Lihong Sun

A Novel Small-Molecule Inhibitor of Transforming Growth Factor β Type I Receptor Kinase (SM16) Inhibits Murine Mesothelioma Tumor Growth In vivo and Prevents Tumor Recurrence after Surgical Resection

Malignant mesothelioma is an aggressive and lethal pleural cancer that overexpresses transforming growth factor beta (TGFbeta). We investigated the efficacy of a novel small-molecule TGFbeta type I receptor (ALK5) kinase inhibitor, SM16, in the AB12 syngeneic model of malignant mesothelioma. SM16 inhibited TGFbeta signaling seen as decreased phosphorylated Smad2/3 levels in cultured AB12 cells (IC(50), approximately 200 nmol/L). SM16 penetrated tumor cells in vivo, suppressing tumor phosphorylated Smad2/3 levels for at least 3 h following treatment of tumor-bearing mice with a single i.p. bolus of 20 mg/kg SM16. The growth of established AB12 tumors was significantly inhibited by 5 mg/kg/d SM16 (P < 0.001) delivered via s.c. miniosmotic pumps over 28 days. The efficacy of SM16 was a result of a CD8+ antitumor response because (a) the antitumor effects were markedly diminished in severe combined immunodeficient mice and (b) CD8+ T cells isolated from spleens of mice treated with SM16 showed strong antitumor cytolytic effects whereas CD8+ T cells isolated from spleens of tumor-bearing mice treated with control vehicle showed minimal activity. Treatment of mice bearing large tumors with 5 mg/kg/d SM16 after debulking surgery reduced the extent of tumor recurrence from 80% to <20% (P < 0.05). SM16 was also highly effective in blocking and regressing tumors when given p.o. at doses of 0.45 or 0.65 g/kg in mouse chow. Thus, SM16 shows potent activity against established AB12 malignant mesothelioma tumors using an immune-mediated mechanism and can significantly prevent tumor recurrence after resection of bulky AB12 malignant mesothelioma tumors. These data suggest that ALK5 inhibitors, such as SM16, offer significant potential for the treatment of malignant mesothelioma and possibly other cancers.

Small Molecular Inhibitor of Transforming Growth Factor-β Protects Against Development of Radiation-Induced Lung Injury

PURPOSE To determine whether an anti-transforming growth factor-beta (TGF-beta) type 1 receptor inhibitor (SM16) can prevent radiation-induced lung injury. METHODS AND MATERIALS One fraction of 28 Gy or sham radiotherapy (RT) was administered to the right hemithorax of Sprague-Dawley rats. SM16 was administered in the rat chow (0.07 g/kg or 0.15 g/kg) beginning 7 days before RT. The rats were divided into eight groups: group 1, control chow; group 2, SM16, 0.07 g/kg; group 3, SM16, 0.15 g/kg; group 4, RT plus control chow; group 5, RT plus SM16, 0.07 g/kg; group 6, RT plus SM16, 0.15 g/kg; group 7, RT plus 3 weeks of SM16 0.07 g/kg followed by control chow; and group 8, RT plus 3 weeks of SM16 0.15 g/kg followed by control chow. The breathing frequencies, presence of inflammation/fibrosis, activation of macrophages, and expression/activation of TGF-beta were assessed. RESULTS The breathing frequencies in the RT plus SM16 0.15 g/kg were significantly lower than the RT plus control chow from Weeks 10-22 (p <0.05). The breathing frequencies in the RT plus SM16 0.07 g/kg group were significantly lower only at Weeks 10, 14, and 20. At 26 weeks after RT, the RT plus SM16 0.15 g/kg group experienced a significant decrease in lung fibrosis (p = 0.016), inflammatory response (p = 0.006), and TGF-beta1 activity (p = 0.011). No significant reduction was found in these measures of lung injury in the group that received SM16 0.7 g/kg nor for the short-course (3 weeks) SM16 at either dose level. CONCLUSION SM16 at a dose of 0.15 g/kg reduced functional lung damage, morphologic changes, inflammatory response, and activation of TGF-beta at 26 weeks after RT. The data suggest a dose response and also suggest the superiority of long-term vs. short-term dosing.

SM16, an Orally Active TGF-β Type I Receptor Inhibitor Prevents Myofibroblast Induction and Vascular Fibrosis in the Rat Carotid Injury Model

Objective—TGF-&bgr; plays a significant role in vascular injury-induced stenosis. This study evaluates the efficacy of a novel, small molecule inhibitor of ALK5/ALK4 kinase, in the rat carotid injury model of vascular fibrosis. Methods and Results—The small molecule, SM16, was shown to bind with high affinity to ALK5 kinase ATP binding site using a competitive binding assay and biacore analysis. SM16 blocked TGF-&bgr; and activin-induced Smad2/3 phosphorylation and TGF-&bgr;-induced plasminogen activator inhibitor (PAI)-luciferase activity in cells. Good overall selectivity was demonstrated in a large panel of kinase assays, but SM16 also showed nanomolar inhibition of ALK4 and weak (micromolar) inhibition of Raf and p38. In the rat carotid injury model, SM16 dosed once daily orally at 15 or 30 mg/kg SM16 for 14 days caused significant inhibition of neointimal thickening and lumenal narrowing. SM16 also prevented induction of adventitial smooth muscle &agr;-actin–positive myofibroblasts and the production of intimal collagen, but did not decrease the percentage of proliferative cells. Conclusion—These results are the first to demonstrate the efficacy of an orally active, small-molecule ALK5/ALK4 inhibitor in a vascular fibrosis model and suggest the potential therapeutic application of these inhibitors in vascular fibrosis.

Type III TGF-β receptor downregulation generates an immunotolerant tumor microenvironment

Cancers subvert the host immune system to facilitate disease progression. These evolved immunosuppressive mechanisms are also implicated in circumventing immunotherapeutic strategies. Emerging data indicate that local tumor-associated DC populations exhibit tolerogenic features by promoting Treg development; however, the mechanisms by which tumors manipulate DC and Treg function in the tumor microenvironment remain unclear. Type III TGF-β receptor (TGFBR3) and its shed extracellular domain (sTGFBR3) regulate TGF-β signaling and maintain epithelial homeostasis, with loss of TGFBR3 expression promoting progression early in breast cancer development. Using murine models of breast cancer and melanoma, we elucidated a tumor immunoevasion mechanism whereby loss of tumor-expressed TGFBR3/sTGFBR3 enhanced TGF-β signaling within locoregional DC populations and upregulated both the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in plasmacytoid DCs and the CCL22 chemokine in myeloid DCs. Alterations in these DC populations mediated Treg infiltration and the suppression of antitumor immunity. Our findings provide mechanistic support for using TGF-β inhibitors to enhance the efficacy of tumor immunotherapy, indicate that sTGFBR3 levels could serve as a predictive immunotherapy biomarker, and expand the mechanisms by which TGFBR3 suppresses cancer progression to include effects on the tumor immune microenvironment.

Small Molecule Inhibition of the TNF Family Cytokine CD40 Ligand Through a Subunit Fracture Mechanism

BIO8898 is one of several synthetic organic molecules that have recently been reported to inhibit receptor binding and function of the constitutively trimeric tumor necrosis factor (TNF) family cytokine CD40 ligand (CD40L, aka CD154). Small molecule inhibitors of protein-protein interfaces are relatively rare, and their discovery is often very challenging. Therefore, to understand how BIO8898 achieves this feat, we characterized its mechanism of action using biochemical assays and X-ray crystallography. BIO8898 inhibited soluble CD40L binding to CD40-Ig with a potency of IC(50) = 25 μM and inhibited CD40L-dependent apoptosis in a cellular assay. A co-crystal structure of BIO8898 with CD40L revealed that one inhibitor molecule binds per protein trimer. Surprisingly, the compound binds not at the surface of the protein but by intercalating deeply between two subunits of the homotrimeric cytokine, disrupting a constitutive protein-protein interface and breaking the proteins 3-fold symmetry. The compound forms several hydrogen bonds with the protein, within an otherwise hydrophobic binding pocket. In addition to the translational splitting of the trimer, binding of BIO8898 was accompanied by additional local and longer-range conformational perturbations of the protein, both in the core and in a surface loop. Binding of BIO8898 is reversible, and the resulting complex is stable and does not lead to detectable dissociation of the protein trimer. Our results suggest that a set of core aromatic residues that are conserved across a subset of TNF family cytokines might represent a generic hot-spot for the induced-fit binding of trimer-disrupting small molecules.

STAT3 Signaling Is Required for Optimal Regression of Large Established Tumors in Mice Treated with Anti-OX40 and TGFβ Receptor Blockade

Triplett, Tucker, and colleagues show that combination cancer therapy using an OX40 agonist and TGFβ receptor blockade depends in part on STAT3 signaling by OX40-expressing T cells; this combination increases intratumoral CD4 and CD8 T-cell functions, which are dampened in the absence of STAT3 signaling. In preclinical tumor models, αOX40 therapy is often successful at treating small tumors, but is less effective once the tumors become large. For a tumor immunotherapy to be successful to cure large tumors, it will most likely require not only an agonist to boost effector T-cell function but also inhibitors of T-cell suppression. In this study, we show that combining αOX40 antibodies with an inhibitor of the TGFβ receptor (SM16) synergizes to elicit complete regression of large established MCA205 and CT26 tumors. Evaluation of tumor-infiltrating T cells showed that SM16/αOX40 dual therapy resulted in an increase in proliferating granzyme B+ CD8 T cells, which produced higher levels of IFNγ, compared with treatment with either agent alone. We also found that the dual treatment increased pSTAT3 expression in both CD4 and CD8 T cells isolated from tumors. Because others have published that STAT3 signaling is detrimental to T-cell function within the tumor microenvironment, we explored whether deletion of STAT3 in OX40-expressing cells would affect this potent combination therapy. Surprisingly, we found that deletion of STAT3 in OX40-expressing cells decreased the efficacy of this combination therapy, showing that the full therapeutic potential of this treatment depends on STAT3 signaling, most likely in the T cells of tumor-bearing mice. Cancer Immunol Res; 3(5); 526–35. ©2015 AACR.

Pyrazolone based TGFβR1 kinase inhibitors

Interruption of TGFbeta signaling through inhibition of the TGFbetaR1 kinase domain may prove to have beneficial effect in both fibrotic and oncological diseases. Herein we describe the SAR of a novel series of TGFbetaR1 kinase inhibitors containing a pyrazolone core. Most TGFbetaR1 kinase inhibitors described to date contain a core five-membered ring bearing N as H-bond acceptor. Described herein is a novel strategy to replace the core structure with pyrazolone ring, in which the carbonyl group is designed as an H-bond acceptor to interact with catalytic Lys 232.

The Use of Virtual Screening in ALK5 Kinase Inhibitor Discovery and Validation of Orally Active ALK5 Kinase Inhibitors in Oncology

The multifunctional cytokine, TGF-β, is often overexpressed in human tumors and in preclinical studies has been demonstrated to have autocrine and paracrine protumor activities including immune evasion, invasiveness, epithelial to mesenchymal transition, angiogenesis, tumor-stromal interactions, survival, induction of tumor interstitial pressure, and decreased drug penetration. These findings suggest that antagonism of the TGF-β pathway may be of benefit in the treatment of cancer. One attractive target, the type I TGF-β receptor (ALK5) has an intracellular serine/threonine kinase, which is required for TGF-β signaling and is amenable to inhibition by small molecule, ATP binding site-targeted kinase inhibitors.

STAT3 signaling is required for anti-OX40/TGF-β receptor blockade-mediated regression of large established tumors

OX40 (CD134, TNFRSF4), a member of the tumor necrosis factor receptor (TNFR) superfamily is expressed on activated CD4+ and CD8+ T cells. In pre-clinical tumor models, agonist OX40 antibody (αOX40) therapy is often successful at treating small tumors but is less effective once the tumors have become established. For a tumor immunotherapy to be successful it will most likely require not only an agonist to boost effector T cell function but also an antagonist to eliminate T cell suppression. In this study, we show that agonist OX40 antibody synergizes with an orally bioavailable inhibitor of TGF-β (SM16) to elicit complete regression of large established tumors, resulting in long-term survival in 40-85% of αOX40/SM16 treated mice in two murine tumor models. Evaluation of tumor infiltrating T cells showed that SM16/αOX40 dual therapy resulted in an increase in OX40 and Granzyme B-expressing CD8+ T cells undergoing proliferation and which produced greater levels of IFNγ. We also found that this dual treatment led to an increase in pSTAT3 staining in both CD4+ and CD8+ T cells isolated from tumors. Therefore, we tested whether deletion of STAT3 in OX40 expressing cells would impact this potent combination therapy, since others have shown that pSTAT3 up-regulation is detrimental to T cell function within the tumor microenvironment. Surprisingly, deletion of STAT3 decreased the therapeutic efficacy of this combination therapy, suggesting that immune enhancement of T cells within tumor-bearing mice is reliant on signals through STAT3 to gain their full therapeutic potential.