DDDR-51. NR4A1/2 dual targeting affects Epithelial to Mesenchymal transition and potentiates Temozolomide response in Glioblastoma Multiforme

Abstract Glioblastoma multiforme (GBM) is an aggressive brain tumor with a median survival of 15–18 months, driven by treatment resistance and intratumoral heterogeneity. A key driver of these challenges is epithelial-to-mesenchymal transition (EMT), which enhances glioma stemness, invasiveness, and DNA repair mechanisms. Although EMT regulation can be difficult, indirect targeting of key transcription factors driving EMT such as Sp1 and TWIST1 offers a novel strategy. We evaluated bis-indole-derived compounds (CDIMs), inverse agonists of NR4A1 and NR4A2, which have shown efficacy in downregulating TWIST1 and enhancing temozolomide (TMZ) sensitivity. Four patient-derived GBM cell lines were tested individually with TMZ and three CDIM compounds (DIM-3-Cl-5-CF3, DIM-3,5-Cl2, and DIM-3,5-Br2) as well as in combination. CDIMs significantly enhanced TMZ efficacy and exhibit strong synergistic effects (Bliss Synergy Score > 1) across all cell lines ranging from 2.7 to 15.5. DIM-3,5-Cl2 emerged as the lead compound, causing an increased TMZ-induced cytotoxicity in GBM4V cell line by an additional 30.9% (p=0.01), 36.8% (p=0.007), and 30.9% (p=0.03) when combined with 125 µM, 250 µM, and 500 µM of TMZ, respectively. DIM-3,5-Cl2 (12.5 µM) also reduced U87 cell migration by over 50% compared to controls (p<0.05). Additionally, western blot analysis showed that combinatorial treatment of DIM-3,5-Cl2 and TMZ more effectively suppressed TWIST1, Sp1, G9a, β1-integrin, and survivin compared to either compound across all cell lines. In vivo, intracranial xenograft models showed early signs of efficacy, with an average survival extension of 3.3 days in CDIM-treated animals and continued survival in TMZ and combination groups at the time of reporting. These findings support DIM-3,5-Cl2 as a promising therapeutic candidate that targets EMT-related resistance pathways, enhances TMZ cytotoxicity, and impairs GBM migration. Ongoing studies are assessing its effects on stemness and EMT gene expression, as well as its potential to improve TMZ efficacy in vivo.