Abstract
Emergence of temozolomide (TMZ) resistance and increased tumor aggressiveness are common in recurrent glioblastoma. However, mechanisms promoting these phenomena are incompletely understood. Previously, we described in vivo models of acquired TMZ resistance in GBM patient derived xenografts (PDXs), including MGMT expressing TMZ resistant subline (GBM12-3080) and TMZ naïve subline (GBM12-5199). To identify mechanisms associated with MGMT upregulation, the epigenetic differences between these lines were analyzed by ChIP-seq. Compared to GBM12-5199, GBM12-3080 displays H3K4me3 within the MGMT promoter region and H3K36me3 within the MGMT gene body region, indicative of an active epigenetic landscape. In addition, a genomic region localized between the promoters of MKI67, which encodes Ki-67 protein, and MGMT acquired active enhancer marks (H3K4me1 and H3K27ac) in GBM12-3080. Similar histone marks increase within this putative enhancer region were identified in paired samples from TMZ naïve and recurrent tumors in three out of eight PDX models and one out of three recurrent patient tumors. Using H3K27ac occupancy as a guide, tiled fragments of the putative enhancer were cloned into a luciferase reporter system, which ultimately identified a 1.5 kb region with robust enhancer activity. In parallel, a chromatin conformation capture assay demonstrated physical interaction between this 1.5 kb enhancer region and the MGMT promoter, which was only detectable in GBM12-3080. Finally, deletion of this enhancer by CRISPR/Cas9 in both GBM12-3080 and MGMT expressing glioblastoma cell line (SKMG3) reduced MGMT expression by greater than 90% and reduced the TMZ IC(50) by 71% and 97%, respectively. Consistently, the expression of the adjacent gene, MKI67, was reduced by approximately 40% in both lines, and tumorigenicity of GBM12-3080 deletion clones was markedly suppressed. In conclusion, K-M enhancer regulates MGMT and Ki-67 expression independent of DNA methylation. Inhibition of this enhancer may sensitize tumors to TMZ and reduce tumor growth in a sub-population of recurrent glioblastoma.