Nitric Oxide is an Epitranscriptomic Regulator of Gene Expression by Inhibiting m6A mRNA Demethylation.

Elevated nitric oxide (NO) is a well-established negative prognostic indicator across multiple cancer types. The aim of this project was to elucidate novel signaling mechanisms by which NO drives aggressive cancer development. N6-methyladenosine (m6A) is the most abundant and conserved internal transcriptional modification in mRNA. m6A plays an essential role in biological function; however, disruption of m6A levels promote aberrant cell behaviors, especially tumorigenesis. Therefore, we sought to expand our understanding of NO as an epigenetic regulator via mediating m6A mRNA methylation and ultimately leading to regulation of oncogenic transcripts in triple negative breast cancer. We discovered a novel mechanism by which NO inhibits m6A demethylases, iron- and alpha-ketoglutarate-dependent dioxygenases FTO and ALKBH5. Inhibition of demethylation by NO markedly increased cellular m6A mRNA levels. Next, we examined the role of NO in regulating gene expression downstream of m6A mRNA methylation changes. By combining RNA sequencing and m6A methylated RNA immunoprecipitation sequencing, we identified genes that were differentially expressed in response to NO-mediated hypermethylation of m6A mRNA, many of which are associated with tumorigenic hallmarks. Taken together, these data have revealed a novel signaling mechanism such that NO acts an epitranscriptomic regulator of m6A mRNA methylation and thus a driver of oncogenic gene expression.