ALKBH5-mediated m⁶A demethylation stabilizes BMP-2 mRNA via YTHDF2 to drive pathological osteogenesis in ankylosing spondylitis-derived mesenchymal stem cells

Pathological osteogenesis contributes significantly to ankylosing spondylitis (AS) progression, with mesenchymal stem cells (MSCs) exhibiting increased osteogenic potential. While BMP-2 overexpression has been observed in AS-MSCs, the precise molecular mechanisms underlying its dysregulation remain elusive. Using m⁶A dot blot and MeRIP-qPCR, we quantified global and BMP-2-specific m⁶A modifications during osteogenesis. Genetic perturbations of the ALKBH5 and YTHDF genes were achieved via siRNA/lentivirus. BMP-2 mRNA dynamics were investigated through actinomycin D chase assays, subcellular fractionation, and polysome profiling. Site-directed mutagenesis confirmed m⁶A functionality, whereas subcutaneous implantation assays validated its pathological relevance. Furthermore, structure-based virtual screening was performed to identify potential ALKBH5 inhibitors, which were subsequently validated in vitro and in a subcutaneous implantation model. AS-MSCs exhibited unique m⁶A dynamics during osteogenesis: diminished m⁶A methylation/YTHDF2 but elevated ALKBH5/BMP-2 expression distinguished AS-MSCs. Functional interrogation demonstrated that ALKBH5 ablation accelerated BMP-2 mRNA decay, attenuated protein output, and impaired mineralization—phenotypes rescued by ALKBH5 reconstitution. Mechanistically, ALKBH5-mediated m⁶A erasure attenuated the YTHDF2-BMP-2 mRNA interaction, consequently subverting transcript degradation. Virtual screening identified lifitegrast and nebivolol as high-affinity ALKBH5 ligands. Functional validation revealed that nebivolol significantly suppressed BMP-2 expression and rescued the pathological osteogenic phenotype both in vitro and in vivo. Our data suggest that ALKBH5 may contribute to BMP-2 mRNA stabilization via regulation of the m⁶A-YTHDF2 axis, potentially participating in pathological ossification processes in AS. Targeting ALKBH5 with nebivolol may represent a potential therapeutic approach for further investigation in AS-related pathological bone formation.