07-276 Sigma-AldrichAnti-PDGFRα Antibody

BACKGROUND: Glioblastoma multiforme is the most lethal brain tumor with limited therapeutic options. Antigens expressed on the surface of malignant cells are potential targets for antibody-mediated gene/drug delivery. PRINCIPAL FINDINGS: In this study, we investigated the ability of genetically modified human mesenchymal stem cells (hMSCs) expressing a single-chain antibody (scFv) on their surface against a tumor specific antigen, EGFRvIII, to enhance the therapy of EGFRvIII expressing glioma cells in vivo. The growth of U87-EGFRvIII was specifically delayed in co-culture with hMSC-scFvEGFRvIII. A significant down-regulation was observed in the expression of pAkt in EGFRvIII expressing glioma cells upon culture with hMSC-scFvEGFRvIII vs. controls as well as in EGFRvIII expressing glioma cells from brain tumors co-injected with hMSC-scFvEGFRvIII in vivo. hMSC expressing scFvEGFRvIII also demonstrated several fold enhanced retention in EGFRvIII expressing flank and intracranial glioma xenografts vs. control hMSCs. The growth of U87-EGFRvIII flank xenografts was inhibited by 50% in the presence of hMSC-scFvEGFRvIII (p<0.05). Moreover, animals co-injected with U87-EGFRvIII and hMSC-scFvEGFRvIII intracranially showed significantly improved survival compared to animals injected with U87-EGFRvIII glioma cells alone or with control hMSCs. This survival was further improved when the same animals received an additional dosage of hMSC-scFvEGFRvIII two weeks after initial tumor implantation. Of note, EGFRvIII expressing brain tumors co-injected with hMSCs had a lower density of CD31 expressing blood vessels in comparison with control tumors, suggesting a possible role in tumor angiogenesis. CONCLUSIONS/SIGNIFICANCE: The results presented in this study illustrate that genetically modified MSCs may function as a novel therapeutic vehicle for malignant brain tumors.

Disruption of signaling pathways such as those mediated by sonic hedgehog (Shh) or platelet-derived growth factor (Pdgf) causes craniofacial abnormalities, including cleft palate. The role that microRNAs play in modulating palatogenesis, however, is completely unknown. We show that, in zebrafish, the microRNA Mirn140 negatively regulates Pdgf signaling during palatal development, and we provide a mechanism for how disruption of Pdgf signaling causes palatal clefting. The pdgf receptor alpha (pdgfra) 3' UTR contained a Mirn140 binding site functioning in the negative regulation of Pdgfra protein levels in vivo. pdgfra mutants and Mirn140-injected embryos shared a range of facial defects, including clefting of the crest-derived cartilages that develop in the roof of the larval mouth. Concomitantly, the oral ectoderm beneath where these cartilages develop lost pitx2 and shha expression. Mirn140 modulated Pdgf-mediated attraction of cranial neural crest cells to the oral ectoderm, where crest-derived signals were necessary for oral ectodermal gene expression. Mirn140 loss of function elevated Pdgfra protein levels, altered palatal shape and caused neural crest cells to accumulate around the optic stalk, a source of the ligand Pdgfaa. These results suggest that the conserved regulatory interactions of mirn140 and pdgfra define an ancient mechanism of palatogenesis, and they provide candidate genes for cleft palate.

To compare PDGF- and insulin/IGF-1-induced class I(A) PI 3-kinase/Akt survival signaling in normal retinas and retinal ganglion cells (RGCs).Normal rat retinas and RGC-5 cells were used for (1) immunohistochemical and immunoblot studies to detect PDGF receptor (PDGFR) subtypes and (2) immunoprecipitation, immunoblot, and in vitro lipid kinase assays to determine basal and PDGF-induced class I(A) PI 3-kinase/Akt survival signaling, in comparison with insulin or IGF-1 responses. Furthermore, RGC-5 cells were exposed to broad-spectrum (LY294002) or p110 isoform-selective (PI-103) PI 3-kinase inhibitors (versus Akt inhibitor) to assess the consequent effects on Akt phosphorylation, caspase-3/PARP cleavage, apoptotic phenotype, and cell viability, as a function of serum trophic factors.PDGFR-alpha and -beta immunoreactivity was observed in rat retinal Müller cells and in the RGC layer and blood vessels, respectively. In addition, PDGFR-alpha and -beta protein expression was observed in RGC-5 cells. Both retinas and RGC-5 cells exhibited a similar pattern of subunit-specific basal class I(A) PI 3-kinase activity, which was stimulated in a temporal and signal-specific manner by PDGF and insulin/IGF-1. Furthermore, RGC-5 cells showed PDGFR-alpha/beta tyrosine phosphorylation that induced the p85alpha regulatory subunit to activate p110alpha/beta-associated class I(A) PI 3-kinase, which in turn enhanced Akt phosphorylation. Exposure of serum-deprived RGC-5 cells to PI 3-kinase or Akt inhibitors increased susceptibility to apoptotic phenotype as revealed by caspase-3 and PARP cleavage.The present findings provide direct evidence of two distinct modes of retinal class I(A) PI 3-kinase activation that occurs in response to PDGF receptor and insulin/IGF-1 receptor stimulation. PDGF-induced PI 3-kinase/PIP3/Akt axis may provide new therapeutic approaches to ameliorate cell death in diabetic retinopathy and other retinal neurodegenerations.

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The FIP1-like-1 (FIP1L1)-platelet-derived growth factor receptor-alpha (FIP1L1-PDGFR-alpha) fusion kinase causes hypereosinophilic syndrome (HES) in a defined subset of patients. Imatinib mesylate is a potent inhibitor of ABL but also of PDGFR-alpha, and has been associated with durable hematologic responses in patients with HES. However, development of mutations in the tyrosine kinase domain may hamper the activity of tyrosine kinase inhibitors (TKIs), which suggests that novel agents are warranted to prevent or overcome resistance. We evaluated the efficacy of the novel TKI EXEL-0862 in FIP1L1-PDGFR-alpha-expressing cell lines and in cells from a patient with HES harboring the FIP1L1-PDGFR-alpha gene. EXEL-0862 inhibited the proliferation of EOL-1 and imatinib-resistant T674I FIP1L1-PDGFR-alpha-expressing cells and resulted in potent inhibition of the phosphorylation of PDGFR-alpha and downstream proteins STAT3 and Erk1/2, both in vitro and ex vivo. Moreover, EXEL-0862 induced apoptotic death in EOL-1 cells and imatinib-resistant T674I FIP1L1-PDGFR-alpha-expressing cells, and resulted in significant downregulation of the antiapoptotic protein Mcl-1 through a caspase-dependent mechanism. Our data establish EXEL-0862 as a solid candidate for the targeted treatment of patients with FIP1L1-PDGFR-alpha-positive HES.

Idiopathic hypereosinophilic syndrome (HES) is a myeloproliferative disorder characterized by tissue involvement and organ dysfunction due to abnormal eosinophil proliferation. In a subset of patients, this is caused by the FIP1L1-PDGFR-alpha fusion tyrosine kinase. Cumulative evidence indicates that the Bcr-Abl tyrosine kinase inhibitor imatinib mesylate (Gleevec) is active for the treatment of patients with HES, particularly those expressing the FIP1L1-PDGFR-alpha oncoprotein. The novel tyrosine kinase inhibitor AMN107 was initially developed as a potent Bcr-Abl inhibitor based on the molecular structure of imatinib. We tested the in vitro efficacy of imatinib and AMN107 in the EOL-1 cell line and in cells from a patient with HES harboring the FIP1L1-PDGFR-alpha fusion kinase. AMN107 was as potent as imatinib in inducing apoptosis and inhibiting proliferation of EOL-1 cells, with IC(50) values of 0.54 and 0.20 nM, respectively. In addition, both drugs inhibited the phosphorylation of PDGFR-alpha tyrosine kinase with equivalent efficacy. We conclude that AMN107 and imatinib are active and equipotent against cells expressing the FIP1L1-PDGFR-alpha fusion gene.

This two-centre phase-II trial aimed at investigating the efficacy of imatinib in metastasised melanoma patients in correlation to the tumour expression profile of the imatinib targets c-kit and platelet-derived growth factor receptor (PDGF-R). The primary study end point was objective response according to RECIST, secondary end points were safety, overall and progression-free survival. In all, 18 patients with treatment-refractory advanced melanoma received imatinib 800 mg day(-1). In 16 evaluable patients no objective responses could be observed. The median overall survival was 3.9 months, the median time to progression was 1.9 months. Tumour biopsy specimens were obtained from 12 patients prior to imatinib therapy and analysed for c-kit, PDGF-Ralpha and -Rbeta expression by immunohistochemistry. In four cases, cell lines established from these tumour specimens were tested for the antiproliferative effects of imatinib and for functional mutations of genes encoding the imatinib target molecules. The tumour specimens stained positive for CD117/c-kit in nine out of 12 cases (75%), for PDGF-Ralpha in seven out of 12 cases (58%) and for PDGF-Rbeta in eight out of 12 cases (67%). The melanoma cell lines showed a heterogenous expression of the imatinib target molecules without functional mutations in the corresponding amino-acid sequences. In vitro imatinib treatment of the cell lines showed no antiproliferative effect. In conclusion, this study did not reveal an efficacy of imatinib in advanced metastatic melanoma, regardless of the expression pattern of the imatinib target molecules c-kit and PDGF-R.