P01.19A GENOME-WIDE RNAI SYNTHETIC LETHAL SCREEN FOR SENSITIZERS OF GLIOBLASTOMA MULTIFORME TO TEMOZOLOMIDE.

Glioblastoma multiforme (GBM) is highly resistant to therapy and carries a dismal prognosis. Standard of care includes surgery followed by radiotherapy (RT) with concurrent and adjuvant chemotherapy with temozolomide (TMZ). The addition of TMZ to RT increases the overall survival by approximately 3 months compared to RT alone, but there are virtually no long-term survivors. In this work we sought to identify small-molecule sensitizers to TMZ in GBM by integrating a genome-wide lentiviral shRNA library screen with the Connectivity Map database. We first transfected the human GBM cell line U87 with a pooled RNAi library targeting approximately 5000 genes, and incubated the cells at three sub-lethal TMZ concentrations or in control medium. DNA was extracted from both treated and untreated cells at 12 days. Barcodes identifying each shRNA were amplified by PCR and quantified using next-generation sequencing. Individual silencing of 292 genes lead to significantly reduced cell growth in the treated population while silencing of 414 genes increased cellular viability compared to the control group. The Connectivity Map database was then interrogated using these results to identify small-molecule inhibitors that would induce analogous changes in gene expression. This integrated high-throughput approach identified a broad range of compounds with potential cytotoxic activity such as the anti-psychotic thioridazine, the phytoestrogen resveratrol, the antibiotic furazolidone, and the proteasomal inhibitor MG-262. Upon validation in low-throughput cytotoxicity assays, we found these compounds effective as single-agent therapies, but also synergistic with concomitant TMZ therapy. Several of these compounds have previously been implicated in anti-cancer therapy, but not as synthetic lethal partners of chemotherapeutic drugs. Interestingly, we found a prominent cytotoxic effect of the dopamine receptor antagonist thioridazine when given as monotherapy and as combination therapy with TMZ. The therapeutic potential of thioridazine and concomitant TMZ in vivo is currently being explored in a clinically relevant intracranial GBM animal model.