Abstract 1328: Using whole-genome CRISPR-Cas9 screening to identify resistance networks in glioblastoma

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Abstract Glioblastoma (GBM) is the most aggressive and common type of adult malignant brain tumor, with 12,000 new diagnoses each year. Even with the current standard of care—surgical resection, radiation, and temozolomide (TMZ)-based chemotherapy—the median survival is about 20 months. This is partly due to the high rate of resistance to conventional therapy, including TMZ, leading to recurrence rates close to 100%. It remains largely unknown what drives the development of this resistance. Many studies have shown differences between primary and recurrent tumors, but a deeper understanding of resistance mechanisms is needed. CRISPR-Cas9 screening is a powerful tool for systematic and unbiased genetic analysis, which we applied to understand TMZ resistance. We performed a genome-wide CRISPR knockout screen in H4 human GBM cells, encompassing over 17,000 genes. A DMSO-treated population was compared with a TMZ-treated population over 14 days. In this drug sensitivity screen, depletion of guides corresponds to a TMZ-resistance gene, whereas enrichment of guides corresponds to a TMZ-sensitivity gene. Analysis showed that there was significant enrichment in guides for known TMZ-sensitivity genes that have been highly cited—ATG14, MSH6, MLH1, and PMS2—thus validating our screen results. However, more importantly, we were able to identify a list of 200 novel genes implicated in TMZ resistance. Pathway analysis revealed that these genes were enriched in Hippo and Notch signaling, both known to play a role in chemoresistance. From this list of novel genes, we identified 4 previously unstudied genes. These genes showed significant elevations in RNA expression (p&lt;0.05) in recurrent tumors when compared to primary tumors in patient datasets, along with significant survival benefits corresponding to low gene expression (p&lt;0.05). To validate the identified genes, we assessed RNA expression in multiple patient-derived xenograft (PDX) lines and found that multiple exposures of TMZ were required to generate a resistant phenotype with gene expression elevation. We validated this at the protein level and showed that multiple exposures of TMZ resulted in target expression elevations compared to the control, thus confirming that the effect of TMZ-resistance gene upregulation is only noted when cells are forced into resistance. Further validation experiments revealed that knocking out these genes in vitro resulted in increased TMZ sensitivity. In summary, a whole-genome CRISPR-Cas9 knockout screen was performed to identify a novel set of genes that contribute to therapeutic resistance in GBM, as validated by in vitro experiments performed on a set of these genes. We have also identified a specific network of enriched pathways that represent novel genetic vulnerabilities. Ultimately, we believe this work will provide critical insight into mechanisms of resistance in GBM—a disease desperately in need of new therapeutic approaches. Citation Format: Shreya Budhiraja, Shivani Baisiwala, Ella Perrault, Li Chen, Cheol Park, Chidiebere Awah, Crismita Dmello, Andrew Zolp, Adam Sonabend, Atique Ahmed. Using whole-genome CRISPR-Cas9 screening to identify resistance networks in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1328.