CRISPR-Based Epigenome Editing and Genome Wide Screening Define Mediators of Chemotherapy Response in Glioblastoma

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Alkylating chemotherapies exhibit survival benefit for patients with glioblastoma (GBM), the most common malignant primary brain tumor. CRISPRoff is a programmable epigenetic memory writer that stably and heritably silences any gene through DNA methylation. Epigenetic silencing of MGMT via promoter methylation is predictive of response to alkylating agents as well as prognostic for progression free and overall survival. Here we performed epigenome editing using CRISPRoff to stably silence MGMT through induced promoter methylation, as a therapeutically tractable approach for potentiating GBM to temozolomide (TMZ) or lomustine (CCNU). We then used genome-wide engineered CRISPR/Cas9 systems to broadly define sensitizers of GBM cells to alkylating agents, as a platform to discover novel sensitizing targets. Targeted epigenome editing was performed through electroporation of modified mRNAs encoding CRISPRoff machinery comprising deactivated Cas9 fused to a DNA methyltransferase complex, combined with sgRNAs, into MGMT unmethylated GBM cell lines (LN18, T98G) and then treated with either vehicle, TMZ, or CCNU. Parallel experiments were performed through electroporation of sgRNA/Cas9 ribonucleoproteins. Gene silencing was assessed using bisulfite targeted sequencing, RT-qPCR, and western blot. Drug sensitization was determined using luminescent cell viability assays. Genome-wide CRISPR interference (CRISPRi) screens were performed in triplicate cultures. Epigenomic silencing of the MGMT promoter through CRISPRoff-induced methylation reduced MGMT transcript levels by 96.7% and generated up to 88-fold sensitization to TMZ mediated cell death in GBM cells, with IC50 superior to GBM cells with baseline methylated MGMT. In addition, CRISPRoff of MGMT induced 20-fold sensitization to CCNU. CRISPRoff methylation of MGMT was equivalent to CRISPR/Cas9 homozygous deletion of the MGMT gene for drug sensitization and was superior to polyclonal Cas9 mediated deletion of MGMT by a factor of 10. To define additional mediators of chemotherapy response in GBM, CRISPRi screens revealed 185 and 266 genetic sensitizers to TMZ treatment in LN18 and T98G cells, respectively, in addition to validating MGMT. One hundred eighty and 238 sensitizers to CCNU were identified in LN18 and T98G cells, respectively. TMZ sensitizing genes conserved across cell lines were enriched for the ATR (i.e., BCRA2), DNA repair (i.e., REV1), and cell cycle pathways (i.e., PSMD13), while CCNU sensitizing genes were enriched for the Fanconi anemia pathway (i.e., FANCI, FANCD2). In contrast, gene hits that resulted in resistance to alkylating agents were enriched for the mismatch repair pathway (i.e., MSH2, PMS2). We integrate targeted epigenome editing with unbiased genome-wide approaches to build a novel discovery and therapeutic platform in glioblastoma, a framework that is well suited for targeting diseases with known or suspected epigenetic vulnerabilities.