RANKL inhibition with denosumab improves fibrous dysplasia by decreasing lesional cell proliferation and increasing osteogenesis

BACKGROUND Fibrous dysplasia (FD) is a rare, disabling disease with no established treatments. Growing evidence supports inhibiting the pro-osteoclastic factor receptor activator of nuclear Kappa-B ligand (RANKL) as a potential treatment strategy. We conducted a phase 2 trial evaluating the anti-RANKL drug denosumab in adults with FD, with an emphasis on investigating post-discontinuation bone turnover rebound, and cellular mechanisms underlying anti-RANKL effects on FD osteoprogenitors. METHODS Eight subjects received denosumab for 6-months and were observed for 8-months post-discontinuation. Efficacy and safety were evaluated using bone turnover markers, 18F-NaF PET/CT, and lesion biopsies. RANKL neutralization effects were assessed by histology, RNASeq, and an FD mouse model. Interplay between osteoclasts and FD osteoprogenitors was assessed in an ex vivo lesion model. RESULTS Denosumab markedly reduced bone turnover and radiographic lesional activity in all subjects. Denosumab was well-tolerated during the treatment period, however post-discontinuation turnover reached or exceeded pre-treatment in six subjects, associated with severe hypercalcemia in one. Histology and whole-exome RNA sequencing showed reduced FD cell proliferation and increased osteogenic maturation, with increased lesional bone formation. The ex vivo model supported the dependence of FD cell proliferation on osteoclast activation. CONCLUSIONS Osteoclast inhibition by anti-RANKL decreased FD cell proliferation and lesional activity, enabling osteogenic maturation and bone formation. These findings provide new understanding of FD pathogenesis as driven by crosstalk between osteoclasts and pre-osteoblast/osteoblasts, and support denosumab as a mechanistically-driven treatment strategy. Marked bone turnover rebound with post-discontinuation hypercalcemia occurs in a subset of patients, particularly younger individuals with high disease burden. TRIAL REGISTRATION [ClinicalTrials.gov][1] [NCT03571191][2] FUNDING This work was supported by the Intramural Research Program of the NIDCR, NICHD, and Clinical Center, National Institutes of Health. Clinical trial [NCT03571191][2] was conducted as an investigator-sponsored study supported by Amgen, Inc. This research was supported in part by the NIDCR Genomics and Computational Biology Core: ZIC DC000086 and Veterinary Resources Core: ZIC DE000740-05. Work in MTC lab and LVC labs were supported by the of Research on Women’s Health (ORWH) through the Bench to Bedside Program award #884515. ### Competing Interest Statement This study was conducted as an investigator-sponsored study supported by Amgen, Inc. ### Clinical Trial NCT03571191 ### Funding Statement This work was supported by the Intramural Research Program of the NIDCR, NICHD, and Clinical Center, National Institutes of Health. Clinical trial [NCT03571191][2] was conducted as an investigator-sponsored study supported by Amgen, Inc. This research was supported in part by the NIDCR Genomics and Computational Biology Core: ZIC DC000086 and Veterinary Resources Core: ZIC DE000740-05. Work in MTC lab and LVC labs were supported by the of Research on Women's Health (ORWH) through the Bench to Bedside Program award #884515. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The IRB of the National Institutes of Health gave ethical approval for this work I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors [1]: http://ClinicalTrials.gov [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT03571191&atom=%2Fmedrxiv%2Fearly%2F2022%2F11%2F02%2F2022.10.24.22281375.atom