Alison Murphy

SD-208, a Novel Transforming Growth Factor β Receptor I Kinase Inhibitor, Inhibits Growth and Invasiveness and Enhances Immunogenicity of Murine and Human Glioma Cells In vitro and In vivo

The cytokine transforming growth factor (TGF)-β, by virtue of its immunosuppressive and promigratory properties, has become a major target for the experimental treatment of human malignant gliomas. Here we characterize the effects of a novel TGF-β receptor (TGF-βR) I kinase inhibitor, SD-208, on the growth and immunogenicity of murine SMA-560 and human LN-308 glioma cells in vitro and the growth of and immune response to intracranial SMA-560 gliomas in syngeneic VM/Dk mice in vivo. SD-208 inhibits the growth inhibition of TGF-β–sensitive CCL64 cells mediated by recombinant TGF-β1 or TGF-β2 or of TGF-β–containing glioma cell supernatant at an EC50 of 0.1 μmol/L. SD-208 blocks autocrine and paracrine TGF-β signaling in glioma cells as detected by the phosphorylation of Smad2 or TGF-β reporter assays and strongly inhibits constitutive and TGF-β–evoked migration and invasion, but not viability or proliferation. Peripheral blood lymphocytes or purified T cells, cocultured with TGF-β–releasing LN-308 glioma cells in the presence of SD-208, exhibit enhanced lytic activity against LN-308 targets. The release of interferon γ and tumor necrosis factor α by these immune effector cells is enhanced by SD-208, whereas the release of interleukin 10 is reduced. SD-208 restores the lytic activity of polyclonal natural killer cells against glioma cells in the presence of recombinant TGF-β or of TGF-β–containing glioma cell supernatant. The oral bioavailability of SD-208 was verified by demonstrating the inhibition of TGF-β–induced Smad phosphorylation in spleen and brain. Systemic SD-208 treatment initiated 3 days after the implantation of SMA-560 cells into the brains of syngeneic VM/Dk mice prolongs their median survival from 18.6 to 25.1 days. Histologic analysis revealed no difference in blood vessel formation, proliferation, or apoptosis. However, animals responding to SD-208 showed an increased tumor infiltration by natural killer cells, CD8 T cells, and macrophages. These data define TGF-β receptor I kinase inhibitors such as SD-208 as promising novel agents for the treatment of human malignant glioma and other conditions associated with pathological TGF-β activity.

Inhibition of Growth and Metastasis of Mouse Mammary Carcinoma by Selective Inhibitor of Transforming Growth Factor-β Type I Receptor Kinase In vivo

Purpose: Transforming growth factor-β (TGF-β) suppresses tumor development by inhibiting cellular proliferation, inducing differentiation and apoptosis, and maintaining genomic integrity. However, once tumor cells escape from the tumor-suppressive effects of TGF-β, they often constitutively overexpress and activate TGF-β, which may promote tumor progression by enhancing invasion, metastasis, and angiogenesis and by suppressing antitumor immunity. The purpose of this study was to test this hypothesis using TGF-β pathway antagonists. Experimental Design: We examined the effects of selective TGF-β type I receptor kinase inhibitors, SD-093 and SD-208, on two murine mammary carcinoma cell lines (R3T and 4T1) in vitro and in vivo. Results: Both agents blocked TGF-β-induced phosphorylation of the receptor-associated Smads, Smad2 and Smad3, in a dose-dependent manner, with IC50 between 20 and 80 nmol/L. TGF-β failed to inhibit growth of these cell lines but stimulated epithelial-to-mesenchymal transdifferentiation, migration, and invasiveness into Matrigel in vitro. These effects were inhibited by SD-093, indicating that these processes are partly driven by TGF-β. Treatment of syngeneic R3T or 4T1 tumor-bearing mice with orally given SD-208 inhibited primary tumor growth as well as the number and size of metastases. In contrast, SD-208 failed to inhibit R3T tumor growth or metastasis in athymic nude mice. Moreover, in vitro anti-4T1 cell cytotoxic T-cell responses of splenocytes from drug-treated animals were enhanced compared with cells from control animals. In addition, SD-208 treatment resulted in a decrease in tumor angiogenesis. Conclusion: TGF-β type I receptor kinase inhibitors hold promise as novel therapeutic agents for metastatic breast cancer.

Inhibition of Transforming Growth Factor β Signaling Reduces Pancreatic Adenocarcinoma Growth and Invasiveness

Transforming growth factor β (TGFβ) is a pleiotropic factor that regulates cell proliferation, angiogenesis, metastasis, and immune suppression. Dysregulation of the TGFβ pathway in tumor cells often leads to resistance to the antiproliferative effects of TGFβ while supporting other cellular processes that promote tumor invasiveness and growth. In the present study, SD-208, a 2,4-disubstituted pteridine, ATP-competitive inhibitor of the TGFβ receptor I kinase (TGFβRI), was used to inhibit cellular activities and tumor progression of PANC-1, a human pancreatic tumor line. SD-208 blocked TGFβ-dependent Smad2 phosphorylation and expression of TGFβ-inducible proteins in cell culture. cDNA microarray analysis and functional gene clustering identified groups of TGFβ-regulated genes involved in metastasis, angiogenesis, cell proliferation, survival, and apoptosis. These gene responses were inhibited by SD-208. Using a Boyden chamber motility assay, we demonstrated that SD-208 inhibited TGFβ-stimulated invasion in vitro. An orthotopic xenograft mouse model revealed that SD-208 reduced primary tumor growth and decreased the incidence of metastasis in vivo. Our findings suggest mechanisms through which TGFβ signaling may promote tumor progression in pancreatic adenocarcinoma. Moreover, they suggest that inhibition of TGFβRI with a small-molecule inhibitor may be effective as a therapeutic approach to treat human pancreatic cancer.

Transforming Growth Factor β Receptor I Kinase Inhibitor Down-Regulates Cytokine Secretion and Multiple Myeloma Cell Growth in the Bone Marrow Microenvironment

Purpose: Transforming growth factors (TGFs) have pleiotropic biological effects on tumor cells and their environment. In multiple myeloma (MM), we have reported that bone marrow stromal cells (BMSCs) from MM patients produce more TGF-β1 than BMSCs from healthy donors, which in turn induces interleukin (IL)-6 secretion. We show here that the TGF-β receptor I kinase inhibitor SD-208 significantly decreases secretion of both IL-6 and vascular endothelial growth factor (VEGF) from BMSCs, as well as tumor cell growth triggered by MM cell adhesion to BMSCs. Experimental Design: Cytokine production and MM cell proliferation triggered by TGF-β1 or adhesion to BMSCs were examined in the presence or absence of SD-208. Effects of SD-208 on TGF-β1–induced signaling pathways triggering IL-6 and VEGF transcription in BMSCs were also delineated. Results: SD-208 significantly inhibits not only transcription but also secretion of both IL-6 and VEGF from BMSCs triggered by either TGF-β1 or adhesion of MM cells to BMSCs. Moreover, SD-208 decreased tumor cell growth triggered by MM cell adhesion to BMSCs. SD-208 works, at least in part, by blocking TGF-β1–triggered nuclear accumulation of Smad2/3 and hypoxia-inducible factor 1α, as well as related production of IL-6 and VEGF, respectively. Conclusions: These studies indicate that SD-208 inhibits production of cytokines mediating MM cell growth, survival, drug resistance, and migration in the BM milieu, thereby providing the preclinical rationale for clinical evaluation of SD-208 to improve patient outcome in MM.

Role of the TGF-β/Alk5 Signaling Pathway in Monocrotaline-induced Pulmonary Hypertension

RATIONALE Pulmonary arterial hypertension is a progressive disease characterized by an elevation in the mean pulmonary artery pressure leading to right heart failure and a significant risk of death. Alterations in two transforming growth factor (TGF) signaling pathways, bone morphogenetic protein receptor II and the TGF-beta receptor I, Alk1, have been implicated in the pathogenesis of pulmonary hypertension (PH). However, the role of TGF-beta family signaling in PH and pulmonary vascular remodeling remains unclear. OBJECTIVES To determine whether inhibition of TGF-beta signaling will attenuate and reverse monocrotaline-induced PH (MCT-PH). METHODS We have used an orally active small-molecule TGF-beta receptor I inhibitor, SD-208, to determine the functional role of this pathway in MCT-PH. MEASUREMENTS AND MAIN RESULTS The development of MCT-PH was associated with increased vascular cell apoptosis, which paralleled TGF-beta signaling as documented by psmad2 expression. Inhibition of TGF-beta signaling with SD-208 significantly attenuated the development of the PH and reduced pulmonary vascular remodeling. These effects were associated with decreased early vascular cell apoptosis, adventitial cell proliferation, and matrix metalloproteinase expression. Inhibition of TGF-beta signaling with SD-208 in established MCT-PH resulted in a small but significant improvement in hemodynamic parameters and medial remodeling. CONCLUSIONS These findings provide evidence that increased TGF-beta signaling participates in the pathogenesis of experimental severe PH.

Transforming growth factor-β receptor type 1 (TGFβRI) kinase activity but not p38 activation is required for TGFβRI-induced myofibroblast differentiation and profibrotic gene expression

Transforming growth factor-β (TGFβ) is a major mediator of normal wound healing and of pathological conditions involving fibrosis, such as idiopathic pulmonary fibrosis. TGFβ also stimulates the differentiation of myofibroblasts, a hallmark of fibrotic diseases. In this study, we examined the underlying processes of TGFβRI kinase activity in myofibroblast conversion of human lung fibroblasts using specific inhibitors of TGFβRI (SD-208) and p38 mitogen-activated kinase (SD-282). We demonstrated that SD-208, but not SD-282, inhibited TGFβ-induced SMAD signaling, myofibroblast transformation, and collagen gel contraction. Furthermore, we extended our findings to a rat bleomycin-induced lung fibrosis model, demonstrating a significant decrease in the number of myofibroblasts at fibroblastic foci in animals treated with SD-208 but not those treated with SD-282. SD-208 also reduced collagen deposition in this in vivo model. Microarray analysis of human lung fibroblasts identified molecular fingerprints of these processes and showed that SD-208 had global effects on reversing TGFβ-induced genes involved in fibrosis, inflammation, cell proliferation, cytoskeletal organization, and apoptosis. These studies also revealed that although the p38 pathway may not be needed for appearance or disappearance of the myofibroblast, it can mediate a subset of inflammatory and fibrogenic events of the myofibroblast during the process of tissue repair and fibrosis. Our findings suggest that inhibitors such as SD-208 may be therapeutically useful in human interstitial lung diseases and pulmonary fibrosis.

Inhibiting TGF-β signaling restores immune surveillance in the SMA-560 glioma model

Transforming growth factor-beta (TGF-beta) is a proinvasive and immunosuppressive cytokine that plays a major role in the malignant phenotype of gliomas. One novel strategy of disabling TGF-beta activity in gliomas is to disrupt the signaling cascade at the level of the TGF-beta receptor I (TGF-betaRI) kinase, thus abrogating TGF-beta-mediated invasiveness and immune suppression. SX-007, an orally active, small-molecule TGF-betaRI kinase inhibitor, was evaluated for its therapeutic potential in cell culture and in an in vivo glioma model. The syngeneic, orthotopic glioma model SMA-560 was used to evaluate the efficacy of SX-007. Cells were implanted into the striatum of VM/Dk mice. Dosing began three days after implantation and continued until the end of the study. Efficacy was established by assessing survival benefit. SX-007 dosed at 20 mg/kg p.o. once daily (q.d.) modulated TGF-beta signaling in the tumor and improved the median survival. Strikingly, approximately 25% of the treated animals were disease-free at the end of the study. Increasing the dose to 40 mg/kg q.d. or 20 mg/kg twice daily did not further improve efficacy. The data suggest that SX-007 can exert a therapeutic effect by reducing TGF-beta-mediated invasion and reversing immune suppression. SX-007 modulates the TGF-beta signaling pathway and is associated with improved survival in this glioma model. Survival benefit is due to reduced tumor invasion and reversal of TGF-beta-mediated immune suppression, allowing for rejection of the tumor. Together, these results suggest that treatment with a TGF-betaRI inhibitor may be useful in the treatment of glioblastoma.

Potential involvement of BMP receptor type IB activation in a synergistic effect of chondrogenic promotion between rhTGFbeta3 and rhGDF5 or rhBMP7 in human mesenchymal stem cells.

Chondrogenic promotion by rhGDF5 with or without rhTGFβ3 was studied in pellet culture of human mesenchymal stem cells (HMSCs). A synergy between rhGDF5 and rhTGFβ3 was observed in promoting chondrogenesis. rhBMP2, rhBMP6, rhBMP7 and rhTGFβ1 were further tested and showed the same effect. To explore the mechanism, the expression of TGFβtype I and II receptors, ALK5, ALK2, ALK3, ALK6, TGFβRII, BMPRII, ActRII was studied. ALK6 showed increase by the rhTGFβ1 or rhTGFβ3 treatment. ALK6 protein expression also showed increase by rhTGFβ3. rhTGFβ1/rhTGFβ3 induced ALK6 up-regulation was inhibited by SD-208, a TGFβ type I receptor inhibitor. Chondrogenesis by rhTGFβ1/rhTGFβ3 or the combination between rhTGFβ1/rhTGFβ3 and rhGDF5 also was diminished by SD-208. SMAD1/5/8 phosphorylation in nascent human mesenchymal stem cells (HMSCs) was stimulated weakly by rhGDF5 but strongly by rhBMP7. The rhGDF5 stimulated SMAD1/5/8 phosphorylation was enhanced by rhTGFβ1/rhTGFβ3 but inhibited by SD-208. The rhBMP7 stimulated SMAD1/5/8 phosphorylation did not show influence by rhTGFβ3 and SD-208. Our results indicated the potential involvement of ALK6 activation by rhTGFβs in the synergy between rhTGFβs and rhBMPs.