Abstract 533: Disruption of cholinergic signaling via inhibition of the M3 muscarinic acetylcholine receptor reduces glioblastoma stemness and enhances cell death

Abstract Background: Glioblastoma (GBM) is the most aggressive primary brain tumor in adults, with a median survival of less than two years despite treatment with surgery, chemotherapy, and radiation. Tumor recurrence, driven in part by treatment-resistant glioblastoma stem cells (GSCs), contributes to this poor prognosis. Understanding the mechanisms that promote GSC resistance may aid in developing targeted therapies. GBMs frequently occur in acetylcholine (ACh)-rich brain regions. ACh is a neurotransmitter responsible for cholinergic signaling through muscarinic acetylcholine receptors (mAChRs) and has been shown to regulate stemness in healthy oligodendrocyte progenitor cells. The M3 mAChR has also been shown to hold potential prognostic value in GBMs. Taken together, we investigated the role of ACh and the M3 mAChR in promoting GSC-like states and therapeutic resistance. Methods: Human GBM LN-229 cells were cultured under stem-like conditions. CRISPR/Cas9 was used to generate an M3 mAChR knockout line. To assess the role of cholinergic signaling in promoting stemness, cell lines were treated with vehicle control or 3µM of carbachol, an ACh mimetic, and sphere formation assays and immunoblotting for stem-like markers (Nestin and CD44) were performed. To assess if M3 mAChR inhibition decreases stemness and tumor cell viability, cell lines were treated with benztropine (BZT; 1 µM and 3 µM), a muscarinic antagonist, and MTT assay for cell viability was performed. All experiments were performed in triplicates. Results: Carbachol treatment increased the number of spheres compared to vehicle control (mean: 34 vs. 20, p=0.0141), as well as the sizes of the spheres (mean: 192 µM vs. 100 µM, p<0.0001). Immunoblotting after carbachol treatment increased protein expression of stemness markers and the M3 mAChR. The M3 mAChR knockout cell line demonstrated a decrease in cell viability after 48 hours of treatment compared to wildtype control (p<0.0001). Also, there was a decrease in protein expression of stemness markers in the M3 mAChR knockout cell line compared to wildtype control. Additionally, BZT treatment reduced cell viability compared to vehicle control in the MTT assay at 1 µM (p<0.0001) and 3 µM (p<0.0001). Conclusion: ACh signaling via M3 mAChR promotes stemness in GBM and disruption of cholinergic signaling through knockout of the M3 receptor decreases stemness and tumor cell viability. Also, pharmacological inhibition of cholinergic signaling with a muscarinic antagonist such as BZT may be useful to target GSCs with the ultimate goal of improving patient outcomes. Citation Format: Godwin Peasah-Darkwah, Ayanfeoluwa S. Kolawole, Tingting Huang, Grace M. Rosner, Johnathan D. Rodgers Gochicoa, Matthew C. Havrda, Kelli B. Pointer. Disruption of cholinergic signaling via inhibition of the M3 muscarinic acetylcholine receptor reduces glioblastoma stemness and enhances cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 533.