HiTIP-seq profiles epigenomic reprogramming of patient-derived diffuse midline glioma stem cells to epigenetic therapy

Abstract

Diffuse midline glioma (DMG), H3K27-altered, is lethal pediatric-type, high-grade, localized to the midline region of the central nervous system. Effective treatment guidelines are absent, and clinical trials are preferred for primary or recurrent DMG patients. Recently, epigenetic agent-based immunotherapy has exhibited promising therapeutic effects in the clinical setting. However, the underlying mechanisms remain a mystery. The rare DMG tumor samples from biopsy or resection largely impede basic research, by using patient-derived tumor cells which better recapitulate the parental tumor's heterogeneity compared to established cell lines. As an epigenetic reprogramming disease, DMG exhibits a global loss of H3K27 trimethylation (H3K27me3) and a gain of H3K27 acetylation (H3K27ac). Analysis of multiple epigenetic marks is fundamentally necessary. However, traditional techniques cannot allow ultra-low input and high-throughput. Herein we have developed a new method called high-throughput in situ tagged immunoprecipitation sequencing (HiTIP-seq), which uses an integrated superhydrophobic microwell array technology (InSMART). We were able to perform 100 parallel assays from as few as 100 cells per microwell on a single chip. We applied the technology to profile epigenetic alterations of three-dimensional (3D) cell cultures derived from DMG patients. Our HiTIP-seq integrated with RNA sequencing (RNA-seq) analysis revealed that the combination of epigenetic agents (panobinostat and tazemetostat), reprogrammed histone modifications and drove transcriptome changes. Among them, Wnt inhibitory factor 1 (WIF1) has a gain of H3K27ac and a loss of H3K27me3, which leads to the upregulated expression. Altogether, HiTIP-seq is a versatile method for high-throughput analysis of histone modifications, suitable for both DMG research and studying rare 3D models.