Activin Signaling Research Articles

Efficacy and Safety of Sotatercept Across Ranges of Cardiac Index in Patients with Pulmonary Arterial Hypertension: A Pooled Analysis of PULSAR and STELLAR

BackgroundThis analysis compared effects of the activin signaling inhibitor, sotatercept, across pulmonary arterial hypertension (PAH) subgroups stratified by baseline cardiac index (CI). MethodsPooled data from PULSAR (N=106; NCT03496207) and STELLAR (N=323; NCT04576988) were analyzed using two different CI thresholds, < and ≥ 2.0 L/min/m2 or 2.5 L/min/m2. Median differences in change from baseline at week 24 were evaluated using Hodges-Lehmann (HL) estimator and mean differences by least squares (LS), with 95% confidence intervals and p-values; p=0.05 was significant. Categorial endpoints and time-to-clinical worsening were analyzed by Cochran-Mantel-Haenszel and Cox model (hazard ratio (HR), respectively, without multiplicity adjustment. ResultsOf 429 participants, 51 and 378 had CI <2.0 L/min/m2 and ≥ 2.0 L/min/m2, respectively, whereas 179 and 250 had CI <2.5 L/min/m2 and ≥ 2.5 L/min/m2, respectively. Across all CI subgroups, sotatercept vs placebo significantly improved median 6-minute walk distance (range: 33.9 to 63.7 m: p<0.001), pulmonary vascular resistance (range: -202.8 to -395.4 dyn•s•cm-5; p≤0.002), and N-terminal pro-B-type natriuretic peptide (range: -317.3 to -1041.2 pg/mL; p<0.001). LS means showed reductions in pulmonary and right atrial pressures, decreased right ventricular size and enhanced tricuspid annular plane systolic excursion/ systolic pulmonary artery pressure in all subgroups. Sotatercept significantly delayed time to first occurrence of death or a worsening event in the subset of participants with CI ≥2.5 (HR 0.12; p<0.001), ≥2.0 (HR 0.13; p<0.001), and <2.5 (HR 0.21; p<0.001) L/min/m2. Improvements in World Health Organization functional class (all p<0.050) and European Society of Cardiology/European Respiratory Society risk score (all p<0.001) were seen within each subgroup. ConclusionsEfficacy and safety were consistent across baseline CI subgroups, supporting use of sotatercept in PAH patients irrespective of baseline cardiac hemodynamics.

A genome-engineered tool set for Drosophila TGF-β/BMP signaling studies.

Ligands of the TGF-β/BMP superfamily are crucially involved in the regulation of growth, patterning and organogenesis and can act as long-range morphogens. Essential for understanding TGF-β/BMP signaling dynamics and regulation are tools that allow monitoring and manipulating pathway components at physiological expression levels and endogenous spatiotemporal patterns. We used genome engineering to generate a comprehensive library of endogenously epitope- or fluorescent-tagged versions of receptors, co-receptors, transcription factors and key feedback regulators of the Drosophila BMP and Activin signaling pathways. We demonstrate that the generated alleles are biologically active and can be used for assessing tissue and subcellular distribution of the corresponding proteins. Furthermore, we show that the genomic platforms can be used for in locus structure-function and cis-regulatory analyses. Finally, we present a complementary set of protein binder-based tools, which allow visualization as well as manipulation of the stability and subcellular localization of epitope-tagged proteins, providing new tools for the analysis of BMP signaling and beyond.

Unravel novel therapeutic targets in heart failure and associated pulmonary hypertension

Abstract Background Heart Failure (HF) is a complex clinical syndrome with poor prognosis. Pulmonary hypertension (PH) often complicates HF but can hardly be addressed therapeutically. Sodium-glucose co-transporter 2 inhibitors (SGLT2i) improve cardiovascular outcomes in patients with HF. Whether SGLT2i positively impact on HF-associated PH is poorly understood. Omics offer great promise in the discovery of novel biomarkers and therapeutic targets for HF and PH. Aims We investigated novel markers associated with HF and PH, as well as how these biomarkers vary in response to HF-modyfing therapies, including SGLT2i. We also explored the impact of SGLT2i, compared with conventional anti-HF therapy, on HF-associated PH and right ventricular function. Methods Seventy-four patients with HF and reduced ejection fraction (HFrEF) with/without diabetes were divided into a gliflozin (G)-group receiving 10 mg/day empagliflozin or dapagliflozin, and a control (C)-group receiving conventional anti-HF therapy. Resting echocardiography, analyses on senescence markers [leucocyte telomere length (LTL), mitochondrial DNA copy number (mtDNAcn)], as well as measurement of two microRNAs (miRNA-21 and miRNA-92, recently implicated in response to treatment in HFpEF patients), were performed. Shotgun proteomic analyses were conducted on peripheral blood mononuclear cells. Results Compared to group C, group G had a significantly higher ejection fraction (EF, 42±13 vs 33±8, p=0.001), reduced left ventricular filling pressures (11±3 vs 15±5, p=0.001), reduced pulmonary artery systolic pressure (PAPs, 31±11 vs 46±15, p=0.001), better right ventricular-pulmonary artery coupling index (0.64±0.31 vs 0.44±0.22, p=0.002), increased LTL (Figure 1 A), higher mtDNAcn (Figure 1 B), reduced miRNA-21 levels (Figure 1 C) and no significant changes of miRNA-92 (Figure 1 D). Functional proteomic analysis showed that, compared with the C-group, the protein cargo from the G-group was able to trigger several biological pathways, showing a significant activation of T lymphocytes immune response (p-value=9.67x10-06, z-score=2.21) chemotaxis of monocytes (p-value=9.39x10-07, z-score=2.0), leukocytes (p-value=9.67x10-10, z-score=2.0)and phagocytes (p-value)=3.10x10-10, z-score=2.03) (Figure 2). Compared with the C-group, the G-group showed a downregulation of activin signaling pathway (z-score=-2.12), which is a novel therapeutic target in pulmonary arterial hypertension (PAH). Conclusions In HF patients, SGLT2i therapy is associated with improved left ventricular function and improved pulmonary hemodynamics. Activin is overactive in HF patients and its serum levels may be reduced by SGLT2i. SGLT2i may preserve LTL and mtDNA-cn as well as modulate the expression of specific miRNA implied in the regulation of endothelial function. SGLT2i may represent an additional therapeutic tool in patients with HF-associated PH, an area with no approved treatment options beyond traditional HF therapy.Figure 1:genomic analysesFigure 2:proteomic analyses

Stepwise release of Activin-A from its inhibitory prodomain is modulated by cysteines and requires furin coexpression to promote melanoma growth

The Activin-A precursor dimer can be cleaved by furin, but how this proteolytic maturation is regulated in vivo and how it facilitates access to signaling receptors is unclear. Here, analysis in a syngeneic melanoma grafting model shows that without furin coexpression, Activin-A failed to accelerate tumor growth, correlating with failure of one or both subunits to undergo cleavage in signal-sending cells, even though compensatory processing by host cells nonetheless sustained elevated circulating Activin-A levels. In reporter assays, furin-independent cleavage of one subunit enabled juxtacrine Activin-A signaling, whereas completion of proteolytic maturation by coexpressed furin or by recipient cells stimulated contact-independent activity, crosstalk with BMP receptors, and signal inhibition by follistatin. Mechanistically, Activin-A processing was modulated by allosteric disulfide bonds flanking the furin site. Disruption of these disulfide linkages with the prodomain enabled Activin-A binding to cognate type II receptors independently of proteolytic maturation. Stepwise proteolytic maturation is a novel mechanism to control Activin-A protein interactions and signaling.

8796 Increased Sertoli Cell Proliferation in FSTL3 Deleted Mouse Testis

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Activin, a TGFβ ligand, plays key roles in Sertoli cell proliferation. Follistatin like 3 (FSTL3) is a glycoprotein inhibitor of activin expressed strongly in the testis. We have shown that FSTL3 deletion (FSTL3 KO) in mice increases testicular size, likely caused by increased spermatogenesis supported by the increased Sertoli cell numbers seen in FSTL3 KO mouse testes. It is not clear, however, whether there is increased proliferation of Sertoli cells postnatally. We, therefore, investigated Sertoli cell proliferation, and molecular pathways that might be activated upon FSTL3 deletion that lead to increased Sertoli cell proliferation in mice. Paired testes weight comparison indicates that there is significant increase in testicular mass in FSTL3 KO between 28d and 56d coinciding with the onset of continued spermatogenesis. Immunofluorescence studies using PCNA as marker for proliferation and SOX9 to identify Sertoli cells, however, found no significant differences between proliferation of Sertoli cells in WT and FSTL3 KO. BrdU injection of mice followed by immunofluorescence labelling of proliferating Sertoli cells also showed no difference between the two genotypes by immunofluorescence. We then proceeded to assess whether there is an alteration of differentiation in FSTL3 KO testes to test the possibility that instead of proliferation there is an increased differentiation of a pre-Sertoli like cell in FSTL3 KO mice using WT1 and SOX9 as markers for early or late Sertoli cells. This experiment revealed that there was no difference between the ratio of WT1 and SOX9 expression in the two genotypes. Flow cytometric analyses using testicular dispersates from mice using Sox9 and Ki67 antibodies to identify proliferating Sertoli cells, however, revealed an approximately two-fold increase in proliferating Sertoli cells in FSTL3 KO compared to WT at 7d. At 56d there were very few proliferating Sertoli cells in either genotype and there was no difference in this number in FSTL3 deleted mice compared to WT. mRNA sequence analysis showed that approximately 1000 genes are differentially regulated between WT and FSTL3 KO at 3d. Gene ontology and pathway analysis revealed that a constellation of meiosis related genes and activin signalling pathway, among others, are enriched in FSTL3 KO testis at this stage. Data presented here demonstrates that FSTL3 deletion induces activin receptor signalling pathway, genes crucial for meiosis and therefore the spermatogenic process, and Sertoli cell proliferation. This opens the possibility that the early arrest of Sertoli cell proliferation seen in mammals including mice and humans might be circumvented by inactivating FSTL3. This could then lead to a therapeutic angle in male infertility that are based on paucity of Sertoli cells. It will be important to address why FSTL3 deletion induces Sertoli cell proliferation only postnatally but not post pubertally. Presentation: 6/1/2024

8796 Increased Sertoli Cell Proliferation in FSTL3 Deleted Mouse Testis

Abstract Disclosure: R. Ballesteros: None. A. Mukherjee: None. Activin, a TGFβ ligand, plays key roles in Sertoli cell proliferation. Follistatin like 3 (FSTL3) is a glycoprotein inhibitor of activin expressed strongly in the testis. We have shown that FSTL3 deletion (FSTL3 KO) in mice increases testicular size, likely caused by increased spermatogenesis supported by the increased Sertoli cell numbers seen in FSTL3 KO mouse testes. It is not clear, however, whether there is increased proliferation of Sertoli cells postnatally. We, therefore, investigated Sertoli cell proliferation, and molecular pathways that might be activated upon FSTL3 deletion that lead to increased Sertoli cell proliferation in mice. Paired testes weight comparison indicates that there is significant increase in testicular mass in FSTL3 KO between 28d and 56d coinciding with the onset of continued spermatogenesis. Immunofluorescence studies using PCNA as marker for proliferation and SOX9 to identify Sertoli cells, however, found no significant differences between proliferation of Sertoli cells in WT and FSTL3 KO. BrdU injection of mice followed by immunofluorescence labelling of proliferating Sertoli cells also showed no difference between the two genotypes by immunofluorescence. We then proceeded to assess whether there is an alteration of differentiation in FSTL3 KO testes to test the possibility that instead of proliferation there is an increased differentiation of a pre-Sertoli like cell in FSTL3 KO mice using WT1 and SOX9 as markers for early or late Sertoli cells. This experiment revealed that there was no difference between the ratio of WT1 and SOX9 expression in the two genotypes. Flow cytometric analyses using testicular dispersates from mice using Sox9 and Ki67 antibodies to identify proliferating Sertoli cells, however, revealed an approximately two-fold increase in proliferating Sertoli cells in FSTL3 KO compared to WT at 7d. At 56d there were very few proliferating Sertoli cells in either genotype and there was no difference in this number in FSTL3 deleted mice compared to WT. mRNA sequence analysis showed that approximately 1000 genes are differentially regulated between WT and FSTL3 KO at 3d. Gene ontology and pathway analysis revealed that a constellation of meiosis related genes and activin signalling pathway, among others, are enriched in FSTL3 KO testis at this stage. Data presented here demonstrates that FSTL3 deletion induces activin receptor signalling pathway, genes crucial for meiosis and therefore the spermatogenic process, and Sertoli cell proliferation. This opens the possibility that the early arrest of Sertoli cell proliferation seen in mammals including mice and humans might be circumvented by inactivating FSTL3. This could then lead to a therapeutic angle in male infertility that are based on paucity of Sertoli cells. It will be important to address why FSTL3 deletion induces Sertoli cell proliferation only postnatally but not post pubertally. Presentation: 6/1/2024

A new day has come: Sotatercept for the treatment of pulmonary arterial hypertension

Despite increasing therapeutic options and evolving treatment strategies, including targeting three therapeutic pathways, in the management of pulmonary arterial hypertension (PAH), morbidity and mortality have remained unacceptably high. Sotatercept is a first-in-class, novel activin signaling inhibitor approved for treating PAH based on evolving efficacy and safety evidence. This state-of-the-art review summarizes the current understanding of the mechanism of action, the impact on outcomes that improve how patients feel, function, and survive, and the safety and adverse event profile to inform readers of this breakthrough novel therapy.

Pulmonary Arterial Hypertension and TGF-β Superfamily Signaling: Focus on Sotatercept.

Pulmonary arterial hypertension (PAH) is a rare and progressive disease that continues to remain highly morbid despite multiple advances in medical therapies. There remains a persistent and desperate need to identify novel methods of treating and, ideally, reversing the pathologic vasculopathy that results in PAH development and progression. Sotatercept is a first-in-class fusion protein that is believed to primarily inhibit activin signaling resulting in decreased cell proliferation and differentiation, though the exact mechanism remains uncertain. Here, we review the currently available PAH therapies, data highlighting the importance of transforming growth factor-β (TGF-β) superfamily signaling in the development of PAH, and the published and on-going clinical trials evaluating sotatercept in the treatment of PAH. We will also discuss preclinical data supporting the potential use of the fusion protein KER-012 in the inhibition of aberrant TGF-β superfamily signaling to ameliorate the obstructive vasculopathy of PAH.

Activin levels correlate with lymphocytic infiltration in epithelial ovarian cancer.

The TGF-β superfamily member activin, a dimer of the gene products of INHBA and/or INHBB, has been implicated in immune cell maturation and recruitment, but its immune impact within epithelial ovarian cancer (EOC) is not well characterized. We sought to explore differences in activin (INHBA/ Inhibin-βA and INHBB/ Inhibin-βB) between malignant and ovarian tissues at the RNA and protein level and assess the relationship between activin and immune cells in EOC. Publicly available RNA sequencing data were accessed from GEO (#GSE143897) with normalization and quantification performed via DESeq2. Immune gene expression profile was further explored within the TCGA-OV cohort derived from The Cancer Genome Atlas (TCGA). Immunohistochemical analysis was performed to evaluate activin A and T-cell markers CD8 and FoxP3 at the protein level. ELISA to activin-A was used to assess levels in the ascites of advanced EOC patients. Kaplan-Meier curves were generated to visualize survival outcomes. Gene expression levels of components of the activin signaling pathway were elevated within EOC when compared to a benign cohort, with differences in activin type I/II receptor gene profiles identified. Additionally, INHBA gene expression was linked to lymphocytic immune markers in EOC samples. Immunohistochemistry analysis revealed a positive correlation of CD8 and FOXP3 staining with activin A at the protein level in both primary and metastatic epithelial ovarian cancer samples. Furthermore, Activin-A (inhibin-βA) is significantly elevated in EOC patient ascites. INHBA expression is elevated within EOC, correlating with worse survival, with activin protein levels correlating with specific immune infiltration. Our findings suggest that activin-A may play a role in suppressing anti-tumor immunity in EOC, highlighting its potential as a therapeutic target.

Identification of ActivinβA and Gonadotropin Regulation of the Activin System in the Ovary of Chinese Sturgeon Acipenser sinensis.

Activin is a dimeric growth factor with diverse biological activities in vertebrates. This study aimed to investigate the regulatory role of the activin signaling pathway in the ovary of the endangered, cultured sturgeon species Acipenser sinensis. One activinβA subunit was identified, with a full-length complementary DNA (cDNA) sequence of 1572 base pairs. Multiple sequence alignment suggested that ActivinβA shared high sequence identities with its counterparts in four other sturgeon species. Phylogenetic analysis indicated the conserved evolution of ActivinβA among vertebrates from mammals to fish species. Transcripts of activinβA were distributed ubiquitously in the liver, kidney, intestine, ovary, midbrain, hypothalamus, and pituitary, with the highest transcription found in the pituitary. In Chinese sturgeon ovarian cells, in vitro human recombinant Activin A incubation stimulated the activin system-related gene transcriptions of activinβA, follistatin, its receptors -activinRIIA and activinRIIB, and drosophila mothers against decapentaplegic proteins (smads) smad2, smad3, and smad4. Ovary development-related mRNA levels of cyp19a1a and aromatase receptors of erα and erβ were enhanced by Activin A or human chorionic gonadotropin (hCG) incubation. Furthermore, 15 IU/mL hCG treatment increased the transcription levels of activinβA, follistatin, activinRIIA, and smad2. This suggested that the activin system was functional for the regulation of ovary development in Chinese sturgeon, possibly under the regulation of gonadotropin, by recruiting activinβA, follistatin, activinRIIA, and smad2. These results were helpful for the molecular exploration of activin signaling in fish species, as well as the ovarian maturation regulation of A. sinensis.

Abstract Tu053: Activin Signaling Inhibition Enhances Cardiac Functional Recovery After Aortic Debanding

Background: Human aortic stenosis (AS) induces adverse cardiac remodeling and heart failure by pressure overload. Reducing pressure overload (e.g., Trans-catheter aortic valve replacement (TAVR) mitigates or reverses adverse remodeling but these benefits are limited in many TAVR patients. Here we established a murine transverse-aortic-constriction (TAC)-debanding model to mimic TAVR and identify ways to enhance reverse remodeling. Methods: 10-week-old wild-type C57BL/6 mice were subjected to TAC surgery and debanding by suture removal, or sham-debanding, between 4 (short duration) and 10 weeks (long duration) after TAC. Echocardiography was performed to assess cardiac function. RNA sequencing (RNAseq) of TAC and debanded hearts demonstrated activation of activin signaling with TAC that was relieved by debanding. Based on this, TAC-debanded animals were treated with an investigational modified activin receptor type IIB ligand trap, RKER-012 (10 mg/kg IP, bi-weekly), to inhibit signaling downstream of activin and related ligands. Results: Serial echocardiography of TAC-debanded animals showed greater dysfunction with longer duration TAC (Fractional shortening [FS]=39.2±3.84 for 4 weeks-TAC vs Sham: 61.2±0.94, p&lt;0.05; FS=18.2±1.80 for 10 weeks-TAC vs Sham-TAC: 64.7±0.43, p&lt;0.05). Cardiac functional recovery was slower and less complete in animals with longer duration TAC (10 weeks after debanding with TAC for 4 weeks, FS=57.8±1.41 vs 46.6±3.4 TAC for 10 weeks). RNAseq analysis of hearts post-debanding (10 weeks TAC+10 weeks debanding) showed a 5.73-fold decrease (p=2.57x10 -16 ) in FSTL3 expression, a biomarker of activin pathway signaling, suggesting a decrease in activin signaling accompanied functional recovery. To assess functional contribution of activin signaling to incomplete recovery and therapeutic potential of pathway inhibitors, we treated debanded animals (debanded 8 weeks post-TAC) with RKER-012 or placebo (TBS) for 4 weeks. RKER-012-treated mice showed significantly increased ejection fraction (54.12±1.50 vs placebo control 42.88±3.21; p=0.001) and reduced heart weight (p=0.01). Conclusions: Debanding after TAC mitigates adverse remodeling that is slower and less complete with longer duration TAC. A ligand trap (RKER-012) that was designed to inhibit signaling downstream of activin enhanced recovery after debanding and warrants further study as a potential therapeutic intervention in patients with incomplete functional recovery after TAVR.

Activin Signaling Pathway Specialization During Embryonic and Skeletal Muscle Development in Rainbow Trout (Oncorhynchus mykiss).

Activin signaling is essential for proper embryonic, skeletal muscle, and reproductive development. Duplication of the pathway in teleost fish has enabled diversification of gene function across the pathway but how gene duplication influences the function of activin signaling in non-mammalian species is poorly understood. Full characterization of activin receptor signaling pathway expression was performed across embryonic development and during early skeletal muscle growth in rainbow trout (RBT, Oncorhynchus mykiss). Rainbow trout are a model salmonid species that have undergone two additional rounds of whole genome duplication. A small number of genes were expressed early in development and most genes increased expression throughout development. There was limited expression of activin Ab in RBT embryos despite these genes exhibiting significantly elevated expression in post-hatch skeletal muscle. CRISPR editing of the activin Aa1 ohnolog and subsequent production of meiotic gynogenetic offspring revealed that biallelic disruption of activin Aa1 did not result in developmental defects, as occurs with knockout of activin A in mammals. The majority of gynogenetic offspring exhibited homozygous activin Aa1 genotypes (wild type, in-frame, or frameshift) derived from the mosaic founder female. The research identifies mechanisms of specialization among the duplicated activin ohnologs across embryonic development and during periods of high muscle growth in larval and juvenile fish. The knowledge gained provides insights into potential viable gene-targeting approaches for engineering the activin receptor signaling pathway and establishes the feasibility of employing meiotic gynogenesis as a tool for producing homozygous F1 genome-edited fish for species with long-generation times, such as salmonids.

The Activin Branch Ligand Daw Regulates the Drosophila melanogaster Immune Response and Lipid Metabolism against the Heterorhabditis bacteriophora Serine Carboxypeptidase.

Despite impressive advances in the broad field of innate immunity, our understanding of the molecules and signaling pathways that control the host immune response to nematode infection remains incomplete. We have shown recently that Transforming Growth Factor-β (TGF-β) signaling in the fruit fly Drosophila melanogaster is activated by nematode infection and certain TGF-β superfamily members regulate the D. melanogaster anti-nematode immune response. Here, we investigate the effect of an entomopathogenic nematode infection factor on host TGF-β pathway regulation and immune function. We find that Heterorhabditis bacteriophora serine carboxypeptidase activates the Activin branch in D. melanogaster adults and the immune deficiency pathway in Activin-deficient flies, it affects hemocyte numbers and survival in flies deficient for Activin signaling, and causes increased intestinal steatosis in Activin-deficient flies. Thus, insights into the D. melanogaster signaling pathways and metabolic processes interacting with H. bacteriophora pathogenicity factors will be applicable to entomopathogenic nematode infection of important agricultural insect pests and vectors of disease.

Contemporary Treatment of Pulmonary Arterial Hypertension: A U.S. Perspective.

Pulmonary arterial hypertension (PAH) is a complex fatal condition that requires aggressive treatment with close monitoring. Significant progress has been made over the last three decades in the treatment of PAH, but, despite this progress, survival has remained unacceptably low. In the quest to improve survival, therapeutic interventions play a central role. In the last few years, there have been remarkable attempts to identify novel treatments. Finally, we have had a breakthrough with the discovery of the fourth treatment pathway in PAH. Activin signaling inhibition distinguishes itself as a potential antiproliferative intervention as opposed to the traditional therapies, which mediate their effect primarily by vasodilatation. With this novel treatment pathway, we stand at an important milestone with an exciting future ahead and the natural question of when to use an activin signaling inhibitor for the treatment of PAH. In this state-of-the-art review, we focus on the placement of this novel agent in the PAH treatment paradigm, based on the available evidence, with special focus on the U.S. patient population. This review also provides an expert opinion of the current treatment algorithm in important subgroups of patients with comorbidities from the U.S. perspective.

Sotatercept: First Approval.

Sotatercept (sotatercept-csrk; WINREVAIRTM) is an activin signalling inhibitor that is being developed by Merck and Co., Inc. (Rahway, NJ, USA) for the treatment of pulmonary arterial hypertension. Sotatercept recently received approval in the USA for the treatment of adults with pulmonary arterial hypertension [World Health Organisation (WHO) Group 1] to increase exercise capacity, improve WHO functional class and reduce the risk of clinical worsening events. This article summarizes the milestones in the development of sotatercept leading to this first approval for pulmonary arterial hypertension.

Therapeutic approaches for pulmonary hypertension in patients with chronic kidney disease.

Pulmonary hypertension is a common comorbidity in patients with chronic kidney disease (CKD), but therapeutic options are limited. We discuss the epidemiology of pulmonary hypertension in patients with CKD and review therapies for pulmonary hypertension with a focus on emerging treatments for pulmonary arterial hypertension (PAH). The definition of pulmonary hypertension has been updated to a lower threshold of mean pulmonary artery pressures of more than 20 mmHg, potentially leading to more patients with CKD to qualify for the diagnosis of pulmonary hypertension. Endothelin receptor antagonists, a class of medications, which demonstrated efficacy in patients with PAH, have been shown to slow progression of CKD, but their efficacy in lowering pulmonary artery pressures and their effects on reducing cardiovascular mortality in this population remains unproven. Sotatercept, a novel activin signaling inhibitor, which was previously studied in dialysis patients has been shown to increase exercise capacity in patients with PAH. These studies may lead to new specific therapies for pulmonary hypertension in patients with CKD. Pulmonary hypertension is common in patients with CKD. Although our understanding of factors leading to pulmonary hypertension in this population have evolved, evidence supporting disease-specific therapy in CKD is limited arguing for larger, long-term studies.

Prognostic genomic signatures in patients with RAS and BRAF wild-type (wt) metastatic colorectal cancer (mCRC) from the phase III study of first-line FOLFIRI/cetuximab versus FOLFIRI/cetuximab followed by cetuximab (Cet) alone (ERMES study).

3590 Background: The ERMES trial did not show non-inferiority of maintenance with Cet alone versus standard treatment. However, preliminary results suggested that a strategy of de-escalation treatment with only Cet might be effective in selected patients. Here we describe preliminary data from comprehensive genomic profiling (CGP) of CRC samples from the ERMES study. Methods: Patients with untreated RAS/BRAF wt mCRC were randomly assigned (1:1) to receive either FOLFIRI/Cet until PD/toxicity (arm A) or FOLFIRI/Cet for 8 cycles followed by Cet alone (arm B). DNA from tumor tissue samples was analyzed with the Oncomine Comprehensive Assay Plus covering hot spot mutations in over 500 genes. Variant calling was performed using the Ion Reporter v5.20 software. The prognostic value of genomic signatures was assessed in the intention-to-treat (ITT) patients’ population with available sequencing data. Results: CGP has been successfully completed so far for 139 patients of the ITT population (68 in arm A and 71 in arm B). This subgroup had a median progression free survival (mPFS) of 9.3 months. The comparison of the genomic profile of cases within the 25th and the 75th mPFS percentile (34 and 36 patients, respectively), identified two signatures that were defined: RES, 14 genes with genomic alterations unique to the 25th percentile poor prognosis group; SENS, 34 genes altered only in the 75th percentile good prognosis cohort. The RES signature was significantly associated with ERBB2, IL-4 and IL-13, MET activated PI3K/AKT, ESR, PI3K and FGFR4 signalling. When the RES signature was applied to the whole cohort, patients with at least one genomic alterations in any gene of the signature showed a mPFS of 2.4 months versus 15.2 months of the wt (Hazard Ratio, HR wt versus mutant 0.07). The SENS signature was associated with pathways related to gene transcription and expression, SMAD 2/3/4 transcription, DNA repair, chromatin modification and Activin signalling. The mPFS of SENS mutant and wt patients was 16.2 and 3.2 months, respectively (HR wt versus mutant 10.09). An exploratory analysis found no significant differences in mPFS for patients with good prognosis based on genomic signatures enrolled in the standard arm (A) versus the experimental arm (B). Conclusions: These preliminary data suggest that CGP of mCRC patients can identify prognostic genomic signatures, which might allow a better stratification of patients and the identification of subgroups that might benefit a treatment de-escalation strategy.

Fluorescence-activated nuclear sorting (FANS) of nuclei from in vitro-generated syncytiotrophoblast

Large, multinucleated cells, like syncytiotrophoblasts (STB), are not readily analyzed by standard methods used for single cells, such as single-cell RNA-sequencing and fluorescence-activated cellular sorting (FACS). Here we have demonstrated that fluorescence-activated nuclear sorting (FANS) is suitable to analyze nuclei from STB. Human pluripotent stem cells (PSCs) can be differentiated into a mixed trophoblast populations comprising approximately 20 % STB by treatment with BMP4 (Bone Morphogenetic Protein-4), plus A83-01 and PD173074, inhibitors of activin and FGF2 signaling, respectively (the BAP model) in about a week. Here we demonstrate that FANS can be used to separate two types of STB nuclei from the nine different clusters of trophoblast nuclei previously identified in the BAP model by single nucleus RNA sequencing (snRNAseq). Rather than using cell surface markers, as in FACS, transcription factors in various combinations were employed to target specific nuclear types. Nuclei were isolated at d 8 of BAP differentiation of H1 human embryonic stem cells and fixed in 4 % paraformaldehyde. After permeabilization in 0.1 % triton X-100, nuclei were incubated for 3 and 1 h at 4 °C with primary and secondary antibodies respectively and nuclear samples were then subjected to FANS. By using markers identified by snRNA and immunohistochemistry, nuclei were first sorted into a Topoisomerase-1, or TOP1, bright population and then into the two STB subpopulations by using antibodies to JUNB (Jun B Proto-Oncogene) and TFCP2L1 (Transcription Factor CP2 Like 1). The protocol established here is simple, straightforward, and efficient and can be used on a relatively large scale to sort individual subtypes of nuclei from mixed populations of trophoblasts for further analysis.

ARID1B maintains mesenchymal stem cell quiescence via inhibition of BCL11B-mediated non-canonical Activin signaling

ARID1B haploinsufficiency in humans causes Coffin-Siris syndrome, associated with developmental delay, facial dysmorphism, and intellectual disability. The role of ARID1B has been widely studied in neuronal development, but whether it also regulates stem cells remains unknown. Here, we employ scRNA-seq and scATAC-seq to dissect the regulatory functions and mechanisms of ARID1B within mesenchymal stem cells (MSCs) using the mouse incisor model. We reveal that loss of Arid1b in the GLI1+ MSC lineage disturbs MSCs’ quiescence and leads to their proliferation due to the ectopic activation of non-canonical Activin signaling via p-ERK. Furthermore, loss of Arid1b upregulates Bcl11b, which encodes a BAF complex subunit that modulates non-canonical Activin signaling by directly regulating the expression of activin A subunit, Inhba. Reduction of Bcl11b or non-canonical Activin signaling restores the MSC population in Arid1b mutant mice. Notably, we have identified that ARID1B suppresses Bcl11b expression via specific binding to its third intron, unveiling the direct inter-regulatory interactions among BAF subunits in MSCs. Our results demonstrate the vital role of ARID1B as an epigenetic modifier in maintaining MSC homeostasis and reveal its intricate mechanistic regulatory network in vivo, providing novel insights into the linkage between chromatin remodeling and stem cell fate determination.

A Lifelike guided journey through the pathophysiology of pulmonary hypertension—from measured metabolites to the mechanism of action of drugs

IntroductionPulmonary hypertension (PH) is a pathological condition that affects approximately 1% of the population. The prognosis for many patients is poor, even after treatment. Our knowledge about the pathophysiological mechanisms that cause or are involved in the progression of PH is incomplete. Additionally, the mechanism of action of many drugs used to treat pulmonary hypertension, including sotatercept, requires elucidation.MethodsUsing our graph-powered knowledge mining software Lifelike in combination with a very small patient metabolite data set, we demonstrate how we derive detailed mechanistic hypotheses on the mechanisms of PH pathophysiology and clinical drugs.ResultsIn PH patients, the concentration of hypoxanthine, 12(S)-HETE, glutamic acid, and sphingosine 1 phosphate is significantly higher, while the concentration of L-arginine and L-histidine is lower than in healthy controls. Using the graph-based data analysis, gene ontology, and semantic association capabilities of Lifelike, led us to connect the differentially expressed metabolites with G-protein signaling and SRC. Then, we associated SRC with IL6 signaling. Subsequently, we found associations that connect SRC, and IL6 to activin and BMP signaling. Lastly, we analyzed the mechanisms of action of several existing and novel pharmacological treatments for PH. Lifelike elucidated the interplay between G-protein, IL6, activin, and BMP signaling. Those pathways regulate hallmark pathophysiological processes of PH, including vasoconstriction, endothelial barrier function, cell proliferation, and apoptosis.DiscussionThe results highlight the importance of SRC, ERK1, AKT, and MLC activity in PH. The molecular pathways affected by existing and novel treatments for PH also converge on these molecules. Importantly, sotatercept affects SRC, ERK1, AKT, and MLC simultaneously. The present study shows the power of mining knowledge graphs using Lifelike’s diverse set of data analytics functionalities for developing knowledge-driven hypotheses on PH pathophysiological and drug mechanisms and their interactions. We believe that Lifelike and our presented approach will be valuable for future mechanistic studies of PH, other diseases, and drugs.