Abstract 1378: Identification of genomic regions associated with temozolomide resistance in glioblastoma through analysis of histone marks on chromatin

Abstract

Glioblastoma (GBM) is an aggressive and fatal primary brain tumor. Temozolomide (TMZ) is a critical component of the standard care of newly diagnosed GBM patients, but unfortunately preexisting primary resistance and rapid emergence of secondary resistance invariably limits the therapeutic benefit of TMZ in GBM. Prior studies have identified genetic and epigenetic alterations that can modulate TMZ sensitivity and treatment outcome. However, comprehensive analysis of histone marks and knowledge of epigenetic regulation of genes associated with TMZ sensitivity or resistance is lacking. To identify epigenetic states associated with TMZ resistance, we performed an epigenetic profiling of eight different histone marks in GBM xenografts. Using chromatin immunoprecipitation combined with high throughput sequencing (ChIP-seq), distribution of H3K4me1, H3K4me3, H3K9ac, H3K9me2, H3K9me3, H3K27ac, H3K27me3 and H3K36me3 histone marks was compared in a panel of GBM patient-derived xenograft sub-lines derived by treating TMZ sensitive GBM12 tumors with placebo (n=2 sublines) or temozolomide (n=6 sub-lines) and then propagating resulting recurrent tumors. Our analysis revealed that H3K4me1 and H3K27ac modification patterns varied globally across individual sub-lines, while distribution of H3K4me3, H3K36me3 histone marks was specifically altered in discrete genomic regions in resistant sub-lines depending on the mechanisms of resistance. To find how epigenetic modifications affect TMZ sensitivity, we analyzed ChIP-Seq data using Hidden Markov Model to test if one or a combination of histone marks relates to TMZ resistance. The effect of histone modifications on transcription was simultaneously determined by RNA sequencing. These analyses helped identify specific histone modifications which could be functionally related to TMZ resistance. Through these analyses we have identified 1142 genomic regions governed by a specific epigenetic pattern. We subsequently analyzed a candidate genomic region on top of the list. By using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated nucleases 9 system (CRISPR/Cas9 system), we have generated clones from an intrinsically TMZ resistant SKMG3 cell line with deletion up to four-kilo base genomic region. Clonogenic growth assays showed that deletion of this genomic region enhanced TMZ sensitivity, reducing the IC50 from 186 μM to less than 60μM, p<0.05. This finding indicates that this genomic region is functionally related with TMZ sensitivity. Taken together, our study reveals epigenetic modifications related to TMZ resistance in GBM cells and a specific genomic region involved in regulating TMZ sensitivity. The analysis of epigenetic state at this genomic region could potentially be useful in predicting treatment response and may help in designing TMZ sensitizing therapy in GBM. Citation Format: Xiaoyue Chen, Haiyun Gan, Jeong Heong Lee, Dong Fang, Gaspar Kitange, Jann Sarkaria, Zhiguo Zhang. Identification of genomic regions associated with temozolomide resistance in glioblastoma through analysis of histone marks on chromatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1378. doi:10.1158/1538-7445.AM2017-1378