N6-methyladenosine modification of FZR1 mRNA promotes gemcitabine resistance in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer with limited therapeutic options, and gemcitabine insensitivity remains a major challenge. N6-methyladenosine (m6A) is a prevalent nucleotide modification in mRNA that has been linked to various biological processes in human diseases, yet its role in drug sensitivity of cancer remains largely unknown. Herein, by detecting the global m6A profile in a panel of gemcitabine-sensitive and gemcitabine-insensitive PDAC cells, we identified the key role of hyper-m6A modification of the master G0/G1 regulator Fizzy and Cell Division Cycle 20 Related 1 (FZR1) in regulating gemcitabine sensitivity. Targeting m6A modification of FZR1 improves the gemcitabine treatment response of gemcitabine-insensitive PDAC cells in vitro and in vivo. Mechanistically, Gem Nuclear Organelle Associated Protein 5 (GEMIN5) was identified as a novel m6A mediator that specifically binds to m6A-modified FZR1 and recruits the eIF3 translation initiation complex to accelerate FZR1 translation. Upregulation of FZR1 maintains the G0/G1 quiescent state and impairs gemcitabine sensitivity of PDAC cells. Further clinical analysis showed that both high levels of FZR1 m6A modification and FZR1 protein indicated a poor response to gemcitabine. These findings unveil the critical function for m6A modification in regulating gemcitabine sensitivity in PDAC and identify the m6A-FZR1 axis as a potential target to enhance gemcitabine response. For m6A-seq, immunoprecipitated and input groups were analyzed with three cell lines for each group for a total of 6 samples. PAR-CLIP using GEMIN5 antibody in PANC-1 cells.