Fig 2: A, ArSulfs cause the degradation of the splicing factor RBM39 by recruiting it to the DCAF15 cullin-ring ligase; loss of RBM39 causes splicing defects. B, The arylsulfonamide compounds proven to degrade RBM39.

Fig 3: KIF20A and KIF20B are downregulated by RBM39 degradation. A, Plot of proteins changes quantified from proteomics as in Fig. 3A with their scores from RBM39 eCLIP enrichment (as colors). As shown in Fig. 3A proteomics was done in Mv4;11 cells treated with indisulam at 3 µmol/L for 8 hours. Strongly up- or downregulated sequences with high eCLIP scores are likely to be transcriptional targets of RBM39, such as KIF20A or KIF20B. B, Change in KIF20A protein levels relative to vehicle treatment (DMSO) in HCT-116 (left) or HCT-116DCAF15-/- (right) cells treated with the sulfonamides at 5 µmol/L for 6 hours (left) and quantified by taking the mean of the level of three unique peptides measured in PRM experiments. Individual dots represent mean levels of the peptides from biological quadruplicates (n = 4). Statistical analysis was performed using t test (*, P < 0.0332; **, P < 0.0021; ***, P < 0.0002). C, As for B but for KIF20B using two unique peptides. D, Western blot analysis of anti-KIF20A, anti-RBM39, and anti-tubulin of HCT-116 cells treated with vehicle (DMSO), E7820 (5 µmol/L) for 6 hours. The numbers on the Western blot analysis indicate the signal intensities of the bands relative to the tubulin bands, normalized to DMSO treatment as quantified with Empiria Studio. E, Comparison of noncoding KIF20B transcript (204) and coding KIF20B transcript (201) shows retained intron which was analyzed by primer pair B, whereby the coding transcript was analyzed by primer pair A (full transcript list Supplementary Fig. S5A). RBM39 loss leads to intron retention and preferential formation of a noncoding transcript in KIF20B splicing. F, Change in amplicon levels from primer pair A relative to vehicle treatment (DMSO) in HCT116 cells treated with the sulfonamides at 5 µmol/L for 6 hours. Values represent mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$ - {\rm{\Delta \Delta }}$\end{document}Ct values ±SEM from biological triplicates (n = 3, dots show individual values). Statistical analysis was performed using parametric unpaired t test with Welch correction (*, P < 0.0332; **, P < 0.0021; ***, P < 0.0002; for an example individual measurement see Supplementary Fig. S5B). G, As in E for primer pair B. H, As in D comparison of the coding transcript and noncoding transcripts show retained introns which were analyzed by primer pair A and B (full transcript list Supplementary Fig S5A). RBM39 loss leads to intron retention and preferential formation of a noncoding KIF20A transcript after splicing. I, As in E for primer pair A. J, As in E for primer pair B.

Fig 4: KIF20A and KIF20B downregulation leads to multinucleated cells. A, Immunofluorescence images of HCT-116 cells treated with DMSO or E7820 (5 µmol/L) for 48 hours and stained for DNA (blue), RBM39 (red), FLAG (cyan), and a-Tubulin (green). For clarification purposes, the images shown for the different channels correspond to different z-slices. B, Amount of multinucleated and mitotic cells observed in vehicle treated (DMSO) and E7820 treated HCT116 cells at 5 µmol/L for 48 hours. Values represent mean values ±SD from counting 100 cells per treatment in biological triplicates (n = 3, dots show individual values). Statistical analysis was performed using parametric unpaired t test with Welch correction (*, P < 0.0332; **, P < 0.0021; ***, P < 0.0002). Example images showed in Supplementary Fig. S3A. C, As for A but in HCT116 cells overexpressing FLAG tagged KIF20A. More example images shown in Supplementary Fig. S3B. D, As for B but in HCT116 cells overexpressing FLAG tagged KIF20A.

Fig 5: RBM39 degradation causes changes in the cell cycle. A, The PRISM dataset reveals that indisulam toxicity across hundreds of cancer cell lines correlates with the toxicity profile observed with mitotic poisons. B, Cell cycle was determined using flow cytometry. The data were analyzed using FlowJo 10.5.3 and the population quantified using the Watson (pragmatic) model. Asynchronous cells were treated with DMSO or E7820 (5 µmol/L) for 24 hours. C, Western blot analysis of anti-phospho-histone H3 (Ser10), anti-cyclin B1, anti-RBM39, and anti-tubulin of HCT-116 cells treated with vehicle (DMSO), E7820 (5 µmol/L), Tasisulam (5 µmol/L), ABT-751 (5 µmol/L), Nocodazole (325 nmol/L), or GSK-923295 (325 nmol/L) for 6 hours (right), 24 hours (middle), or 48 hours (left). The numbers on the Western blot indicate the signal intensities of the bands relative to the tubulin bands, normalized to DMSO treatment as quantified with Image Studio Lite.