Abstract A068: Targeting glioblastoma stem cells through a MET inhibitor

Background: Glioblastoma (GBM) is the most common and aggressive human brain cancer with high relapse rate, lacking any targeted therapy. The current therapy consisting of surgery in combination with radiotherapy and the DNA-alkylating agent temozolomide has led to median survival increase. Nevertheless, many patients, after early treatment response, experience drug resistance and tumor progression. Recent advances identified cancer stem cell subpopulation as the cause of treatment failure, suggesting that the direct targeting of this cell subset may represent an innovative approach to improve the therapy efficacy. Based on studies indicating the role of the tyrosine kinase receptor MET in GBM stem cells, we examined the effects of the rational combination of radiotherapy with a MET inhibitor against stem-like cells from a human GBM cell line. Methods: Stem-like cells from U87MG cell line were sorted using PKH-67-labeling method for isolating slow-dividing cells (PKH67 + ), and analyzed for cell proliferation index by WST assay, clonogenic potential, and expression of stemness-related transcription factors by RT-PCR. Effects by the MET inhibitor alone and in combination with radiotherapy (IR) were evaluated on cell proliferation by Trypan Blue exclusion test, on apoptosis, cell cycle progression, GFAP protein expression by flow cytometry and on neurosphere-forming efficiency. The mechanisms underlying the effects induced by the combined treatment were dissected by biochemical and RT-PCR assays. Results: The PKH-67 + cells recapitulated the functional properties of stem-like cells, as slower cell proliferation index, higher clonogenic capacity, and expression of stemness-transcription factors than the negative counterpart. They also displayed a marked radioresistance dependent on MET signalling activation and characterized by the enrichment in stem-like cells as indicated by the upregulation of stemness-related transcription factor expression Nanog and Sox2, and enhanced susceptibility to MET inhibitor. The single-agent therapy with the inhibitor induced cell proliferation inhibition with an early cell cycle arrest in G2/M phase and a late cytotoxic effect. The drug alone negatively affected the neurosphere-forming efficiency and the clonogenic capacity. Following IR, the daily treatment with the inhibitor of MET significantly caused time-dependent cell proliferation inhibition, associated with an increased percentage of apoptotic cells and cells positive for GFAP, a glial differentiation marker, as compared to single-agent treatments. The biochemical analysis confirmed that MET inhibition remarkably impaired the radioresistance, and parallelly reduced the content of stem-like cells, as indicated either by the negative regulation of Nanog and Sox2 genes or the positive regulation of GFAP. Conclusions: Our results provide evidence that targeting MET with a specific inhibitor might interfere with MET-mediated radiotherapy resistance, thus sensitizing the GBM-stem-like cells to radiation, depleting the pool, and parallelly inducing their reprogramming towards more differentiated subtype. Citation Format: Anna Rossini, Evelyn Oliva Savoia, Eleonora Cicoria, Lorenzo Castagnoli, Serenella Pupa, Filippo de Braud, Massimo Di Nicola. Targeting glioblastoma stem cells through a MET inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A068.