dCas9/CRISPR-based methylation of O-6-methylguanine-DNA methyltransferase enhances chemosensitivity to temozolomide in malignant glioma.
dCas9/CRISPR-based methylation of O-6-methylguanine-DNA methyltransferase enhances chemosensitivity to temozolomide in malignant glioma.
复制标题
基于dCas9/ crispr的o -6-甲基鸟嘌呤- dna甲基转移酶甲基化增强了恶性胶质瘤对替莫唑胺的化学敏感性。
DOI：
10.1007/s11060-023-04531-z
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发表时间：
2024-01
影响因子：
3.9
通讯作者：
Lai, Albert
中科院分区：
文献类型：
--
作者：
Rinonos, Serendipity Zapanta;Li, Tie;Pianka, Sean Thomas;Prins, Terry J.;Eldred, Blaine S. C.;Kevan, Bryan M.;Liau, Linda M.;Nghiemphu, Phioanh Leia;Cloughesy, Timothy F.;Lai, Albert
关键词：
Malignant glioma carries a poor prognosis despite current therapeutic modalities. Standard of care therapy consists of surgical resection, fractionated radiotherapy concurrently administered with temozolomide (TMZ), a DNA-alkylating chemotherapeutic agent, followed by adjuvant TMZ. O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, removes alkylated lesions from tumor DNA, thereby promoting chemoresistance. MGMT promoter methylation status predicts responsiveness to TMZ; patients harboring unmethylated MGMT (~60% of glioblastoma) have a poorer prognosis with limited treatment benefits from TMZ. Via lentiviral-mediated delivery into LN18 glioma cells, we employed deactivated Cas9-CRISPR technology to target the MGMT promoter and enhancer regions for methylation, as mediated by the catalytic domain of the methylation enzyme DNMT3A. Methylation patterns were examined at a clonal level in regions containing Differentially Methylation Regions (DMR1, DMR2) and the Methylation Specific PCR (MSP) region used for clinical assessment of MGMT methylation status. Correlative studies of genomic and transcriptomic effects of dCas9/CRISPR-based methylation were performed via Illumina 850K methylation array platform and bulk RNA-Seq analysis. We used the dCas9/DNMT3A catalytic domain to achieve targeted MGMT methylation at specific CpG clusters in the vicinity of promoter, enhancer, DMRs and MSP regions. Consequently, we observed MGMT downregulation and enhanced glioma chemosensitivity in survival assays in vitro, with minimal off-target effects. dCas9/CRISPR is a viable method of epigenetic editing, using the DNMT3A catalytic domain. This study provides initial proof-of-principle for CRISPR technology applications in malignant glioma, laying groundwork for subsequent translational studies, with implications for future epigenetic editing-based clinical applications. The online version contains supplementary material available at 10.1007/s11060-023-04531-z.
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