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 transcription (via recruitment of transcriptional co-activators) of BMAL1, a master circadian transcription factor frequently deregulated in human cancers.
Material and 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). MYCN inducible overexpression and knock down lines were generated and Q-PCR assays used to assess MYCN-mediated disruption of molecular clock genes’ expression. Growth-suppressive and pro-apoptotic effects of the RORα agonist SR1078 were tested in a panel of MYCN-amplified and non-amplified lines, as well as in MYCN inducible MYCN-3 cells (Tet-ON), using MTT and caspase 3/7 activation. MYCN-amplified xenografts were used to test in vivo therapeutic response to SR1078.
Results and Conclusions: The circadian clock is profoundly disrupted in MYCN-amplified NB lines and tumors. Specifically, RORα and BMAL1 expression are uniformly repressed in MYCN-amplified NB lines and tumors (p<0.0001) and their reduced levels strongly correlate with poor survival (890 patients, p<0.0001). Moreover, RORα re-activation via SR1078 reduces cell viability and induces apoptotic cell death to a higher extent in MYCN-amplified compared to MYCN-non amplified lines. Importantly, RORα re-activation strongly suppresses MYCN transcription and induces BMAL1 expression in MYCN-amplified cells and tumors, inhibiting cell survival. Lastly, tumor weights of xenografts treated with SR1078 were significantly (p=0.020) smaller than control. Together, our data suggest that: 1) reactivating RORα may represent an effective way to inhibit MYCN-mediated metabolic functions, and 2) RORα agonists can be developed as effective therapy for MYCN-amplified NB.