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.