In Vivo ORF Overexpression Screening Identifies CCN4 as a Regulator of Glioblastoma Growth Validated Across Multiple Models.

Despite current multimodal therapies for glioblastoma (GBM), its prognosis remains grim. Thus, a tremendous need exists to identify new genetic drivers that may serve as potential therapeutic targets in glioblastoma (GBM). We describe an in vivo overexpression screening strategy to identify drivers of glioblastoma where we have leveraged TCGA datasets to conduct a functional genomics screen of prioritized open reading frames (ORFs) that are overexpressed and/or amplified in GBM. To interrogate these potential drivers within a more relevant physiological context, the screening was accomplished in vivo in an orthotopic patient-derived glioma stem-like cell (GSC) model. Among 5 positive "hits" from the screen, Cellular Communication Network factor 4 (CCN4) was prioritized for further evaluation. Our functional analyses demonstrated that CCN4 overexpression drives tumor growth in multiple GBM models. Depletion of CCN4 reduced growth in vitro and in vivo and markedly decreased colony formation with the growth phenotype restored upon ectopic expression of CCN4. Structural functional analysis of CCN4 was also conducted. We believe that this screening strategy can serve as a platform for further identification and validation of drivers of GBM.