Abstract A38: Genome-wide CRISPR-Cas9 screens reveal modulators of temozolomide sensitivity in glioblastoma

Abstract

Abstract Glioblastoma (GBM) is a prevalent and highly lethal form of primary brain tumour. Presently, median survival time for GBM patients is only 15 months and thus improved treatment methods are in great need. Treatment of GBM consists of surgery, radiotherapy and the chemotherapeutic agent temozolomide (TMZ). Unfortunately, many GBMs are refractory to TMZ and most others ultimately develop resistance leading to localized disease recurrence and brain tumor invasion. With the aim of identifying new therapeutic targets for GBM we performed genome-wide CRISPR-Cas9 screens to identify genes that modulate TMZ sensitivity in GBM. The TKO library of single-guide RNAs (gRNAs) targeting over 89,000 exons in 17,232 human genes was used to screen a panel of 5 patient-derived human GBM stem cell (GSC) lines. GSCs treated with DMSO were grown in parallel with GSCs treated with either a lethal, or sub-lethal dose of TMZ. Next-generation sequencing was used to identify gRNAs that were increased/decreased in abundance in TMZ treated pools of cells. Positive selection screening revealed that loss of key components of the mismatch repair pathway–MLH1, MSH2, MSH6 and PMS2 confers resistance to lethal doses of TMZ, as has previously been observed in GBM patients. Negative selection screening using sub-lethal doses of TMZ showed that loss of a conserved set of 15 genes that conferred TMZ hypersensitivity in four MGMT-negative GSC lines while the lone MGMT-positive GSC line in our set had a completely different set of hypersensitivity genes. Functional annotation revealed enrichment for genes involved in multiple DNA repair pathways, most prominently Fanconi Anemia and interstrand cross-link repair. Further studies validated that deletion of either member of the MCM8/9 helicase complex sensitizes GBM cells to TMZ and characterized the previously undescribed gene ZC3H7A which was revealed to be a cytoplasmic, stress-granule associated protein. In addition, our results revealed that in drug resistant MGMT expressing GSCs, TMZ resistance can be restored via the PARP inhibitor ABT-888. In conclusion, our results have identified a core set of genes that can be targeted to increase sensitivity to the chemotherapeutic agent TMZ responsive GBMs and elucidated opportunities for restoring TMZ sensitivity in resistant GBMs. These genes, many of which are targetable enzymes represent promising therapeutic targets for increasing efficacy of chemotherapy and decreasing lethality in GBM. Citation Format: Graham MacLeod, Nishani Rajakulendran, Traver Hart, Helen Yu, Peter B. Dirks, Stephane Angers. Genome-wide CRISPR-Cas9 screens reveal modulators of temozolomide sensitivity in glioblastoma [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A38.