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. Author manuscript; available in PMC: 2016 Nov 2.
Published in final edited form as: Nat Med. 2016 May 2;22(6):672–678. doi: 10.1038/nm.4097

Figure 3. Splicing and gene expression changes in myeloid leukemias treated with E7107 in SRSF2-wildtype or mutant backgrounds.

Figure 3

(a) Schematic of secondary transplantation experimentation with timed sacrifice following E7107 for RNA-seq analyses. Sub-lethally irradiated mice were transplanted with MLL-AF9;Srsf2+/+ or MLL-AF9;Srsf2P95H/+ primary leukemias followed by 5 days of E7107 (4 mg/kg/d) or vehicle treatment. GFP+ Mac1+ double-positive BM cells were then sorted 3 hours after the fifth treatment for RNA-seq (n = 5 mice per group). (b) Multidimensional scaling analysis of all 20 mice from (a) based on alternatively spliced cassette exons (top, N = 6,369), alternatively spliced retained introns (middle, N = 1,791), and expressed protein-coding genes (bottom, N = 9,339). Scatter plots of cassette exon splicing (top rows, isoform ratios represented as Percent Spliced In (PSI, Ψ) values), retained introns (middle rows, Ψ values), and gene expression (bottom rows, normalized expression values) from (c) MLL-AF9;Srsf2+/+ or (d) MLL-AF9;Srsf2P95H/+ mice treated with vehicle or E7107. Percentages indicate the fraction of differentially spliced cassette exons or retained introns (inclusion rates increased or decreased by absolute magnitude ≥ 10% with P < 0.01), or number of differentially expressed genes (fold change ≥ 2.5 with P < 0.01). Red and blue dots represent individual splicing events or coding genes that are promoted or repressed in E7107 versus vehicle-treated cells, respectively. (e) Sashimi plots28,29 across splice junctions surrounding differentially spliced cassette exons in Dot1l and Meis1, with reads summarized across five replicate mice. Barplots represent the percent spliced (Ψ) cassette exon inclusion ratios across all samples. (f) RT-PCR analysis of the effect of acute exposure to E7107 (10 nM) on splicing of Dot1l and Meis1 in MLL-AF9;Srsf2+/+ and MLL-AF9;Srsf2P95H/+ leukemia cells in vitro. (g) Quantitative RT-PCR (qRT-PCR) analysis quantifying the relative levels of exclusion (EX) and inclusion (IN) isoforms of Dot1l and Meis1 following acute exposure to E7107 (10 nM) in vitro. Error bars represent mean ± SD. *P < 0.05; **P < 0.01.