Morphogenetic Pathway Research Articles

Bone morphogenetic protein signaling pathway– Ethanol interactions disrupt palate formation independent of gata3

Fetal Alcohol Spectrum Disorders (FASD) describes a wide array of neurological defects and craniofacial malformations, associated with ethanol teratogenicity. While there is growing evidence for a genetic component to FASD, little is known of the genes underlying these ethanol-induced defects. Along with timing and dosage, genetic predispositions may help explain the variability within FASD. From a screen for gene-ethanol interactions, we found that mutants for Bmp signaling components are ethanol-sensitive leading to defects in the zebrafish palate. Loss of Bmp signaling results in reductions in gata3 expression in the maxillary domain of the neural crest in the 1st pharyngeal arch, leading to palate defects while upregulation of human GATA3 rescues these defects. Here, we show that ethanol-treated Bmp mutants exhibit misshaped and/or broken trabeculae. Surprisingly, up regulation of GATA3 does not rescue ethanol-induced palate defects and gata3 expression was not altered in ethanol-treated Bmp mutants or dorsomorphin-treated larvae. Timing of ethanol sensitivity shows that Bmp mutants are ethanol sensitive from 10 to 18 hours post-fertilization (hpf), prior to Bmp’s regulation of gata3 in palate formation. This is consistent with our previous work with dorsomorphin-dependent knock down of Bmp signaling from 10 to 18 hpf disrupting endoderm formation and subsequent jaw development. Overall, this suggests that ethanol disrupts Bmp-dependent palate development independent of and earlier than the role of gata3 in palate formation by disrupting epithelial development. Ultimately, these data demonstrate that zebrafish is a useful model to identify and characterize gene-ethanol interactions and this work will directly inform our understanding of FASD.

A defined tubby domain β-barrel surface region of TULP3 mediates ciliary trafficking of diverse cargoes.

The primary cilium is a paradigmatic subcellular compartment at the nexus of numerous cellular and morphogenetic pathways. The tubby family protein TULP3 acts as an adapter of the intraflagellar transport complex-A in transporting integral membrane and membrane-associated lipidated proteins into cilia. However, the mechanisms by which TULP3 coordinates ciliary transport of diverse cargoes is not well understood. Here, we provide molecular insights into TULP3-mediated ciliary cargo recognition. We screened for critical TULP3 residues by proximity biotinylation-mass spectrometry, structural analysis, and testing TULP3 variants in human patients with hepatorenal fibrocystic disease and spina bifida. The TULP3 residues we identified (a) were located on one side of the β-barrel of the tubby domain away from the phosphoinositide binding site, (b) mediated ciliary trafficking of lipidated and transmembrane cargoes, and (c) determined proximity with these cargoes in vivo without affecting ciliary localization, phosphoinositide binding or hydrodynamic properties of TULP3. Overall, these findings implicate a specific region of one of the surfaces of the TULP3 β-barrel in ciliary trafficking of diverse cargoes. This region overlooks the β strands 8-12 of the β-barrel and is away from the membrane anchoring phosphoinositide binding site. Targeting the TULP3-cargo interactions could provide therapeutics in ciliary trafficking diseases.

Bone Morphogenetic Protein signaling pathway - ethanol interactions disrupt palate formation independent of gata3.

Bmp pathway mutants are ethanol sensitive resulting in palate defects. Ethanol disrupts Bmp-dependent palate development independent of gata3 . Timing of ethanol sensitivity suggests ethanol disrupts Bmp-dependent epithelial morphogenesis.

Mutations in the Bone Morphogenetic Protein signaling pathway sensitize zebrafish and humans to ethanol-induced jaw malformations.

In this study, we apply a unique combination of zebrafish-based approaches and human genetic and facial dysmorphology analyses to resolve the cellular mechanisms driven by the ethanol-sensitive Bmp pathway.

Spatial transcriptomics of human and murine Neurofibromatosis Type I fracture pseudarthroses reveal impaired BMP signaling

Abstract Introduction/Objective In Neurofibromatosis Type 1 (NF1), caused by mutations in the NF1 tumor-suppressor gene, children form fibrotic nonunions known as pseudarthroses following long bone fractures. Here, we integrate spatial transcriptomics of human and murine samples to investigate the pathogenesis underlying pseudoarthrosis development. Methods/Case Report We performed spatial transcriptomics on 10-day post-fracture calluses from control and periosteum-specific Nf1-deficient (Nf1Postn) mice as well as a human NF1 patient pseudarthrosis specimen. Differential gene expression and SpatialTime analyses were performed. BMP signaling was validated by immunohistochemical staining of tissues for phospho-SMAD1/5/8. Results (if a Case Study enter NA) Spatial transcriptomics detected eight cell clusters, including bone, cartilage, and muscle within mouse fracture calluses. In the control, clusters involved in fracture repair were enriched for genes associated with extracellular matrix and collagen formation (progenitor and cartilage clusters), cartilage and endochondral bone processes (ossifying perichondrium cluster), and bone remodeling (woven bone cluster). Expression of TGF-beta pathway genes was highest near the fracture plane, while BMP and Wnt pathways were activated further from the fracture. In the Nf1Postn fracture, healing was significantly delayed, lacking a robust callus and with a lower abundance of reparative skeletal cell clusters. While expression of TGF-beta pathway genes was similar to control, BMP pathway expression was minimal within the Nf1Postn fracture, which was further demonstrated by a lack of phospho-SMAD1/5/8 staining. In the human pseudarthrosis, while TGF-beta pathway genes were expressed near the fracture plane as expected, there was no enrichment of BMP pathway expression in the pseudarthrosis tissue and no significant phospho-SMAD1/5/8 staining. Conclusion Spatial transcriptomics allows for the simultaneous unbiased analysis of multiple signal transduction pathways within their native tissue environment. Our analyses 1) revealed the cellular diversity required for fracture healing, 2) demonstrated the spatially-restricted expression of key morphogenetic pathways within a fracture callus, and 3) provides in situ evidence for impaired BMP pathway activation associated with NF1 fracture pseudarthroses.

Histone-like transcription factor Hfl1p in Candida albicans harmonizes nuclear and mitochondrial genomic network in regulation of energy metabolism and filamentation development

ABSTRACT Candida albicans is an opportunistic fungal pathogen known for surviving in various nutrient-limited conditions within the host and causing infections. Our prior research revealed that Hfl1p, an archaeal histone-like or Hap5-like protein, is linked to mitochondrial ATP generation and yeast-hyphae morphogenesis. However, the specific roles of Hfl1p in these virulence behaviours, through its function in the CBF/NF-Y complex or as a DNA polymerase II subunit, remain unclear. This study explores Hfl1p’s diverse functions in energy metabolism and morphogenesis. By combining proteomic analysis and phenotypic evaluations of the hfl1Δ/hfl1Δ mutant with ChIP data, we found that Hfl1p significantly impacts mitochondrial DNA-encoded CI subunits, the tricarboxylic acid (TCA) cycle, and morphogenetic pathways. This influence occurs either independently or alongside other transcription factors recognizing a conserved DNA motif (TAXXTAATTA). These findings emphasize Hfl1p’s critical role in linking carbon metabolism and mitochondrial respiration to the yeast-to-filamentous form transition, enhancing our understanding of C. albicans’ metabolic adaptability during morphological transition, an important pathogenic trait of this fungus. This could help identify therapeutic targets by disrupting the relationship between energy metabolism and cell morphology in C. albicans.

Hitting the target: cell signaling pathways modulation by extracellular vesicles.

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released outside the cell. EVs have drawn attention not only for their role in cell waste disposal, but also as additional tools for cell-to-cell communication. Their complex contents include not only lipids, but also proteins, nucleic acids (RNA, DNA), and metabolites. A large part of these molecules are involved in mediating or influencing signal transduction in target cells. In multicellular organisms, EVs have been suggested to modulate signals in cells localized either in the neighboring tissue or in distant regions of the body by interacting with the cell surface or by entering the cells via endocytosis or membrane fusion. Most of the EV-modulated cell signaling pathways have drawn considerable attention because they affect morphogenetic signaling pathways, as well as pathways activated by cytokines and growth factors. Therefore, they are implicated in relevant biological processes, such as embryonic development, cancer initiation and spreading, tissue differentiation and repair, and immune response. Furthermore, it has recently emerged that multicellular organisms interact with and receive signals through EVs released by their microbiota as well as by edible plants. This review reports studies investigating EV-mediated signaling in target mammalian cells, with a focus on key pathways for organism development, organ homeostasis, cell differentiation and immune response.

Optimal performance objectives in the highly conserved bone morphogenetic protein signaling pathway

Throughout development, complex networks of cell signaling pathways drive cellular decision-making across different tissues and contexts. The transforming growth factor β (TGF-β) pathways, including the BMP/Smad pathway, play crucial roles in determining cellular responses. However, as the Smad pathway is used reiteratively throughout the life cycle of all animals, its systems-level behavior varies from one context to another, despite the pathway connectivity remaining nearly constant. For instance, some cellular systems require a rapid response, while others require high noise filtering. In this paper, we examine how the BMP-Smad pathway balances trade-offs among three such systems-level behaviors, or “Performance Objectives (POs)”: response speed, noise amplification, and the sensitivity of pathway output to receptor input. Using a Smad pathway model fit to human cell data, we show that varying non-conserved parameters (NCPs) such as protein concentrations, the Smad pathway can be tuned to emphasize any of the three POs and that the concentration of nuclear phosphatase has the greatest effect on tuning the POs. However, due to competition among the POs, the pathway cannot simultaneously optimize all three, but at best must balance trade-offs among the POs. We applied the multi-objective optimization concept of the Pareto Front, a widely used concept in economics to identify optimal trade-offs among various requirements. We show that the BMP pathway efficiently balances competing POs across species and is largely Pareto optimal. Our findings reveal that varying the concentration of NCPs allows the Smad signaling pathway to generate a diverse range of POs. This insight identifies how signaling pathways can be optimally tuned for each context.

Enhancing microshoot regeneration in "Turiaçu" pineapple through nodular cluster induction with putrescine

Nodular clusters are a morphogenetic pathway used in the in vitro propagation of Bromeliaceae due to their high responsiveness in the regeneration of microshoots. Additionally, endogenous polyamines, especially putrescine, may contribute to the induction of these nodular clusters. However, the potential effects of this regulator on the regeneration of microshoots when applied to explants are unclear, especially in traditional pineapple cultivars such as "Turiaçu". Therefore, our objective here was to induce and characterize the morphoanatomy of nodular clusters in combinations of 6-benzylaminopurine (BAP) and putrescine (PUT) in "Turiaçu" pineapple. Leaf segments induced in culture medium supplemented with 4 μM naphthaleneacetic acid (NAA), 8 μM BAP, and 100 μM PUT resulted in an induction rate of 56 %, with the formation of microshoots via nodular clusters, increased length, and the number of leaves per microshoot. Morphoanatomical studies revealed that in the medium with 4 μM NAA, 8 μM BAP, and 100 μM PUT, the clusters presented a voluminous size, microshoots with leaves, and the development of new leaves along the base of the explant, and the formation of meristemoids with small cells arranged in a spiral. The results indicate that auxins and cytokinins combined with exogenous putrescine are efficient for inducing nodule clusters. Thus, exogenous putrescine improves the efficiency of microshoot regeneration by nodular clusters.

Spatial transcriptomics implicates impaired BMP signaling in NF1 fracture pseudarthrosis in murine and patient tissues.

The neurofibromatosis type 1 (NF1) RASopathy is associated with persistent fibrotic nonunions (pseudarthrosis) in human and mouse skeletal tissue. Here, we performed spatial transcriptomics to define the molecular signatures occurring during normal endochondral healing following fracture in mice. Within the control fracture callus, we observed spatially restricted activation of morphogenetic pathways, such as TGF-β, WNT, and BMP. To investigate the molecular mechanisms contributing to Nf1-deficient delayed fracture healing, we performed spatial transcriptomic analysis on a Postn-cre;Nf1fl/- (Nf1Postn) fracture callus. Transcriptional analyses, subsequently confirmed through phospho-SMAD1/5/8 immunohistochemistry, demonstrated a lack of BMP pathway induction in Nf1Postn mice. To gain further insight into the human condition, we performed spatial transcriptomic analysis of fracture pseudarthrosis tissue from a patient with NF1. Analyses detected increased MAPK signaling at the fibrocartilaginous-osseus junction. Similar to that in the Nf1Postn fracture, BMP pathway activation was absent within the pseudarthrosis tissue. Our results demonstrate the feasibility of delineating the molecular and tissue-specific heterogeneity inherent in complex regenerative processes, such as fracture healing, and reconstructing phase transitions representing endochondral bone formation in vivo. Furthermore, our results provide in situ molecular evidence of impaired BMP signaling underlying NF1 pseudarthrosis, potentially informing the clinical relevance of off-label BMP2 as a therapeutic intervention.

Podocyte Pathogenic Bone Morphogenetic Protein-2 Pathway and Immune Cell Behaviors in Primary Membranous Nephropathy.

Primary membranous nephropathy (PMN) is one of the leading causes of end-stage renal disease, and the most frequent cause of massive proteinuria in nondiabetic adults, resulting in fatal complications. However, the underlying pathomechanisms of PMN remain largely unclear. Here, single-cell RNA sequencing is employed to analyze kidney biopsies from eleven PMN patients and seven healthy subjects. Profiling 44060 cells from patients allowed us to characterize the cellular composition and cell-type-specific gene expression in the PMN kidney. The complement-induced BMP2/pSMAD1/COL4 pathway is identified as the pathogenic pathway in podocytes, bridging two key events, i.e., complement system activation and glomerular basement membrane thickening in PMN. Augmented infiltration and activation of myeloid leukocytes and B lymphocytes are found, profiling delicate crosstalk of immune cells in PMN kidneys. Overall, these results provide valuable insights into the roles of podocytes and immune cells in PMN, and comprehensive resources toward the complete understanding of PMN pathophysiology.

Gene Expression, Morphology, and Electrophysiology During the Dynamic Development of Human Induced Pluripotent Stem Cell-Derived Atrial- and Ventricular-Like Cardiomyocytes.

Gene expression, morphology, and electrophysiological combination are essential for assessing the dynamic development of human induced pluripotent stem cell-derived atrial- and ventricular-like cardiomyocytes (iPS-AM and iPS-VM, respectively). For iPS-AM/VM differentiation, we performed the small molecule-based temporal modulation of the retinoic acid and bone morphogenetic protein signaling pathways. We investigated the gene expression and morphology using immunofluorescence, quantitative real-time polymerase chain reaction, flow cytometry, and transmission electron microscopy as well as registered electrophysiological functions using a whole-cell patch clamp on days 20, 30, and 60 post-differentiations. Pan-cardiomyocyte marker, including troponin T2 (TNNT2) and alpha-actinin-2 (ACTN2), expressions increased both in iPS-AMs and iPS-VMs. Similarly, the mRNA expression of both iPS-AM-specific markers, ie, natriuretic peptide A (NPPA), myosin light chain 7 (MYL7), and K+ channel Kir3.4 (KCNJ5), and iPS-VM-specific markers, ie, gap junction α-1 (GJA1), myosin light chain 2 (MYL2), and alpha-1-subunit of a voltage-dependent L-type calcium channel (CACNA1C), increased from 0 to 20 days, and then decreased from 30 to 60 days. Concerning morphology, cardiac troponin-T (cTnT) arrangement was progressively organized and developed from a disorderly myofibrillar distribution to an organized sarcomere pattern both in iPS-AMs and iPS-VMs. Mitochondrial numbers gradually increased and those of lipid droplets decreased during dynamic development. Regarding physiological function, the resting and action potential amplitudes remained statistically indifferent in both cell types, and the action potential duration was prolonged during the development. IPS-AMs/VMs displayed dynamic development concerning their gene expression, morphology, and electrophysiological function. The discoveries of this study could provide novel insights into heart development and encourage further research.

Comprehensive deciphering the alternative splicing patterns involved in leaf morphogenesis of Liriodendron chinense

Alternative splicing (AS), a pivotal post-transcriptional regulatory mechanism, profoundly amplifies diversity and complexity of transcriptome and proteome. Liriodendron chinense (Hemsl.) Sarg., an excellent ornamental tree species renowned for its distinctive leaf shape, which resembles the mandarin jacket. Despite the documented potential genes related to leaf development of L. chinense, the underlying post-transcriptional regulatory mechanisms remain veiled. Here, we conducted a comprehensive analysis of the transcriptome to clarify the genome-wide landscape of the AS pattern and the spectrum of spliced isoforms during leaf developmental stages in L. chinense. Our investigation unveiled 50,259 AS events, involving 10,685 genes (32.9%), with intron retention as the most prevalent events. Notably, the initial stage of leaf development witnessed the detection of 804 differentially AS events affiliated with 548 genes. Although both differentially alternative splicing genes (DASGs) and differentially expressed genes (DEGs) were enriched into morphogenetic related pathways during the transition from fishhook (P2) to lobed (P7) leaves, there was only a modest degree of overlap between DASGs and DEGs. Furthermore, we conducted a comprehensively AS analysis on homologous genes involved in leaf morphogenesis, and most of which are subject to post-transcriptional regulation of AS. Among them, the AINTEGUMENTA-LIKE transcript factor LcAIL5 was characterization in detailed, which experiences skipping exon (SE), and two transcripts displayed disparate expression patterns across multiple stages. Overall, these findings yield a comprehensive understanding of leaf development regulation via AS, offering a novel perspective for further deciphering the mechanism of plant leaf morphogenesis.

Osteoblast differentiation of Gli1⁺ cells via Wnt and BMP signaling pathways during orthodontic tooth movement

ObjectivesFactors that induce bone formation during orthodontic tooth movement (OTM) remain unclear. Gli1 was recently identified as a stem cell marker in the periodontal ligament (PDL). Therefore, we evaluated the mechanism of differentiation of Cre/LoxP-mediated Gli1/Tomato+ cells into osteoblasts during OTM. MethodsAfter the final administration of tamoxifen to 8-week-old Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato mice for 2 days, nickel–titanium closed coil springs were attached between the upper anterior alveolar bone and the first molar. Immunohistochemical localizations of β-catenin, Smad4, and Runx2 were observed in the PDL on 2, 5, and 10 days after OTM initiation. ResultsIn the untreated tooth, few Gli1/Tomato+ cells were detected in the PDL. Two days after OTM initiation, the number of Gli1/Tomato+ cells increased in the PDL on the tension side. On this side, 49.3 ± 7.0% of β-catenin+ and 48.7 ± 5.7% of Smad4+ cells were found in the PDL, and Runx2 expression was detected in some Gli1/Tomato+ cells apart from the alveolar bone. The number of positive cells in the PDL reached a maximum on day 5. In contrast, on the compression side, β-catenin and Smad4 exhibited less immunoreactivity. On day 10, Gli1/Tomato+ cells were aligned on the alveolar bone on the tension side, with some expressing Runx2. ConclusionsGli1+ cells in the PDL differentiated into osteoblasts during OTM. Wnt and bone morphogenetic proteins signaling pathways may be involved in this differentiation.

Structural and functional validation of a highly specific Smurf2 inhibitor.

Smurf1 and Smurf2 are two closely related member of the HECT (homologous to E6AP carboxy terminus) E3 ubiquitin ligase family and play important roles in the regulation of various cellular processes. Both were initially identified to regulate transforming growth factor-β and bone morphogenetic protein signaling pathways through regulating Smad protein stability and are now implicated in various pathological processes. Generally, E3 ligases, of which over 800 exist in humans, are ideal targets for inhibition as they determine substrate specificity; however, there are few inhibitors with the ability to precisely target a particular E3 ligase of interest. In this work, we explored a panel of ubiquitin variants (UbVs) that were previously identified to bind Smurf1 or Smurf2. In vitro binding and ubiquitination assays identified a highly specific Smurf2 inhibitor, UbV S2.4, which was able to inhibit ligase activity with high potency in the low nanomolar range. Orthologous cellular assays further demonstrated high specificity of UbV S2.4 toward Smurf2 and no cross-reactivity toward Smurf1. Structural analysis of UbV S2.4 in complex with Smurf2 revealed its mechanism of inhibition was through targeting the E2 binding site. In summary, we investigated several protein-based inhibitors of Smurf1 and Smurf2 and identified a highly specific Smurf2 inhibitor that disrupts the E2-E3 protein interaction interface.

Effects of Recombinant Human Bone Morphogenetic Protein-2 Mediated Wingless-Related Integration Site Beta-Catenin Pathway on Bone Defect Repair and Angiogenesis in Rabbits

Effects of Recombinant Human Bone Morphogenetic Protein-2 Mediated Wingless-Related Integration Site Beta-Catenin Pathway on Bone Defect Repair and Angiogenesis in Rabbits

Development of Physiologically Relevant Skin Organoids from Human Induced Pluripotent Stem Cells.

The development of skin organs for studying developmental pathways, modeling diseases, or regenerative medicine purposes is a major endeavor in the field. Human induced pluripotent stem cells (hiPSCs) are successfully used to deriveskin cells, but the field is still far from meeting the goal of creating skin containing appendages, such as hair follicles and sweat glands. Here, the goal is to generate skin organoids (SKOs) from human skin fibroblast or placental CD34+ cell-derived hiPSCs. With all three hiPSC lines, complex SKOs with stratified skin layers and pigmented hair follicles are generated with different efficacies. In addition, the hiPSC-derived SKOs develop sebaceous glands, touch-receptive Merkel cells, and more importantly eccrine sweat glands. Together, physiologically relevant skin organoids are developed by direct induction of embryoid body formation, along with simultaneous inactivation of transforming growth factor beta signaling, activation of fibroblast growth factor signaling, and inhibition of bone morphogenetic protein signaling pathways. The skin organoids created in this study can be used as valuable platforms for further research into human skin development, disease modeling, or reconstructive surgeries.

Context-dependent ciliary regulation of hedgehog pathway repression in tissue morphogenesis.

A fundamental problem in tissue morphogenesis is identifying how subcellular signaling regulates mesoscale organization of tissues. The primary cilium is a paradigmatic organelle for compartmentalized subcellular signaling. How signaling emanating from cilia orchestrates tissue organization-especially, the role of cilia-generated effectors in mediating diverse morpho-phenotypic outcomes-is not well understood. In the hedgehog pathway, bifunctional GLI transcription factors generate both GLI-activators (GLI-A) and GLI-repressors (GLI-R). The formation of GLI-A/GLI-R requires cilia. However, how these counterregulatory effectors coordinate cilia-regulated morphogenetic pathways is unclear. Here we determined GLI-A/GLI-R requirements in phenotypes arising from lack of hedgehog pathway repression (derepression) during mouse neural tube and skeletal development. We studied hedgehog pathway repression by the GPCR GPR161, and the ankyrin repeat protein ANKMY2 that direct cAMP/protein kinase-A signaling by cilia in GLI-R generation. We performed genetic epistasis between Gpr161 or Ankmy2 mutants, and Gli2/Gli3 knockouts, Gli3R knock-in and knockout of Smoothened, the hedgehog pathway transducer. We also tested the role of cilia-generated signaling using a Gpr161 ciliary localization knock-in mutant that is cAMP signaling competent. We found that the cilia-dependent derepression phenotypes arose in three modes: lack of GLI-R only, excess GLI-A formation only, or dual regulation of either lack of GLI-R or excess GLI-A formation. These modes were mostly independent of Smoothened. The cAMP signaling-competent non-ciliary Gpr161 knock-in recapitulated Gpr161 loss-of-function tissue phenotypes solely from lack of GLI-R only. Our results show complex tissue-specific GLI-effector requirements in morphogenesis and point to tissue-specific GLI-R thresholds generated by cilia in hedgehog pathway repression. Broadly, our study sets up a conceptual framework for rationalization of different modes of signaling generated by the primary cilium in mediating morphogenesis in diverse tissues.

A Phase 1b Double-Blind, Placebo-Controlled Study of DISC-0974, an Anti-Hemojuvelin Antibody, in Patients with Non-Dialysis Dependent Chronic Kidney Disease and Anemia

Hepcidin, a central regulator of iron homeostasis, is pathologically elevated in patients with non-dialysis-dependent chronic kidney disease (NDD-CKD) and anemia. Elevations in hepcidin contribute to the onset, maintenance, and severity of anemia. DISC-0974 is an investigational, first-in-class, monoclonal antibody that blocks hemojuvelin (HJV), a co-receptor in the bone morphogenetic protein-signaling pathway regulating hepcidin expression. In a healthy volunteer study, DISC-0974 demonstrated dose-dependent reductions in serum hepcidin, increases in serum iron with doses up to 56 mg administered subcutaneously (SC), and increasing trends in reticulocyte count, reticulocyte hemoglobin, mean corpuscular hemoglobin, total hemoglobin (Hgb), and red blood cell count. In a rat model of anemia in CKD, DISC-0974 decreased hepcidin, increased serum iron, and normalized hemoglobin (Wu et al., ASH 2022). DISC-0974-103 is a Phase 1b, double-blind, placebo-controlled, single-ascending dose study (NCT05745883) to assess the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of DISC-0974 in patients with NDD-CKD and anemia. Eligible participants include patients over 18 years of age with NDD-CKD, stages 2-5, hemoglobin (Hgb) <10.5 g/dL in women and <11.0 g/dL in men, serum ferritin ≥100 μg/L, and transferrin saturation (TSAT) ≤30%. Major exclusionary criteria include: concomitant treatment with therapeutic oral iron, intravenous iron, erythropoietin-stimulating agents, or blood transfusions; splenectomy; and nutritional, genetic, infectious, or autoimmune causes of anemia. Approximately 32 participants will be enrolled. On Day 1, participants will receive a single dose of DISC-0974 SC with evaluations for safety, PK, and PD through 57 days of follow-up. Dose escalation is planned across 4 cohorts consisting of 8 participants each, randomized 3:1 active treatment to placebo, treated at single doses of 28, 40, 60, and 90 mg SC DISC-0974. Safety Review Committee assessments are planned to gate escalation decisions. Primary endpoints include adverse events (AEs), clinical laboratory assessments, vital signs, physical examinations, and electrocardiograms. Secondary endpoints include changes in serum hepcidin-25, iron, TSAT, ferritin, Hgb, and other hematology biomarkers. Enrollment is currently ongoing, and available study results will be presented at the meeting.

A Phase 1b Trial of DISC-0974, an Anti-Hemojuvelin Antibody, in Patients with Myelofibrosis and Anemia

Hepcidin, a central regulator of iron homeostasis, is pathologically elevated in patients with myelofibrosis (MF) and anemia. Chronic elevations in hepcidin contribute to the onset and severity of anemia. DISC-0974 is an investigational, first-in-class, monoclonal antibody that blocks hemojuvelin, a co-receptor in the bone morphogenetic protein-signaling pathway driving hepcidin expression. Preclinical studies have shown that DISC-0974 suppresses hepcidin and increases serum iron. A healthy volunteer study has demonstrated dose-dependent reductions in serum hepcidin, increases in serum iron with doses up to 56 mg administered subcutaneously, and increasing trends in reticulocyte count, reticulocyte hemoglobin, mean corpuscular hemoglobin, total hemoglobin (Hgb), and red blood cell count. In this Phase 1b/2a, open-label, multiple-ascending dose study (NCT05320198), we are assessing the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of DISC-0974 in patients with MF and anemia. Eligible participants include patients over 18 years of age with intermediate-2 or higher-risk MF and anemia. MF is required to be confirmed using the World Health Organization 2016 criteria. Anemia is defined per protocol as Hgb < 10 g/dL or transfusion dependence, as defined by the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT). A stable dose of hydroxyurea and/or Janus kinase (JAK) inhibitor is allowed on trial. Major exclusion criteria include: liver iron concentration ≥ 7 mg/g dry weight; splenectomy; nutritional, genetic, infectious, or autoimmune causes of anemia. Dose escalation is based on a Bayesian optimal interval design with accelerated titration. Safety Review Committee assessments gate escalation decisions. Dosing occurs monthly for a total of 6 doses. Primary endpoints include safety and tolerability assessments of adverse events (AEs), clinical laboratory assessments, vital signs, physical examinations, and electrocardiograms. Secondary endpoints include anemia response as defined by the IWG-MRT, transfusion response as defined by a decrease of 50% in transfusion requirement over any 8-week period, standard PK parameters and PD metrics including serum hepcidin-25, iron, transferrin saturation, ferritin, and hematology biomarkers. All data are summarized using descriptive statistics. Initial data show a dose-related increase in pharmacokinetic parameters. DISC-0974 treatment led to dose-dependent reductions in serum hepcidin-25 and increases in serum iron levels. There have been no serious AEs or AEs leading to study withdrawal. Most AEs were mild, transient, and unrelated to DISC-0974. As of August 1, 2023, enrollment and dose escalation are ongoing. Safety, PK, and PD data from at least the first two cohorts will be presented.