Specific exercise patterns generate an epigenetic molecular memory window that drives long-term memory formation and identifies ACVR1C as a bidirectional regulator of memory in mice.

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.

SEMA3G regulates BMP9 inhibition of VEGF-mediated migration and network formation in pulmonary endothelial cells.

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.

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

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

Hemojuvelin-mediated hepcidin induction requires both bone morphogenetic protein type I receptors ALK2 and ALK3.

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.

Gut microbiota regulates the ALK5/NOX1 axis by altering glutamine metabolism to inhibit ferroptosis of intrahepatic cholangiocarcinoma cells.

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 Fe2+ 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.

Cell Senescence in Heterotopic Ossification.

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

Matrix metalloproteinase-9 deficiency confers resilience in fibrodysplasia ossificans progressiva in a man and mice.

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.

A healthy 35-year-old man (patient-R) with the classic fibrodysplasia ossificans progressiva (FOP) mutation and the congenital great toe malformation of FOP had extreme lack of heterotopic ossification (HO) and nearly normal mobility. We hypothesized that patient-R lacked a sufficient inflammatory trigger for HO. Blood tests revealed a reduction in the level of an inflammatory protein called matrix metalloproteinase-9 (MMP-9) compared to other individuals with FOP as well as healthy controls. DNA analysis in patient-R identified mutations in MMP-9, one of which predicted decreased activity of MMP-9 which was confirmed by further testing. Inflammatory cells (macrophages) expressing the MMP-9 mutations identified in patient-R produced significantly less Activin A, an obligate stimulus for HO in FOP. In order to determine if MMP-9 deficiency was a cause of HO prevention in FOP, we inhibited MMP-9 activity by genetic, biologic, or pharmacologic means in FOP mouse models and showed that MMP-9 inhibition prevented or dramatically decreased trauma-induced HO in FOP, locked-up Activin A in the extracellular matrix, and induced regeneration of injured skeletal muscle. Our data show that MMP-9 links inflammation to HO and illustrate that one patient's clinical picture can reveal critical molecular mechanisms of disease that unveil new treatment strategies.

Silibinin is a suppressor of the metastasis-promoting transcription factor ID3.

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 IC50 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.

Human ACVR1C missense variants that correlate with altered body fat distribution produce metabolic alterations of graded severity in knock-in mutant mice.

BACKGROUND & AIMS: Genome-wide studies have identified three missense variants in the human gene ACVR1C, encoding the TGF-ß 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.

BMP-ACVR1 Axis is Critical for Efficacy of PRC2 Inhibitors in B-Cell Lymphoma.

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.

TP-0184 inhibits FLT3/ACVR1 to overcome FLT3 inhibitor resistance and hinder AML growth synergistically with venetoclax.

We identified activin A receptor type I (ACVR1), a member of the TGF-ß 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.

A fibrodysplasia ossificans progressiva patient with a rare missense mutation in ACVR1 detected on 18F-FDG PET/CT.

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). 18F-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 18F-FDG uptake, whereas end-stage ossifications do not. Collectively, 18F-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.

Circular RNA ZNF800 (hsa_circ_0082096) regulates cancer stem cell properties and tumor growth in colorectal cancer.

BACKGROUND: Cancer stem cells form a rare cell population in tumors that contributes to metastasis, recurrence and chemoresistance in cancer patients. Circular RNAs (circRNAs) are post-transcriptional regulators of gene expression that sponge targeted microRNA (miRNAs) to affect a multitude of downstream cellular processes. We previously showed in an expression profiling study that circZNF800 (hsa_circ_0082096) was up-regulated in cancer stem cell-enriched spheroids derived from colorectal cancer (CRC) cell lines. METHODS: Spheroids were generated in suspension spheroidal culture. The ZNF800 mRNA, pluripotency stem cell markers and circZNF800 levels were determined by quantitative RT-PCR. CircZNF800-miRNA interactions were shown in RNA pulldown assays and the miRNA levels determined by stem-loop qRT-PCR. The effects of circZNF800 on cell proliferation were tested by EdU staining followed by flowcytometry. Expression of stem cell markers CD44/CD133, Lgr5 and SOX9 was demonstrated in immunofluorescence microscopy. To manipulate the cellular levels of circZNF800, circZNF800 over-expression was achieved via transfection of in vitro synthesized and circularized circZNF800, and knockdown attained using a CRISPR-Cas13d-circZNF800 vector system. Xenografted nude mice were used to demonstrate effects of circZNF800 over-expression and knockdown on tumor growth in vivo. RESULTS: CircZNF800 was shown to be over-expressed in late-stage tumor tissues of CRC patients. Data showed that circZNF800 impeded expression of miR-140-3p, miR-382-5p and miR-579-3p while promoted the mRNA levels of ALK/ACVR1C, FZD3 and WNT5A targeted by the miRNAs, as supported by alignments of seed sequences between the circZNF800-miRNA, and miRNA-mRNA paired interactions. Analysis in CRC cells and biopsied tissues showed that circZNF800 positively regulated the expression of intestinal stem cell, pluripotency and cancer stem cell markers, and promoted CRC cell proliferation, spheroid and colony formation in vitro, all of which are cancer stem cell properties. In xenografted mice, circZNF800 over-expression promoted tumor growth, while circZNF800 knockdown via administration of CRISPR Cas13d-circZNF800 viral particles at the CRC tumor sites impeded tumor growth. CONCLUSIONS: CircZNF800 is an oncogenic factor that regulate cancer stem cell properties to lead colorectal tumorigenesis, and may be used as a predictive marker for tumor progression and the CRISPR Cas13d-circZNF800 knockdown strategy for therapeutic intervention of colorectal cancer.

AAV-Mediated Targeting of the Activin A-ACVR1R206H Signaling in Fibrodysplasia Ossificans Progressiva.

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 ACVR1R206H (Acvr1R206H/+) 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 (ACVR1opt) and artificial miRNAs targeting Activin A and its receptor ACVR1R206H ablated the aberrant activation of BMP-Smad1/5 signaling and the osteogenic differentiation of Acvr1R206H/+ 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 Acvr1R206H/+ 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.

Pacritinib is a potent ACVR1 inhibitor with significant anemia benefit in patients with myelofibrosis.

In patients with cytopenic myelofibrosis, treatment with the JAK2/IRAK1 inhibitor pacritinib was associated with anemia benefit in the phase 3 PERSIST-2 study. The impact of pacritinib on transfusion independence (TI) has not been previously described, nor has the mechanism by which pacritinib improves anemia been elucidated. Because it has been previously postulated that inhibition of activin A receptor, type 1 (ACVR1)/activin receptor-like kinase-2 improves anemia in patients with myelofibrosis via suppression of hepcidin production, we assessed the relative inhibitory potency of pacritinib compared with other JAK2 inhibitors against ACVR1. Pacritinib inhibited ACVR1 with greater potency (half-maximal inhibitory concentration [IC50] = 16.7 nM; Cmax:IC50 = 12.7) than momelotinib (IC50 = 52.5 nM; Cmax:IC50 = 3.2), fedratinib (IC50 = 273 nM; Cmax:IC50 = 1.0), or ruxolitinib (IC50 > 1000; Cmax:IC50 < 0.01). Pacritinib's inhibitory activity against ACVR1 was corroborated via inhibition of downstream SMAD signaling in conjunction with marked suppression of hepcidin production. Among patients on PERSIST-2 who were not transfusion independent at baseline based on Gale criteria, a significantly greater proportion achieved TI on pacritinib compared with those treated on best available therapy (37% vs 7%, P = .001), and significantly more had a ≥50% reduction in transfusion burden (49% vs 9%, P < .0001). These data indicate that the anemia benefit of the JAK2/IRAK1 inhibitor pacritinib may be a function of potent ACVR1 inhibition.

Polypeptide Substrate Accessibility Hypothesis: Gain-of-Function R206H Mutation Allosterically Affects Activin Receptor-like Protein Kinase Activity.

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.

Activin E-ACVR1C cross talk controls energy storage via suppression of adipose lipolysis in 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 ß-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.

Reduced GS Domain Serine/Threonine Requirements of Fibrodysplasia Ossificans Progressiva Mutant Type I BMP Receptor ACVR1 in the Zebrafish.

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).

Multifocal heterotopic ossification in a man with germline variants of LIM Mineralization Protein-1 (LMP-1).

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-1T161I or the LMP-1D181G 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.

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

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