AbstractBackgroundDuring development, excessive osteogenic differentiation of mesenchymal progenitor cells (MPC) within the cranial sutures can lead to premature suture fusion or craniosynostosis, leading to craniofacial and cognitive issues. Saethre-Chotzen syndrome (SCS) is a common form of craniosynostosis, caused byTWIST-1gene mutations. Currently, the only treatment option for craniosynostosis involves multiple invasive cranial surgeries, which can lead to serious complications.MethodsThe present study utilizedTwist-1haploinsufficient (Twist-1^del/+) mice as SCS mouse model to investigate the inhibition of Kdm6a and Kdm6b activity using the pharmacological inhibitor, GSK-J4, on calvarial cell osteogenic potential.ResultsThis study showed that the histone methyltransferaseEZH2, an osteogenesis inhibitor, is downregulated in calvarial cells derived fromTwist-1^del/+mice, whereas the counter histone demethylases,Kdm6aandKdm6b, known promoters of osteogenesis, were upregulated. In vitro studies confirmed that siRNA-mediated inhibition ofKdm6aandKdm6bexpression suppressed osteogenic differentiation ofTwist-1^del/+calvarial cells. Moreover, pharmacological targeting of Kdm6a and Kdm6b activity, with the inhibitor, GSK-J4, caused a dose-dependent suppression of osteogenic differentiation byTwist-1^del/+calvarial cells in vitro and reduced mineralized bone formation inTwist-1^del/+calvarial explant cultures. Chromatin immunoprecipitation and Western blot analyses found that GSK-J4 treatment elevated the levels of the Kdm6a and Kdm6b epigenetic target, the repressive mark of tri-methylated lysine 27 on histone 3, on osteogenic genes leading to repression ofRunx2andAlkaline Phosphataseexpression. Pre-clinical in vivo studies showed that local administration of GSK-J4 to the calvaria ofTwist-1^del/+mice prevented premature suture fusion and kept the sutures open up to postnatal day 20.ConclusionThe inhibition of Kdm6a and Kdm6b activity by GSK-J4 could be used as a potential non-invasive therapeutic strategy for preventing craniosynostosis in children with SCS.Graphical abstractPharmacological targeting of Kdm6a/b activity can alleviate craniosynostosis in Saethre-Chotzen syndrome. Aberrant osteogenesis by Twist-1 mutant cranial suture mesenchymal progenitor cells occurs via deregulation of epigenetic modifiers Ezh2 and Kdm6a/Kdm6b. Suppression of Kdm6a- and Kdm6b-mediated osteogenesis with GSK-J4 inhibitor can prevent prefusion of cranial sutures.