A Pt(IV)-conjugated brain penetrant macrocyclic peptide shows pre-clinical efficacy in glioblastoma.

Glioblastoma (GBM) is the most aggressive primary malignant brain tumor, with a median survival of approximately 15 months. Treatment is limited by the blood-brain barrier (BBB) which restricts the passage of most drugs to the brain. We previously reported the design and synthesis of a BBB-penetrant macrocyclic cell-penetrating peptide conjugate (M13) covalently linked at the axial position of a Pt(IV) cisplatin prodrug. Here we show the Pt(IV)-M13 conjugate releases active cisplatin upon intracellular reduction and effects potent in vitro GBM cell killing. Pt(IV)-M13 significantly increased platinum uptake in an in vitro BBB spheroid model and intravenous administration of Pt(IV)-M13 in GBM tumor-bearing mice led to higher platinum levels in brain tissue and intratumorally compared with cisplatin. Pt(IV)-M13 administration was tolerated in naïve nude mice at higher dosage regimes than cisplatin and significantly extended survival above controls in a murine GBM xenograft model (median survival 33 days for Pt(IV)-M13 vs 24 days for Pt(IV) prodrug, 22.5 days for cisplatin and 22 days for control). Increased numbers of γH2AX nuclear foci, biomarkers of DNA damage, were observed in tumors of Pt(IV)-M13-treated mice, consistent with elevated platinum levels. The present work provides the first demonstration that systemic injection of a Pt(IV) complex conjugated to a brain-penetrant macrocyclic peptide can lead to increased platinum levels in the brain and extend survival in mouse GBM models, supporting further development of this approach and the utility of brain-penetrating macrocyclic peptide conjugates for delivering non-BBB penetrant drugs to the central nervous system.

Profiling cytotoxic microRNAs in pediatric and adult glioblastoma cells by high-content screening, identification, and validation of miR-1300.

MicroRNAs play an important role in the regulation of mRNA translation and have therapeutic potential in cancer and other diseases. To profile the landscape of microRNAs with significant cytotoxicity in the context of glioblastoma (GBM), we performed a high-throughput screen in adult and pediatric GBM cells using a synthetic oligonucleotide library representing all known human microRNAs. Bioinformatics analysis was used to refine this list and the top seven microRNAs were validated in a larger panel of GBM cells using state-of-the-art in vitro assays. The cytotoxic effect of our most relevant candidate was assessed in a preclinical model. Our screen identified ~100 significantly cytotoxic microRNAs with 70% concordance between cell lines. MicroRNA-1300 (miR-1300) was the most potent and robust candidate. We observed a striking binucleated phenotype in miR-1300 transfected cells due to cytokinesis failure followed by apoptosis. This was also observed in two stem-like patient-derived cultures. We identified the physiological role of miR-1300 as a regulator of endomitosis in megakaryocyte differentiation where blockade of cytokinesis is an essential step. In GBM cells, where miR-1300 is normally not expressed, the oncogene Epithelial Cell Transforming 2 (ECT2) was validated as a direct key target. ECT2 siRNA phenocopied the effects of miR-1300, and ECT2 overexpression led to rescue of miR-1300 induced binucleation. We showed that ectopic expression of miR-1300 led to decreased tumor growth in an orthotopic GBM model. Our screen provides a resource for the neuro-oncology community and identified miR-1300 as a novel regulator of endomitosis with translatable potential for therapeutic application.

In vitro screening of clinical drugs identifies sensitizers of oncolytic viral therapy in glioblastoma stem-like cells.

Oncolytic viruses (OV) have broad potential as an adjuvant for the treatment of solid tumors. The present study addresses the feasibility of clinically applicable drugs to enhance the oncolytic potential of the OV Delta24-RGD in glioblastoma. In total, 446 drugs were screened for their viral sensitizing properties in glioblastoma stem-like cells (GSCs) in vitro. Validation was done for 10 drugs to determine synergy based on the Chou Talalay assay. Mechanistic studies were undertaken to assess viability, replication efficacy, viral infection enhancement and cell death pathway induction in a selected panel of drugs. Four viral sensitizers (fluphenazine, indirubin, lofepramine and ranolazine) were demonstrated to reproducibly synergize with Delta24-RGD in multiple assays. After validation, we underscored general applicability by testing candidate drugs in a broader context of a panel of different GSCs, various solid tumor models and multiple OVs. Overall, this study identified four viral sensitizers, which synergize with Delta24-RGD and two other strains of OVs. The viral sensitizers interact with infection, replication and cell death pathways to enhance efficacy of the OV.

Cell migration in paediatric glioma; characterisation and potential therapeutic targeting.

BACKGROUND: Paediatric high grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are highly aggressive brain tumours. Their invasive phenotype contributes to their limited therapeutic response, and novel treatments that block brain tumour invasion are needed. METHODS: Here, we examine the migratory characteristics and treatment effect of small molecule glycogen synthase kinase-3 inhibitors, lithium chloride (LiCl) and the indirubin derivative 6-bromoindirubin-oxime (BIO), previously shown to inhibit the migration of adult glioma cells, on two pHGG cell lines (SF188 and KNS42) and one patient-derived DIPG line (HSJD-DIPG-007) using 2D (transwell membrane, immunofluorescence, live cell imaging) and 3D (migration on nanofibre plates and spheroid invasion in collagen) assays. RESULTS: All lines were migratory, but there were differences in morphology and migration rates. Both LiCl and BIO reduced migration and instigated cytoskeletal rearrangement of stress fibres and focal adhesions when viewed by immunofluorescence. In the presence of drugs, loss of polarity and differences in cellular movement were observed by live cell imaging. CONCLUSIONS: Ours is the first study to demonstrate that it is possible to pharmacologically target migration of paediatric glioma in vitro using LiCl and BIO, and we conclude that these agents and their derivatives warrant further preclinical investigation as potential anti-migratory therapeutics for these devastating tumours.

Methylation-specific multiplex ligation-dependent probe amplification identifies promoter methylation events associated with survival in glioblastoma.

DNA methylation plays an important role in cancer biology and methylation events are important prognostic and predictive markers in many tumor types. We have used methylation-specific multiplex ligation-dependent probe amplification to survey the methylation status of MGMT and 25 tumor suppressor genes in 73 glioblastoma cases. The data obtained was correlated with overall survival and response to treatment. The study revealed that methylation of promoter regions in TP73 (seven patients), THBS1 (eight patients) and PYCARD (nine patients) was associated with improved outcome, whereas GATA5 (21 patients) and WT1 (24 patients) promoter methylation were associated with poor outcome. In patients treated with temozolomide and radiation MGMT and PYCARD promoter methylation events remained associated with improved survival whereas GATA5 was associated with a poor outcome. The identification of GATA5 promoter methylation in glioblastoma has not previously been reported. Furthermore, a cumulative methylation score separated patients into survival groups better than any single methylation event. In conclusion, we have identified specific methylation events associated with patient outcome and treatment response in glioblastoma, and these may be of functional and predictive/prognostic significance. This study therefore provides novel candidates and approaches for future prospective validation.

Reprogramming of the tumour microenvironment by stromal PTEN-regulated miR-320.

PTEN (Phosphatase and tensin homolog deleted on chromosome 10) expression in stromal fibroblasts suppresses epithelial mammary tumours, but the underlying molecular mechanisms remain unknown. Using proteomic and expression profiling, we show that Pten loss from mammary stromal fibroblasts activates an oncogenic secretome that orchestrates the transcriptional reprogramming of other cell types in the microenvironment. Downregulation of miR-320 and upregulation of one of its direct targets, ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2) are critical events in Pten-deleted stromal fibroblasts responsible for inducing this oncogenic secretome, which in turn promotes tumour angiogenesis and tumour-cell invasion. Expression of the Pten-miR-320-Ets2-regulated secretome distinguished human normal breast stroma from tumour stroma and robustly correlated with recurrence in breast cancer patients. This work reveals miR-320 as a critical component of the Pten tumour-suppressor axis that acts in stromal fibroblasts to reprogramme the tumour microenvironment and curtail tumour progression.

MicroRNAs and glioblastoma; the stem cell connection.

Recent data draw close parallels between cancer, including glial brain tumors, and the biology of stem and progenitor cells. At the same time, it has become clear that one of the major roles that microRNAs play is in the regulation of stem cell biology, differentiation, and cell 'identity'. For example, microRNAs have been increasingly implicated in the regulation of neural differentiation. Interestingly, initial studies in the incurable brain tumor glioblastoma multiforme strongly suggest that microRNAs involved in neural development play a role in this disease. This encourages the idea that certain miRs allow continued tumor growth through the suppression of differentiation and the maintenance of the stem cell-like properties of tumor cells. These concepts will be explored in this article.

Genetic strategies for brain tumor therapy.

Gene therapy is a potentially useful approach in the treatment of human brain tumors, which are notoriously refractory to conventional approaches. Most human clinical trials to date have been unsuccessful in terms of improving patient outcome. Recent improvements in viral vectors, the development of stem cell technology, and increased understanding of the mechanism of action of therapeutic transgenes provide hope that the next generation of gene therapeutics may show increased efficacy in treatment of this devastating disease.

HLA class II-induced translocation of PKC alpha and PKC beta II isoforms is abrogated following truncation of DR beta cytoplasmic domains.

Several studies have attempted to identify regions of the MHC class II molecule that participate in signal transduction. The importance of intact murine I-A cytoplasmic domains, either to tether the class II molecule to the cytoskeleton or to recruit signal-transducing proteins, is now well established. Recent data have also suggested that residues of the I-A beta transmembrane are involved in a second distinct signaling pathway. In the light of data suggesting that the second messengers activated on ligation of human and mouse class II molecules may differ, we set out to investigate whether the structural requirements for signaling for the human DR molecule are similar to those already described for the murine I-A molecule. We show that mutant DR class II molecules lacking 12 amino acids of the beta-chain cytoplasmic tail fail to translocate the protein kinase C alpha (PKC alpha) and PKC beta II isoenzymes following stimulation with an anti-class II mAb. In contrast, truncation of either or both cytoplasmic domains of the DR molecule has no effect on class II-induced tyrosine kinase activity. PKC translocation following class II stimulation has been observed in both human and murine B lymphocytes, whereas tyrosine kinase activation is present in human B lymphocytes but absent in resting murine B lymphocytes. Therefore, we conclude that the DR beta cytoplasmic tail is requisite for the principal signaling pathway initiated via MHC class II. These data suggest that the signaling pathways seen in resting vs primed murine B cells may also operate in human APCs.