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Osteopenia is frequently observed in patients with iron overload, especially in those with HFE-dependent hereditary hemochromatosis (HH). Interestingly, not all mouse models of HH show bone loss, suggesting that iron overload alone may not suffice to induce bone loss. In this study, the bone phenotypes of Hjv -/- and hepatocyte-specific Alk2- and Alk3-deficient mice as additional mouse models of HH were investigated to further clarify, how high iron levels lead to bone loss and which signaling mechanisms are operational. Neither male nor female 12-week-old Hjv -/- mice had an altered trabecular or cortical bone mass or bone turnover, despite severe iron overload. Male 12-month-old Hjv -/- mice even presented with a higher femoral trabecular bone volume compared to wildtype mice. Similarly, female mice with hepatocyte-specific Alk2 or Alk3 deficiency did not show an altered bone phenotype at 3, 6, and 12 months of age. Male hepatocyte-specific Alk3-deficient mice also had a normal trabecular bone mass at all ages analyzed, despite showing increased bone resorption and decreased bone formation parameters. Interestingly, hepatocyte-specific Alk2-deficient mice showed reduced femoral trabecular bone at 6 months of age due to suppressed bone formation. Cortical thickness at the femur was reduced in both, 6-month-old male hepatocyte-specific Alk2- and Alk3-deficient mice. Raising hepatocyte-specific Alk2-deficient male mice on an iron-deficient diet rescued the bone phenotype. Taken together, despite iron overload, trabecular bone microarchitecture was not altered in mice deficient of Hjv or Alk3. Only male hepatocyte-specific Alk2-deficient mice showed site-specific lower trabecular and cortical bone mass at the femur, which was dependent on iron. Thus, bone loss does not correlate with the extent of iron overload in these mouse models, but may relate to the amount of iron-loaded macrophages, as precursors of osteoclasts, in the bone marrow., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Activin receptor type 1 (ACVR1; ALK2) and activin receptor like type 1 (ACVRL1; ALK1) are transforming growth factor beta family receptors that integrate extracellular signals of bone morphogenic proteins (BMPs) and activins into Mothers Against Decapentaplegic homolog 1/5 (SMAD1/SMAD5) signaling complexes. Several activating mutations in ALK2 are implicated in fibrodysplasia ossificans progressiva (FOP), diffuse intrinsic pontine gliomas, and ependymomas. The ALK2 R206H mutation is also present in a subset of endometrial tumors, melanomas, non-small lung cancers, and colorectal cancers, and ALK2 expression is elevated in pancreatic cancer. Using DNA-encoded chemistry technology, we screened 3.94 billion unique compounds from our diverse DNA-encoded chemical libraries (DECLs) against the kinase domain of ALK2. Off-DNA synthesis of DECL hits and biochemical validation revealed nanomolar potent ALK2 inhibitors. Further structure-activity relationship studies yielded center for drug discovery (CDD)-2789, a potent [NanoBRET (NB) cell IC 50 : 0.54 μM] and metabolically stable analog with good pharmacological profile. Crystal structures of ALK2 bound with CDD-2281, CDD-2282, or CDD-2789 show that these inhibitors bind the active site through Van der Waals interactions and solvent-mediated hydrogen bonds. CDD-2789 exhibits high selectivity toward ALK2/ALK1 in KINOMEscan analysis and NB K192 assay. In cell-based studies, ALK2 inhibitors effectively attenuated activin A and BMP-induced Phosphorylated SMAD1/5 activation in fibroblasts from individuals with FOP in a dose-dependent manner. Thus, CDD-2789 is a valuable tool compound for further investigation of the biological functions of ALK2 and ALK1 and the therapeutic potential of specific inhibition of ALK2., Competing Interests: Competing interests statement:A provisional patent was submitted by R.J., D.M., K.M.B., F.L., J.-Y.L., Z.T. D.W.Y., S.S.P., and M.M.M. from Baylor College of Medicine.

The acquisition of invasive properties is a prerequisite for tumor progression and metastasis. Molecular subtypes of KRAS-driven lung cancer exhibit distinct modes of invasion that contribute to unique growth properties and therapeutic susceptibilities. Despite this, preclinical strategies designed to exploit growth within the context of invasion are lacking. To address this, we designed an experimental system to screen for targetable signaling pathways linked to active early 3D invasion phenotypes in different molecular subtypes of KRAS-driven lung adenocarcinoma. Combined live-cell imaging of human bronchial epithelial cells in a 3D invasion matrix and transcriptomic profiling identified mutant LKB1-specific upregulation of BMP6. LKB1 loss increased BMP6 signaling, which induced the canonical iron regulatory hormone hepcidin. Intact LKB1 was necessary to maintain BMP6 signaling homeostasis and restrict ALK2/BMP6-fueled growth. Preclinical studies in a Kras/Lkb1-mutant syngeneic mouse model and in a xenograft model showed potent growth suppression by inhibiting the ALK2/BMP6 signaling axis with single-agent inhibitors that are currently in clinical trials. Lastly, BMP6 expression was elevated in tumors of patients with LKB1-mutant early-stage lung cancer. These results are consistent with those of a model in which LKB1 acts as a "brake" to iron-regulated growth and suggest that ALK2 inhibition can be used for patients with LKB1-mutant tumors. Significance: Three-dimensional invasion-linked gene expression analysis reveals a therapeutic vulnerability to inhibition of ALK2/BMP6 signaling in LKB1-mutant lung cancer that can be rapidly translated to the clinic., (©2024 The Authors; Published by the American Association for Cancer Research.)

The proliferation of the endothelium is a highly coordinated process to ensure the emergence, expansion, and homeostasis of the vasculature. While Bone Morphogenetic Protein (BMP) signaling fine-tunes the behaviors of endothelium in health and disease, how BMP signaling influences the proliferation of endothelium and therefore, modulates angiogenesis remains largely unknown. Here, we evaluated the role of Activin A Type I Receptor (ACVR1/ALK2), a key BMP receptor in the endothelium, in modulating the proliferation of endothelial cells. We show that ACVR1/ALK2 is a key modulator for the proliferation of endothelium in the retinal vessels. Loss of endothelial ALK2 leads to a significant reduction in endothelial proliferation and results in fewer branches/endothelial cells in the retinal vessels. Interestingly, venous endothelium appears to be more susceptible to ALK2 deletion. Mechanistically, ACVR1/ALK2 inhibits the expression of CDKN1A/p21, a critical negative regulator of cell cycle progression, in a SMAD1/5-dependent manner, thereby enabling the venous endothelium to undergo active proliferation by suppressing CDKN1A/p21. Taken together, our findings show that BMP signaling mediated by ACVR1/ALK2 provides a critical yet previously underappreciated input to modulate the proliferation of venous endothelium, thereby fine-tuning the context of angiogenesis in health and disease., Competing Interests: Declarations Competing interests None., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Pediatric diffuse midline gliomas (pDMG) are an aggressive type of childhood cancer with a fatal outcome. Their major epigenetic determinism has become clear, notably with the identification of K27M mutations in histone H3. However, the synergistic oncogenic mechanisms that induce and maintain tumor cell phenotype have yet to be deciphered. In 20 to 30% of cases, these tumors have an altered BMP signaling pathway with an oncogenic mutation on the BMP type I receptor ALK2, encoded by ACVR1 . However, the potential impact of the BMP pathway in tumors non-mutated for ACVR1 is less clear. By integrating bulk, single-cell, and spatial transcriptomic data, we show here that the BMP signaling pathway is activated at similar levels between ACVR1 wild-type and mutant tumors and identify BMP2 and BMP7 as putative activators of the pathway in a specific subpopulation of cells. By using both pediatric isogenic glioma lines genetically modified to overexpress H3.3K27M and patients-derived DIPG cell lines, we demonstrate that BMP2/7 synergizes with H3.3K27M to induce a transcriptomic rewiring associated with a quiescent but invasive cell state. These data suggest a generic oncogenic role for the BMP pathway in gliomagenesis of pDMG and pave the way for specific targeting of downstream effectors mediating the K27M/BMP crosstalk., Competing Interests: PH, SM, CB, MH, AB, AR, LM, JT, NL, PL, MC, LB, AD, IR, JB, CD, VA, AM, ML, EP, AV, PG, SM, PL, VR, EC, MC No competing interests declared, (© 2023, Huchede et al.)

Rare diseases and conditions have thus far received relatively less attention in the field of precision/personalized medicine than common chronic diseases. There is a dire need for orphan drug discovery and therapeutics in ways that are informed by the precision/personalized medicine scholarship. Moreover, people with rare conditions, when considered collectively across diseases worldwide, impact many communities. In this overarching context, Activin A Receptor Type 1 (ACVR1) is a transmembrane kinase from the transforming growth factor-β superfamily and plays a critical role in modulating the bone morphogenetic protein signaling. Missense variants of the ACVR1 gene result in modifications in structure and function and, by extension, abnormalities and have been predominantly linked with two rare conditions: fibrodysplasia ossificans progressiva and diffuse intrinsic pontine glioma. We report here an extensive bioinformatic analyses assessing the pool of 50,951 variants and forecast seven highly destabilizing mutations (R206H, G356D, R258S, G328W, G328E, R375P, and R202I) that can significantly alter the structure and function of the native protein. Protein-protein interaction and ConSurf analyses revealed the crucial interactions and localization of highly deleterious mutations in highly conserved domains that may impact the binding and functioning of the protein. cBioPortal, CanSAR Black, and existing literature affirmed the association of these destabilizing mutations with posterior fossa ependymoma, uterine corpus carcinoma, and pediatric brain cancer. The current findings suggest these deleterious nonsynonymous single nucleotide polymorphisms as potential candidates for future functional annotations and validations associated with rare conditions, further aiding the development of precision medicine in rare diseases.

Activin E activates brown and beige adipocytes and has been controversially implicated as a factor that induces obesity and fatty liver. Here, we sought to address this controversial issue by producing recombinant human activin E to evaluate its effects on HB2 brown adipocytes in vitro. Activin E increased uncoupling protein 1 (Ucp1) and fibroblast growth factor 21 (Fgf21) mRNA expression in the adipocytes. This upregulation was suppressed by SB431542, an inhibitor of activin receptor-like kinase (Alk) TGF-β type I receptors. SB431542 also inhibited the activin E-induced phosphorylation of Smad2/3. A promoter assay using a CAGA-Luc reporter and Alk expression vectors revealed that activin E activated the TGF-β/activin pathway via Alk7. The upregulation of Ucp1 and Fgf21 mRNA might be mediated through Alk7 and Smad2/3 phosphorylation. Activin E is a potential stimulator of energy expenditure by activating brown adipocytes and highlights its potential as a therapeutic target for treating obesity., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The diagnosis of fibrodysplasia ossificans progressiva is missed or delayed because of its insidious precursors, especially in uncharacteristic cases. Fibrodysplasia ossificans progressiva, which mostly displayed the mutation c.617G > A, p.R206H, is characterized by congenital malformation of the great toe and progressive extra-skeletal ossification of ligaments, tendons and muscles. The mutation c.774G > C, p.R258S (HGVS: NC_000002.11:g.158626896 C > G) in activin A receptor type I is an infrequent etiology of fibrodysplasia ossificans progressiva and can present different clinical features. Awareness of these multiple clinical features will help endocrinologists in the early diagnosis of fibrodysplasia ossificans progressiva. We report a case of fibrodysplasia ossificans progressiva with the activin A receptor type I mutation c.774G > C, p.R258S, which was diagnosed before its ossifying period., (© 2024. The Author(s).)

A 2-year-and-10-month-old boy presented with multiple masses in the neck and chest for over 3 months. The child had a history of unstable walking, with hard lumps visible at the injury sites after falls, which would resolve on their own. Following a recent injury, a mass was discovered in the posterior neck, protruding above the skin surface and accompanied by limited joint movement. Gradually, new masses were found on the left side of the neck, back near the scapular lower angle, in the scapular fossa, and along the left axillary midline. Magnetic resonance imaging examination showed diffuse low signal on T1-weighted images and high signal on T2-weighted images in the bilateral posterior neck and back muscles two months ago. A CT scan revealed muscle swelling, with areas of patchy low density and multiple nodular high-density ossifications within some muscles. Genetic testing results indicated a mutation in the ACVR1 gene, leading to the final diagnosis of progressive ossifying myositis in this patient. This article summarizes the etiology, diagnosis, and treatment of one case of progressive ossifying myositis, providing a reference for clinicians.

When a genetic disease is characterized by the abnormal activation of normal molecular pathways and cellular events, it is illuminating to critically examine the places and times of these activities both in health and disease. Therefore, because heterotopic ossification (HO) in fibrodysplasia ossificans progressiva (FOP) is by far the disease's most prominent symptom, attention is also directed toward the pathways and processes of bone formation during skeletal development. FOP is recognizable by effects of the causative mutation on skeletal development even before HO manifests, specifically in the malformation of the great toes. This signature skeletal phenotype is the most highly penetrant, but is only one among several skeletal abnormalities associated with FOP. Patients may present clinically with joint malformation and ankylosis, particularly in the cervical spine and costovertebral joints, as well as characteristic facial features and a litany of less common, non-skeletal symptoms, all stemming from missense mutations in the ACVR1 gene. In the same way that studying the genetic cause of HO advanced our understanding of HO initiation and progression, insight into the roles of ACVR1 signaling during tissue development, particularly in the musculoskeletal system, can be gained from examining altered skeletal development in individuals with FOP. This review will detail what is known about the molecular mechanisms of developmental phenotypes in FOP and the early role of ACVR1 in skeletal patterning and growth, as well as highlight how better understanding these processes may serve to advance patient care, assessments of patient outcomes, and the fields of bone and joint biology.

Abstract: Hemojuvelin (HJV) is a glycosylphosphatidylinositol-anchored protein of the repulsive guidance molecule family acting as a bone morphogenetic protein (BMP) coreceptor to induce the hepatic iron regulatory protein hepcidin. Hepcidin causes ubiquitination and degradation of the sole known iron exporter ferroportin, thereby limiting iron availability. The detailed signaling mechanism of HJV in vivo has yet to be investigated. In the current manuscript, we used an established model of adeno-associated virus (AAV)-mediated liver-specific overexpression of HJV in murine models of hepatocyte-specific deficiency of the BMP type I receptors Alk2 or Alk3. In control mice, HJV overexpression increased hepatic Hamp messenger RNA (mRNA) levels, soluble HJV (sHJV), splenic iron content (SIC), as well as phosphorylated small mothers against decapentaplegic protein (pSMAD1/5/8) levels. In contrast, in Alk2fl/fl;Alb-Cre and Alk3fl/fl;Alb-Cre mice, which present with moderate and severe iron overload, respectively, the administration of AAV-HJV induced HJV and sHJV. However, it did not rescue the iron overload phenotypes of those mice. Serum iron levels were induced in Alk2fl/fl;Alb-Cre mice after HJV overexpression. In phosphate-buffered saline-injected Alk3fl/fl;Alb-Cre mice, serum iron levels and the expression of duodenal ferroportin remained high, whereas Hamp mRNA levels were decreased to 1% to 5% of the levels detected in controls. This was reduced even further by AAV-HJV overexpression. SIC remained low in mice with hepatocyte-specific Alk2 or Alk3 deficiency, reflecting disturbed iron homeostasis with high serum iron levels and transferrin saturation and an inability to induce hepcidin by HJV overexpression. The data indicate that ALK2 and ALK3 are both required in vivo for the HJV-mediated induction of hepcidin., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The functional role of TGFβ type I receptor, activin-like kinase (ALK)-1 in post-myocardial infarction (MI) cardiac remodeling is unknown. We hypothesize that reduced ALK1 activity reduces survival and promotes cardiac fibrosis after MI. MI was induced in wild-type (WT), and ALK +/- mice by left coronary ligation. After 14 days ALK1 +/- mice had reduced survival with a higher rate of cardiac rupture compared to WT mice. ALK1 +/- left ventricles (LVs) had increased volumes at the end of systole and at the end of diastole. After MI ALK1 +/- LVs had increased profibrotic SMAD3 signaling, type 1 collagen, and fibrosis as well as increased levels of TGFβ1 co-receptor, endoglin, VEGF, and ALK1 ligands BMP9 and BMP10. ALK1 +/- LVs had decreased levels of stromal-derived factor 1α. These data identify the critical role of ALK1 in post-MI survival and cardiac remodeling and implicate ALK1 as a potential therapeutic target to improve survival after MI., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Intrahepatic cholangiocarcinoma (ICC) is a kind of hepatobiliary tumor that is increasing in incidence and mortality. The gut microbiota plays a role in the onset and progression of cancer, however, the specific mechanism by which the gut microbiota acts on ICC remains unclear. In this study, feces and plasma from healthy controls and ICC patients were collected for 16S rRNA sequencing or metabolomics analysis. Gut microbiota analysis showed that gut microbiota abundance and biodiversity were altered in ICC patients compared with controls. Plasma metabolism analysis showed that the metabolite glutamine content of the ICC patient was significantly higher than that of the controls. KEGG pathway analysis showed that glutamine plays a vital role in ICC. In addition, the use of antibiotics in ICC animals further confirmed that changes in gut microbiota affect changes in glutamine. Further experiments showed that supplementation with glutamine inhibited ferroptosis and downregulated ALK5 and NOX1 expression in HuCCT1 cells. ALK5 overexpression or NOX1 overexpression increased NOX1, p53, PTGS2, ACSL4, LPCAT3, ROS, MDA and Fe 2+ and decreased FTH1, SLC7A11 and GSH. Knockdown of NOX1 suppressed FIN56-induced ferroptosis. In vivo, supplementation with glutamine promoted tumor growth. Overexpression of ALK5 repressed tumor growth and induced ferroptosis in nude mice, which could be reversed by the addition of glutamine. Our results suggested that the gut microbiota altered glutamine metabolism to inhibit ferroptosis in ICC by regulating the ALK5/NOX1 axis., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aims: Bone morphogenetic protein-9 (BMP9) is critical for bone morphogenetic protein receptor type-2 (BMPR2) signalling in pulmonary vascular endothelial cells. Furthermore, human genetics studies support the central role of disrupted BMPR2 mediated BMP9 signalling in vascular endothelial cells in the initiation of pulmonary arterial hypertension (PAH). In addition, loss-of-function mutations in BMP9 have been identified in PAH patients. BMP9 is considered to play an important role in vascular homeostasis and quiescence., Methods and Results: We identified a novel BMP9 target as the class-3 semaphorin, SEMA3G. Although originally identified as playing a role in neuronal development, class-3 semaphorins may have important roles in endothelial function. Here we show that BMP9 transcriptional regulation of SEMA3G occurs via ALK1 and the canonical Smad pathway, requiring both Smad1 and Smad5. Knockdown studies demonstrated redundancy between type-2 receptors in that BMPR2 and ACTR2A were compensatory. Increased SEMA3G expression by BMP9 was found to be regulated by the transcription factor, SOX17. Moreover, we observed that SEMA3G regulates VEGF signalling by inhibiting VEGFR2 phosphorylation and that VEGF, in contrast to BMP9, negatively regulated SEMA3G transcription. Functional endothelial cell assays of VEGF-mediated migration and network formation revealed that BMP9 inhibition of VEGF was abrogated by SEMA3G knockdown. Conversely, treatment with recombinant SEMA3G partially mimicked the inhibitory action of BMP9 in these assays., Conclusions: This study provides further evidence for the anti-angiogenic role of BMP9 in microvascular endothelial cells and these functions are mediated at least in part via SOX17 and SEMA3G induction., Competing Interests: Declaration of competing interest P.D.U. and N.W.M. have published US (US10336800) and EU (EP3166628B1) patents entitled: ‘Therapeutic Use of Bone Morphogenetic Proteins’. All other authors declare no conflicts of interests. The funders had no role in the study's design, in the collection, analyses, interpretation of data or writing of the manuscript., (Copyright © 2023. Published by Elsevier Inc.)

Background: ID3 (inhibitor of DNA binding/differentiation-3) is a transcription factor that enables metastasis by promoting stem cell-like properties in endothelial and tumor cells. The milk thistle flavonolignan silibinin is a phytochemical with anti-metastatic potential through largely unknown mechanisms., Hypothesis/purpose: We have mechanistically investigated the ability of silibinin to inhibit the aberrant activation of ID3 in brain endothelium and non-small cell lung cancer (NSCLC) models., Methods: Bioinformatic analyses were performed to investigate the co-expression correlation between ID3 and bone morphogenic protein (BMP) ligands/BMP receptors (BMPRs) genes in NSCLC patient datasets. ID3 expression was assessed by immunoblotting and qRT-PCR. Luciferase reporter assays were used to evaluate the gene sequences targeted by silibinin to regulate ID3 transcription. In silico computational modeling and LanthaScreen TR-FRET kinase assays were used to characterize and validate the BMPR inhibitory activity of silibinin. Tumor tissues from NSCLC xenograft models treated with oral silibinin were used to evaluate the in vivo anti-ID3 effects of silibinin., Results: Analysis of lung cancer patient datasets revealed a top-ranked positive association of ID3 with the BMP9 endothelial receptor ACVRL1/ALK1 and the BMP ligand BMP6. Silibinin treatment blocked the BMP9-induced activation of the ALK1-phospho-SMAD1/5-ID3 axis in brain endothelial cells. Constitutive, acquired, and adaptive expression of ID3 in NSCLC cells were all significantly downregulated in response to silibinin. Silibinin blocked ID3 transcription via BMP-responsive elements in ID3 gene enhancers. Silibinin inhibited the kinase activities of BMPRs in the micromolar range, with the lower IC 50 values occurring against ACVRL1/ALK1 and BMPR2. In an in vivo NSCLC xenograft model, tumoral overexpression of ID3 was completely suppressed by systematically achievable oral doses of silibinin., Conclusions: ID3 is a largely undruggable metastasis-promoting transcription factor. Silibinin is a novel suppressor of ID3 that may be explored as a novel therapeutic approach to interfere with the metastatic dissemination capacity of NSCLC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Exercise has beneficial effects on cognition throughout the lifespan. Here, we demonstrate that specific exercise patterns transform insufficient, subthreshold training into long-term memory in mice. Our findings reveal a potential molecular memory window such that subthreshold training within this window enables long-term memory formation. We performed RNA-seq on dorsal hippocampus and identify genes whose expression correlate with conditions in which exercise enables long-term memory formation. Among these genes we found Acvr1c, a member of the TGF ß family. We find that exercise, in any amount, alleviates epigenetic repression at the Acvr1c promoter during consolidation. Additionally, we find that ACVR1C can bidirectionally regulate synaptic plasticity and long-term memory in mice. Furthermore, Acvr1c expression is impaired in the aging human and mouse brain, as well as in the 5xFAD mouse model, and over-expression of Acvr1c enables learning and facilitates plasticity in mice. These data suggest that promoting ACVR1C may protect against cognitive impairment., (© 2024. The Author(s).)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

EZH2 is the catalytic subunit of the histone methyltransferase Polycomb Repressive Complex 2 (PRC2), and its somatic activating mutations drive lymphoma, particularly the germinal center B-cell type. Although PRC2 inhibitors, such as tazemetostat, have demonstrated anti-lymphoma activity in patients, the clinical efficacy is not limited to EZH2-mutant lymphoma. In this study, Activin A Receptor Type 1 (ACVR1), a type I Bone Morphogenetic Protein (BMP) receptor, is identified as critical for the anti-lymphoma efficacy of PRC2 inhibitors through a whole-genome CRISPR screen. BMP6, BMP7, and ACVR1 are repressed by PRC2-mediated H3K27me3, and PRC2 inhibition upregulates their expression and signaling in cell and patient-derived xenograft models. Through BMP-ACVR1 signaling, PRC2 inhibitors robustly induced cell cycle arrest and B cell lineage differentiation in vivo. Remarkably, blocking ACVR1 signaling using an inhibitor or genetic depletion significantly compromised the in vitro and in vivo efficacy of PRC2 inhibitors. Furthermore, high levels of BMP6 and BMP7, along with ACVR1, are associated with longer survival in lymphoma patients, underscoring the clinical relevance of this study. Altogether, BMP-ACVR1 exhibits anti-lymphoma function and represents a critical PRC2-repressed pathway contributing to the efficacy of PRC2 inhibitors., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Fibrodysplasia ossificans progressiva (FOP) is an exceedingly rare human genetic disorder characterized by the progressive and incapacitating formation of ectopic bone outside the skeleton. We report a case of FOP patient with mutations within the ACVR1 gene (c.982G>A; p.G328R). 18 F-FDG positron emission tomography/computed tomography (PET/CT) was carried out for disease assessment. Previous studies have shown increased FDG uptake in regions of heterotopic ossification (HO) in FOP. However, in our study, the PET/CT features demonstrate that active ossificans exhibit increased 18 F-FDG uptake, whereas end-stage ossifications do not. Collectively, 18 F-FDG PET/CT emerges as a prospective approach to evaluate medication efficacy in the early stages, directing early intervention and pharmacological management of FOP before ossifications formation., (Copyright © 2023 Société française de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.)

Background & Aims: Genome-wide studies have identified three missense variants in the human gene ACVR1C, encoding the TGF-β superfamily receptor ALK7, that correlate with altered waist-to-hip ratio adjusted for body mass index (WHR/BMI), a measure of body fat distribution., Methods: To move from correlation to causation and understand the effects of these variants on fat accumulation and adipose tissue function, we introduced each of the variants in the mouse Acvr1c locus and investigated metabolic phenotypes in comparison with a null mutation., Results: Mice carrying the I195T variant showed resistance to high fat diet (HFD)-induced obesity, increased catecholamine-induced adipose tissue lipolysis and impaired ALK7 signaling, phenocopying the null mutants. Mice with the I482V variant displayed an intermediate phenotype, with partial resistance to HFD-induced obesity, reduction in subcutaneous, but not visceral, fat mass, decreased systemic lipolysis and reduced ALK7 signaling. Surprisingly, mice carrying the N150H variant were metabolically indistinguishable from wild type under HFD, although ALK7 signaling was reduced at low ligand concentrations., Conclusion: Together, these results validate ALK7 as an attractive drug target in human obesity and suggest a lower threshold for ALK7 function in humans compared to mice., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the results reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

We identified activin A receptor type I (ACVR1), a member of the TGF-β superfamily, as a factor favoring acute myeloid leukemia (AML) growth and a new potential therapeutic target. ACVR1 is overexpressed in FLT3-mutated AML and inhibition of ACVR1 expression sensitized AML cells to FLT3 inhibitors. We developed a novel ACVR1 inhibitor, TP-0184, which selectively caused growth arrest in FLT3-mutated AML cell lines. Molecular docking and in vitro kinase assays revealed that TP-0184 binds to both ACVR1 and FLT3 with high affinity and inhibits FLT3/ACVR1 downstream signaling. Treatment with TP-0184 or in combination with BCL2 inhibitor, venetoclax dramatically inhibited leukemia growth in FLT3-mutated AML cell lines and patient-derived xenograft models in a dose-dependent manner. These findings suggest that ACVR1 is a novel biomarker and plays a role in AML resistance to FLT3 inhibitors and that FLT3/ACVR1 dual inhibitor TP-0184 is a novel potential therapeutic tool for AML with FLT3 mutations., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Background & Aims: Pancreatic ductal adenocarcinoma can develop from precursor lesions, including pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasm (IPMN). Previous studies indicated that loss of Acvr1b accelerates the Kras-mediated development of papillary IPMN in the mouse pancreas; however, the cell type predominantly affected by these genetic changes remains unclear., Methods: We investigated the contribution of cellular origin by inducing IPMN associated mutations (KRAS G12D expression and Acvr1b loss) specifically in acinar (Ptf1a CreER ;Kras LSL-G12D ;Acvr1b fl/fl mice) or ductal (Sox9CreER;Kras LSL-G12D ;Acvr1b fl/fl mice) cells in mice. We then performed magnetic resonance imaging and a thorough histopathologic analysis of their pancreatic tissues., Results: The loss of Acvr1b increased the development of pancreatic intraepithelial neoplasia and IPMN-like lesions when either acinar or ductal cells expressed a Kras mutation. Magnetic resonance imaging, immunohistochemistry, and histology revealed large IPMN-like lesions in these mice that exhibited features of flat, gastric epithelium. In addition, cyst formation in both mouse models was accompanied by chronic pancreatitis. Experimental acute pancreatitis accelerated the development of large mucinous cysts and pancreatic intraepithelial neoplasia when acinar, but not ductal, cells expressed mutant Kras and lost Acvr1b., Conclusions: These findings indicate that loss of Acvr1b in the presence of the Kras oncogene promotes the development of large and small precancerous lesions from both ductal and acinar cells. However, the IPMN-like phenotype was not equivalent to that observed when these mutations were made in all pancreatic cells during development. Our study underscores the significance of the cellular context in the initiation and progression of precursor lesions from exocrine cells., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare genetic disorder characterized by progressive disabling heterotopic ossification (HO) at extra-skeletal sites. Here, we developed adeno-associated virus (AAV)-based gene therapy that suppresses trauma-induced HO in FOP mice harboring a heterozygous allele of human ACVR1 R206H ( Acvr1 R206H/+ ) while limiting the expression in non-skeletal organs such as the brain, heart, lung, liver, and kidney. AAV gene therapy carrying the combination of codon-optimized human ACVR1 (ACVR1 opt ) and artificial miRNAs targeting Activin A and its receptor ACVR1 R206H ablated the aberrant activation of BMP-Smad1/5 signaling and the osteogenic differentiation of Acvr1 R206H/+ skeletal progenitors. The local delivery of AAV gene therapy to HO-causing cells in the skeletal muscle resulted in a significant decrease in endochondral bone formation in Acvr1 R206H/+ mice. These mice showed little to no expression in a major AAV-targeted organ, the liver, due to liver-abundant miR-122-mediated repression. Thus, AAV gene therapy is a promising therapeutic strategy to explore in suppressing HO in FOP.

Fibrodysplasia ossificans progressiva (FOP) is a rare human genetic condition characterized by altered skeletal development and extraskeletal bone formation. All cases of FOP are caused by mutations in the type I bone morphogenetic protein (BMP) receptor gene ACVR1 that result in overactivation of the BMP signaling pathway. Activation of the wild-type ACVR1 kinase requires assembly of a tetrameric type I and II BMP receptor complex followed by phosphorylation of the ACVR1 GS domain by type II BMP receptors. Previous studies showed that the FOP-mutant ACVR1-R206H required type II BMP receptors and presumptive glycine/serine-rich (GS) domain phosphorylation for overactive signaling. Structural modeling of the ACVR1-R206H mutant kinase domain supports the idea that FOP mutations alter the conformation of the GS domain, but it is unclear how this leads to overactive signaling. Here we show, using a developing zebrafish embryo BMP signaling assay, that the FOP-mutant receptors ACVR1-R206H and -G328R have reduced requirements for GS domain phosphorylatable sites to signal compared to wild-type ACVR1. Further, ligand-independent and ligand-dependent signaling through the FOP-mutant ACVR1 receptors have distinct GS domain phosphorylatable site requirements. ACVR1-G328R showed increased GS domain serine/threonine requirements for ligand-independent signaling compared to ACVR1-R206H, whereas it exhibited reduced serine/threonine requirements for ligand-dependent signaling. Remarkably, while ACVR1-R206H does not require the type I BMP receptor partner, Bmpr1, to signal, a ligand-dependent GS domain mutant of ACVR1-R206H could signal independently of Bmpr1 only when Bmp7 ligand was overexpressed. Of note, unlike human ACVR1-R206H, the zebrafish paralog Acvr1l-R203H does not show increased signaling activity. However, in domain-swapping studies, the human kinase domain, but not the human GS domain, was sufficient to confer overactive signaling to the Acvr1l-R203H receptor. Together these results reflect the importance of GS domain activation and kinase domain functions in regulating ACVR1 signaling and identify mechanisms of reduced regulatory constraints conferred by FOP mutations. © 2023 American Society for Bone and Mineral Research (ASBMR)., (© 2023 American Society for Bone and Mineral Research (ASBMR).)

ALK7, a type I receptor for the transforming growth factor-β superfamily, is known to be predominantly expressed in adipocytes in both mice and humans. The present review describes recent findings suggesting that ALK7 plays a major role in regulating lipid metabolism and fat mass. Furthermore, the ligands and upstream regulators that activate ALK7 signaling are discussed. The focus is on findings in mice and their derivative tissues and cells that harbor the mutations of ALK7 and related molecules. Particular attention is paid to the contradictory nature of the current literature about the loss-of-function phenotypes and the relationship with insulin secretion and sensitivity. Additional attention is paid to the ALK7 gene variants found in humans and their associated traits. The goal is to seek a parsimonious, and preferably singular and unified, description of the underlying mechanism. This review also introduces recent promising findings about ALK7 neutralizing treatment to obese mice.

Body fat distribution is a heritable risk factor for cardiovascular and metabolic disease. In humans, rare Inhibin beta E ( INHBE , activin E) loss-of-function variants are associated with a lower waist-to-hip ratio and protection from type 2 diabetes. Hepatic fatty acid sensing promotes INHBE expression during fasting and in obese individuals, yet it is unclear how the hepatokine activin E governs body shape and energy metabolism. Here, we uncover activin E as a regulator of adipose energy storage. By suppressing β-agonist-induced lipolysis, activin E promotes fat accumulation and adipocyte hypertrophy and contributes to adipose dysfunction in mice. Mechanistically, we demonstrate that activin E elicits its effect on adipose tissue through ACVR1C, activating SMAD2/3 signaling and suppressing PPARG target genes. Conversely, loss of activin E or ACVR1C in mice increases fat utilization, lowers adiposity, and drives PPARG-regulated gene signatures indicative of healthy adipose function. Our studies identify activin E-ACVR1C as a metabolic rheostat promoting liver-adipose cross talk to restrain excessive fat breakdown and preserve fat mass during prolonged fasting, a mechanism that is maladaptive in obese individuals.

A 54-year-old man with a history of unimelic, post-traumatic multifocal heterotopic ossification (HO) and normal genetic analysis of ACVR1 and GNAS had variants of unknown significance (VUS) in PDLIM-7 (PDZ and LIM Domain Protein 7), the gene encoding LMP-1 (LIM Mineralization Protein-1), an intracellular protein involved in the bone morphogenetic protein (BMP) pathway signaling and ossification. In order to determine if the LMP-1 variants were plausibly responsible for the phenotype observed, a series of in vitro experiments were conducted. C2C12 cells were co-transfected with a BMP-responsive reporter as well as the LMP-1 wildtype (wt) construct or the LMP-1 T161I or the LMP-1 D181G constructs (herein designated as LMP-161 or LMP-181) corresponding to the coding variants detected in the patient. A significantly increased BMP-reporter activity was observed in LMP-161 or LMP-181 transfected cells compared to the wt cells. The LMP-181 variant exhibited BMP-reporter activity with a four-fold increase over the LMP-1 wt protein. Similarly, mouse pre-osteoblastic MC3T3 cells transfected with the patient's LMP-1 variants expressed higher levels of osteoblast markers both at mRNA and protein levels and preferentially mineralized when stimulated with recombinant BMP-2 compared to control cells. Presently, there are no pathogenic variants of LMP-1 known to induce HO in humans. Our findings suggest that the germline variants in LMP-1 detected in our patient are plausibly related to his multifocal HO (LMP1-related multifocal HO). Further observations will be required to firmly establish this gene-disease relationship., (© 2023 Wiley Periodicals LLC.)

Although structurally similar to type II counterparts, type I or activin receptor-like kinases (ALKs) are set apart by a metastable helix-loop-helix (HLH) element preceding the protein kinase domain that, according to a longstanding paradigm, serves passive albeit critical roles as an inhibitor-to-substrate-binding switch. A single recurrent mutation in the codon of the penultimate residue, directly adjacent the position of a constitutively activating substitution, causes milder activation of ACVR1/ALK2 leading to sporadic heterotopic bone deposition in patients presenting with fibrodysplasia ossificans progressiva, or FOP. To determine the protein structural-functional basis for the gain of function, R206H mutant, Q207D (aspartate-substituted caALK2) and HLH subdomain-truncated (208 Ntrunc) forms were compared to one another and the wild-type enzyme through in vitro kinase and protein-protein interaction analyses that were complemented by signaling read-out (p-Smad) in primary mouse embryonic fibroblasts and Drosophila S2 cells. Contrary to the paradigm, the HLH subdomain actively suppressed the phosphotransferase activity of the enzyme, even in the absence of FKBP12. Unexpectedly, perturbation of the HLH subdomain elevated kinase activity at a distance, i.e., allosterically, at the ATP-binding and polypeptide-interacting active site cleft. Accessibility to polypeptide substrate (BMP Smad C-terminal tails) due to allosterically altered conformations of type I active sites within heterohexameric cytoplasmic signaling complexes-assembled noncanonically by activin-type II receptors extracellularly-is hypothesized to produce a gain of function of the R206H mutant protein responsible for episodic heterotopic ossification in FOP.

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., (© 2023. The Author(s).)

Background: Fibrodysplasia ossificans progressiva (FOP) is an ultrarare and disabling genetic disorder of connective tissue characterized by congenital malformation of the great toes, and progressive heterotopic ossification (HO) in soft connective tissues. A gain-of-function mutation of activin A receptor type I (ACVR1) enables ACVR1 to recognize activin A as an agonist with bone morphogenetic protein (BMP) signalling that leads to HO. Previous studies confirmed that activin A stimulates BMP signalling in vitro and drives HO in mouse models of FOP. However, the roles for BMP4 and BMP6 in FOP are supported only by correlative evidence in vitro. Thus, it remains unclear whether the circulating levels of activin A, BMP4 and BMP6 correlate with flare-ups in FOP patients. Hence, we investigated the protein levels of activin A, BMP4 and BMP6 in the serum of FOP patients., Results: We recruited 16 untreated FOP patients and 16 age- and sex- matched healthy control subjects in this study. The 16 FOP patients were retrospectively divided into the flare-up group (n = 8) and remission group (n = 8) depending on whether they had flare-ups or worsening of any joint movement in the last 6 months. The serum activin A, BMP4 and BMP6 levels were detected by enzyme-linked immunosorbent assay. The serum activin A, BMP4 and BMP6 levels were slightly higher in FOP patients (median: 434.05 pg/mL, 459.48 pg/mL and 67.84 pg/mL) versus healthy control subjects (median: 364.14 pg/mL, 450.39 pg/mL and 55.36 pg/mL). However, there were no statistically significant differences between the two groups (p > 0.05 for all items), nor were there significant differences between the flare-up and remission groups of FOP (p > 0.05 for all items). Univariate and multivariate logistic regression analyses showed that age, sex, and serum activin A, BMP4 and BMP6 levels were not related to flare-up in FOP patients., Conclusions: There were no significant differences in the serum levels of activin A, BMP4 and BMP6 in FOP patients compared with healthy control subjects. Serum activin A, BMP4 and BMP6 proteins might not be the stimulators for FOP flare-up, and may not be biomarkers for FOP diagnosis., (© 2023. The Author(s).)

Background: Fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by progressive heterotopic ossification of muscle and connective tissues, is caused by autosomal dominant activating mutations in the type I receptor, ACVR1/ALK2. The classic human FOP variant, ACVR1 R206H , shows increased bone morphogenetic protein (BMP) signaling and activation by activins., Results: Here, we performed in vivo functional characterization of human ACVR1 R206H and orthologous zebrafish Acvr1l R203H using early embryonic zebrafish dorsoventral patterning as a phenotypic readout for receptor activity. Our results showed that human ACVR1 R206H and zebrafish Acvr1l R203H exhibit functional differences in early embryonic zebrafish, and that human ACVR1 R206H retained its signaling activity in the absence of a ligand-binding domain (LBD). We also showed, for the first time, that zebrafish Acvr2ba/Acvr2bb receptors are required for human ACVR1 R206H signaling in early embryonic zebrafish., Conclusions: Together, these data provide new insight into ACVR1 R206H signaling pathways that may facilitate the design of new and effective therapies for FOP patients., (© 2022 American Association for Anatomy.)

Activin A receptor type I (ACVR1), a transmembrane serine/threonine kinase, belongs to the transforming growth factor-β superfamily, which signals via phosphorylating the downstream effectors and SMAD transcription factors. Its central role in several biological processes and intracellular signaling is well known. Genetic variation in ACVR1 has been associated with a rare disease, fibrodysplasia ossificans progressive, and its somatic alteration is reported in rare cancer diffuse intrinsic pontine glioma. Furthermore, altered expression or variation of ACVR1 is associated with multiple pathologies such as polycystic ovary syndrome, congenital heart defects, diffuse idiopathic skeletal hyperostosis, posterior fossa ependymoma and other malignancies. Recent advancements have witnessed ACVR1 as a potential pharmacological target, and divergent promising approaches for its therapeutic targeting have been explored. This review highlights the structural and functional characteristics of receptor ACVR1, associated signaling pathways, genetic variants in several diseases and cancers, protein-protein interaction, gene expression, regulatory miRNA prediction and potential therapeutic targeting approaches. The comprehensive knowledge will offer new horizons and insights into future strategies harnessing its therapeutic potential., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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.

Background: Adult-type diffuse gliomas, CNS WHO grade 4 are the most aggressive primary brain tumors and represent a particular challenge for therapeutic intervention., Methods: In a single-center retrospective study of matched pairs of initial and post-therapeutic glioma cases with a recurrence period greater than 1 year, we performed whole exome sequencing combined with mRNA and microRNA expression profiling to identify processes that are altered in recurrent gliomas., Results: Mutational analysis of recurrent gliomas revealed early branching evolution in 75% of the patients. High plasticity was confirmed at the mRNA and miRNA levels. SBS1 signature was reduced and SBS11 was elevated, demonstrating the effect of alkylating agent therapy on the mutational landscape. There was no evidence for secondary genomic alterations driving therapy resistance. ALK7/ACVR1C and LTBP1 were upregulated, whereas LEFTY2 was downregulated, pointing towards enhanced Tumor Growth Factor β (TGF-β) signaling in recurrent gliomas. Consistently, altered microRNA expression profiles pointed towards enhanced Nuclear Factor Kappa B and Wnt signaling that, cooperatively with TGF-β, induces epithelial to mesenchymal transition (EMT), migration, and stemness. TGF-β-induced expression of pro-apoptotic proteins and repression of antiapoptotic proteins were uncoupled in the recurrent tumor., Conclusions: Our results suggest an important role of TGF-β signaling in recurrent gliomas. This may have clinical implications since TGF-β inhibitors have entered clinical phase studies and may potentially be used in combination therapy to interfere with chemoradiation resistance. Recurrent gliomas show high incidence of early branching evolution. High tumor plasticity is confirmed at the level of microRNA and mRNA expression profiles., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

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 TGFβ1 to be an FOP-specific stimulant of Activin A, shown by the upregulation of the INHBA gene and protein expression. Activin A and TGFβ1 were both induced by BMP4 in FOP and control fibroblasts. Treatment with TNFα and IL6 produced negligible levels of Activin A and TGFβ1 in both cell groups. We present for the first time TGFβ1 as a triggering factor of Activin A production in FOP. As TGFβ1 can promote the induction of the main driver of FOP, TGFβ1 could also be considered a possible therapeutic target in FOP treatment.

Background: Changes in gene-specific promoter methylation may result from aging and environmental influences. Atherosclerosis is associated with aging and environmental effects. Thus, promoter methylation profiling may be used as an epigenetic tool to evaluate the impact of aging and the environment on atherosclerosis development. However, gene-specific methylation changes are currently inadequate epigenetic markers for predicting atherosclerosis and cardiovascular disease pathogenesis., Results: We profiled and validated changes in gene-specific promoter methylation associated with atherosclerosis using stenosis radiophenotypes of cranial vessels and blood inflammatory cells rather than direct sampling of atherosclerotic plaques. First, we profiled gene-specific promoter methylation changes using digital restriction enzyme analysis of methylation (DREAM) sequencing in peripheral blood mononuclear cells from eight samples each of cranial vessels with and without severe-stenosis radiophenotypes. Using DREAM sequencing profiling, 11 tags were detected in the promoter regions of the ACVR1C, ADCK5, EFNA2, ENOSF1, GLS2, KNDC1, MTNR1B, PACSIN3, PAX8-AS1, TLDC1, and ZNF7 genes. Using methylation evaluation, we found that EFNA2, ENOSF1, GLS2, KNDC1, MTNR1B, PAX8-AS1, and TLDC1 showed > 5% promoter methylation in non-plaque intima, atherosclerotic vascular tissues, and buffy coats. Using logistic regression analysis, we identified hypomethylation of MTNR1B as an independent variable for the stenosis radiophenotype prediction model by combining it with traditional atherosclerosis risk factors including age, hypertension history, and increases in creatinine, lipoprotein (a), and homocysteine. We performed fivefold cross-validation of the prediction model using 384 patients with ischemic stroke (50 [13%] no-stenosis and 334 [87%] > 1 stenosis radiophenotype). For the cross-validation, the training dataset included 70% of the dataset. The prediction model showed an accuracy of 0.887, specificity to predict stenosis radiophenotype of 0.940, sensitivity to predict no-stenosis radiophenotype of 0.533, and area under receiver operating characteristic curve of 0.877 to predict stenosis radiophenotype from the test dataset including 30% of the dataset., Conclusions: We identified and validated MTNR1B hypomethylation as an epigenetic marker to predict cranial vessel atherosclerosis using stenosis radiophenotypes and blood inflammatory cells rather than direct atherosclerotic plaque sampling., (© 2023. The Author(s).)

Abstract: Altered bone morphogenetic protein (BMP) signaling is associated with many musculoskeletal diseases. However, it remains unknown whether BMP dysfunction has direct contribution to debilitating pain reported in many of these disorders. Here, we identified a novel neuropathic pain phenotype in patients with fibrodysplasia ossificans progressiva (FOP), a rare autosomal-dominant musculoskeletal disorder characterized by progressive heterotopic ossification. Ninety-seven percent of these patients carry an R206H gain-of-function point mutation in the BMP type I receptor ACVR1 (ACVR1 R206H ), which causes neofunction to Activin A and constitutively activates signaling through phosphorylated SMAD1/5/8. Although patients with FOP can harbor pathological lesions in the peripheral and central nervous system, their etiology and clinical impact are unclear. Quantitative sensory testing of patients with FOP revealed significant heat and mechanical pain hypersensitivity. Although there was no major effect of ACVR1 R206H on differentiation and maturation of nociceptive sensory neurons (iSNs) derived from FOP induced pluripotent stem cells, both intracellular and extracellular electrophysiology analyses of the ACVR1 R206H iSNs displayed ACVR1-dependent hyperexcitability, a hallmark of neuropathic pain. Consistent with this phenotype, we recorded enhanced responses of ACVR1 R206H iSNs to TRPV1 and TRPA1 agonists. Thus, activated ACVR1 signaling can modulate pain processing in humans and may represent a potential target for pain management in FOP and related BMP pathway diseases., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the International Association for the Study of Pain.)

Accumulated studies have suggested that bone morphogenetic proteins (BMPs) are critical for skin development. However, it remains elusive how BMP signaling via ALK2 (aka ACVR1), one of the important BMP type I receptors, regulates keratinocyte differentiation. To address this question, we utilized a genetic system that enhances BMP signaling via ALK2 in an epidermis-specific manner in mice (hereafter ca-Alk2:K14-Cre). Ca-Alk2:K14-Cre mice displayed a sticky and hairless skin phenotype with a thinner epidermis incapable of differentiating. Although cellular proliferation and survival were comparable between wild-type and ca-Alk2:K14-Cre mice, skin differentiation was severely hampered in ca-Alk2:K14-Cre mice. To uncover the mechanism of altered keratinocyte differentiation, we performed a transcriptome analysis. As a result, we found that the expression levels of cell cycle inhibitor p21 were increased in ca-Alk2:K14-Cre mice. Our findings suggest that aberrant BMP signaling via ALK2 positively regulates p21 expression that attenuates keratinocyte differentiation, and further highlights the critical role of BMP signaling in skin development., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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 ACVR1 R206H 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 BMPR1A R443C and ACVR2A V173I as candidate modifiers. Molecular modeling predicted significant structural perturbations. Neither variant decreased BMP signaling in ACVR1 R206H HEK 293T cells at baseline or after stimulation with BMP4 or activin A (AA), ligands that activate ACVR1 R206H signaling. Overexpression of BMPR1A R443C in a Tg(ACVR1-R206Ha) embryonic zebrafish model, in which overactive BMP signaling yields ventralized embryos, did not alter ventralization severity, while ACVR2A V173I exacerbated ventralization. Co-expression of both variants did not affect dorsoventral patterning. In contrast, BMPR1A knockdown in ACVR1 R206H 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 ACVR1 R206H HEK cells and had no effect in Tg(ACVR1-R206Ha) zebrafish. Co-knockdown in ACVR1 R206H 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 ACVR1 R206H signaling, particularly in response to AA, and that ACVR2A V173I unexpectedly increased ACVR1 R206H -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)., (© 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 devastating disease of progressive heterotopic bone formation for which effective treatments are currently unavailable. FOP is caused by dominant gain-of-function mutations in the receptor ACVR1 (also known as ALK2), which render the receptor inappropriately responsive to activin ligands. In previous studies, we developed a genetic mouse model of FOP that recapitulates most clinical aspects of the disease. In this model, genetic loss of the wild-type Acvr1 allele profoundly exacerbated heterotopic ossification, suggesting the hypothesis that the stoichiometry of wild-type and mutant receptors dictates disease severity. Here, we tested this model by producing FOP mice that conditionally overexpress human wild-type ACVR1. Injury-induced heterotopic ossification (HO) was completely blocked in FOP mice when expression of both the mutant and wild-type receptor were targeted to Tie2-positive cells, which includes fibro/adipogenic progenitors (FAPs). Perinatal lethality of Acvr1 R206H/+ mice was rescued by constitutive ACVR1 overexpression, and these mice survived to adulthood at predicted Mendelian frequencies. Constitutive overexpression of ACVR1 also provided protection from spontaneous abnormal skeletogenesis, and the incidence and severity of injury-induced HO in these mice was dramatically reduced. Analysis of pSMAD1/5/8 signaling both in cultured cells and in vivo indicates that ACVR1 overexpression functions cell-autonomously by reducing osteogenic signaling in response to activin A. We propose that ACVR1 overexpression inhibits HO by decreasing the abundance of ACVR1(R206H)-containing signaling complexes at the cell surface while increasing the representation of activin-A-bound non-signaling complexes comprised of wild-type ACVR1. © 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)., (© 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).)

Heterotopic ossification is the most disabling feature of fibrodysplasia ossificans progressiva, an ultra-rare genetic disorder for which there is currently no prevention or treatment. Most patients with this disease harbor a heterozygous activating mutation (c.617 G > A;p.R206H) in ACVR1. Here, we identify recombinant AAV9 as the most effective serotype for transduction of the major cells-of-origin of heterotopic ossification. We use AAV9 delivery for gene replacement by expression of codon-optimized human ACVR1, ACVR1R206H allele-specific silencing by AAV-compatible artificial miRNA and a combination of gene replacement and silencing. In mouse skeletal cells harboring a conditional knock-in allele of human mutant ACVR1 and in patient-derived induced pluripotent stem cells, AAV gene therapy ablated aberrant Activin A signaling and chondrogenic and osteogenic differentiation. In Acvr1(R206H) knock-in mice treated locally in early adulthood or systemically at birth, trauma-induced endochondral bone formation was markedly reduced, while inflammation and fibroproliferative responses remained largely intact in the injured muscle. Remarkably, spontaneous heterotopic ossification also substantially decreased in in Acvr1(R206H) knock-in mice treated systemically at birth or in early adulthood. Collectively, we develop promising gene therapeutics that can prevent disabling heterotopic ossification in mice, supporting clinical translation to patients with fibrodysplasia ossificans progressiva., (© 2022. The Author(s).)

ALK7 (Activin receptor-like kinase 7) is a member of the TGF-β receptor superfamily predominantly expressed by cells and tissues involved in endocrine functions, such as neurons of the hypothalamus and pituitary, pancreatic β-cells and adipocytes. Recent studies have begun to delineate the processes regulated by ALK7 in these tissues and how these become integrated with the homeostatic regulation of mammalian metabolism. The picture emerging indicates that ALK7's primary function in metabolic regulation is to limit catabolic activities and preserve energy. Aside of the hypothalamic arcuate nucleus, the function of ALK7 elsewhere in the brain, particularly in the cerebellum, where it is abundantly expressed, remains to be elucidated. Although our understanding of the basic molecular events underlying ALK7 signaling has benefited from the vast knowledge available on TGF-β receptor mechanisms, how these connect to the physiological functions regulated by ALK7 in different cell types is still incompletely understood. Findings of missense and nonsense variants in the Acvr1c gene, encoding ALK7, of some mouse strains and human subjects indicate a tolerance to ALK7 loss of function. Recent discoveries suggest that specific inhibitors of ALK7 may have therapeutic applications in obesity and metabolic syndrome without overt adverse effects., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

During vertebrate embryogenesis, the germ layers are patterned by secreted Nodal signals. In the classical model, Nodals elicit signaling by binding to a complex comprising Type I/II Activin receptors (Acvr) and the co-receptor Tdgf1. However, it is currently unclear whether receptor binding can also affect the distribution of Nodals themselves through the embryo, and it is unknown which of the putative Acvr paralogs mediate Nodal signaling in zebrafish. Here, we characterize three Type I (Acvr1) and four Type II (Acvr2) homologs and show that - except for Acvr1c - all receptor-encoding transcripts are maternally deposited and present during zebrafish embryogenesis. We generated mutants and used them together with combinatorial morpholino knockdown and CRISPR F0 knockout (KO) approaches to assess compound loss-of-function phenotypes. We discovered that the Acvr2 homologs function partly redundantly and partially independently of Nodal to pattern the early zebrafish embryo, whereas the Type I receptors Acvr1b-a and Acvr1b-b redundantly act as major mediators of Nodal signaling. By combining quantitative analyses with expression manipulations, we found that feedback-regulated Type I receptors and co-receptors can directly influence the diffusion and distribution of Nodals, providing a mechanism for the spatial restriction of Nodal signaling during germ layer patterning., Competing Interests: HP, AK, DM, DČ, GS, KR, HM, MA, PM No competing interests declared

Background: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by progressive heterotopic ossification (HO) in soft tissues due to a heterozygous mutation of the ACVR1A gene (FOP-ACVR1A), which erroneously transduces the BMP signal by Activin-A. Although inflammation is known to trigger HO in FOP, the role of FOP-ACVR1A on inflammatory cells remains to be elucidated., Results: We generated immortalized monocytic cell lines from FOP-iPSCs (FOP-ML) and mutation rescued iPSCs (resFOP-ML). Cell morphology was evaluated during the monocyte induction and after immortalization. Fluorescence-activated cell sorting (FACS) was performed to evaluate the cell surface markers CD14 and CD16 on MLs. MLs were stimulated with lipopolysaccharide or Activin-A and the gene expression was evaluated by quantitative PCR and microarray analysis. Histological analysis was performed for HO tissue obtained from wild type mice and FOP-ACVR1A mice which conditionally express human mutant ACVR1A gene by doxycycline administration. Without any stimulation, FOP-ML showed the pro-inflammatory signature of CD16+ monocytes with an upregulation of INHBA gene, and treatment of resFOP-ML with Activin-A induced an expression profile mimicking that of FOP-ML at baseline. Treatment of FOP-ML with Activin-A further induced the inflammatory profile with an up-regulation of inflammation-associated genes, of which some, but not all, of which were suppressed by corticosteroid. Experiments using an inhibitor for TGFβ or BMP signal demonstrated that Activin-A-induced genes such as CD16 and CCL7, were regulated by both signals, indicating Activin-A transduced dual signals in FOP-ML. A comparison with resFOP-ML identified several down-regulated genes in FOP-ML including LYVE-1, which is known to suppress matrix-formation in vivo. The down-regulation of LYVE-1 in HO tissues was confirmed in FOP model mice, verifying the significance of the in vitro experiments., Conclusion: These results indicate that FOP-ML faithfully recapitulated the phenotype of primary monocytes of FOP and the combination with resFOP-ML is a useful tool to investigate molecular events at the initial inflammation stage of HO in FOP., (© 2022. The Author(s).)

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.

Identifying genetic variants associated with lower waist-to-hip ratio can reveal new therapeutic targets for abdominal obesity. We use exome sequences from 362,679 individuals to identify genes associated with waist-to-hip ratio adjusted for BMI (WHRadjBMI), a surrogate for abdominal fat that is causally linked to type 2 diabetes and coronary heart disease. Predicted loss of function (pLOF) variants in INHBE associate with lower WHRadjBMI and this association replicates in data from AMP-T2D-GENES. INHBE encodes a secreted protein, the hepatokine activin E. In vitro characterization of the most common INHBE pLOF variant in our study, indicates an in-frame deletion resulting in a 90% reduction in secreted protein levels. We detect associations with lower WHRadjBMI for variants in ACVR1C, encoding an activin receptor, further highlighting the involvement of activins in regulating fat distribution. These findings highlight activin E as a potential therapeutic target for abdominal obesity, a phenotype linked to cardiometabolic disease., (© 2022. The Author(s).)