CRISPR/Cas9 dropout screen in two glioblastoma stem cell lines with a domain-focused sgRNA library targeting 1387 human proteins involved in epigenetic regulation.

Poor survival and lack of response to current treatment modalities in adult human glioblastomas is attributed to the persistence of a subpopulation of glioma stem cells (GSCs). To identify novel approaches to therapeutically target GSCs, we performed CRISPR/Cas9 knockout screens in GSCs and identified the kinase TAK1 as an important selective survival factor in 50% of the tested GSCs. In sensitive cells, knockout of TAK1 leads to induction of caspase-dependent apoptosis via the RIPK1/Caspase 8/FADD complex due to constitutive, low-level secretion of tumor necrosis factor alpha (TNF-a) and stimulation of TNF receptor 1. Furthermore, we show that expression of genes involved in immune signaling and a mesenchymal signature predict the sensitivity and response of GSCs to TAK1 inhibition. In summary, we have identified TAK1 as a new therapeutic target for mesenchymal GBM and a potential gene signature for selection of patients benefitting from TAK1 targeted therapy. The domain-focused epigenetic sgRNA library was used in lentiviral pooled format to transduce Cas9-BSD expressing glioma stem cell lines (G166 and U3013) at ∼500-fold representation of the library at an MOI of 0.3. Two days after transduction, 1–2 μg/ml puromycin was added for 5 days. A portion of cells was harvested as day 0 time point after selection was completed. The rest of the cells were then passaged to maintain 500-fold representation and cultured for an additional 35–38 days (eight to ten cell doublings). During the screening period antibiotic selection for Cas9 and sgRNA expression was maintained at 2.5 μg/ml blasticidine and 0.5 μg/ml puromycin, respectively. Each screen was performed in two biological replicates. Drop-out of sgRNAs over the culture period was determined by comparing abundance of sgRNAs between day 35 (G166) or day 38 (U3013) and day 0 by next-generation sequencing.