Noncoding regulatory mutations as a driving event for the oncogenic core regulatory circuitries of neuroblastoma

Introduction

Neuroblastoma (NB) is a pediatric tumor composed of adrenergic (ADRN) and mesenchymal-like (MES) cells which derive from the dysregulation of normal cell differentiation imposed by NB Core Regulatory Circuitries (CRCs). We hypothesize that somatic single-nucleotide variants (SNVs) in active CRCs transcription factor binding sites (aTFBSs) may underlie such perturbation, promoting NB tumorigenesis. We aim to investigate such patterns of regulatory elements and identify putative driver SNVs, exploring their role in NB.

Methods

MES and ADRN aTFBSs were identified by integrating 42 ChIP-seq and 12 ATAC-seq experiments in 7 ADRN and 2 MES NB cell lines. Using the Fisher test, we tested these regions for an enrichment of somatic SNVs obtained from the WGS data of 397 NB patients. SNVs were selected based on their impact on CRC TF binding through the FABIAN-variant tool. Next, aTFBS target genes were identified by analyzing the promoter capture HiC (CHiC) in 2 ADRN and 2 MES NB cell lines and their expression values were correlated with clinical and survival data of a second cohort of 498 NBs.

Results

We found six aTFBS sets of significantly enriched TFs (FDR ≤ 0.1) in SNVs, i.e., 5 ADRN (GATA3, HAND2, ISL1, MYCN, and TBX2) and 1 MES (FOSL2), with 689 mutations impacting the binding of CRC TFs (Fabian ≠ 0). Mutated aTFBSs were found to interact with genes of neuronal differentiation pathways and proliferated MES cells , suggesting the potential impact of SNVs on NB cell identities. By focusing on genes of developmental and differentiation processes interacting with aTFBSs carrying SNVs with the highest (cut-off ≥ 0.1) or lowest (cut-off ≤ -0.1) Fabian score, we found targets (ROBO2, CACNB1, PIK3R1, MDGA1, HES6, LDLRAD4, DGUOK, IRX1, and SPOCK2) whose expression significantly correlated with worse NB outcomes (FDR ≤ 0.05).

Conclusions

These results demonstrated that somatic noncoding SNVs may act synergistically to affect NB CRCs and thus contribute to tumorigenesis.