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high-throughput screening for high-grade glioma

Final Report Summary - HTS-4-GBM (high-throughput screening for high-grade glioma)

Glioblastoma multiforme (GBM) is the most common and most aggressive form of brain tumour, which can affect both children and adults. It is associated with a dismal prognosis, with only 5% of patients alive 5 years after diagnosis. More effective therapies are therefore urgently needed for GBM patients.

PROJECT OBJECTIVES: The primary objective of the HTS-4-GBM project is to identify and validate novel therapeutic targets in GBM, using a unique combination of drug and small interfering RNA (siRNA) screening. This will allow us to identify already-approved drugs that could be useful in the treatment of GBM, together with biomarkers that may be used for patient selection in future clinical trials.

WORK COMPLETED TO DATE: Three libraries (Tocris, Lopac and Prestwick), containing a total of 3707 already-approved drugs and pharmacologically active molecules, were screened alone and in combination with either the standard of care for GBM (temozolomide chemotherapy) or a targeted agent (EGFR inhibitor, erlotinib) in 3 GBM cell lines. This led to the identification of several active compounds, whose main molecular targets were determined by bioinformatic analysis. These molecular targets were then validated by siRNA screening, where 3 independent sequences of siRNA for each target were screened alone and in combination with temozolomide and erlotinib in 3 GBM cell lines. The top genes identified by this approach, and their pathophysiological role in GBM, are now being validated. Furthermore, the results of the siRNA screen were used to select the most promising drugs to test in pre-clinical models of GBM and ultimately use in future clinical trials.

MAIN RESULTS: The high-throughput drug screening has led to the identification of 123 molecules showing promising activity against GBM cell lines in vitro. These include 81 “GBM killers” (i.e. drugs that were able to kill GBM cells when used alone) and 42 “Sensitivity enhancers” (i.e. drugs that were able to increase the sensitivity of GBM cells to temozolomide and/or erlotinib). The bioinformatic analysis of these 123 active compounds using different databases (e.g. Pubmed Compound, Drugbank, Drugsurv and GOSTAR) revealed a total of 1257 molecular targets, whose top 412 were evaluated by siRNA screening. This led to the discovery of 158 putative therapeutic targets in GBM (i.e. genes that either decreased the viability of GBM cells or increased their sensitivity to temozolomide and/or erlotinib, when their expression was silenced by siRNA). These 158 molecular targets fall into 3 main categories: i) genes that are known therapeutic targets in oncology (e.g. TUBB, CDK1, FRAP1), ii) genes that were recently shown to represent novel therapeutic targets in GBM (e.g. AURKA, AURKB, PLK1), and iii) genes that were not known as therapeutic targets in oncology until now (e.g. SIGMAR1, DRD5, HIPK3). This result demonstrates the potential of our approach to unveil novel therapeutic targets in hard-to-treat cancers. The 158 targets were then ranked based on the extent of the effects and the number of cell lines affected by the gene knockdown, and the top genes are being validated in vitro and in vivo. The expression level of the top 26 genes has been evaluated in established GBM cell lines and in patient-derived GBM stem cells by quantitative real-time PCR. This analysis showed significant differences between established cell lines and stem cells in at least 15 of the 26 genes tested, thus potentially uncovering novel markers of GBM stem cells. Furthermore, the role of the top 2 genes in regulating GBM cell proliferation and response to therapy has already been confirmed in vitro using siRNA. The functional validation of additional top genes is currently under way.

Elsewhere, the results of the siRNA screen were used to select the most promising drugs based on the following criteria: i) drug approved for any medical use in Europe and/or US, ii) minimally toxic (chemotherapy agents were therefore excluded), iii) able to target the key genes involved in the pathophysiology of GBM (as identified by siRNA screening) and iv) able to cross the blood-brain barrier. Of the 16 compounds selected for further characterization (including anti-helminthics, statins and anti-psychotics), 15 were found to display potent anti-proliferative properties in vitro against GBM cell lines. The 5 most efficacious drugs have now been selected and are being tested in association in vitro and in vivo to develop novel combination treatments for GBM patients.

EXPECTED FINAL RESULTS AND POTENTIAL IMPACT: The overall objective of this project is to discover, validate and ultimately implement in the clinic, novel therapeutic strategies for GBM patients. Despite a number of important challenges, progress has been substantial. Within the timeframe of the Fellowship, we have been able to identify already-approved drugs that could be repurposed for the treatment of GBM, together with key genes involved in GBM proliferation and response to therapy, which may represent novel therapeutic targets and/or biomarkers for patient selection for future clinical trials. As such, we expect to initiate the clinical validation of our findings within the next 2-3 years, and hopefully impact on clinical practice and improve the outcome of GBM patients.