Cis elements within EVI1 required for generation of the EVI1+18 bp splice variant by mutant SF3B1. (A) EVI1 gene structure and protein domains (top). Inset illustrates the transcripts when +18 nucleotides (red rectangle) are excluded (top) or included (bottom). Green A and red A indicate the branchpoint for canonical and aberrant transcripts, respectively. Single underlining indicates sequence motifs that were subsequently mutated in the minigene assay (each individual minigene construct is named “MT1” to “MT13”). aa, amino acid. (B) RT-PCR analysis of the +18 nucleotides inclusion in a minigene (top) or endogenous (bottom) context following transfection of minigenes with the illustrated mutations into SF3B1-K666N knocked-in K562 cells and SF3B1-WT K562 cells. (C) Schematic of the model proposed by which EVI1 rearrangements and SF3B1 mutations promote leukemia development. As previously demonstrated, structural rearrangements at chromosome 3q reposition the GATA2 distal enhancer to upregulate EVI1 expression while simultaneously downregulating GATA2. As shown in this study, approximately one-third of patients with EVI1 rearrangements harbor concomitant change-of-function mutations in SF3B1, which promote use of an aberrant intron-proximal branch site within intron 12 of EVI1. This splicing alteration generates a stable unannotated transcript of EVI1 (“EVI1+18”), which is translated to express an EVI1 protein with insertion of 6 amino acids at the C-terminal end of the second ZF domain of EVI1. The EVI1+18 isoform is expressed whenever any recurrent cancer hotspot mutations in SF3B1 is present in cells with human EVI1 expression. Although EVI1+18 is not sufficient for leukemia transformation on its own, EVI1+18 enhances leukemogenicity in the setting of the EVI1 rearrangement and alters the chromatin localization of EVI1 to loci well known to be involved in leukemia development (such as MEIS1 and the HOXB locus).