Abstract 197: Identifying a novel network of driver genes in glioblastoma

Abstract

Abstract The purpose of this study is to identify novel genes that drive proliferation in glioblastoma (GBM) and likely contribute to gliomagenesis, therefore facilitating the aggressive nature of the tumor. GBM is an aggressive primary malignancy of the brain with almost a 100% recurrence rate. It is the most common malignant brain tumor in adults, with an average survival of 21 months after diagnosis, with the current standard of care. As such, new advances in therapy are desperately needed. Large scale genomic approaches have shown that GBM has a complex genetic architecture, with a great deal of intratumoral heterogeneity. However, a more comprehensive understanding of determinants of growth is required to identify new targets and provide new therapeutic strategies. CRISPR-Cas9 screening technology has enabled whole-genome screens that allow for systematic and objective identification of genes governing cell viability and proliferation. These genes may represent unique vulnerabilities that can be targeted with novel therapeutics. Here, we performed a genome-wide CRISPR knockout screen in SNB19 human GBM cells, covering 17,000 genes with 4 guides per gene. In this type of screen, we anticipate that those guides that are depleted over time are the ones that correspond to genes driving growth and proliferation of GBM cells. Initial analysis showed that there was a reduction in guides for known driver genes, as we might expect. However, in addition to known drivers, we were able to identify a list of approximately 150 new genes that showed significant depletion in our screen (p<0.01), meaning that they likely contribute to the viability of GBM cells. From this list, we identified 5 previously unstudied genes, which show significant elevations in expression at the RNA and protein levels (p<0.05), and show significant survival benefit in patient datasets (p<0.05). For these genes, we first validated expression in multiple patient-derived xenograft (PDX) lines. We were able to confirm that compared to a neural stem cell (NSC) control, all genes showed a significant elevation in protein expression in the PDX lines. Furthermore, we were able to generate CRISPR-Cas9 knockouts in our NSC line and in each of our PDX lines. These knockouts showed a significant reduction in viability in PDX lines (p<0.01) while showing no significant effect on viability in the NSC line. In addition, intracranial implantation of the knockout PDX cells in mice shows an increase in survival compared to control. In addition to identifying genes that may represent therapeutic vulnerabilities, we have further been able to identify pathways that may be potential targets. Specifically, proteasomal degradation, mRNA transport, and ribosomal processes show significant enrichment in our set of genes (FDR < .25). In summary, we have used a whole-genome CRISPR-Cas9 knockout screen to identify a large novel set of genes that contributes to glioma viability and proliferation. Of these genes, we have been able to further validate a limited set to show that they do in fact contribute to the survival of cells in vitro and animal survival in vivo. Finally, we have been able to show enrichment of specific pathways across these gene sets, giving us a set of genes and pathways that represent novel genetic vulnerabilities. We believe this work will contribute significantly to developing new therapies for a disease that is desperately in need of additional therapeutic options. Citation Format: Shivani Baisiwala, Cheol Park, Chidibere Awah, Jack M. Shireman, Miranda R. Saathoff, Adam Sonabend, Atique U. Ahmed. Identifying a novel network of driver genes in glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 197.