Figure 4 from Targeting PRMT5 Inhibitor–Induced Adaptation in Pancreatic Cancer with the RBM39 Degrader Indisulam

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Synergistic response to PRMT5 and RBM39 inhibitors. A, Clonogenic assays of PSN1 cells treated with the indicated dosages of indisulam and JNJ-64619178 (above) or GSK-3326595 (below) for 9 days. One of three biological replicates is shown. B, Most synergistic area scores according to the ZIP, Loewe, and HSA models for the combination of indisulam with PRMT5 inhibition [JNJ-64619178 (left) or GSK-3326595 (right)] in four PDAC cell lines: PSN1, PaTu-8988T, HPAC, and Panc-1. Response values are from the corresponding clonogenic growth assays (n = 3) as determined in (A), and concentrations were adapted to the responsiveness of each cell line. C, Synergy distribution plots showing the ZIP synergy score of PSN1 (left) and MiaPaCa2 (right) cells treated with MRTX1719 and indisulam as indicated. Values are based on clonogenic growth assays (n = 3). D, Scheme of the generation of PDCLs. PDX, patient-derived xenotransplant. E, ZIP, Loewe, and HAS most synergistic area scores of six GoeCDX cell lines treated with JNJ-64619178 and indisulam. The experiment corresponds to at least three independent biological replicates for every cell line. F, Clonogenic assay of GoeCDX4 cells with the indicated dosages of indisulam and JNJ-64619178. One of six biological replicates is shown. G, Western blot of PRMT5 expression in GoeCDX cells. HSP90 was used as the loading control. One of three biological replicates is shown. H, Quantification of G. I, Scheme of the experimental setup of the ISRA. PSN1 cells were seeded at a low density and treated with the individual GI80 value of the indicated drugs and drug combinations (8 nmol/L JNJ-64619178, 800 nmol/L indisulam, 800 nmol/L GSK-3326595, 200 nmol/L GSK-3326595, and 400 nmol/L indisulam or 4 nmol/L JNJ-64619178 and 70 nmol/L indisulam; DMSO was used as the control). The medium with fresh therapies was changed weekly, and confluence was measured weekly for 10 weeks. Wells with a confluence more than 50% were considered resistant. J, Results of the ISRA plotted as a Kaplan–Meier curve Statistical analysis was performed with a log-rank test: ****, P < 0.0001. The results from three biological replicates (3 × 30 wells per condition) are shown. K, ISRA outgrown cell populations from the indicated conditions were further cultured in medium containing the respective drug. RBM39 protein levels were assessed by Western blotting. β-Actin served as a loading control. L, Quantification of the RBM39/β-actin ratio in control (n = 3 outgrown wells) and PRMT5i (n = 3 GSK3326595 and n = 2 JNJ-64619178 outgrown wells). Mann–Whitney test: *, P < 0.05. M, Cell populations that grew out in the control ISRA, GSK-3326595 ISRA, and indisulam ISRA were treated with vehicle (DMSO), 800 nmol/L indisulam, or 800 nmol/L GSK-3326595. Relative clonogenic growth was assessed after 7 days. The bar graph shows the percentage inhibition of clonogenic growth normalized to DMSO-treated controls, and each dot represents one population derived from a single well. For each biological replicate of the ISRA (J), three populations were analyzed, yielding a total of n = 9 population tested in one biological replicate. Statistical analysis was performed using the Kruskal–Wallis test with Dunn multiple-comparison test: **, P < 0.01. [D, Created in BioRender. Schneider, G. (2026) https://BioRender.com/0w6dd14; I, Created in BioRender. Schneider, G. (2026) https://BioRender.com/xsbxjh0.]

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