Retinoic acid rewires the adrenergic core regulatory circuitry of childhood neuroblastoma

Abstract

Neuroblastoma cell identity depends on a core regulatory circuit (CRC) of transcription factors that collaborate with MYCN to drive the oncogenic gene expression program. For neuroblastomas dependent on the adrenergic CRC, treatment with retinoids can inhibit cell growth and induce differentiation in both primary neuroblastomas and cell lines; however, the underlying mechanisms remain unclear. Here we show that when MYCN-amplified neuroblastomas cells are treated with all-trans retinoic acid (ATRA), histone H3K27 acetylation and methylation become redistributed to decommission super-enhancers driving the expression of PHOX2B and GATA3, together with the activation of new super-enhancers that drive high levels of MEIS1, HIC1 and SOX4 expression. These findings indicate that treatment with ATRA can reprogram the enhancer landscape to collapse the adrenergic CRC, resulting in downregulation of MYCN expression, while upregulating a new retino-sympathetic CRC that causes proliferative arrest and sympathetic differentiation. Thus, we provide mechanisms that account for the beneficial effects of retinoids against high-risk neuroblastoma and explain the rapid downregulation of expression of MYCN despite massive levels of gene amplification.

Super-enhancer associated core regulatory circuits mediate susceptibility to retinoic acid in neuroblastoma cells

“…This suggest that activation of retinoic acid signaling is not stably maintained in both the responsive cell lines, although even in SH-SY5Y retinoic acid signaling pathway genes remain positively enriched compared to control conditions (Figure 2D withdrawn vs. control) after 96 h of withdrawal, suggesting some level of maintenance or a slow decrease in retinoic acid signaling. This further indicates that BE2C could also revert to the control state after a longer duration, consistent with what was shown recently (Zimmerman et al, 2021). Notably, the normalized enrichment score measuring retinoic acid signaling pathway gene set downregulation is greater in SH-SY5Y than BE2C, reflecting the observation that SH-SY5Y cells retract ATRA-induced neuronal projections and revert, whereas BE2C maintain their differentiated morphology.…”

“…Then, to identify CRC components that define ATRA responsiveness, we identified the regulators in both BE2C and SH-SY5Y cells (Figure 6A). The resulting ATRA responsive CRC contained previously reported ADRN CRC components, including PHOX2B, TBX2, and KLF7 (Boeva et al, 2017;Durbin et al, 2018;Zimmerman et al, 2021), as well as novel candidates CEBPG, KLF13, TWIST1, and FEV, which were identified in the responsive cell lines but not in the ATRA-resistant SH-EP cell line (Figure 6A, Supplementary Figure S6B). According to Boeva et al (2017) andvan Groningen et al (2017), KLF7 is a member of the ADRN CRC.…”

“…Our analyses identified several transcription factors as superenhancer-associated targets, which appear to be activated by ATRA treatment in responsive cell lines, including HIC1, SMAD3, RARB, and HIF1A (Figure 3D). We also saw the suppression of established ADRN core transcription regulatory circuitry (CRC) components PHOX2B, and MYCN in BE2C cells, which stabilize the ADRN CRC in MYCN-amplified cell lines (Supplementary Figure S6A) (Durbin et al, 2018;Zeid et al, 2018;Zimmerman et al, 2021). This prompted us to investigate the effect of ATRA treatment on the CRC of responsive neuroblastoma cell lines.…”

“…In neuroblastoma, two phenotypically distinct subtypes of neuroblastoma cells-adrenergic (ADRN) and mesenchymal (MES), have been identified based on the super-enhancer landscape and associated core transcriptional regulatory circuitries (CRC) (Boeva et al, 2017;van Groningen et al, 2017). It was recently demonstrated that in MYCN-amplified neuroblastoma cell lines, retinoic acid induces reprogramming of the ADRN CRC, leading to massive downregulation of MYCN expression, tumour suppression, and cell differentiation (Zimmerman et al, 2021).…”

Molecular Mechanisms of MYCN Dysregulation in Cancers

“…Importantly, transcriptional and posttranscriptional regulation determines the final level of N-MYC protein in both MYCN amplified and non-amplified tumors. For instance, enhancer hijacking that repositions a super enhancer close to the affected genes through chromosomal translocation accounts for the high level of C-MYC or N-MYC expression in some neuroblastoma cells without MYCC amplification or without a high MYCN copy number, respectively ( 68 , 69 ).…”