Introduction: Glioblastoma, despite harboring frequent tyrosine kinase receptor amplification, is notoriously resistant to targeted therapy. Quiescent cancer cells within a tumor are thought to be responsible, but the transition from actively cycling to quiescent requires significant changes in gene expression. Chromatin organization dictates gene expression by regulating regions of the genome that are accessible. Cancer cells have deregulated chromatin organization, making them more plastic and able to transition between cycling and quiescent states. A better understanding of the chromatin organization of glioblastomas and the gene expression changes that occur during resistance could shed light on potential susceptibilities of resistant cells.
Methods: We generated and characterized a model of glioblastoma resistance by treating a PDGFRA amplified glioblastoma stem cell line (GSC8) with the tyrosine kinase inhibitor dasatinib (Fig. 1A) and doing RNA-seq and ChIP-seq (H3K4me3, H3K9me3, H3K27me3, H3K27ac). We identified and tested two small molecule inhibitors that preferentially target persister cells.
Results: Treatment of GSC8 with dasatinib resulted in 90% death, but 10% reproducible survivors with 4 day (GSCST) or 1 month (GSCper) treatment (Fig. 1B). The cycling rate of persisters was very low (Fig. 1C), suggesting a quiescent phenotype. RNA-seq identified upregulation of several chromatin modifying enzymes including the H3K27me3 demethylase KDM6A. ChIP-seq for H3K27me3 showed widespread decreases in this repressive chromatin mark (Fig. 1D). Moreover, the KDM6A inhibitor GSKJ4 showed increased killing of persister GBM cells compared to untreated (Fig 1E). Pathway analysis of persister gene expression profiles identified a Notch signature. Treatment of GSCper with a gamma-secretase inhibitor also resulted in preferential killing of GSCper relative to GSC (Fig. 1F).
Conclusions: Glioblastoma resistance to targeted therapy is mediated by chromatin reorganization and resultant gene expression changes favoring a quiescent phenotype. This phenotype is targetable using small molecule inhibitors of chromatin modifying enzymes such as KDM6A or quiescent signaling pathways such as Notch.
Patient Care: By identifying pathways and potential susceptibilities of resistant glioblastoma cells, we hope to guide future clinical trials for combination therapies.
Learning Objectives: By the conclusion of this session, participants should be able to: 1.) Describe the importance of quiescence in drug resistance in glioblastoma 2.) Understand how chromatin modifying enzymes allow cells to switch from cycling to quiescent 3.)Identify possible novel treatments of resistant glioblastoma