Retinoic acid and TGF-β signalling cooperate to overcome MYCN-induced retinoid resistance

David J. Duffy, Aleksandar Krstic, Melinda Halasz, Thomas Schwarzl, Anja Konietzny, Kristiina Iljin, Desmond G. Higgins, Walter Kolch

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.