zeid R, Lawlor MA, Poon E, Reyes JM, Fulciniti M, Lopez MA, et al. Enhancer 31. Kremer CL, Klein RR, Mendelson J, Browne W, Samadzedeh LK, Vanpatten K, et al. invasion shapes MYCN-dependent transcriptional amplification in neuro- Expression of mTOR signaling pathway markers in prostate cancer progression. blastoma. Nat Genet. 2018;50:515–23. https://doi.org/10.1038/s41588-018-0044-9 Prostate. 2006;66:1203–12. https://doi.org/10.1002/pros.20410 Cell Death Discovery (2022) 8:157 K. Voeltzke et al. 32. Lee M, Kim EJ, Jeon MJ. MicroRNAs 125a and 125b inhibit ovarian cancer cells 53. Yang S, Zheng J, Ma Y, Zhu H, Xu T, Dong K, et al. Oct4 and Sox2 are over- through post-transcriptional inactivation of EIF4EBP1. Oncotarget. expressed in human neuroblastoma and inhibited by chemotherapy. Oncol Rep. 2016;7:8726–42. https://doi.org/10.18632/oncotarget.6474 2012;28:186–92. https://doi.org/10.3892/or.2012.1765 33. Cha Y-L, Li P-D, Yuan L-J, Zhang M-Y, Zhang Y-J, Rao H-L, et al. EIF4EBP1 over- 54. Gifford CA, Ziller MJ, Gu H, Trapnell C, Donaghey J, Tsankov A, et al. Transcrip- expression is associated with poor survival and disease progression in patients tional and epigenetic dynamics during specification of human embryonic stem with hepatocellular carcinoma. PLoS ONE. 2015;10:e0117493. https://doi.org/ cells. Cell. 2013;153:1149–63. https://doi.org/10.1016/j.cell.2013.04.037 10.1371/journal.pone.0117493 55. Čančer M, Hutter S, Holmberg KO, Rosén G, Sundström A, Tailor J, et al. Huma- 34. Fransson S, Abel F, Kogner P, Martinsson T, Ejeskär K. Stage-dependent expres- nized stem cell models of pediatric medulloblastoma reveal an Oct4/mTOR axis sion of PI3K/Akt‑pathway genes in neuroblastoma. Int J Oncol. 2013;42:609–16. that promotes malignancy. Cell Stem Cell. 2019;25:855–870.e11. https://doi.org/ https://doi.org/10.3892/ijo.2012.1732 10.1016/j.stem.2019.10.005 35. Meng X, Li H, Fang E, Feng J, Zhao X. Comparison of stage 4 and stage 4s 56. Påhlman S, Mohlin S. Hypoxia and hypoxia-inducible factors in neuroblastoma. neuroblastoma identifies autophagy-related gene and LncRNA signatures asso- Cell Tissue Res. 2018;372:269–75. https://doi.org/10.1007/s00441-017-2701-1 ciated with prognosis. Front Oncol. 2020;10:1411. https://doi.org/10.3389/ 57. Schaaf MB, Garg AD, Agostinis P. Defining the role of the tumor vasculature in fonc.2020.01411 antitumor immunity and immunotherapy. Cell Death Dis. 2018;9:115. https://doi. 36. Balakumaran BS, Porrello A, Hsu DS, Glover W, Foye A, Leung JY, et al. MYC org/10.1038/s41419-017-0061-0 activity mitigates response to rapamycin in prostate cancer through eukaryotic 58. Lugano R, Ramachandran M, Dimberg A. Tumor angiogenesis: causes, con- initiation factor 4E-binding protein 1-mediated inhibition of autophagy. Cancer sequences, challenges and opportunities. Cell Mol Life Sci. 2020;77:1745–70. Res. 2009;69:7803–10. https://doi.org/10.1158/0008-5472.CAN-09-0910 https://doi.org/10.1007/s00018-019-03351-7 37. Tameire F, Verginadis II, Leli NM, Polte C, Conn CS, Ojha R, et al. ATF4 couples 59. Leprivier G, Rotblat B, Khan D, Jan E, Sorensen PH. Stress-mediated translational MYC-dependent translational activity to bioenergetic demands during tumour control in cancer cells. Biochim. Biophys. Acta. 2015;1849:845–60. https://doi.org/ progression. Nat Cell Biol. 2019;21:889–99. https://doi.org/10.1038/s41556-019- 10.1016/j.bbagrm.2014.11.002 0347-9 60. Nishida Y, Adati N, Ozawa R, Maeda A, Sakaki Y, Takeda T. Identification and 38. Liu Y, Horn JL, Banda K, Goodman AZ, Lim Y, Jana S et al. The androgen receptor classification of genes regulated by phosphatidylinositol 3-kinase- and TRKB- regulates a druggable translational regulon in advanced prostate cancer. Sci mediated signalling pathways during neuronal differentiation in two subtypes of Transl Med. 2019;11. https://doi.org/10.1126/scitranslmed.aaw4993 the human neuroblastoma cell line SH-SY5Y. BMC Res Notes. 2008;1:95. https:// 39. Yamaguchi S, Ishihara H, Yamada T, Tamura A, Usui M, Tominaga R, et al. ATF4- doi.org/10.1186/1756-0500-1-95 mediated induction of 4E-BP1 contributes to pancreatic beta cell survival under endoplasmic reticulum stress. Cell Metab. 2008;7:269–76. https://doi.org/10.1016/ j.cmet.2008.01.008 40. Azar R, Lasfargues C, Bousquet C, Pyronnet S. Contribution of HIF-1α in 4E-BP1 ACKNOWLEDGEMENTS gene expression. Mol. Cancer Res. 2013;11:54–61. https://doi.org/10.1158/1541- We would like to thank Dr. Bastian Malzkorn (Institute of Neuropathology, Heinrich 7786.MCR-12-0095 Heine University Düsseldorf) for helpful discussions. GL was supported by funding 41. Cheung CHY, Hsu C-L, Tsuei C-Y, Kuo T-T, Huang C-T, Hsu W-M, et al. Combina- from the Elterninitiative Düsseldorf e.V., the Research Commission of the Medical torial targeting of MTHFD2 and PAICS in purine synthesis as a novel therapeutic Faculty of Heinrich Heine University, the Deutsche Forschungsgemeinschaft (Grant LE strategy. Cell Death Dis. 2019;10:786. https://doi.org/10.1038/s41419-019-2033-z 3751/2-1), and the German Cancer Aid (Grant 70112624). The laboratory of TGPG is 42. Hsu C-L, Chang H-Y, Chang J-Y, Hsu W-M, Huang H-C, Juan H-F. Unveiling supported by the Barbara und Wilfried Mohr Foundation. BR is supported by the MYCN regulatory networks in neuroblastoma via integrative analysis of het- Israel Science Foundation (grant No. 1436/19). erogeneous genomics data. Oncotarget. 2016;7:36293–310. https://doi.org/ 10.18632/oncotarget.9202 43. SEQC/MAQC consortium. SEQC/MAQC consortium: a comprehensive assessment of RNA-seq accuracy, reproducibility and information. Nat Biotechnol. AUTHOR CONTRIBUTIONS 2014;32:903–14. https://doi.org/10.1038/nbt.2957 Conception and design: KV and GL. Provision of study material and patients: IE and 44. Kocak H, Ackermann S, Hero B, Kahlert Y, Oberthuer A, Juraeva D, et al. Hox-C9 TK. Financial and administrative support: GR. Data analysis and interpretation: KV, activates the intrinsic pathway of apoptosis and is associated with spontaneous TGPG, AS, and GL. Critical review and discussion: BR, MR, AS, GR, and GL. Experimental regression in neuroblastoma. Cell Death Dis. 2013;4:e586. https://doi.org/ support: KV, KS, CF, AK, DP, LH, and MFO. Manuscript writing: KV, GR, and GL. Final 10.1038/cddis.2013.84 approval of the manuscript: all authors. 45. Rajbhandari P, Lopez G, Capdevila C, Salvatori B, Yu J, Rodriguez-Barrueco R, et al. Cross-cohort analysis identifies a TEAD4-MYCN positive feedback loop as the core regulatory element of high-risk neuroblastoma. Cancer Discov. 2018;8:582–99. FUNDING https://doi.org/10.1158/2159-8290.CD-16-0861 Open Access funding enabled and organized by Projekt DEAL. 46. Armengol G, Rojo F, Castellví J, Iglesias C, Cuatrecasas M, Pons B, et al. 4E-binding protein 1: a key molecular “funnel factor” in human cancer with clinical implica- tions. Cancer Res. 2007;67:7551–5. https://doi.org/10.1158/0008-5472.CAN-07-0881 47. Balamuth NJ, Wood A, Wang Q, Jagannathan J, Mayes P, Zhang Z, et al. Serial COMPETING INTERESTS transcriptome analysis and cross-species integration identifies centromere- TK received honoraria for Consulting/Advisory by Amgen, AstraZeneca, BMS, Merck associated protein E as a novel neuroblastoma target. Cancer Res. KGaA, MSD, Novartis, Pfizer, Roche, for Research Funding by Merck KGaA and Roche; 2010;70:2749–58. https://doi.org/10.1158/0008-5472.CAN-09-3844 for talks by Merck KGaA, AstraZeneca. The other authors declare no conflict of 48. Rugolo F, Bazan NG, Calandria J, Jun B, Raschellà G, Melino G, et al. The interest. expression of ELOVL4, repressed by MYCN, defines neuroblastoma patients with good outcome. Oncogene. 2021;40:5741–51. https://doi.org/10.1038/s41388-021- 01959-3 49. Pieraccioli M, Nicolai S, Pitolli C, Agostini M, Antonov A, Malewicz M, et al. ZNF281 ADDITIONAL INFORMATION inhibits neuronal differentiation and is a prognostic marker for neuroblastoma. Proc. Natl Acad. Sci. USA. 2018;115:7356–61. https://doi.org/10.1073/pnas.1801435115 Supplementary information The online version contains supplementary material 50. Lee JW, Son MH, Cho HW, Ma YE, Yoo KH, Sung KW, et al. Clinical significance of available at https://doi.org/10.1038/s41420-022-00963-0. MYCN amplification in patients with high-risk neuroblastoma. Pediatr Blood Cancer. 2018;65:e27257. https://doi.org/10.1002/pbc.27257 Correspondence and requests for materials should be addressed to Gabriel Leprivier. 51. Qing G, Li B, Vu A, Skuli N, Walton ZE, Liu X, et al. ATF4 regulates MYC-mediated neuroblastoma cell death upon glutamine deprivation. Cancer Cell. Reprints and permission information is available at http://www.nature.com/ 2012;22:631–44. https://doi.org/10.1016/j.ccr.2012.09.021 reprints 52. Ren P, Yue M, Xiao D, Xiu R, Gan L, Liu H, et al. ATF4 and N-Myc coordinate glutamine metabolism in MYCN-amplified neuroblastoma cells through ASCT2 Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims activation. J Pathol. 2015;235:90–100. https://doi.org/10.1002/path.4429 in published maps and institutional affiliations. Cell Death Discovery (2022) 8:157 K. Voeltzke et al. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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