Abstract B025: The DNA methyltransferase gene MGMT is regulated by Nuclear Receptor 4A1 (NR4A1) and NR4A2 in glioblastoma cells and is a druggable target

Abstract Background: Approximately 13, 000 glioblastoma (GBM) patients are diagnosed each year in the United States and the 5-year survival rate is 12%. GBM patients are initially treated with surgery and adjuvant chemotherapy with the alkylating prodrug Temozolomide (TMZ). Responsiveness to TMZ is variable and dependent on molecular characteristics of the tumor with GBM patients expressing mutant O6-methylguanine-DNA methyltransferase (MGMT) having a more favorable status. Unfortunately, many patients do not respond well to TMZ and development of drug-resistance is common. Several studies have identified genes associated with resistance of GBM to TMZ, the primary of these being MGMT. A previous study indicated Specificity Protein 1 (Sp1) as being a transcriptional regulator of MGMT and given the established relationship between the NR4A and Sp gene families in the regulation of other genes, for example transferrin receptor (CD71), we hypothesized that there might be some involvement from the NR4As in the regulation of MGMT activity and could serve as a druggable target through the use of our novel bis-indole derived dual NR4A1/2 inverse agonists. Methods: The NR4A ligands used in this study include a series of 1, 1-bis (3′-indolyl) -1- (3, 5-disubstitutedphenyl) methane analogs (DIM-3, 5) that are potent anticancer agents. Both human- (U87-MG, A172) and mouse-derived (CT-2A, Akt/Ras) glioblastoma cell lines were used. Cell proliferation was determined by Resazurin assay and knockdown of NR4A1, NR4A2 and Sp1 was carried out using small inhibitory RNAs (siRNAs) targeted to each receptor. mRNA inhibition was quantified using quantitative real-time polymerase chain reaction (QRT-PCR). Western blot analysis was used to evaluated the protein expression of MGMT and related gene products. Luciferase and chromatin immunoprecipitation (ChIP) assays were used to evaluate the effects of the DIM treatments on the promoter region of the MGMT gene. Results: Each of the DIM-3, 5 analogs in dose response from 2. 5-12. 5 µM exhibited potent cytotoxic effects with an IC50 of 10 µM respectively in all tested cell lines. Western blot analysis revealed a significant reduction in the protein level of MGMT when treated with either the DIM-3, 5 analogs or the siRNAs. Furthermore, QRT-PCR revealed a significant decrease (70%) in the expression of MGMT at the mRNA level after 4 and 8 hours of treatment with each of the respective DIM-3, 5 analogs, or the siRNA treatments. Luciferase assays showed a 85% decrease in the p-MGMT reporter after treatment with any of the DIM-3, 5 analogs, and the same was seen in the siRNA treated cells as well. The ChIP results are ongoing but are promising and further functional assay testing is ongoing. Conclusions: NR4A1/2 play a role alongside Sp1 in the transcriptional regulation of the MGMT gene and are druggable targets of the DIM-3, 5 analogs in glioblastoma cell, presenting a novel therapy for the treatment of resistant phenotypes of glioblastoma and the potential for a synergistic relationship between the DIM-3, 5 analogs and TMZ. Citation Format: Evan Farkas, Srijana Upadhyay, Stephen Safe. The DNA methyltransferase gene MGMT is regulated by Nuclear Receptor 4A1 (NR4A1) and NR4A2 in glioblastoma cells and is a druggable target abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (6Suppl): Abstract nr B025.