Pharmacological targeting of KDM6A and KDM6B, as a novel therapeutic strategy for treating craniosynostosis in Saethre-Chotzen syndrome

BackgroundDuring 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 by TWIST-1 gene mutations. Currently, the only treatment option for craniosynostosis involves multiple invasive cranial surgeries, which can lead to serious complications.MethodsThe present study utilized Twist-1 haploinsufficient (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 methyltransferase EZH2, an osteogenesis inhibitor, is downregulated in calvarial cells derived from Twist-1(del/+) mice, whereas the counter histone demethylases, Kdm6a and Kdm6b, known promoters of osteogenesis, were upregulated. In vitro studies confirmed that siRNA-mediated inhibition of Kdm6a and Kdm6b expression suppressed osteogenic differentiation of Twist-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 by Twist-1(del/+) calvarial cells in vitro and reduced mineralized bone formation in Twist-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 of Runx2 and Alkaline Phosphatase expression. Pre-clinical in vivo studies showed that local administration of GSK-J4 to the calvaria of Twist-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.