Investigating Epigenetic Regulation of GD2 Expression in Glioblastoma

Jhalak Trivedi

Mentors: Dr. Robert Suter and Dr. Nagi Ayad, Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center.

Date/Time: August 23rd, 2024 at 3:20pm.

Abstract: Glioblastoma (GBM) is an aggressive and lethal brain cancer, characterized by rapid growth, invasive behaviour, and resistance to standard therapies, leading to a dismal prognosis with median survival times often less than 15 months (Penn Medicine). Recent advances in Chimeric Antigen Receptor T-cell (CAR-T) therapy have emerged as a promising approach, particularly targeting GD2, a glycosphingolipid expressed on GBM cells (Land et al.) However, the clinical success of GD2-targeting CAR-T therapy in GBM has been limited due to an incomplete understanding of the molecular and epigenetic mechanisms that regulate GD2 expression. The Ayad laboratory has identified Polycomb Group Factor 1 (PCGF1) as a major regulator of GD2 expression in GBM cells (unpublished findings). This project focuses on unravelling the role of PCGF1, a component of the Polycomb Repressive Complex 1 (PRC1), in modulating GD2 expression. We hypothesize that the knockdown of PCGF1, a component of a non-canonical PRC1 complex, will alter global histone methylation and ubiquitination in tumour cells, altering the expression of genes regulating tumor cell GD2 levels.

To test this hypothesis, we employed a multi-tiered approach integrating findings from RNA and Cut&Run sequencing of wild-type and PCGF1 knockdown LN229 GBM cells to understand better the transcriptional and epigenetic regulation of GD2 by PCGF1. RNA sequencing data was aligned, and counts were generated with STAR. Differential expression was assessed using DESeq2 using MSigDB.. In addition to changes in gene expression, Cut&Run was performed for IGG, H3K4me3, H3K27me3, and H2AK119ub1 to evaluate changes in PRC1-related histone modifications. Cut&Run data alignment was performed using bowtie2, peak-calling via Macs2 and SEACR with bedtools, and motif analysis via MEME. Enrichment analysis for identified consensus peaks was performed using the R package Chipseeker and msigdb.

The analysis revealed distinct changes in gene expression and histone modifications at genes involved in various significant pathways including glycosphingolipids catabolism and metabolism pathway, suggesting a critical role for PCGF1 in the transcriptional regulation of key pathways involved in GD2 expression and uptake. These observations hint at a potential role for PCGF1 in modulating GD2 uptake by cells, suggesting that further exploration of PCGF1’s impact on the epigenetic landscape of GBM could uncover new avenues for enhancing GD2-targeted therapies.