Abstract A047: Bis-indole derived dual NR4A1/2 inverse agonists synergistically enhance temozolomide-induced cytotoxicity in glioblastoma cells through downregulation of drug-resistance genes

Abstract Backgroud and Purpose: Glioblastoma (GBM) is the most common form of adult brain cancer and the standard of care, temozolomide (TMZ) and radiation therapy, is subject to drug resistance. The orphan nuclear receptors 4A1 (NR4A1) and NR4A2 are highly expressed in GBM patients and their growth is inhibited by a series of bis-indole derived compounds (CDIMs) that bind both receptors and act as inverse agonists. It has been reported that CDIMs decrease expression of several NR4A-regulated genes such as G9a, several integrins and survivin in cancer cells, which have been shown to play a role in TMZ resistance in GBM. The objective of our study is to determine whether CDIMs synergistically enhance the cytotoxic effects of TMZ treatment through the downregulation of NR4A-regulated TMZ-resistance genes. Methods: The CellTiter-Blue assay kit from Promega was used as a measure for metabolic capacity and cell-viability. Small interfering RNAs (siRNAs) were used to transiently silence the expression of NR4A1/2. The Bliss-Independence model and SynergyFinder tool were used to quantify synergism in select combinations of CDIMs and TMZ. Western blot analysis was used to determine treatment-related effects on the NR4A-regulated TMZ-resistance genes. Results: Initial testing showed that several human-derived GBM cell-lines exhibited varied responsiveness to treatment with up to 1000 µM TMZ. The knockdown (KD) of NR4A1/2 by RNA interference (siRNAs) potentiates the cytotoxicity of TMZ in U87-MG GBM cells, which were the most resistant of the GBM cell lines tested. The increase in TMZ-induced cytotoxicity in N4RA1/2 deficient GBM cells was as high as 5-fold in combination. An array of combination doses of TMZ plus several CDIM analogs identified several treatments that exhibited a synergistic growth inhibitory effect in the GBM cell-lines, with some exhibiting inhibitory effects 25% greater or more than expected. When lysates of cells treated with these synergistic combinations were analyzed using western blot analysis and compared to controls, TMZ treatment alone, and CDIM treatment alone, there was a decreased expression of the TMZ-resistance genes: G9a, β1-integrin and surviving after cotreatment with CDIMs plus TMZ. Furthermore, NR4A1/2 KD treated cells showed decreased expression in the TMZ-resistance genes when compared to control and TMZ treatment alone. Conclusions: The CDIM-3,5 dual NR4A1/2 inverse agonists represent a novel mechanism-based approach for treating GBM by targeting the NR4A-mediated pro-oncogenic pathways. The CDIMs decrease expression of NR4A-mediated drug resistance genes and synergistically enhance the cytotoxicity of TMZ treatment. The battery of cell-lines are being used to investigate the synergistic responses and mechanisms. This will include studies on the effects of individual knockdown and pharmacologic inhibition of the NR4A-regulated resistance genes to determine their relative contributions to enhancing the potential therapeutic efficacy of TMZ and our ongoing in vivo study comparing the different treatments effects on survival. Citation Format: Evan Farkas, Ana Gutierrez, Andrei Mikheev, Robert Rostomily, Stephen Safe. Bis-indole derived dual NR4A1/2 inverse agonists synergistically enhance temozolomide-induced cytotoxicity in glioblastoma cells through downregulation of drug-resistance genes [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A047.