Mutations in the RNA Splicing Factor SF3B1 Promote Tumorigenesis through MYC Stabilization

Activation of MYC signaling in SF3B1-mutant CLL. A, Transcriptional regulation network of significantly enriched master regulators. Nodes indicate TFs or target genes differentially expressed between SF3B1-mutant and WT CLL. B, Venn diagram showing the overlap of MYC targets and differentially expressed genes in SF3B1-mutant samples (hypergeometric test; P = 8.1e-08 for positive regulated genes; P = 9.5e-29 for negative regulated genes). C, Results of preranked GSEA on MYC target genes. Genes up/downregulated in SF3B1-mutant CLL were tested against a preranked gene list which was generated based on the correlation with MYC. D, Heat map of unsupervised clustering of differentially expressed genes based on SF3B1 genotype. Analyses in B, C, and D were performed using an expression matrix of 294 patients with CLL from the International Cancer Genome Consortium Data Portal (Z-score > 0 represents upregulation in SF3B1-mutant samples). ***, P < 0.001.

SF3B1 mutations promote MYC-driven lymphomagenesis. A and D, Kaplan–Meier curves of survival of primary (A) and recipient (D) mice transplanted with bone marrow (BM) cells from mice with indicated genotype [Log-rank (Mantel–Cox) test]. B and C, Hemoglobin (Hb; B) and platelet (PLT; C) counts of recipient mice at day 28 after transplant (the mean value ± SD is shown). E and F, Representative pictures of spleen and liver of recipient mice (E; an inch ruler is shown together) and spleen weights of recipient mice (F) at day 28 (Cd19-Cre^+/− control and Cd19-Cre^+/− Sf3b1^K700E/+ mice were sacrificed for comparison; the mean value ± SD is shown). G, Hematoxylin and eosin staining of tissues from representative moribund primary mice [scale bar, 200 μm: Low power field (LPF); 50 μm: High power field (HPF)]. H, Representative cytomorphology of BM mononuclear cells of recipient mice (scale bar, 20 μm). **, P < 0.01; ***, P < 0.001.

Aberrant splicing of PPP2R5A leads to MYC activation via post-translational regulation. A and B, (Half) volcano plot showing alternative 3′ss events in SF3B1-mutant CLL (A) and mouse Sf3b1^K700E splenic B cells (B). Genes highlighted in green represent genes that are misspliced in both human SF3B1-mutant CLL and mouse Sf3b1^K700E splenic B cells. C, Venn diagram of numbers of differentially spliced genes in indicated datasets (Fisher exact test). D, Venn diagram of numbers of differentially spliced and downregulated genes in SF3B1-mutant CLL and Nalm-6 samples. E, Sashimi plots of PPP2R5A 3′ss in isogenic Nalm-6 cells (top) and representative CLL samples (bottom) with or without SF3B1 mutations. F, Representative RT-PCR results of aberrantly spliced transcripts in BM samples from patients with CLL with or without SF3B1 mutations. G, Western blot (WB) analysis of Nalm-6 SF3B1^K700E knock-in cells transduced with shRNAs against UPF1 (representative results from three biologically independent experiments with similar results). H, Half-lives of PPP2R5A transcripts with alternative 3′ss were measured by qPCR (n = 3; the mean value ± SD is shown; P values* compared with shControl). I, Box plot of PPP2R5A expression from patients with CLL with or without SF3B1 mutations based on RNA-seq data (sources indicated with different colors; the mean value ± SD is shown). J and K, Quantitative real-time PCR (J) and WB (K) analysis of primary CLL patient samples with or without SF3B1 mutations (the mean value ± SD is shown). L and N, WB analysis of protein lysates from isogenic Nalm-6 cells (representative results from three independent experiments are shown). M, Isogenic Nalm-6 cells were treated with 100 μg/mL cycloheximide, and cell lysates were prepared at the indicated time points after treatment. The amount of MYC protein at each time point (left) was quantified using ImageJ, and protein half-life was calculated using Prism 7 (right). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Impaired apoptosis in SF3B1-mutant cells linked to increased BCL2 S70 phosphorylation. A, Volcano plot showing differentially expressed genes in isogenic SF3B1-mutant Nalm-6 cells |Log₂ (Fold change) | = 0.5 and −log₁₀(P value) = 2 are used as thresholds; genes with |x| > 5 and/or y > 30 are shown on |x| = 5 or y = 30]. B–E, qPCR of BAD (B), BIK (C), BMF (D), and BBC3 (PUMA; E) in isogenic Nalm-6 cells upon treatment with CPT at indicated concentrations for 5 hours (n = 3; the mean value ± SD is shown; P values are shown for comparisons of K700K vs. each mutant). F, Representative flow cytometry analysis of isogenic Nalm-6 cells upon treatment with CPT for 5 hours. G and H, Percentages of cells in early (Annexin V⁺ PI⁻; top, G) and late (Annexin V⁺ PI⁻; bottom, H) apoptotic phases with different concentrations of CPT (n = 3; the mean value ± SD is shown; P values are shown for comparisons of K700K vs. each mutant). I, Heat map was made using Prism 7 based on the cell growth upon CPT treatment at 1 μmol/L (n = 3; scale represents the relative cell number at each day relative to BCL2 WT–expressing cells). J, Parental Nalm-6 cells were transduced with either WT, S70A-mutant, or S70E-mutant BCL2 (all HA-tagged), treated with 5 μmol/L CPT for 5 hours, and protein lysates were immunoprecipitated with an HA antibody. Eluted protein (95% was used for BAK blotting and 5% was used for HA blotting) as well as 5% input protein were analyzed by WB. K, Isogenic Nalm-6 cells from J were treated with 5 μmol/L CPT for 5 hours, and cytochrome C release was evaluated by intracellular flow cytometry staining [left; representative flow cytometry analysis from four independent experiments; mean fluorescence intensity (MFI) is provided on the right; n = 4; the mean ± SD]. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Restoration of PPP2R5A expression in Sf3b1-mutant Eμ-Myc tumors reduces tumorigenicity. A, Schematic representation of the study design. B, Confirmation of PPP2R5A expression in Cd19-cre^+/− Sf3b1^K700E/+ Eμ-Myc^Tg/+ cells with expression of an empty vector (EV) or PPP2R5A cDNA. C and D, white blood cell (WBC; C) and PLT (D) counts of recipients (the mean value ± SD is shown). E, Representative photo of spleen (left) and spleen weight of recipients (right; the mean value ± SD is shown; an inch ruler is shown together). F, Percentages of GFP⁺ DAPI⁻ cells in BM, spleen, liver, and peripheral blood (PB; right) with representative flow cytometry analysis (left; the mean value ± SD is shown). G, Kaplan–Meier curves of survival of recipients transplanted with Sf3b1^K700E/+ Eμ-Myc tumor ± PPP2R5A expression [Log-rank (Mantel–Cox) test]. *, P < 0.05; **, P < 0.01; ***, P < 0.001.