Epigenetic remodeling of the immune contexture in primary and recurrent glioblastoma to enhance immunotherapy efficacy

Glioblastoma (GBM) is one of the most aggressive and lethal primary brain tumors in adults. It is characterized by rapid invasive growth and strong resistance to therapy. Despite the current standard of care with chemoradiotherapy, the median overall survival (OS) remains very low. Furthermore, due to its aggressive nature, approximately 70% of GBM patients experience disease progression within one year from of diagnosis, with a median OS less than 1 year. Although preclinical studies have shown promising results, recent data from phase III clinical trial revealed that immunotherapy with checkpoint antibodies has not yet provided significant clinical benefits in GBM. Consequently, there is an urgent need for new therapeutic strategies to improve patient outcomes and overcome resistance to immunotherapy. Emerging evidence suggests that epigenetic dysregulation plays a critical role in driving tumor progression, immune evasion, and therapy resistance. Among epigenetic mechanisms, DNA methylation, is an epigenetic escape mechanism, frequently exploited by tumors to create a strongly immunosuppressive environment and evade immune surveillance. In this context, drugs targeting epigenetic abnormalities, such as DNA hypomethylating agents (DHA), have demonstrated encouraging results in various cancer types. In this study, we aimed to characterize the epigenetic and transcriptional landscapes of paired primary and recurrent GBM cell lines, comparing them with melanoma brain metastases (MM-BM), and to investigate the effect of guadecitabine treatment on these cell lines. Methylome analysis with EPIC arrays revealed significant epigenetic divergence between primary and recurrent GBM cells, identifies 891 differentially methylated (DM) genes. Recurrent GBM lines showed increased methylation in genes involved in apoptosis inhibition and cell motility, consistent with a more aggressive and therapy-resistant phenotype. In contrast, primary GBM cells exhibited methylation changes linked to immune suppression and extracellular matrix remodeling. Comparison with MM-BM cell lines uncovered over 3,000 DM genes in each case, with MM-BM showing distinct methylation signatures related to neural differentiation and sensory perception—likely reflecting their non-glial origin and brain microenvironment adaptation. LINE-1 analysis confirmed higher global methylation level in recurrent GBM cell lines compared to primary ones. Treatment with guadecitabine effectively reduced global DNA methylation levels across all cell types, with primary GBM cells showing the greatest degree of demethylation, suggesting higher epigenetic plasticity. Transcriptomic analysis following DHA treatment revealed substantial transcriptional reprogramming, with over 1,000 differentially expressed genes per cell line. Functional enrichment analysis showed that guadecitabine activated immune-related pathways across all investigated groups. In primary GBM, it upregulated cytokine signaling and lymphocyte migration; in recurrent GBM, it promoted antigen presentation via major histocompatibility complex (MHC) class I and T cell activation. MM-BM cells exhibited enhanced innate and adaptive immune signaling, including MHC class II expression and NF-κB pathway activation, alongside reduced pro-apoptotic signaling. Methylome analysis confirmed widespread epigenetic remodeling after DHA treatment. Guadecitabine induced methylation changes associated with inflammatory responses, chemotaxis, and immune activation in both GBM subtypes. Functional assays showed that primary GBM cells had the highest baseline proliferation and migration activity, but guadecitabine significantly reduced proliferation and motility across all cell lines, with the strongest effects observed in primary GBM cells. Migration inhibition was detected as early as 24 hours post DHA treatment, indicating a robust anti-invasive effect. Overall, our findings demonstrate that guadecitabine exerts multifaceted anti-tumor effects in primary and recurrent GBM as well as MM-BM cell lines. Globally, DHA treatment is able to reactivate immune-related gene expression, suppresses key malignant features such as proliferation and invasion, and reshapes the epigenetic landscape in a tumor subtype-specific manner. These results in addition to previous findings, support the use of DHA into combination therapeutic strategies for GBM, particularly in the recurrent setting where resistance to treatment remains a major clinical challenge. Future in vivo and translational studies will be essential to validate these findings and explore how epigenetic therapy can enhance immunogenicity and therapeutic responsiveness in GBM.