MYCN Amplifications and Metabolic Rewiring in Neuroblastoma

Simple Summary Transcription factors (TFs) can reprogram cellular states by modulating the expression of their target genes and establishing gene expression programs under homeostasis and diseases manifestation. In neuroblastoma, the TF MYCN has been recorded as dysregulated, presenting both aberrant expression and genomic abnormalities across its coding locus. Herein, we computationally investigated the gene expression characteristics that distinguish neuroblastoma-MYCN-amplified from neuroblastoma non-MYCN-amplified cancer cells, and we addressed the upregulation of several metabolism-related TF-encoding genes. Moreover, cistromic computational assessments of MYCN revealed its direct binding across regulatory sequences that reside in cis proximity to several of those genes. These results illuminate substantial mechanistic interrelationships between the key driver of neuroblastoma and a wealth of transcriptional regulators in cancer cells. Abstract Cancer is a disease caused by (epi)genomic and gene expression abnormalities and characterized by metabolic phenotypes that are substantially different from the normal phenotypes of the tissues of origin. Metabolic reprogramming is one of the key features of tumors, including those established in the human nervous system. In this work, we emphasize a well-known cancerous genomic alteration: the amplification of MYCN and its downstream effects in neuroblastoma phenotype evolution. Herein, we extend our previous computational biology investigations by conducting an integrative workflow applied to published genomics datasets and comprehensively assess the impact of MYCN amplification in the upregulation of metabolism-related transcription factor (TF)-encoding genes in neuroblastoma cells. The results obtained first emphasized overexpressed TFs, and subsequently those committed in metabolic cellular processes, as validated by gene ontology analyses (GOs) and literature curation. Several genes encoding for those TFs were investigated at the mechanistic and regulatory levels by conducting further omics-based computational biology assessments applied on published ChIP-seq datasets retrieved from MYCN-amplified- and MYCN-enforced-overexpression within in vivo systems of study. Hence, we approached the mechanistic interrelationship between amplified MYCN and overexpression of metabolism-related TFs in neuroblastoma and showed that many are direct targets of MYCN in an amplification-inducible fashion. These results illuminate how MYCN executes its regulatory underpinnings on metabolic processes in neuroblastoma.


Introduction
Cancer is defined by genomic, epigenomic, and gene expression abnormalities [1] and is characterized by the evolution of metabolic phenotypes that are substantially deviated correlate with the age of diagnosis [29]. They are primarily classified into four groups, as very low-, low-, intermediate-, and high-risk, based on their disease stage (according to the International Neuroblastoma Risk Group Staging System (INRGSS)), their age at the time of diagnosis, their histological type (maturing ganglioneuroma versus ganglioneuroblastoma, intermixed versus ganglioneuroblastoma, or nodular versus neuroblastoma), their grade, their MYCN gene status, their 11q chromosomal status, and their tumor cell ploidy [28,30]. Stage L1 refers to tumors located only in the area where they started, L2 tumors are spread both to nearby and other areas, M tumors have spread to other parts of the body, and MS tumors have spread to only the liver, skin, and/or bone marrow in patients younger than 18 months [30].
Based on the INRGSS, very low-risk tumors encompass stage L1/L2 maturing ganglioneuroma or intermixed ganglioneuroblastoma, stage L1 tumors with non-amplified MYCN, and stage MS in children younger than 18 months of age with no 11q aberration. Low-risk neuroblastomas include stage L2 in children younger than 18 months of age with no 11q aberration; L2 in children older than 18 months of age with ganglioneuroblastoma, nodular, or differentiated neuroblastoma with no 11q aberration; and stage M in children younger than 18 months without MYCN amplification and hyperdiploidy. Intermediaterisk neuroblastomas include stage L2 in children younger than 18 months without MYCN amplification but with 11q aberration; stage L2 in patients older than 18 months with ganglioneuroblastoma, nodular, or neuroblastoma with differentiating histology and 11q aberration; stage L2 in patients older than 18 months with ganglioneuroblastoma, nodular, or poorly differentiated or undifferentiated neuroblastoma; stage M in children younger than 12 months with diploidy; and stage M in children 12 months to 18 months old with diploidy. High-risk neuroblastomas include stage L1 and L2 tumors with MYCN amplification, stage M in patients younger and older than 18 months of age with MYCN amplification, stage MS in children younger than 18 months with 11q aberration, and stage MS in children younger than 18 months of age with MYCN amplification [30,31].
Neuroblastoma treatment is designed based on the risk assessment, with most patients with very low-and low-risk tumors undergoing surgery alone, intermediate-risk patients receiving both surgery and chemotherapy (determined by the child's age, stage, tumor histology, and ploidy, as well as the genetic changes in chromosomes 1p and 11q), and high-risk patients requiring a more intensive treatment including surgery, chemotherapy, radiation, retinoid therapy, immunotherapy, and stem cell transplant [31].
Less than 50% of high-risk patients survive, in contrast to low-risk patients who are cured in the majority of cases [29]. Neuroblastoma tumors exhibit extreme phenotypic heterogeneity that might indicate alternative sensitivity during therapeutic drug applications [32]. Importantly, spontaneous regression of the disease has often been detected in infants [27]. Neuroblastoma cells exhibit divergent metabolic features compared to normal sympathetic neurons [28]. Mechanistically, the disease development is highly connected with, among others, a well-studied genomic abnormality: the amplification of MYCN [27][28][29]33], which nowadays has been assigned as a prognostic factor or/and considered as a causal mechanism that drives the disease phenotype evolution [33]. As mentioned above, MYCN amplification is frequent in high-risk patients [28] and is utilized for patient-risk classification [34]; however, this is not the only mechanism responsible for the disease, since non-MYCN-amplified neuroblastoma is abundantly recorded in patients. Chromosomal abnormalities, such as the gain of 17q, the loss of 1p, and the loss of 11q, have also been associated with worse prognoses [35].
The biology of neuroblastoma and its underlying developmental mechanisms have been progressively elucidated by advanced studies [36][37][38] beyond the context of MYCN amplifications that have been mapped both across the endogenous locus and in structures of extrachromosomal DNA amplicons (ecDNA) [1,39]. Recent studies have shown that RAS/MAPK pathway alterations and telomere maintenance mechanisms may also be useful for the discrimination of the high-risk subgroups or for the identification of intermediate-risk subgroups with possible disease recurrence [40,41]. Additionally, muta-tions in the paired-like homeobox 2B (PHOX2B) and anaplastic lymphoma kinase (ALK) genes have been detected in patients with rare familial neuroblastoma [42,43]. In many cases, neuroblastoma phenotypes are characterized by the aberrant expression of MYCN and its downstream interrelationships with critical metabolism-related genes. For example, it has been shown that MYCN confers metabolic reprogramming in neuroblastoma cells in cooperation with ATF4, and together, they assemble a positive feedback loop which is critical for the activation of the serine-glycine-one carbon (SCOG) biosynthetic pathway in MYCN-amplified neuroblastoma cells [44]. In addition, MYCN was recently shown to promote de novo lipogenesis in neuroblastoma through direct activation of lipogenic enzymes that orchestrate fatty acid synthesis, a key hallmark of metabolic reprogramming in cancer supporting tumor survival under nutrient deprivation conditions [45]. The engagement of MYCN in neuroblastoma metabolic rewiring was further underscored by the finding of its positive feedback loop with enzyme aldehyde dehydrogenase family 18 member A1 (ALDH18A1), a critical component in glutamine metabolism. Increased ALDH18A1 in MYCN-amplified neuroblastoma is associated with amino acid and nucleotide metabolism, thus supporting the cell proliferation and self-renewal potency of cancer cells [46].
The role(s) of MYCN in metabolic reprogramming-related gene expression program evolution in neuroblastoma has remained largely elusive. This work evaluates the impact of MYCN amplification in metabolic reprogramming in neuroblastoma. Two workflows were applied to address this altered metabolic state in the contexts of MYCN amplifications and transgenic-based enforced expression of the oncoprotein. We meta-analyzed published datasets to address the impact of MYCN overexpression [26,29]. Our findings distinguish a collection of metabolically committed and cancer-related TF-encoding genes that become overexpressed and are "molecularly flagged" by direct MYCN binding across their cisacting regulatory elements. Importantly, comparative analysis of the results reciprocally confirmed our findings and highlighted the functional significance of MYCN for neuroblastoma development. Hence, TFs are comprehensively studied at the mechanistic and regulatory level in the context of neuroblastoma metabolism. The emerging new knowledge describes previously unspecified direct targets of MYCN oncoprotein that encode for TFs with regulatory capacities, thus facilitating a better understanding of how genomic alterations drive metabolic phenotypes in cancers.

Transcriptomics Analyses
RNA-seq analyses in neuroblastoma cell lines: Computational meta-analysis comparing MYCN-amplified and non-MYCN-amplified neuroblastoma cell lines was conducted by utilizing publicly available RNA-seq data described in Boeva et al. [47] (GEO accession number GSE90683). The detailed RNA-seq analysis pipeline and results can be found in Koutsi et al. [1]. In brief, quality control of sequencing reads was conducted using FastQC (Galaxy Version 0.73 + galaxy0). Then, reads were mapped to the hg19 reference human genome using the HISAT2 alignment tool (Galaxy Version 2.2.1 + galaxy0) with "paired-end data from a single interleaved dataset", "stranded", and "reverse" parameters [48]. Gene expression quantification was performed using the htseq-count tool with "union mode", "stranded", "reverse," and "minimum alignment quality 10" parameters (Galaxy Version 0.9.1 + galaxy1) [49]. The EdgeR algorithm was employed to identify differentially expressed genes (DEGs) between MYCN-amplified and non-MYCN-amplified neuroblastoma cell lines with a p-value threshold of <0.01 and |log 2 [fold change (FC)]| ≥ 1 considering only the genes with more than ten counts in at least five individual samples. Differentially expressed TFs were identified by utilizing the official list of human TFs [50]. Gene ontology analyses (GOs) of upregulated DEGs (uDEGs) were performed through the gProfiler tool [51]. Selected statistically significant biological processes (p-adjusted < 0.05) were visualized as dotplots using the ggplot2 package. gProfiler was selected because it provides information for the experimental or computational annotation origins of the genes assigned to each biological term. This allows the demarcation of metabolism-related genes. Heatmaps of DEGs were constructed using pheatmap and assessing log 2 (CPM) normalized counts.
RNA-seq analyses in neuroblastoma patient-derived samples: (A) We utilized publicly available RNA-seq datasets, described in Rifatbegovic et al. (GEO accession number GSE94035) [52]. The clinical samples were derived from neuroblastoma patients with primary stage 4 metastatic (M) tumors. The total cohort of 16 samples was subjected to rigorous computational processing in order to identify the portion/ratio of immune cells within each sample, thus allowing us to exclude highly heterogeneous datasets from our computational investigation. This step is important to avoid any transcriptional noise that originates from highly "contaminated" samples to dominate our downstream analyses. This was conducted by the application of ABsolute Immune Signal deconvolution (ABIS), an efficient tool for clinical sample profiling that assesses gene expression values and is available at https://giannimonaco.shinyapps.io/ABIS/ (accessed on 13 September 2023) [53]. Six samples that met the criteria and displayed low "contamination" from immune cells were selected for further transcriptomics analysis. Three of those were MYCNamplified and three were non-MYCN-amplified and they shared similar-limited enrichment of immune cells (≤10%). RNA-seq analyses were conducted as described above with minor modifications outlined below: Sequencing reads were mapped to the hg19 reference human genome by utilizing the HISAT2 alignment tool (Galaxy Version 2.2.1 + galaxy0) and applying "single-end" and "unstranded" options [48]. The calculation of reads that were mapped across the genes was performed by the application of the htseq-count tool (Galaxy Version 0.9.1 + galaxy1) in union mode with "unstranded" and "minimum alignment quality 10" options [49]. The EdgeR algorithm was employed to identify DEGs between MYCN-amplified and non-MYCN-amplified neuroblastoma patient-derived samples with a p-value threshold of <0.05 and |log 2 (FC)| ≥ 0.6, considering only the genes with more than ten counts in at least two individual samples. (B) Neuroblastoma datasets from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) project were also assessed to evaluate the effect of MYCN amplification in the metabolic rewiring of neuroblastoma [54]. Raw RNA-seq counts of gene expression quantification (n = 162) accompanied by the clinical and histological characteristics of the specimens were retrieved from the National Cancer Institute GDC Data Portal. The samples for which information regarding MYCN amplification status was not available were excluded from further analysis. In a second layer of classification, 66 primary tumor samples with undifferentiated or poorly differentiated histological status derived from high-risk and stage 4 patients were subjected to ABIS deconvolution of immune cells. Twenty MYCN-amplified and twenty-five non-MYCN-amplified samples displayed ≤10% enrichment for immune cells and were utilized for differential gene expression analysis through EdgeR (p-value < 0.05 and |log2(FC)| ≥ 0.58). Genes with at least ten counts in twelve samples were processed in the analysis.

ChIP-Seq Data Analyses
To investigate the role of MYCN in human neuroblastoma, we utilized ChIP-sequencing FASTQ files obtained from the Gene Expression Omnibus database (GEO; https://www. ncbi.nlm.nih.gov/geo/, accessed on 21 June 2023) (GEO accession number GSE138315). Meta-analyses were conducted by integrating publicly available data from two distinct groups of neuroblastoma cell lines based on MYCN amplification status. The analysis was conducted using three MYCN-amplified neuroblastoma cell lines (COG-N-415, LA-N-5, and NB-1643) and one non-MYCN-amplified neuroblastoma cell line (NB-69) as a control. Detailed information about these samples is provided in [29]. Furthermore, we utilized publicly available MYCN-ChIP-seq data from the SHEP MYCN-ER cell line, in which MYCN overexpression is achieved under a 4-hydroxytamoxifen (4-OHT)activating MYCN, while the control cells were treated with DMSO (GEO accession number GSE199086) [26]. Moreover, for monitoring the ASCL1-binding profile in a human MYCNamplified neuroblastoma cell line, we utilized a publicly accessible ASCL1-ChIP-seq FASTQ file from Kelly cells, which was obtained from the GEO database (GEO accession number GSE120074) [55]. ChIP-sequencing data were analyzed by utilizing the Galaxy online platform (https://usegalaxy.org/, accessed on 21 June 2023) [56], a widely used tool for bioinformatics analysis. Initial quality assessment of the sequencing reads was performed using FastQC (Galaxy Version 0.73 + galaxy0). Adaptors and low-quality sequences were trimmed, using Trim Galore (Galaxy Version 0.6.7 + galaxy0) with default parameters. Trimmed reads were then aligned to the hg19 reference human genome using the Bowtie2 tool (Galaxy Version 2.5.0 + galaxy0) with "single-end" or "paired-end" and "very sensitive end-to-end" parameters [57]. Duplicate reads were removed using the RmDup tool (Galaxy Version 2.0.1) from the SAMtools package [58]. To ensure same-scale comparisons, the samples were normalized for their sequencing depth using the Downsample SAM/BAM tool (Galaxy Version 2.18.2.1). Peak calling was performed by MACS2 (Galaxy Version 2.2.7.1 + galaxy0), with the input data serving as control files and default parameters [59]. Peaks were considered significant using a q-value cutoff of 0.05. To ensure data reliability, peaks detected in chromosome Y and unplaced contigs (e.g., chrUn and chrM) as well as those found in hg19 ENCODE blacklisted regions known to have low sequencing assurance were excluded [60]. The reads that mapped within the identified peaks were counted using MultiCovBed (Galaxy Version 2.30.0) [61]. The quantification of the signal intensity was performed using the bamCoverage tool from the deepTools package (Galaxy Version 3.5.1.0.0) with the "normalize to reads per kilobase per million (RPKM)" and "average fragment size = 300 bp" options, which generated bigwig files [62]. For the identification of inducible peaks, a comparative analysis was conducted by calculating the FC values between the MYCN-amplified neuroblastoma cell lines (COG-N-415, LA-N-5, and NB-1643) and non-MYCN-amplified neuroblastoma cell line . Similarly, in the case of MYCN overexpression [26], inducible peaks were identified according to their FC value between SHEP MYCN-ER 4-OHT and SHEP MYCN-ER DMSO. We set a minimum FC cutoff of ≥2 and then the inducible peaks were filtered based on a minimum number of reads, specifically, 20 reads for MYCN-amplified neuroblastoma cell lines and 40 reads for SHEP MYCN-ER 4-OHT. Common MYCN-inducible peaks between the different MYCN-amplified neuroblastoma cell lines and/or SHEP MYCN-ER 4-OHT were identified using the "Intersect" function of the bx-python package (Version 0.7.1), which was accessed through Galaxy. This analysis allowed us to identify the overlapping inducible peaks that were consistently shaped by MYCN across the different experimental conditions. Additionally, we employed the "Intersect" function (Version 0.7.1) to identify ASCL1 peaks that overlapped with the common MYCN-inducible peaks across the different MYCN-amplified neuroblastoma cell lines and SHEP MYCN-ER 4-OHT over-expressing system. Furthermore, the assignment of ChIP-seq peaks to the nearest transcription start site (TSS) of the hg19 reference human genome was determined by the Genomic Regions Enrichment of Annotations Tool (GREAT) (version 4.0.4), employing the whole genome as background and the "single nearest gene" parameter within 1000 kb [63]. In addition, the identification and analysis of transcription factor binding motifs (TFBMs) within the ChIP-seq peaks were carried out using the MEME-ChIP [64] and AME (Analysis of Motif Enrichment) [65] tools, with motifs from the JASPAR 2022 CORE non-redundant vertebrate database as inputs (http://jaspar.genereg.net, accessed on 22 June 2023). For MEME-ChIP, the options "DNA -mod zoops or -mod anr -minw 4 -maxw 15" were applied, and for AME, the options "-scoring average -method fisher-hit-lo-fraction 0.25-control shuffle" were used to identify enriched motifs within the ChIP-seq peaks. In both MEME-ChIP and AME analyses, an E-value threshold for motif enrichment and a p-value cutoff of < 0.05 for statistical significance were considered, ensuring the identification of TFBMs that exhibited biological relevance and statistical significance within the ChIP-seq peaks. Furthermore, the identification of E-boxes was centered on the inducible peaks located in close proximity to BCL6, DLX1, NRF2, ATF4, and STAT5B genes. To conduct this analysis, we utilized the MAST (Motif Alignment and Search Tool) and FIMO (Find Individual Motif Occurrences) tools, accessed through the MEME-Suite [64,66,67], applying default parameters. The E-box motifs used as inputs for scanning were sourced from the JASPAR 2022 CORE non-redundant vertebrate database (MA0059.1 and MA0819.2) [68]. The MAST tool was also utilized for the computational analysis of TFBMs recognized by KLF4, STAT5B, STAT5A::STAT5B, and BCL6 across the MYCN locus. Specifically, the relevant TFBMs used as inputs for this scanning were sourced from the JASPAR 2022 CORE non-redundant vertebrate database (MA0039.4, MA1625.1, MA0519.1, and MA0463.2). Finally, the visualization and exploration of signals in specific genomic regions were performed using the IGV (Integrative Genomics Viewer version 2.16.1) browser [69].

Gene Expression Rewiring and Metabolic Signatures in MYCN-Amplified Neuroblastoma
To monitor the gene expression in human neuroblastoma and address the impact of MYCN amplification in in vivo metabolic reconstitution, we utilized our established workflow that was previously applied [1] for intratumoral computational transcriptional profiling in neuroblastoma published results [47]. We applied stringent criteria to avoid any transcriptional noise dominating our downstream analyses, according to published strategies [70]. The results indicated the profile of activation and repression of DEGs (FC ≥ 2; p-value < 0.01), which then were evaluated according to the function of their encoded products (Figure 1(Ai)). We fished out 286 uDEGs in MYCN-amplified compared to non-MYCN-amplified neuroblastoma cell lines and processed them through GOs (Supplementary Figure S1A). Next, we distinguished 38 that encode for TFs according to the official TF list [50] (Table S1). At this stage, the functional significance of those TFs in metabolism was assessed primarily based on their classification to specific processes via GOs (Supplementary Figure S1A and Figure 1(Aii)). We selected 36 TFs that have been previously recorded in metabolism-related regulatory functions (Supplementary Figure S1B). In particular, GOs highlighted metabolic pathways such as the cellular metabolic process and macromolecule biosynthetic process (Figure 1 Figure 1B). Thus, the above integrative analyses captured metabolism-related TF upregulation in neuroblastoma cell lines that bear copy number genomic variations of MYCN.

MYCN-Binding Landscape in Neuroblastoma Illuminating Novel TF-Encoding Target-Genes
To investigate how MYCN amplification mechanistically imposes the metabolic rewiring of neuroblastoma cells, we first attended to the in cis distribution of its encoded product ( Figure 2A upper and lower panels). MYCN binding was monitored at the whole chromosomal and regional scales. We utilized published ChIP-seq datasets (workflow A) and conducted genome topology assessments from the ground state (see Materials and Methods). We applied our in-house workflow; published ChIP-seq datasets obtained from studies in human neuroblastoma cell lines, which carried (COG-N-415, LA-N-5, and NB-1643) or lacked (NB-69) genomic amplifications of the MYCN as described [29], were subjected to rigorous bioinformatics processing (see Materials and Methods). Initially, MYCN-ChIP-seq peaks were called, followed by individual pairwise comparisons of their signals' intensities implemented between NB-69 and COG-N-415, LA-N-5, and NB-1643. A list of inducible signals was generated for each assessment (FC ≥ 2; enriched in MYCN-amplified cell lines compared to non-MYCN-amplified) and showed 32,749 for COG-N-415/NB-69, 38,777 for LA-N-5/NB-69, and 30,809 for NB-1643/NB-69 comparisons, respectively (Table S2). Next, a step of evaluation of our ChIP-seq meta-analyses was oriented to validate MYCN-binding events across known target loci where the oncogenic TF was captured [71], as well as additional off-targets; this process serves as an internal control. For instance, robust MYCN binding was mapped across the SLC1A5 (ASCT2) locus, which encodes a known regulator of glutamine metabolism, a key feature of neuroblastoma tumorigenesis [71,72] (Figure 2A upper panel). In addition, MYCN binds to the NCL locus that encodes the nuclear RNAbinding protein nucleolin, essential for rRNA processing and ribosome biogenesis [26,73], and the NPM1 locus which encodes the multifunctional nuclear protein nucleophosmin, known for ribosome biogenesis and genome stability regulation (Figure 2A upper panel). In sharp contrast, off-target loci (e.g., AFM, OCT4) were scanned and demonstrated as inattentive/not significant in MYCN binding (Figure 2A lower panel). IGVs sharply display the above findings in high-resolution, thus ensuring the validity of our approach. In another layer of evaluation, we detected MYCN binding across the ATF4 locus, an anticipated result since the MYCN-ATF4 axis is one of the well-addressed TF interrelationships in neuroblastoma [28,44]. This established feedback loop is crucial for increasing the amino acid availability and, consequently, sustaining MYCN-mediated growth in neuroblastoma cells. Thus, MYCN collaborates with ATF4 in regulating ASCT2 expression, but in addition, it targets the ATF4 cis-acting regulatory region (IGVs) (Figure 2A upper panel). The above results authenticate both the novelty and the accuracy of our approach.  Moreover, we investigated the enrichment of the MYCN genomic locus for TFBMs recognized by the above TFs. We applied MAST-tool-based analysis and processed a genomic region of ~4.3 kb that flanks the TSS of MYCN (chr2:16,077,951-16,082,248) ( Figure 3). We mapped several motifs for KLF4, multiple for STAT5B, and a single one for BCL6, randomly distributed across this DNA sequence. This profile poises for the binding of those MYCN-targeted TFs across MYCN. Given that MYCN amplifications result in the in cis expansion of additional copies of the amplified DNA sequence, the above TFBMs are anticipated to become multiplied. Hence, the possibility of the assembly of an auto- Next, we examined the cistrome of MYCN across chromatin landscapes that host genomic loci that encode for several of the upregulated metabolism-related TFs demarcated above. We zoomed in across the chromatin microenvironments of residence and monitored the patterns of MYCN genomic distribution. IGVs depict in high-resolution that the BCL6 locus hosts enhanced MYCN binding, predominantly enriched~5 kb downstream of the TSS and at a lesser but significant intensity on the promoter of the gene (Figure 2A  upper panel). Indicatively, precisely shaped ChIP-seq signals are mounted across these vicinities. These binding profiles are mapped in all three MYCN-amplified, but not in the non-MYCN-amplified, neuroblastoma cell lines. Transcription factor binding motif analyses (TFBMs) demonstrated that MYCN masks regulatory sequences enriched for TF binding sites (TFBSs), including E-boxes recognized from MYCN::MAX (Figure 2A upper panel), as evaluated by both MAST tool-and FIMO-based analyses [64,66,67]. Interestingly, these genomic coordinates reconstitute in vivo chromatin landscapes that coincide with the well-addressed SE of the gene, described in other types of cancers [19]. Hence, BCL6 is tagged by MYCN binding, a profile that mechanistically is in line with its transcriptional upregulation in MYCN-amplified neuroblastoma cells. DLX1 hosts enhanced MYCN binding, predominantly enriched across a DNA stretch of~1 kb that flanks its TSS and encompasses the core promoter, as well as TFBSs for FOXM1 that were previously characterized in ovarian cancer [74] (Figure 2A  Evidently, our results underscore that MYCN is substantially committed to metabolic rewiring in neuroblastoma cells through its attachment to transcriptional regulatory ciselements that neighbor metabolism-related TF-encoding genes. To gain further mechanistic confirmation, we applied the same workflow to an alternative system of study that relies on the enforced expression of MYCN (workflow B). More specifically, Wang et al. [26] conducted an advanced study by utilizing the SHEP MYCN-ER cell line [34], which bears a single copy of a 4-hydroxytamoxifen (4-OHT)-inducible MYCN transgene. This allows for a fine-tuned overexpression of MYCN based on the addition of 4-OHT. SHEP MYCN-ER cells treated with DMSO were utilized as a control. MYCN-ChIP-seq datasets were analyzed according to our in-house protocol described above and subjected to rigorous bioinformat-ics processing (see Materials and Methods). In brief, MYCN-ChIP-seq peaks were called, followed by pairwise comparison of their signal intensities between 4-OHT-treated and DMSO-treated SHEP MYCN-ER experiments. A list of 11,308 inducible signals was generated (FC ≥ 2; enriched in 4-OHT-treated cells compared to DMSO-treated cells) (Table S2). Next, we monitored in high-resolution the distribution of these signals across the loci that host known-and off-targets, described above, and we verified to a significant extent the results in both systems, thus confirming the specificity of our findings (Figure 2A upper and lower panels). IGVs substantially validate the above findings, display in high-resolution MYCN-binding profiles that match those obtained from the above analyses, and demonstrate at the genomic loci-specific level that TSS-proximal cis-acting elements of the genes of investigation are occupied by MYCN in both systems of study. Overall, the binding profiles shaped recapitulate those that emerged from the analyses of the MYCN-amplified versus non-MYCN-amplified neuroblastoma cells and depict robust binding of the MYCN oncoprotein upstream of or flanking the TSSs of the above target-genes and control loci ( Figure 2A upper and lower panels). Indeed, these patterns of genomic distributions of MYCN assembled during its attachment to the respective chromatin landscapes, in both cell systems of study, are overlapping or nearly identical. Moreover, our computational investigation was complemented with another layer of evaluation; we assessed the endogenous MYCN for MYCN binding and identified that (a) MYCN exhibits auto-regulatory capabilities facilitated by direct targeting of its own coding locus by its encoded oncoprotein. This phenomenon is evident in both systems of study. This is consistent with previous studies that mapped MYCN binding across extended genomic loci that harbor MYCN in neuroblastoma [75]. Interestingly, it has been shown that this type of autoregulation can be disrupted in amplified, but not in single-copy, neuroblastoma cell lines [76]. This underscores the impact of genomic amplification in the establishment of diverse mechanistic rules for the same disease. (b) Furthermore, our results show that in neuroblastoma cell lines that carry (COG-N-415, LA-N-5, and NB-1643) genomic amplifications of MYCN, robust binding of the TF is mapped across an extended (≥10 kb) genomic region. This pattern is inattentive/not significant, or even abolished, from the non-MYCN-amplified cells (NB-69). Given that MYCN amplification routinely spans several or even hundreds of kb in cis [77,78], the multiple copies generated can first provide an amplified chromatin landscape that serves as an anchored site for MYCN binding and, in addition, can multiply intact amplicons of coding genes resulting in overexpression of MYCN oncoprotein. Thus, it is realistic to argue that MYCN amplifications footprint the endogenous loci at the (epi)genomic level. (c) On the other hand, in the SHEP MYCN-ER cell line system of study, 4-OHT-inducible binding of MYCN is centered around the TSS-proximal intronic and exonic sequences of MYCN and mounts a precisely shaped peak that masks a DNA stretch of~1 kb. This sounds realistic since this cell line harbors a single copy MYCN transgene and lacks MYCN amplicons. The above results imply that in both systems an auto-regulatory mechanism facilitates MYCN overexpression with or without affecting the (epi)genome architecture of its residential chromosomal coordinates. This is made more intriguing when considering that outside of chromosomal landscapes MYCN-ecDNA-amplicons still work and encode MYCN efficiently. Finally, an additional layer of verification was achieved by the intersection of the individual spectrums of the inducible MYCN peaks that emerged from the whole meta-analysis. We sequentially overlayed the individual spectrums of MYCN-ChIP-seq inducible peaks following a gradual decreasing mode of analysis. The results depicted 7971 common signal instances, which then were subjected to GOs ( Figure 2B). The outcome of this comparative assessment sharply illustrates strong correlation between the genomic distribution of MYCN oncoprotein with metabolism-related processes in neuroblastoma cells ( Figure 2B). More specifically, GOs highlighted, among others, cellular functions related to RNA metabolism, cellular macromolecule catabolic processes, ribonucleoprotein complex and ribosome biogenesis, peptide biosynthetic processes, etc. Given that those genomic loci are randomly distributed across chromosomal landscapes and that they were ChIPed through MYCN binding, a clear correlation of the TF with metabolic rewiring is underscored. This suggests the assembly and function of an MYCN axis that involves additional metabolism-related TFs, such as those comprehensively discussed above and in the discussion section. The pleiotropic commitment of those regulators of gene expression in vital cellular processes implies the in vivo reconstitution of a newly evolved metabolic state in neuroblastoma.
Moreover, we investigated the enrichment of the MYCN genomic locus for TFBMs recognized by the above TFs. We applied MAST-tool-based analysis and processed a genomic region of~4.3 kb that flanks the TSS of MYCN (chr2:16,077,951-16,082,248) ( Figure 3). We mapped several motifs for KLF4, multiple for STAT5B, and a single one for BCL6, randomly distributed across this DNA sequence. This profile poises for the binding of those MYCNtargeted TFs across MYCN. Given that MYCN amplifications result in the in cis expansion of additional copies of the amplified DNA sequence, the above TFBMs are anticipated to become multiplied. Hence, the possibility of the assembly of an auto-regulatory loop between those transcriptional regulatory proteins and MYCN cannot be excluded. This is in line with the biology of TFs' expression regulation, per se. Finally, we investigated the binding profile of ASCL1, a basic helix-loop-helix TF that is expressed in neuroblastoma cells and is required for cell growth and arrest of differentiation [55]. ASCL1 cis-regulatory elements are targeted by MYCN and additional TF members of the adrenergic (ADRN) neuroblastoma core regulatory circuitry (CRC) [55]. We meta-analyzed one ASCL1-ChIP-seq dataset derived from experiments in Kelly cells and an MYCN-amplified neuroblastoma cell line, described in [55]. First, we called 10,661 ASCL1-ChIP-seq peaks and intersected them with the common 7971 MYCN-ChIP-seq-inducible peaks, described above (Supplementary Figure S2A). We retrieved 2081 common peaks, and we processed those in GOs. The results show statistically and biologically significant metabolic processes, such as ncRNA metabolic processes, cellular macromolecule metabolic processes, ribonucleoprotein complex biogenesis, etc. (Supplementary Figure S2A). Next, we monitored the profile of ASCL1 binding across several genomic loci, including MYCN and other upregulated metabolismrelated TFs, described above. The results highlight proximal binding events of ASCL1 in many of those genes, including KLF4, NRF2, GLI2, and MYCN (Supplementary Figure S2B). This analysis strengthens our findings, since it illuminates that several of the upregulated metabolism-related TFs described in our computational study are targeted by an additional TF that is critical for neuroblastoma and is also an MYCN target. ASCL1 was captured enriched, but not statistically significant upregulated, in our transcriptomics meta-analysis between MYCN-amplified and non-MYCN-amplified cell lines, but this cannot exclude the possibility for a regulatory role in fine-tuning the transcription of several of the upregulated metabolism-related TFs genes discussed in our work. These findings open new avenues for further investigations in the neuroblastoma field.

Computational Validation in Neuroblastoma Patient-Derived Samples
To further confirm our findings and to gain additional insights into the biology of the disease, we assessed gene expression profiles in clinical samples derived from neuroblastoma patients. Transcriptomics profiling in patient-derived surgically removed solid tumors frequently suffers from intrinsic tumor heterogeneity and/or enrichment of the specimens with irrelevant cells, e.g., blood/immune cells, stroma cells, etc. These matter against the precise recording of any differentiation between the transcriptomes of individual samples [79]. In cases of TF investigations, this becomes more challenging, since they are broadly expressed in multitudes of tissue types and, consequently, the monitoring of the expression states of their coding genes, in such systems of study, can be hindered when "non-cancerous"/irrelevant cell types are significantly represented within the samples of examination. Those drawbacks can lead to the domination of the entire computational analyses by "background" signals. To bypass these barriers, we applied a rigorous computational rationale that significantly facilitates the abolishment of "non-cancerous"derived/non-specific RNA-seq signals yet maintains the balance between the samples and the sensitivity of the analyses (see Materials and Methods). We utilized publicly available RNA-seq clinical datasets and performed transcriptomics meta-analyses centered on both the metabolic rewiring and the 22 metabolism-related TFs identified. First, we curated datasets from stage 4/M metastatic neuroblastoma patients [52] and processed them, with emphasis on the deconvolution from immune cells, by the application of the ABIS tool [53]. From the entire pool of sixteen samples evaluated, six met the applied criteria, and were meticulously selected for further investigation. An equal number of datasets derived from patient samples from both categories (three MYCN-amplified neuroblastoma and three non-MYCN-amplified neuroblastoma) that are characterized by similar-low enrichment for immune cells (≤10%) were comparatively studied. The results depicted 2726 DEGs [|log 2 (FC)| ≥ 0.6 and p-value < 0.05] between these groups. Of those, 1396 DEGs were upregulated (Table S3). GOs applied on the 1396 uDEGs highlighted, among others, several ontologies and functions connected to metabolism, including peptide biosynthetic, amide metabolic, and cellular macromolecule processes, etc. (Supplementary Figure S3(Ai)). Next, we intersected the spectrum of 1396 uDEGs with the one of 286 uDEGs initially identified in cell lines and revealed 77 common genes (Table S3). Of those, eight encode for metabolismrelated TFs included in the pool of the 22 TFs initially identified, such as MYCN, BCL6, KLF4, SIX4, NR2F2, GLI2, GLI3, and WT1. Two additional metabolism-related TF-encoding genes, ATF4 and DLX1, were captured that were commonly upregulated according to FC but not statistically significant according to the p-value threshold and were thus excluded from the heatmap (Supplementary Figure S3(Aii)). These findings significantly validate the knowledge derived from the studies on cell lines. MYCN-amplified neuroblastoma cell line, described in [55]. First, we called 10,661 ASCL1-ChIP-seq peaks and intersected them with the common 7971 MYCN-ChIP-seq-inducible peaks, described above (Supplementary Figure S2A). We retrieved 2081 common peaks, and we processed those in GOs. The results show statistically and biologically significant metabolic processes, such as ncRNA metabolic processes, cellular macromolecule metabolic processes, ribonucleoprotein complex biogenesis, etc. (Supplementary Figure  S2A). Next, we monitored the profile of ASCL1 binding across several genomic loci, including MYCN and other upregulated metabolism-related TFs, described above. The results highlight proximal binding events of ASCL1 in many of those genes, including KLF4, NRF2, GLI2, and MYCN (Supplementary Figure S2B). This analysis strengthens our findings, since it illuminates that several of the upregulated metabolism-related TFs described in our computational study are targeted by an additional TF that is critical for neuroblastoma and is also an MYCN target. ASCL1 was captured enriched, but not statistically significant upregulated, in our transcriptomics meta-analysis between MYCNamplified and non-MYCN-amplified cell lines, but this cannot exclude the possibility for a regulatory role in fine-tuning the transcription of several of the upregulated metabolismrelated TFs genes discussed in our work. These findings open new avenues for further investigations in the neuroblastoma field.

Computational Validation in Neuroblastoma Patient-Derived Samples
To further confirm our findings and to gain additional insights into the biology of the disease, we assessed gene expression profiles in clinical samples derived from neuroblastoma patients. Transcriptomics profiling in patient-derived surgically removed solid tumors frequently suffers from intrinsic tumor heterogeneity and/or enrichment of the specimens with irrelevant cells, e.g., blood/immune cells, stroma cells, etc. These matter against the precise recording of any differentiation between the transcriptomes of individual samples [79]. In cases of TF investigations, this becomes more challenging, since they are broadly expressed in multitudes of tissue types and, consequently, the monitoring of the expression states of their coding genes, in such systems of study, can be hindered when "non-cancerous"/irrelevant cell types are significantly represented within the samples of examination. Those drawbacks can lead to the domination of the entire Next, we explored another cohort of samples derived from neuroblastoma patients annotated in TARGET-NBL (neuroblastoma project) [54]. Rigorous criteria were applied and gated the samples that derived from primary, undifferentiated or poorly differentiated, high-risk, and stage 4 neuroblastoma tumors. The pool of 66 samples was then processed for deconvolution of absolute immune signals, as described above, and resulted in a sub-cohort of 45 samples composed of 20 MYCN-amplified and 25 non-MYCN-amplified neuroblastoma tumors that are characterized by similar-low enrichment from immune cells (≤10%). Comparative transcriptomics analysis highlighted 3424 uDEGs (Table S3), and GOs revealed, among others, cellular biosynthetic, peptide metabolic, and rRNA metabolic processes (Supplementary Figure S3(Bi)). Next, we intersected the spectrums of 3424 uDEGs and 286 uDEGs and identified 110 common genes. Of those, ten encode for metabolism-related TFs included in the pool of the 22 TFs initially identified, such as FOXF1, OSR2, WT1, MYCN, NR2F2, SIX4, GLI2, NR1D1, HLF, and CREBZF. Interestingly, manual curation of ATF4 depicts a statistically significant p-value threshold even with lower induction (FC 1.42), whereas DLX1 was captured while upregulated according to its FC but was not statistically significant according to the p-value threshold; thus, both were excluded from the heatmap (Supplementary Figure S3(Bii)). In line with the results described above, these findings also validate the knowledge emerging from the studies on cell lines. Overall, our findings illuminate previously unspecified elements of the complex transcriptional regulatory landscape(s) assembled and underscore the essential commitment of TFs during the metabolic rewiring occurring in neuroblastoma.

Discussion
Human nervous system metabolism has been elucidated both under normal and oncogenic states [3]. Herein, we conducted alternative workflows and succeeded in addressing the interrelationship of several TFs with MYCN overexpression, in neuroblastoma. Among them, BCL6, KLF4, DLX1, NRF2, and STAT5B are prominent and involved in metabolic regulation. In more detail, the BCL6 gene, also known as B-Cell Lymphoma 6, resides at Chromosome 3q27.3 in a genomic region of 24.3 kb and encodes a TF that is a member of the Broad-Complex, Tramtrack, and Bric-a-Brac/Pox Virus and Zinc Finger (BTB/POZ) family [80,81]. It was initially identified as a genomic region susceptible to chromosomal abnormalities in cases involving the diffuse large B-cell lymphoma (DLBCL) family [80] and follicular lymphoma (FL) [19,82,83]. BCL6 is a transcriptional repressor that binds DNA through six zinc fingers and can interact with several other factors in order to regulate transcription. It is crucial for B cell development and overexpressed in lymphomas, while it also serves as a diagnostic and prognostic marker, facilitating patient stratification and treatment decision-making [84,85]. Mutations which disrupt two neighboring binding sites that are recognized by transcriptional repressors of the gene diminish the capacity of BCL6 to effectively bind to its own promoter, thereby disrupting the negative autoregulatory mechanism that maintains control over the expression levels of BCL6 [81,86]. In addition, an intragenic super-enhancer (iSE) of approximately 33 kb, which spans the first intron of BCL6, exhibits a high frequency of mutations in primary DLBCL cases [1,19]. In addition, knock-in mouse models with constitutive expression of BCL6 in B cells, which resemble the 3q27 chromosomal translocation of the human DLBCL, have been studied. More specifically, Cattoretti et al. [87] showed that mice developed increased GC formation and lymphomas that closely resembled human DLBCL. These findings highlight the critical role of BCL6 dysregulation in DLBCL [87]. Metabolic reprogramming of B cells has been shown to integrate epigenetic activation of BCL6 gene to induce germinal center B cell differentiation, thus confirming its crucial pathogenic role in lymphomagenesis and immune diseases [88]. Indeed, BCL6 has been involved in the pathogenesis of B-acute lymphoblastic leukemia (B-ALL), chronic myeloid leukemia (CML), breast cancer, and non-small cell lung cancer (NSCLC) [85,[89][90][91], and thus its dysregulated gene expression is an attractive target for therapeutic interventions. Furthermore, the balance between MYC and BCL6 expression in pre-B-ALL has been previously suggested as a key determinant of cell survival [92].
Notably, BCL6 has also been proposed to act as a metabolic switch in mouse liver cells, regulating genes involved in lipid metabolism. It functions as a repressor of genes involved in fat burning, being a negative regulator of oxidative metabolism and a promising target for fatty liver disease [93]. Novel compounds, including peptide and small molecule inhibitors, have been developed to target BCL6 effectively and disrupt its interaction with co-repressor complexes, counteracting the effects on its target genes [85].
In neuroblastoma cell lines, differential BCL6 mRNA expression has been recorded between neuroblastic (N-type) and Schwannian stromal (S-type) cells, being higher in S-type cells [32]. However, BCL6 expression in the neuroblastic regions of neuroblastoma tumors was associated with enhanced relapse time and increased overall patient survival compared to regions without BCL6 expression. This positive association of BCL6 with clinical outcome has also been observed in primary central nervous system lymphoma, follicular lymphoma, and DLBCL, possibly because BCL6 enhances chemotherapy responses. Although detailed mechanistic studies on the role of BCL6 expression in neuroblastoma are missing, it can be speculated that, analogous to lymphomas, BCL6 can mediate the arrest of neural crest cell differentiation involved in tumor development, as well as potentially contribute to the genetic instability observed in neuroblastomas, possibly due to attenuation of the DNA damage response [32].
In turn, the KLF4 gene, also known as Krüppel-like factor 4, resides at Chromosome 9q31.2 in a genomic region of 4.9 kb. KLF4 encodes for a DNA binding and zinc fingercontaining TF that functions as a master regulator of the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) along with OCT4, SOX2, and c-MYC (Yamanaka factors) [94]. KLF4 s unique structural features confer pioneer regulatory activities: its three zinc fingers bind to a 10 bp cognate DNA sequence (5 -GAGGCGTGGC-3 ) [95] and, in addition, it exhibits a low complexity surface efficient in interacting with chromatin remodelers and TFs [96]. KLF4 functions either as a tumor suppressor or as an oncogene in a tissue-specific fashion. For instance, it has been identified as overexpressed in early breast cancer and squamous cell carcinomas [97]. Oppositely, KLF4 loss in early colorectal cancers due to targeted proteasome-dependent degradation designates its function as a tumor-suppressor through inhibiting tumorigenesis via regulation of cell-cycle arrest [98]. Regarding its commitment to tumor metabolic rewiring, in breast cancer, elevated KLF4 expression has been detected and contributes to activating phosphofructokinase (PFKP), a key enzyme involved in the glycolysis pathway [99]. Conversely, in pancreatic cancer, KLF4 executes an alternative role and inhibits tumor progression by transcriptionally repressing the lactate dehydrogenase (LDHA) enzyme. This operation restricts the glycolysis-dependent altered metabolic reprogramming of cancer cells, suggesting a potential tumor-suppressive function for KLF4 in this context [100]. In a recent study, a novel interaction between monomethyltransferase SET8 and KLF4 was identified. The SET8/KLF4 complex formation negatively affects the transcriptional activation of SIRT4 by KLF4, thus promoting the proliferation-favorable Warburg effect of hepatocellular carcinoma cells [101]. In glioblastoma, KLF4 methylation-dependent transcriptional activation of UDP-glucose 6-dehydrogenase (UGDH) increases the synthesis of glycosaminoglycans (GAGs), which constitute essential structural polymers/components of the extracellular matrix (ECM), thus facilitating tumor growth and invasion [102]. Interestingly, KLF4 has been described as a putative MYCN-target gene [103].
In neuroblastoma tumors, low expression of KLF4 has been associated with poor survival of patients, while its overexpression in neuroblastoma cell line SH-SY-5Y was shown to inhibit cell proliferation and induce cell arrest by upregulating the cell cycleinhibitor p21Waf1/Cip1 as well as activating cell differentiation [104]. In our study, many neuroblastoma cell lines with MYCN amplification exhibited increased expression of KLF4, indicating a potential contribution to the cell cycle dysregulation and the transformation process underlying neuroblastoma development.
The DLX1 gene, also known as Distal-Less Homeobox 1, resides at Chromosome 2q31.1 in a genomic region of 4.1 kb. DLX1 encodes for a homeobox-containing TF, a homolog of the renowned TF that is encoded by the Distal-less (Dll) gene in Drosophila melanogaster. Dll controls the development of limb appendages, such as the distal part of the leg, the assembly of structures of the peripheral nervous system, etc., in fruit flies [105][106][107][108][109]. DLX1 in humans exerts crucial roles in the assembly of craniofacial structures, and its dysregulation is engaged in distinct types of cancer [110]. For instance, DLX1 is a reliable biomarker for prostate cancer (PCa), and its elevated expression accompanies the development of aggressive phenotypes of the disease and poor patient survival [111,112]. At the molecular level, DLX1 establishes protein-protein interactions with beta-catenin and activates betacatenin/TCF signaling pathways, thus promoting growth, migration, and cell colony assembly in Pca [112]. In addition, in normal hematopoiesis, DLX1 is expressed in a lineagespecific manner, interacts with SMAD4 via its homeodomain, and inhibits BMP-4-induced transcriptional activation [110,113]. In aberrant hematopoiesis, such as in acute myeloid leukemia (AML), upregulated expression of DLX1 has been recorded as a downstream effect of activating mutations of fms-like tyrosine kinase 3 (FLT3), a frequent genomic alteration (~30%) that accompanies AML phenotypes [114,115]. Mechanistically, this is served by MAPK and JNK pathways [114]. Finally, in ovarian cancer, DLX1 is a direct target of Forkhead Box M1 (FOXM1) TF. FOXM1 recognizes two conserved binding sites that flank the TSS of DLX1, thus upregulating its expression in high-grade ovarian cancer [74]. Consequently, DLX1 upregulation promotes cell proliferation, migration, and metastasis. The above findings imply a pleiotropic commitment of DLX1 in various mechanisms of tumorigenesis. Although there are no studies available elucidating the expression and function of DLX1 in neuroblastoma, a possible role in the regulation of neural crest cell differentiation mechanisms cannot be excluded, given its expression in neural crest cells [116].
Furthermore, the NRF2 gene, also known as Nuclear Factor Erythroid 2-related factor 2 (NFE2L2), resides at Chromosome 2q31.2 in a genomic region of 34.8 kb. It encodes for a TF that is a cap'n'collar (CNC) member of a family of basic leucine zipper proteins (bZIP) and is composed of 7 Neh domains [117]. NRF2 can be activated by toxic electrophilic stimuli and oxidant factors to induce the transcription of antioxidant and detoxifying genes, but also genes involved in immune and inflammatory responses, tissue remodeling, and carcinogenesis [118]. However, the primary function of NRF2 is cell adaptation to stress [119]. It is well known that oxidative stress and metabolic dysfunction are highly interconnected, leading to severe pathogenic conditions. NRF2, being a master regulator of stress responses, has been shown to be activated in diverse tumor types where it is implicated in tumor formation, progression, and metastasis [120,121]. NRF2 forms heterodimers with MAF proteins and regulates the expression of multitudes of genes that are under the control of antioxidant response elements (ARE) [122]. The functions of NRF2 in cancer include both protective and promoting roles. Interestingly, transient activation of NRF2 works against cancer development, while its permanent activation is committed to carcinogenesis [123], metastasis, and resistance to therapies [117,124].
In neuroblastoma, NRF2 has been described to promote cell proliferation and resistance to retinoic acid (RA) cytotoxicity [125], as well as to a variety of other therapeutic agents. In respect to MYCN, a study showed that the aggressive neuroblastoma cell line HTLA-230 bearing MYCN amplification was slightly sensitive to bortezomib (BTZ), a selective inhibitor of the proteasome, due to NRF2 activation and subsequent upregulation of the antioxidant heme oxygenase-1 (HO-1). After treatment, these cells downregulated p53 and upregulated p21, favoring cell survival. However, the SH-SY-5Y cells without MYCN amplification exhibited a higher sensitivity to BTZ because they were unable to upregulate HO-1, indicating the importance of NRF2 in targeting neuroblastoma chemoresistance and driving cell adaption to oxidative stress [126].
The STAT5B gene, also known as Signal Transducer and Activator of Transcription 5B, resides at Chromosome 17q21.2 in a genomic region of 77.2 kb. STAT5B encodes for a TF that optimally binds to tandemly arranged interferon gamma-activated sequence (GAS) motifs [127]. Prior to activation, STAT5B is located in the cytoplasm. Upon activation, it becomes phosphorylated, translocates into the nucleus, and regulates the transcriptional activation of its target genes [128]. STAT5B is executive in hematopoiesis and in the differentiation of pluripotent stem cells (PSCs) in adult bone marrow. STAT5 plays an oncogenic role through the activation of c-Myc, cyclin D1, and Bcl-xl, inducing cellular transformation and progression of the cell cycle [129].
Its aberrant activation has been associated with myeloid malignancies such as chronic and acute myelogenous leukemia (CML and AML) [130]. STAT5's contribution to metabolic rewiring in cancer has been well-studied. It has been linked to oxidative metabolism in pre-B cells [131] as well as to a metabolic shift towards aerobic glycolysis in tumor cells [132]. Additionally, there is evidence of STAT5's direct activation of the glutamine synthetase (GS) enzyme upon radiotherapy. The GS promoter region harbors STAT5 response elements, and GS elevated expression in radioresistant cancer cells is suggested to enhance their DNA repair capacity and their survival advantage [133]. Moreover, an interaction of STAT5B with the δ-opioid receptors has been shown to increase neuronal survival, as well as neurite outgrowth and differentiation [134,135]. In neuroblastoma cell lines, STAT5B has been reported as a target of 5-aza-2 -deoxycytidine treatment to induce cell apoptosis and inhibit cell proliferation by changing the expression level of genes involved in the JAK/STAT pathway [136], indicating its important role.

Conclusions
Taken altogether, it is evident that MYCN amplification exhibits a crucial regulatory role in neuroblastoma cells, and our findings provide additional mechanistic insights through the regulation of pivotal transcription factors such as BCL6, KLF4, DLX1, NRF2, and STAT5B, which confer altered gene expression programs. It is known that inducible gene expression requires the synergistic operation of TFs and their counterparts that function together with respect to signal integration and epigenomic dependencies, e.g., accessibility of histone-DNA structures and nucleosome packaging, modifications on histones and DNA, coactivators and chromatin modifier/remodeler integration in chromosomal landscapes, etc. [16,137]. In many cases, a TF is sufficient to reprogram a cellular state or even to transform entire tissues or organs, e.g., HOX TFs [138]. Such transformations are gained through the establishment of newly developed gene expression programs that efficiently convert the phenotypic characteristics of origin. In neuroblastoma, this phenotypic conversion primarily relies, among others, on the overexpression of MYCN oncoprotein, which accounts for the altered transcriptional regulation of dozens of genes. Elegant studies have shown that, even when "chopped out" from the endogenous chromatin microenvironment, MYCN can be assembled in ecDNA amplicons, which therefore episomally drive the overexpression of MYCN [39]. Here, we show that occasionally in MYCN-amplified oncogenotypes, some of the upregulated MYCN target-genes encode for additional TFs, and many of those share the capacity to implement metabolism-related functions. Given the capability of TFs to establish regulatory networks [18], to fine-tune their own expression by assembling auto-regulatory loops [16], and to execute distinct modalities depending, among others, on the epigenetic landscape of incorporation, it becomes evident that neuroblastoma oncophenotype evolution might be mechanistically controlled in multiple layers. Moreover, it is well respected that an advanced approach to developing therapeutic applications targets the fundamental components that regulate the dysregulated gene expression programs on which the oncogenic phenotype relies [16]. A class of such functional candidate molecules is the TFs, which in many cases function as the master regulators of tumorigenesis or downstream targets of the oncogenic drivers. This provides tremendous capabilities for designing therapeutic approaches based on synthetic lethality [139]. Such vulnerabilities cannot be identified or precisely predicted by plain DNA sequence examinations but require an in-depth understanding of how cancer cells establish their vital gene expression programs. Hence, we envision that monitoring of the TFs' mechanistic dependencies and functional interrelationships is essential in order that such perplexing biological phenomena of human pathogenesis will become adequately disentangled.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cancers15194803/s1, Figure S1: Differential gene expression analyses in neuroblastoma: (A) The dotplot illustrates the most statistically and biologically significant processes associated with the total spectrum of 286 uDEGs, as defined by GOs. The metabolismrelated ontologies are highlighted within boxes. (B) The heatmap depicts the transcriptional profile of TF-encoding genes side by side in non-MYCN-amplified and MYCN-amplified neuroblastoma cell lines. Figure [47]. RNA-sequencing FASTQ files of human neuroblastoma patient-derived samples were downloaded from the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/, accessed on 21 June 2023) database (GEO accession number GSE94035), and information about samples can be found in Rifatbegovic et al., 2018 [52]. RNAseq data for TARGET-NBL datasets with related clinical information were retrieved from the National Cancer Institute GDC Data Portal. ChIP-sequencing FASTQ files for transcription factor MYCN in human neuroblastoma cell lines were obtained from the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/, accessed on 21 June 2023) database (GEO accession numbers GSE138315 and GSE199086). Further information regarding the samples can be found in Upton et al., 2020 [29] and Wang et al., 2023 [26]. The ChIP-sequencing FASTQ file for transcription factor ASCL1 in human neuroblastoma cell line with MYCN amplification was sourced from the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/, accessed on 21 June 2023) database (GEO accession number GSE120074). Detailed sample information can be found in Wang et al., 2019 [55].