DDDR-32. DNMTs epigenetically regulate MGMT expression and contribute to TMZ resistance in glioblastoma

Abstract Glioblastomas (GBM) are aggressive brain tumors characterized by significant heterogeneity and notable resistance to treatment, particularly to temozolomide (TMZ), a standard chemotherapeutic agent utilized in clinical practice. A key mechanism underlying this resistance is the upregulation of the MGMT (O6-methylguanine-DNA methyltransferase) gene. Prior studies have shown that alterations in MGMT promoter methylation status influence MGMT expression levels. However, the regulatory mechanisms governing this process remain unclear. To identify the molecular changes associated with TMZ resistance, we established eight TMZ-resistant cell lines derived from patient-derived glioma stem cells (PDGs). Quantitative PCR (qPCR) and Western blot (WB) analyses confirmed elevated MGMT expression in these resistant cells. RNA sequencing of both treatment-naïve and resistant cells revealed significant alterations in key epigenetic enzymes, including DNA methyltransferases (DNMTs) DNMT1 and DNMT3A, as well as various DNA demethylation enzymes and methyl-binding domains, such as TETs and MBDs. qPCR further validated the downregulation of UHRF1 and DNMT1 mRNA in resistant cells. Besides, treatment-naïve cells exhibiting high methyltransferase expression demonstrated increased susceptibility to 5-Azacytidine (5-AZA), a pan DNMT inhibitor, compared to their resistant counterparts, which displayed reduced methylation capacity. Moreover, 5-AZA-mediated epigenetic reprogramming enhanced the resistance of treatment-naïve cells by inhibiting cell proliferation and upregulating MGMT expression. Notably, withdrawal of 5-AZA reverted MGMT expression levels to those observed in untreated cells. Retrospective analysis of sequencing data from paired initial and recurrent tumors in adult diffuse glioma patients revealed a consistent negative correlation between DNMTs expression and MGMT expression, with DNMT1 emerging as the most significant factor. Finally, specific inhibition of DNMT1 resulted in a marked upregulation of MGMT expression. In summary, our work highlights the role of MGMT in glioma TMZ resistance and identifies the downregulation of DNMTs as a critical mechanism facilitating MGMT upregulation, thereby contributing to chemotherapy resistance.