title: Retinoic acid and TGF-β signalling cooperate to overcome MYCN-induced retinoid resistance.
abstract: Retinoid therapy is widely employed in clinical oncology to differentiate malignant cells into their more benign counterparts. However, certain high-risk cohorts, such as patients with MYCN-amplified neuroblastoma, are innately resistant to retinoid therapy. Therefore, we employed a precision medicine approach to globally profile the retinoid signalling response and to determine how an excess of cellular MYCN antagonises these signalling events to prevent differentiation and confer resistance.
  
  
  
  We applied RNA sequencing (RNA-seq) and interaction proteomics coupled with network-based systems level analysis to identify targetable vulnerabilities of MYCN-mediated retinoid resistance. We altered MYCN expression levels in a MYCN-inducible neuroblastoma cell line to facilitate or block retinoic acid (RA)-mediated neuronal differentiation. The relevance of differentially expressed genes and transcriptional regulators for neuroblastoma outcome were then confirmed using existing patient microarray datasets.
  
  
  
  We determined the signalling networks through which RA mediates neuroblastoma differentiation and the inhibitory perturbations to these networks upon MYCN overexpression. We revealed opposing regulation of RA and MYCN on a number of differentiation-relevant genes, including LMO4, CYP26A1, ASCL1, RET, FZD7 and DKK1. Furthermore, we revealed a broad network of transcriptional regulators involved in regulating retinoid responsiveness, such as Neurotrophin, PI3K, Wnt and MAPK, and epigenetic signalling. Of these regulators, we functionally confirmed that MYCN-driven inhibition of transforming growth factor beta (TGF-β) signalling is a vulnerable node of the MYCN network and that multiple levels of cross-talk exist between MYCN and TGF-β. Co-targeting of the retinoic acid and TGF-β pathways, through RA and kartogenin (KGN; a TGF-β signalling activating small molecule) combination treatment, induced the loss of viability of MYCN-amplified retinoid-resistant neuroblastoma cells.
  
  
  
  Our approach provides a powerful precision oncology tool for identifying the driving signalling networks for malignancies not primarily driven by somatic mutations, such as paediatric cancers. By applying global omics approaches to the signalling networks regulating neuroblastoma differentiation and stemness, we have determined the pathways involved in the MYCN-mediated retinoid resistance, with TGF-β signalling being a key regulator. These findings revealed a number of combination treatments likely to improve clinical response to retinoid therapy, including co-treatment with retinoids and KGN, which may prove valuable in the treatment of high-risk MYCN-amplified neuroblastoma.
authors.affiliation: Systems Biology Ireland, University College Dublin, Belfield, Dublin 4, Ireland. duffy@whitney.ufl.edu.|Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland. duffy@whitney.ufl.edu.|School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland. duffy@whitney.ufl.edu.|The Whitney Laboratory for Marine Bioscience and Sea Turtle Hospital, University of Florida, St. Augustine, Florida, 32080, USA. duffy@whitney.ufl.edu.
authors.forename: David J
authors.initials: DJ
authors.lastname: Duffy
authors.affiliation: Systems Biology Ireland, University College Dublin, Belfield, Dublin 4, Ireland.|Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.|School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Krstic
authors.affiliation: Systems Biology Ireland, University College Dublin, Belfield, Dublin 4, Ireland.|Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.|School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Melinda
authors.initials: M
authors.lastname: Halasz
authors.affiliation: Systems Biology Ireland, University College Dublin, Belfield, Dublin 4, Ireland.|European Molecular Biology Laboratory (EMBL), Meyerhofstraße 1, 69117, Heidelberg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schwarzl
authors.affiliation: Systems Biology Ireland, University College Dublin, Belfield, Dublin 4, Ireland.|Present address: Department of Biology, University of Konstanz, Konstanz, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Konietzny
authors.affiliation: VTT Technical Research Centre of Finland, Tietotie 2, FI-02044 VTT, Espoo, Finland.
authors.forename: Kristiina
authors.initials: K
authors.lastname: Iljin
authors.affiliation: Systems Biology Ireland, University College Dublin, Belfield, Dublin 4, Ireland.|Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.|School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Desmond G
authors.initials: DG
authors.lastname: Higgins
authors.affiliation: Systems Biology Ireland, University College Dublin, Belfield, Dublin 4, Ireland.|Conway Institute of Biomolecular & Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.|School of Medicine, University College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Walter
authors.initials: W
authors.lastname: Kolch
mesh_terms: D000813:Anilides / Q000627:therapeutic use*; D000970:Antineoplastic Agents / Q000627:therapeutic use; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D000071447:N-Myc Proto-Oncogene Protein / Q000187:drug effects*; D009447:Neuroblastoma / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism; D010795:Phthalic Acids / Q000627:therapeutic use*; D057285:Precision Medicine; D012176:Retinoids / Q000627:therapeutic use; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000187:drug effects*; D014212:Tretinoin / Q000627:therapeutic use*
chemical_list: D000813:Anilides; D000970:Antineoplastic Agents; C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D010795:Phthalic Acids; D012176:Retinoids; D016212:Transforming Growth Factor beta; D014212:Tretinoin; C572342:kartogenin
keywords: Cancer; ChIP sequencing (ChIP-seq); Differentiation; Differentiation therapy; Genome medicine; Interaction proteomics; Kartogenin (KGN); MYC (c-MYC); Neuroblastoma; Neuronal differentiation; Precision medicine; RepSox; Systems medicine; Transforming growth factor beta (TGF-β) signalling; Wnt β-catenin signalling; mRNA sequencing (mRNA-seq)