Role of noradrenergic core regulatory circuitry transcription factors in neuroblastoma cell identity

Introduction: The heterogeneity of neuroblastoma cell identity has been recently revealed with the characterization of noradrenergic and NCC (neural crest cell)-like/mesenchymal cells, each cell type being governed by a core regulatory circuitry (CRC) of specific transcription factors (Boeva et al., 2017; van Groningen et al., 2017). Whereas the noradrenergic CRC includes the PHOX2B, HAND2, and GATA3 transcription factors, the CRC associated to the NCC-like/mesenchymal identity is composed of AP-1 transcription factors among others. The present study aims at deciphering the role of the identity-related transcription factors, PHOX2B and GATA3, in the establishment and maintenance of the noradrenergic identity. Experimental Procedures: Using the CRISPR-Cas9 gene editing approach, we performed the knockout of PHOX2B and GATA3 genes in the SH-SY5Y neuroblastoma cell line. The obtained clones were characterized in terms of growth, phenotype, and identity, using in particular transcriptomic (RNA-seq) and epigenomic (H3K27ac ChIP-seq) analysis. Results:PHOX2B knockout in the SH-SY5Y cell line did not change its identity. PHOX2B−/− cells keep their noradrenergic phenotype, demonstrating that PHOX2B expression is not essential for the maintenance of the noradrenergic CRC. Nevertheless, the absence of PHOX2B expression in these cells decreased their proliferation both in vitro and in vivo, after mouse xenografts. In contrast to PHOX2B knockout cells, GATA3 knockout cells exhibit a mesenchymal phenotype: GATA3−/− cells harbor actin stress fibers and display migration and invasion capacities. Compared to the parental cells, they exhibit a higher resistance to chemotherapy in vitro. In a hierarchical unsupervised clustering, GATA3−/− cells show a transcriptomic profile close to neuroblastoma mesenchymal cell lines, with a weak expression of the noradrenergic set of transcription factors, including PHOX2B, and a high expression of factors related to the mesenchymal identity. Consistently with their transcriptomic profile, GATA3−/− cells are characterized by a super-enhancer landscape close to that of the SH-EP mesenchymal cell line. Finally, GATA3 knockout cells exhibit a decreased proliferation both in vitro and in vivo in xenograft experiments. Further experiments are ongoing to explore cell identity in the obtained tumors. Conclusions: PHOX2B and GATA3, which are two members of the noradrenergic CRC, play different roles in the maintenance of the noradrenergic identity in neuroblastoma cells. We hypothesize that PHOX2A could compensate for the absence of PHOX2B, thus explaining why PHOX2B knockout cells maintain their noradrenergic identity. In contrast, GATA3 knockout cells exhibit a mesenchymal identity. Bioinformatics analyses are ongoing to decipher the reprogramming process of these cells. Citation Format: Agathe Peltier, Caroline Louis-Brennetot, Cecile Pierre-Eugene, Cecile Thirant, Simon Durand, Divya Sahu, Sandrine Grossetete-Lalami, Virginie Raynal, Sylvain Baulande, Olivier Delattre, Valentina Boeva, Isabelle Janoueix-Lerosey. Role of noradrenergic core regulatory circuitry transcription factors in neuroblastoma cell identity [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B78.