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.

TGF-Beta Induces Activin A Production in Dermal Fibroblasts Derived from Patients with Fibrodysplasia Ossificans Progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a catastrophic, ultra-rare disease of heterotopic ossification caused by genetic defects in the ACVR1 gene. The mutant ACVR1 receptor, when triggered by an inflammatory process, leads to heterotopic ossification of the muscles and ligaments. Activin A has been discovered as the main osteogenic ligand of the FOP ACVR1 receptor. However, the source of Activin A itself and the trigger of its production in FOP individuals have remained elusive. We used primary dermal fibroblasts from five FOP patients to investigate Activin A production and how this is influenced by inflammatory cytokines in FOP. FOP fibroblasts showed elevated Activin A production compared to healthy controls, both in standard culture and osteogenic transdifferentiation conditions. We discovered TGFß1 to be an FOP-specific stimulant of Activin A, shown by the upregulation of the INHBA gene and protein expression. Activin A and TGFß1 were both induced by BMP4 in FOP and control fibroblasts. Treatment with TNFα and IL6 produced negligible levels of Activin A and TGFß1 in both cell groups. We present for the first time TGFß1 as a triggering factor of Activin A production in FOP. As TGFß1 can promote the induction of the main driver of FOP, TGFß1 could also be considered a possible therapeutic target in FOP treatment.

Transcriptomic Differences Underlying the Activin-A Induced Large Osteoclast Formation in Both Healthy Control and Fibrodysplasia Ossificans Progressiva Osteoclasts.

Fibrodysplasia Ossificans Progressiva (FOP) is a very rare genetic disease characterized by progressive heterotopic ossification (HO) of soft tissues, leading to immobility and premature death. FOP is caused by a mutation in the Activin receptor Type 1 (ACVR1) gene, resulting in altered responsiveness to Activin-A. We recently revealed that Activin-A induces fewer, but larger and more active, osteoclasts regardless of the presence of the mutated ACVR1 receptor. The underlying mechanism of Activin-A-induced changes in osteoclastogenesis at the gene expression level remains unknown. Transcriptomic changes induced by Activin-A during osteoclast formation from healthy controls and patient-derived CD14-positive monocytes were studied using RNA sequencing. CD14-positive monocytes from six FOP patients and six age- and sex-matched healthy controls were differentiated into osteoclasts in the absence or presence of Activin-A. RNA samples were isolated after 14 days of culturing and analyzed by RNA sequencing. Non-supervised principal component analysis (PCA) showed that samples from the same culture conditions (e.g., without or with Activin-A) tended to cluster, indicating that the variability induced by Activin-A treatment was larger than the variability between the control and FOP samples. RNA sequencing analysis revealed 1480 differentially expressed genes induced by Activin-A in healthy control and FOP osteoclasts with p(adj) < 0.01 and a Log2 fold change of ≥±2. Pathway and gene ontology enrichment analysis revealed several significantly enriched pathways for genes upregulated by Activin-A that could be linked to the differentiation or function of osteoclasts, cell fusion or inflammation. Our data showed that Activin-A has a substantial effect on gene expression during osteoclast formation and that this effect occurred regardless of the presence of the mutated ACVR1 receptor causing FOP.

[A PhD completed. The use of periodontal ligament fibroblasts in research on fibrodysplasia ossificans progressiva]. / Serie: Hora est. Het gebruik van parodontale-ligamentfibroblasten in onderzoek naar fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva is a rare hereditary bone disease characterized by so-called heterotopic bone formation: the formation of new bone in areas of the body where bone normally never develops. Due to the formation of this heterotopic bone, approximately 70% of patients eventually also experience limitations in the mobility of the jaw, which in many cases results in a significantly reduced maximum mouth opening. Because of these jaw-related problems, teeth are sometimes extracted in these patients. Periodontal ligament fibroblasts can be isolated from these teeth, cells that play a role in both bone formation and bone breakdown. The location in the jaw area where heterotopic bone formation takes place determines the effect on maximal mouth opening. In addition, periodontal ligament fibroblasts are shown to be very useful for (fundamental) research into exceptional bone diseases such as fibrodysplasia ossificans progressiva.

Dysregulated BMP signaling through ACVR1 impairs digit joint development in fibrodysplasia ossificans progressiva (FOP).

The development of joints in the mammalian skeleton depends on the precise regulation of multiple interacting signaling pathways including the bone morphogenetic protein (BMP) pathway, a key regulator of joint development, digit patterning, skeletal growth, and chondrogenesis. Mutations in the BMP receptor ACVR1 cause the rare genetic disease fibrodysplasia ossificans progressiva (FOP) in which extensive and progressive extra-skeletal bone forms in soft connective tissues after birth. These mutations, which enhance BMP-pSmad1/5 pathway activity to induce ectopic bone, also affect skeletal development. FOP can be diagnosed at birth by symmetric, characteristic malformations of the great toes (first digits) that are associated with decreased joint mobility, shortened digit length, and absent, fused, and/or malformed phalanges. To elucidate the role of ACVR1-mediated BMP signaling in digit skeletal development, we used an Acvr1R206H/+;Prrx1-Cre knock-in mouse model that mimics the first digit phenotype of human FOP. We have determined that the effects of increased Acvr1-mediated signaling by the Acvr1R206H mutation are not limited to the first digit but alter BMP signaling, Gdf5+ joint progenitor cell localization, and joint development in a manner that differently affects individual digits during embryogenesis. The Acvr1R206H mutation leads to delayed and disrupted joint specification and cleavage in the digits and alters the development of cartilage and endochondral ossification at sites of joint morphogenesis. These findings demonstrate an important role for ACVR1-mediated BMP signaling in the regulation of joint and skeletal formation, show a direct link between failure to restrict BMP signaling in the digit joint interzone and failure of joint cleavage at the presumptive interzone, and implicate impaired, digit-specific joint development as the proximal cause of digit malformation in FOP.

Recent progress in drug development for fibrodysplasia ossificans progressiva.

Fibrodysplasia Ossificans Progressiva (FOP) is a rare genetic disease caused by heterozygous missense mutations in Activin A receptor type I which is also known as Activin-like kinase 2 (ALK2), a type I receptor of Bone Morphogenetic Proteins(BMP). Patients with FOP usually undergo episodic flare-ups and the heterotopic ossification in soft and connective tissues. Molecular mechanism study indicates that Activin A, the ligand which normally transduces Transforming Growth Factor Beta signaling, abnormally activates BMP signaling through ALK2 mutants in FOP, leading to heterotopic bone formation. To date, effective therapies to FOP are unavailable. However, significant advances have recently been made in the development of FOP drugs. In this article, we review the recent advances in understanding the FOP mechanism and drug development, with a focus on the small-molecular and antibody drugs currently in the clinical trials for FOP treatment.

Modeling human somite development and fibrodysplasia ossificans progressiva with induced pluripotent stem cells.

Somites (SMs) comprise a transient stem cell population that gives rise to multiple cell types, including dermatome (D), myotome (MYO), sclerotome (SCL) and syndetome (SYN) cells. Although several groups have reported induction protocols for MYO and SCL from pluripotent stem cells, no studies have demonstrated the induction of SYN and D from SMs. Here, we report systematic induction of these cells from human induced pluripotent stem cells (iPSCs) under chemically defined conditions. We also successfully induced cells with differentiation capacities similar to those of multipotent mesenchymal stromal cells (MSC-like cells) from SMs. To evaluate the usefulness of these protocols, we conducted disease modeling of fibrodysplasia ossificans progressiva (FOP), an inherited disease that is characterized by heterotopic endochondral ossification in soft tissues after birth. Importantly, FOP-iPSC-derived MSC-like cells showed enhanced chondrogenesis, whereas FOP-iPSC-derived SCL did not, possibly recapitulating normal embryonic skeletogenesis in FOP and cell-type specificity of FOP phenotypes. These results demonstrate the usefulness of multipotent SMs for disease modeling and future cell-based therapies.

Novel bicyclic pyrazoles as potent ALK2 (R206H) inhibitors for the treatment of fibrodysplasia ossificans progressiva.

Mutant activin receptor-like kinase-2 (ALK2) is associated with the pathogenesis of fibrodysplasia ossificans progressiva, making it an attractive target for therapeutic intervention. We synthesized a new series of bicyclic pyrazoles and evaluated their mutant ALK2 enzyme inhibitory activities, leading to the identification of 8 as the most potent inhibitor. This compound showed moderate microsomal metabolic stability and human ether-a-go-go related gene (hERG) safety. In C2C12 cells carrying mutant ALK2 (R206H), 8 efficiently inhibited the bone morphogenetic protein (BMP)-induced alkaline phosphatase activity.

Do Interactions of Vitamin D3 and BMP Signaling Hold Implications in the Pathogenesis of Fibrodysplasia Ossificans Progressiva?

PURPOSE OF REVIEW: Fibrodysplasia ossificans progressiva (FOP) is a debilitating rare disease known for episodic endochondral heterotopic ossification (HO) caused by gain-of-function mutations in ACVR1/ALK2. However, disease severity varies among patients with identical mutations suggesting disease-modifying factors, including diet, may have therapeutic implications. The roles of vitamin D3 in calcium metabolism and chondrogenesis are known, but its effects on BMP signaling and chondrogenesis are less studied. This review attempts to assess the possibility of vitamin D's effects in FOP by exploring relevant intersections of VD3 with mechanisms of FOP flares. RECENT FINDINGS: In vitro and in vivo studies suggest vitamin D suppresses inflammation, while clinical studies suggest that vitamin D3 protects against arteriosclerosis and inversely correlates with non-genetic intramuscular HO. However, the enhancement of chondrogenesis, BMP signaling, and possibly Activin A expression by vitamin D may be more relevant in FOP. There appears to be little potential for vitamin D to reduce HO in FOP, but testing the potential for excess vitamin D to promote HO may be warranted.

Screening for Small Molecule Inhibitors of BMP-Induced Osteoblastic Differentiation from Indonesian Marine Invertebrates.

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder with heterotopic ossification (HO) in soft tissues. The abnormal activation of bone morphogenetic protein (BMP) signaling by a mutant activin receptor-like kinase-2 (ALK2) leads to the development of HO in FOP patients, and, thus, BMP signaling inhibitors are promising therapeutic applications for FOP. In the present study, we screened extracts of 188 Indonesian marine invertebrates for small molecular inhibitors of BMP-induced alkaline phosphatase (ALP) activity, a marker of osteoblastic differentiation in a C2C12 cell line stably expressing ALK2(R206H) (C2C12(R206H) cells), and identified five marine sponges with potent ALP inhibitory activities. The activity-guided purification of an EtOH extract of marine sponge Dysidea sp. (No. 256) resulted in the isolation of dysidenin (1), herbasterol (2), and stellettasterol (3) as active components. Compounds 1-3 inhibited ALP activity in C2C12(R206H) cells with IC50 values of 2.3, 4.3, and 4.2 µM, respectively, without any cytotoxicity, even at 18.4-21.4 µM. The direct effects of BMP signaling examined using the Id1WT4F-luciferase reporter assay showed that compounds 1-3 did not decrease the reporter activity, suggesting that they inhibit the downstream of the Smad transcriptional step in BMP signaling.

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.

The effect of Activin-A on periodontal ligament fibroblasts-mediated osteoclast formation in healthy donors and in patients with fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a genetic disease characterized by heterotopic ossification (HO). The disease is caused by a mutation in the activin receptor type 1 (ACVR1) gene that enhances this receptor's responsiveness to Activin-A. Binding of Activin-A to the mutated ACVR1 receptor induces osteogenic differentiation. Whether Activin-A also affects osteoclast formation in FOP is not known. Therefore we investigated its effect on the osteoclastogenesis-inducing potential of periodontal ligament fibroblasts (PLF) from teeth of healthy controls and patients with FOP. We used western blot analysis of phosphorylated SMAD3 (pSMAD3) and quantitative polymerase chain reaction to assess the effect of Activin-A on the PLF. PLF-induced osteoclast formation and gene expression were studied by coculturing control and FOP PLF with CD14-positive osteoclast precursor cells from healthy donors. Osteoclast formation was also assessed in control CD14 cultures stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANK-L). Although Activin-A increased activation of the pSMAD3 pathway in both control and FOP PLF, it increased ACVR1, FK binding protein 12 (FKBP12), an inhibitor of DNA binding 1 protein (ID-1) expression only in FOP PLF. Activin-A inhibited PLF mediated osteoclast formation albeit only significantly when induced by FOP PLF. In these cocultures, it reduced M-CSF and dendritic cell-specific transmembrane protein (DC-STAMP) expression. Activin-A also inhibited osteoclast formation in M-CSF and RANK-L mediated monocultures of CD14+ cells by inhibiting their proliferation. This study brings new insight on the role of Activin A in osteoclast formation, which may further add to understanding FOP pathophysiology; in addition to the known Activin-A-mediated HO, this study shows that Activin-A may also inhibit osteoclast formation, thereby further promoting HO formation.

Therapeutic advances for blocking heterotopic ossification in fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease in which heterotopic bone forms in muscle and soft tissue, leading to joint dysfunction and significant disability. FOP is progressive and many patients are wheelchair-bound by the 3rd decade of life. FOP is caused by an activating mutation in the ACVR1 gene, which encodes the activin A Type 1 receptor. Aberrant signalling through this receptor leads to abnormal activation of the pSMAD 1/5/8 pathway and triggers the formation of bone outside of the skeleton. There is no curative therapy for FOP; however, exciting advances in novel therapies have developed recently. Here, we review the clinical and translational pharmacology of three drugs that are currently in clinical trials (palovarotene, REGN 2477 and rapamycin) as well as other emerging treatment strategies for FOP.

Neofunction of ACVR1 in fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor point mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Two mechanisms of mutated ACVR1 (FOP-ACVR1) have been proposed: ligand-independent constitutive activity and ligand-dependent hyperactivity in BMP signaling. Here, by using FOP patient-derived induced pluripotent stem cells (FOP-iPSCs), we report a third mechanism, where FOP-ACVR1 abnormally transduces BMP signaling in response to Activin-A, a molecule that normally transduces TGF-ß signaling but not BMP signaling. Activin-A enhanced the chondrogenesis of induced mesenchymal stromal cells derived from FOP-iPSCs (FOP-iMSCs) via aberrant activation of BMP signaling in addition to the normal activation of TGF-ß signaling in vitro, and induced endochondral ossification of FOP-iMSCs in vivo. These results uncover a novel mechanism of extraskeletal bone formation in FOP and provide a potential new therapeutic strategy for FOP.

Discovery of Heterotopic Bone-Inducing Activity in Hard Tissues and the TGF-ß Superfamily.

Bone is a unique organ because it can be experimentally induced in soft tissues by implanting a single growth factor, bone morphogenetic protein (BMP). Heterotopic bone-inducing activity was found in demineralized bone matrix in 1965. The characterization of this activity in bone enabled the purification and molecular cloning of BMPs and showed that they are members of the transforming growth factor-ß (TGF-ß) superfamily. Assay systems developed for this bone-inducing activity revealed the molecular mechanisms of the intracellular signaling of members of the superfamily, including BMPs. Moreover, they are being applied to elucidate molecular mechanisms and to develop novel therapeutics for a disease caused by an abnormality in BMP signaling.

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.

Fibrodysplasia ossificans progressiva. A case report and focus on the BMP signaling pathway.

Fibrodysplasia ossificans progressiva is a very rare heritable disease characterized by a progressive heterotopic endochondal ossification, occurring in the first decade of life, and leading thereafter to a severe ankylosis of the spine, limbs and jaw, with a progressive and severe functional disability. To date the cause of the disease remains unknown and no medical treatment has been proved efficient. It has recently been shown that a recurrent mutation in activation domain of the activin-receptor IA (ACVR1), a BMP receptor, could lead to an abnormal signalling pathway of BMP-4 and contribute to the occurrence of the devastating lesions characteristic of the disease.

Alk2 regulates early chondrogenic fate in fibrodysplasia ossificans progressiva heterotopic endochondral ossification.

Bone morphogenetic protein (BMP) signaling is a critical regulator of cartilage differentiation and endochondral ossification. Gain-of-function mutations in ALK2, a type I BMP receptor, cause the debilitating disorder fibrodysplasia ossificans progressiva (FOP) and result in progressive heterotopic (extraskeletal) endochondral ossification within soft connective tissues. Here, we used murine mesenchymal progenitor cells to investigate the contribution of Alk2 during chondrogenic differentiation and heterotopic endochondral ossification (HEO). Alk2(R206H/+) (gain-of-function), Alk2(CKO) (loss-of-function), and wild-type mouse embryonic fibroblasts were evaluated for chondrogenic potential. Chondrogenic differentiation was accelerated in Alk2(R206H/+) cells, due in part to enhanced sensitivity to BMP ligand. In vivo, Alk2(R206H/+) cells initiated robust HEO and recruited wild-type cell contribution. Despite expression of other type I BMP receptors (Alk3 and Alk6), chondrogenesis of Alk2(CKO) cells was severely impaired by absence of Alk2 during early differentiation. Alk2 is therefore a direct regulator of cartilage formation and mediates chondrogenic commitment of progenitor cells. These data establish that at least one effect of ALK2 gain-of-function mutations in FOP patients is enhanced chondrogenic differentiation which supports formation of heterotopic endochondral bone. This establishes ALK2 as a plausible therapeutic target during early chondrogenic stages of lesion formation for preventing heterotopic bone formation in FOP and other conditions.

Establishment of a novel model of chondrogenesis using murine embryonic stem cells carrying fibrodysplasia ossificans progressiva-associated mutant ALK2.

Fibrodysplasia ossificans progressiva (FOP) is a genetic disorder characterized by heterotopic endochondral ossification in soft tissue. A mutation in the bone morphogenetic protein (BMP) receptor ALK2, R206H, has been identified in patients with typical FOP. In the present study, we established murine embryonic stem (ES) cells that express wild-type human ALK2 or typical mutant human ALK2 [ALK2(R206H)] under the control of the Tet-Off system. Although wild-type ALK2 and mutant ALK2(R206H) were expressed in response to a withdrawal of doxycycline (Dox), BMP signaling was activated only in the mutant ALK2(R206H)-expressing cells without the addition of exogenous BMPs. The Dox-dependent induction of BMP signaling was blocked by a specific kinase inhibitor of the BMP receptor. The mutant ALK2(R206H)-carrying cells showed Dox-regulated chondrogenesis in vitro, which occurred in co-operation with transforming growth factor-ß1 (TGF-ß1). Overall, our ES cells are useful for studying the molecular mechanisms of heterotopic ossification in FOP in vitro and for developing novel inhibitors of chondrogenesis induced by mutant ALK2(R206H) associated with FOP.