RNA-binding proteins such as LIN28B and IGF2BP1 which reduce miRNA attack of oncogenic-linked transcripts were previously reported as oncogenic themselves in animal models of neuroblast-derived cancers. This suggests that post-transcriptional regulation intervention represents a novel strategy against neuroblastoma.
We previously reported that FDA-approved copper chelation therapy has in vivo efficacy against neuroblastoma. RNA-seq and Mass spectrometry analyses in neuronal cancer cells lines suggested that several methylated RNA (m6A)-binding proteins were deregulated by copper chelator treatment. Indeed, copper chelation treated neuroblastoma cells lines showed a decrease in YTHDF2 protein expression. YTHDF2 knock out (DepMAP Chronos dependency score, 0.32) and knock-down studies (siRNA pool, 38% decrease in cell confluence) in neuroblastoma cell lines, indicated that YTHDF2 depletion is selective against neuroblastoma cell growth. Despite YTHDF2 being essential in glioblastoma cancer stem cell population maintenance and tumorigenesis, no specific inhibitor is reported. Thus, to elucidate tool YTHDF2 inhibitors we utilized bioinformatic docking of small molecules against the YTHDF2 RNA-binding domains. Two molecules yielded highly significant virtual binding. In vitro studies, showed both compounds enhanced copper chelation clonogenicity effects in neuroblastoma cells. This suggested that YTHDF2 function has molecular redundancies with copper chelation. YTHDF2 RNA-binding inhibitors downregulated MYCN protein expression. High throughput PAR-CLIP pull downs of YTHDF2 and interacting RNAs demonstrated MYCN binding at three sequence locations (PARalyzer Peak calling, POSTAR3 online tool), confirmatory studies demonstrated YTHDF2 protein-MYCN mRNA binding.
Ongoing work will involve unbiased RNA-sequencing strategies to better define the interplay between YTHDF2 and copper signalling in neuroblastoma. YTHDF2 is yet to be pharmaceutically targeted or studied in the neuroblastoma context. Our work therefore represents a significant breakthrough in drugging oncogenic post-transcriptional processes either indirectly through depleting copper, or with our small molecule inhibitors.