The KAT module of the SAGA complex maintains the oncogenic gene expression program in MYCN-amplified neuroblastoma.

Pediatric cancers are frequently driven by genomic alterations that result in aberrant transcription factor activity. Here, we used functional genomic screens to identify multiple genes within the transcriptional coactivator Spt-Ada-Gcn5-acetyltransferase (SAGA) complex as selective dependencies for MYCN-amplified neuroblastoma, a disease of dysregulated development driven by an aberrant oncogenic transcriptional program. We characterized the DNA recruitment sites of the SAGA complex in neuroblastoma and the consequences of loss of SAGA complex lysine acetyltransferase (KAT) activity on histone acetylation and gene expression. We demonstrate that loss of SAGA complex KAT activity is associated with reduced MYCN binding on chromatin, suppression of MYC/MYCN gene expression programs, and impaired cell cycle progression. Further, we showed that the SAGA complex is pharmacologically targetable in vitro and in vivo with a KAT2A/KAT2B proteolysis targeting chimeric. Our findings expand our understanding of the histone-modifying complexes that maintain the oncogenic transcriptional state in this disease and suggest therapeutic potential for inhibitors of SAGA KAT activity in MYCN-amplified neuroblastoma.

Targeting c-Myc transactivation by LMNA inhibits tRNA processing essential for malate-aspartate shuttle and tumour progression.

BACKGROUND: A series of studies have demonstrated the emerging involvement of transfer RNA (tRNA) processing during the progression of tumours. Nevertheless, the roles and regulating mechanisms of tRNA processing genes in neuroblastoma (NB), the prevalent malignant tumour outside the brain in children, are yet unknown. METHODS: Analysis of multi-omics results was conducted to identify crucial regulators of downstream tRNA processing genes. Co-immunoprecipitation and mass spectrometry methods were utilised to measure interaction between proteins. The impact of transcriptional regulators on expression of downstream genes was measured by dual-luciferase reporter, chromatin immunoprecipitation, western blotting and real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) methods. Studies have been conducted to reveal impact and mechanisms of transcriptional regulators on biological processes of NB. Survival differences were analysed using the log-rank test. RESULTS: c-Myc was identified as a transcription factor driving tRNA processing gene expression and subsequent malate-aspartate shuttle (MAS) in NB cells. Mechanistically, c-Myc directly promoted the expression of glutamyl-prolyl-tRNA synthetase (EPRS) and leucyl-tRNA synthetase (LARS), resulting in translational up-regulation of glutamic-oxaloacetic transaminase 1 (GOT1) as well as malate dehydrogenase 1 (MDH1) via inhibiting general control nonrepressed 2 or activating mechanistic target of rapamycin signalling. Meanwhile, lamin A (LMNA) inhibited c-Myc transactivation via physical interaction, leading to suppression of MAS, aerobic glycolysis, tumourigenesis and aggressiveness. Pre-clinically, lobeline was discovered as a LMNA-binding compound to facilitate its interaction with c-Myc, which inhibited aminoacyl-tRNA synthetase expression, MAS and tumour progression of NB, as well as growth of organoid derived from c-Myc knock-in mice. Low levels of LMNA or elevated expression of c-Myc, EPRS, LARS, GOT1 or MDH1 were linked to a worse outcome and a shorter survival time of clinical NB patients. CONCLUSIONS: These results suggest that targeting c-Myc transactivation by LMNA inhibits tRNA processing essential for MAS and tumour progression.

Exploring the role of HLA variants in neuroblastoma susceptibility through whole exome sequencing.

Although a number of susceptibility loci for neuroblastoma (NB) have been identified by genome-wide association studies, it is still unclear whether variants in the HLA region contribute to NB susceptibility. In this study, we conducted a comprehensive genetic analysis of variants in the HLA region among 724 NB patients and 2863 matched controls from different cohorts. We exploited whole-exome sequencing data to accurately type HLA alleles with an ensemble approach on the results from three different typing tools, and carried out rigorous sample quality control to ensure a fine-scale ancestry matching. The frequencies of common HLA alleles were compared between cases and controls by logistic regression under additive and non-additive models. Population stratification was taken into account adjusting for ancestry-informative principal components. We detected significant HLA associations with NB. In particular, HLA-DQB1*05:02 (OR = 1.61; padj = 5.4 × 10-3) and HLA-DRB1*16:01 (OR = 1.60; padj = 2.3 × 10-2) alleles were associated to higher risk of developing NB. Conditional analysis highlighted the HLA-DQB1*05:02 allele and its residue Ser57 as key to this association. DQB1*05:02 allele was not associated to clinical features worse outcomes in the NB cohort. Nevertheless, a risk score derived from the allelic combinations of five HLA variants showed a substantial predictive value for patient survival (HR = 1.53; p = 0.032) that was independent from established NB prognostic factors. Our study leveraged powerful computational methods to explore WES data and HLA variants and to reveal complex genetic associations. Further studies are needed to validate the mechanisms of these interactions that contribute to the multifaceted pattern of factors underlying the disease initiation and progression.

A human neural crest model reveals the developmental impact of neuroblastoma-associated chromosomal aberrations.

Early childhood tumours arise from transformed embryonic cells, which often carry large copy number alterations (CNA). However, it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains, which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives, the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN, whose amplification co-occurs with CNAs in NB. Moreover, CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together, our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours.

NR1D1-transactivated lncRNA NUTM2A-AS1 promotes chemoresistance and immune evasion in neuroblastoma via inhibiting B7-H3 degradation.

Neuroblastoma (NB), a common solid tumour in young children originating from the sympathetic nervous system during embryonic development, poses challenges despite therapeutic advances like high-dose chemotherapy and immunotherapy. Some survivors still grapple with severe side effects and drug resistance. The role of lncRNA NUTM2A-AS1 has been explored in various cancers, but its function in drug-resistant NB progression is unclear. Our study found that NUTM2A-AS1 expression in cisplatin-resistant NB cells increased in a time- and dose-dependent manner. Knockdown of NUTM2A-AS1 significantly improved NB cell sensitivity to cisplatin and inhibited metastatic abilities. Additionally, we identified B7-H3, an immune checkpoint-related protein, as a NUTM2A-AS1-associated protein in NB cells. NUTM2A-AS1 was shown to inhibit the protein degradation of B7-H3. Moreover, NUTM2A-AS1 modulated immune evasion in cisplatin-resistant NB cells through B7-H3. Furthermore, NUTM2A-AS1 expression in cisplatin-resistant NB cells was transactivated by NR1D1. In summary, our results unveil the molecular or biological relationship within the NR1D1/NUTM2A-AS1/B7-H3 axis in NB cells under cisplatin treatment, providing an intriguing avenue for fundamental research into cisplatin-resistant NB.

A multi-omics approach for biomarker discovery in neuroblastoma: a network-based framework.

Neuroblastoma (NB) is one of the leading causes of cancer-associated death in children. MYCN amplification is a prominent genetic marker for NB, and its targeting to halt NB progression is difficult to achieve. Therefore, an in-depth understanding of the molecular interactome of NB is needed to improve treatment outcomes. Analysis of NB multi-omics unravels valuable insight into the interplay between MYCN transcriptional and miRNA post-transcriptional modulation. Moreover, it aids in the identification of various miRNAs that participate in NB development and progression. This study proposes an integrated computational framework with three levels of high-throughput NB data (mRNA-seq, miRNA-seq, and methylation array). Similarity Network Fusion (SNF) and ranked SNF methods were utilized to identify essential genes and miRNAs. The specified genes included both miRNA-target genes and transcription factors (TFs). The interactions between TFs and miRNAs and between miRNAs and their target genes were retrieved where a regulatory network was developed. Finally, an interaction network-based analysis was performed to identify candidate biomarkers. The candidate biomarkers were further analyzed for their potential use in prognosis and diagnosis. The candidate biomarkers included three TFs and seven miRNAs. Four biomarkers have been previously studied and tested in NB, while the remaining identified biomarkers have known roles in other types of cancer. Although the specific molecular role is yet to be addressed, most identified biomarkers possess evidence of involvement in NB tumorigenesis. Analyzing cellular interactome to identify potential biomarkers is a promising approach that can contribute to optimizing efficient therapeutic regimens to target NB vulnerabilities.

KAP1 stabilizes MYCN mRNA and promotes neuroblastoma tumorigenicity by protecting the RNA m6A reader YTHDC1 protein degradation.

BACKGROUND: Neuroblastoma (NB) patients with amplified MYCN often face a grim prognosis and are resistant to existing therapies, yet MYCN protein is considered undruggable. KAP1 (also named TRIM28) plays a crucial role in multiple biological activities. This study aimed to investigate the relationship between KAP1 and MYCN in NB. METHODS: Transcriptome analyses and luciferase reporter assay identified that KAP1 was a downstream target of MYCN. The effects of KAP1 on cancer cell proliferation and colony formation were explored using the loss-of-function assays in vitro and in vivo. RNA stability detection was used to examine the influence of KAP1 on MYCN expression. The mechanisms of KAP1 to maintain MYCN mRNA stabilization were mainly investigated by mass spectrum, immunoprecipitation, RIP-qPCR, and western blotting. In addition, a xenograft mouse model was used to reveal the antitumor effect of STM2457 on NB. RESULTS: Here we identified KAP1 as a critical regulator of MYCN mRNA stability by protecting the RNA N6-methyladenosine (m6A) reader YTHDC1 protein degradation. KAP1 was highly expressed in clinical MYCN-amplified NB and was upregulated by MYCN. Reciprocally, KAP1 knockdown reduced MYCN mRNA stability and inhibited MYCN-amplified NB progression. Mechanistically, KAP1 regulated the stability of MYCN mRNA in an m6A-dependent manner. KAP1 formed a complex with YTHDC1 and RNA m6A writer METTL3 to regulate m6A-modified MYCN mRNA stability. KAP1 depletion decreased YTHDC1 protein stability and promoted MYCN mRNA degradation. Inhibiting MYCN mRNA m6A modification synergized with chemotherapy to restrain tumor progression in MYCN-amplified NB. CONCLUSIONS: Our research demonstrates that KAP1, transcriptionally activated by MYCN, forms a complex with YTHDC1 and METTL3, which in turn maintain the stabilization of MYCN mRNA in an m6A-dependent manner. Targeting m6A modification by STM2457, a small-molecule inhibitor of METTL3, could downregulate MYCN expression and attenuate tumor proliferation. This finding provides a new alternative putative therapeutic strategy for MYCN-amplified NB.

A novel APP splice variant-dependent marker system to precisely demarcate maturity in SH-SY5Y cell-derived neurons.

SH-SY5Y, a neuroblastoma cell line, can be converted into mature neuronal phenotypes, characterized by the expression of mature neuronal and neurotransmitter markers. However, the mature phenotypes described across multiple studies appear inconsistent. As this cell line expresses common neuronal markers after a simple induction, there is a high chance of misinterpreting its maturity. Therefore, sole reliance on common neuronal markers is presumably inadequate. The Alzheimer's disease (AD) central gene, amyloid precursor protein (APP), has shown contrasting transcript variant dynamics in various cell types. We differentiated SH-SY5Y cells into mature neuron-like cells using a concise protocol and observed the upregulation of total APP throughout differentiation. However, APP transcript variant-1 was upregulated only during the early to middle stages of differentiation and declined in later stages. We identified the maturity state where this post-transcriptional shift occurs, terming it "true maturity." At this stage, we observed a predominant expression of mature neuronal and cholinergic markers, along with a distinct APP variant pattern. Our findings emphasize the necessity of using a differentiation state-sensitive marker system to precisely characterize SH-SY5Y differentiation. Moreover, this study offers an APP-guided, alternative neuronal marker system to enhance the accuracy of the conventional markers.

Beyond Clinical Trials: Understanding Neurotrophic Tropomyosin Receptor Kinase Inhibitor Challenges and Efficacy in Real-World Pediatric Oncology.

PURPOSE: Our study aimed to explore real-world treatment scenarios for children and adolescents with neurotrophic tropomyosin receptor kinase (NTRK)-fused tumors, emphasizing access, responses, side effects, and outcomes. PATIENTS AND METHODS: Pooled clinical data from 17 pediatric cases (11 soft-tissue sarcomas, five brain tumors, and one neuroblastoma) treated with larotrectinib and radiologic images for 14 patients were centrally reviewed. Testing for gene fusions was prompted by poor response to treatment, tumor progression, or aggressiveness. RESULTS: Six different NTRK fusion subtypes were detected, and various payment sources for testing and medication were reported. Radiologic review revealed objective tumor responses (OR) in 11 of 14 patients: Complete responses: two; partial responses: nine; and stable disease: three cases. Grades 1 or 2 Common Terminology Criteria for Adverse Events adverse effects were reported in five patients. Regarding the entire cohort's clinical information, 15 of 17 patients remain alive (median observation time: 25 months): four with no evidence of disease and 11 alive with disease (10 without progression). One patient developed resistance to the NTRK inhibitor and died from disease progression while another patient died due to an unrelated cause. CONCLUSION: This real-world study confirms favorable agnostic tumor OR rates to larotrectinib in children with NTRK-fused tumors. Better coordination to facilitate access to medication remains a challenge, particularly in middle-income countries like Brazil.

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

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

In vivo cisplatin-resistant neuroblastoma metastatic model reveals tumour necrosis factor receptor superfamily member 4 (TNFRSF4) as an independent prognostic factor of survival in neuroblastoma.

Neuroblastoma is the most common solid extracranial tumour in children. Despite major advances in available therapies, children with drug-resistant and/or recurrent neuroblastoma have a dismal outlook with 5-year survival rates of less than 20%. Therefore, tackling relapsed tumour biology by developing and characterising clinically relevant models is a priority in finding targetable vulnerability in neuroblastoma. Using matched cisplatin-sensitive KellyLuc and resistant KellyCis83Luc cell lines, we developed a cisplatin-resistant metastatic MYCN-amplified neuroblastoma model. The average number of metastases per mouse was significantly higher in the KellyCis83Luc group than in the KellyLuc group. The vast majority of sites were confirmed as having lymph node metastasis. Their stiffness characteristics of lymph node metastasis values were within the range reported for the patient samples. Targeted transcriptomic profiling of immuno-oncology genes identified tumour necrosis factor receptor superfamily member 4 (TNFRSF4) as a significantly dysregulated MYCN-independent gene. Importantly, differential TNFRSF4 expression was identified in tumour cells rather than lymphocytes. Low TNFRSF4 expression correlated with poor prognostic indicators in neuroblastoma, such as age at diagnosis, stage, and risk stratification and significantly associated with reduced probability of both event-free and overall survival in neuroblastoma. Therefore, TNFRSF4 Low expression is an independent prognostic factor of survival in neuroblastoma.

Small-molecule inhibition of the METTL3/METTL14 complex suppresses neuroblastoma tumor growth and promotes differentiation.

The N6-methyladenosine (m6A) RNA modification is an important regulator of gene expression. m6A is deposited by a methyltransferase complex that includes methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14). High levels of METTL3/METTL14 drive the growth of many types of adult cancer, and METTL3/METTL14 inhibitors are emerging as new anticancer agents. However, little is known about the m6A epitranscriptome or the role of the METTL3/METTL14 complex in neuroblastoma, a common pediatric cancer. Here, we show that METTL3 knockdown or pharmacologic inhibition with the small molecule STM2457 leads to reduced neuroblastoma cell proliferation and increased differentiation. These changes in neuroblastoma phenotype are associated with decreased m6A deposition on transcripts involved in nervous system development and neuronal differentiation, with increased stability of target mRNAs. In preclinical studies, STM2457 treatment suppresses the growth of neuroblastoma tumors in vivo. Together, these results support the potential of METTL3/METTL14 complex inhibition as a therapeutic strategy against neuroblastoma.

Differential RNA Expression Between Metastatic and Primary Neuroblastoma Cells.

INTRODUCTION: Neuroblastoma (NB) is the most common extra-cranial malignancy in children. Poor survival in high-risk NB is attributed to recurrent metastatic disease. To better study metastatic disease, we used a novel mouse model to investigate differential gene expression between primary tumor cells and metastatic cells. We hypothesized that metastatic NB cells have a different gene expression profile from primary tumor cells and cultured cells. METHODS: Using three human NB cell lines (NGP, CHLA255, and SH-SY5Y), orthotopic xenografts were established in immunodeficient nod/scid gamma mice via subcapsular renal injection. Mice were sacrificed and NB cells were isolated from the primary tumor and from sites of metastasis (bone marrow, liver). RNA sequencing, gene set analysis, and pathway analysis were performed to identify differentially expressed genes and molecular pathways in the metastatic cells compared to primary tumor cells. RESULTS: There were 266 differentially expressed genes in metastatic tumor cells (bone marrow and liver combined) compared to primary tumor cells. The top upregulated gene was KCNK1 and the top downregulated genes were PDE7B and NEBL. Top upregulated pathways in the metastatic cells were involved in ion transport, cell signaling, and cell proliferation. Top downregulated pathways were involved in DNA synthesis, transcription, and cellular metabolism. CONCLUSIONS: In metastatic NB cells, our study identified the upregulation of biologic processes involved in cell cycle regulation, cell proliferation, migration, and invasion. Ongoing studies aim to validate downstream translation of these genomic alterations, as well as target these pathways to more effectively suppress and inhibit recurrent metastatic disease in NB.

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

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

Generation of Orthotopic and Subcutaneous Patient-Derived Xenograft Models from Diverse Clinical Tissue Samples of Pediatric Extracranial Solid Tumors.

Realistic and renewable laboratory models that accurately reflect the distinct clinical features of childhood cancers have enormous potential to speed research progress. These models help us to understand disease biology, develop new research methods, advance new therapies to clinical trial, and implement personalized medicine. This chapter describes methods to generate patient-derived xenograft models of neuroblastoma and rhabdomyosarcoma, two tumor types for which children with high-risk disease have abysmal survival outcomes and survivors have lifelong-debilitating effects from treatment. Further, this protocol addresses model development from diverse clinical tumor tissue samples, subcutaneous and orthotopic engraftment, and approaches to avoid model loss.

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

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

Review of Patient Gene Profiles Obtained through a Non-Negative Matrix Factorization-Based Framework to Determine the Role Inflammation Plays in Neuroblastoma Pathogenesis.

Neuroblastoma is the most common extracranial solid tumor in children. It is a highly heterogeneous tumor consisting of different subcellular types and genetic abnormalities. Literature data confirm the biological and clinical complexity of this cancer, which requires a wider availability of gene targets for the implementation of personalized therapy. This paper presents a study of neuroblastoma samples from primary tumors of untreated patients. The focus of this analysis is to evaluate the impact that the inflammatory process may have on the pathogenesis of neuroblastoma. Eighty-eight gene profiles were selected and analyzed using a non-negative matrix factorization framework to extract a subset of genes relevant to the identification of an inflammatory phenotype, whose targets (PIK3CG, NFATC2, PIK3R2, VAV1, RAC2, COL6A2, COL6A3, COL12A1, COL14A1, ITGAL, ITGB7, FOS, PTGS2, PTPRC, ITPR3) allow further investigation. Based on the genetic signals automatically derived from the data used, neuroblastoma could be classified according to stage rather than as a "cold" or "poorly immunogenic" tumor.

Cell communication pathway prognostic model identified detrimental neurodevelopmental pathways in neuroblastoma.

Neurodevelopmental cell communication plays a crucial role in neuroblastoma prognosis. However, determining the impact of these communication pathways on prognosis is challenging due to limited sample sizes and patchy clinical survival information of single cell RNA-seq data. To address this, we have developed the cell communication pathway prognostic model (CCPPM) in this study. CCPPM involves the identification of communication pathways through single-cell RNA-seq data, screening of prognosis-significant pathways using bulk RNA-seq data, conducting functional and attribute analysis of these pathways, and analyzing the post-effects of communication within these pathways. By employing the CCPPM, we have identified ten communication pathways significantly influencing neuroblastoma, all related to axongenesis and neural projection development, especially the BMP7-(BMPR1B-ACVR2B) communication pathway was found to promote tumor cell migration by activating the transcription factor SMAD1 and regulating UNK and MYCBP2. Notably, BMP7 expression was higher in neuroblastoma samples with distant metastases. In summary, CCPPM offers a novel approach to studying the influence of cell communication pathways on disease prognosis and identified detrimental communication pathways related to neurodevelopment.