PO-082 BMP4-induced differentiation does not enhance chemosensitivity of glioma stem-like cells to temozolomide

Introduction Glioblastoma (GBM) is the most aggressive primary brain tumour, with a median patient survival of 14 months. Accumulating evidence suggests that glioma stem-like cells (GSCs) are responsible for glioblastoma propagation, recurrence and resistance to therapy. Differentiation therapy is consider as a promising approach to eliminate this cell population in GBM. Bone morphogenic proteins (BMPs) are members of TGF-β superfamily, which control various biological functions. BMP4 plays an important role in the differentiation and proliferation of neural stem cells as well as GSCs. Therefore, the aim of this study was to investigate whether BMP4-induced differentiation would increase sensitivity to a chemotherapeutic drug, temozolomide (TMZ). Material and methods To explore the therapeutic potential of BMP4-induced differentiation, we used 4 patient-derived GSCs, with different expression of epidermal growth factor receptor (EGFR). Markers of differentiation, pluripotency, apoptosis and cell cycle were determined by western blotting, qPCR, next-generation sequencing (NGS) and flow cytometry. Results and discussions We show that BMP4 activates their cognate receptors (BMPRs) and triggers Smad signalling cascade in GSCs independently of the EGFR expression. Four days following exposure to BMP4 in three of four cell lines we found upregulation of astrocytic (GFAP) and neuronal (b-Tubulin III) markers with the concomitant reduction of OLIG2, CD133 and SOX2 (pluripotency markers, as determined at mRNA and protein levels). Transcriptome analysis by NGS (RNAseq) confirmed these changes. Next, we investigated whether a BMP4-based treatment would increased TMZ response in GSCs. TMZ treatment alone resulted in accumulation of the cleaved caspase 3, caspase 7 and PARP, which evidenced induction of apoptosis. Surprisingly, pre-treatment with BMP4 did not enhance sensitivity to TMZ. Evaluation of the cell cycle by flow cytometry showed that TMZ induced growth arrest in the G2 phase, which was associated with the accumulation of CYCLIN B1. Moreover, BMP4 treatment alone or in combination with TMZ resulted in reduction of the level of phospho-RB and increased the levels of two cyclin-dependent kinase inhibitors, p21 and p 27 as determined by western blotting. Conclusion These results demonstrate that BMP4 induces differentiation of GSCs along astrocytic and neuronal lineages but differentiated glioma cells are not more sensitive to TMZ chemotherapy. Studies supported by grant 2016/23/P/NZ2/04111 from The National Science Centre, Poland.