Alternative splicing in lineage-switched and MPAL cases. (A) Pie charts showing the classification of nondifferential (non-DEEj) and differential (DEEj) exon-exon junctions. Shown are the percentages of splicing events assigned to a particular mode of splicing. A complex splicing event corresponds to several (2 or more) alternative splicing incidents occurring simultaneously in the same sample. (B) Volcano plots demonstrating differential usage of exon-exon junctions in the transcriptome of AML/myeloid vs ALL/lymphoid cells of cases of lineage switching (LS01, LS03, and LS04) or MPAL. The vertical dashed lines represent twofold differences between the AML and ALL cells, and the horizontal dashed line shows the FDR-adjusted q-value threshold of .05 (left). Venn diagrams (right) showing distribution of splice variants identified as significantly changed in AML (or myeloid fraction of patients with MPAL), including DEEjs, differential exon usage (DEU), and retained introns (RI). (C) Enrichment analysis of affected signaling pathways by the DEEjs and DEU in the lineage-switched acute leukemia (LSAL) AML relapse and myeloid compartment of patients with MPAL. Pathway enrichment analysis was performed with g:Profiler (https://biit.cs.ut.ee/gprofiler/gost) at the highest significance threshold, with multiple-testing correction (g:SCS algorithm). (D) Fold log₂ change of total transcript levels among genes affected by alternative splicing (left) and of differentially spliced variants in lineage-switched and myeloid compartments of patients with MPAL (right). (E) Representation of impact of alternative splicing on mRNA composition and open reading frames (ORFs) of select genes. Column graphs on the right indicate corresponding fold changes of variant expression between AML (or myeloid) and ALL (or lymphoid) populations in 2 tested lineage-switched cases (LS03 and LS04) and 1 case of MPAL.