650 Target the activin receptor 1c on CD4+ T cells to achieve anti-tumor therapeutic effects

BackgroundActivins, members of the transforming growth factor-ß (TGF-ß) superfamily, were isolated and identified in endocrine system, and have been widely studied in endocrine-related cancers,1 2 but not substantially in the context of immune system and endocrine-unrelated cancers.3–5 It has been reported that upon binding to the receptors, activins cause the intracellular recruitment and phosphorylation of smad proteins, which mediate the expression of Foxp3.6–9 Therefore, we hypothesized that the blockade of the interaction of activins and their receptors will inhibit the activins-mediated Foxp3 induction in CD4+ T cells, thus modify the immune suppressive tumor microenvironment and achieve the goal of cancer immunotherapy.MethodsELISA (enzyme-linked immunosorbent assay) has been performed to determine the plasma level of Activin A in tumor-bearing mice and cancer patients. In vitro iTreg (induced regulatory T cells) differentiation has been done to naïve CD4+ cells isolated from wild type mice in the presence or absence of Activin A, and the percentage of Foxp3+ cells was demonstrated by flow cytometric analysis. qRT-PCR analysis has been conducted to determine the mRNA level of activin receptor isotypes in the immune subpopulations sorted from Foxp3-YFP mice. In the end, in vivo subcutaneous transplanted tumor studies have been done to evaluate the anti-tumor therapeutic effects of activin-receptor 1c blockade.ResultsWe show here that tumor-bearing mice had elevated Activin A levels, which correlated directly with tumor burden. Likewise, cancer patients had elevated plasma Activin A compared to healthy controls. Importantly, our in vitro studies suggested that Activin A promoted differentiation of conventional CD4+ cells into Foxp3-expressing induced Tregs, especially when TGF-ß was limited. Database and qRT-PCR analysis of sorted major immune cell subsets in mice revealed that activin receptor 1C (Acvr1c) was uniquely expressed on Tregs and was highly upregulated during iTreg differentiation. Mice deficient in Acvr1c were more resistant to cancer progression compared to wild type mice. This phenotype correlated with reduced expression of the FoxP3 transcription factor in CD4+ cells. Similar phenomena were observed when we treated the mice with anti-Acvr1c antibody after tumor inoculation. This anti-tumor therapeutic effect was more significant when anti-Acvr1c antibody was administrated in combination with anti-PD-1 antibody.ConclusionsBlocking Activin A signaling through its receptor 1c is a promising and disease-specific strategy for preventing the accumulation of immunosuppressive iTregs in cancer. Hence it represents a potential candidate for cancer immunotherapy.AcknowledgementsThis research is supported by the Bloomberg-Kimmel Institute (Immunometabolism Program & Immune Modulation Program), the Melanoma Research Alliance, the NIH (RO1AI099300, RO1AI089830, and R01AI137046), and The DoD (PC130767).ReferencesRisbridger GP, Schmitt JF, Robertson DM. Activins and inhibins in endocrine and other tumors. Endocr Rev 2001;22(6):836–858.Cui X, et al. Perspectives of small molecule inhibitors of activin receptor-like kinase in anti-tumor treatment and stem cell differentiation (Review). Mol Med Rep 2019;19(6):5053–5062.Michael IP, et al. ALK7 signaling manifests a homeostatic tissue barrier that is abrogated during tumorigenesis and metastasis. Dev Cell 2019;49(3):409–424.Wu B, et al. The TGF-ß superfamily cytokine Activin-A is induced during autoimmune neuroinflammation and drives pathogenic Th17 cell differentiation. Immunity 2021;54(2):308–323.Antsiferova M, et al. Activin promotes skin carcinogenesis by attraction and reprogramming of macrophages. MBO Mol Med 2017;9(1):27–45.Tsuchida K, et al. Activin isoforms signal through type I receptor serine/threonine kinase ALK7. Mol Cell Endocrinol 2004;220(1–2):59–65.Khalil AM, et al. Differential binding activity of TGF-ß family proteins to select TGF-ß receptors. J Pharmacol Exp Ther 2016;358(3):423–430.Huber S, et al. Activin a promotes the TGF-beta-induced conversion of CD4+CD25- T cells into Foxp3+ induced regulatory T cells. J Immunol 2009;182(8):4633–4640.Iizuka-Koga M, et al. Induction and maintenance of regulatory T cells by transcription factors and epigenetic modifications. J Autoimmun 2017;83:113–121.Ethics ApprovalAll animal experiments were performed under protocols approved by the Johns Hopkins University Institutional Animal Care and Use Committee (IACUC).

Functionally diverse heteromeric traps for ligands of the transforming growth factor-β superfamily

Ligands of the transforming growth factor-β (TGF-β) superfamily are important targets for therapeutic intervention but present challenges because they signal combinatorially and exhibit overlapping activities in vivo. To obtain agents capable of sequestering multiple TGF-β superfamily ligands with novel selectivity, we generated soluble, heterodimeric ligand traps by pairing the extracellular domain (ECD) of the native activin receptor type IIB (ActRIIB) alternately with the ECDs of native type I receptors activin receptor-like kinase 4 (ALK4), ALK7, or ALK3. Systematic analysis of these heterodimeric constructs by surface plasmon resonance, and comparison with their homodimeric counterparts, revealed that each type I receptor partner confers a distinct ligand-binding profile to the heterodimeric construct. Additional characterization in cell-based reporter gene assays confirmed that the heterodimeric constructs possessed different profiles of signaling inhibition in vitro, which translated into altered patterns of pharmacological activity when constructs were administered systemically to wild-type mice. Our results detail a versatile platform for the modular recombination of naturally occurring receptor domains, giving rise to inhibitory ligand traps that could aid in defining the physiological roles of TGF-β ligand sets or be directed therapeutically to human diseases arising from dysregulated TGF-β superfamily signaling.

Myostatin/Activin Receptor Ligands In Muscle And The Development Status Of Attenuating Drugs

Muscle wasting disease indications are among the most debilitating and often deadly noncommunicable disease states. As a comorbidity, muscle wasting is associated with different neuromuscular diseases and myopathies, cancer, heart failure, chronic pulmonary and renal diseases, peripheral neuropathies, inflammatory disorders and of course, musculoskeletal injuries. Current treatment strategies are relatively ineffective and can at best only limit the rate of muscle degeneration. This includes nutritional supplementation and appetite stimulants as well as immunosuppressants capable of exacerbating muscle loss. Arguably, the most promising treatments in development attempt to disrupt myostatin and activin receptor signaling as these circulating factors are potent inhibitors of muscle growth and regulators of muscle progenitor cell differentiation. Indeed, several studies demonstrated the clinical potential of “inhibiting the inhibitors”, increasing muscle cell protein synthesis, decreasing degradation, enhancing mitochondrial biogenesis and preserving muscle function. Such changes can prevent muscle wasting in various disease animal models yet many drugs targeting this pathway failed during clinical trials, some from serious treatment-related adverse events and off-target interactions. More often, however, failures resulted from the inability to improve muscle function despite preserving muscle mass. Drugs still in development include antibodies and gene therapeutics, all with different targets and thus, safety, efficacy and proposed use profiles. Each is unique in design and, if successful, could revolutionize the treatment of both acute and chronic muscle wasting. They could also be used in combination with other developing therapeutics for related muscle pathologies or even metabolic diseases.

Accumulated Knowledge of Activin Receptor-Like Kinase 2 (ALK2)/Activin A Receptor, Type 1 (ACVR1) as a Target for Human Disorders

Activin receptor-like kinase 2 (ALK2), also known as Activin A receptor type 1 (ACVR1), is a transmembrane kinase receptor for members of the transforming growth factor-β family. Wild-type ALK2/ACVR1 transduces osteogenic signaling in response to ligand binding. Fifteen years ago, a gain-of-function mutation in the ALK2/ACVR1 gene was detected in patients with the genetic disorder fibro-dysplasia ossificans progressiva, which is characterized by heterotopic ossification in soft tissues. Additional disorders, such as diffuse intrinsic pontin glioma, diffuse idiopathic skeletal hyperostosis, primary focal hyperhidrosis, and congenital heart defects, have also been found to be associated with ALK2/ACVR1. These findings further expand in vitro and in vivo model system research and promote our understanding of the molecular mechanisms of the pathogenesis and development of novel therapeutics and diagnosis for disorders associated with ALK2/ACVR1. Through aggressive efforts, some of the disorders associated with ALK2/ACVR1 will be overcome in the near future.

Myostatin regulates the production of fibroblast growth factor 23 (FGF23) in UMR106 osteoblast–like cells

Myostatin is a signaling molecule produced by skeletal muscle cells (myokine) that inhibits muscle hypertrophy and has further paracrine and endocrine effects in other organs including bone. Myostatin binds to activin receptor type 2B which forms a complex with transforming growth factor-β type I receptor (TGF-βRI) and induces intracellular p38MAPK and NFκB signaling. Fibroblast growth factor 23 (FGF23) is a paracrine and endocrine mediator produced by bone cells and regulates phosphate and vitamin D metabolism in the kidney. P38MAPK and NFκB-dependent store-operated Ca2+ entry (SOCE) are positive regulators of FGF23 production. Here, we explored whether myostatin influences the synthesis of FGF23. Fgf23 gene expression was determined by qRT-PCR and FGF23 protein by ELISA in UMR106 osteoblast–like cells. UMR106 cells expressed activin receptor type 2A and B. Myostatin upregulated Fgf23 gene expression and protein production. The myostatin effect on Fgf23 was significantly attenuated by TGF-βRI inhibitor SB431542, p38MAPK inhibitor SB202190, and NFκB inhibitor withaferin A. Moreover, SOCE inhibitor 2-APB blunted the myostatin effect on Fgf23. Taken together, myostatin is a stimulator of Fgf23 expression in UMR106 cells, an effect at least partially mediated by downstream TGF-βRI/p38MAPK signaling as well as NFκB-dependent SOCE.