gdc-0449 and Glioblastoma

Neoplastic cells of Glioblastoma multiforme (GBM) may or may not show sustained response to temozolomide (TMZ) chemotherapy. We hypothesize that TMZ chemotherapy response in GBM is predetermined in its neoplastic clones via a specific set of mutations that alter relevant pathways. We describe exome-wide enrichment of variant allele frequencies (VAFs) in neurospheres displaying contrasting phenotypes of sustained versus reversible TMZ-responses in vitro. Enrichment of VAFs was found on genes ST5, RP6KA1 and PRKDC in cells showing sustained TMZ-effect whereas on genes FREM2, AASDH and STK36, in cells showing reversible TMZ-effect. Ingenuity pathway analysis (IPA) revealed that these genes alter cell-cycle, G2/M-checkpoint-regulation and NHEJ pathways in sustained TMZ-effect cells whereas the lysine-II&V/phenylalanine degradation and sonic hedgehog (Hh) pathways in reversible TMZ-effect cells. Next, we validated the likely involvement of the Hh-pathway in TMZ-response on additional GBM neurospheres as well as on GBM patients, by extracting RNA-sequencing-based gene expression data from the TCGA-GBM database. Finally, we demonstrated TMZ-sensitization of a TMZ non-responder neurosphere in vitro by treating them with the FDA-approved pharmacological Hh-pathway inhibitor vismodegib. Altogether, our results indicate that the Hh-pathway impedes sustained TMZ-response in GBM and could be a potential therapeutic target to enhance TMZ-response in this malignancy.

Topics: Alleles; Anilides; Cell Division; Cell Line, Tumor; Dacarbazine; Exome; Female; Follow-Up Studies; G2 Phase; Gene Frequency; Glioblastoma; Hedgehog Proteins; Humans; Male; Neoplasm Proteins; Pyridines; Radiography; Spheroids, Cellular; Temozolomide

Gliomagenesis and resistance of glioblastoma (GBM) are believed to be mediated by glioma stem cells (GSC). Evidence suggests that SHH signaling promotes GSC proliferation and self-renewal.. ABTC-0904 was a two-arm, multicenter phase 0/II study of GDC-0449, an oral inhibitor of Smoothened (SMO) in patients undergoing resection for recurrent GBM. All patients (Arms I and II) had surgery and received drug post-operatively. Only patients in Arm I received drug prior to surgery. The primary objective was to determine 6-month progression free survival (PFS-6). Secondary endpoints include median PFS (mPFS) and overall survival (mOS), response rate, and toxicity. Correlative studies included bioanalysis of GDC-0449, and inhibition of SHH signaling, GSC proliferation and self-renewal.. Forty-one patients were enrolled. Pharmacokinetics of GDC-0449 in plasma demonstrated levels within expected therapeutic range in 75% of patients. The proportion of tumorcells producing CD133. GDC-0449 was well tolerated, reached tumor, and inhibited CD133

Topics: Antineoplastic Agents; Brain Neoplasms; Glioblastoma; Glioma; Hedgehog Proteins; Humans; Neoplasm Recurrence, Local; Neoplastic Stem Cells

Scientists working in translational oncology regularly conduct multigroup studies of mice with serially measured tumors. Longitudinal data collected can feature mid-study dropouts and complex nonlinear temporal response patterns. Parametric statistical models such as ones assuming exponential growth are useful for summarizing tumor volume over ranges for which the growth model holds, with the advantage that the model's parameter estimates can be used to summarize between-group differences in tumor volume growth with statistical measures of uncertainty. However, these same assumed growth models are too rigid to recapitulate patterns observed in many experiments, which in turn diminishes the effectiveness of their parameter estimates as summary statistics. To address this problem, we generalized such models by adopting a nonparametric approach in which group-level response trends for logarithmically scaled tumor volume are estimated as regression splines in a generalized additive mixed model. We also describe a novel summary statistic for group level splines over user-defined, experimentally relevant time ranges. This statistic reduces to the log-linear growth rate for data well described by exponential growth and also has a sampling distribution across groups that is well approximated by a multivariate Gaussian, thus facilitating downstream analysis. Real-data examples show that this nonparametric approach not only enhances fidelity in describing nonlinear growth scenarios but also improves statistical power to detect interregimen differences when compared with the simple exponential model so that it generalizes the linear mixed effects paradigm for analysis of log-linear growth to nonlinear scenarios in a useful way. SIGNIFICANCE: This work generalizes the statistical linear mixed modeling paradigm for summarizing longitudinally measured preclinical tumor volume studies to encompass studies with nonlinear and nonmonotonic group response patterns in a statistically rigorous manner.

Topics: Anilides; Animals; Antineoplastic Agents, Alkylating; Bias; Decision Making; Disease Models, Animal; Female; Genes, Tumor Suppressor; Glioblastoma; Heterografts; Humans; Medical Oncology; Mice; Mice, Nude; Models, Statistical; Neoplasm Transplantation; Neoplasms; Normal Distribution; Patched-1 Receptor; Piperazines; Pyridines; Random Allocation; Statistics, Nonparametric; Temozolomide; Translational Research, Biomedical; Tumor Burden

The treatment of glioblastoma is a critical health issue, owing to its resistance to chemotherapy. The current standard of treatment is surgical resection, followed by adjuvant radiotherapy and temozolomide treatment. Long‑term local treatment of glioblastoma is rarely achieved and the majority of the patients undergo relapse. Resistance to temozolomide emerges from numerous signalling pathways that are altered in glioblastoma, including the Hedgehog signalling pathway. Hence, further research is required to identify effective treatment modalities. We investigated the effect of vismodegib, arsenic trioxide and temozolomide on glioblastoma in vitro and in vivo to apply our findings to the clinical setting. WST‑1 assay revealed that glioblastoma proliferation was inhibited following treatment with these drugs either in single or in combination; this synergistic effect was confirmed by CalcuSyn software. Western blot analysis revealed an increase in the expression of cleaved caspase‑3 and γH2AX. Furthermore, there was marked inhibition and decreased tumour growth in mice that received combination therapy, unlike those that received single agent or vehicle treatment. Our results revealed that the combination of arsenic trioxide/vismodegib and temozolomide may be an attractive therapeutic method for the treatment of glioblastoma.

Topics: Anilides; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Arsenic Trioxide; Caspase 3; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Mice; Neoplasm Recurrence, Local; Pyridines; Signal Transduction; Temozolomide; Xenograft Model Antitumor Assays

Hedgehog (Hh) signaling pathway is a valid therapeutic target in a wide range of malignancies. We focus here on glioblastoma multiforme (GBM), a lethal malignancy of the central nervous system (CNS). By analyzing RNA-sequencing based transcriptomics data on 149 clinical cases of TCGA-GBM database we show here a strong correlation (r = 0.7) between GLI1 and PTCH1 mRNA expression--as a hallmark of the canonical Hh-pathway activity in this malignancy. GLI1 mRNA expression varied in 3 orders of magnitude among the GBM patients of the same cohort showing a single continuous distribution-unlike the discrete high/low-GLI1 mRNA expressing clusters of medulloblastoma (MB). When compared with MB as a reference, the median GLI1 mRNA expression in GBM appeared 14.8 fold lower than that of the "high-Hh" cluster of MB but 5.6 fold higher than that of the "low-Hh" cluster of MB. Next, we demonstrated statistically significant up- and down-regulation of GLI1 mRNA expressions in GBM patient-derived low-passage neurospheres in vitro by sonic hedgehog ligand-enriched conditioned media (shh-CM) and by Hh-inhibitor drug vismodegib respectively. We also showed clinically achievable dose (50 μM) of vismodegib alone to be sufficient to induce apoptosis and cell cycle arrest in these low-passage GBM neurospheres in vitro. Vismodegib showed an effect on the neurospheres, both by down-regulating GLI1 mRNA expression and by inducing apoptosis/cell cycle arrest, irrespective of their relative endogenous levels of GLI1 mRNA expression. We conclude from our study that this single continuous distribution pattern of GLI1 mRNA expression technically puts almost all GBM patients in a single group rather than discrete high- or low-clusters in terms of Hh-pathway activity. That is suggestive of therapies with Hh-pathway inhibitor drugs in this malignancy without a need for further stratification of patients on the basis of relative levels of Hh-pathway activity among them.

Topics: Anilides; Apoptosis; Cell Cycle Checkpoints; Dose-Response Relationship, Drug; Down-Regulation; Gene Expression Regulation, Neoplastic; Glioblastoma; Hedgehog Proteins; Humans; Medulloblastoma; Oncogene Proteins; Pyridines; RNA, Messenger; Signal Transduction; Trans-Activators; Up-Regulation; Zinc Finger Protein GLI1

Glioblastoma multiforme (GBM) is composed of heterogeneous and genetically different cells, which are highly invasive and motile. The standard chemotherapeutic agent, temozolomide, affects GBM cell proliferation but is generally unable to prevent tumor recurrence. Hedgehog pathway activation has been reported to be relevant in GBM and different pharmacological pathway modulators have been identified. We report that by growing a commercially available recurrent GBM cell line (DBTRG-05MG) without serum and in the presence of defined growth factors; we obtained a less differentiated cell population, growing in suspension as neurospheres, in which the Hedgehog pathway is activated. Furthermore, the expression profile of Hedgehog pathway components found in DBTRG-05MG neurospheres is similar to primary stem-like cells derived from recurrent GBM patients. We report the effect of our novel specific Smoothened receptor antagonist (SEN450) on neurosphere growing cells and compared its effect to that of well known benchmark compounds. Finally, we showed that SEN450 is both antiproliferative on its own and further reduces tumor volume after temozolomide pretreatment in a mouse xenograft model using DBTRG-05MG neurosphere cells. Altogether our data indicate that the Hedgehog pathway is not irreversibly switched off in adherent cells but can be reactivated when exposed to well-defined culture conditions, thus restoring the condition observed in primary tumor-derived material, and that pharmacological modulation of this pathway can have profound influences on tumor proliferation. Therefore, pharmacological inhibition of the Hedgehog pathway is a potentially useful therapeutic approach in GBM.

Topics: Anilides; Animals; Cell Line, Tumor; Cell Proliferation; Dacarbazine; Gene Expression Profiling; Glioblastoma; Hedgehog Proteins; Humans; Mice; Mice, Nude; Pyridines; Receptors, G-Protein-Coupled; Signal Transduction; Smoothened Receptor; Temozolomide; Transcription Factors; Veratrum Alkaloids; Xenograft Model Antitumor Assays; Zinc Finger Protein GLI1