Postdoctoral Scientist University of Oxford, United Kingdom
Introduction: Multiple myeloma (MM) is the second most common hematologic malignancy and remains an incurable disease. There is a significant unmet need to identify and characterise biology associated with high-risk and therapy resistance. The Polycomb repressive complex 2 (PRC2) is a chromatin regulatory complex which acts as an epigenetic mediator to control gene transcription by its enzymatic activity to catalyse tri-methylation of histone 3 lysine 27 residues (H3K27me3). The expression of PRC2 members EZH2 and cofactors PHF19 have been associated with inferior overall survival in MM patients. Following this observation, using data from over 1500 MM patients, we investigated the genomic and transcriptomic landscape of the PRC2 complex in high-risk MM patients with a view to identify novel therapeutic targets. We utilized the EZH2 inhibitor Tazemetostat (Taz) to identify MM cell lines sensitive and resistant to PRC2 inhibition. Furthermore, we map key transcriptomic differences in the sensitive lines. Through both chemical and genetic disruption, we investigate phenotypic differences in PRC2 activity in MM cell lines with different genetic backgrounds.
Methods: The Myeloma Genome Project data was used to map the expression of PRC2 genes in MM patients. A panel of MM cell lines were treated with Taz to determine cells with sensitivity to inhibition of EZH2 methyltransferase. For subsequent experiments, cells were treated with either 1µM Taz or DMSO control for 5 days. RNAseq was performed to identify a gene signature associated with sensitivity to EZH2 inhibition and significant pathways associated with EZH2 inhibition in sensitive and resistant cell lines were identified. Using the MMRF patient dataset, a gene set variance analysis was used to assign a PRC2 associated activity score for each patient. ChIP-seq was performed and histone marks (H3K27ac, H3K27me3 and H3K4me3) were analyzed in differentially express genes. Gene expression in interferon response genes across a panel of MM lines was validated by qPCR. The expression of PRC2 components were modulated in MM cell lines by CRISPR-Cas9. Taz sensitivity and phenotypic differences in apoptosis and cell growth were measured.
Results: Patients with a PRC2 dependant gene signature were identified by mapping genomic and transcriptomic activity of PRC2 and its interactions across MM patients. In addition, MM cell lines showed varying levels of sensitivity to Taz treatment. RNAseq experiments revealed several pathways significantly altered upon EZH2 inhibition in sensitive cell lines including upregulation of IFN response genes and MYC downregulation. Furthermore, upregulation of H3K27ac and H3K4me3, active histone marks, were identified in promoters of IFN response genes in Taz sensitive cells. These significant transcriptional changes of IFN expression were confirmed across a panel of MM cell lines.
Conclusions: PRC2 plays a role in IFN-related gene expression and may constitute a therapeutic mechanism in high-risk MM patients.
