Introduction

Neuroblastoma (NB) is the most frequent extracranial solid tumor in children, accounting for 15% of all childhood cancer deaths. Although the 5-year survival rate of patients with high-risk disease has increased in recent decades, NB remains a challenge in pediatric oncology, and identifying novel therapeutic targets and agents is an urgent clinical need. N6-methyladenosine (m6A), the most abundant post-transcriptional mRNA modification in mammals, is tightly regulated by 'writers' and 'erasers', which deposit and remove the modification, respectively, and by 'readers', which can detect changes in mRNA modification status and influence downstream cellular processes. m6A and related proteins have been found frequently altered in different tumors and may represent a good therapeutic target also in NB.

Materials and methods

By modulating the expression of the writer complex component METTL14 or the demethylase ALKBH5, we evaluated the impact of m6A mRNA modification on NB tumor aggressiveness in vitro and in vivo. We also investigated the effect of inhibiting m6A recognition by knocking out YTHDF reader proteins. Next, to identify a molecule capable of interfering with YTHDF m6A mRNA recognition, we screened in silico by molecular docking a library of 1.5 million compounds against the YTHDF1 protein and subsequently evaluated the 113 best-scoring compounds by homogeneous time-resolved fluorescence. We selected compound A as a low micromolar pan-YTHDF inhibitor and evaluated its effects on NB cells also by mass spectrometry analysis. Finally, we synthesized and tested more than 30 analogs in a structure-activity relationship (SAR) study.

Results and discussion

METTL14 overexpression promotes cell proliferation and invasion in vitro and tumor progression in vivo, leading to faster tumor growth and larger tumor masses. Conversely, ALKBH5 overexpression or METTL14 knockout leads to opposite results, with a significant decrease in cell proliferation, an increase in apoptosis, and reduced invasion ability in vitro, while dramatically slowing tumor growth in vivo. Consistent with these observations, knockout of YTHDF paralogs or YTHDF inhibition with compound A also results in decreased NB cell proliferation and reduced clonogenic potential. We are now finalizing our SAR study, also guided by docking analyses. 

Conclusion

Our results show that high levels of m6A correlate with higher NB aggressiveness and that targeting m6A deposition or m6A recognition on mRNAs could be an effective therapeutic strategy for NB treatment.

Laboratory of Translational Genomics