Cell Senescence in Heterotopic Ossification.

The formation of bone outside the normal skeleton, or heterotopic ossification (HO), occurs through genetic and acquired mechanisms. Fibrodysplasia ossificans progressiva (FOP), the most devastating genetic condition of HO, is due to mutations in the ACVR1/ALK2 gene and is relentlessly progressive. Acquired HO is mostly precipitated by injury or orthopedic surgical procedures but can also be associated with certain conditions related to aging. Cellular senescence is a hallmark of aging and thought to be a tumor-suppressive mechanism with characteristic features such as irreversible growth arrest, apoptosis resistance, and an inflammatory senescence-associated secretory phenotype (SASP). Here, we review possible roles for cellular senescence in HO and how targeting senescent cells may provide new therapeutic approaches to both FOP and acquired forms of HO.

Pay Attention to the Osteochondromas in Fibrodysplasia Ossificans Progressiva.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is an extremely rare disease characterized by malformation of the bilateral great toes and progressive heterotopic ossification. The clinical features of FOP occur due to dysfunction of the bone morphogenetic protein (BMP) signaling pathway induced by the mutant activin A type I receptor/activin-like kinase-2 (ACVR1/ALK2) which contributes to the clinical features in FOP. Dysregulation of the BMP signaling pathway causes the development of osteochondroma. Poor awareness of the association between FOP and osteochondromas always results in misdiagnosis and unnecessary invasive operation. CASE PRESENTATION: In this study, we present a case of classical FOP involving osteochondroma. An 18-year-old male adolescent, born with deformity of bilateral big toes, complained multiple masses on his back for 1 year. The mass initially emerged with a tough texture and did not cause pain. It was misdiagnosed as an osteochondroma. After two surgeries, the masses became hard and spread around the entire back region. Meanwhile, extensive heterotopic ossification was observed around the back, neck, hip, knee, ribs, and mandible during follow-up. Osteochondromas were observed around the bilateral knees. No abnormalities were observed in the laboratory blood test results. Whole exome sequencing revealed missense mutation of ACVR1/ALK2 (c.617G > A; p.R206H) in the patient and confirmed the diagnosis of FOP. CONCLUSION: In summary, classical FOP always behaves as a bilateral deformity of the big toes, as well as progressive ectopic ossification and osteochondromas in the distal femur and proximal tibia. An understanding of the association between osteochondromas and FOP aids in diagnosis and avoids unnecessary invasive management in patients.

A blocking monoclonal antibody reveals dimerization of intracellular domains of ALK2 associated with genetic disorders.

Mutations in activin receptor-like kinase 2 (ALK2) can cause the pathological osteogenic signaling seen in some patients with fibrodysplasia ossificans progressiva and other conditions such as diffuse intrinsic pontine glioma. Here, we report that intracellular domain of wild-type ALK2 readily dimerizes in response to BMP7 binding to drive osteogenic signaling. This osteogenic signaling is pathologically triggered by heterotetramers of type II receptor kinases and ALK2 mutant forms, which form intracellular domain dimers in response to activin A binding. We develop a blocking monoclonal antibody, Rm0443, that can suppress ALK2 signaling. We solve the crystal structure of the ALK2 extracellular domain complex with a Fab fragment of Rm0443 and show that Rm0443 induces dimerization of ALK2 extracellular domains in a back-to-back orientation on the cell membrane by binding the residues H64 and F63 on opposite faces of the ligand-binding site. Rm0443 could prevent heterotopic ossification in a mouse model of fibrodysplasia ossificans progressiva that carries the human R206H pathogenic mutant.

Bone morphogenetic protein signaling is a possible therapeutic target in gynecologic cancer.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor ß (TGFß) superfamily. BMPs play crucial roles in embryogenesis and bone remodeling. Recently, BMP signaling has been found to have diverse effects on different types of tumors. In this review, we summarized the effects of BMP signaling on gynecologic cancer. BMP signaling has tumor-promoting effects on ovarian cancer (OC) and endometrial cancer (EC), whereas it has tumor-suppressing effects on uterine cervical cancer (UCC). Interestingly, EC has frequent gain-of-function mutations in ACVR1, encoding one of the type I BMP receptors, which are also observed in fibrodysplasia ossificans progressiva and diffuse intrinsic pontine glioma. Little is known about the relationship between BMP signaling and other gynecologic cancers. Tumor-promoting effects of BMP signaling in OC and EC are dependent on the promotion of cancer stemness and epithelial-mesenchymal transition (EMT). In accordance, BMP receptor kinase inhibitors suppress the cell growth and migration of OC and EC. Since both cancer stemness and EMT are associated with chemoresistance, BMP signaling activation might also be an important mechanism by which OC and EC patients acquire chemoresistance. Therefore, BMP inhibitors are promising for OC and EC patients even if they become resistant to standard chemotherapy. In contrast, BMP signaling inhibits UCC growth in vitro. However, the in vivo effects of BMP signaling have not been elucidated in UCC. In conclusion, BMP signaling has a variety of functions, depending on the types of gynecologic cancer. Therefore, targeting BMP signaling should improve the treatment of patients with gynecologic cancer.

RARγ: The Bone of Contention for Endothelial Cells in Prostate Cancer Metastasis.

Excessive bone deposition associated with prostate cancer bone metastases is believed to aid in metastatic progression. One mechanism of osteoblast expansion is the transdifferentiation of bone marrow endothelial cells. Prostate cancer cells contribute several secreted factors, including bone morphogenetic protein 4 (BMP4), to the microenvironment that support osteoblastic transdifferentiation. In this issue of Cancer Research, Yu and colleagues share their findings of how BMP-mediated endothelial conversion can be inhibited by treatment with retinoic acid receptor (RAR) agonists. Using agonists like the all-trans retinoic acid or palovarotene, the authors demonstrated the role of the interaction of BMP-activated SMAD1 with RARγ for osteoblastic differentiation. RARγ agonists potentiated the proteasomal degradation of the Smad1-RARγ complex, blocking BMP signaling. Because palovarotene is clinically effective in the treatment of aberrant bone formation found in fibrodysplasia ossificans progressiva, its repurposing for the treatment of osteoblastic cancer metastasis is promising. However, patient selection and dose-finding studies will be critical for the translation of these findings to complement standard of care for patients with bone metastatic prostate cancer. See related article by Yu et al., p. 3158.

ALK2: A Therapeutic Target for Fibrodysplasia Ossificans Progressiva and Diffuse Intrinsic Pontine Glioma.

Fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG) are diseases that typically manifest in childhood and are associated with severely reduced life expectancy. However, there are currently no effective therapies for these diseases, which remain incurable. Activin receptor-like kinase-2 (ALK2), encoded by the ACVR1 gene, is a bone morphogenetic protein (BMP) type-I receptor subtype that plays an important physiological role in the development of bones, muscles, brain, and other organs. Constitutively active mutants of ALK2 have been identified as causative of FOP and involved in the tumorigenesis of DIPG owing to abnormal activation of BMP signaling, and therefore have emerged as promising treatment targets. Here, we describe these two diseases, along with the link to ALK2 signal transduction, and highlight potential ALK2 inhibitors that are under development to offer new hope for patients with FOP and DIPG.

ACVR1 Function in Health and Disease.

Activin A receptor type I (ACVR1) encodes for a bone morphogenetic protein type I receptor of the TGFß receptor superfamily. It is involved in a wide variety of biological processes, including bone, heart, cartilage, nervous, and reproductive system development and regulation. Moreover, ACVR1 has been extensively studied for its causal role in fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder characterised by progressive heterotopic ossification. ACVR1 is linked to different pathologies, including cardiac malformations and alterations in the reproductive system. More recently, ACVR1 has been experimentally validated as a cancer driver gene in diffuse intrinsic pontine glioma (DIPG), a malignant childhood brainstem glioma, and its function is being studied in other cancer types. Here, we review ACVR1 receptor function and signalling in physiological and pathological processes and its regulation according to cell type and mutational status. Learning from different functions and alterations linked to ACVR1 is a key step in the development of interdisciplinary research towards the identification of novel treatments for these pathologies.

Elevated BMP and Mechanical Signaling Through YAP1/RhoA Poises FOP Mesenchymal Progenitors for Osteogenesis.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by the formation of extraskeletal bone, or heterotopic ossification (HO), in soft connective tissues such as skeletal muscle. All familial and sporadic cases with a classic clinical presentation of FOP carry a gain-of-function mutation (R206H; c.617 G > A) in ACVR1, a cell surface receptor that mediates bone morphogenetic protein (BMP) signaling. The BMP signaling pathway is recognized for its chondro/osteogenic-induction potential, and HO in FOP patients forms ectopic but qualitatively normal endochondral bone tissue through misdirected cell fate decisions by tissue-resident mesenchymal stem cells. In addition to biochemical ligand-receptor signaling, mechanical cues from the physical environment are transduced to activate intracellular signaling, a process known as mechanotransduction, and can influence cell fates. Utilizing an established mesenchymal stem cell model of mouse embryonic fibroblasts (MEFs) from the Acvr1R206H/+ mouse model that mimics the human disease, we demonstrated that activation of the mechanotransductive effectors Rho/ROCK and YAP1 are increased in Acvr1R206H/+ cells. We show that on softer substrates, a condition associated with low mechanical signaling, the morphology of Acvr1R206H/+ cells is similar to the morphology of control Acvr1+/+ cells on stiffer substrates, a condition that activates mechanotransduction. We further determined that Acvr1R206H/+ cells are poised for osteogenic differentiation, expressing increased levels of chondro/osteogenic markers compared with Acvr1+/+ cells. We also identified increased YAP1 nuclear localization in Acvr1R206H/+ cells, which can be rescued by either BMP inhibition or Rho antagonism. Our results establish RhoA and YAP1 signaling as modulators of mechanotransduction in FOP and suggest that aberrant mechanical signals, combined with and as a result of the increased BMP pathway signaling through mutant ACVR1, lead to misinterpretation of the cellular microenvironment and a heightened sensitivity to mechanical stimuli that promotes commitment of Acvr1R206H/+ progenitor cells to chondro/osteogenic lineages.

The evolving therapeutic landscape of genetic skeletal disorders.

BACKGROUND: Rare bone diseases account for 5% of all birth defects yet very few have personalised treatments. Developments in genetic diagnosis, molecular techniques and treatment technologies however, are leading to unparalleled therapeutic advance. This review explores the evolving therapeutic landscape of genetic skeletal disorders (GSDs); the key conditions and there key differentials. METHODS: A retrospective literature based review was conducted in December 2018 using a systematic search strategy for relevant articles and trials in Pubmed and clinicaltrials.gov respectively. Over 140 articles and 80 trials were generated for review. RESULTS: Over 20 personalised therapies are discussed in addition to several novel disease modifying treatments in over 25 GSDs. Treatments discussed are at different stages from preclinical studies to clinical trials and approved drugs, including; Burosumab for X-linked hypophosphatemia, Palovarotene for Hereditary Multiple Exostoses, Carbamazepine for Metaphyseal Chondrodysplasia (Schmid type), Lithium carbonate and anti-sclerostin therapy for Osteoporosis Pseudoglioma syndrome and novel therapies for Osteopetrosis. We also discuss therapeutic advances in Achondroplasia, Osteogenesis Imperfecta (OI), Hypophosphotasia (HPP), Fibrodysplasia Ossificans Progressiva, and RNA silencing therapies in preclinical studies for OI and HPP. DISCUSSION: It is an exciting time for GSD therapies despite the challenges of drug development in rare diseases. In discussing emerging therapies, we explore novel approaches to drug development from drug repurposing to in-utero stem cell transplants. We highlight the improved understanding of bone pathophysiology, genetic pathways and challenges of developing gene therapies for GSDs.

An Adult Zebrafish Model of Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare human skeletal disease caused by constitutively activating mutations in the gene ACVR1, which encodes a type I BMP/TGFß family member receptor. FOP is characterized by progressive heterotopic ossification (HO) of fibrous tissues, including skeletal muscle, tendons, and ligaments, as well as malformation of the big toes, vertebral fusions, and osteochondromas. Surgical interventions in patients often result in enhanced HO, which can exacerbate rather than improve diagnostic outcomes. As a result of these difficulties, a variety of animal models are needed to study human FOP. Here we describe the methods for creating and characterizing zebrafish conditionally expressing Acvr1lQ204D, the first adult zebrafish model for FOP.

Inhibition of overactive TGF-ß attenuates progression of heterotopic ossification in mice.

Acquired heterotopic ossification (HO) is a painful and debilitating disease characterized by extraskeletal bone formation after injury. The exact pathogenesis of HO remains unknown. Here we show that TGF-ß initiates and promotes HO in mice. We find that calcified cartilage and newly formed bone resorb osteoclasts after onset of HO, which leads to high levels of active TGF-ß that recruit mesenchymal stromal/progenitor cells (MSPCs) in the HO microenvironment. Transgenic expression of active TGF-ß in tendon induces spontaneous HO, whereas systemic injection of a TGF-ß neutralizing antibody attenuates ectopic bone formation in traumatic and BMP-induced mouse HO models, and in a fibrodysplasia ossificans progressive mouse model. Moreover, inducible knockout of the TGF-ß type II receptor in MSPCs inhibits HO progression in HO mouse models. Our study points toward elevated levels of active TGF-ß as inducers and promoters of ectopic bone formation, and suggest that TGF-ß might be a therapeutic target in HO.

Cartilage-derived retinoic acid-sensitive protein (CD-RAP): A stage-specific biomarker of heterotopic endochondral ossification (HEO) in fibrodysplasia ossificans progressiva (FOP).

BACKGROUND: Genesis of a cartilaginous scaffold is an obligate precursor to bone formation in heterotopic endochondral ossification (HEO). We tested the hypothesis that cartilage-derived retinoic acid-sensitive protein (CD-RAP) can serve as a plasma biomarker for the pre-osseous cartilaginous stage of HEO. Palovarotene, a retinoic acid receptor-gamma (RARγ) agonist, has been proposed as a possible treatment for fibrodysplasia ossificans progressiva (FOP) and is a potent inhibitor of HEO in mouse models. Current drug development for FOP mandates the identification of stage-specific biomarkers to facilitate the evaluation of clinical trial endpoints. RESULTS: Here we show in an injury-induced, constitutively-active transgenic mouse model of FOP that CD-RAP levels peaked between day-7 and day-10 during the zenith of histologically-identified chondrogenesis, preceded radiographically apparent HEO, and were diminished by palovarotene. Cross-sectional analysis of CD-RAP levels in plasma samples from FOP patients demonstrated a statistically non-significant trend toward higher levels in the recent flare-up period (three weeks to three months within onset of symptoms). However, in a longitudinal subgroup analysis of patients followed for at least six months after resolution of flare-up symptoms, there was a statistically significant decrease of CD-RAP when compared to levels in the same patients at the time of active or recent exacerbations. CONCLUSIONS: These data support the further exploration of CD-RAP as a stage-specific biomarker of HEO in FOP.

Mast cell inhibition as a therapeutic approach in fibrodysplasia ossificans progressiva (FOP).

BACKGROUND: Episodic flare-ups of fibrodysplasia ossificans progressiva (FOP) are characterized clinically by severe, often posttraumatic, connective tissue swelling and intramuscular edema, followed histologically by an intense and highly angiogenic fibroproliferative reaction. This early inflammatory and angiogenic fibroproliferative response is accompanied by the presence of abundant mast cells far in excess of other reported myopathies. RESULTS: Using an injury-induced, constitutively-active transgenic mouse model of FOP we show that mast cell inhibition by cromolyn, but not aprepitant, results in a dramatic reduction of heterotopic ossification. Cromolyn, but not aprepitant, significantly decreases the total number of mast cells in FOP lesions. Furthermore, cromolyn specifically diminishes the number of degranulating and resting degranulated mast cells in pre-osseous lesions. CONCLUSIONS: This work demonstrates that consideration of FOP as a type of localized mastocytosis may offer new therapeutic interventions for treatment of this devastating condition.

Heterotopic ossification: Mechanistic insights and clinical challenges.

Bone formation is exquisitely controlled both spatially and temporally. Heterotopic ossification (HO) is pathological bone formation in soft tissues that often leads to deleterious outcomes. Inherited genetic forms of HO can be life-threatening and can happen as early as in infancy. However, there is currently no effective treatment for HO as the underlying cellular and molecular mechanisms have not been completely elucidated. Trauma-induced non-genetic forms of HO often occur as a common complication after surgeries or accidents, and the location of HO occurrence largely determines the symptom and outcome. While it has been difficult to determine the complicated factors causing HO, recent advancement in identifying cellular and molecular mechanism causing the genetic forms of HO may provide important insights in all HO. Here in this review, we summarize recent studies on HO to provide a current status of both clinical options of HO treatments and mechanical understanding of HO.

Retinoid roles and action in skeletal development and growth provide the rationale for an ongoing heterotopic ossification prevention trial.

The majority of skeletal elements develop via endochondral ossification. This process starts with formation of mesenchymal cell condensations at prescribed sites and times in the early embryo and is followed by chondrogenesis, growth plate cartilage maturation and hypertrophy, and replacement of cartilage with bone and marrow. This complex stepwise process is reactivated and recapitulated in physiologic conditions such as fracture repair, but can occur extraskeletally in pathologies including heterotopic ossification (HO), Ossification of the Posterior Longitudinal Ligament (OPLL) and Hereditary Multiple Exostoses (HME). One form of HO is common and is triggered by trauma, invasive surgeries or burns and is thus particularly common amongst severely wounded soldiers. There is also a congenital and very severe form of HO that occurs in children with Fibrodysplasia Ossificans Progressiva (FOP) and is driven by activating mutations in ACVR1 encoding the type I bone morphogenetic protein (BMP) receptor ALK2. Current treatments for acquired HO, including NSAIDs and local irradiation, are not always effective and can have side effects, and there is no effective treatment for HO in FOP. This review article describes the research path we took several years ago to develop a new and effective treatment for both congenital and acquired forms of HO and specifically, the testing of synthetic retinoid agonists to block the initial and critical chondrogenic step leading to HO onset and progression. We summarize studies with mouse models of injury-induced and congenital HO demonstrating the effectiveness and mode of action of the retinoid agonists, including Palovarotene. Our studies have provided the rationale for, directly led to, an ongoing phase 2 FDA clinical trial to test efficacy and safety of Palovarotene in FOP. Top-line results released a few months ago by the pharmaceutical sponsor Clementia are very encouraging. Given shared developmental pathways amongst pathologies of extraskeletal tissue formation, Palovarotene may also be effective in HME as preliminary in vitro data suggest.

Early clinical observations on the use of imatinib mesylate in FOP: A report of seven cases.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is an ultrarare genetic disorder of progressive, disabling heterotopic ossification (HO) for which there is presently no definitive treatment. Research studies have identified multiple potential targets for therapy in FOP, and novel drug candidates are being developed for testing in clinical trials. A complementary approach seeks to identify approved drugs that could be re-purposed for off-label use against defined targets in FOP. One such drug is imatinib mesylate, a tyrosine kinase inhibitor originally developed for use in patients with chronic myeloid leukemia (CML). Imatinib has the desirable effect of attacking multiple targets involved in the early hypoxic and inflammatory stages of FOP flare-ups, including HIF1-α, PDGFRα, c-KIT, and multiple MAP kinases. RESULTS: Based on compelling biologic rationale, strong preclinical data, and a favorable safety profile, imatinib has been prescribed on an off-label basis in a non-trial setting in seven children with continuous FOP flare-ups, predominantly in the axial regions, and which were not responsive to standard-of-care regimens. Anecdotal reports in these seven isolated cases document that the medication was well-tolerated with a ubiquitous reported decrease in the intensity of flare-ups in the six children who took the medication. CONCLUSIONS: These early clinical observations support the implementation of clinical trials in children with uncontrolled FOP flare-ups to determine if imatinib may ameliorate symptoms or alter the natural history of this debilitating and life-threatening disease.

Activin-A enhances mTOR signaling to promote aberrant chondrogenesis in fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare and intractable disease characterized by extraskeletal bone formation through endochondral ossification. Patients with FOP harbor point mutations in ACVR1, a type I receptor for BMPs. Although mutated ACVR1 (FOP-ACVR1) has been shown to render hyperactivity in BMP signaling, we and others have uncovered a mechanism by which FOP-ACVR1 mistransduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-ß signaling. Although Activin-A evokes enhanced chondrogenesis in vitro and heterotopic ossification (HO) in vivo, the underlying mechanisms have yet to be revealed. To this end, we developed a high-throughput screening (HTS) system using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) to identify pivotal pathways in enhanced chondrogenesis that are initiated by Activin-A. In a screen of 6,809 small-molecule compounds, we identified mTOR signaling as a critical pathway for the aberrant chondrogenesis of mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs). Two different HO mouse models, an FOP model mouse expressing FOP-ACVR1 and an FOP-iPSC-based HO model mouse, revealed critical roles for mTOR signaling in vivo. Moreover, we identified ENPP2, an enzyme that generates lysophosphatidic acid, as a linker of FOP-ACVR1 and mTOR signaling in chondrogenesis. These results uncovered the crucial role of the Activin-A/FOP-ACVR1/ENPP2/mTOR axis in FOP pathogenesis.

ACVR1-Fc suppresses BMP signaling and chondro-osseous differentiation in an in vitro model of Fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare and devastating genetic disease of heterotopic endochondral ossification (HEO), and currently no effective therapies are available for this disease. A recurrent causative heterozygous mutation (c.617 G>A; R206H) for FOP was identified in activin receptor type IA (ACVR1), a bone morphogenetic protein (BMP) type I receptor. This mutation aberrantly activates the BMP-Smad1/5/8 signaling pathway and leads to HEO in FOP patients. Here we report development of a soluble recombinant ACVR1-Fc fusion protein by combining the extracellular domain of human wild type ACVR1 and the Fc portion of human immunoglobulin gamma 1 (IgG1). The ACVR1-Fc fusion protein significantly down-regulated the dysregulated BMP signaling caused by the FOP ACVR1 mutation and effectively suppressed chondro-osseous differentiation in a previously described cellular FOP model, human umbilical vein endothelial cells (HUVECs) that were infected with adenovirus-ACVR1R206H (HUVECR206H). This ACVR1-Fc fusion protein holds great promise for prevention and treatment of HEO in FOP and related diseases.

Novel asymptomatic CNS findings in patients with ACVR1/ALK2 mutations causing fibrodysplasia ossificans progressiva.

BACKGROUND: Fibrodysplasia ossificans progressiva is an autosomal dominant disorder due to germline mutations of ACVR1/ALK2 causing progressive heterotopic endochondral ossifications. Evidence of central nervous system involvement has emerged only recently. METHODS: We performed an observational cross-sectional brain MRI study in 13 patients (8 females, mean age 20 years), examining the relationship of clinical and neuroradiological findings. RESULTS: All patients presented small asymptomatic lesions similar to hamartomas at the level of the dorsal medulla and ventral pons, associated with minor brainstem dysmorphisms and abnormal origin of the vestibulocochlear and facial nerves. The size of the brainstem lesions did not correlate with patient's age (p=0.061), age at first flare-up (p=0.733), severity of disability (p=0.194), history of head trauma (p=0.415) or hearing loss (p=0.237). The radiologic features and the absence of neurological symptoms were consistent with a benign process. Variable signal abnormalities and/or calcifications of the dentate nuclei were noted in all patients, while basal ganglia abnormalities were present in nine subjects. Brain calcifications positively correlated with patient's age (p<0.001) and severity of disability (p=0.002). CONCLUSIONS: Our data support the hypothesis that the effects of mutation of the ACVR1/ALK2 gene are extended to the central nervous system. Brainstem hamartomatous lesions and dysmorphisms, variably associated with dentate nucleus and basal ganglia signal abnormalities and/or calcifications, may represent useful disease hallmarks.