An ALK2 inhibitor, BLU-782, prevents heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease driven by gain-of-function variants in activin receptor-like kinase 2 (ALK2), the most common variant being ALK2R206H. In FOP, ALK2 variants display increased and dysregulated signaling through the bone morphogenetic protein (BMP) pathway resulting in progressive and permanent replacement of skeletal muscle and connective tissues with heterotopic bone, ultimately leading to severe debilitation and premature death. Here, we describe the discovery of BLU-782 (IPN60130), a small-molecule ALK2R206H inhibitor developed for the treatment of FOP. A small-molecule library was screened in a biochemical ALK2 binding assay to identify potent ALK2 binding compounds. Iterative rounds of structure-guided drug design were used to optimize compounds for ALK2R206H binding, ALK2 selectivity, and other desirable pharmacokinetic properties. BLU-782 preferentially bound to ALK2R206H with high affinity, inhibiting signaling from ALK2R206H and other rare FOP variants in cells in vitro without affecting signaling of closely related homologs ALK1, ALK3, and ALK6. In vivo efficacy of BLU-782 was demonstrated using a conditional knock-in ALK2R206H mouse model, where prophylactic oral dosing reduced edema and prevented cartilage and heterotopic ossification (HO) in both muscle and bone injury models. BLU-782 treatment preserved the normal muscle-healing response in ALK2R206H mice. Delayed dosing revealed a short 2-day window after injury when BLU-782 treatment prevented HO in ALK2R206H mice, but dosing delays of 4 days or longer abrogated HO prevention. Together, these data suggest that BLU-782 may be a candidate for prevention of HO in FOP.

Sex as a Critical Variable in Basic and Pre-Clinical Studies of Fibrodysplasia Ossificans Progressiva.

Heterotopic ossification (HO) is most dramatically manifested in the rare and severely debilitating disease, fibrodysplasia ossificans progressiva (FOP), in which heterotopic bone progressively accumulates in skeletal muscles and associated soft tissues. The great majority of FOP cases are caused by a single amino acid substitution in the type 1 bone morphogenetic protein (BMP) receptor ACVR1, a mutation that imparts responsiveness to activin A. Although it is well-established that biological sex is a critical variable in a range of physiological and disease processes, the impact of sex on HO in animal models of FOP has not been explored. We show that female FOP mice exhibit both significantly greater and more variable HO responses after muscle injury. Additionally, the incidence of spontaneous HO was significantly greater in female mice. This sex dimorphism is not dependent on gonadally derived sex hormones, and reciprocal cell transplantations indicate that apparent differences in osteogenic activity are intrinsic to the sex of the transplanted cells. By circumventing the absolute requirement for activin A using an agonist of mutant ACVR1, we show that the female-specific response to muscle injury or BMP2 implantation is dependent on activin A. These data identify sex as a critical variable in basic and pre-clinical studies of FOP.

An anti-ACVR1 antibody exacerbates heterotopic ossification by fibro-adipogenic progenitors in fibrodysplasia ossificans progressiva mice.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by progressive and catastrophic heterotopic ossification (HO) of skeletal muscle and associated soft tissues. FOP is caused by dominantly acting mutations in the gene encoding the bone morphogenetic protein (BMP) type I receptor, ACVR1 (ALK2), the most prevalent of which results in an arginine to histidine substitution at position 206 (ACVR1[R206H]). The fundamental pathological consequence of FOP-causing ACVR1 receptor mutations is to enable activin A to initiate canonical BMP signaling in fibro-adipogenic progenitors (FAPs), which drives HO. We developed a monoclonal blocking antibody (JAB0505) against the extracellular domain of ACVR1 and tested its effect on HO in 2 independent FOP mouse models. Although JAB0505 inhibited BMP-dependent gene expression in wild-type and ACVR1(R206H)-overexpressing cell lines, JAB0505 treatment profoundly exacerbated injury-induced HO. JAB0505-treated mice exhibited multiple, distinct foci of heterotopic lesions, suggesting an atypically broad anatomical domain of FAP recruitment to endochondral ossification. This was accompanied by dysregulated FAP population growth and an abnormally sustained immunological reaction following muscle injury. JAB0505 drove injury-induced HO in the absence of activin A, indicating that JAB0505 has receptor agonist activity. These data raise serious safety and efficacy concerns for the use of bivalent anti-ACVR1 antibodies to treat patients with FOP.

Activin-dependent signaling in fibro/adipogenic progenitors causes fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal-dominant disorder characterized by progressive and profoundly disabling heterotopic ossification (HO). Here we show that fibro/adipogenic progenitors (FAPs) are a major cell-of-origin of HO in an accurate genetic mouse model of FOP (Acvr1 tnR206H ). Targeted expression of the disease-causing type I bone morphogenetic protein (BMP) receptor, ACVR1(R206H), to FAPs recapitulates the full spectrum of HO observed in FOP patients. ACVR1(R206H)-expressing FAPs, but not wild-type FAPs, activate osteogenic signaling in response to activin ligands. Conditional loss of the wild-type Acvr1 allele dramatically exacerbates FAP-directed HO, suggesting that mutant and wild-type ACVR1 receptor complexes compete for activin ligands or type II BMP receptor binding partners. Finally, systemic inhibition of activin A completely blocks HO and restores wild-type-like behavior to transplanted Acvr1 R206H/+ FAPs. Understanding the cells that drive HO may facilitate the development of cell-specific therapeutic approaches to inhibit catastrophic bone formation in FOP.