1331 Validation of a New Therapeutic Target Using Tissues From Tumour Resection Surgery to Re-Establish Cell Cycle Checkpoint Response in Glioblastoma

INTRODUCTION: The extremely poor response to therapy, resulting in relapse of patients with GBM, is attributed to the existence of Glioma Stem Cells (GSCs) and their ability to resist the standard of care therapy. Spy1 is a cyclin like protein which plays a role in expansion of GSCs and unlike classical cyclins can stimulate cell cycle progression against cell cycle checkpoint control. METHODS: Twelve GBM patient-derived tumour samples were subjected to cell extraction. Cells were cultured as neurospheres to enrich for GSC populations in vitro. Spy1 levels were manipulated/depleted in GSCs using lentiviral system. Drug response was tested using plate reader viability assay and/or trypan blue exclusion assay. The impact of Spy1 depletion on tumour burden in vivo using syngeneic mouse model and tumour response to treatment was assessed using zebrafish patient derived xenograft (PDX) drug screening platform. RESULTS: In vitro assays demonstrate significant upregulation of Spy1 expression levels in chemotherapy treated cultures in comparison to vehicle control, across all samples tested (n= 12, p<0.05 - p=0.001). Consequently, downregulation of Spy1 resulted in decreased proliferation in 8/12 patient cultures and a significant increase of sensitivity to selected chemotherapeutics (n=6, p<0.01). Mouse glioma xenografts with depleted levels of Spy1 result in significantly lower tumour burden in comparison to control (n=8, p<0.01). In vivo treatment of glioblastoma PDXs in zebrafish demonstrated significantly decreased tumour growth over time in glioma samples with downregulated levels of Spy1 in comparison to controls (n=50, p<0.001). CONCLUSIONS: Our data demonstrate that Spy1 downregulation results in decreased aggressiveness of patient-derived GSCs in vitro and in vivo suggesting that design of Spy1-targeting therapeutics could potentially elucidate novel, effective strategies in the treatment of glioblastoma.