Neutralization of Receptor activator of nuclear factor-κB ligand reduces fibrosis and promotes osteoblast differentiation in a mouse model of fibrous dysplasia driven by somatic expression of Gnas(R201H).

Fibrous dysplasia (FD) is a rare disorder caused by somatic activating mutations in GNAS, encoding the alpha subunit of the Gs protein. Activating GNAS mutations result in focal expansile bone lesions, which cause pain, deformity, and increased risk of fracture. Somatic mosaicism in FD leads to both GNAS mutant and genetically WT osteoprogenitor cells, which jointly contribute to the formation of fibrotic lesions within the bone. Additionally, these lesions contain numerous osteoclasts formed in response to robust lesional expression of RANKL. Neutralizing antibody to RANKL is effective in reducing lesion growth in patients with FD and in preclinical models. To determine the effect of RANKL neutralization specifically on mutant cells early after onset of FD, we used a murine model of C57BL/6 Sox9(CreERT);Gnas((R201H)fl/+);Rosa26(LSL-tdTomato) mice, which recapitulates the somatic mosaicism of FD bone lesions and in which mutant cells are lineage traced. Analysis of Gnas((R201H)fl/+) mice showed a diffuse accumulation of SMA(+) early osteoblastic cells, with contribution from both tdTomato(+) mutant and tdTomato(-) WT populations. Anti-RANKL treatment of Gnas((R201H)fl/+) mice inhibited osteoclast formation and substantially reduced fibrosis, detected by Masson's trichrome staining within the proximal metaphysis of the femur and the femoral head. Treatment with anti-RANKL decreased the accumulation of both mutant and WT SMA(+) cells, accompanied by an increased number of mutant cells expressing the mature osteoblast marker osteocalcin, and an increase in overall osteoblast density. To elucidate the role of RANKL expression by mutant cells in the formation of FD lesions, we generated Sox9(CreERT);Gnas((R201H)fl/+);Rosa26(LSL-tdTomato);Rankl(fl/fl) mice. Deletion of Rankl in Gnas((R201H)fl/+) mutant cells did not prevent fibrosis in this model. The results suggest that while anti-RANKL treatment promotes osteoprogenitor differentiation to reduce fibrosis, the loss of RANKL expression from GNAS mutant cells alone is not sufficient to reverse the pathology of FD bone lesions.