Neuroblastoma is a tumor of the peripheral sympathetic nervous system, derived from multipotent neural crest cells (NCCs). To define core regulatory circuitries (CRCs) controlling the gene expression program of neuroblastoma, we established and analyzed the neuroblastoma super-enhancer landscape. We discovered three types of identity in neuroblastoma cell lines: a sympathetic noradrenergic identity, defined by a CRC module including the PHOX2B, HAND2 and GATA3 transcription factors (TFs); an NCC-like identity, driven by a CRC module containing AP-1 TFs; and a mixed type, further deconvoluted at the single-cell level. Treatment of the mixed type with chemotherapeutic agents resulted in enrichment of NCC-like cells. The noradrenergic module was validated by ChIP-seq. Functional studies demonstrated dependency of neuroblastoma with noradrenergic identity on PHOX2B, evocative of lineage addiction. Most neuroblastoma primary tumors express TFs from the noradrenergic and NCC-like modules. Our data demonstrate a previously unknown aspect of tumor heterogeneity relevant for neuroblastoma treatment strategies.
Two cell identities, noradrenergic and mesenchymal, have been characterized in neuroblastoma cell lines according to their epigenetic landscapes relying on specific circuitries of transcription factors. Yet, their relationship and relative contribution in patient tumors remain poorly defined. Here, we demonstrate that the knock-out of GATA3, but not of PHOX2A or PHOX2B, in noradrenergic cells induces a mesenchymal phenotype. Our results document spontaneous plasticity in several models between both identities and show that plasticity relies on epigenetic reprogramming. We demonstrate that an in vivo microenvironment provides a powerful pressure towards a noradrenergic identity for these models. Consistently, tumor cells with a mesenchymal identity are not detected in a series of PDX models. Further study of the intra-tumor noradrenergic heterogeneity reveals two distinct cell populations exhibiting features of chromaffin-like or sympathoblast-like cells. This work emphasizes that both external cues of the environment and intrinsic factors control plasticity and cell identity in neuroblastoma.
Long intergenic non-coding RNAs (lincRNAs) are emerging as integral components of signaling pathways in various cancer types. In neuroblastoma, only a handful of lincRNAs are known as upstream regulators or downstream effectors of oncogenes. Here, we exploit RNA sequencing data of primary neuroblastoma tumors, neuroblast precursor cells, neuroblastoma cell lines and various cellular perturbation model systems to define the neuroblastoma lincRNome and map lincRNAs up- and downstream of neuroblastoma driver genes MYCN , ALK and PHOX2B . Each of these driver genes controls the expression of a particular subset of lincRNAs, several of which are associated with poor survival and are differentially expressed in neuroblastoma tumors compared to neuroblasts. By integrating RNA sequencing data from both primary tumor tissue and cancer cell lines, we demonstrate that several of these lincRNAs are expressed in stromal cells. Deconvolution of primary tumor gene expression data revealed a strong association between stromal cell composition and driver gene status, resulting in differential expression of these lincRNAs. We also explored lincRNAs that putatively act upstream of neuroblastoma driver genes, either as presumed modulators of driver gene activity, or as modulators of effectors regulating driver gene expression. This analysis revealed strong associations between the neuroblastoma lincRNAs MIAT and MEG3 and MYCN and PHOX2B activity or expression. Together, our results provide a comprehensive catalogue of the neuroblastoma lincRNome, highlighting lincRNAs up- and downstream of key neuroblastoma driver genes. This catalogue forms a solid basis for further functional validation of candidate neuroblastoma lincRNAs.
Noradrenergic and mesenchymal identities have been characterized in neuroblastoma cell lines according to their epigenetic landscapes and core regulatory circuitries. However, their relationship and relative contribution in patient tumors remain poorly defined. We now document spontaneous and reversible plasticity between the two identities, associated with epigenetic reprogramming, in several neuroblastoma models. Interestingly, xenografts with cells from each identity eventually harbor a noradrenergic phenotype suggesting that the microenvironment provides a powerful pressure towards this phenotype. Accordingly, such a noradrenergic cell identity is systematically observed in single-cell RNA-seq of 18 tumor biopsies and 15 PDX models. Yet, a subpopulation of these noradrenergic tumor cells presents with mesenchymal features that are shared with plasticity models, indicating that the plasticity described in these models has relevance in neuroblastoma patients. This work therefore emphasizes that intrinsic plasticity properties of neuroblastoma cells are dependent upon external cues of the environment to drive cell identity.
In contrast to adult cancers, most pediatric malignancies display limited genetic heterogeneity. In Ewing sarcoma, the major driver of tumor development is the expression of the chimeric EWSR1-FLI1 transcription factor, which results from a chromosome translocation. Additional recurrent genetic alterations include the CDKN2A, STAG2, and TP53 genes. Through its binding to specific elements including GGAA microsatellite sequences, EWSR1-FLI1 has a major impact on epigenetic landscape of the cancer cells and consequently, obscures the epigenetic makeup of the Ewing cell-of-origin. The Ewing cell is somehow a “neomorphic” cell, the biologic properties of which are tightly dependent upon the expression level of EWSR1-FLI1. When a cell harbors a low level of expression of EWSR1-FLI1, it loses this neomorphic identity and retrieve its cell-of-origin, mesenchymal phenotype. This leads us to propose a new model of phenotypic plasticity whereby the dynamic fluctuation of the expression level of a dominant oncogene is an intrinsic characteristic of its oncogenic potential. The causes of EWSR1-FLI1 expression fluctuations are presently unknown. They may be partly stochastic as they are observed in cell lines in vitro but also dependent upon signals from the surrounding microenvironment. The situation is very different in neuroblastoma where the blueprint of the cell-of-origin, most frequently a noradrenergic progenitor, can be clearly detected whatever the genetic abnormalities of the cancer cells. Our recent data nevertheless suggest that neuroblastoma cells may also exhibit some plasticity and transition between different epigenetic states that witness different cell identities. In conclusion, pediatric cancers constitute simplified cancer systems to investigate the relative and interdependent contributions of genetic and nongenetic factors and to document identity shifts. Citation Format: Olivier Delattre, G.A. Franzetti, K. Laud-Duval, A. Brisac, Valentina Boeva, Caroline Louis-Brennetot, Agathe Peltier, Simon Durand, Cécile Pierre-Eugène, Didier Surdez, Eve Lapouble, Gaelle Pierron, Sandrine Grossetête-Lalami, Isabelle Janoueix-Lerosey. Identity shifts in pediatric cancers [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr IA02.
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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, Cécile Pierre-Eugène, Cécile Thirant, Simon Durand, Divya Sahu, Sandrine Grossetête-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.
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