FTO depletion does not alter m6A stoichiometry in AML mRNA: a reassessment using direct RNA nanopore sequencing.

The RNA demethylase FTO has been proposed to promote acute myeloid leukemia (AML) by demethylating N 6-methyladenosine (m6A) from oncogenic transcripts, especially MYC. However, the evidence that supports the idea that FTO demethylates m6A in AML relies on methods that are non-quantitative and unable to reveal m6A stoichiometry changes before or after FTO depletion. To directly test whether FTO regulates m6A in mRNA, we employed Oxford Nanopore direct RNA sequencing to map and quantify m6A at single-nucleotide resolution. We find that the stoichiometry of m6A sites throughout the transcriptome and especially at MYC-specific sites are unaffected despite depletion of FTO activity by knockout, knockdown, or pharmacologic inhibition. This pattern was seen in AML cell lines MONOMAC-6 and MOLM-13, as well as in the non-AML cell line HEK293T. We also find that the anti-leukemia effect of the small-molecule FTO inhibitor FB23-2 is not due to FTO inhibition since it remains cytotoxic to FTO-deficient cells. Instead of regulating m6A, we find that FTO depletion markedly increases N 6,2'-O-dimethyladenosine (m6Am) in snRNAs, consistent with m6Am in snRNA being a target of FTO. Overall, our findings do not support an 'm6A eraser' role for FTO in AML cell lines under the conditions tested, and they suggest that the reported demethylation functions of FTO on m6A should be reinvestigated using quantitative m6A mapping methods.