Kaplan, Frederick S.

Abstract Single case studies of extraordinary disease resilience may provide therapeutic insight into conditions for which no definitive treatments exist. An otherwise healthy 35-year-old man (patient-R) with the canonical pathogenic ACVR1R206H variant and the classic congenital great toe malformation of fibrodysplasia ossificans progressiva (FOP) had extreme paucity of post-natal heterotopic ossification (HO) and nearly normal mobility. We hypothesized that patient-R lacked a sufficient post-natal inflammatory trigger for HO. A plasma biomarker survey revealed a reduction in total matrix metalloproteinase-9 (MMP-9) compared to healthy controls and individuals with quiescent FOP. Whole exome sequencing identified compound heterozygous variants in MMP-9 (c.59C > T, p.A20V and c.493G > A, p.D165N). Structural analysis of the D165N variant predicted both decreased MMP-9 secretion and activity that were confirmed by enzyme-linked immunosorbent assay and gelatin zymography. Further, human proinflammatory M1-like macrophages expressing either MMP-9 variant produced significantly less Activin A, an obligate ligand for HO in FOP, compared to wildtype controls. Importantly, MMP-9 inhibition by genetic, biologic, or pharmacologic means in multiple FOP mouse models abrogated trauma-induced HO, sequestered Activin A in the extracellular matrix (ECM), and induced regeneration of injured skeletal muscle. Our data suggest that MMP-9 is a druggable node linking inflammation to HO, orchestrates an existential role in the pathogenesis of FOP, and illustrates that a single patient’s clinical phenotype can reveal critical molecular mechanisms of disease that unveil novel treatment strategies.

Fibrodysplasia ossificans progressiva (FOP; MIM# 135100) is an ultra-rare congenital disorder caused by gain-of-function point mutations in the Activin receptor A type I (ACVR1, also known as ALK2) gene. FOP is characterized by episodic heterotopic ossification (HO) in skeletal muscles, tendons, ligaments, or other soft tissues that progressively causes irreversible loss of mobility. FOP mutations cause mild ligand-independent constitutive activation as well as ligand-dependent bone morphogenetic protein (BMP) pathway hypersensitivity of mutant ACVR1. BMP signaling is also a key pathway for mediating acquired HO. However, HO is a highly complex biological process involving multiple interacting signaling pathways. Among them, the hypoxia-inducible factor (HIF) and mechanistic target of rapamycin (mTOR) pathways are intimately involved in both genetic and acquired HO formation. HIF-1α inhibition or mTOR inhibition reduces HO formation in mouse models of FOP or acquired HO in part by de-amplifying the BMP pathway signaling. Here, we review the recent progress on the mechanisms of the HIF-1α and mTOR pathways in the amplification of HO lesions and discuss the future directions and strategies to translate the targeting of HIF-1α and the mTOR pathways into clinical interventions for FOP and other forms of HO.

Abstract Disclosure: K.M. Dahir: Consulting Fee; Self; Alexion Pharmaceuticals, Inc., Ultragenyx, Inozyme, AM Pharma. Grant Recipient; Self; Regeneron Pharmaceuticals, Alexion Pharmaceuticals, Inc., AstraZeneca, Ultragenyx. J. McGinniss: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. E. Forleo-Neto: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. S. Mellis: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. R.J. Sanchez: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. M. Di Rocco: Research Investigator; Self; Regeneron Pharmaceuticals, Ipsen. R. Keen: Advisory Board Member; Self; International Clinical Council on FOP and IFOPA Registry. Research Investigator; Self; Ipsen, Regeneron Pharmaceuticals, Clementia. P. Orcel: Research Investigator; Self; Regeneron Pharmaceuticals. C. Roux: Consulting Fee; Self; Alexion Pharmaceuticals, Inc., Amgen Inc. Grant Recipient; Self; Regeneron Pharmaceuticals, Kyowa, Kirin Brewery, Alexion Pharmaceuticals, Inc. J. Tabarkiewicz: Speaker; Self; Merck, Novartis Pharmaceuticals. Other; Self; SoftSystem. J. Bachiller-Corral: Research Investigator; Self; Regeneron Pharmaceuticals. A.M. Cheung: Consulting Fee; Self; Ipsen. Grant Recipient; Self; Ipsen, Incyte, Regeneron Pharmaceuticals. M. Al Mukaddam: Grant Recipient; Self; Ipsen, Clementia, Incyte, Regeneron Pharmaceuticals. K. Mohammadi: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. D. Srinivasan: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. A. Rankin: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. A.N. Economides: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. D. Gonzalez Trotter: Employee; Self; Regeneron Pharmaceuticals. Stock Owner; Self; Regeneron Pharmaceuticals. F.S. Kaplan: Research Investigator; Self; Regeneron Pharmaceuticals, Clementia, Ipsen. M.W. Eekhoff: Grant Recipient; Self; Regeneron Pharmaceuticals, Ipsen, AstraZeneca, IMI, IFOPA. R.J. Pignolo: Grant Recipient; Self; Regeneron Pharmaceuticals, Clementia, Ipsen, Incyte. Background: Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare autosomal dominant disorder driven by missense mutations in ACVR1. This results in inappropriate activation by activin-A. FOP is characterized by progressive heterotopic ossification and painful soft tissue inflammatory events known as flare-ups. Garetosmab, an investigational, fully human monoclonal antibody against activin-A, prevents formation of new heterotopic ossification lesions in FOP. Here we describe the impact of garetosmab on flare-up events in the LUMINA-1 study (NCT03188666). Methods: This was a post hoc analysis of phase 2 LUMINA-1, a randomized double-blind placebo-controlled study that evaluated the safety and efficacy of garetosmab 10 mg/kg/every 4 weeks intravenous vs placebo in adult patients with FOP over 28 weeks (Period 1), followed by a 28-week open-label treatment period (Period 2) and subsequent open-label extension (Period 3). Patient-reported flare-ups were collected via a patient diary and severity of symptoms was reported as mild, moderate, or severe. Clinician-reported flare-ups were collected as adverse events. Results: In Period 1, there was a significant reduction in the proportion of patients reporting one or more flare-ups (35% vs 71%, p=0.032) and clinician-reported flare-ups (10% vs 42%, p=0.039) with garetosmab vs placebo, respectively. The overall number of patient-reported flare-ups (13 vs 34) and mean days experiencing new flare-ups (42.6 days vs 65.4 days) were also reduced with garetosmab vs placebo, respectively. Most flare-ups occurred in the back in garetosmab-treated patients, and most flare-ups occurred in the lower extremities and back for those on placebo. Pain was the most frequent symptom among both cohorts. Flare-up associated joint stiffness and severity of flare-up associated symptoms were nominally reduced at both patient and flare-up level of assessment in those treated with garetosmab. No patients reported severe swelling or severe decrease in movement. One patient reported severe pain and joint stiffness in the garetosmab cohort. In Period 2, there were significant reductions in the proportion of patients experiencing flare-ups (68% vs 14%, p=0.0002) and number of patient-reported flares (31 vs 11) among those who crossed over from placebo in Period 1 to garetosmab. Patients who continued on garetosmab through Period 2 maintained a sustained reduction in number of flare-ups (12 vs 6) and proportion of patients experiencing flare-ups (33.3% vs 22.2%). Reductions were maintained through the open-label extension. Conclusions: Patients treated with garetosmab experienced significant and sustained reductions in the frequency, duration, and severity of flare-ups. The ability of garetosmab to reduce flare-up events may provide a clinically meaningful benefit for patients with FOP. Presentation: Sunday, June 18, 2023

Abstract Background Fibrodysplasia ossificans progressiva (FOP) is an ultrarare genetic disorder with episodic and progressive heterotopic ossification. Tissue trauma is a major risk factor for flareups, heterotopic ossification (HO), and loss of mobility in patients with FOP. The International Clinical Council on FOP generally recommends avoiding surgery in patients with FOP unless the situation is life-threatening, because soft tissue injury can trigger an FOP flareup. Surprisingly little is known about flareups, HO formation, and loss of mobility after fractures of the normotopic (occurring in the normal place, distinct from heterotopic) skeleton when treated nonoperatively in patients with FOP. Questions/purposes (1) What proportion of fractures had radiographic evidence of union (defined as radiographic evidence of healing at 6 weeks) or nonunion (defined as the radiographic absence of a bridging callus at 3 years after the fracture)? (2) What proportion of patients had clinical symptoms of an FOP flareup because of the fracture (defined by increased pain or swelling at the fracture site within several days after closed immobilization)? (3) What proportion of patients with fractures had radiographic evidence of HO? (4) What proportion of patients lost movement after a fracture? Methods We retrospectively identified 36 patients with FOP from five continents who sustained 48 fractures of the normotopic skeleton from January 2001 to February 2021, who were treated nonoperatively, and who were followed for a minimum of 18 months after the fracture and for as long as 20 years, depending on when they sustained their fracture during the study period. Five patients (seven fractures) were excluded from the analysis to minimize cotreatment bias because these patients were enrolled in palovarotene clinical trials (NCT02190747 and NCT03312634) at the time of their fractures. Thus, we analyzed 31 patients (13 male, 18 female, median age 22 years, range 5 to 57 years) who sustained 41 fractures of the normotopic skeleton that were treated nonoperatively. Patients were analyzed at a median follow-up of 6 years (range 18 months to 20 years), and none was lost to follow-up. Clinical records for each patient were reviewed by the referring physician–author and the following data for each fracture were recorded: biological sex, ACVR1 gene pathogenic variant, age at the time of fracture, fracture mechanism, fracture location, initial treatment modality, prednisone use at the time of the fracture as indicated in the FOP Treatment Guidelines for flare prevention (2 mg/kg once daily for 4 days), patient-reported flareups (episodic inflammatory lesions of muscle and deep soft connective tissue characterized variably by swelling, escalating pain, stiffness, and immobility) after the fracture, follow-up radiographs of the fracture if available, HO formation (yes or no) as a result of the fracture determined at a minimum of 6 weeks after the fracture, and patient-reported loss of motion at least 6 months after and as long as 20 years after the fracture. Postfracture radiographs were available in 76% (31 of 41) of fractures in 25 patients and were independently reviewed by the referring physician–author and senior author for radiographic criteria of fracture healing and HO. Results Radiographic healing was noted in 97% (30 of 31) of fractures at 6 weeks after the incident fracture. Painless nonunion was noted in one patient who sustained a displaced patellar fracture and HO. In seven percent (three of 41) of fractures, patients reported increased pain or swelling at or near the fracture site within several days after fracture immobilization that likely indicated a site-specific FOP flareup. The same three patients reported a residual loss of motion 1 year after the fracture compared with their prefracture status. HO developed in 10% (three of 31) of the fractures for which follow-up radiographs were available. Patient-reported loss of motion occurred in 10% (four of 41) of fractures. Two of the four patients reported noticeable loss of motion and the other two patients reported that the joint was completely immobile (ankylosis). Conclusion Most fractures treated nonoperatively in individuals with FOP healed with few flareups, little or no HO, and preservation of mobility, suggesting an uncoupling of fracture repair and HO, which are two inflammation-induced processes of endochondral ossification. These findings underscore the importance of considering nonoperative treatment for fractures in individuals with FOP. Physicians who treat fractures in patients with FOP should consult with a member of the International Clinical Council listed in the FOP Treatment Guidelines (https://www.iccfop.org). Level of Evidence Level IV, therapeutic study.

ABSTRACT Fibrodysplasia ossificans progressiva (FOP) is an ultra‐rare, severely disabling genetic disorder of progressive heterotopic ossification (HO). The single‐arm, open‐label, phase 3 MOVE trial (NCT03312634) assessed efficacy and safety of palovarotene, a selective retinoic acid receptor gamma agonist, in patients with FOP. Findings were compared with FOP natural history study (NHS; NCT02322255) participants untreated beyond standard of care. Patients aged ≥4 years received palovarotene once daily (chronic: 5 mg; flare‐up: 20 mg for 4 weeks, then 10 mg for ≥8 weeks; weight‐adjusted if skeletally immature). The primary endpoint was annualized change in new HO volume versus NHS participants (by low‐dose whole‐body computed tomography [WBCT]), analyzed using a Bayesian compound Poisson model (BcPM) with square‐root transformation. Twelve‐month interim analyses met futility criteria; dosing was paused. An independent Data Monitoring Committee recommended trial continuation. Post hoc 18‐month interim analyses utilized BcPM with square‐root transformation and HO data collapsed to equalize MOVE and NHS visit schedules, BcPM without transformation, and weighted linear mixed‐effects (wLME) models, alongside prespecified analysis. Safety was assessed throughout. Eighteen‐month interim analyses included 97 MOVE and 101 NHS individuals with post‐baseline WBCT. BcPM analyses without transformation showed 99.4% probability of any reduction in new HO with palovarotene versus NHS participants (with transformation: 65.4%). Mean annualized new HO volume was 60% lower in MOVE versus the NHS. wLME results were similar (54% reduction fitted; nominal p = 0.039). All palovarotene‐treated patients reported ≥1 adverse event (AE); 97.0% reported ≥1 retinoid‐associated AE; 29.3% reported ≥1 serious AE, including premature physeal closure (PPC)/epiphyseal disorder in 21/57 (36.8%) patients aged <14 years. Post hoc computational analyses using WBCT showed decreased vertebral bone mineral density, content, and strength, and increased vertebral fracture risk in palovarotene‐treated patients. Thus, post hoc analyses showed evidence for efficacy of palovarotene in reducing new HO in FOP, but high risk of PPC in skeletally immature patients. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

ABSTRACT Bone morphogenetic protein (BMP) signaling is critical in skeletal development. Overactivation can trigger heterotopic ossification (HO) as in fibrodysplasia ossificans progressiva (FOP), a rare, progressive disease of massive HO formation. A small subset of FOP patients harboring the causative ACVR1R206H mutation show strikingly mild or delayed-onset HO, suggesting that genetic variants in the BMP pathway could act as disease modifiers. Whole-exome sequencing of one such patient identified BMPR1AR443C and ACVR2AV173I as candidate modifiers. Molecular modeling predicted significant structural perturbations. Neither variant decreased BMP signaling in ACVR1R206H HEK 293T cells at baseline or after stimulation with BMP4 or activin A (AA), ligands that activate ACVR1R206H signaling. Overexpression of BMPR1AR443C in a Tg(ACVR1-R206Ha) embryonic zebrafish model, in which overactive BMP signaling yields ventralized embryos, did not alter ventralization severity, while ACVR2AV173I exacerbated ventralization. Co-expression of both variants did not affect dorsoventral patterning. In contrast, BMPR1A knockdown in ACVR1R206H HEK cells decreased ligand-stimulated BMP signaling but did not affect dorsoventral patterning in Tg(ACVR1-R206Ha) zebrafish. ACVR2A knockdown decreased only AA-stimulated signaling in ACVR1R206H HEK cells and had no effect in Tg(ACVR1-R206Ha) zebrafish. Co-knockdown in ACVR1R206H HEK cells decreased basal and ligand-stimulated signaling, and co-knockdown/knockout (bmpr1aa/ab; acvr2aa/ab) decreased Tg(ACVR1-R206Ha) zebrafish ventralization phenotypes. Our functional studies showed that knockdown of wild-type BMPR1A and ACVR2A could attenuate ACVR1R206H signaling, particularly in response to AA, and that ACVR2AV173I unexpectedly increased ACVR1R206H-mediated signaling in zebrafish. These studies describe a useful strategy and platform for functionally interrogating potential genes and genetic variants that may impact the BMP signaling pathway. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

ABSTRACT Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare genetic disorder characterized by progressive heterotopic ossification (HO), often heralded by flare-ups, leading to reduced movement and life expectancy. This placebo-controlled, double-blind trial (NCT02190747) evaluated palovarotene, an orally bioavailable selective retinoic acid receptor gamma agonist, for prevention of HO in patients with FOP. Patients experiencing a flare-up were enrolled in two cohorts: (1) patients ≥15 years were randomized 3:1 to palovarotene 10/5 mg (weeks 1–2/3–6) or placebo; (2) patients ≥6 years were randomized 3:3:2 to palovarotene 10/5 mg, palovarotene 5/2.5 mg (weeks 1–2/3–6), or placebo. Cohort data were pooled. The primary endpoint was the proportion of responders (no/minimal new HO at flare-up body region by plain radiograph) at week 6. Change from baseline in HO volume and new HO incidence were assessed by computed tomography (CT) at week 12. Tissue edema was assessed by magnetic resonance imaging (MRI) or ultrasound. Forty patients (aged 7–53 years) were enrolled (placebo: n = 10; palovarotene 5/2.5 mg: n = 9; palovarotene 10/5 mg: n = 21). Disease history was similar between groups. In the per-protocol population, the proportion of responders at week 6 by plain radiograph was 100% with palovarotene 10/5 mg; 88.9% with palovarotene 5/2.5 mg; 88.9% with placebo (Cochran-Armitage trend test: p = 0.17). At week 12, the proportions were 95.0% with palovarotene 10/5 mg; 88.9% with palovarotene 5/2.5 mg; 77.8% with placebo (Cochran-Armitage trend test: p = 0.15). Week 12 least-squares mean (LSmean) new HO volume, assessed by CT, was 3.8 × 103 mm3 with palovarotene 10/5 mg; 1.3 × 103 mm3 with palovarotene 5/2.5 mg; 18.0 × 103 mm3 with placebo (pairwise tests versus placebo: p ≤ 0.12). Palovarotene was well-tolerated. No patients discontinued treatment or required dose reduction; one patient had dose interruption due to elevated lipase. Although these findings were not statistically significant, they support further evaluation of palovarotene for prevention of HO in FOP in larger studies. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Fibrodysplasia ossificans progressiva (FOP) is a rare and devastating genetic disease, in which soft connective tissue is converted into heterotopic bone through an endochondral ossification process. Patients succumb early as they gradually become trapped in a second skeleton of heterotopic bone. Although the underlying genetic defect is long known, the inherent complexity of the disease has hindered the discovery of effective preventions and treatments. New developments in the gene therapy field have motivated its consideration as an attractive therapeutic option for FOP. However, the immune system's role in FOP activation and the as-yet unknown primary causative cell, are crucial issues which must be taken into account in the therapy design. While gene therapy offers a potential therapeutic solution, more knowledge about FOP is needed to enable its optimal and safe application.

ABSTRACT Fibrodysplasia ossificans progressiva (FOP) is a progressive, debilitating genetic disease in which skeletal muscle and connective tissue is episodically replaced by heterotopic bone. Discovery of surrogate biomarkers of disease (genotype)-related and flare-up-associated activity of FOP in a readily accessible matrix, such as plasma, would facilitate an understanding of the complex pathophysiology of FOP, aid patient care, and provide a valuable tool for the development and monitoring of potential therapeutics. In a case–control study, using a carefully collected and curated set of plasma samples from 40 FOP patients with the classic ACVR1R206H mutation and 40 age- and sex-matched controls, we report the identification of disease-related and flare-up-associated biomarkers of FOP using a multiplex analysis of 113 plasma-soluble analytes. Adiponectin (implicated in hypoxia, inflammation, and heterotopic ossification) as well as tenascin-C (an endogenous activator of innate immune signaling through the TLR4 pathway and a substrate for kallikrein-7) were highly correlated with FOP genotype, while kallikrein-7 was highly correlated with acute flare-up status. Plasma-soluble biomarkers for FOP support a flare-up-related acute inflammatory phase of disease activity superimposed on a genotypic background of chronic inflammation. © 2021 American Society for Bone and Mineral Research (ASBMR).