Abstract 4239: Effect of MTAP expression on PRMT5 inhibitor sensitivity in bladder urothelial carcinoma models

Abstract Introduction: Methylthioadenosine phosphorylase (MTAP) is a metabolic enzyme integral to the methionine/adenine salvage pathway, whose expression is lost in 30% of bladder cancer (BCa). The cellular changes associated with MTAP loss, such as accumulation of its substrate, MTA, increase the dependence of cancer cells on other metabolic enzymes, opening opportunities for synthetically lethal targeted therapy. One such target is protein arginine methyltransferase 5 (PRMT5), which relies on the methionine-cycle intermediate SAM to methylate proteins. An emerging class of PRMT5 inhibitors exploits the increased dependence of MTAP-deficient tumors on PRMT5 to induce tumor-specific death. The present study investigates the sensitivity of genetically engineered BCa cell lines and patient-derived organoids (PDOs) to the PRMT5 inhibitor AZ14209703. Methods: MTAP was knocked out of BCa cell line 5637 (MTAP+) by 48h reverse transfection with recombinant Cas9 and MTAP or CTRL sgRNAs. MTAP was overexpressed in MTAP-null BCa cell lines RT4 and UM-UC-3 and a patient-derived organoid by lentiviral transduction with a stable MTAP overexpression (OE) plasmid or empty vector (EV). Protein expression was determined by western blot (WB). For viability assays, cells were plated at 1000 cells/well in a 96 well plate and allowed to adhere overnight before treatment. Viability was assessed 7d after AZ14209703 treatment by Cell-TiterGlo® assay, and IC50 and area under the curve (AUC) were computed. Results: WB analysis showed no detectable MTAP in 5637 cells transfected with the MTAP sgRNA-Cas9 complex and, conversely, robust overexpression in RT4, UM-UC-3, and the PDO following lentiviral transduction. In viability assays, MTAP expression conferred protection from PRMT5i in all models tested. 5637 MTAP-/- cells exhibited reduced area under the curve (AUC) and IC50 compared to the sgCTRL. MTAP OE in UM-UC-3 and RT4 increased the AUC and IC50 versus the EV. Conclusions: The present findings suggest that PRMT5i is highly selective to MTAP-deficient BCa models. Future work involving these models will characterize the sensitivity of the MTAP-OE PDO to PRMT5i, study the effects of MTAP and PRMT5i on T and NK cell killing, and determine whether MTAP status affects the potency of PRMT5i in combination with other therapies. Citation Format: Connor Purcell, Leiqing Zhang, Maryam Ghandali, Maximilian Pinho-Schwerrman, Paola Gyuliane, Brian K. Baker, Lanlan Zhou, Dinara Ryspayeva, Benedito A. Carneiro, Wafik S. El-Deiry. Effect of MTAP expression on PRMT5 inhibitor sensitivity in bladder urothelial carcinoma models [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 4239.