Identification of an alternatively spliced RBM39 transcript in ALL. (A) Pie chart showing differential splicing events in B-ALL (n = 35) vs normal human CD19⁺ B cells (n = 4). (B) Pie chart showing differential splicing events in NALM6 cells treated with DMSO or with EHT1610 (5 μM) for 4 hours. (C) GO analysis of overlapping differentially spliced genes between (B) and supplemental Figure 1A. (D) Scatter plot comparison of the differential splicing events analyzed in (B) and supplemental Figure 1A. Red gene names depict the top differentially spliced events. Transcripts presenting ΔPSI > 25% are shown. (E) Fold-change (day 20/day 4) in sgRNA abundance in pooled RBP-focused negative selection screen in NALM6 cells. Red dots indicate RBPs that are the top differentially spliced genes shown in (D). Each dot represents the average of all sgRNAs that targeted an RBP. (F) Sashimi plots depicting splicing and exon-exon junctions for an alternative splicing event in the RBM39 transcript in NALM6 cells treated with EHT1610 for 4 hours. The gene is shown along the horizontal axis. Thicker sections represent exons that code for protein sequence. Numbers over the lines that are connecting exons represent the number of reads mapped to that exon-exon junction. (G) NALM6 cells were treated with EHT1610 (5 μM) or GNF2133 (5 μM) for 4 hours. PCR reactions were performed for the detection of an RBM39 splicing event (upper). Schematic plot showing the detection of a PTC insertion between exon 2 and exon 3 of RBM39. Red and black dots represent the NMD and canonical isoforms, respectively (lower). (H) Western blot for UPF1 upon silencing of UPF1 (shUPF1.1 and shUPF1.2) in NALM6 cells (left). PCR reactions were performed for the detection of RBM39 splicing events in indicated cells with EHT1610 (1 μM) for 4 hours (right). (I) Inclusion level in normal human CD19⁺ B cells (n = 4) and B-ALL (n = 35) for the identical alternative spliced event in RBM39 transcript shown in (F). ∗P < .05. (J) Western blot analysis of RBM39 in CD19⁺ B cells from 3 different donors, ALL cell lines, and B-ALL PDX samples (upper). Quantification of the RBM39 protein normalized to GAPDH is shown (lower). (K) B-ALL PDX samples were treated with EHT1610 (5 μM) and GNF2133 (5 μM) for 4 hours. PCR reactions were performed for the detection of RBM39 splicing events. (L) Inclusion level in thymocytes (n = 3), CD4⁺ (n = 2), CD3⁺ (n = 3), and T-ALL (n = 3) for the identical alternative spliced event in the RBM39 transcript is shown in (F). ∗P < .05. Data were acquired from GSE139622.⁹ A3SS, alternative 3′ splice sites; A5SS, alternative 5′ splice sites; AS, alternative splicing; FDR, false discovery rate; PSI, percent spliced in; PTC, premature stop codon; RI, intron retention.