FTO promotes human NK cell proliferation yet restricts effector functions.

Human embryonic stem cell (hESC)- or induced pluripotent stem cell (iPSC)-derived natural killer (NK) cells serve as promising adoptive cell therapies for cancer patients and as valuable models for studying the regulation of human NK cell development and effector functions. In this study, we used shRNA to knock down the expression of FTO in hESCs and differentiated these cells into NK cells to explore the role of FTO in regulating human NK cell development and effector functions. Our data revealed that the FTO knockdown in hESCs resulted in a comparable effect on haematopoietic progenitor cell generation but significantly impaired CD45⁺CD56⁺ NK cell production, which might be attributed to impaired proliferation, as indicated by reduced Ki-67 expression. Functional assays demonstrated that FTO knockdown in hESC-NK cells effectively increased per-cell cytotoxicity, including the expression of perforin, granzyme B, and IFN-γ, and increased degranulation (as indicated by the CD107a level). Integrated MeRIP-seq and RNA-seq analyses identified ADAMTS1 and ZAP70 as key target genes of FTO, demonstrating that FTO knockdown increased m⁶A methylation near the 3'UTR regions of ADAMTS1 and ZAP70 mRNAs and upregulated their transcript levels. ADAMTS1 is a known proliferation inhibitor, whereas increased ZAP70 expression is accompanied by increased phosphorylation of ERK1/2, a downstream component of ZAP70 signalling cascades, which positively regulates the effector functions of NK cells. Overall, our results demonstrate that FTO maintains human NK cell proliferation but restricts their per-cell effector functions, possibly through regulating the m⁶A methylation of ADAMTS1 and ZAP70 mRNAs. Exploring methods to achieve stage-specific FTO knockdown in mature NK cells may provide a potential strategy to optimize hESC-NK cell-based immunotherapy.