Background. MYCN activation is a hallmark of advanced tumor stage in neuroblastoma (NB), characterizing high-risk patients prone to resistant disease. MYCN is also a potent regulator of metabolic reprogramming that favors NB adaptation to its microenvironment. The retinoic acid receptor-related orphan receptor α (RORα) is a key regulator of cell metabolism, immunity, as well as the circadian rhythm. Importantly, RORα activates the transcriptio of BMAL1, a master circadian transcription factor frequently deregulated in human cancers. Moreover, synthetic ligands RORα agonists (i.e. SR1078) have been recently optimized with great therapeutic potential.
Methods. Multivariate logistic regression analysis identified low levels of RORα as independent predictors of EFS and OS survival in large NB patients’ cohorts (n=890 patients). MYCN inducible over-expression and knock-down NB lines were generated and Q-PCR assays used to assess MYCN-mediated disruption of central components of the clock machinery. Growth-suppressive and pro-apoptotic effects of genetic RORα over-expression and RORα activation (via SR1078) were tested in a panel of MYCN-amplified and non-amplified lines, as well as in MYCN-inducible MYCN-3 cells (Tet-ON). MYCN-amplified xenografts were used to test in vivo therapeutic response to SR1078.
Results. Ectopic MYCN expression upregulates the circadian repressor REV-ERBα and suppresses the circadian activator RORα and the central clock BMAL1. In contrast, MYCN silencing effectively restores their expression levels. Interestingly, the expression of the main regulators of the clock machinery (RORα, REV-ERBα, and BMAL1) are also profoundly altered in MYCN-amplified NB patient samples. Specifically, RORα and BMAL1 are uniformly repressed in MYCN-amplified NB and their reduced levels independently correlate with poor survival in large patients’ cohorts (n=890 patients total, p<0.0001). Importantly, re-activation of RORα (via SR1078) strongly suppresses MYCN transcription and restores BMAL1 levels, inhibiting cell survival. Moreover, SR1078 reduces cell viability and induces apoptotic cell death to a higher extent in MYCN-amplified compared to MYCN-non amplified lines. Lastly, both SR1078 treatment and inducible RORα over-expression significantly (p=0.020) inhibit tumor growth in MYCN NB xenografts. Together, our data suggest that RORα loss promotes MYCN-induced disruption of molecular clock, contributing to NB tumorigenesis. Restoration of RORα opposes these MYCN functions, representing an effective strategy for MYCN-amplified NB.
Citation Format: Myrthala Moreno-Smith, Ling Tao, Ronald Bernardi, Kathleen De Preter, Mario Capasso, Sanjeev Vasudevan, Jason M. Shohet, Eveline Barbieri. RORα activation opposes MYCN signaling and restores the circadian clock in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1951. doi:10.1158/1538-7445.AM2017-1951