Targeting Transforming Growth Factor-β Research Articles

TGF-β receptor-specific NanoBRET Target Engagement in living cells for high-throughput kinase inhibitor screens

Targeting transforming growth factor-β (TGF-β) receptors is a promising pharmacological approach to normalize aberrant signaling in genetic and non-genetic TGF-β associated diseases including fibrosis, cancer, cardiovascular and musculoskeletal disorders. To identify novel TGF-β receptor kinase inhibitors, methods like in vitro kinase assays, western blot or transcriptional reporter assays are often used for screening purposes. While these methods may have certain advantages, the lack of integration of key features such as receptor specificity, high-throughput capability, and cellular context resemblance remains a major disadvantage. This deficiency could ultimately hinder the translation of study outcomes into later (clinical) stages of drug development. In this study, we introduce an adjusted and optimized live cell NanoBRET Target Engagement (TE)-based method to identify TGF-β receptor specific kinase inhibitors. This comprehensive toolkit contains various TGF-β type I and type II receptors, with corresponding nanoBRET tracers, and disease-related cell lines, including novel non-commercially available materials. The nanoBRET capacity and kinase inhibitory window can be significantly enhanced for functional measurements when stable expression cell lines and substantially low tracer concentrations are used. In addition, this system can be tailored to study TGF-β associated genetic disorders and possibly be used to screen for disease-specific therapeutics. Therefore, the use of this optimized, live cell, antibody-independent nanoBRET Target Engagement assay is highly encouraged for future high-throughput compound screens targeting TGF-β/BMP receptors.

Efficacy and Safety of Bintrafusp Alfa, a Bifunctional Fusion Protein Targeting Transforming Growth Factor-β and Programmed Death-Ligand 1, Plus Chemotherapy in Patients With Stage IV NSCLC

IntroductionIn a phase 1 study, bintrafusp alfa was found to have an encouraging clinical activity in patients with previously treated advanced NSCLC. This study evaluated the safety and efficacy of bintrafusp alfa with chemotherapy in patients with stage IV NSCLC regardless of the programmed death-ligand 1 (PD-L1) expression status. MethodsIn this open-label, phase 1b/2 study (NCT03840915), eligible patients were assigned to one of four cohorts. Patients with previously untreated metastatic NSCLC (cohorts A, B, and C) received bintrafusp alfa with chemotherapy as first-line treatment, whereas patients whose disease progressed on previous treatment with programmed cell death protein 1/PD-L1 inhibitors (cohort D) received bintrafusp alfa with chemotherapy as second-line treatment. The primary objective of this study was to evaluate the safety and tolerability of bintrafusp alfa with chemotherapy. ResultsFour serious and one nonserious treatment-emergent adverse events were considered dose-limiting toxicities, none of which were assessed as related to bintrafusp alfa by the investigator. Any-grade bintrafusp alfa-related adverse events occurred in 20.7% of patients in cohorts A+B+C and in 16.7% of patients in cohort D. Keratoacanthoma was the most common transforming growth factor-β inhibition-mediated skin lesion (cohorts A+B+C: 12.1% and cohort D: 8.3%). In cohorts A+B+C, the overall response rate was 48.3%, and in patients with PD-L1 tumor proportion score of more than or equal to 50.0%, it was 71.4%. On the basis of an interim analysis, the data were considered mature, and no further analysis has been planned. ConclusionBintrafusp alfa with chemotherapy was found to have a manageable safety profile and encouraging clinical activity in patients with stage IV NSCLC.

Efficacy, safety, and biomarker analyses of bintrafusp alfa, a bifunctional fusion protein targeting TGF-β and PD-L1, in patients with advanced non-small cell lung cancer

BackgroundBintrafusp alfa, a first-in-class bifunctional fusion protein targeting transforming growth factor-β (TGF-β) and programmed cell death ligand 1, has demonstrated encouraging efficacy as second-line treatment in patients with non-small cell...

Mesenchymal Stem Cell Derived Exosomes Repair Uterine Injury by Targeting Transforming Growth Factor-β Signaling.

Intrauterine adhesions (IUA) refer to adhesions within the uterine cavity and cervix caused by injuries from uterine surgery. They are a significant cause of female infertility. Exosomes derived from mesenchymal stem cells (MSCs) play an active role in the treatment of IUA. However, the mechanism by which they reduce fibrosis in the damaged endometrium remains unclear. In this paper, we demonstrate that exosomes derived from placental mesenchymal stem cells (PMSCs) can restore uterine functions and improve the fertility rate of injured animals. This is achieved by promoting cell proliferation, increasing endometrial thickness, and reversing fibrosis. Regarding the molecular mechanism behind these therapeutic effects, we identify three specific miRNAs, namely, miR-125b-5p, miR-30c-5p, and miR-23a-3p, enriched in PMSC-exosomes, as the key players in the treatment of IUA. Specifically, miR-125b-5p/miR-30c-5p and miR-23a-3p inhibit the expression of smad2 and smad3 by targeting their 3'-untranslated regions, resulting in the downregulation of the transforming growth factor-β (TGF-β)/smad signaling pathway and the reversal of fibrosis. Notably, the safety of PMSC-exosomes in intrauterine treatment was also been confirmed. In conclusion, we illustrate that exosomes derived from PMSCs possess the capability to repair endometrial damage and enhance fertility in injured animals by regulating the TGF-β/smad pathway via miR-125b-5p, miR-30c-5p, and miR-23a-3p. This provides insights into the precision treatment of IUA through exosome-based cell-free therapy.

Immunomodulatory Layered Double Hydroxide Nanoparticles Enable Neurogenesis by Targeting Transforming Growth Factor-β Receptor 2.

Immune microenvironment amelioration and reconstruction by functional biomaterials has become a promising strategy for spinal cord injury (SCI) recovery. In this study, we evaluated the neural regeneration and immunoregulation functions of Mg/Al layered double hydroxide (Mg/Al-LDH) nanoparticles in completely transected and excised mice and revealed the immune-related mechanisms. LDH achieved significant performance in accelerating neural stem cells (NSCs) migration, neural differentiation, L-Ca2+ channel activation, and inducible action potential generation. In vivo, the behavioral and electrophysiological performance of SCI mice was significantly improved by LDH implantation, with BrdU+ endogenous NSCs and neurons clearly observed in the lesion sites. According to RNA-seq and ingenuity pathway analysis, transforming growth factor-β receptor 2 (TGFBR2) is the key gene through which LDH inhibits inflammatory responses and accelerates neural regeneration. Significant colocalization of TGFBR2 and LDH was found on the cell membranes of NSCs both in vitro and in vivo, and LDH increased the expression of TGF-β2 in NSCs and activated the proliferation of precursor neural cells. LDH decreased the expression of M1 markers and increased the expression of M2 markers in both microglia and bone marrow-derived macrophages, and these effects were reversed by a TGFBR2 inhibitor. In addition, as a carrier, LDH loaded with NT3 exhibited better recovery effects with regard to the basso mouse scale score, motor evoked potential performance, and regenerated neural cell numbers than LDH itself. Thus, we have developed Mg/Al-LDH that can be used to construct a suitable immune microenvironment for SCI recovery and have revealed the targeted receptor.

Targeting transforming growth factor-β signaling for enhanced cancer chemotherapy

During the past decades, drugs targeting transforming growth factor-β (TGFβ) signaling have received tremendous attention for late-stage cancer treatment since TGFβ signaling has been recognized as a prime driver for tumor progression and metastasis. Nonetheless, in healthy and pre-malignant tissues, TGFβ functions as a potent tumor suppressor. Furthermore, TGFβ signaling plays a key role in normal development and homeostasis by regulating cell proliferation, differentiation, migration, apoptosis, and immune evasion, and by suppressing tumor-associated inflammation. Therefore, targeting TGFβ signaling for cancer therapy is challenging. Recently, we and others showed that blocking TGFβ signaling increased chemotherapy efficacy, particularly for nanomedicines. In this review, we briefly introduce the TGFβ signaling pathway, and the multifaceted functions of TGFβ signaling in cancer, including regulating the tumor microenvironment (TME) and the behavior of cancer cells. We also summarize TGFβ targeting agents. Then, we highlight TGFβ inhibition strategies to restore the extracellular matrix (ECM), regulate the tumor vasculature, reverse epithelial-mesenchymal transition (EMT), and impair the stemness of cancer stem-like cells (CSCs) to enhance cancer chemotherapy efficacy. Finally, the current challenges and future opportunities in targeting TGFβ signaling for cancer therapy are discussed.

Targeting transforming growth factor-β receptors in pulmonary hypertension

The transforming growth factor-β (TGF-β) superfamily includes several groups of multifunctional proteins that form two major branches, namely the TGF-β-activin-nodal branch and the bone morphogenetic protein (BMP)-growth differentiation factor (GDF) branch. The response to the activation of these two branches, acting through canonical (small mothers against decapentaplegic (Smad) 2/3 and Smad 1/5/8, respectively) and noncanonical signalling pathways, are diverse and vary for different environmental conditions and cell types. An extensive body of data gathered in recent years has demonstrated a central role for the cross-talk between these two branches in a number of cellular processes, which include the regulation of cell proliferation and differentiation, as well as the transduction of signalling cascades for the development and maintenance of different tissues and organs. Importantly, alterations in these pathways, which include heterozygous germline mutations and/or alterations in the expression of several constitutive members, have been identified in patients with familial/heritable pulmonary arterial hypertension (PAH) or idiopathic PAH (IPAH). Consequently, loss or dysfunction in the delicate, finely-tuned balance between the TGF-β-activin-nodal branch and the BMP-GDF branch are currently viewed as the major molecular defect playing a critical role in PAH predisposition and disease progression. Here we review the role of the TGF-β-activin-nodal branch in PAH and illustrate how this knowledge has not only provided insight into understanding its pathogenesis, but has also paved the way for possible novel therapeutic approaches.

Abstract P3-09-13: Identification of a tumor biomarker in advanced triple-negative breast cancer that predicts response to bintrafusp alfa (M7824), a bifunctional fusion protein targeting transforming growth factor-β and programmed death ligand 1

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Identification of biomarkers that are predictive of response to investigational therapies will help identify patients that are more likely to clinically benefit from treatment. Bintrafusp alfa (M7824) is an innovative first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. In this expansion cohort of a global, phase 1, open-label trial (NCT02517398), an extensive tumor biomarker analysis was conducted to identify potential markers correlating with efficacy in patients with advanced TNBC treated with bintrafusp alfa. Methods: Patients received infusions of bintrafusp alfa 1200 mg every 2 weeks until confirmed progression, unacceptable toxicity, or trial withdrawal. Tumor archival material was collected from each patient for biomarker analysis. All biomarker analyses were exploratory. PD-L1 expression and immune phenotype were determined by immunohistochemistry. Tumor samples were also processed for RNA sequencing (RNAseq) and resulting data were analyzed to identify potential associations between gene expression and efficacy of bintrafusp alfa treatment. Results: As of August 24, 2018, 33 patients with heavily pretreated TNBC were treated with bintrafusp alfa (54.5% of patients received ≥4 prior regimens). The objective response rate was 9.1% (n=3) and the disease control rate was 15.2% in a total of 5 patients. PD-L1 expression levels in tumor cells and in the tumor microenvironment were generally low in this cohort with the majority of patients having 0% PD-L1+ tumor cells and/or ≤10% PD-L1+ immune cells in their tumor microenvironment. Moreover, several patients had an immune desert phenotype. PD-L1 expression was not associated with response to treatment. Importantly, exploratory analysis of RNAseq data (n=26 passing quality control) identified differential expression of the high mobility group AT-hook 2 (HMGA2) gene with 32.0-fold higher expression (as computed by DESeq2; q=2.23e−13) in patients who experienced disease control (response or stable disease) compared to those who had progressive disease. HMGA2 is a transcriptional regulator whose expression is upregulated by TGF-β signaling. Furthermore, HMGA2 is known to be an important factor in mediating TGF-β-induced epithelial-mesenchymal transition. In The Cancer Genome Atlas dataset, approximately 12% of subjects annotated as TNBC have high expression of HMGA2. Clinical efficacy and safety data for this expansion cohort are presented in a separate abstract. Conclusions: We identified high expression of HMGA2 as a potential predictive biomarker of response in TNBC patients treated with bintrafusp alfa. HMGA2 assay development is on-going to confirm this finding in a future clinical trial. Citation Format: George Locke, Yue Zhang, Laureen S Ojalvo, Christoph Helwig, Alex Rolfe, Olaf Christensen, Isabelle Dussault. Identification of a tumor biomarker in advanced triple-negative breast cancer that predicts response to bintrafusp alfa (M7824), a bifunctional fusion protein targeting transforming growth factor-β and programmed death ligand 1 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-09-13.

Abstract P3-09-06: Bintrafusp alfa (M7824), a bifunctional fusion protein targeting transforming growth factor-β and programmed death ligand 1, in advanced triple-negative breast cancer: Preliminary results from a phase 1 cohort

Abstract Background: Approximately 10%-20% of breast cancers (BCs) are classified as triple-negative breast cancer (TNBC). TNBC lacks targeted treatment options and has a poor prognosis. Bintrafusp alfa (M7824) is an innovative first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. We report interim results in patients with advanced TNBC treated with bintrafusp alfa in an expansion cohort of an ongoing, open-label, phase 1 trial (NCT02517398). Methods: Eligible patients had confirmed TNBC (ER: IHC <1%; PR: IHC <1%; HER2: FISH non-amplified, IHC 0/1+, or IHC 2+ and FISH non-amplified) that progressed during or after first-line chemotherapy with an ECOG performance status of ≤1 and measurable disease by RECIST 1.1. Prior treatment with immune checkpoint inhibitors was not permitted. Patients received bintrafusp alfa 1200 mg every 2 weeks until confirmed progression, unacceptable toxicity, or trial withdrawal. The primary endpoint was confirmed best overall response (BOR) as assessed by independent review committee (IRC) according to RECIST 1.1. Key secondary endpoints included investigator-assessed BOR and safety. Exploratory endpoints included an extensive integrated biomarker evaluation of patient samples. These data are presented in a separate, submitted abstract. Results: As of August 24, 2018, 33 patients with heavily pretreated TNBC (54.5% of patients had ≥4 prior regimens) were enrolled in this study of bintrafusp alfa. Patients received a median of 3 doses of bintrafusp alfa (range, 1-24). Confirmed responses as assessed by IRC and investigator occurred in 3 patients (1 complete response and 2 partial responses as assessed by IRC; ORR, 9.1% [95% CI, 1.9%-24.3%]); disease control was achieved in a total of 5 patients (15.2% [95% CI, 5.1%-31.9%]) by both IRC and investigator read. The median progression-free survival per IRC was 1.3 months (95% CI, 1.2-1.4 months), and the median overall survival was 7.8 months (95% CI, 2.1-12.8 months). Importantly, a biomarker related to TGF-β biology was identified via RNAseq analysis of tumor samples with 32.0-fold higher expression in patients who experienced disease control (response or stable disease) compared with those who had progressive disease (biomarker results for this expansion cohort are presented in a separate abstract). PD-L1 expression was not associated with response to treatment. In the entire cohort, 6 patients (18.2%) experienced ≥1 grade 3 treatment-related adverse event (TRAE), including anemia (n=3), asthenia, decreased appetite, generalized rash, hypophysitis, and increased transaminases (all n=1). No grade 4 TRAEs were reported. Three patients discontinued treatment due to TRAEs, including 1 death that was assessed by the investigator as related to treatment. Dyspnea, hemolysis, and thrombocytopenia were reported as grade 5 TRAEs in this patient, who had extensive disease at trial entry and was noted to have multiple pulmonary emboli, progressive disease, and expanding pleural effusion after 3 doses. No autoantibodies mediating hemolysis or thrombocytopenia were identified on workup. Conclusions: In summary, bintrafusp alfa was well tolerated, with a safety profile consistent with expectations in this heavily pretreated, advanced TNBC cohort. We identified high expression of a potential predictive biomarker of response in patients with advanced TNBC treated with bintrafusp alfa. Detailed biomarker data are presented in the separate abstract and warrant further investigation of bintrafusp alfa in TNBC. Citation Format: Alexander Spira, Ahmad Awada, Nicolas Isambert, David Lorente Estellés, John Nemunaitis, Nicolas Penel, Laureen S Ojalvo, Christoph Helwig, Christian Borel. Bintrafusp alfa (M7824), a bifunctional fusion protein targeting transforming growth factor-β and programmed death ligand 1, in advanced triple-negative breast cancer: Preliminary results from a phase 1 cohort [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-09-06.

MiR-17-5p promotes cervical cancer cell proliferation and metastasis by targeting transforming growth factor-β receptor 2.

MicroRNAs (miRNAs) play critical roles in post-translational gene expression. The aim of the current study was to investigate the effects of miR-17-5p in cervical cancer. Fifteen clinical cervical cancer tissue samples, as well as their paired adjacent noncancerous tissues, were collected. The microarray was performed to identify differential miRNAs in cervical cancer. Luciferase reporter assay was conducted to identify the target gene of selected miRNA. SiHa was transfected with mimics, inhibitors as well as negative controls of miR-17-5p and Targeting Transforming Growth Factor-β Receptor 2 (TGFBR2) open reading frame or siRNA. Cell counting kit-8 (CCK-8) assay and transwell experiment were performed to detect the proliferation rate and metastasis, respectively. Western blotting and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis were used to analyze TGFBR2 expression. Balb/c nude mice were utilized to verify the effect of miR-17-5p in vivo. Microarray analysis identified miR-17-5p as our interesting miRNA, and luciferase reporter assay identified TGFBR2 as its target gene. MiR-17-5p overexpression significantly enhanced cervical cancer cell proliferation and metastasis. In-vivo study also verified that miR-17-5p overexpression stimulated cervical cancer growth. MiR-17-5p enhances cervical cancer proliferation and metastasis via targeting TGFBR2. It is proposed that targeting miR-17-5p may be a promising therapeutic approach for cervical cancer.

Targeting Transforming Growth Factor-β Signaling in Aortopathies in Marfan Syndrome

Targeting Transforming Growth Factor-β Signaling in Aortopathies in Marfan Syndrome

Targeting transforming growth factor-β signaling in liver metastasis of colon cancer

Despite a primary tumor suppressor role, there is compelling evidence suggesting that TGF-β can promote tumor growth, invasion and metastasis in advanced stages of colorectal cancer. Blocking these tumor-promoting effects of TGF-β provides a potentially important therapeutic strategy for the treatment of colorectal cancer. However, little is known about how the inhibitors of TGF-β receptor kinases affect colorectal carcinogenesis in vivo. Here, we have observed that a novel dual kinase inhibitor of TGF-β type I and type II receptors, LY2109761, inhibits TGF-β-mediated activation of Smad and non-Smad pathways in CT26 colon adenocarcinoma cells having K-Ras mutation. The inhibitor attenuates the oncogenic effects of TGF-β on cell migration, invasion and tumorigenicity of CT26 cells. Furthermore, LY2109761 decreases liver metastases and prolongs survival in an experimental metastasis model. These findings suggest that the dual kinase inhibitor LY2109761 has potential therapeutic value for metastatic colorectal cancer.

D.P.3.13 A small-molecule inhibitor targeting transforming growth factor-β type I receptor kinase ameliorates muscular atrophy in a mouse model of caveolin-3-deficient muscular dystrophy

D.P.3.13 A small-molecule inhibitor targeting transforming growth factor-β type I receptor kinase ameliorates muscular atrophy in a mouse model of caveolin-3-deficient muscular dystrophy

Targeting transforming growth factor-β signaling

Most cancers are characterized by excessive transforming growth factor-beta production by tumors, which can promote tumor growth and mediate epithelial-to-mesenchymal transition. Transforming growth factor-beta also has the ability to overproduce extracellular matrix components in response to injury and other stimuli. There are many strategies undergoing current evaluation for inhibiting the deleterious biological effects of transforming growth factor-beta by disrupting its signaling at various levels. The current review focuses on the recent advances made in this area, and the potential of these strategies in the clinical treatment of cancer and fibrosis. Four main strategies used most recently for disrupting transforming growth factor-beta signaling are brought into focus in this review: inhibition or sequestration of the transforming growth factor-beta protein ligands, inhibition of transforming growth factor-beta receptor kinase activity, inhibition of SMAD signaling downstream of transforming growth factor-beta kinase activity and restoration of antitumor immunity upon transforming growth factor-beta inhibition. Various techniques currently used to employ these four strategies are discussed. Several lines of evidence suggest that altered transforming growth factor-beta signaling contributes to tumor progression and metastasis as well as development of fibrosis. Accumulating data from preclinical and clinical studies indicate that antagonizing aberrant transforming growth factor-beta signaling is a promising novel therapeutic approach in cancer and fibrotic disorders.

A novel immunotherapeutic modality with direct hemoperfusion targeting transforming growth factor-β prolongs the survival of tumor-bearing rats

Immune responses are frequently depressed in patients with cancer. One of the reasons for a poor immune response is the presence of increased levels of immunosuppressive substances associated with tumor growth. Transforming growth factor-beta (TGF-beta), a representative immunosuppressive cytokine, plays various roles in the progression of cancer. To remove immunosuppressive substances from tumor-bearing hosts, we developed an immunosuppressive substance adsorption (ISA) column for direct hemoperfusion (DHP) treatment. It is filled with extra-fine fibers that can adsorb TGF-beta. In this study, we investigated the effects of this DHP treatment on serum levels and activities of TGF-beta, cellular immune responses, and anti-tumor effects in KDH-8 (TGF-beta-producing hepatocellular carcinoma cell line)-bearing rats. We further studied the ability of ISA fibers to adsorb tumor-associated immunosuppressive cytokines [TGF-beta, interleukin (IL)-6 and vascular endothelial growth factor (VEGF)] in samples of body fluids obtained from patients with metastatic cancer. DHP treatment decreased serum levels and activities of TGF-beta in tumor-bearing rats and restored T lymphocyte response to mitogen. Tumor growth in rats treated by DHP was significantly slower than that in untreated rats. The survival time of treated rats was significantly longer than that of untreated rats. The concentrations of TGF-beta, IL-6, and VEGF in the samples of human body fluids were decreased markedly by in vitro treatment with ISA fibers. These results suggest that DHP treatment with an ISA column, which removes TGF-beta and other immunosuppressive substances from the sera of tumor-bearing hosts, is potentially a new immunotherapeutic strategy for cancer.

Pioglitazone Inhibits In-Stent Restenosis in Atherosclerotic Rabbits by Targeting Transforming Growth Factor-β and MCP-1

Although emerging data from preclinical and clinical studies suggests a reduction of in-stent restenosis with peroxisome proliferator-activated receptor (PPAR)-gamma agonists, the reduction of neointimal growth via anti-inflammatory mechanisms has not been explored. Hypercholesterolemic New Zealand White rabbits (n=45) received bilateral balloon-expandable stents implanted into atherosclerotic iliac arteries. Animals were randomized to oral pioglitazone 3 (low dose) or 10 mg/kg per day (high dose) started on the day of stent implantation; control rabbits received placebo. Tissue harvest was performed 28 days after stenting, and stented segments underwent histology, morphometry, immunostaining for macrophages, and scanning electron microscopy. In selected animals, stented arterial segments were placed in organoid culture for 48 hours, and the conditioned media was assayed for 23 different cytokines. There was a 21% reduction in neointimal area for high-dose pioglitazone treated versus placebo rabbits (P<0.005), which was associated with a significant reduction of neointimal macrophages. Analysis of conditioned media revealed an 82% and 74% reduction in the release of monocyte chemoattractant protein-1 (MCP-1) (P<0.007) and transforming growth factor (TGF)-beta1 (P<0.01), respectively, in stented segments from animals treated with 10 mg/kg per day pioglitazone versus placebo. Oral pioglitazone suppresses in-stent neointimal growth by limiting local inflammatory pathways and may be useful as an adjunctive therapy in patients undergoing percutaneous interventions.