MS-275 Rescues the Development of Cleidocranial Dysplasia in Runx2^+/- Mice

Objectives: Cleidocranial dysplasia (CCD) is a genetic skeletal disorder caused by haploinsufficiency of RUNX2. We previously identified that HDAC inhibitors (HDACi) stimulated RUNX2 protein stability and its transcriptional activity. Specific aim of this study was to examine whether genetic RUNX2 haploinsufficiency could be overcome by treatment of HDACi.
Methods: Wildtype and Runx2^+/-mice were mated and pregnant mice were injected with MS-275 or vehicle at 14.5d.p.c. Skeletal staining was performed with E16.5 embyos and micro CT analysis was achieved with P0 mice. Primary cells were isolated from calvaria of Runx2^+/+ and Runx2^+/- mice at P0. We analyzed osteoblast differentiation, cell proliferation, Runx2 transacting activity, and Runx2 protein stability and epigenetic landscape by using those cells. Immunohistochemistry was also performed to confirm the quantity of Runx2 protein. uCT analysis was carried out to investigate in vivo calvarial mineralization degree.
Results: The MS-275 neither caused pregnant mice to die or show toxic behaviors nor did it affect littermate numbers. Systemic administration of MS-275 remarkably improved most of the skeletal deformities associated with Runx2 haploinsufficiency; cranial bone development and clavicle maturation, spinal bone maturations and mandible growth. With the several in vitro studies, we demonstrated MS-275 has positive effects on primary osteoblasts differentiation and it can also restore delayed differentiation pattern of Runx2^+/- calvaria cells. In the molecular level, MS-275 administration in Runx2^+/- mice stimulated Runx2 acetylation, thereby recovering hypomorphic expression of active Runx2 through enhanced stabilization of the protein. In addition, Runx2 transacting activity in Runx2^+/-calvarial osteoblasts was substantially recovered by the MS-275 treatment.
Conclusions: MS-275 could be applied for the intervention of CCD phenotype development through combination of post-translational modification activity and epigenetic regulation. It is meaningful because we showed that genetic disease can be rescue through therapeutic regimen in this study. Moreover, the mechanism of action we identified in this study strongly suggests that HDACi could also be applied for bone regeneration.