Exth-78. crisproff epigenome editing and functional genomics define mediators of chemotherapy resistance in glioblastoma

Abstract Alkylating chemotherapies exhibit survival benefit for patients with glioblastoma (GBM). CRISPRoff is a programmable epigenetic memory writer that stably and heritably silences genes through DNA methylation. Epigenetic silencing of MGMT via promoter methylation predicts response to chemotherapy and is prognostic for 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 chemotherapy. We then used genome-wide CRISPR interference (CRISPRi) screens to broadly define novel chemosensitizing targets of GBM cells. Epigenome editing was performed through electroporation of CRISPRoff mRNA and sgRNAs into MGMT hypomethylated GBM cell lines (LN18 and T98G) and primary GBM cells. Whole genome bisulfite sequencing, RT-qPCR, and western blot assessed gene silencing. Cell viability assays measured drug sensitivity. GBM xenografts with CRISPRoff-induced silencing of MGMT were intracranially transplanted in mice and treated with temozolomide. Genome-wide functional genomics screens were performed using CRISPR interference. CRISPRoff against MGMT reduced MGMT mRNA and protein levels by 99.97%, generating up to 150-fold sensitization to temozolomide and 9-fold sensitization to lomustine in GBM cells. Silencing remained stable and heritable in clonally isolated GBM cells at 145 days after transient delivery of CRISPRoff, and gene repression and drug sensitization were equivalent to Cas9-mediated homozygous deletion of MGMT in vitro and in vivo. CRISPRoff of MGMT in intracranial xenografts exhibited significant reduction in tumor growth with temozolomide treatment compared to temozolomide treatment with control CRISPRoff (p = 0.0047). CRISPRi screens validated MGMT and defined 266 and 238 additional modifiers of temozolomide and lomustine response, respectively, including the ATR (BRCA2), DNA repair (REV1), cell cycle (PSMD13), and Fanconi anemia pathways (FANCI, FANCD2). In summary, we integrate targeted epigenome editing with unbiased genome-wide approaches to establish a novel discovery and therapeutic platform against glioblastoma.