2285 - N-Terminal Androgen Receptor Antagonists Offer Superior Tumor Suppression on Glioblastoma than C-Terminal Inhibitors

Purpose/Objective(s):

Emerging evidence, including our own, implicates androgen receptor (AR) signaling in promoting glioblastoma (GBM) cell survival, stemness, and therapy resistance. While our preliminary data demonstrated good tolerance of the C-terminal AR antagonist (C-ARA) bicalutamide when added to chemoradiotherapy in GBM patients, and animal studies showed excellent tumor control with bicalutamide combined with brain radiotherapy (RT) as a radiosensitizer, bicalutamide (BIC) or enzalutamide (ENZA) in the C-ARA category, when used alone, has resulted in notable failures in mice with orthotopically implanted GBM tumors. This suggests intrinsic resistance of GBM to these drugs as monotherapy. Previous studies indicate that GBM cells express full-length AR (FL-AR) and truncated AR variants (AR-Vs) lacking the ligand-binding domain (LBD), which collectively contribute to genomic and non-genomic AR signaling and may drive therapeutic resistance. Building on our findings with C-ARAs, which showed therapeutic potential against FL-AR, we further evaluated novel N-terminal AR antagonists (N-ARAs) to target truncated AR proteoforms and compared their effects to those of C-ARAs.

Materials/Methods:

GBM cell lines, including primary cell lines established from patients' GBM specimens, were cultured in vitro. Cell proliferation assays were performed after 48 hours of culture with or without drug treatment. ENZA, BIC, and apalutamide (APA) were tested in the C-ARA category. EPI-7386 (EPI) and cinobufagin (CINO) were tested in the N-ARA category. RNA-seq analyses were conducted on total mRNA extracted from U87MG cells cultured with medium only (control), BIC (C-ARA), or CINO (N-ARA) at IC₅₀ concentrations for each drug, with samples collected at 0, 4, 24, and 48 hours.

Results:

IC₅₀ values for each drug were calculated after 48 hours of treatment in GBM cells: 25 µM (ENZA), 40 µM (BIC), 108 µM (APA), 0.1 µM (CINO), and 1.2 µM (EPI) for U87MG cells; versus 40 µM (ENZA), 55 µM (BIC), 0.5 µM (CINO), and 2.5 µM (EPI) for a primary human GBM cell line. RNA-seq analyses demonstrated suppression of cancer stem cell (CSC) marker genes, including Nestin, CD133, SOX9, and AR, by CINO and BIC, but with more prominent and durable effects observed with CINO treatment. Gene Set Enrichment Analysis (GSEA) revealed much more profound immunostimulatory effects of CINO than BIC, with TNFa signaling via NF?B, IL6-JAK-STAT3 signaling, interferon ? response, and IL2-STAT5 signaling pathways ranked among the top 15 enriched pathways.

Conclusion:

N-ARAs demonstrate higher potency in suppressing GBM proliferation in vitro, with superior effects over C-ARAs in suppressing cancer stem cells and promoting immunostimulation, both of which are essential for GBM therapy. Future in vivo and clinical studies on N-ARAs are warranted.