SUN-728 Methotrexate as a Potential Inhibitor of New Heterotopic Ossification in a Mouse Model of Fibrodysplasia Ossificans Progressiva (FOP)

Abstract Disclosure: I.G. Mamikunian: None. G. Ganesh: None. C. De Cunto: None. R. Morhart: None. E.C. Hsiao: None. Background: FOP is an autosomal dominant genetic disorder classified by the development of heterotopic ossification (HO) following musculoskeletal injury. We previously found that primary and induced IPSC skeletal muscle stem cells with the FOP ACVR1 R206H mutation regenerate at a lower capacity than wildtype equivalents, suggestive of a significant mechanistic change in muscle repair. Activation of the immune system plays key roles in driving new HO formation. Immune changes include altered pro-inflammatory cytokine profiles, as well as increased macrophage activation and sensitivity to activin A. Given the significant role of inflammation in driving FOP-related HO formation, and recent clinical case series suggesting that anti-IL1 and JAK inhibitor therapies may have benefit for FOP patients, we hypothesized that suppression of the immune system by methotrexate (MTX), an antimetabolite that inhibits cell proliferation, used anecdotally in patients with FOP, may decrease new HO formation. Methods: We used a mouse model of FOP where the ACVR1 R206H receptor mutation that causes FOP was expressed in fibroadipocyte cells (PDGFRα-cre/ACVR1R206H mice). 7 FOP and 14 control mice between the ages of 3.5 and 5 weeks were used in the experiment. HO was measured after delivery of skeletal muscle trauma with administration of 1mg/kg/day of MTX over the course of 3 weeks. Mice were split into 3 groups, 2 given MTX either starting treatment before or after trauma, and the third as a standard control. Limbs were harvested and stored in PBS for subsequent radiologic imaging. Brooker classifications (ranging from (4) bridging ossification to (0) no ossification) were used to determine the severity of HO and demonstrate significant decreases in bone volume. Results: Intraperitoneal MTX treatment, given either before or after the skeletal muscle injury, could decrease the Brooker score severity of new trauma-induced HO in FOP mice. Analysis was done on 13 mice (Average Brooker scores: 2 Untreated mice=4, 4 pre-injury treatment mice=1.25; 6 post-injury treatment mice=2.71). All FOP mice developed bridging HO three weeks post-trauma. Mice with pre-injury treatment developed less severe HO than mice without pre-treatment. P values of (<0.08) were found between untreated mice and post-injury treatment mice, and (<0.017) between untreated and pre-injury treatment mice. Significant results were found between untreated mice and pre-treatment mice. Conclusion: MTX, a cost-effective and widely-available anti-inflammatory and immunosuppressive drug can reduce the severity of HO formation in a mouse model of FOP. These results provide support for the further study of MTX to see if these effects show clinical benefit for patients with FOP or other forms of HO formation. Presentation: Sunday, July 13, 2025