Design, synthesis and evaluation of a pyrazolo[3,4-d]pyrimidine derivative as a novel and potent TGFß1R1 inhibitor.

The transforming growth factor ß1 (TGFß1)/SMAD signaling pathway regulates many vital physiological processes. The development of potent inhibitors targeting activin receptor-like kinase 5 (ALK5) would provide potential treatment reagents for various diseases. A significant number of ALK5 inhibitors have been discovered, and they are currently undergoing clinical evaluation at various stages. However, the clinical demands were far from being met. In this study, we utilized an alternative conformation-similarity-based virtual screening (CSVS) combined with a fragment-based drug designing (FBDD) strategy to efficiently discover a potent and active hit with a novel chemical scaffold. After structural optimization in the principle of group replacement, compound 57 was identified as the most promising ALK5 inhibitor. Compound 57 demonstrated significant inhibitory effects against the TGF-ß1/SMAD signaling pathway. It could markedly attenuate the production of extracellular matrix (ECM) and deposition of collagen. Also, the lead compound showed adequate pharmacokinetic (PK) properties and good in vivo tolerance. Moreover, treatment with compound 57 in two different xerograph models showed significant inhibitory effects on the growth of pancreatic cancer cells. These results suggested that lead compound 57 refers as a promising ALK5 inhibitor both in vitro and in vivo, which merits further validation.

Targeting the TGF-ß signaling pathway: an updated patent review (2021-present).

INTRODUCTION: The TGF-ß signaling pathway is a complex network that plays a crucial role in regulating essential biological functions and is implicated in the onset and progression of multiple diseases. This review highlights the recent advancements in developing inhibitors targeting the TGF-ß signaling pathway and their potential therapeutic applications in various diseases. AREA COVERED: The review discusses patents on active molecules related to the TGF-ß signaling pathway, focusing on three strategies: TGF-ß activity inhibition, blocking TGF-ß receptor binding, and disruption of the signaling pathway using small molecule inhibitors. Combination therapies and the development of fusion proteins targeting multiple pathways are also explored. The literature search was conducted using the Cortellis Drug Discovery Intelligence database, covering patents from 2021 onwards. EXPERT OPINION: The development of drugs targeting the TGF-ß signaling pathway has made significant progress in recent years. However, addressing challenges such as specificity, systemic toxicity, and patient selection is crucial for their successful clinical application. Targeting the TGF-ß signaling pathway holds promise as a promising approach for the treatment of various diseases.

First-in-human study of GFH018, a small molecule inhibitor of transforming growth factor-ß receptor I inhibitor, in patients with advanced solid tumors.

BACKGROUND: Transforming growth factor-ß (TGF-ß) is a cytokine with multiple functions, including cell growth regulation, extracellular matrix production, angiogenesis homeostasis adjustment and et al. TGF-ß pathway activation promotes tumor metastasis/progression and mediates epithelial-mesenchymal transmission suppressing immunosurveillance in advanced tumors. GFH018, a small molecule inhibitor blocking TGF-ß signal transduction, inhibits the progression and/or metastasis of advanced cancers. This first-in-human study evaluated the safety, tolerability, pharmacokinetics (PK), and efficacy of GFH018 monotherapy in patients with advanced solid tumors. METHODS: This phase I, open-label, multicenter study used a modified 3+3 dose escalation and expansion design. Adult patients with advanced solid tumors failing the standard of care were enrolled. Starting at 5 mg, eight dose levels up to 85 mg were evaluated. Patients received GFH018 BID (14d-on/14d-off) starting on the 4th day after a single dose on cycle 1, day 1. Subsequent cycles were defined as 28 days. The study also explored the safety of 85 mg BID 7d-on/7d-off. Adverse events were graded using NCI criteria for adverse events (NCI-CTCAE v5.0). PK was analyzed using a noncompartmental method. Efficacy was evaluated using RECIST 1.1. Blood samples were collected for biomarker analysis. RESULTS: Fifty patients were enrolled and received at least one dose of GFH018. No dose-limiting toxicity occurred, and the maximum tolerated dose was not reached. Forty-three patients (86.0%) had at least one treatment-related adverse event (TRAE), and three patients (6.0%) had ≥ G3 TRAEs. The most common TRAEs (any grade/grade ≥3) were AST increased (18%/0%), proteinuria (14%/2%), anemia (14%/2%), and ALT increased (12%/0%). No significant cardiotoxicity or bleeding was observed. GFH018 PK was linear and dose-independent, with a mean half-life of 2.25-8.60 h from 5 - 85 mg. Nine patients (18.0%) achieved stable disease, and one patient with thymic carcinoma achieved tumor shrinkage, with the maximum target lesion decreased by 18.4%. Serum TGF-ß1 levels were not associated with clinical responses. The comprehensive recommended dose for Phase II was defined as 85 mg BID 14d-on/14d-off. CONCLUSIONS: GFH018 monotherapy presented a favorable safety profile without cardiac toxicity or bleeding. Modest efficacy warrants further studies, including combination strategies. TRIAL REGISTRATION: ClinicalTrial. gov ( https://www. CLINICALTRIALS: gov/ ), NCT05051241. Registered on 2021-09-02.

Therapeutic Effect of Cyclin-Dependent Kinase 4/6 Inhibitor on Dermal Fibrosis in Murine Models of Systemic Sclerosis.

OBJECTIVE: One of the histologic characteristics of systemic sclerosis (SSc) is an increased number of dermal myofibroblasts, and transforming growth factor ß (TGFß) plays a crucial role in the promotion of myofibroblast differentiation from fibroblasts, leading to dermal fibrosis. This study was undertaken to 1) examine whether inhibition of the cell cycle with a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor suppresses the proliferation of fibroblasts and their differentiation into myofibroblasts, and 2) assess the therapeutic effects of a CDK4/6 inhibitor, administered as monotherapy or in combination with a TGFß receptor (TGFßR) inhibitor, on dermal fibrosis in murine models of SSc. METHODS: Fibroblasts obtained from the skin of patients with SSc were cultured in the presence or absence of TGFß. The effects of palbociclib, a CDK4/6 inhibitor, on fibroblast proliferation and TGFß-induced differentiation into myofibroblasts were examined using bromodeoxyuridine uptake assays as well as immunofluorescence and immunoblotting analyses. Murine models of HOCl- and bleomycin-induced dermal fibrosis were used to study the effect of a CDK4/6 inhibitor on dermal fibrosis, with the CDK4/6 inhibitor treatment administered as monotherapy or in combination with galunisertib, a TGFßR inhibitor. RESULTS: Addition of a CDK4/6 inhibitor to the cell cultures suppressed the proliferation of human dermal SSc fibroblasts and their TGFß-induced differentiation into myofibroblasts, without inhibiting canonical and noncanonical TGFß signals. In murine models of dermal fibrosis, treatment of mice with a CDK4/6 inhibitor decreased dermal thickness and collagen content, as well as dermal fibroblast proliferation and the numbers of myofibroblasts. Combination therapy with the CDK4/6 inhibitor and TGFßR inhibitor resulted in additive antifibrotic effects. Mechanistically, the CDK4/6 inhibitor suppressed the expression of cellular communication network 2 and cadherin-11, which are proteins that have important roles in the development and progression of fibrosis. CONCLUSION: Results of this study demonstrate the therapeutic effect of a CDK4/6 inhibitor on dermal fibrosis when administered as monotherapy or in combination with a TGFßR inhibitor. CDK4/6 inhibitors, including palbociclib used in the present study, may represent novel agents for the treatment of SSc, which, if used in combination with a TGFßR inhibitor, might result in increased efficacy.

Disulfiram Sensitizes a Therapeutic-Resistant Glioblastoma to the TGF-ß Receptor Inhibitor.

Despite neurosurgery following radiation and chemotherapy, residual glioblastoma (GBM) cells develop therapeutic resistance (TR) leading to recurrence. The GBM heterogeneity confers TR. Therefore, an effective strategy must target cancer stem cells (CSCs) and other malignant cancer cells. TGF-ß and mesenchymal transition are the indicators for poor prognoses. The activity of aldehyde dehydrogenases (ALDHs) is a functional CSC marker. However, the interplay between TGF-ß and ALDHs remains unclear. We developed radiation-resistant and radiation-temozolomide-resistant GBM models to investigate the underlying mechanisms conferring TR. Galunisertib is a drug targeting TGF-ß receptors. Disulfiram (DSF) is an anti-alcoholism drug which functions by inhibiting ALDHs. The anti-tumor effects of combining DSF and Galunisertib were evaluated by in vitro cell grow, wound healing, Transwell assays, and in vivo orthotopic GBM model. Mesenchymal-like phenotype was facilitated by TGF-ß in TR GBM. Additionally, TR activated ALDHs. DSF inhibited TR-induced cell migration and tumor sphere formation. However, DSF did not affect the tumor growth in vivo. Spectacularly, DSF sensitized TR GBM to Galunisertib both in vitro and in vivo. ALDH activity positively correlated with TGF-ß-induced mesenchymal properties in TR GBM. CSCs and mesenchymal-like GBM cells targeted together by combining DSF and Galunisertib may be a good therapeutic strategy for recurrent GBM patients.

Blockade of TGF-ßR improves the efficacy of doxorubicin by modulating the tumor cell motility and affecting the immune cells in a melanoma model.

TGF-ß contributes to drug resistance and the invasiveness of tumor cells and weakens the anti-tumor immune responses. The present study aimed at examining the efficacy of the combination of SB431542, as a specific inhibitor of TGF-ßR, and doxorubicin in controlling the melanoma tumor in mice. The impact of the combination of the doxorubicin and SB431542 on the cell growth, apoptosis, migration, and invasiveness of B16-F10 cells was examined. Besides, the B16-F10 tumor was induced in C57BL/6 mice, and the effects of the mentioned treatment on the tumor volume, survival, and the exhaustion state of T cells were evaluated. Although the combination of doxorubicin and SB431542 did not exhibit synergism in the inhibition of cell growth and apoptosis induction, it efficiently prohibited the migration and the epithelial to mesenchymal transition of B16-F10 cells, and the combination of doxorubicin and SB431542 caused an increase in mRNA levels of E-cadherin and, on the other hand, led to a decline in the expression of Vimentin. Tumor volume and the survival of tumor-bearing mice were efficiently controlled by the combination therapy. This treatment also eventuated in a decrease in the percentage of PD-L1+, TCD4+, and TCD8+ cells as indicators of exhausted T cells within the spleens of tumor-bearing mice. Blockade of TGF-ßR also propelled the RAW 264.7 cells towards an anti-tumor M1 macrophage phenotype. The inhibition of TGF-ßR demonstrated a potential to increase the efficacy of doxorubicin chemotherapy by the means of affecting cellular motility and restoring the anti-tumor immune responses.

Reducing tumor invasiveness by ramucirumab and TGF-ß receptor kinase inhibitor in a diffuse-type gastric cancer patient-derived cell model.

BACKGROUND: Diffuse-type gastric cancer (GC) is known to be more aggressive and relatively resistant to conventional chemotherapy. Hence, more optimized treatment strategy is urgently needed in diffuse-type GC. METHODS: Using a panel of 10 GC cell lines and 3 GC patient-derived cells (PDCs), we identified cell lines with high EMTness which is a distinct feature for diffuse-type GC. We treated GC cells with high EMTness with ramucirumab alone, TGF-ß receptor kinase inhibitor (TEW-7197) alone, or in combination to investigate the drug's effects on invasiveness, spheroid formation, EMT marker expression, and tumor-induced angiogenesis using a spheroid-on-a-chip model. RESULTS: Both TEW-7197 and ramucirumab treatments profoundly decreased invasiveness of EMT-high cell lines and PDCs. With a 3D tumor spheroid-on-a-chip, we identified versatile influence of co-treatment on cancer cell-induced blood vessel formation as well as on EMT progression in tumor spheroids. The 3D tumor spheroid-on-a-chip demonstrated that TEW-7197 + ramucirumab combination significantly decreased PDC-induced vessel formation. CONCLUSIONS: In this study, we showed TEW-7197 and ramucirumab considerably decreased invasiveness, thus EMTness in a panel of diffuse-type GC cell lines including GC PDCs. Taken together, we confirmed that combination of TEW-7197 and ramucirumab reduced tumor spheroid and GC PDC-induced blood vessel formation concomitantly in the spheroid-on-a-chip model.

Pancreatic Adenocarcinoma Therapeutics Targeting RTK and TGF Beta Receptor.

Despite the improved overall survival rates in most cancers, pancreatic cancer remains one of the deadliest cancers in this decade. The rigid microenvironment, which majorly comprises cancer-associated fibroblasts (CAFs), plays an important role in the obstruction of pancreatic cancer therapy. To overcome this predicament, the signaling of receptor tyrosine kinases (RTKs) and TGF beta receptor (TGFßR) in both pancreatic cancer cell and supporting CAF should be considered as the therapeutic target. The activation of receptors has been reported to be aberrant to cell cycle regulation, and signal transduction pathways, such as growth-factor induced proliferation, and can also influence the apoptotic sensitivity of tumor cells. In this article, the regulation of RTKs/TGFßR between pancreatic ductal adenocarcinoma (PDAC) and CAFs, as well as the RTKs/TGFßR inhibitor-based clinical trials on pancreatic cancer are reviewed.

Role of neutrophil extracellular traps in regulation of lung cancer invasion and metastasis: Structural insights from a computational model.

Lung cancer is one of the leading causes of cancer-related deaths worldwide and is characterized by hijacking immune system for active growth and aggressive metastasis. Neutrophils, which in their original form should establish immune activities to the tumor as a first line of defense, are undermined by tumor cells to promote tumor invasion in several ways. In this study, we investigate the mutual interactions between the tumor cells and the neutrophils that facilitate tumor invasion by developing a mathematical model that involves taxis-reaction-diffusion equations for the critical components in the interaction. These include the densities of tumor and neutrophils, and the concentrations of signaling molecules and structure such as neutrophil extracellular traps (NETs). We apply the mathematical model to a Boyden invasion assay used in the experiments to demonstrate that the tumor-associated neutrophils can enhance tumor cell invasion by secreting the neutrophil elastase. We show that the model can both reproduce the major experimental observation on NET-mediated cancer invasion and make several important predictions to guide future experiments with the goal of the development of new anti-tumor strategies. Moreover, using this model, we investigate the fundamental mechanism of NET-mediated invasion of cancer cells and the impact of internal and external heterogeneity on the migration patterning of tumour cells and their response to different treatment schedules.

Network model-based screen for FDA-approved drugs affecting cardiac fibrosis.

Cardiac fibrosis is a significant component of pathological heart remodeling, yet it is not directly targeted by existing drugs. Systems pharmacology approaches have the potential to provide mechanistic frameworks with which to predict and understand how drugs modulate biological systems. Here, we combine network modeling of the fibroblast signaling network with 36 unique drug-target interactions from DrugBank to predict drugs that modulate fibroblast phenotype and fibrosis. Galunisertib was predicted to decrease collagen and α-SMA expression, which we validated in human cardiac fibroblasts. In vivo fibrosis data from the literature validated predictions for 10 drugs. Further, the model was used to identify network mechanisms by which these drugs work. Arsenic trioxide was predicted to induce fibrosis by AP1-driven TGFß expression and MMP2-driven TGFß activation. Entresto (valsartan/sacubitril) was predicted to suppress fibrosis by valsartan suppression of ERK signaling and sacubitril enhancement of PKG activity, both of which decreased Smad3 activity. Overall, this study provides a framework for integrating drug-target mechanisms with logic-based network models, which can drive further studies both in cardiac fibrosis and other conditions.

Cell surface thermal proteome profiling tracks perturbations and drug targets on the plasma membrane.

Numerous drugs and endogenous ligands bind to cell surface receptors leading to modulation of downstream signaling cascades and frequently to adaptation of the plasma membrane proteome. In-depth analysis of dynamic processes at the cell surface is challenging due to biochemical properties and low abundances of plasma membrane proteins. Here we introduce cell surface thermal proteome profiling for the comprehensive characterization of ligand-induced changes in protein abundances and thermal stabilities at the plasma membrane. We demonstrate drug binding to extracellular receptors and transporters, discover stimulation-dependent remodeling of T cell receptor complexes and describe a competition-based approach to measure target engagement of G-protein-coupled receptor antagonists. Remodeling of the plasma membrane proteome in response to treatment with the TGFB receptor inhibitor SB431542 leads to partial internalization of the monocarboxylate transporters MCT1/3 explaining the antimetastatic effects of the drug.

TGF-ß Activity of a Demineralized Bone Matrix.

Allografts consisting of demineralized bone matrix (DBM) are supposed to retain the growth factors of native bone. However, it is not clear if transforming growth factor ß1 (TGF-ß1) is maintained in the acid-extracted human bone. To this aim, the aqueous solutions of supernatants and acid lysates of OraGRAFT® Demineralized Cortical Particulate and OraGRAFT® Prime were prepared. Exposing fibroblasts to the aqueous solution caused a TGF-ß receptor type I kinase-inhibitor SB431542-dependent increase in interleukin 11 (IL11), NADPH oxidase 4 (NOX4), and proteoglycan 4 (PRG4) expression. Interleukin 11 expression and the presence of TGF-ß1 in the aqueous solutions were confirmed by immunoassay. Immunofluorescence further confirmed the nuclear translocation of Smad2/3 when fibroblasts were exposed to the aqueous solutions of both allografts. Moreover, allografts released matrix metalloprotease-2 activity and blocking proteases diminished the cellular TGF-ß response to the supernatant. These results suggest that TGF-ß is preserved upon the processing of OraGRAFT® and released by proteolytic activity into the aqueous solution.

TGF-ßR inhibitor SB431542 restores immune suppression induced by regulatory B-T cell axis and decreases tumour burden in murine fibrosarcoma.

The contribution of immune cells in soft tissue sarcomas (STS) is not completely known and understanding their role is very essential for employing immunotherapy strategies. Here, we show that murine fibrosarcoma-conditioned medium promoted total spleen cell proliferation but inhibited T cell responses to mitogenic and allo-antigen-mediated stimulation. This increased proliferation was found to be in B cells resulting in generation of Breg further leading to Treg population. This was found to be the same in vitro and in vivo. The phenotype of these B cells was CD19+CD81+CD27+CD25+PD-L1hi and they secreted both IL-10 and TGF-ß. These tumor evoked Bregs (tBreg), when co-cultured with B depleted T cells, suppressed their proliferation in response to anti-CD3/CD28 stimulation. tBreg-induced suppression of T cell responses was not abrogated by the inhibition or neutralization of IL-10 but by the small molecule inhibitor of TGFß Receptor type I, SB431542. While SB531542 per se was not cytotoxic to tumor cells, administration of SB431542 in tumor-bearing mice (TBM) significantly reduced the tumor burden. In addition, the treatment significantly reduced Treg cells and rescued proliferation of T cells in response to mitogen and allo-antigen. Collectively, our results identify that tumor evoked Breg cells mediate T cell immune suppression through TGFß-mediated pathway and that targeting the Breg-Treg axis can be potentially used as an immunotherapy agent.

Recent progress in TGF-ß inhibitors for cancer therapy.

Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine that is involved in proliferation, metastasis, and many other important processes in malignancy. Inhibitors targeting TGF-ß have been considered by pharmaceutical companies for cancer therapy, and some of them are in clinical trial now. Unfortunately, several of these programs have recently been relinquished, and most companies that remain in the contest are progressing slowly and cautiously. This review summarizes the TGF-ß signal transduction pathway, its roles in oncogenesis and fibrotic diseases, and advancements in antibodies and small-molecule inhibitors of TGF-ß.

A novel approach for monitoring TGF-ß signaling in vivo in colon cancer.

The TGF-ß receptor kinase inhibitors (TRKI) have been reported to inhibit tumorigenicity in colon cancer. However, there is no direct evidence showing that these inhibitors function through inhibiting the TGF-ß- mediated tumor-promoting effects in vivo. We established a TGF-ß inducible reporter system by inserting a luciferase reporter gene to the vector downstream of TGF-ß-inducible promoter elements, and transfected it into colon cancer cell lines. TRKIs SB431542 and LY2109761 were used to treat TGF-ß inducible cells in vitro and in vivo. The luciferase activity was induced 5.24-fold by TGF-ß in CT26 inducible cells, while it was marginally changed in MC38 inducible cells lacking Smad4 expression. Temporary treatment of mice with SB431542 inhibited the TGF-ß pathway and TGF-ß induced bioluminescence activity in vivo. Long-term treatment with LY2109761 inhibited tumorigenicity and liver metastasis in vivo in concomitant with reduced luciferase activity in the tumor. In this study, we established a model to monitor the TGF-ß pathway in vivo and to compare the antitumor effects of TRKIs. Based on this novel experimental tool, we provided direct evidences that LY2109761 inhibits tumorigenicity and liver metastasis by blocking the pro-oncogenic functions of TGF-ß in vivo.

Roles of TGF-ß signaling pathway in tumor microenvirionment and cancer therapy.

Transforming growth factor ß (TGF- ß) signaling pathway has pleiotropic effects on cell proliferation, differentiation, adhesion, senescence, and apoptosis. TGF-ß can be widely produced by various immune or non-immune cells and regulate cell behaviors through autocrine and paracrine. It plays essential roles in biological processes including embryological development, immune response, and tumor progression. Few cell signalings can contribute to so many pleiotropic functions as the TGF- ß signaling pathway in mammals. The significant function of TGF-ß signaling in tumor progression and evasion leading it to draw great attention in scientific and clinical research. Understanding the mechanism of TGF- ß signaling provides us with chances to potentiate the effectiveness and selectivity of this therapeutic method. Herein, we review the molecular and cellular mechanisms of TGF-ß signaling in carcinomas and tumor microenvironment. Then, we enumerate main achievements of TGF-ß blockades used or being evaluated in cancer therapy, providing us opportunities to improve therapeutical approaches in the tumor which thrive in a TGF-ß-rich environment.

Promotion of liver regeneration and anti­fibrotic effects of the TGF­ß receptor kinase inhibitor galunisertib in CCl4­treated mice.

The cytokine transforming growth factor­ß (TGF­ß) serves a key role in hepatic fibrosis and has cytostatic effects on hepatocytes. The present study investigated the anti­fibrogenic and regenerative effects of the TGF­ß receptor type I kinase inhibitor galunisertib (LY2157299) in mice with carbon tetrachloride (CCl4)­induced liver cirrhosis and in vitro. Mice were intraperitoneally treated with CCl4 for 8 weeks. At week 5, the mice were divided randomly into four treatment groups: Vehicle­treated; and treated with low­; middle­; and high­dose galunisertib, which was administered from weeks 5­8. The mice were sacrificed after 8 weeks of CCl4 treatment. Liver fibrosis, as evaluated by histology and determination of hydroxyproline content, progressed during week 4­8 of CCl4 treatment in the vehicle­treated mice. Galunisertib treatment dose­dependently prevented liver fibrosis, as demonstrated by the direct inhibition of α­smooth muscle actin­positive activated hepatic stellate cells (HSCs) after 8 weeks of CCl4 treatment. The levels of active matrix metalloproteinase (MMP)­9 in galunisertib­treated livers were significantly increased compared with the vehicle­treated livers. In the high­dose group, the number of PCNA­positive hepatocytes and endothelial cells markedly increased compared with the vehicle group. Reverse transcription­quantitative PCR analysis verified that interleukin­6 and epiregulin expression levels were significantly increased in livers from the group treated with high­dose galunisertib compared with the vehicle­treated group. Galunisertib inhibited the proliferation of activated HSCs and collagen synthesis in addition to restoring MMP activity. Moreover, galunisertib promoted liver remodeling by proliferating hepatocytes and vascular endothelial cells, while significantly increasing liver weight. These results are consistent with the cytostatic action of TGF­ß that negatively regulates liver regeneration, and demonstrated that galunisertib inhibited TGF­ß signaling, halted liver fibrosis progression and promoted hepatic regeneration. The results of the present study suggest that galunisertib may be an effective treatment for liver cirrhosis.

Inhibition of TGF-ß-receptor signaling augments the antitumor function of ROR1-specific CAR T-cells against triple-negative breast cancer.

BACKGROUND: Immunotherapy with chimeric antigen receptor (CAR)-engineered T-cells is effective in some hematologic tumors. In solid tumors, however, sustained antitumor responses after CAR T-cell therapy remain to be demonstrated both in the pre-clinical and clinical setting. A perceived barrier to the efficacy of CAR T-cell therapy in solid tumors is the hostile tumor microenvironment where immunosuppressive soluble factors like transforming growth factor (TGF)-ß are thought to inhibit the cellular immune response. Here, we analyzed whether CAR T-cells specific for the receptor tyrosine kinase-like orphan receptor 1 (ROR1) antigen, that is frequently expressed in triple-negative breast cancer (TNBC), are susceptible to inhibition by TGF-ß and evaluated TGF-ß-receptor signaling blockade as a way of neutralizing the inhibitory effect of this cytokine. METHODS: CD8+ and CD4+ ROR1-CAR T-cells were prepared from healthy donors and their antitumor function analyzed using the TNBC cell line MDA-MB-231 in vitro and in a microphysiologic 3D tumor model. Analyses were performed in co-culture assays of ROR1-CAR T-cells and MDA-MB-231 cells with addition of exogenous TGF-ß. RESULTS: The data show that exposure to TGF-ß engages TGF-ß-receptor signaling in CD8+ and CD4+ ROR1-CAR T-cells as evidenced by phosphorylation of small mothers against decapentaplegic homolog 2. In the presence of TGF-ß, the cytolytic activity, cytokine production and proliferation of ROR1-CAR T-cells in co-culture with MDA-MB-231 TNBC cells were markedly impaired, and the viability of ROR1-CAR T-cells reduced. Blockade of TGF-ß-receptor signaling with the specific kinase inhibitor SD-208 was able to protect CD8+ and CD4+ ROR1-CAR T-cells from the inhibitory effect of TGF-ß, and sustained their antitumor function in vitro and in the microphysiologic 3D tumor model. Combination treatment with SD-208 also led to increased viability and lower expression of PD-1 on ROR1-CAR T-cells at the end of the antitumor response. CONCLUSION: We demonstrate the TGF-ß suppresses the antitumor function of ROR1-CAR T-cells against TNBC in preclinical models. Our study supports the continued preclinical development and the clinical evaluation of combination treatments that shield CAR T-cells from TGF-ß, as exemplified by the TGF-ß-receptor kinase inhibitor SD-208 in this study.

Differential kinase activity of ACVR1 G328V and R206H mutations with implications to possible TßRI cross-talk in diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is a lethal pediatric brain cancer whose median survival time is under one year. The possible roles of the two most common DIPG associated cytoplasmic ACVR1 receptor kinase domain mutants, G328V and R206H, are reexamined in the context of new biochemical results regarding their intrinsic relative ATPase activities. At 37 °C, the G328V mutant displays a 1.8-fold increase in intrinsic kinase activity over wild-type, whereas the R206H mutant shows similar activity. The higher G328V mutant intrinsic kinase activity is consistent with the statistically significant longer overall survival times of DIPG patients harboring ACVR1 G328V tumors. Based on the potential cross-talk between ACVR1 and TßRI pathways and known and predicted off-targets of ACVR1 inhibitors, we further validated the inhibition effects of several TßRI inhibitors on ACVR1 wild-type and G328V mutant patient tumor derived DIPG cell lines at 20-50 µM doses. SU-DIPG-IV cells harboring the histone H3.1K27M and activating ACVR1 G328V mutations appeared to be less susceptible to TßRI inhibition than SF8628 cells harboring the H3.3K27M mutation and wild-type ACVR1. Thus, inhibition of hidden oncogenic signaling pathways in DIPG such as TßRI that are not limited to ACVR1 itself may provide alternative entry points for DIPG therapeutics.

A Novel Way of Preventing Postoperative Pancreatic Fistula by Directly Injecting Profibrogenic Materials into the Pancreatic Parenchyma.

This paper aims to validate if intrapancreatic injection of penicillin G can enhance hardness and suture holding capacity (SHC) of the pancreas through prompting the fibrosis process. Soft pancreatic texture is constantly mentioned as one of the most contributory predictors of postoperative pancreatic fistula (POPF). Soft pancreas has poor SHC and higher incidence of parenchymal tearing, frequently leading to POPF. From a library of 114 antibiotic compounds, we identified that penicillin G substantially enhanced pancreatic hardness and SHC in experimental mice. Specifically, we injected penicillin G directly into the pancreas. On determined dates, we measured the pancreatic hardness and SHC, respectively, and performed molecular and histological examinations for estimation of the degree of fibrosis. The intrapancreatic injection of penicillin G activated human pancreatic stellate cells (HPSCs) to produce various fibrotic materials such as transforming growth factor-ß1 (TGF-ß1) and metalloproteinases-2. The pancreatic hardness and SHC were increased to the maximum at the second day after injection and then it gradually subsided demonstrating its reversibility. Pretreatment of mice with SB431542, an inhibitor of the TGF-ß1 receptor, before injecting penicillin G intrapancreatically, significantly abrogated the increase of both pancreatic hardness and SHC caused by penicillin G. This suggested that penicillin G promotes pancreatic fibrosis through the TGF-ß1 signaling pathway. Intrapancreatic injection of penicillin G promotes pancreatic hardness and SHC by enhancing pancreatic fibrosis. We thus think that penicillin G could be utilized to prevent and minimize POPF, after validating its actual effectiveness and safety by further studies.